**The Fabrication of Calcium Alginate Beads as a Green Sorbent for Selective Recovery of Cu(II) from Metal Mixtures**

**Niannian Yang 1, Runkai Wang 1,\*, Pinhua Rao 1,\*, Lili Yan 1, Wenqi Zhang 2, Jincheng Wang <sup>1</sup> and Fei Chai <sup>1</sup>**


Received: 3 April 2019; Accepted: 13 May 2019; Published: 17 May 2019

**Abstract:** Calcium alginate (CA) beads as a green sorbent were easily fabricated in this study using sodium alginate crosslinking with CaCl2, and the crosslinking pathway was the exchange between the sodium ion of α-L-guluronic acid and Ca(II). The experimental study was conducted on Cu(II), Cd(II), Ni(II) and Zn(II) as the model heavy metals and the concentration was determined by inductively coupled plasma optical emission spectrometry (ICP-OES). The characterization and sorption behavior of the CA beads were analyzed in detail via using scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The adsorption experiments demonstrated that the CA beads exhibited a high removal efficiency for the selective adsorption of Cu(II) from the tetra metallic mixture solution and an excellent adsorption capacity of the heavy metals separately. According to the isotherm studies, the maximum uptake of Cu(II) could reach 107.53 mg/g, which was significantly higher than the other three heavy metal ions in the tetra metallic mixture solution. Additionally, after five cycles of adsorption and desorption, the uptake rate of Cu(II) on CA beads was maintained at 92%. According to the properties mentioned above, this material was assumed to be applied to reduce heavy metal pollution or recover valuable metals from waste water.

**Keywords:** alginate beads; green sorbent; selective adsorption; heavy metals

#### **1. Introduction**

In recent years, agriculture and the mining, chemical fertilizer, leather, battery and paper industries have developed vigorously, and the phenomenon of heavy metal wastewater directly or indirectly being discharged into the environment has become more and more serious, particularly in developing countries [1,2]. Heavy metal ions in wastewater are mainly comprised of zinc (Zn), nickel (Ni), cadmium (Cd), copper (Cu) and so on. Among them, copper is widely used in industrial production, such as in printed circuit boards (PCBs), and it is also one of the indispensable nutrients (trace elements) in the human body [3,4]. When copper-containing wastewater is discharged into the environment beyond its self-purification range, the high toxicity and non-biodegradability of copper ions poses a serious threat to animal and human health. Hence, investigating how to effectively remove copper ions from wastewater is very important to the ecological environment. In addition, the recovery of copper from wastewater also has certain economic benefits.

To make the recovery of Cu more meaningful, the literature reported many methods. For example, Coruh et al. [5] selected vitrification as the technology to deal with industrial copper, mixing it

with other inorganic wastes and materials and sintering them into glass for reuse. Printed circuit boards (PCBs) are widely used in the electronics industry [6,7], resulting in a large amount of copper-containing wastewater. Liu et al. [8] used electrolysis to recover 97% copper from waste PCBs, and Mdlovu et al. [9] reported the use of the microemulsion process to recover copper nanoparticles with diameters of 20–50 nm. In addition to the methods reported above, adsorption was also selected to treat copper-containing wastewater due to its advantages, such as low initial cost and process simplicity [10,11]. However, the instability of the adsorbents and difficulty in the separation still limit their practical applications. Overall, for the treatment of heavy metals in wastewater, availability and cost effectiveness play an important role in the synthesis of the adsorbents. This has made people pay attention to abundant, renewable and environmentally-friendly marine resources, such as bio-sorbents [12]. Torres-Caban et al. [13] proved that a calcium alginate/spent coffee grounds composite bead was an effective biological absorbent for the removal of copper ions, which first made us notice the role of alginate in the process of adsorption.

Alginate derived from brown algae is a highly popular material for the biosorption of heavy metals due to its advantages such as low cost and high affinity via gelation [14,15]. Abundant functional groups have been found in sodium alginate, such as carboxyl and hydroxyl groups, which can crosslink with cations [16,17]. The reason for this is that the carboxyl group is a negative group, and it can adsorb electrostatically with heavy metal ions and produce chelation at the same time. Sodium alginate reacts with divalent cations such as Ca(II), Ba(II) and Sr(II), to form insoluble hydrogels, which are crosslinked to form a reticular structure called the "egg box" structure, and the crosslinking pathway is the exchange between the sodium ions of α-L-guluronic acid and divalent ions [18,19]. In all types of alginate materials, alginate beads can be easily recovered from water [20,21]. Consequently, calcium alginate (CA) is a promising biomaterial for the biosorption of heavy metals [22]. In previous studies, it has been found that CA has a selective adsorption effect on some metal ions, which is extremely important for the recovery and re-utilization of metal ions. Hence, we want to investigate whether CA has selective adsorption on copper under the interference of cadmium, zinc and nickel metal ions.

In our study, the alginate beads as a green sorbent were easily fabricated with the Ca(II) crosslink, maintaining the high efficiency of the selective recovery of Cu(II) from the metal mixtures and the good adsorption of the heavy metals separately. The experimental study was conducted on Cu(II), Cd(II), Ni(II) and Zn(II) as the model heavy metals. The morphology and structure, functional groups, adsorption mechanism of CA beads were investigated by various characterization methods, such as scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). All pH measurements were adopted using a LEICI PHS-2F pH meter. Additionally, the alginate beads showed good reusability after five rounds of simple sorption–desorption procedures.

#### **2. Materials and Methods**

#### *2.1. Materials*

Sodium alginate and calcium(II) chloride (CaCl2) were purchased from Adamas-beta. Calcium chloride was used for crosslinking of the alginate beads and sodium alginic acid was used to fabricate the alginate beads. Four types of heavy metals, Cu(NO3)2·3H2O, Zn(NO3)2·6H2O, Ni(NO3)2·6H2O and Cd(NO3)2·4H2O, were purchased from Aladdin. To adjust the pH of the solution, 1 M hydrochloric acid (HCl) and 1 M sodium hydroxide (NaOH) were applied, which were acquired from Sinopharm Chemical Reagent Co., Ltd. All other chemicals used in this study were of analytical grade without purification. Distilled water (DW) with specific resistivity greater than 18 MΩ·cm was used in all experiments.

#### *2.2. Preparation of Calcium Alginate Beads*

Sodium alginate powder was added to distilled water to obtain a yellow viscous sodium alginate solution. The obtained solution remained stationary until there were no air bubbles. To synthesize the spherical bio-sorbent, sodium alginate solution was added dropwise into CaCl2 (1%, w/v) solution under gentle stirring with a dropper and the bead was formed immediately. After 2 hours of curing, the sphere became compact, and a CA bead with a diameter of about 3 mm was obtained. The gel ball was rinsed with distilled water several times to remove free Ca(II) and stored in distilled water for further use.

#### *2.3. Material Characterizations*

The concentration of Cu(II), Zn(II), Ni(II) and Cd(II) used in all experiments was determined by inductively coupled plasma optical emission spectrometry (ICP-OES, Varian 700-ES, Walnut Creek, CA, USA), using 2% HNO3 as the medium. The standard solution and the solution of heavy metal ions to be measured were acidic. Scanning electron microscopy (SEM, Hitachi SU8010, Ibaraki, Japan) was used to obtain the information of the physical structure and morphology of alginate beads. Fourier transform infrared spectroscopy (FTIR, PerkinElmer Spectrum Two, Waltham, MA, USA) was recorded in the 400–4000 cm−<sup>1</sup> region on a FTIR spectrophotometer using a KBr disk method. Thermogravimetric analysis (TGA, TA Q600 SDT, New Castle, DE, USA) was carried out in a nitrogen gas flow from room temperature to 600 ◦C with a heating rate of 10 K/min. The point of zero charge (STARTER 2100, Parsippany, NJ, USA) was measured within the pH range from 3.0 to 10.0 by addition of 0.1 N HCl and NaOH. The samples were further analyzed by X-ray photoelectron spectroscopy (XPS, Thermo Fisher 250XI, Waltham, MA, USA) for the Cu 2p, Cd 3d, Zn 2p and Ni 2p regions. The charging shifts of the spectra were calibrated by placing the C1s peak at 284.8 eV from the adventitious carbon. Then, the results obtained from XPS were analyzed using the non-linear least squares curve fitting program (XPSPEAK4.1, software, Hong Kong, China).

#### *2.4. Adsorption Experiments*

For kinetic studies, an adsorption experiment was carried out using 200 mg/L of Cu(II), Zn(II), Ni(II) and Cd(II) in distilled water, respectively, of which the initial pH was 5.5, 6.3, 6.6 and 6.6, respectively. The saturated adsorption time was determined, and the samples were taken at a predetermined time interval. The adsorption isotherms of CA beads in a monomer solution and a tetra-metallic mixture solution were studied to evaluate their saturated adsorption capacity. The beads (1 g) were fully dispersed in 50 mL of monometallic solution and tetra-metallic mixture solution with different concentrations ranging from 50–800 mg/L at 120 r/min for 12 h at a temperature of 25 ◦C in a thermostabilized warm bath. In the pH effect experiment, five groups of tetra-metallic mixture solutions with a pH of 2.0, 3.0, 4.0, 5.0 and 6.0, respectively, were prepared and adjusted by adding NaOH (0.1 mol/L) or HNO3 (0.1 mol/L).

The suspended impurities in the solution were removed by membrane filtration. The concentration of the metal ions was determined by ICP-OES and the amount of adsorption could be calculated using the following equation:

$$\mathbf{q} = \frac{(\mathbf{C}\_0 - \mathbf{C})\mathbf{V}}{\mathbf{M}} \tag{1}$$

where q is the adsorption of the metal ions (mg/g), C0 and C are the initial and final metal ion concentrations, respectively (mg/L), V is the total volume of suspension (L), and M is the dry mass of adsorbent (g).

#### *2.5. Desorption and Reuse Experiments*

Desorption experiments were also conducted using 1% CaCl2 solutions and 0.1 M HNO3 as the desorption reagents. In this experiment, 1 g of CA beads were added to 50 mL tetra-metallic mixture solutions with pH 5.7. After sorption, the heavy metal loaded CA beads were subsequently suspended in a 0.1 M HNO3 eluting agent to evaluate the desorption performance. Then, the CA beads were washed with 1% CaCl2 solutions to make them neutral and distilled water several times to remove the free Ca(II) ions from the beads. Four sorption–desorption cycles were conducted to assess the reusability of CA beads.

#### **3. Results and Discussion**

#### *3.1. Characterization of CA Beads*

The CA beads were synthesized through crosslinking with a diameter of 3 mm. Figure 1a shows the overall appearance of CA beads after drying; the diameter was reduced to about 1 mm. Figure 2b,c shows that there were a lot of ravines on the surface of CA beads, which increased the surface area and provided more adsorption sites. The surface of the CA bead was composed of wire-like Ca-alginate and a honeycomb network could be observed in the internal structure of CA (Figure 1d).

**Figure 1.** Scanning electron microscopy (SEM) images of the outer surface (**a**–**c**) and the internal structure (**d**) of the dried calcium alginate (CA) bead.

**Figure 2.** Thermogravimetric analysis (TGA) curves of the CA beads.

To evaluate the hydration and thermal decomposition of the CA beads, thermogravimetric analysis (TGA) was applied. As seen in Figure 2, the first mass loss was attributed to the dehydration process and a 12.6 wt% loss up to 210 ◦C. The second part was a thermal degradation stage, which resulted in pyrolysis from 210 ◦C to 510 ◦C with a weight loss of 45.8 wt%. The third part represented the conversion of the remaining materials to carbon residues and CaCO3 and the residual weights were 40.2 wt% [23].

#### *3.2. Adsorption Kinetics*

In industrial applications, adsorption kinetics are important for process design and operation, which required us to achieve adsorption equilibrium under certain system conditions. The kinetic behavior of the metal ions removed by CA beads is presented in Figure 3. The experimental data were fitted with pseudo-first-order and pseudo-second-order kinetics [24,25].

$$\text{Pseudo} - \text{first} - \text{order model}: \ q\_{\text{lt}} = q\_1 (1 - \exp(-\mathbf{k}\_1 \mathbf{t})) \tag{2}$$

$$\text{Pseudo} - \text{second} - \text{order model}: \ q\_t = \frac{\mathbf{q}\_2^2 \mathbf{k}\_2 \mathbf{t}}{1 + \mathbf{q}\_2 \mathbf{k}\_2 \mathbf{t}} \tag{3}$$

where both q1 and q2 are the amount of metal ions adsorbed at equilibrium (mg/g), qt is the amount of metal ions adsorbed at any time t (mg/g), k1 and k2 are equilibrium rate constants of the pseudo-first-order and pseudo-second-order, respectively (g/mg min).

**Figure 3.** Kinetic adsorption plots of Cu(II), Zn(II), Ni(II) and Cd(II) by CA beads, respectively. Reaction conditions: T = 293 K, [Cu(II)]0 = [Zn(II)]0 = [Ni(II)]0 = [Cd(II)]0 = 200 mg/L.

The results for the kinetic model parameters are summarized in Table 1. The higher coefficient of determination (R2) value of the pseudo-second-order model demonstrated that the adsorption rate was dominated by the rate of the chemical reaction [26]. In this experiment, the ion exchange occurred between the metal ions and Ca(II) and the adsorption rate of the CA was dependent on the exchange rate. The obtained k2 values for Cu(II), Zn(II), Ni(II) and Cd(II) adsorption onto CA beads indicated that the adsorption rate was Ni(II) > Zn(II) > Cd(II) ≈ Cu(II).


**Table 1.** Parameters of pseudo-first-order and pseudo-second-order kinetic models. q1 and q2 = the amount of metal ions adsorbed at equilibrium; k1 and k2 = equilibrium rate constants of the pseudo-first-order and pseudo-second-order, R2 = coefficient of determination, respectively.

#### *3.3. Adsorption Isotherm*

In order to evaluate the maximum adsorption capacity of CA beads, the adsorption isotherms were measured in the initial metal concentrations ranging from 0–800 mg/L. The adsorption equilibrium data were fitted with Langmuir and Freundlich isotherm models [27,28].

$$\text{Langmuri model}: \ \mathbf{q}\_{\text{e}} = \frac{\mathbf{q}\_{\text{m}} \mathbf{K}\_{\text{L}} \mathbf{C}\_{\text{e}}}{1 + \mathbf{K}\_{\text{L}} \mathbf{C}\_{\text{e}}} \tag{4}$$

$$\text{Freundlich model : } \mathbf{q}\_{\mathbf{c}} = \mathbf{K}\_{\mathbf{F}} \mathbf{C}\_{\mathbf{c}}^{1/\mathbf{n}} \tag{5}$$

where qe is the equilibrium adsorption capacity of heavy metal ions (mg/g), qm indicates the maximum adsorption capacity for heavy metal ions (mg/g), Ce is the equilibrium concentration after adsorption (mg/L), KL and KF denote equilibrium constants of Langmuir (L/mg) and Freundlich (mg/g) (L/g), respectively, and n is the Freundlich exponent.

Figure 4 shows the adsorption isotherm of Cu(II), Zn(II), Ni(II) and Cd(II) separately on the CA beads and Table 2 lists the parameter values along with their correlation coefficients. The CA beads showed good adsorption towards Cu(II), Zn(II), Ni(II) and Cd(II), with a maximum adsorption of 140.55 mg/g, 174.60 mg/g, 114.69 mg/g and 216.82 mg/g, respectively. As shown in Table 3, calcium alginate beads exhibited significant advantages over other low-cost biosorption materials in terms of their maximum adsorption capacity for Cu(II).

Figure 5 shows the selective removal of Cu(II) on CA beads. With the increase of equilibrium concentration, the adsorption effect of CA beads on Cu(II) first increased rapidly and then tended to slow until equilibrium was reached, while the adsorption capacity of Zn(II), Ni(II) and Cd(II) decreased slowly. The fitting parameters of the Langmuir and Freundlich models are shown in Table 2 and the adsorption data were more consistent with the Langmuir model (R<sup>2</sup> = 0.9920) than the Freundlich model (R<sup>2</sup> = 0.8126) according to the Cu(II)\* adsorption coefficient, which suggested that the adsorption of Cu(II) was the mono-layer sorption during the sorption process. Due to the competitive sorption between Cu(II) and other metals, the isotherm data of Zn(II), Ni(II) and Cd(II) were unable to be fit by the Langmuir or Freundlich models. Therefore, the CA beads showed better sorption capacity toward Cu(II) selectively than the other three heavy metals, indicating that the CA beads could be applied to the selective recovery of Cu(II) from polymetallic solutions.


**Table 2.** Parameters of the Langmuir and Freundlich models. qm = the maximum adsorption capacity for heavy metal ions; KL and KF = equilibrium constants of Langmuir and Freundlich, respectively; n = Freundlich exponent; R2 = coefficient of determination.

\* Parameters of Langmuir and Freundlich models for selective adsorption of Cu(II) by CA in mixed solution.

**Table 3.** Comparison of adsorption capacity of low-cost adsorbents for Cu(II).


Where, IDA, EDTA and DTPA represent iminodiacetic acid, ethylene diamine tetraacetic acid, and diethylenetriamine pentaacetic acid, respectively.

**Figure 4.** The adsorption isotherm by CA beads in the Cu(II), Zn(II), Ni(II) and Cd(II) heavy metal solution at different initial concentrations. Reaction conditions: T = 293 K, [Cu(II)]0 = [Zn(II)]0 = [Ni(II)]0 = [Cd(II)]0 = 0–800 mg/L, reaction time = 12 h.

**Figure 5.** The selective removal and adsorption isotherm by CA beads in the mixed heavy metals solution at different initial concentrations. Reaction conditions: T = 293 K, [Cu(II)]0 = [Zn(II)]0 = [Ni(II)]0 = [Cd(II)]0 = 0–800 mg/L, reaction time = 12 h. qe = the equilibrium adsorption capacity of heavy metal ions; Ce = the equilibrium concentration after adsorption.

A dimensionless RL constant was introduced to reveal the essential characteristics of the Langmuir model, and the adsorption process was further evaluated. The correlation values of RL could be calculated from the following equation.

$$R\_L = \frac{1}{1 + \mathbf{b}C\_i} \tag{6}$$

where *Ci* is the initial concentration of Cu(II) (mg/L) and b is the dimensionless constant of Langmuir. The values of *RL* indicated that the adsorption process may be an unfavorable trend (*RL* > 1), linear (*RL* = 1), favorable (0 < *RL* < 1) or irreversible (*RL* = 0) [34]. Here, the adsorption process of Cu(II) by CA beads showed that the b value was 0.0639, the calculated RL value was within the range of 0.0175–0.2434 and just fell within the range of 0–1, indicating that CA beads were favorable to the Cu(II) adsorption process.

#### *3.4. E*ff*ect of pH*

The effect of pH on Cu(II) adsorption by CA beads in the mixed metal solutions was investigated with the pH value in the range of 2–6. Figure 6 shows the uptake capacities of heavy metals using the CA beads at various pH values. When the pH value was lower than 2.0, few heavy metal ions were adsorbed due to the competition of the many H<sup>+</sup> ions. With the increase of initial pH value from 2 to 6, the adsorption capacity of CA beads to Cu(II) increased significantly, and when the pH was 6, the maximum adsorption was observed. However, there was no obvious change in the adsorption amount of the other three kinds of metal ions, which were only slightly increased.

The point of zero charge (PZC) was employed to analyze the surface charges of the CA beads, and the measured value was 8.2. When the pH was lower than 8.2, the surface of the material was positively charged because of the introduction of calcium ions. At a pH above 8.2, there was a negative charge due to the reaction between the -OH on the surface and the OH· in the solution, leading to the formation of a negatively charged functional group, O−. Therefore, the adsorption process of heavy metal ions by CA beads could be affected by pH, and there is a competition mechanism between H<sup>+</sup> and metal ions with carboxyl groups in acidic environments. Additionally, the carboxylic (-COOH) and hydroxyl (-OH) groups of the CA beads were changed to the protonated forms and the Ca(II) was released into the solution under acidic conditions [35].

**Figure 6.** Effect of pH in a quaternary metal system. Reaction conditions: T = 293 K, [Cu(II)]0 = [Zn(II)]0 = [Ni(II)]0 = [Cd(II)]0 = 200 mg/L, reaction time = 12 h.

#### *3.5. Adsorption Mechanism Analysis*

FTIR was used to analyze the molecular structure of chemical compounds and the FTIR spectra of CA beads is shown before and after metal ion adsorption in Figure 7. For the CA beads before adsorption, the dominant peak at 3425.28 cm−<sup>1</sup> was due to the vibration stretching of the O-H bond, indicating that hydroxyl (-OH) groups existed in the beads. Bands at 2926.01 cm−<sup>1</sup> referred to the vibration stretching of -CH. The absorption peaks around 1610.24 cm−1, 1418.32 cm−<sup>1</sup> and 1034.37 cm−<sup>1</sup> could be attributed to the asymmetric and symmetric stretching vibrations of -COO (carboxylate) and the stretching vibration of C-O, respectively [36]. After the sorption of metal ions, the FTIR spectra displacement slightly changed. This may be because the increased ionic volume weakened the stretching and torsional vibration of the functional groups, thus causing the displacement of adsorption peaks. The decrease of peak vibration intensity indicated that metal ions bind to -OH and -COO formed in the CA beads. In addition, no new peaks were produced, indicating that the functional groups of the adsorbents did not change, and the ion exchange process between the metal ions and the CA beads was possible.

**Figure 7.** FTIR images of CA beads before and after the adsorption.

Figure 8 shows the XPS diagram of Cu 2p, Cd 3d, Zn 2p and Ni 2p (after sorption), further exploring the adsorption mechanism. The binding energy of Cu 2p, Cd 3d, Zn 2p and Ni 2p were 933.55/953.44 eV, 405.59/412.34 eV, 1021.97/1044.94 eV and 856.96 eV, respectively, after deconvolution, indicating that the adsorbed metal ions were in chemical states without further oxidation or reduction and the valence states were not changed during the ion exchange process [37–40].

**Figure 8.** X-ray photoelectron spectroscopy (XPS) spectra of CA beads after the adsorption of mixed metal ions at a concentration of 500 mg/L in aqueous solution.

Cu(II) preferentially bound to the active sites of CA beads when the four ions were coexistent at the same concentration (Figure 9). The selective adsorption mechanism of copper ions on CA beads was via divalent metal ions exchanging ions with Ca(II) in the "egg box" structure [41]. The adsorption sites were mainly negative groups (COO-) that could adsorb the cation in the solution, and the stability of the bond with Cu(II) was better [42]. Therefore, compared with Zn(II), Ni(II) and Cd(II), the affinity for Cu(II) was the strongest in the ion exchange process between Ca(II) and metal ions in CA beads. This competitive relationship made the CA bead a potential material that could be applied to the recovery the copper ions from heavy metal mixtures.

**Figure 9.** Effect of Cu(II) competitive adsorption in a quaternary metal system. Reaction conditions: T = 293 K, pH = 5, [Cu(II)]0 = [Zn(II)]0 = [Ni(II)]0 = [Cd(II)]0 = 1 mM/10 mM, reaction time = 12 h.

#### *3.6. Desorption and Reusability Experiment*

Regeneration of loaded sorbent is a key factor in water treatment processes and the desorption of the adsorbed Cu(II), Zn(II), Ni(II) and Cd(II) ions with 0.1 M HNO3 was investigated. Figure 10 showed the reusability of CA beads with four heavy metal ions. The results revealed that after five cycles, the CA beads still had selective adsorption for Cu(II) and the adsorption capacity was more than 92%. Therefore, it can be explained that CA beads have good reusability during reuse experiments and have economic potential in wastewater treatment.

**Figure 10.** Effect of recycling on Cu(II), Zn(II), Ni(II) and Cd(II) ion adsorption. Reaction conditions: T = 293 K, [Zn(II)]0 = [Ni(II)]0 = [Cd(II)]0 = 200 mg/L; reaction time = 12 h.

#### **4. Conclusions**

A spherical CA bead with a diameter of 3 mm was prepared by crosslinking the hydroxyl and carboxyl groups of sodium alginate with Ca(II) to form an insoluble hydrogel; there were a large number of active sites in the porous honeycomb structure for metal ions to attach. According to the pseudo-second-order and Langmuir isotherm model, the adsorption mechanism was explained well and the chemical reaction dominated the rate of the adsorption process. The maximum uptake of Cu(II) could reach 107.53 mg/g in the mixed heavy metal solution and the valence state of the four metal ions was not changed according to XPS analysis during the adsorption process. Cu(II) exchanged ions with Ca(II), binding with α-L-guluronic acid in the "egg box" structure. The selective adsorption was indicated through the isotherm experiments, giving this material a high potential for the continuous treatment of the selective recovery of copper from multi-metal solutions.

**Author Contributions:** Conceptualization, R.W. and P.R.; methodology, N.Y. and F.C.; software, L.Y. investigation, N.Y., R.W. and W.Z.; resources, R.W. and J.W.; writing—original draft preparation, N.Y.; writing—review and editing, N.Y., R.W. and P.R.; supervision, R.W. and P.R.; project administration, R.W., J.W. and P.R.; funding acquisition, R.W. and J.W.

**Funding:** This research was funded by Shanghai Sailing Program (Grant No. 17YF1407200), the "Capacity Building Project of Some Local Colleges and Universities in Shanghai" (Grant No. 17030501200), SUES Sino-foreign cooperative innovation center for city soil ecological technology integration (Grant No. 2017PT03) and the Project of Shanghai University Young Teacher Training Scheme (Grant No. ZZGCD16018).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Luminescent Layered Double Hydroxides Intercalated with an Anionic Photosensitizer via the Memory Effect**

#### **Alexandre C. Teixeira 1, Alysson F. Morais 1, Ivan G.N. Silva 1, Eric Breynaert <sup>2</sup> and Danilo Mustafa 1,\***


Received: 12 February 2019; Accepted: 9 March 2019; Published: 14 March 2019

**Abstract:** Layered double hydroxides (LDHs) containing Eu3+ activators were synthesized by coprecipitation of Zn2+, Al3+, and Eu3+ in alkaline NO3 −-rich aqueous solution. Upon calcination, these materials transform into a crystalline ZnO solid solution containing Al and Eu. For suitably low calcination temperatures, this phase can be restored to LDH by rehydration in water, a feature known as the memory effect. During rehydration of an LDH, new anionic species can be intercalated and functionalized, obtaining desired physicochemical properties. This work explores the memory effect as a route to produce luminescent LDHs intercalated with 1,3,5-benzenetricarboxylic acid (BTC), a known anionic photosensitizer for Eu3+. Time-dependent hydration of calcined LDHs in a BTC-rich aqueous solution resulted in the recovery of the lamellar phase and in the intercalation with BTC. The interaction of this photosensitizer with Eu3+ in the recovered hydroxide layers gave rise to efficient energy transfer from the BTC antennae to the Eu3+ ions, providing a useful tool to monitor the rehydration process of the calcined LDHs.

**Keywords:** layered double hydroxide; memory effect; rare earth; europium; 1,3,5-benzenetricarboxylic acid

#### **1. Introduction**

Layered double hydroxides (LDHs), also called cationic clays, are a class of anion-exchange materials with a general chemical formula of [MII1−xMIIIx(OH)2] x+[An−x/n] <sup>x</sup>−·yH2O (M: metal, A: anion). The isomorphic substitution of divalent metal cations (MII) in otherwise neutral brucite-like MII(OH)2 sheets with trivalent cations (MIII) introduces positive charges in the hydroxide layers. In the overall LDH structure, these charges are compensated by the intercalation of anions (An−) in the interlamellar space, as illustrated in Figure 1.

Synthetic LDHs exhibit a wide flexibility in their composition, as a score of metal cations and polyvalent anions can be introduced in the structure, tuning their chemico-physical properties [1–3]. By changing both the interlayer and the metal components, LDHs have been tuned towards different applications, serving as heterogeneous catalysts, catalyst supports [4], water treatment agents [5], luminescent materials [6–9], etc.

**Figure 1.** Layered double hydroxides (LDHs) are built up from sheets of [MII1−xMIIIx(OH)2] x+ octahedral units intercalated with anions. Each octahedron is formed from a metal cation (MII or MIII) 6-coordinated to OH<sup>−</sup> groups (red spheres).

The introduction of trivalent rare earth elements (RE3+) in LDH matrixes has revealed a series of 2D structured materials that have promising luminescent properties [7,10]. Rare earth elements form a subgroup of column 3 in the periodic table and exhibit very low influence of the ligand field in the energy of their spectral lines. This characteristic optical property results from the shielding of the 4f sub-shell by their filled 5d and 6s most external sub-shells [11,12]. Additionally, RE3+ ions present very low molar extinction coefficients because their 4f intraconfigurational transitions are forbidden, as demonstrated by Laporte's rule (Δ- = ± 1). To increase the total observable luminescence, it is necessary to embed these elements in highly light-absorbing matrixes that serve as harvesting antennae, transferring the harvested energy to the luminescent center (antenna effect). This increases the excited state population of the RE3+, thereby increasing the overall luminescence [13–15].

The antenna effect has been demonstrated in RE-containing LDHs synthesized by direct coprecipitation with anionic antenna molecules or by exploiting anion exchange to intercalate anionic sensitizer ligands in the interlamellar space. A series of anionic sensitizers, including 4-biphenylacetate [8], sulfonates, and other carboxylates [16,17], have been intercalated in LDHs to incorporate RE3+ in their hydroxide layers. Several authors have also reported LDHs intercalated with Eu3+ complexes, where not only the ligand, but also the RE3+ is located in the interlamellar space [6,18].

Controlled thermal decomposition of LDHs converts their structure into a mixed metal oxyhydroxide phase, before reaching a fully oxidic end product [5,9,19,20]. Starting from the oxyhydroxide phase, the lamellar LDH structure can be recovered by re-hydrating the product in aqueous solutions containing anions, a phenomenon known as the memory effect [5,9,20–23]. During this process, selective adsorption of dissolved anionic species An<sup>−</sup> can efficiently occur, as indicated by the large anion exchange capacity observed for thermally treated samples [24]. The most frequently investigated application of the memory effect in LDHs involves the adsorption and subsequent removal of anionic dyes from waste water [5,25]. A less explored application is its use for the intercalation of anionic photosensitizing molecules.

The memory effect of the layered double hydroxides has been described in the literature for a large number of combinations of metal cations [21,23,26,27]. Different applications can be explored for different chemical compositions. For instance, Wong et al. [27] have shown that the memory effect of LDHs containing Li and Al can be used to sense water uptake in organic coatings. Ni et al. [25] have explored the memory effect of Zn/Al LDHs in the removal of methyl orange from aqueous solutions. Targeting environmental remediation, Gao et al. [23] have investigated the influence of humic acid on

the memory effect of Mg/Al and Zn/Al LDHs. The removal of boron species from waste water was also explored [28].

In this work, an underused strategy for generating luminescent LDHs via the memory effect was explored. The luminescent and structural properties of thermally treated [Zn2Al0.95Eu0.05(OH)6]·(NO3 −) LDHs after hydration in the presence of 1,3,5-benzenetricarboxilate (BTC) were investigated. Calcination was performed at 350 and 600 ◦C, followed by a time-dependent rehydration process in a BTC-rich aqueous solution, equilibrating the samples over a period between 1 min and 5 days. The resulting hydrated phases were characterized by both powder X-ray diffraction and luminescence spectroscopy.

#### **2. Materials and Methods**

H3BTC (97 mol%, Sigma-Aldrich), Zn(NO3)2·6H2O (98 mol%, Vetec), Al(NO3)3·9H2O (98 mol%, LabSynth), and NaOH (97 mol%, Vetec) were used without further purification. Eu(NO3)3·6H2O was prepared by dissolving Eu2O3 (CSTARM, 99,99 mol%) in concentrated nitric acid, which led to the subsequent crystallization of Eu(NO3)3·6H2O in accordance to the procedure described by Silva et al. [29].

#### *2.1. Sample Preparation*

Layered double hydroxides with nominal molar composition [Zn2Al0.95Eu0.05(OH)6]·(NO3 −) (ZnAlEu-NO3 LDH) and [Zn2Al0.95Eu0.05(OH)6]·(BTC3−)0.33 (ZnAlEu-BTC LDH) were synthesized by coprecipitation. A volume of 10 mL of a 1 mol L−<sup>1</sup> solution containing the metal precursors Zn(NO3)2·6H2O, Al(NO3)2·9H2O, and Eu(NO3)2·6H2O in the ratio 2:0.95:0.05 was added dropwise (~10 mL h−1) to 200 mL of an alkaline (pH 8) solution containing the dissolved ligands. During the synthesis, this solution was continuously stirred, purged with N2(g), and pH-stated at pH 8 using an automatic titrator (Metrohm 785 DMP Titrino). The resulting suspension was equilibrated in a closed vessel at 60 ◦C for two days, followed by centrifugation and subsequent rinsing with distilled water. The resulting solid phase was dried in air at 60 ◦C for three days.

For the LDHs intercalated with NO3 −, the nitrate anions originated from the precursor metal salts. For LDHs intercalated with BTC3−, the amount of BTC (2.3 × <sup>10</sup>−<sup>3</sup> mol) was chosen to exceed the positive charges in the hydroxide layers by a factor of at least 2, with BTC/Al = 2:3.

Calcination of the dried ZnAlEu-NO3 LDHs was performed by heating the LDH in a muffle furnace for four hours at, respectively, 200 ◦C (C-LDH-200), 350 ◦C (C-LDH-350), or 600 ◦C (C-LDH-600), ramping up the oven temperature from ambient to the final temperature at a rate of 5 ◦C min<sup>−</sup>1.

Rehydration of the calcined samples was achieved by suspending 100 mg of the calcined LDHs in 10 mL of a 12 mmol L−<sup>1</sup> BTC aqueous solution. This solution was prepared by dissolving BTC in a heated (60 ◦C) aqueous solution, subsequently titrated with a 1M NaOH solution (ca. 0.3 mL) to finally reach a pH between 3.5 and 4.0. Rehydration of the LDH was performed under continuous stirring (~300 rpm), varying the rehydration time from 1 min up to five days.

#### *2.2. Characterization*

A thermogravimetric analysis was performed on 13 mg of the ZnAlEu–NO3 LDH using a TGA Q500 (TA Instruments, New Castle, DE, USA), by ramping up the temperature from room temperature to 800 ◦C at a rate of 5 ◦C min−<sup>1</sup> using an air flow of 60 mL min<sup>−</sup>1.

Powder X-ray diffraction (PXRD) analyses were performed using a D8 Discover (Bruker, Atibaia, Brazil) diffractometer in Bragg–Brentano geometry using Cu Kα radiation (λ = 1.5418 Å). Data were recorded from 4◦ to 70◦ 2θ in steps of 0.05◦ using an integration time of 1 s.

Luminescence spectra were obtained on a SPEX® Fluorolog® 3 (Horiba, São Paulo, SP, Brazil) equipped with a 450 W Xenon lamp as an excitation source.

#### **3. Results and Discussion**

X-ray diffraction patterns of the as-prepared ZnAlEu–NO3 LDHs (Figure 2A) readily revealed the characteristic (003) and (006) basal reflections at 9.90◦ and 19.85◦ 2θ, which indicated a basal spacing (see Figure 1) of 8.96 Å, typical for NO3 −-intercalated LDHs [30]. From the partially overlapped (110) and (113) reflections around 60.45 2θ, the metal-to-metal distance within the hydroxide layers could also be estimated as 'a = 2d(110) = 3.06 Å' [3]. The (00*l*) basal reflections of the ZnAlEu–BTC LDHs at 6.6◦, 13.2◦, 20.2◦, and 26.2◦ 2θ confirmed the basal spacing of 13.4 Å previously reported for BTC-intercalated LDHs (as illustrated in Figure 2C) [7].

**Figure 2.** Powder X-ray diffraction patterns of (**A**) the as-prepared ZnAlEu LDHs and (**B**) ZnAlEu–NO3 LDHs calcined at 200 ◦C (C-LDH-200), 350 ◦C (C-LDH-350), and 600 ◦C (C-LDH-600). Starred peaks are related to the formation of a ZnO-like phase. The diffractogram of ZnO is shown for comparison. (**C**) Intercalation scheme and basal spacing for LDHs with 1,3,5-benzenetricarboxilate (BTC) and NO3 − [30].

LDHs can be dehydrated and dehydroxylated by calcination at temperatures as low as 200 ◦C [31]. For the ZnAlEu–NO3 LDHs, thermogravimetric analysis showed three main mass loss events (Figure 3). The first one, centered around 90 ◦C, is related to the desorption of physisorbed and weakly bound surface water. A more pronounced mass loss was observed at 220 ◦C, related to the removal of crystal water, the dehydroxylation of vicinal OH groups within the same layer, and the subsequent

condensation of OH groups from adjacent layers when the layer structure collapsed. At this time, a mixed metal oxyhydroxide phase was obtained, containing a series of oxygen sites O2<sup>−</sup>(C–LDH) and oxygen vacancies resulting from the following endothermic transformation [21], which occurred above 180 ◦C:

$$\text{2OH}^-\text{(LDH)} \rightarrow \text{H}\_2\text{O}\_{\text{(g)}} + \text{O}^{2-}\text{(C-LDH)}$$

The PXRD pattern of the sample calcined at 200◦C (C-LDH-200) (Figure 2B) indicated the formation of a ZnO-like phase. The disappearance of the basal reflections of the LDHs with the retention of the partially overlapped (110) and (113) reflections indicated the collapse of the layers, but with incomplete dehydroxylation (as illustrated in Figure 4).

**Figure 3.** Thermogravimetric analysis of the ZnAlEu–NO3 LDHs.

**Figure 4.** Schematic overview of the dehydroxylation of LDHs at low temperatures.

The final mass loss event observed in the TGA over a broad temperature range from 300 to 620 ◦C (Figure 3) was assigned to further dehydroxylation, continuous decomposition, and release of the intercalated NO3 −. As described by Valente et al. [32], the extent of this last event is related to the close packing induced by the collapse of the interlayer structure, thereby hindering the release of the interlayer products. The experimental mass loss observed at 600 ◦C in the TG experiment is in agreement with a theoretical loss of 16 wt % that was expected upon dehydroxylation and complete removal of NO3 <sup>−</sup> from a [Zn2Al0.95Eu0.05(OH)6]·(NO3)·(H2O) LDH phase.

By calcining ZnAlEu–NO3 LDH at 350 ◦C, i.e., above the second mass loss event in the thermogravimetric analysis (Figure 3), the material was converted into ZnO-like crystals, exhibiting no signs of segregation of crystalline Al2O3 or Eu2O3 [33]. Some authors attribute the absence of diffraction lines for Al2O3 or Eu2O3 to the formation of a ZnO-based solid solution embedding the trivalent ions [34–36]. Even upon calcination at 600 ◦C, no indication for Al2O3 or Eu2O3 could be observed.

Figure 5 shows the evolution of the X-ray diffraction patterns as a function of the rehydration time of calcined LDHs suspended BTC-rich aqueous solutions. Overall, this rehydration process can be expressed by the following equation [23]:

$$\text{M}^{\text{II}}\text{l}\_{1-\text{x}}\text{M}^{\text{III}}\text{\_{x}\text{O}\_{1+\text{x}/2}} + \text{(x/n)}\text{A}^{\text{n}-} + (\text{m} + 1 + \text{x/2})\text{H}\_{2}\text{O} \rightarrow \text{M}^{\text{II}}\text{}\_{1:\text{x}}\text{M}^{\text{III}}\text{\_{x}(\text{OH})} \\ \text{\_{2}(\text{A}^{\text{n}-})}\text{\_{x/n}\text{-m}\text{H}\_{2}\text{O} + \text{xOH}^{-} $$

For samples calcined at 350 ◦C (Figure 5 left), the emergence of the reflection at 9.90◦ 2θ indicated a recovery of a lamellar structure after 1 min of rehydration. As described by Valente et al. [32], NO3 − could become partially confined upon the collapse of the LDH structure, which could assist the recovery of the structure upon rehydration. After 30 min, BTC started to become intercalated in the previously recovered layered phase. The (00*l*) basal reflections of the recovered BTC-intercalated lamellar phase presented a slight shift to higher 2θ, indicating that the as-synthesized and recovered phases possessed different hydration contents. After three days of rehydration, the reflections resulting from the oxide phase broadened considerably, which indicated a breakdown of the oxide domains in the sample.

**Figure 5.** Powder X-ray diffraction patterns for different hydration times of ZnAlEu–NO3 LDH samples calcined at 350 ◦C (**left**) and 600 ◦C (**right**). Starred peaks relate to the formation of a ZnO phase. Reflections associated with the reappearance of a layered phase are marked with open squares, while the basal reflections of LDH intercalated with BTC (7.0◦, 12.7 Å) are marked with an open triangle. The PXRD patters of the samples ZnAlEu–NO3 LDH and ZnAlEu–BTC LDH are shown for comparison.

For specimens treated at 600 ◦C (Figure 5 right), the recovery of the lamellar phase was delayed compared to that of samples treated at 350 ◦C. After five days, a lower degree of regeneration was observed for the LDH calcined at 600 ◦C (C-LDH-600).

The photoluminescence properties of the samples were investigated by measuring their excitation and emission spectra (Figure 6). The excitation spectra (Figure 6 left) of all samples were collected at room temperature, monitoring the (Eu3+) 5D0→7F2 emission at 614 nm. In the calcined samples, no zinc oxide O2−→Eu3+ Ligand-to-Metal-Charge Transfer band (LMCT) was found, in accordance to other studies of Eu3+-doped ZnO [37]. In the hydrated materials, the broad excitation band in the higher energy region (235–315 nm) was composed by two bands owing to the energy transference from the BTC to Eu3+. One band was centered around 265 nm and was attributed to (BTC)O2-→Eu3+ LMCT [29,38–40]. The other band, centered at 290 nm, corresponded to the BTC singlet transition S0→Sn [41–43] with subsequent energy transfer to Eu3+.

**Figure 6.** (**left**) Normalized excitation spectra recorded at 300 K, monitoring the (Eu3+) 5D0→7F2 emission at 614 nm of the ZnAlEu–NO3 LDH (as-prepared) together with the calcined and rehydrated samples. (**right**) Normalized emission spectra (300 K) under excitation at the BTC ligand-to-metal transference band (290 nm) for the rehydrated samples and under excitation at the (Eu3+) 7F0→5L6 excitation band at 395 nm for the as-prepared and calcined samples. The labels of the 4f-4f intraconfigurational transitions of Eu3+ are shown. The spectra of the sample ZnAlEu–BTC LDH are shown for comparison.

The emission spectrum under direct excitation of the (Eu3+) 7F0→5L6 excitation band (395 nm) of the as-synthesized ZnAlEu–NO3 LDHs (Figure 6 right) showed the characteristic emission bands of Eu3+. Similar emission spectra were observed for the samples calcined at 350 and 600 ◦C, but with broader emission bands (especially for C-LDH-350). The broadening of the emission bands arose from slightly different sites occupied by Eu3+ in the matrix, which produced slightly different emission profiles [44]. This indicated that the number of sites occupied by Eu3+ increased after calcination. Compared to the 5D0→7F1 transition, the higher intensity of the 5D0→7F2 transition was enabled by forced electric dipole [45–48], which indicated the absence of inversion symmetry around the average Eu3+ site.

After rehydrating in a BTC-rich aqueous solution, the calcined samples started to recover their lamellar structure. At the same time, these samples also started to transfer energy from the BTC antenna to Eu3+. Figure <sup>6</sup> shows the emission spectra under excitation of the BTC S0→Sn transition after time-dependent hydration of the samples C-LDH-350 and C-LDH-600. Although no intercalation with BTC was found after hydrating the sample C-LDH-350 for 1 min, the photosensitizer was still able to transfer energy to Eu3+. This effect was probably due to BTC molecules adsorbed on the outer surface of the particles, which recovered to the LDH phase faster than the bulk [23]. This effect was seen in the sample C-LDH-600 only for longer hydration times, which indicated that the rehydration of this sample was less efficient than that of C-LDH-350, as was also inferred from the PXRD results.

#### **4. Conclusions**

In summary, to produce luminescent LDH phases, ZnAlEu–NO3 LDHs calcined at up to 600 ◦C can only partially be restored to the LDH phase through rehydration in BTC-rich aqueous solutions. The calcination temperature appears to be an important factor for an efficient rehydration, as samples calcined at 350 ◦C showed a higher degree of regeneration compared to those treated at 600 ◦C. For samples treated at 350 ◦C, the recovery of the LDH structure started after 1 min of rehydration, while for the samples treated at 600 ◦C the regeneration only started after 5 min. After 30 min, the pH of the rehydration solution increased as a result of the dissolution of the oxides, and this facilitated the intercalation of BTC. The interaction of this photosensitizer with Eu3+ in the recovered hydroxide layers gave rise to efficient energy transfer from the BTC antenna molecule to the Eu3+ ions, which proved to be a useful tool to monitor the rehydration process of the calcined LDHs.

**Author Contributions:** Conceptualization, E.B. and D.M.; methodology, A.F.M., I.G.N.S., E.B. and D.M.; validation, A.C.T. and A.F.M.; formal analysis, A.C.T, A.F.M. and I.G.N.S.; investigation, A.C.T., A.F.M. and I.G.N.S.; resources, D.M.; data curation, A.C.T., A.F.M., I.G.N.S. and D.M.; writing—original draft preparation, A.F.M., I.G.N.S., E.B. and D.M.; writing—review and editing, A.F.M., I.G.N.S., E.B. and D.M.; visualization, A.F.M. and I.G.N.S.; supervision, E.B. and D.M.; project administration, D.M.; funding acquisition, D.M.

**Funding:** This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2015/19210-0), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, 1723707, Finance Code 001), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 142127/2014-0 and 403055/2016-4).

**Acknowledgments:** The authors acknowledge the Laboratory of Crystallography (IFUSP, São Paulo) for assistance with the PXRD measurements.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Layered Double Hydroxides in Bioinspired Nanotechnology**

#### **Giuseppe Arrabito 1,\*, Riccardo Pezzilli 2, Giuseppe Prestopino 2,\* and Pier Gianni Medaglia <sup>2</sup>**


Received: 26 May 2020; Accepted: 6 July 2020; Published: 11 July 2020

**Abstract:** Layered Double Hydroxides (LDHs) are a relevant class of inorganic lamellar nanomaterials that have attracted significant interest in life science-related applications, due to their highly controllable synthesis and high biocompatibility. Under a general point of view, this class of materials might have played an important role for the origin of life on planet Earth, given their ability to adsorb and concentrate life-relevant molecules in sea environments. It has been speculated that the organic–mineral interactions could have permitted to organize the adsorbed molecules, leading to an increase in their local concentration and finally to the emergence of life. Inspired by nature, material scientists, engineers and chemists have started to leverage the ability of LDHs to absorb and concentrate molecules and biomolecules within life-like compartments, allowing to realize highly-efficient bioinspired platforms, usable for bioanalysis, therapeutics, sensors and bioremediation. This review aims at summarizing the latest evolution of LDHs in this research field under an unprecedented perspective, finally providing possible challenges and directions for future research.

**Keywords:** origin of life; DNA; layer double hydroxide; synthetic biology; bioinspired devices; biosensors; bioanalysis

#### **1. Introduction**

Layered double hydroxides (LDHs) are an important class of two-dimensional (2D) layered materials belonging to the group of hydrotalcite-like (HT) compounds [1–4]. They are constituted by stacks of positively charged hydroxyl layers of bivalent ions (e.g., Ca2+, Zn2+, Mg2<sup>+</sup> and Ni2<sup>+</sup>) and trivalent metallic ions (e.g., Al3<sup>+</sup>, Fe3<sup>+</sup>, Cr3<sup>+</sup> and In3<sup>+</sup>). The isomorphic substitution of some of the bivalent metal ions by the trivalent metal ions forms a positive residual charge on the metal-hydroxide framework, which is in turn balanced by exchangeable interlayer anions to maintain the global electroneutrality. The general formula of LDHs is [M1−*<sup>x</sup>* <sup>2</sup>+M*<sup>x</sup>* <sup>3</sup><sup>+</sup>(OH)2] *<sup>x</sup>*+·[A*x*/*n*] *<sup>n</sup>*−·*m*H2O, where M<sup>2</sup><sup>+</sup> and M3<sup>+</sup> are the divalent and trivalent metal ions, respectively; A*n*<sup>−</sup> are inorganic or organic anions; *m* is the number of interlayer water; and *x* = M<sup>3</sup>+/(M2<sup>+</sup> + M3+) is the layer charge density, or molar ratio [5]. It is worth pointing out that, in LDHs, the electric charge of the layers and of the interlayer ions is the opposite of that found in the vast majority of layered materials such as silicate clays (cationic clays), which feature negatively charged host layers and exchangeable cations in the interlayer spaces. Indeed, LDHs are also usually known as anionic clays [6]. Their unique physicochemical properties [7], i.e., biocompatibility, lamellar structure and compositional diversity, make them suitable for adsorption processes of a large variety of molecules, ranging from organic molecules to biomacromolecules. LDHs also play a prominent role in photocatalysis [8] and, more recently, in many life sciences related applications [9], especially for biosensors [10], drug delivery [11] and tissue bioengineering [12]. In the

last years, the application of LDHs and their composites in biological, chemical and environmental processes have been extensively reviewed [13–16].

In particular, the role of LDHs as catalysts in relevant organic chemistry reactions, as photocatalytic centers, and their emerging application in cellular biology were extensively summarized in a previous review from our group [5]. Notably, the well-investigated chemistry- and biology-related applications of LDHs summarized in that review article highlight the conspicuous studies focused on the synthesis, characterization and applications of LDH-based systems. In fact, similar to ZnO-materials [17,18], LDHs can be synthesized under mild conditions [19] following the coprecipitation method, which consists in the addition of a basic substance to an aqueous solution containing the salts of two different metals, namely M2<sup>+</sup> and M3<sup>+</sup>, leading to the precipitation of the metal hydroxides, with the subsequent formation of LDHs [20–22]. Such synthetic approach can be leveraged to produce LDH materials on demand, with the desired chemical composition, aggregation state and particle size [15]. The latter can be tuned ranging from nanoclusters [23] to micron scale [24]. Notably, highly porous and highly dispersed LDHs with high surface area allow the full potential of LDH-based compounds to be efficiently exploited in many applications, such as heterogeneous catalysis and catalyst supports. Single layer LDHs are also ideal building blocks for functional assembly. However, the LDHs synthesized by conventional coprecipitation methods tend to easily aggregate, due to electrostatic interactions, interlamellar hydrogen bonding networks and their hydrophilic nature [25], thus resulting in low surface area and large particle size. To overcome these issues, many research efforts have optimized down to single- or few-layer nanosheets. Delamination of LDHs into single layers in polar solvents, such as formamide, butanol and acrylate, is a well-known process, as extensively reviewed by Wang and O'Hare [26]. In this context, it is worth mentioning that carbonate (CO2<sup>−</sup> <sup>3</sup> ) contamination during both synthesis and post-treatment processes complicates LDH delamination, to such an extent that the direct exfoliation of a CO2<sup>−</sup> <sup>3</sup> -LDH is believed to be almost impossible [27]. The conventional approach to overcome this consists in the development of anion-exchange protocols to replace carbonate ions with other intercalating anions featuring weaker electrostatic interactions with the LDH structure [25,28]. Recently, a direct exfoliation method of CO2<sup>−</sup> <sup>3</sup> -LDHs, which avoids anion-exchange reactions, using liquid phase delamination via ultrasonic tip has been demonstrated [29]. Ecofriendly delamination approaches constituted by washing/stirring the LDH dispersion in decarbonated water have also been reported [30–32]. Isolation and recovery of delaminated LDHs without aggregation is a big challenge hindering practical application and commercial exploitation. In the last years, with the aim to increase LDH dispersion and organophilicity and to fabricate stable delaminated LDH dry powders, O'Hare and coworkers developed post-treatment routes for LDHs using both aqueous miscible organic (AMO-LDHs) and aqueous immiscible (AIM-LDHs) organic solvents, yielding highly porous, highly dispersed LDH powders of exfoliated nanosheets [33,34]. Some experimental protocols also permit films of interconnected LDH nanoplatelets to be grown onto aluminum surfaces as hierarchically nanostructured coatings by soaking aluminum thick foils in an aqueous solution containing a soluble zinc salt [35–39]. In this case, the trivalent ions (Al3<sup>+</sup>) are provided by the sacrificial aluminum foil, whose role is both reactant and substrate, and ultimately allows the LDH film adhesion to be improved. LDH-based materials have triggered conspicuous studies in the field of organic chemistry, mainly due to their excellent ability as heterogeneous catalysts for the preparation of fine chemicals, intermediates and valuable molecules, benefiting from the simplicity of their preparation from cheap and non-harmful precursors, and the low toxicity of their possibly produced decomposition products [5]. The high reactivity of LDHs is related to their layered structure which features a high anion-exchange capability [40–44], and to their high basicity, due to either structural hydroxyl anions in hydrated material or O2−-Mn<sup>+</sup> pairs in the case of water-free calcined material [45]. A crucial feature is also represented by the metal ratio. In fact, the incorporation of specific metals in the octahedral layer can produce LDH compounds with unexpected and novel properties, as well as modulate the existing chemical reactivity. It is worth mentioning literature where the arrangement of cations within the LDH lattice is discussed. As a matter of fact, the ordering of cations is believed to have crucial effects on

many of the physicochemical properties of LDHs, affecting the charge density of the metal hydroxide sheets, and the overall bonding, mobility, orientation and reactivity of the chemical species in the interlayer space and on the surface [46–49]. In particular, the M2+-M3<sup>+</sup> cation order is important for the catalytic activity of LDHs [50], and the different distribution of the M3<sup>+</sup> cation in the M2<sup>+</sup> matrix regulates interlayer anionic charge and, in turn, intercalation reactions [51].

The intriguing catalytic properties and the high compatibility with biological systems of LDHs continuously open new research directions. Among them, up-to-date research topics of interest both for fundamental research and applications come from the presumed role of LDHs in the evolution of natural systems and, in turn, from the realization of a novel class of LDH-based life-inspired devices. As a matter of fact, the path permitting a complete understanding of the role of LDHs within the origin of life is still unclear and needs more research. However, some studies point out their possible role in this exciting research field. In fact, cationic clays and LDHs [52,53] might have played an important role for the origin of life on planet Earth, having triggered the formation of life-relevant prebiotic settings through the evolution of molecular systems, leading to the formation of polymerized molecules and finally to the emergence of life [54]. In a pre-biological Earth model, the ion exchange properties arising from the positively charged sites in LDHs allowed the latter to concentrate amino acids, and to safely promote biopolymer formation, offering also protection from the effects of UV irradiation. A mechanism of prebiotic information storage and transfer in LDH matrices based on replication and conservation of M2<sup>+</sup>-M3<sup>+</sup> cation arrangements was also proposed by Greenwell and Coveney [55]. In a sort of reverse evolutionary process, in the recent years, learning from nature, researchers have conceived the concepts of biomimetism and bioinspiration [56] in nanomaterials science to achieve unprecedented advanced functionalities based on designs that referred to structures in living creatures [57–61]. Under this exciting perspective, the most recent studies in the field of LDHs are striving to recapitulate life-like scenarios in the construction of hybrid LDH-based platforms, mimicking compositions or structures of natural species (e.g., biological macromolecules and microorganisms), with the aim to further improve their efficiency and their sustainability in the context of the natural environment [62,63]. Indeed, the low cost, biocompatibility and extraordinary adaptability of LDHs in many different purposes have allowed the effective application of bioinspired synthesis routes to LDH-based assembly of nanostructured materials and devices, which combine the eco-friendly nature of these materials and their versatility in the context of materials science. Another intriguing strategy to achieve bio-functionalization consists in associating biological species (e.g., amino acids, nucleosides, oligo nucleotides, DNA, proteins and enzymes) to LDH host matrices, as recently reviewed by Forano et al. [15] for LDH-biohybrids based on immobilization of bacteria and algae.

Motivated by the emerging role of bioinspired nanomaterials research, we felt the need to review the role of LDHs in this scenario, by investigating the fundamental science aspects and the possible emerging nanotechnology-related applications. At first, the present review provides an insight into the link between the role of LDHs in the origin of life, in particular focusing on their catalytic activities that promoted the formation of pre-biotic molecules and the possibility to fabricate artificial LDHs-based artificial compartments to mimic prebiotic platforms. These studies are speculative, mostly driven by the willingness to explore basic aspects of LDHs in their possible but still not completely clear role within the topic of origin of life. Secondly, this review updates and extends our previously review article [5], covering different aspects and, more in general, aiming at bridging fundamental science with new approaches that may support the design of LDH-based systems for the fabrication of life-like and life-inspired devices, especially in the fields of composites and coatings, sensors, life-inspired catalysis and bioremediation. To guide the reader, this review is divided into the following sections: the role of clays in the origin of life (Section 2), LDH interaction with biomolecules (Section 3), LDH-based compartments fabrication (Section 4), LDH-bioinspired devices assembly (Section 5) and finally the conclusions and perspectives (Section 6).

#### **2. The Role of Clays in the Origin of Life**

The origin of life (or abiogenesis) is among the most intriguing questions in science [64]. Although it is not possible to know all the details of this process, some scientific hypotheses attempt to explain the synthesis of the molecules which were necessary for the emergence of primitive forms of life in planet Earth 3.8 billion years ago [65] as deriving from a reducing atmosphere (Miller's experiment [66]), meteorites impacts [67] or from metal sulfides in deep-sea hydrothermal vents, as explained in the famous article of Russel [68]. Recent studies from Das at al. [69] leveraged computational methods to shed light on the role of hydrogen cyanide (HCN) as a source of ribonucleic acid (RNA) and protein precursors. The authors also argued that just the interaction of HCN with water would have sufficed to trigger a series of reactions leading to the life-essential precursors (nucleic acids and proteins), as reported in Figure 1.

**Figure 1.** The synthesis of life relevant molecules (RNA, proteins) starting from inorganic materials and leading to the onset of chemical evolution. Figure reproduced from Ref. [70] https://pubs.acs.org/doi/10. 1021/acscentsci.9b00832; Copyright (2019) American Chemical Society, further permissions related to the material excerpted should be directed to the ACS.

Once prebiotic molecules formed in water, cationic and anionic clays were also present on the surface of the Earth and could then interact with prebiotic molecules. In fact, whereas the probability that molecules could encounter in a 3D liquid environment is quite low, the solid mineral surfaces permitted to adsorbed molecules and permit them to interact and react [71]. According to some theories, radionuclide-induced defect sites in iron-rich minerals could have triggered the formation of prebiotic life [72]. Among all the possible hypothesis for the origin of life, one of the most relevant is that of hydrothermal vents [73], since they provided the possible evolutionary transition from geochemical to biochemical processes in absence of sun light. Hydrothermal vents are crevices in the seafloor from which geothermally heated water pours into ocean water. Among them, alkaline vents (such as the Lost City hydrothermal field) could have acted as ideal electrochemical flow reactors in the ocean during the Hadean period [74], in which the ocean waters were acidic since they were rich in CO2. More specifically, within these hydrothermal vents, a pH gradient was formed between inorganic micropores barriers containing catalytic Fe(Ni)S minerals and the acidic ocean water, resulting in proton gradients similar to the proton-motive force required for carbon fixation in bacteria and archaea. The alkaline hydrothermal field provided serpentinization reactions that produced methane and hydrogen. Such pH energy input, in addition to the catalysis provided by the Fe(Ni)S minerals, triggered the reduction of CO2 with H2 and the relative production of life-essential organic molecules, such as hydrocarbons, finally leading to the first primitive cellular systems. Within this scenario, the question that can arise is then if LDHs were present in the hydrothermal vents. To this regard, Cairns-Smith and Braterman speculated that inorganic Fe2+/Fe3<sup>+</sup> LDHs (also defined green rust) [75] were at the sites of Archean oceanic hydrothermal vents, constituting one of the most-common components of early ocean sediments. The presence of this peculiar LDH can be ascribed to the lack of oxygen in the Archean atmosphere, permitting the presence of Fe2<sup>+</sup> in aqueous environments and oxidation and precipitation as FeIIIOOH. As explained by Russell [76], the green rust might have played a fundamental role in the formation of prebiotic life. In particular, within the alkaline vent model, the green rust precipitated forming individual compartments that permitted the formation of ionic gradients and catalytic centers for endergonic reactions, aided by sulfides and trace elements acting as catalytic promoters that led to the formation of pre-biotic molecules.

In this context, as first speculated by Bernal [77], clays likely played an important role in promoting the molecular organization and the formation of polymerized biomolecules (nucleotides and amino acids), given their potentially large adsorption capacity, the ability to shield molecules against ultraviolet radiation, concentrate organic chemicals and catalyze chemical polymerization reactions. Following the seminal proposal by Cairns-Smith [78], who postulated a concept of "genetic takeover", according to which carbon-based life may have gradually evolved from organic–inorganic complexes of clays with organic molecules, some remarkable experiments demonstrated that clay minerals played an active role in the abiotic origin of life. For instance, as elegantly demonstrated by Ferris [79], montmorillonite clays could have a role in the polymerization of short RNA oligomers (up to 50 mer). Similarly, the fluctuation of temperature and humidity could have triggered the amino acids polymerization in the presence of clay minerals [80]. Finally, clays could have increased the rate of formation of vesicles from fatty acid micelles, along with the possibility to encapsulate clay particles inside the vesicles in addition to RNA molecules adsorbed on the clay surfaces, as demonstrated by Szostak et al. using montmorillonite clay [81]. Once formed, such vesicles could grow by incorporating fatty acids and divide, thus mediating vesicle replication through cycles of growth and division. In this scenario, since the first hypotheses by Kuma et al. [52] and Arrhenius [53], there are not so many studies in this field that regard the role of LDHs, even if they are positively charged clays that can interact with negatively charged molecules. LDHs might have played a role in selecting DNA over RNA for genetic information, due to its ability in enhancing the Watson–Crick hydrogen-bonding when intercalated within the LDH interlayers, in comparison to RNA [55,82]. Grégoire et al. [83] also explored the possible role of LDHs in early Earth chemistry, leading to the formation of peptides. Very recently, Vasti et al. demonstrated that LDHs strongly interact with model lipid membranes [84]; these experiments could also highlight the role of LDHs in partitioning within the membranes of primitive cellular-like compartments. In a very recent hypothesis paper, Bernhardt [85] proposed that LDH clays might have promoted the synthesis of prebiotic precursors and also played other roles in the origin of life, such as in the formation of the first lipid bilayers.

The fundamental reaction that triggered aerobic life was the photosynthetic oxygen evolution, in which two water molecules produce four electrons and four protons used for the photosynthetic processes and molecular oxygen [86]. This reaction is catalyzed by a manganese-containing cofactor contained in photosystem II known as the oxygen-evolving complex. Mimicking natural systems, studies have demonstrated that LDHs can constitute ideal low-cost catalytic materials for the oxygen evolution [87], in which both the M2<sup>+</sup> and M3<sup>+</sup> take part to the redox reactions, as reported in Figure 2. Some relevant applications of LDHs as catalysts for oxygen evolution reaction (OER) are discussed in Section 5.3.

**Figure 2.** Simplified scheme for the oxygen evolution mechanism involving the formation of a peroxide (M–OOH) intermediate under acid (blue line) and alkaline (red line) reaction conditions. Reproduced from Ref. [87] under a Creative Commons Attribution 4.0 International License.

#### **3. LDH Interaction with Biomolecules and Relevant Applications**

#### *3.1. Nucleic Acids*

Nucleotides are the key molecules for the storage and transmission of genetic information [88]. The chemistry of their synthesis and polymerization is still a matter of debate [89]; however, based on the observation from Bernal [77], they could have had a role in concentrating and even acting as catalyst to form oligonucleotides. Moreover, they could have also been involved in protecting nucleotide against decomposition in presence of ionizing radiation [90]. Nucleic acids are composed of nitrogenous organic bases, pentose and phosphoric acids, are a typical polyanion within hydrogen-bonding groups, and can therefore enter inside the LDH interlayer, forming a nucleic acid/inorganic composite stabilized by electrostatic interactions. Inspired by the ability to protect and store nucleotides and oligomers, DNA-LDH hybrids have found remarkable applications in the field of gene delivery to cells. The reason for this was investigated by Li et al. [91], who employed dissipative particle dynamic simulations for elucidating the interaction with cellular membranes at different levels (i.e., total or partial penetration vs. anchorage—see Figure 3), as a function of the lateral size and layer number. These results shed light on the very important role of layer thickness—i.e., the lower is the thickness, the higher the probability that DNA-LDHs hybrids can be internalized, and hence serve as gene delivery tool.

**Figure 3.** Interaction of DNA-LDH hybrids of different lateral layer sizes and thicknesses with model

cellular membranes. Relation between the LDH lateral size and LDH layer number at two different time points: 6.4 μs (**a**); and 32 μs (**b**). (**c**) Effect of the size of the DNA-LDH hybrids on the internalization. Reproduced from Ref. [91]. Copyright (2020), with permission from Elsevier.

#### *3.2. Phospholipids*

Cationic clays can effectively interact with living cells producing hybrid half inorganic viable structures, as demonstrated by Murase and Gonda [92] and Konnova et al. [93] in previous seminal studies on montmorillonite and hallosite clay nanotubes, respectively. Indeed, it was clarified that such clays can interact with phospholipids via electrostatic interactions [92], giving rise to apparently ordered phospholipids self-assembled layers, which can lead to "*armored cells"* if exposed to living cell membranes [93]. Notably, in the case of LDHs, the structural analysis from Itoh et al. [94] interestingly demonstrated, by means of spectroscopic and crystallographic characterizations, that stearate ions can form ordered bilayer structures within MgAl-LDH layers with a tilting angle of approximately 29◦ from the right angle of the layers (Figure 4).

**Figure 4.** Scheme of stearate ions ordered layer assembly into LDH: (**a**) tilted bilayer, and (**b**) packing of stearate ions in the LDH framework. Reprinted with permission from Ref. [94] Copyright (2003) American Chemical Society.

#### *3.3. Amino Acids*

Amino acid intercalation inside LDHs is also a well-known topic [95]. An interesting application of LDHs-intercalated with amino acids is constituted by drug delivery, specifically because amino acid molecules provide the interaction sites to effectively improve drug-loading [96]. Apart from the interest in elucidating the structural features, the interaction between anionic clays and amino acids is very interesting and may offer an insight into the active role of LDHs in the polymerization of amino acids, in addition to the well-known characterization of the intercalation [95]. Differently from nucleotides, for which the prebiotic synthesis route is still speculative [89], there is a little more evidence of the possibility that amino acids were present in prebiotic systems. Accordingly, both cationic clays and LDHs might have facilitated the polymerization of amino acids, thanks to the possibility to absorb them via their negative C-termini. Interestingly, as argued by Paecht-Horowitz [97], the former can catalyze polymerization only when the amino acid is small enough to enter the interlayer space of the clay. As for the latter, the computational study from Erastova et al. [54] demonstrated the possibility that LDHs could be a functional system for amino acid adsorption and polymerization on the positively charged LDH surface. Intriguingly, all the amino acids they investigated (alanine, aspartate, histidine, leucine, lysine and tyrosine) showed an increase in adsorption upon dehydration, with the only exception of lysine. Dehydration, in turn, decreases the interlayer spacing, in accord with our experimental findings [37], favoring both the crowing of amino acids and the alignment of the N- and C-termini, and also permitting the formation of a peptide bond (see Figure 5). This reaction leads to the loss of charged group, which, in turn, allows for the introduction of a new amino acid to an adjacent site, where it then can react with the peptide's C-terminus. The growing peptide chain remains tethered at the LDH surface via the C-terminus of the latest amino acid added. Differently to nucleic acids, amino acids do not form strong association on the LDH surface. Even more, the authors observe that, within the clay layers, peptide chains should be able to undergo hydrophobic collapse, an essential mechanism of protein folding, in perfect resemblance to the ribosome systems.

**Figure 5.** Amino acids on LDHs. Ensembles of (**a**) amino acids; (**b**) short; and (**c**) long peptides intercalated into LDH interlayers via their C-terminal. (**d**) Proposed mechanism for peptide bond formation inside LDH interlayers. Reproduced from Ref. [54] under the terms of the Creative Commons CC BY license.

#### *3.4. Carbohydrates and Cellulose*

Carbohydrates are among the most abundant biomolecules in nature. They are formed by plants through photosynthesis and serve as energy sources, structural building blocks and biorecognition elements in organisms. They can exist as oligomers and polymers; the latter are formed by linking single monomeric units with glycosidic bonds. LDHs have been leveraged as systems to prepare biomimetic composites with carbohydrates molecules showing enhanced mechanical properties, mimicking the natural structure of nacre, a biological organic–inorganic composite material produced by some mollusks and formed by the ordered stacking of calcium carbonate and crosslinked protein, resulting in very good mechanical properties (high tensile strength, about 150 MPa). To this aim, it is worth mentioning some previous studies on LDH composites with carboxymethyl cellulose (CMC) [98] and cellulose nanofibrils [99], which exhibited improved mechanical properties. As reported in Figure 6a, the interaction of CMC with LDHs is purely electrostatic, being driven by the negative electrostatic charge of the CMC that permits its insertion into MgAl-LDH galleries. In addition, alginate was also used as composite with LDHs [100], following a bioinspired approach that mimicked the mechanical properties of nacre. In particular, NiAl-LDH is prepared by coprecipitation and is subsequently exfoliated mechanically; after that, it is mixed with an alginate solution permitting to

obtain alginate-coated LDH, which is subsequently crosslinked with Ca2<sup>+</sup> (see Figure 6b). Importantly, the ionic crosslinking of alginate by calcium ions along with the hydrogen bonds between NiAl-LDH and alginate, allows the nacre film to be mimicked, achieving a tensile strength as high as 194 MPa, in addition to transparency higher than 70% in the visible light range.

**Figure 6.** (**a**) Stabilization of the MgAl-LDH-CMC hybrid particle by electrostatic-driven interactions. Reprinted from Ref. [98]. Copyright (2014) with permission from Elsevier. (**b**) Production process of hybrid NiAl-LDH/Alginate-Ca2<sup>+</sup> film. Reproduced from Ref. [100]—Published by The Royal Society of Chemistry.

#### **4. Life-like Clays Based Artificial Compartments**

The fabrication of artificial compartments that mimic life-like properties is a topic of large interest and has been boosted by the development of new microfluidic platforms containing droplet-based and vesicle-based artificial cells [101,102], and by the employment of printing methodologies, as shown also by our group [103,104]. The systems can be realized at solid surfaces mimicking the processes of DNA condensation [105] or in membrane-enclosed liquid compartments [106,107]. Importantly, such artificial biosystems permit to recapitulate some of the features of primitive sub-micron sized protocells, such as effect of droplet size on the molecular behavior, molecular confinement, molecular crowding, and liquid–liquid phase separation. In the context of clay-based compartments, the studies are mainly based on the fabrication of 100–10 μm sized colloidosomes or Pickering emulsions [108], showing intriguing membrane permeability [109,110], and even some life-like behavior, such as enzyme-powered motility [111]. Regarding the fabrication of synthetic compartments, printing methods are gaining increasing importance [112,113], given their rapidity, low-cost and adaptability to biomolecular, organic and inorganic inks [114] or oil droplets [115], opening up the possibility to print artificial cell-like systems with higher degree of flexibility and possibility to assemble complex predesigned droplet networks. The development of compartments containing LDHs is still at its infancy. However, the possible application of LDH-based compartments would lead to the mimicking of nanocomposites, as for instance mimicking the structure and the mechanical properties of the high strength seashell nacre. A fundamental study by Zhang and Evans [116] explored the dynamic assembly of LDH platelet-rich droplets drying onto solid surfaces, taking into consideration the interplay of capillary flows, colloidal stability and pH on the final CoAl-LDH composite deposited on a silicone coated paper. They prepared pH 3 and 10 dispersions resulting in well-dispersed and flocculated CoAl-LDH platelet suspensions, respectively. The pH-dependent colloidal stability of CoAl-LDH dispersions in the pH range from 3 to 10 was discussed by the same authors in another report [117], showing that the suspension was most stable at pH 3, due to the protonation of the OH groups resulting in repulsion forces between LDH platelets, positively charged on both basal and edge surfaces. It is worth pointing out that both stronger acid and basic conditions (i.e., pH < 3 and pH > 10) lead to LDH structural dissolution [118,119]. In the case of deposition of droplets at pH 3 (where

LDHs are well dispersed), a central hole was observed in the LDH deposit (see Figure 7a), along with a ring of higher thickness at 0.6 *r* from the center (where *r* is the radius of the droplet). The authors explained this structure by considering possible capillary effects between LDH platelets. A completely different situation occurred in the case the LDH platelets were deposited as flocculated at pH 10. In this case, the suspension produced a flatter structure due to the lack of capillary flows. As the authors state, these results could be applied to the understanding of droplet morphologies produced by inkjet printing [120], with the aim to optimize both the flow processes in a drying drop and the stacking interactions among LDHs, ultimately permitting to obtain ordered stacking and high uniformity in the LDH coverage. An elegant demonstration of LDH printing was given by Zhu et al. [121] who leveraged exfoliation processes of CoAl-LDH and NiAl-LDH nanosheets in formamide to obtain printable LDH formulations, leading to stable LDH-based solid films (see Figure 7b), in which the LDH stacking is able to mimic the structures of nacre, potentially leading to good mechanical properties. Notably, the authors also demonstrated the possibility to tune the optical and mechanical properties of the LDH films by tuning the anions that can be stacked into the galleries.

**Figure 7.** (**a**) Photographs of a drying drop of 2.0 vol% LDH dispersion at pH 3 as a function of drying time. Reprinted from Ref. [116]. Copyright (2013) with permission from Elsevier. (**b**) Schematic diagram of the preparation of LDH thin films and subsequent printing on substrates. Reproduced from Ref. [121] with permission from The Royal Society of Chemistry.

#### **5. LDH-Bioinspired Devices**

#### *5.1. Applications in Composites and Coatings*

Billions of years of evolution have produced optimized natural materials with outstanding properties resulting by the combination of organic and inorganic elements, which inspire scientists and engineers to design artificial materials. In recent years, the development of bioinspired composites has become an exciting direction for the fabrication of novel multifunctional materials, which exhibit a combination of outstanding mechanical, fire-shielding, optical and oxygen barrier properties. In this field, LDHs are receiving increasing attention since they offer numerous advantages over other inorganic layered materials, also in the field of nanocomposites [122,123]

Shu et al. [124] reported on a nacre-like film based on heparine/NiAl-LDH with a layered nano/microscale-hierarchical structure fabricated by the vacuum-filtration method (Figure 8a). The realized hybrid film showed a reduced elastic modulus (Er ≈ 23.4 GP) and a hardness (H ≈ 0.27 GPa) remarkably higher than those of other reported polymer/LDH composites. In addition to such

outstanding mechanical characteristics, the LDH composite exhibited interesting UV-blocking, flame retardant, and heat-shielding properties. In a successive work, the same group [125] studied a hierarchical structure based on a CoAl-LDH/Poly(vinyl alcohol) film, prepared for the first time by bottom-up layer by layer assembly (Figure 8b). A combination of high tensile strength and elastic modulus is obtained through an appropriate CoAl-LDH aspect ratio, which well compares to those of nacre and lamellar bone.

**Figure 8.** (**a**) Fabrication of the artificial nacre-like HEP/LDH film by three steps: (i) LDH delamination; (ii) assembly of exfoliated LDHs and HEP; and (iii) HEP-LDH hybrid building blocks are aligned by self-assembly induced by vacuum filtration. Reproduced with permission from Ref. [124]. Copyright year (2014) American Chemical Society. (**b**) Fabrication of artificial multilayered PVA/LDH hybrid films by: (i) modification of the surface of LDH platelets with slightly hydrophobic amine-terminated APTES; (ii) spin coating of a layer of PVA; (iii) gradual formation of Langmuir film at the air–water interface; and (iv) sequential repetition to fabricate multilayer films. Reproduced with permission from Ref. [125]. Copyright year (2014) American Chemical Society.

Meng et al. [126] reported another example of brick-and-mortar structure to enhance the mechanical performance of a composite. They developed a PTFE/MLDH (modified ZnAl-LDH)/GF (glass fiber) composite inspired by layer–layer structure and prepared via the multi-layered dipping method, which consists of GF acting as a reinforcement, PTFE acting as matrix and MLDH acting as the layer–layer structure. The results show that the tensile strength and the strain value of PTFE/MLDH/GF composites, with MLDH content around 1.6 wt%, increased to 163.57 MPa and 8.33%, respectively. These values resulted better than those of pure PTFE/GF composites, showing a tensile strength of 112.72 MPa and a fracture strain of 4.99%.

Interestingly, the use of LDH-based compounds is not only limited to mechanical properties, as mentioned above. Liu et al. [127] demonstrated that the combination of two or more diverse materials in a unique composite is a winning strategy, realizing a durable superhydrophobic sponge for oil and organic collector with magnetic-responsive and fire retarding properties. To achieve this result, by means of a two-step process, they combined polydopamine (PDA), ZnAl-LDH, Fe3O4 nanoparticles and n-octadecyl mercaptan (OM), acting, respectively, as a "bio-glue", a fire retardant agent, magnetic material and hydrophobic reagent. First, PDA-modified ZnAl-LDH and Fe3O4 were anchored on the skeleton of polyurethane (PU) sponge by self-polymerization. Next, OM containing thiol groups were covalently combined with PDA by means of Michael addition reaction. The resulting sponge (Figure 9a,b) exhibited remarkable hydrophobicity (water contact angle of 163◦) and highly oleophilic (oil contact angle of 0◦). As a result, it could absorb various kinds of oils and organics up to 53.6 times of its own weight, and the absorbed oils could be collected through a simple squeezing process (Figure 9c). In addition, the presence of magnetic material, Fe3O4, allows easy drive by an external magnetic bar, thus reducing any contaminations.

**Figure 9.** (**a**) SEM images of pristine PU sponge, (**b**) SEM images of PU-LDH-Fe3O4-PDA-OM sponge, (**c**) A series of photographs for the process of absorption and collection toluene (dyed with Sudan III) from the surface of water. Reprinted from Ref. [127] with permission from Elsevier.

The composite materials are also considerably advantageous in the realization of coating films in a large number of fields, leading to an increase in performance and multifunctionality. For example, Wang et al. [128] fabricated superhydrophobic MgAl-LDH coatings on medium density fiberboards with flame retardancy, obtained by the deposition of polydimethylsiloxane (PDMS) and 1H, 1H, 2H, 2H perfluorodecyltrichlorosilane (FDTS)-modified LDH particles. The PDMS@FDTS-Mg/Al LDH coating exhibited superhydrophobicity with a water contact angle of 155◦ and self-cleaning property. The flame retardant character was evaluated by limiting oxygen index (LOI) and cone calorimeter test. LOI value of superhydrophobic MDFs increased by 60.4% as compared to that of the pristine MDFs, from 24.0 to 38.5. The peak heat release rate (PHRR) and total heat release (THR) of MDFs coated with PDMS@FDTS-Mg/Al LDH reduced by 24.7% and 11.2% as compared to MDFs alone. This result demonstrates that the presence of inorganic coating improved the flame retardancy of the MDFs. Wu et al. [129], inspired by the micro- and nanostructure of lotus leaves, or the Cassie state surface, developed a self-layered coating on magnesium alloy through a hydrothermal treatment, followed by radiofrequency (RF) magnetron sputtering of polytetrafluoroethylene (PTFE). A super-hydrophobic (water contact angle of 170◦) and acid resistant surface is created on the alloy. Recently, Yu et al. [130] reported on the possibility to use an environment-friendly LDH-base coating as high oxygen barrier coating in flexible food packaging. The coating film is realized by the reconstruction of MgAl-LDH from MgAl-layered double oxide (LDO) in concentrated aqueous glycine solutions, resulting in a coating solution of LDH and poly(vinyl alcohol) (PVA). It is worth observing the key role of the aspect ratio of reconstructed LDH in the coating permeability performance. Indeed, the relative permeability (P/P0) of the LDH/PVA coated layer decreases as the aspect ratio of LDH nanosheets are increased, from 0.0065 with aspect ratio 87 ± 17 to 0.0038 at 336 ± 170. This is explained by the highest aspect ratio of LDH nanosheets, which leads to better alignment in the coated layer. Thus, through the fine control of aspect ratio, the coating film reached an oxygen transmission rate <sup>&</sup>lt;0.005 mL·m−2·day−<sup>1</sup> at a film thickness of 1175 ± 101 nm.

#### *5.2. Nanogenerators and Physical Sensors*

In the era of smart devices and bioinspired sensor networks, LDHs and related compounds have been demonstrated to play multiple roles, starting, for example, from green nanogenerators for realizing self-powered sensors. In this field, it is worth to mention the work by Cui et al. [131], and those by Sun et al. [132] and Tian et al. [133], who realized a water-driven triboelectric nanogenerator (WD-TENG) and a natural water evaporation (NWE) driven generator, respectively, to harvest energy from water, the most abundant substance on our planet. Briefly, in the first approach, the realization followed a bottom-up strategy, by directly growing a well oriented MgAl-LDH nanosheet network on a metal substrate (Figure 10a). To reduce the surface energy, a further surface modification was performed via crosslinking of fluorine contained silanes, which in turn changed the morphology of the LDH network into a flower-like shape (Figure 10b). The fabricated WD-TENG relied on triboelectric effect, collecting energy from water droplets through the functionalized LDH film as triboelectric layer. The working mechanism is based on a contact-electrification and electrostatic induction at the liquid–solid interface (Figure 10c,d). In the second approach, a NWE-driven generator (NWEG) was realized via a spray-deposition of NiAl-LDHs on a polyethylene terephthalate (PET) substrate [132] (Figure 10e). It operates by means of an NWE-driven gradient of water which flows across the naturally formed surface-charged nanochannels between the LDH flakes, which were evenly spaced on the PET surface, with an ordered layer-by-layer stacking on the substrate (average channel width less than 50 nm) (Figure 10f,g). The performance of the NWEG was reasonably supposed to be regulated by the surface charge density (*d*c), the hydrophilic character and the presence of nanochannels or pores in the NG active layer, which are easily tunable intrinsic properties in the case of LDHs. In a successive work [133] the same group reported on the relationship between *d*<sup>c</sup> and the NWEG performance by precisely tuning *d*<sup>c</sup> of NiAl-LDHs in the range of 2.52–4.59 e/nm2, by adjusting the molar ratio of Al3<sup>+</sup> to Ni2<sup>+</sup>.

**Figure 10.** LDH-based water driven nanogenerators. (**a**) Schematic illustration of the fabrication process for the LDH-based triboelectric layer of the WD-TENG, showing (from left to right) the bare aluminum foil substrate and the MgAl-LDH nanosheet network before and after surface modification.

(**b**) Scanning electron microscope (SEM) images of the as grown (left) and surface modified (right) LDH film. (**c**) Schematic diagram of the modified LDH- (green) based WD-TENG collecting energy from water droplets. (**d**) Output voltage and current density of the WD-TENG under water droplets impacting. Reproduced from Ref. [131] with permission from Elsevier. (**e**) Photo (left) and schematic illustration (right) of the NiAl-LDH NWEG. (**f**) Top-view (top) and cross-sectional (bottom) SEM images of the NiAl-LDH film. (**g**) Schematic diagram of the working principle of the NWEG on a microscopic level. (**h**) (left) Evolution of the open circuit voltage (VOC, blue line) in response to the variation of the relative humidity (RH, red line) due to periodic opening and sealing of the container. (right) Response of VOC to periodic variations of RH between 96% and 28%. Reproduced from Ref. [132] with permission from Elsevier.

Another fundamental element in the fabrication of sensor systems is represented by the energy storage units. In a recent study, Xu et al. [134] reported on the realization of a coplanar asymmetric microscale hybrid device (MHD) supercapacitor based on MXene and CoAl-LDHs using a two-step screen-printing process. The LDH with its faradaic pseudo capacitance behavior was used as positive electrode material to enhance energy density and potential window. The resulting MHD exhibited very interesting electrical properties, such as outstanding cycling stability (92% retention of areal capacitance after 10,000 cycles), potential window extended to 1.45 V, and enhanced energy density (10.80 and 8.84 μWh cm−<sup>2</sup> in 6 M KOH and with PVA-KOH, respectively). In a more recent work [135], a CoMn-LDH doped with polypyrrole (PPy) was used as positive electrode for asymmetric supercapacitor (ASC) with multilayer graphene as cathode, exhibiting strong electrochemical performance. This supercapacitor showed properties comparable to or better than those of similar devices recently reported, with good cycle performance (99.5% retention after 8000 cycles) and energy density as high as 29.6 Wh kg−<sup>1</sup> at a power density of 0.5 kW kg<sup>−</sup>1.

The application of LDH in the field of bioinspired sensors was successfully demonstrated by Ren et al. [136], who developed a skin-inspired sensor based on a multifunctional nanocomposite hydrogel consisting of sodium alginate/sodium polyacrylate/layered rare-earth hydroxide (LRH) (SA/PAAS/LRH), fabricated through a 3D printing system (Figure 11a,b). The device showed a promising multifunctional behavior, such as humidity-dependent electromechanical properties, a sensitivity to mechanical deformation, thanks to a strain-dependent conductivity (Figure 11c), and tunable fluorescence (Figure 11d), while maintaining the characteristics of transparency and stretchability indispensable for the realization of a device for human motion detection. Such versatility makes it a good candidate for future soft wearable equipment. The use of LDH was also demonstrated to drastically improve the performance of composite materials for sensors. For example, Beigi et al. [137] reported on a modified ionic polymer metal composite (IPCM) humidity-sensor based on a nafion polymeric matrix doped with CoAl-LDH nanoparticles, with a significant improvement of sensitivity and responsivity due to the intrinsic hydrophilic characteristic of LDH.

**Figure 11.** Skin-inspired multifunctional device. (**a**) Scheme of the preparation of SA/PAAS/LRH hydrogel. (**b**) Top and side photos of the printed hydrogel wafer with a grid structure. (**c**) Response to

sensor strain. First row: Sensor attached on the forefinger of a human hand responding to bending angles from 0◦ to 90◦ (left); and sensor attached on the back of the hand responding when making a fist (right). Second row: Relative changes of the hydrogel films in sensing wrist bending (left); and elbow bending (right). (**d**) Fluorescent colors of various hydrogels, with different rare-earth ratios, under 254 nm in wavelength ultraviolet irradiation. Reprinted with permission from Ref. [136] Copyright (2020) American Chemical Society.

#### *5.3. Applications in Oxygen Evolution Reaction*

The process of oxygen evolution reaction (OER), 4 OH<sup>−</sup> + energy → O2 + 2H2O + 4e−, is one of the most critical steps of electrochemical water splitting, which is widely recognized as a promising and sustainable method to convert the intermittent electrical energy from the nature into stable and storable energy (hydrogen) [138,139]. However, OER, which is a multistep 4e- process, is thermodynamically not favored and thus kinetically sluggish [140]. Indeed, an OER catalyst should be able to overcome both the activation energy barrier (Ea) and the standard free energy charge (ΔG<sup>0</sup> = 1.23 eV). In recent years, substantial research efforts have been devoted to develop novel non-noble metal-based active OER catalysts based on efficient, low-cost and earth-abundant substitutes for conventionally used precious metal compounds [141]. Different strategies have been conceived to improve the catalyst performance, which may be categorized on three scale levels [142]: (i) at the atomic-scale (e.g., alteration of oxidation state, doping, coordination and composition of metal composites); (ii) at the nano-scale, including different material combinations templated on nanostructures (e.g., nanowires, nanosheets and nanotubes), to increase OER activity by means of high surface area and number of active sites; and (iii) at the meso-scale, i.e., the creation of a porous supporting architecture to enhance mass transport to electrolytes and structural stability. As intriguingly noted in recent studies [142–144] such optimization problem presents many analogies with the demand of photosynthesis in the evolutionary development of plant leaves, like the need of maximized surface area to capture as much light as possible, and sufficient space between neighbors to promote good gas exchange and surface reactions. In addition the 1D hollow tubular structures under the leaves facilitate the transport of nutrients and water to each leaf [142].

In the last decade, LDHs have been proven to be highly active, cost-effective and durable OER catalysts, exhibiting electrocatalytic activity and stability for OER, comparable to or higher than commercial precious metal-based catalysts [145–147]. In their reports, the scholars leveraged all the above-mentioned optimization routes to improve OER activity of LDHs. In particular, in 2014, Lu et al. developed OER electrodes based on a three-dimensional (3D) porous film of vertically aligned NiFe-LDH nanoplates loaded on a nickel foam. Excellent OER performance was demonstrated, with a small onset overpotential (~230 mV), large anodic current density (30 mA cm−2) and outstanding electrochemical durability, benefiting from the intrinsic high activity of the NiFe-LDH catalyst [148] and the unique 3D architecture, whose surface area was increased by the highly porous nickel foam. In the following, many research efforts were focused to improve the OER activity of LDHs (typically NiFe- and NiCo-LDHs), as increasing the number of active sites as well as increasing the activity of the individual active site. Song and Hu [149] and Liang et al. [150] demonstrated that liquid exfoliation of LDHs to single-layer nanosheets leads to greatly enhanced OER activity, due to an increase in the number of active edges sites and higher electronic conductivity, while preserving material composition. Next, Wang et al. [151] demonstrated that dry exfoliation of bulk CoFe LDHs into ultrathin LDH nanosheets through Ar plasma etching also resulted in the formation of multiple vacancies (including O, Co and Fe vacancies), thereby producing a dual effect to OER enhancement, due to the great number of exposed active sites in the 2D LDH nanosheets and their improved activity due to multiple vacancies. The dry-exfoliated CoFe-LDH performed very well in the OER with an overpotential of 266 mV to reach a current density of 10 mA cm−<sup>2</sup> vs. 321 mV required for the untreated pristine CoFe-LDH. In a further report, the same group [152] dry-exfoliated bulk CoFe-LDHs in a N2 plasma into edge-rich ultrathin nanosheets featuring, again, multiple vacancies, as well as nitrogen doping, which facilitated absorption of OER intermediates by altering the electronic density of the adjacent Co or Fe atoms. The ultrathin N-doped CoFe LDH nanosheets loaded on Ni foam exhibited excellent OER performance, with an overpotential of 233 mV at a current density of 10 mA cm<sup>−</sup>2. However, despite such atomic- to nano-scale optimization strategies, the OER performance of LDHs was limited by their intrinsically poor electrical conductivity. Some reports have proposed combining LDHs with conducive materials, e.g. carbon nanotubes [153], graphene [154], graphene oxide [155] and, more recently, silver [156] and CuO nanowires [157]. As for the latter, inspired by the monocot leaf structure in nature, Chen and coworkers conceived an engaging biomimetic nanoleaf based on ultrathin NiCo-LDH nanosheets in situ grown on Cu nanowires, forming a NiCo-LDH lamina featuring large electrochemical surface area (ECSA) and numerous active edge sites for OER reaction (see Figure 12). The CuO nanowires served as veins to support the LDH lamina, while providing mechanical support as well as improving the LDH conductivity, further increasing the OER activity. An enhanced OER performance was then achieved, significantly improved with respect to that of conventional NiCo-LDHs, with a quite small overpotential of 262 mV at 10 mA cm<sup>−</sup>2, good stability and flexibility.

**Figure 12.** Biomimetic LDH nanoleaf for OER. (**a**) Schematic illustration of the biomimetic NiCo-LDH based nanoleaf, showing (top) a photograph of the monocot leaf and a sketch of the biomimetic nanoleaf, with indication of the OER process, and (bottom) the fabrication procedure, with in situ growth of NiCo-LDH on the CuO nanowires/Cu mesh substrate. (**b**) SEM images of the CuO nanowires on the Cu mesh (upper panel, scale bar 10 μm), and of the biomimetic nanoleaves (bottom) (scale bar 1 μm). (**c**) OER performance. First row: Linear sweep voltammetry (LSV) polarization curves (left); and corresponding Tafel plots (right). Second row: Capacitive current vs. scan rates (left); and Nyquist plots at an overpotential of 300 mV (0.1 Hz to 100 kHz) (right). Reprinted from Ref. [157] with permission from Elsevier.

#### *5.4. Peroxidase-Like Activity*

The peroxidase enzymes are widespread in natural systems from bacteria to plants and humans, given their fundamental role in decomposing hydrogen peroxide (H2O2), which is a toxin produced as a byproduct of oxygen during aerobic respiration processes. These proteins contain a heme group in their active site that uses hydrogen peroxide as the electron acceptor to catalyze oxidative reactions. Such electron transfer ability between reducing substrate and H2O2 can be conveniently mimicked by many metal oxide nanoparticles—e.g., iron oxides, cerium oxides, metal sulfides and carbon nanodots. In this regard, many reports have focused on the realization of hybrid structures in which LDHs increase the surface/volume ratio and favor the dispersion of the nanoparticles, thereby allowing the peroxidase activity to be enhanced, as demonstrated by Yang et al. with CoAl-LDH/MFe2O4 (M = Ni, Zn, Co) [158] or CoFe-LDH/CeO2 hybrids [159], as well as core–shell Fe3O4/CoFe-LDH [160], finding applications in the field of analytical chemistry, especially for the determination of glucose, H2O2 and glucose and ascorbic acid, respectively. The mechanism of reaction is based on the electron transfer from the molecule 3,3- ,5,5- -tetramethylbenzidine (TMB) to H2O2. In the presence of glucose oxidase enzyme, glucose is oxidized to gluconolactone and oxygen is reduced to H2O2. As a result, the higher

is the glucose concentration, the higher is the concentration of H2O2 and, hence, the amount of oxidized TMB. Conversely, ascorbic acid is able to convert the oxidized form TMB to the reduced state. As a result, the obtained sensor is based on a colorimetric readout (see Figure 13a) and can be effectively employed in the field of low-cost glucose sensing.

Interestingly, LDH materials with intrinsic peroxidase activity were also engineered by simply introducing metal species featuring peroxidase-like activity mimicking the natural heme group. The first examples of such bioinspired systems were shown by Zhang et al. [161] who leveraged CoFe-LDH nanoplates to obtain a colorimetric sensor for H2O2 (based on the oxidation of TMB), reaching the optimal detection limit of 0.6 μM. Other similar examples are those from Su et al. [162], who leveraged NiCo-LDHs for acetylcholine detection (limit of detection equal to 1.62 μM), and from Zhan et al. [163], who investigated NiFe-LDHs as a sensor for H2O2, reaching the limit of detection of 4.4 ± 0.2 μM (see Figure 13b). Along with colorimetric detection, electrochemistry can also be applied to improve the detection limit of the sensor. In this regard, an example of an electrochemical sensor based on peroxidase mimicking LDHs worth mentioning was recently shown by Fazli et al. [164], who fabricated a PdAl-LDH/carboxymethyl cellulose (CMC) nanocomposite (CMC@Pd/Al-LDH) on a glassy carbon electrode to realize a sensor for H2O2 (limit of detection equal to 0.3 μM). Interestingly, this work shows how CMC is suitable for improving the sensitivity and the exposed active surface area, finally enabling a high number of available sites for electrochemical reactions. An intriguing application of peroxidase mimics for sensing acetylcholine (limit of detection equal to 1.7 μM) was reported by Wang et al. [165] who prepared NiAl-LDH/Carbon dot nanocomposites onto glassy carbon electrodes.

**Figure 13.** LDH-based peroxidase mimics. (**a**) CoAl-LDH/Fe2O4 hybrid materials catalyze the oxidation of TMB in presence of H2O2, mimicking the peroxidase activity. Reprinted from Ref. [158] with permission from Elsevier. (**b**) Intrinsic peroxidase-like activity in NiFe-LDH can be leveraged to build up colorimetric sensors for H2O2 and glucose. Reprinted from Ref. [163] with permission from Elsevier.

#### *5.5. LDHs on Biotemplates for Bioremediation*

The intrinsically high surface to volume ratio of LDHs, possibly enhanced by recently proposed ultrathin LDH synthesis routes [166–168], has been leveraged for many applications that require physical processes of absorption and interaction with molecules for bioremediation of contaminated sites, such heavy metals, toxic substances, pesticides or even favoring their photo-induced degradation [169]. An approach for further improving the efficiency of LDH-empowered devices for bioremediation is the realization of 3D biomimetic structures or the coupling with organic biotemplates (typically of plant origin) aiming at the realization of life-mimicking 3D high-surface hierarchical organization, miniaturization, eco-friendly characteristics and even specificity for the absorption of targeted molecular systems. For instance, typical examples of biotemplates are constituted by diatomaceous earth, leaves and cellulose fibers, which are prone to be easily functionalized by surface functionalization with LDHs.

A plethora of research efforts have been focused on the removal of oil, metal ions, pesticides and agrochemicals from contaminated water by using this combined approach. An interesting example comes from the research of Zhu et al. who showed a one-step synthesis of a biomimetic cactus-like hierarchical architecture for oil/water separation [170], based on a CoNi-LDH coated stainless steel mesh. The role of the 3D LDH morphology appears to be the key factor to induce an outstanding water locking capability into the cactus-like hierarchical structure. The trapped water is, in turn, able to form a stable water layer on the surface, ultimately forming a bioinspired barrier against oil penetration into the mesh, since the resulting surface would be superhydrophilic and underwater superoleophobic. The authors tested different types of oils (diesel oil, lubricating oil, silicon and n-hexane), finding excellent oil rejection ratio and high separation capability and outstanding recyclability over 20 cycles.

Abolghasemi and coworkers used the hierarchical structure of boehmite decorated withMgAl-LDH and porous carbon on a steel fiber for solid phase microextraction of fifteen different pesticides [171]. Interesting examples have also been provided for the removal of Congo red (CR), a dye which is widely employed in many textiles and biotechnological based industries. MgAl-LDH modified diatoms [172] and bioinspired magnetic ZnFe2O4 microspheres synthetized using pine pollen and covered with MgAl-LDH [173] have been used with this purpose. The presence of LDH significantly improved the absorption capacity of the composite material toward the dye, reaching values of about 300 mg/g in both cases. Whereas the 3D structure of the diatom triggered a multilayer CR dye adsorption, in the case of the magnetic ZnFe2O4 microspheres, the adsorption was better fitted following a Langmuir model. A very clever bioinspired approach for CR and Doxycycline (DC) removal and photodegradation under simulated sun light irradiation was proposed by Bing et al. [174], who combined photoactive bismute oxides, MgAl-LDH synthesized onto lotus pollen used as template, and calcination (C) treatments, for the fabrication of Bi2O3/Bi2WO6/MgAl-CLDH heterojunction hybrids with a 3D hierarchically porous structure. The adsorption of both CD and DC was very efficient, reaching 205.3 and 204.3 mg·g<sup>−</sup>1, respectively. Another relevant application of LDH triggered bioremediation comes from the examples in which toxic heavy metals ions can be absorbed on the LDH surfaces. To this aim, NiFe-LDHs [175] have been shown in combination with graphene oxide nanocomposite for the efficient removal of Pb(II) and Cd(II) ions from water, obtaining a maximum adsorption capacity of 986 and 971 mg·g<sup>−</sup>1, respectively, following a Langmuir model. A truly bioinspired system that efficiently removes Cu2<sup>+</sup> ions is the one shown by Dou et al. [176]. The authors leveraged functionalization of MgAl-LDH surface by using the Kabachnik–Fields reaction. More specifically, the MgAl-LDHs were modified with polydopamine, which was further used as source of reactive amino groups used for functionalization with diethyl phosphite, terephthalaldehyde and thiourea, finally allowing the introduction of phosphate groups in the LDHs which are able to specifically adsorb Cu2<sup>+</sup> ions, up to 105.44 mg·g<sup>−</sup>1. In another interesting report, Wang et al. [177] prepared a hybrid material that combined sulfide (derived from (NH4)2MoS4) intercalated NiFe-LDHs with alginate for the extraction of Pb2<sup>+</sup> in aqueous environments. The authors combined the high surface/volume ratio of the bioinspired alginate hydrogel with the LDH sulfide specific interaction with Pb2+, leading to maximum adsorption capacity of about 18 mg·g<sup>−</sup>1.

An outstanding bioinspired example of phosphate removal from water was shown by Lai et al. [178] who prepared a highly porous composite combining graphene oxide/MgMn-LDH (GO/MgMn-LDH) onto *Garcinia subelliptica* leaves, that constituted a natural bio-template (see Figure 14). The authors grew MgMn-LDH in-situ on the leaf-templated GO (L-GO) to obtain L-GO/MgMn-LDH. Interestingly, after calcination at 300 ◦C (L-GO/MgMn-LDH-300), the flavonoids which derived from the leaves were able to intercalate into the LDHs, avoiding the collapse of its structure. In addition, these biomolecules provided an outstanding specificity towards the interaction with phosphate ions, which was quantified as 244.08 mg·g−<sup>1</sup> at pH 3. The phosphate adsorption was very much dependent on the pH. Whereas optimal values were obtained at acidic pHs, the higher was the pH, the higher became the competition between OH- groups and phosphate in the interaction with the LDHs, finally reducing the efficiency in phosphate adsorption. Another clever example of biotemplated approach for the removal of antibiotic, i.e., doxycycline, from water was shown by the intriguing approach of Bing et al. [179] who realized 3D hierarchical tubular micromotors from kapok fibers which were functionalized with Br-intercalated MgAl-LDH/Mn3O4 hybrid. The manganese oxide permitted to

catalyze H2O2 decomposition generating oxygen bubbles for self-propulsion. The Br<sup>−</sup> anions acted as initiator to form an imprinted polymer to specifically absorb doxycycline up to 224.23 mg·g<sup>−</sup>1.

**Figure 14.** *Garcinia subelliptica* leaves templates for GO/MgMn-LDH based removal of phosphate anions. (**a**) Scheme of the templated assembly. (**b**) Phosphate anion adsorption capacity of L-GO/MgMn-LDH-300 compared to that of the calcined LDH composite without leaf-template (GO/MgMn-LDH-300) (left) and anion selectivity of L-GO/MgMn-LDH-300 (right). Figure reprinted from Ref. [178] with permission from Elsevier.

#### **6. Conclusions and Perspectives**

The field of LDHs is rapidly evolving from the initial investigations dealing with the materials discovery for new applications in chemistry, materials science and biomedicine. In fact, leveraging their outstanding simplicity in the synthesis, low cost, reusability and biocompatibility, LDHs have already proved to be a key player for in the field of nanotechnology.

Given their extraordinarily combination of properties and biocompatibility, as well as their capability to catalyze reactions under prebiotic conditions, LDH clays might be a material chosen by the molecular evolution of living systems. However, the approach correlating the existence of LDHs with the origin of life is very bold, and the question whether there could be a relationship between LDH clays (including green rust) and prebiotic synthesis remains unclear, thus continues being a hypothesis that would need to be addressed in depth in future studies. In general, it is believed that clays have been key systems for the development of prebiotic conditions, given their ability in concentrating and protecting life-essential molecules, creating autonomous semipermeable compartments, as well as catalyzing polymerization reactions. In this context, the specific role of LDHs is still underestimated, but it is growing steadily, especially considering the relevant role of these materials in the context of prebiotic peptides synthesis. Motivated by this, the review analyzes the currently state of the art concerning the interactions of life-relevant molecules (DNA, phospholipids, amino acids and carbohydrates) with LDHs, focusing on the structural point of view, in particular analyzing the possible formation of ordered and crystalline systems and, if possible, providing some insights into relevant biological role. Then, after briefly describing the fabrication of LDHs-based life-mimicking compartments, the review focuses on the technologically relevant applications of life-like and life-inspired devices. More specifically, we focus our attention on important applications where bioinspired LDHs play an important role, i.e., composites and coatings, physical and chemical sensors, catalysis and bioremediation.

The low cost and the eco-friendly synthetic approaches for LDHs make them suitable materials for relevant applications, as the formulation of paints, flame retardants, phytosanitary and pharmaceutical products, given their key role as dispersing or encapsulation agents for anionic molecular systems. To our knowledge, Kyowa Chemical Industry was the first to produce synthetic LDHs, since 1966, among their vast product portfolio, obtaining a good market success, especially in the field of resin stabilizers, residual catalyst removal and chlorine absorbers. The path to bring to the market the innovations and approaches reviewed in the present work is clearly challenging, and still many efforts

are requested of researchers to facilitate a more extended development at a larger industrial scale. As a matter of fact, today, many hybrid organic–inorganic nanomaterials are entering a variety of markets [180]. New materials must aim toward higher levels of sophistication, be recyclable and environmentally-friendly and consume less energy or enable energy harvesting. We hope that the bioinspired approach showed in this review could trigger further technological transfer from academia to the industry, highlighting how bio-friendly LDH products can tackle the need of sustainable chemistry approaches for product manufacturing.

This review does not pretend to cover all the possible literature actually available in the field; however, it can be considered as an effort to review the most recent and exciting advancements in the field of bioinspired applications of LDHs. We hope that it can trigger future LDH-based studies, both on the fundamental science and on the applications, with the ultimate aim to favor a further development of LDHs-based nanotechnology under a novel eco-friendly, bioinspired perspective.

**Author Contributions:** Methodology, G.A. and R.P.; writing—original draft preparation, G.A. and G.P.; writing—review and editing, G.A. and G.P.; and supervision, G.A., G.P. and P.G.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** The article processing charges were funded by SIMITECNO SRL, Sistemi e misure per tecnologie, Via Gallian, 62, 00133 Roma (www.simitecno.com).

**Acknowledgments:** We acknowledge the Università degli Studi di Palermo for hospitality and support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Layered Double Hydroxides: A Toolbox for Chemistry and Biology**

#### **Giuseppe Arrabito 1,\*, Aurelio Bonasera 1, Giuseppe Prestopino 2,\*, Andrea Orsini 3, Alessio Mattoccia 2, Eugenio Martinelli 4, Bruno Pignataro <sup>1</sup> and Pier Gianni Medaglia <sup>2</sup>**


Received: 20 June 2019; Accepted: 13 July 2019; Published: 15 July 2019

**Abstract:** Layered double hydroxides (LDHs) are an emergent class of biocompatible inorganic lamellar nanomaterials that have attracted significant research interest owing to their high surface-to-volume ratio, the capability to accumulate specific molecules, and the timely release to targets. Their unique properties have been employed for applications in organic catalysis, photocatalysis, sensors, drug delivery, and cell biology. Given the widespread contemporary interest in these topics, time-to-time it urges to review the recent progresses. This review aims to summarize the most recent cutting-edge reports appearing in the last years. It firstly focuses on the application of LDHs as catalysts in relevant chemical reactions and as photocatalysts for organic molecule degradation, water splitting reaction, CO2 conversion, and reduction. Subsequently, the emerging role of these materials in biological applications is discussed, specifically focusing on their use as biosensors, DNA, RNA, and drug delivery, finally elucidating their suitability as contrast agents and for cellular differentiation. Concluding remarks and future prospects deal with future applications of LDHs, encouraging researches in better understanding the fundamental mechanisms involved in catalytic and photocatalytic processes, and the molecular pathways that are activated by the interaction of LDHs with cells in terms of both uptake mechanisms and nanotoxicology effects.

**Keywords:** layered double hydroxides; cellular biology; catalysis; DNA; drug delivery; hydrotalcite; osteogenesis; photocatalysis; RNA.

#### **1. Introduction**

Nanostructured materials or nanomaterials (NMs) represent an important area of research and a technological sector with full expansion for many different applications. They have long been considered of paramount importance due to their tunable physicochemical properties such as melting point, wettability, electrical and thermal conductivity, catalytic activity, light absorption, and scattering, which ultimately result in better performance compared to their bulk counterparts. In general, NMs are described as materials with length of 1–100 nm in at least one dimension, and can be roughly classified according to their:


Of particular importance is the classification according to their size [1]. *0D nanomaterials* are systems that are at nanoscale in all their *x, y, z* sizes. Remarkable examples include carbon nanodots [2] and metallic nanoparticles (NPs) [3]. *1D nanomaterials* have one dimension greater than nanoscale, e.g., length on the micron scale in one direction only (for instance carbon nanotubes [4], silicon nanowires [5], or ZnO nanowires [6,7]). They have shown important applications as it both interconnects and has key units with a nanoscale dimension in electronics and optoelectronics. On the other hand, *2D nanomaterials* have at least two dimensions on the micron scale: Examples include nanoplatelets and nanoribbons with length or diameter on the micron scale and a nanoscale thickness. In particular, the synthesis and the application of 2D nanomaterials have become a fundamental research area in materials science, due to their many intriguing low-dimensional features that are different from the bulk properties, making them more attractive for subsequent utilization as key building blocks of nanodevices [8]. Besides the basic understanding of new physico-chemical phenomena, 2D NMs are also particularly interesting for investigating and developing novel applications in sensors, photocatalysts, nanocontainers, nanoreactors, and templates for 2D structures of other materials [9]. Three-dimensional nanomaterials (3D) can be defined as materials whose characteristic x, y, z dimensions are well beyond the nanoscale (i.e., >100 nm). These systems are typically not included in the category of nanostructured materials, unless their internal structure is nanostructured. Typical examples of this class of materials are the following: dispersions of nanoparticles, bundles of nanowires, as well as multi-nanolayers, nanocrystalline, or nanoporous materials [10].

Among NMs, layered double hydroxides (LDHs) represent an emerging class of 2D layered materials belonging to the group of hydrotalcite-like (HT) compounds, or anionic clays [11]. LDH structure can be described based on the stacking of charged brucite-like layers consisting of a divalent metal ion M2<sup>+</sup> (e.g., Ca2<sup>+</sup>, Zn2+, Mg2<sup>+</sup>, Ni2<sup>+</sup>), octahedrally coordinated to six OH<sup>−</sup> hydroxyl groups, in which part of the divalent cations (M2<sup>+</sup>) are substituted by the trivalent ions M3<sup>+</sup> (e.g., Al3<sup>+</sup> Fe3<sup>+</sup>, Cr3+, In3+). Such replacement leads to the formation of positively charged layers, whose net charge is compensated, to maintain the global electroneutrality, by the presence of exchangeable anions (An<sup>−</sup>, such as hydroxyl groups, nitrates, carbonates, and sulfates) in between the layers conjointly with water molecules. The general formula of LDHs is the following: [M1–*<sup>x</sup>* <sup>2</sup><sup>+</sup> M*<sup>x</sup>* <sup>3</sup><sup>+</sup>(OH)2] *<sup>x</sup>*+·[A*x*/*n*] <sup>n</sup>−·*m*H2O, where M2<sup>+</sup> and M3<sup>+</sup> are the divalent and trivalent metal ions, respectively, An<sup>−</sup> are inorganic or organic anions, *m* is the number of interlayer water, and *x* = M3+/(M<sup>2</sup>++M3+) is the layer charge density, or molar ratio. LDHs are characterized by a low dimensional opened structure that is suitable for physico-chemical intercalation and adsorption processes with a large variety of molecules ranging from organic molecules to biomacromolecules.

The conventional approach to the synthesis of LDHs is based on the coprecipitation method [12–14], which can be briefly described as follows. The addition of a base to a water solution containing the salts of two different metals, namely M2<sup>+</sup> and M3<sup>+</sup>, causes the precipitation of the metal hydroxides and the formation of LDHs. The precipitates are collected, washed and then dried to be deposited on a solid substrate, or dispersed in solution phase. In some applications it is necessary to produce homogenous films onto solid substrates [15,16]. For this, two different research groups [17,18] demonstrated the possibility of growing stable films of well-formed interconnected LDH nanoplatelets directly onto aluminum surfaces, by immersing aluminum thick foils in a water solution of zinc nitrate or acetate. Differently from the conventional growth method, a single salt is employed in the growth solution providing the divalent metal (Zn2<sup>+</sup>), while the trivalent ion (Al3<sup>+</sup>) is provided by the sacrificial aluminum foil, which behaves as both reactant and substrate, finally improving the adhesion as well. In this regard, the growth of regular ZnAl LDH nanoplates [19] on Al-coated silicon substrates was demonstrated, along with the influence of the reacting aluminum layer thickness on the final morphology and composition of the LDH nanostructures. These kind of LDHs found application as gas sensors [20], Li-ion battery electrodes [16], for enhanced oxygen evolution catalysis [21], and surface enhanced fluorescence [22]. LDH systems are subjected to the so-called "memory effect", i.e., for suitably low calcination temperatures, the resulting phase can be restored to that of LDH by rehydration in water. Remarkably, during the rehydration new anionic species can be intercalated and functionalized, obtaining desired physicochemical properties [23]. In the following, the M2<sup>+</sup>:M3<sup>+</sup>

stoichiometric ratio in LDH will be precisely mentioned and discussed, only if definitely stated by the authors to be meaningful for the application of the LDHs. Otherwise, the LDH formula will be simply indicated as M2+M3<sup>+</sup> LDH (see Figure 1).

**Figure 1.** Schematic view of the M2<sup>+</sup>M3<sup>+</sup> LDH general structure, with Cl−, NO**<sup>3</sup>** <sup>−</sup> and CO3 <sup>2</sup><sup>−</sup> anions intercalated in the brucite-like structure. Other possible chemical species eventually present in the interlamellar space are reported, i.e., hydroxyl groups and interlayer water molecules. The figure is reprinted from Ref. [24] under a Creative Commons Attribution Non-Commercial No Derivatives License (CC BY-NC-ND).

The aim of this review is to introduce the emerging role of LDH materials in chemistry and biology, providing the reader with a broad up-to-date view of the role of these materials in these fields. Indeed, given the widespread contemporary interest in these issues, the topic is certainly hot, and from time-to-time, it is necessary to review the recent progresses. Differently to the previously published reviews, the aim of the present work is to provide some fundamental references concerning earlier investigations and also providing recent researches that are establishing new trends. As to the chemistry-related topics, the interest for LDHs is reviewed especially focusing on relevant organic chemistry reactions and recent advances in photocatalysis, with the ultimate goal to elucidate the key role of LDHs that is rarely investigated in literature. As to the biology-related field, LDHs are reviewed focusing within the well-explored applications in electrochemical and optical biosensors, along with DNA delivery. In addition to these, the review summarizes novel and barely explored topics, namely optical sensors, RNA delivery, cellular differentiation, and fundamental biological pathways relevant to the LDHs uptake into cells. The critical and unprecedented overview provided by the present review is hopefully able to motivate new researchers interest in these emerging fields.

Initially, we will discuss the immense potentiality of these 2D materials in organic chemistry, in particular focusing on their role as catalysts in highly relevant chemical reactions, namely the Baeyer–Villiger reaction, Knoevenagel reaction, Michael addition, and transesterification (Section 2). We will then focus on the outstanding role as photocatalytic centers, discussing their applications to organic molecule degradation, water splitting reaction, CO2 conversion/reduction, and other examples (Section 3). Subsequently, we will analyze the intriguing role of these materials in biology, where their biocompatibility and the suitability as powerful drug vectors have triggered an enormous research interest in the last few years (more than 1000 papers published in 2018 regarding drug and DNA delivery, according to data extracted from the Scopus database). In this regard, we will provide a focus on the research results of the last two years, regarding applications of LDHs as biosensors (Section 4), especially focusing on the novel exciting applications as optical sensors. We will then analyze their

important role as nucleic acid delivery agents (Section 5), providing an unprecedented view on the novel applications in siRNA therapy. Lastly, a specific view on LDHs as key materials in cellular differentiation, drug delivery, and contrast agents will also be provided (Section 6).

#### **2. LDH Applications in Organic Catalysis**

This section will focus on the role of LDH-based materials in organic chemistry, mainly as catalyst for the preparation of fine chemicals, intermediates, and valuable molecules. The interest for LDHs in chemical synthesis is due to some common features they share with classic heterogeneous catalysts; once a reaction has been catalyzed by LDHs, these layered materials can be easily removed from the crude reaction mixture (e.g., by filtration) and recovered for next reaction cycles, with significant simplification of the work-up procedure for the chemist. Among the plethora of available heterogeneous catalysts, LHDs have some additional appealing features, such as the simplicity of their preparation from cheap precursors, the involvement of non-harmful precursors, low toxicity of their possibly produced decomposition products, and no need of expensive rare elements. For details on synthesis routes, the reader is advised to go back to the previous Section, however some other aspects relevant to LDH fabrication strategies will be briefly discussed here as well, in order to clarify why scientists devote increasing attention towards specific LDH compounds.

Chemists' interest over clays does not go so back in time: early in the 80s the works of Kruissink and Reichle on methanation [25] and acetone oligomerization [26], respectively, pointed out that LDHs could be employed as precursors for catalytic species, after thermal degradation. In those works, the properties of the pristine LDHs were not addressed, but shortly after Martin and Pinnavaia revealed how LDHs could be useful also in their pristine form [27]. From there on, the number of publications on catalytic LDHs quickly grew. The aforementioned paper not only had a historical relevance, but it allows us to discuss the different strategies that researchers have when dealing with LDHs as catalysts (see Figure 2):


We are going to restrict our discussion on the latter case. Indeed, in the first case, LDH materials derive their reactivity from the hosted species, and the LDH crystal lattice has a relatively marginal role, boosting up the catalytic activity of the absorbed species but not participating directly. Several reviews have already been published focusing over specific examples where carbon nanoforms [28,29], metal nanoparticles [29,30], organic guests [31,32], and oxometalates [33,34] are used for the preparation of interesting LDH-based hybrids. In the second group, LDHs typically undergo thermal stress, with a consequent loss of structural features and transformation into mixed metal oxides (MMOs) [35,36]. Such a procedure is useful when the lattice is limiting the interaction between the metal centers and the reactants, thus the catalytic behavior arises when the layered clay framework, that is the key feature of a LDH-material, is lost. MMOs are used for a wide range of reactions, mostly in the gas phase [37–40], and various alkylation protocols, condensations and transesterifications [41]. The easiest way to reduce LDHs into MMOs is calcination, but this procedure should not be considered as a protocol since it

points only to the loss of LDH structural features. Firstly, it could be a simple way to remove excess of water, which is detrimental for several catalytic routes [42]. Secondly, operating a strict control over calcination temperature, the so-prepared MMOs can regenerate LDHs after rehydration [43–46]. This is the so-called "memory effect", and published data reported that calcination temperature (Tc) should be below 500 ◦C, as well as compatible with thermal stability of eventual other species introduced/grafted on LDHs [47].

**Figure 2.** Representation of different possible approaches for the development of LDH-based catalysts for organic synthesis.

The reactivity intrinsically ascribed to LDHs is related to their layered structure formed by cations, anions and structural water molecules, but there are other ruling parameters; the most crucial ones are summarized here:


Intriguingly, the metal cation composition strongly contributes to the specific reactivity of LDHs, but it tunes as well the strength of basic moieties. The incorporation of specific metals in the octahedral layer can produce LDH compounds with unexpected and novel properties, as well as modulate the existing chemical reactivity [51,52]. It is worthy to mention literature where the arrangement of cations within the LDH lattice is discussed. As a matter of fact, the ordering of cations is believed to have crucial effects on many of the physicochemical properties of LDHs, affecting the charge density of the metal hydroxide sheets, and the overall bonding, mobility, orientation, and reactivity of the chemical species in the interlayer spaces and on the surface [53–58]. It is therefore desirable to have

homogeneous distribution of cations without segregation of phases with specific composition, which would result in varied reactivity, and in catalytic processes, generation of nonspecific products [59]. Similarly, the composition of the anionic species can be varied [60]. Some other clear advantages bring researchers to consider LDHs as catalysts of choice instead of more traditional ones [57,58]. Besides their activity and/or selectivity, in heterogeneous catalysis LDHs offers the opportunity of minor production of waste and an easier removal of the catalytic species from the crude reaction mixture. One example is given by the work of Lü et al., who fabricated LDH-based catalytic films over porous alumina membrane [61]. Interestingly, the authors revealed superior activity of the catalytic film in the aldol condensation of acetone, compared to the LDH-powders prepared under identical conditions. The authors also pointed out that those LDH-crystallites grown over the alumina support retained hexagonal plate-shape perpendicularly on the substrate, which favored the diffusion of the reactants throughout the substrate, despite the more disordered organization of the LDH-powders.

In the next paragraphs, we will consider some organic reactions of interest, and we will try to give an overview of the most relevant and recent contributions of LDH compounds in organic synthesis. Our aim is to give to the general audience, not only to organic chemists, the feeling why so many researchers focused on specific reaction pathways. We will report, as well, some examples regarding industrially relevant molecules and/or precursors whose synthesis can be improved applying LDHs chemistry. Our attention regards the following classes:


We would like to recommend a review by Sels et al. [62], whose analysis embraced several classes of reactions, although the reader should notice that no distinction was made among LDHs, MMOs derived from LDHs, intercalated materials and else. With an insight into life sciences, it is worth to point out that the synthesis of therapeutic molecules (i.e., drugs, active compounds) typically requires a high number of transformation and purification steps. A straightforward strategy to improve the sustainability of complex chemical synthesis is to consider how nature synthesizes biologically active compounds using enzymatic catalysis. Unfortunately, enzymes that catalyze C-C and C -N bond forming reactions employed in the synthesis of active pharmaceutical ingredients (such as Knoevenagel condensation, Henry reaction, Michael addition, and Friedel-Crafts alkylation) are quite rare [63]. In this regard, chemists have looked into more robust and cost-effective catalytic systems that can be adapted to a broad range of reaction conditions, in order to decrease the production costs of synthesis.

#### *2.1. Baeyer–Villiger Reaction*

The Baeyer–Villiger (BV) reaction (see Figure 3) is an oxidative pathway bringing to the formation of esters and lactones by oxidation of carbonyl compounds with a peroxide derivative [64]. The reaction takes the name of its two developers, Adolf von Baeyer (previously known for the synthesis of indigo) and his student Victor Villiger, reporting the use of Caro's acid (KHSO5) as a new oxidant for the conversion of cyclic ketones to the corresponding lactones [65]. This versatile protocol was revised in the last century of applications [66]; the interest towards this reaction increased when the work by Fried et al. provided the first definitive evidence of lactone formation in living organisms according to the Baeyer–Villiger pathway [67]. Moreover, the enzymatic B-V oxidation was also explored to facilitate biotransformation of sterically demanding ketones [68]. These evidences opened the way to the search of new oxidants, with major urge in the last 20 years due to the necessity of greener protocols [69,70].

**Figure 3.** Baeyer-Villiger reaction protocols, and the evolution of commercially available oxidative reagents.

Some interesting papers came out in the middle of the nineties from Kaneda et al., who reported heterogeneous Baeyer-Villiger oxidation of ketones catalyzed by MgAl-CO3 LDHs using an oxidant combination of molecular oxygen and aldehydes [71]. They also reported higher activity for oxidation of five-membered ring ketones than for that of six-membered ones, with improved yields using mild reaction conditions (40 ◦C). Successive work reported multi-metallic MgAl LDHs containing Fe ions efficiently oxidizing various cyclic ketones in the same mild conditions, while the exchange of Fe with Cu gave superlative yields with bicyclic ketones [72]. The intuition of the beneficial effects due to the presence of iron in the LDH lattice was also confirmed by Kawabata and coworkers, in the frame of a wider investigation including LDHs with different metal species, such as Fe, Co, Ni, or Cu [73]. An attempt to adapt the procedure to other commercially available oxidants was reported as well [74].

A step towards greener chemistry with no or less harmful chemicals was detailed by Pillai et al., who reported Sn-doped LDHs as efficient and relatively cheap catalysts for BV oxidation reactions, in combination with classic H2O2 as oxidizing agent [75]. The mechanism of oxidation was tentatively explained as due to the carbonyl group activation of ketone by the Sn present in the interstitial spaces of LDHs, followed by a subsequent nucleophilic attack by the active peroxide species (peroxycarboximidic acid by reaction of acetonitrile and H2O2); the so formed Criegee adduct rearranges to give the corresponding lactone as a final product. Lately, Jiménez-Sanchidrián et al. [76] confirmed the choice of Sn as a primary element to be considered in the design of BV-active LDHs, with reactions running under very mild conditions (atmospheric pressure and a temperature of 70 ◦C), conversion yields sometimes higher than 80%, and 100% selectivity after 6 h. Among their findings, it is worth noting the evidence that solids containing Zr promoted the decomposition of the hydrogen peroxide and hence adversely influenced the oxidation reaction.

Recent investigations by Olszówka et al. showed that the catalytic performance of MgAl LDHs in cyclohexanone oxidation with H2O2 /nitrile system could be significantly improved by the use of Mg-rich catalysts (Mg/Al = 3.69) [77]. Yields up to 50% ε-caprolactone could be achieved in a single-phase reaction medium based on the acetonitrile solvent. The use of acetonitrile rather than the more expensive, more toxic and more difficult to handle benzonitrile, represented a greener and a more economically viable option. Successive structural studies revealed that a lowering of the microcrystalline domains was beneficial for the BV reaction as it improved catalyst's selectivity [78]. This effect was attributed to the observed higher hydrophilicity of less crystalline materials. While it was argued that the enhanced hydrophilicity of poorly crystalline HT samples facilitates the approach and activation of H2O2 on surface basic centers, it makes the catalysts more prone to random interactions with the organic substrate, and to its subsequent non-selective transformation [79].

#### *2.2. Knoevenagel Reaction*

The Knoevenagel condensation (see Figure 4) is one of the most diffused tools in the hands of organic chemists. Such versatile reaction can be briefly defined as the product of interaction between a carbonyl compound (aldehyde or ketone) and any compound having an active methylene group. When we say active, we mean that the presence of ancillary electron withdrawing moieties (nitro, cyano, or acyl group) weakens C-H bonds in close proximity. The reaction terminates with a dehydration reaction in which a molecule of water leaves the molecular skeleton. The primary product of a Knoevenagel reaction is usually an α,β-unsaturated ketone [80]. The Knoevenagel condensation is frequently used when building biologically active scaffolds, and it finds application in the synthesis of several biologically-active molecules [81], such as steroids [82].

**Figure 4.** Knoevenagel reaction general scheme. Middle and bottom: examples of some pharmaceutically relevant molecules, whose synthesis involves Knoevenagel condensation. In evidence, the newly formed chemical bond (orange) and the residues belonging to the starting carbonyl (red) and 1,3-diketo (blue) moieties.

An interesting application of LDHs in Knoevenagel reaction goes back to 1999, when Rousselot et al. reported the synthesis of mixed Ga/Al-containing LDHs using different counter-ions (CO3 2−, F−, and NO3−) and doped with different divalent and trivalent cations (Cu2<sup>+</sup>, Mg2<sup>+</sup>, Zn2<sup>+</sup>, Al3<sup>+</sup>, Ga3<sup>+</sup>, Mn3<sup>+</sup>, and Sc3<sup>+</sup>) [83]. In their systematic study, they reported on the relative amounts of metallic ions necessary for achieving the best performing material. Besides the structural characterization, the most interesting results regarded the reactivity performance for the Knoevenagel condensation of ethyl cyanoacetate with benzaldehyde. Both calcined and uncalcined materials showed high reactivity; a rehydration process of the calcined samples during the catalytic reaction may explain the similarity. Water, formed together with ethyl cyanocinnamate, would react with the catalysts to give back the original LDH structure.

Fluorinated LDHs (LDH-F) were later developed by Choudary and collaborators in the effort to obtain a more basic (and efficiently) Knoevenagel catalyst [84]. The catalytic clay was prepared starting from a MgAl–NO3 LDH, which underwent calcination (450 ◦C) followed by rehydration in presence of KF aqueous solution. The highly polarized basic fluoride ions displayed high catalytic activity both in Knoevenagel condensation (tested the reaction between 2-methoxybenzaldehyde and malononitrile) and 1,4-Michael addition (tested the reaction between acetylacetone and methyl vinyl ketone), under mild liquid phase conditions. The other advantages of LDH-F include easy separation of the catalyst by simple filtration, high atom economy to enable waste minimization, reduced corrosion, and reusability. Such features are certainly appealing for the industrial world converting to greener protocols.

In the general attempt of finding the best combination of experimental parameters, a step forward came from Constantino et al. [85]. In their work, they focused on NiAl-CO3 LDHs applied to the Knoevenagel reaction involving malononitrile and ethylcyanoacetate, in neat conditions, since the reagents could work as a mixing liquid phase. Mild reaction conditions (60 ◦C) were sufficient to bring the reaction to completion. Interestingly, during preparation of the heterogeneous catalyst, they performed a thermal pre-treatment (150 ◦C per 1 h) claiming that the excellent reaction yields could be due to the anhydrous catalyst that should be able to co-intercalate water molecules generated in the Knoevenagel condensation, and withdraw the reaction equilibrium to the formation of the adduct. Dimethylmalonate, having methylene groups of very low acid strength (pKa > 13) did not give the benzaldehyde adduct, opening the room to chemoselective reactions. Shortly later, the results from Li et al. confirmed the intuition that calcined materials could work in a more profitable way in the context of Knoevenagel protocols [86]. In their study, carbonate-containing LDHs with different combinations of Al3<sup>+</sup>, In3<sup>+</sup>, and Mg2<sup>+</sup> cations were investigated in the condensation of ethyl cyanoacetate with benzaldehyde. The structural characterization pointed out that other relevant parameters should be taken in account, i.e., porosity, surface area, and basicity of the materials, which always exhibited higher values in the case of the more performing calcined materials. Lei et al. moved little further, pointing at the crystallinity of the material [87]. For their MgAl-OH LDHs obtained by calcination/rehydration of MgAl-CO3 LDH-precursors with high crystallinity, a base-acid catalytic mechanism was proposed to interpret the catalytic behavior. Activated MgAl LDHs synthesized by urea hydrolysis showed a much higher activity in aldol and Knoevenagel reactions than the corresponding material synthesized by the co-precipitation method, suggesting that the presence of acid-base hydroxyl pairs was required, and that catalytic activity was maximized for highly crystalline structures with an ordered array of surface hydroxyl groups. Other groups pursued different approaches towards the improvement of catalytic performance, not related to structural features. Among them, one option was to maximize the basicity of the material, by the introduction of new chemical motifs. In a recent example [88], MgAl LDH-nanosheets were functionalized by grafting aminopropyltriethoxysilane (APTS) onto the clay surface, introducing free amino groups. The prepared MgAl-NH2 LDH-nanosheets exhibited excellent performance in the Knoevenagel condensation compared to the homogeneous catalyst APTS and the heterogeneous catalysts with Al2O3 and SiO2. These results find reasonable justification in the high surface area of the hybrid nanosheets, and synergistic effects among MgAl LDHs, the grafted -NH2 groups, and reaction substrates.

Some recent reports tried to conjugate the advantages of heterogeneous catalysis with greener approaches. An example is grafting of LDHs with ionic liquids (ILs). Khan et al. opened the way testing widely described MgAl LDHs (Mg:Al = 3:1), synthesized following published procedures and avoiding pre-treatments, immersed in ILs [89]. Besides the advantageous efficiency of LDH-catalysts and the presence of ILs as a safe and reusable media, they reported also significant alteration in diastereoselectivity in the case of the nitroaldol reaction with nitroethane. In a recent report, Li et al. grafted ILs with different length of their alkyl chains (IL-Cn with n = 4, 8, or 12) onto MgAl-NO3 LDHs, revealing that the grafting approach helped to adjust the distribution of basic sites, and also induced flexibility to the catalyst, allowing easy accessibility of the active centers by the substrates [90]. Application of LDH-ILs-Cn for Knoevenagel condensation of various aldehydes with ethyl cyanoacetate/malononitrile results in excellent yields, high selectivity, and efficacy in aqueous solution at room temperature, and recyclability as well.

It is here worth mentioning a less conventional approach proposed by Zhou and coworkers, who reported on LDHs developed for the catalysis of two different reactions, which happened in sequence in their case, but that could be easily controlled by the presence/absence of specific chemicals [91]. In more detail, they investigated NiGa LDHs, which could catalyze oxidation reactions and Knoevenagel condensations; in the specific case, alcohols were oxidized to the corresponding carbonyl compound (in the study, benzyl alcohol to benzaldehyde), which reacted in the next step with a partner molecule (benzoylacetonitrile). The introduction of the second partner necessarily ruled the occurrence of the second step, otherwise the simple oxidation would result at the end of the treatment. A good tolerance for the catalyst to various substrates, and excellent recyclability were also reported.

#### *2.3. Michael Addition*

The Michael addition reaction (see Figure 5) is generally described as a base-catalyzed addition of a nucleophile, such as an enolate anion, to an activated α,β-unsaturated carbonyl-containing compound, resulting in the formation of a new C-C bond [92]. Since the pioneering work by Arthur Michael [93], this versatile protocol was widely developed for several different fields, ranging from basic research [94] to industrial applications [95].

**Figure 5.** Michael Addition Reaction General Scheme. Middle and bottom: examples of some industrially relevant materials, whose synthesis takes advantage of Michael Addition. In evidence, the newly formed chemical bond (orange) and the residues belonging to the starting carbonyl (red) and 1,3-diketo (blue) moieties.

LDHs play again an interesting role in the recent development of new synthetic routes. A first combination of LDH chemistry and Michael additions came with the work by Choudary et al. [96]. They reported selective 1,4-additions on methyl vinyl ketone, methyl acrylate, simple and substituted chalcones by donors such as nitroalkane, malononitrile, diethylmalonate, cyanoacetamide, and thiols, catalyzed by MgAl LDHs. In their work LDHs, as synthesized or just calcined, showed no relevant activity for the reactions described here, but only the material obtained by decarbonation and subsequent rehydration was an efficient and very selective catalyst. Products of undesirable side reactions resulting from 1,2-addition, polymerization and bis-addition were not observed. Later [97], they discussed the activity of MgAl-OtBu LDHs prepared by incorporating tert-butoxide anion into the interlayer of the clay structure to enhance the basicity of the material. The results were encouraging; reaction yields were reported always to be >85% with mild reaction conditions (room temperature in methanol). Compared with already reported procedures based on non-LDH catalysts, Michael reaction does not entail anymore very long reaction times (in the worst presented case, 2 h vs. 75 h for traditional protocols), high catalyst loading and low yields of the adducts. Waste minimization without any side reactions, use of non-toxic and inexpensive catalysts and recyclability of catalyst are other advantageous features of this procedure. Survey over several reaction parameters (solvent, temperature, and time) was reported by Naciuk et al. [98] who investigated Michael reaction for the synthesis of γ-aminobutyric acid (GABA). Another study on the Michael additions of 2-methylcyclohexane-1,3-dione, 2-acetylcyclopentanone, and 2-acetylcyclohexanone to methyl vinyl ketone interestingly revealed that Mg/Al molar ratio was strongly related to the catalytic performance, pointing out a nonlinear correlation between the Mg content and catalytic activity [99]. This finding would suggest that pure MgO would be more efficient than MgAl LDHs, as they tested and reported, but it causes consecutive reactions of the Michael addition products, which detrimentally reduce product selectivity and yield. Kaneda et al. reported

further insights into the reaction mechanism and the quantification of LDH basic sites [100]. LDH compounds were also used as catalysts in green protocols for Aza-Michael addition. In this regard, in the effort to determine the LDH-catalyst with the highest activity and selectivity, Kantam et al. discussed the reaction between dibutylamine and methyl acrylate (Aza-Michael reaction, due to the amino-group acting as nucleophile) comparing several LDH compounds obtained from calcination and rehydration of different precursors, as well as pure metal oxides [101]. CuAl LDHs proved to be the best candidates, with HT catalyst at room temperature in very good yields. The Cu-Al hydrotalcite showed enhanced activity over the other tested solid catalysts, and it was reused for several cycles with consistent activity and selectivity. One last interesting report concerns LDH-catalysts with anchored L-proline [102]; the catalytic activity was comparable to what reported for other LDHs, but the novelty lay in the asymmetric induction on the Michael addition products. Reaction between β-nitrostyrene and acetone with an inversion in the asymmetric induction was observed when compared to the reaction using pure L-proline catalysis, opening the way to the production of enantiomers using catalysts where the chiral (and expensive) enantiomer is recycled for both the two reactions.

#### *2.4. Transesterification (Biodiesel Production)*

The transesterification reaction is one of the universal and well-known tool in the hands of organic chemists. It consists in the exchange of the alkoxy-group of an ester with another one generated by an alcohol; the reaction is reversible, and can be acid-base catalyzed [103]. Besides basic research, it has a never ending number of industrial applications, such as in plastics and polymer technologies [104,105], and biodiesel conversion of biomasses [106].

The importance of basic sites for the catalysis of transesterification induced the scientific community to verify the suitability and performance of LDH-materials. Cantrell et al. reported a deeply detailed investigation over a series of MgAl-CO3 LDHs [107]. All materials revealed to be effective catalysts for the liquid phase transesterification of glyceryl tributyrate with methanol for biodiesel production. The rate increased steadily with Mg content, with the Mg rich Mg2.93Al catalyst an order of magnitude more active than MgO (comparable results were lately reported by Xie et al. and Zeng et al., in two separate studies using as starting biomass soybean and rape oils, respectively [108,109]). Pure Al2O3 (completely inert) was investigated as well for a complete comparison. Their structural investigation resulted in a correlation between reaction rates and intralayer electron density, which could be associated to increased basicity. Some other interesting aspects were revealed by Liu et al. [110]. In their investigation on poultry fat transesterification with methanol, they determined that rehydration of the calcined catalyst before reaction using wet nitrogen decreased catalytic activity, and also, methanol had to be contacted with the catalyst before the reaction took place, otherwise catalyst activity was seriously impaired by strong adsorption of triglycerides on the active sites. Both increased temperature and methanol-to-lipid molar ratio favorably affected the reaction rate. Navajas et al. confirmed these observations about rehydration, and achieved outstanding sunflower oil conversions up to 96% c with 2% w/w of catalyst [111].

Kondawar et al. worked on glycerol transesterification preparing MgAl LDHs, pure or doped with Ca and La [112] (see Figure 6). While the study also interested the simple metal oxides, and CaO showed the highest activity, the formation of soluble calcium glycerate prevented its recovery and recyclability. For this reason, the study justified Ca-doped LDHs as a good compromise between performance and regenerability of the catalyst. Higher activity for Ca-based materials could be traced in the higher basicity obtained for these entries.

**Figure 6.** Mechanism of solid-base-catalyzed transesterification of glycerol. Adapted from Ref. [112]. Copyright 2017 American Chemical Society.

Looking for alternative approaches, Yagiz et al. developed MgAl-NO3 LDHs functionalized with lipase enzymes [113]. The influence of temperature, pH, time and particle sizes was comparatively evaluated on enzyme activity, and the optimum was reached with the immobilization of 13 mg/g of enzyme, working at pH 8.5 and 4 ◦C. It is reported that the immobilized lipase on HT yielded a lipolytic activity equivalent to 36% of initial activity of lipase, with the relevant advantage of an easy removal of the catalytic hybrid clay at the end of the process and possible recyclability.

#### **3. LDHs Applications in Photocatalysis**

In the previous Section, we have discussed in detail some classic organic reactions, and the contribution that LDH-based compounds offer as a catalyst in the development of alternative, greener, and/or more efficient protocols.

A separate section is here dedicated to LDHs in photocatalysis motivated by the following reasons. First, LDHs have been always tested and studied for several applications, e.g., catalysis, with a regular production of literature; in the case of photocatalytic applications, the trend in the publications appears quite anomalous. The first evidences came late in the 80s, with two pioneering reports from the research unit lead by Pinnavaia [114,115]; after that, a negligible number of publications (4 papers in 15 years) was recorded, until the new century coming, with a boom of reports starting from 2008 when two well recognized papers from the team of Vansant received attention from the scientific

community [116,117]. Thereafter, the urge to find answers to the global climate and environmental crisis pushed the accelerator for a new reinterpretation of LDH chemistry. One of the most active researchers in the field of sunlight-driven water splitting, Michael Grätzel, advertised NiFe LDHs as efficient photoactive catalyst electrodes [118]. The volume of publications released in the time window 2016-2019 on photocatalytically active LDHs grew to >150 items. A second reason led us to the creation of a separate section in the present review: although several detailed reviews are available in the recent literature, for example the report from Mohapatra and Parida [119], the number of new available papers calls for a new up-to-date overview.

This section will address three fields of applications, which are hot topics in the recent literature:


#### *3.1. Organic Molecules Degradation*

Dye molecules are intimately connected with history of humankind, from the prehistoric age to the industrial civilization. Nowadays, although countless applications of dyes, their ubiquitous presence calls for deeper investigations over their implications in environment pollution [120,121]. Many reports closely connect dyes to insurgency of allergic phenomena [122]. It sounds like a paradox, the search for colorants with magnificent properties, for example persistence, ended up in molecules that are difficult to be degraded. The reason why is that chemists pursued different approaches for solving this urgent issue [123–125].

In the last decade LDHs found increasing application in this variegated field, starting from the first report, again from the team of Vansant [126], with increasing attention gained after the publication by Parida and Mohapatra [127], dealing with efficient and cheap ZnFe-CO3 LDHs. Herein, attention is given to specific LDH compositions focusing on the most recent literature. Zn and Al are recurrent elements in the photocatalytic HT clays formulation [128–135]. Many works report that pristine LDHs can be very efficient without any further treatment, such as calcination, providing decoloration percentage of 90% for solutions of methylene blue (MB) after 1 h irradiation time (better than that of commercial ZnO nanoparticles) [128]. In order to improve the photodegradation performance, additives can be included in the LDH-lattice; cerium doping appeared an interesting option to be considered, as it provided an efficiency 1.5 times higher than pure ZnAl LDH [131] (see Figure 7).

**Figure 7.** Proposed photocatalytic mechanism for the cerium presence in ZnAl LDH materials and its role as charge separator. Adapted from Reference [131]. Copyright 2016 Elsevier.

A proposed operation mechanism considers Ce4<sup>+</sup> cations able to: i) Collect e<sup>−</sup> photogenerated by the LDH lattice; ii) convert to \*Ce3+; and iii) transfer the excitation energy (and electron) to an O2 adsorbed molecule. The so-generated O2 − radicals can react with water molecules and generate H2O2; UV light will break the peroxide and generate highly reactive OH radicals, which are deputed to decompose the organic pollutant (e.g., phenol). In a similar way, lanthanum provided beneficial effect in MB photodegradation operated by doped ZnCr LDHs [130]. However, the integration of TiO2 in LDHs [134] looks more appealing, since it has the relevant advantage to be a well-known photosensitizer with an extremely low price on the market, making its choice more affordable and convenient for large scale production/application. Another interesting manipulation is the anionic exchange and decoration with Ag2CO3, which provided higher selectivity over anionic dyes, as revealed in comparative tests between MB and Red X-3B [129].

In order to find alternative materials where synergic effects could be helpful, Mohamed et al. reported a study over polypyrrole nanofibers coated with ZnFe LDHs [135]. The novel hybrid material was tested for the photocatalytic removal of Safranin dye, or Basic Red 2, a biological stain. The adsorption capability was reported to be about 22% higher than the naked nanofibers, and 31% higher than the pristine ZnFe LDHs. The photocatalytic removal was improved by 42% and 54% higher than the nanofibers and the starting LDHs, respectively.

As a general remark, to be kept in mind throughout the following sections, almost all the specific photocatalytic reactions that we decided to mention in the present work deal with Zn(II)-based LDHs. While the presence of Zn(II) is constant in the reported examples, where Zn seems to be a component of fundamental importance, some works focus their attention over different elemental species. One example is reported by Timár et al. [136]; in their work, the photocatalytic degradation of MB was reported using Mn2Cr LDHs as photocatalysts. The performance of such LDHs was comparable with commercially available Degussa P25 TiO2, and remained unaltered over five consecutive runs. Further analyses over materials derived from calcination of the pristine LDHs showed inferior activity; changes in the performance could be traced in the gradual collapse of the layered structure, until no activity could be observed for the photocatalytically inactive double oxide.

#### *3.2. Water Splitting Reaction*

LDHs have emerged as highly active photocatalysts for water splitting reaction due to some appealing features. The large surface area and semiconductor characteristics captured the attention of several groups. Some limitations stimulated researchers to exploit modifications of LDH-based photocatalysts. Among them, Fu et al. reported on the doping procedure of ZnCr LDHs with terbium cations, demonstrating an improved photocatalytic performance [137]. Photoluminescence and photoelectrochemistry measurements on the ZnCrTb LDH samples revealed a more efficient charge carrier separation and higher injection efficiency, compared to the pristine non-doped ZnCr LDHs. Optimal performances were observed for a specific doping level (0.5%), with a 2-fold increase in photocatalytic O2 production efficiency. Oxygen generation through photocatalytic water splitting under visible light irradiation was studied by Gomes Silva et al., who investigated a series of ZnTi, ZnCe, and ZnCr LDHs at different Zn-to-metal atomic ratio (from 4:2 to 4:0.25) [138]. ZnCr LDHs proved once again to be the best matching case, with an atomic ratio of 4:2. The authors focused on the apparent quantum yields for oxygen generation, with values well comparable with previous results by Fu et al. It is worthwhile to point out that these quantum yields (60.9% and 12.2% at 410 nm and 570 nm, respectively) were among the highest values ever determined with visible light for solid materials in the absence of a light harvesting dye. Interestingly, in a field of rapid growth, it is appreciable that different research groups arrived to the confirmation of similar findings, further supporting and consolidating the trend in photocatalytic exploitation of LDHs.

Lee and coworkers followed a different approach to the improvement of photocatalytic performances, with specific focus over the other half-reaction, bringing the evolution of H2 [139]. In their case, the material consisted of a combination of graphitic carbon nitride (*g*-C3N4), which acted

as a support for ZnCr LDH nanocrystals. Structural characterization and investigation of surface area confirmed the localization of the nanocrystals in the mesopores of the graphene-like lattice; an efficient electronic coupling between both the two components of the hybrid material gave rise to the observed enhanced visible light absorptivity and suppression of electron−hole recombination.

In two different reports [140,141], photoanodes were successfully prepared by the integration of bismuth vanadate BiVO4 with Co-based LDHs (see Figure 8). The design presented by Vo and collaborators was more complex, bringing to the preparation of a CoMnZn trimetallic anionic clay, but several details are herein discussed. The evidences suggested synergistic effect of the three-metal composition towards the enhanced photoelectrochemical performance. Surface modification of photoanodes with trimetallic hydroxides greatly improved the migration of holes from bismuth vanadate to LDH, facilitating fast separation and transport of holes, thus retarding the recombination of photogenerated charges.

**Figure 8.** Schematic representation of charge carrier dynamics in water oxidation on BVO (**a**) and BVO/CMZ-LDH (**b**) photoelectrodes. Adapted from Reference [141]. Copyright 2019 Elsevier.

#### *3.3. CO2 Conversion*/ *Reduction*

Photocatalytic conversion of CO2 into alcohols is a chemical pathway widely pursued in order to answer two relevant issues. From one side, to find application to an inert molecule which is the last step of carbon oxidative chain, and a dangerous greenhouse gas as well. From the other side, it is a way to produce liquid fuels in an environmentally compatible manner and in a less energy demanding way. Several different approaches and materials were tested [142–144], and LDHs resulted to be an intriguing option also in this case. Some interesting achievements concerned MgAl LDHs, which are the simplest, most common, and deeply studied HT compounds. Their high CO2 adsorption performance, even at room temperature, was already reported [145]. Among the latest reports, Flores-Flores et al. focused on the development of a microwave-assisted protocol for catalytically active LDHs [146]. As the authors claimed, MW-irradiation was necessary to produce samples with high crystallinity, which had a strong impact over methanol production rate. During the photocatalytic experiments, under halogen lamp irradiation, MgAl LDHs showed higher selectivity for CH3OH production in liquid phase than in gas phase, due to a more negative flat band potential to carry out the CO2 reduction. Iguchi et al. played with the composition of MgAl LDHs, introducing fluorine species in the lattice (e.g., AlF6 3− anions) [147]. In their reports, they observed that the reduction cascade led to the selective production of CO under UV-light irradiation. A highly selective compound for CO2 photoreduction into methanol employed ZnCuGa-CO3 LDHs [148]. In the paper, the photocatalytic material was required to be heated at 150 ◦C in vacuum in order to reduce the content of interlayer water by 31%; after this stage, if LDHs never got in contact with air prior to the photoreduction tests, methanol production selectivity was verified to be >97% in all the studied cases.

A change in the divalent cation (Ni instead of Mg) was demonstrated to modify the outcome of the catalysis; e.g., the resulting NiAl LDHs showed 80% selectivity towards CO among the reduction products after 20 h of UV light irradiation. The substitution of metal cation came from another broad investigation over 16 different kinds of transition metal containing M2<sup>+</sup>M3<sup>+</sup> LDHs (M2<sup>+</sup> = Co, Ni, Cu, Zn; M3<sup>+</sup> = V, Cr, Mn, Fe) applied to the photocatalytic conversion of CO2 to CO in an aqueous solution of NaCl [149]. A *summa* of these results can be finally seen in the investigation reported by Tokudome et al., where NiAl LDHs were tested as nanocrystals [150]; the remarkable rate of photocatalytic CO2 reduction by the nano LDH catalyst was reported to be almost one order of magnitude (7 times) higher than that measured by means of LDH catalyst prepared through conventional methods. It looks like the research unit made a step backward, ignoring the best performances offered by NiV LDHs, which were supposed to benefit from the implementation of nanoforms. It can be argued, however, that vanadium is more expensive and exotic than well established (and cheap) aluminum.

Crystallinity is a recurrent keyword in the work of Zhao et al. [151]. In their work, they compared the synthetic protocols for MgAlTi LDHs (co-precipitation, co-precipitation + hydrothermal, and co-precipitation + calcination + reconstruction). Crystalline TiO2 domains were present in the LDHs obtained by hydrothermal or reconstruction processes. The material hydrothermally treated at 150-200◦C demonstrated the highest CO production due to a well-balanced TiO2 crystallinity and specific surface area. Compared with commercial TiO2-P25 nanoparticles, MgAlTi LDHs demonstrated 2 to 4 times higher catalytic activity in CO2 photoreduction to CO.

Another example calls back what was already described for LDH application in water splitting reaction. The work from Tonda et al. exploited the possibility to take advantage of a two-component hybrid combining *g*-C3N4 with LDHs [152] (see Figure 9). In the present case, a detailed characterization and description of the photocatalytic mechanism in the *g*-C3N4/NiAl-LDH heterojunction culminated in the observation of a CO production rate 5 times higher than that of pure *g*-C3N4, and 9 times higher than what was revealed for pure NiAl LDHs.

**Figure 9.** Top. Schematic illustration of the synthesis process of *g*-C3N4/NiAl-LDH hybrid heterojunctions. Bottom. Proposed mechanism for CO2 photoreduction in the *g*-C3N4/NiAl-LDH heterojunctions. Adapted from Reference [152]. Copyright 2018 American Chemical Society.

#### *3.4. Others*

In this section, a couple of interesting papers focusing on other relevant topics are discussed. In the previous Section, several words have been spent over transesterification as an important route to produce biodiesel. Again, LDH materials are used, as well, in photocatalytic applications close to the petroleum chemistry, as Gao et al. reported [153] (see Figure 10). LDHs were employed in the mitigation of polluting emissions of diesel oil, mostly related to the high concentration of sulphur-containing hydrocarbons [154]. Their LaZnAl-MoO4 LDHs were reported to promote desulfurization of diesel oil under UV irradiation, favoring the oxidation of dibenzothiophene (DBT) to the corresponding sulphone. The conversion rate, based on the quantity of sulfone that remained adsorbed to the LDH surface, was determined as being up to 84% in 1 h.

**Figure 10.** Catalytic oxidation of dibenzothiophene (DBT) on LaZnAl3-MoO4 LDH-catalyst. Adapted from Reference [153]. Copyright 2018 Taylor and Francis.

To justify their evidences, they claimed that MoO4 <sup>2</sup><sup>−</sup> anions increased the interlayer space promoting the adsorption of dibenzothiophene (DBT), which is one of the most relevant sources of sulphur in the oil. Synergistically, MoO4 <sup>2</sup><sup>−</sup> acted as the active sites for the oxidation of DBT, resulting in the high desulfurization efficiency. These statements were justified by comparing the molibdate LDH-photocatalyst with an equivalent one with carbonate anions, which showed inferior desulfurization performance. The presence of La3<sup>+</sup> cations was mandatory, since they brought more positive charge to the brucite-like sheets, leading to an improved adsorption of molibdates on the surface of the layers.

Photocatalytic LDHs found application in the improvement of another fundamental process: N2 fixation. The reaction consists in the reduction of gaseous N2 to ammonia, NH3; it is an essential mechanism for the production of nitrogen containing biological molecules [155], as well as industry relevant products, such as fertilizers [156]. The Haber-Bosch process [157], one of the most important processes of human history, produces ammonia under extreme conditions cycle (400–500 ◦C, 200–250 bar), leaving room for improvements and discovery of more convenient alternatives. Zhao et al. reported the photocatalytic activity of ultrathin LDH nanosheets (NSs) made with different combinations of di- and trivalent metal cations (M2<sup>+</sup> = Mg, Zn, Ni, Cu; M3<sup>+</sup> = Al, Cr) [158]. The most promising samples were the CuCr-LDH NSs; photocatalytic reduction of N2 to NH3 was observed in water at 25 ◦C under visible-light irradiation, with quantum yield of about 0.44% at 380 nm and 0.10% at 500 nm. Monochromatic light of wavelength 500 nm afforded a NH3 evolution rate of about 7.1 μmol L<sup>−</sup>1, which was a great achievement considering that the authors were far away from the UV and significantly closer to the maximum of solar emission spectrum. The photocatalytic activity was attributed to the

distorted structure in the LDH NSs, which is supposed to enhance N2 chemisorption and to promote NH3 formation.

#### **4. LDH Applications in Biosensors**

LDHs have been largely employed as efficient biosensors, owing to their excellent biocatalytic properties and to the possibility of producing hybrid materials with enzymes. Starting from the first example of urea biosensors based on the immobilization of urease into oppositely charged clays [159], most of the research in the field of LDH-based biosensors was devoted to the fabrication of oxidoreductase enzyme/LDH amperometric biosensors, where typically employed enzymes were transketolase, acetylcholinesterase, horseradish peroxidase, and glucose oxidase [160]. The researchers also showed the possibility to prepare hybrid LDHs containing redox active molecules as enzyme immobilization matrices, such as anthraquinone sulfonate, ferrocene, and 2,2'-azinobis 3-ethylbenzothiazoline-6-sulphonate.

In general, the class of enzyme-based biosensors is featured with high costs and low stability, being their response potentially affected by factors such as temperature, pH, and ionic strength [161,162]. For these reasons, recent research efforts were focused on the fabrication of enzyme-free biosensors mostly based on the functionalization of electrodes with functional nanomaterials, benefiting from low costs, rapid response, high sensitivity [163], and from the possibility to enhancing electrode activity providing much more accessible exposed active sites as well as to provide convenient ion/electron transport channels for electrochemical detection of analyte molecules. In this scenario, LDHs have been explored as convenient materials for the fabrication of enzyme-less glucose biosensors. As a remarkable example, Cui et al. [164] reported on the fabrication of a bifunctional non-enzymatic flexible glucose microsensor based on CoFe LDHs by directly growing a CoFe layered double hydroxide nanosheet array (LDH-NSA) on a Ni wire. The LDH system showed high sensitivity and high selectivity in electrochemical and colorimetric detection of glucose, with linear ranges from 10 to 1000 mM and from 1 to 20 mM, and detection limits of 0.27 μM and 0.51 μM, respectively. The electrocatalytic glucose oxidation mechanism of CoFe LDH was tentatively explained by the authors, as follows:

#### LDH-Co(II) + OH<sup>−</sup> → LDH(OH−)Co(III) + e<sup>−</sup> LDH(OH−)Co(III) + glucose → LDH-Co(II) + gluconolactone

As further examples, Ai et al. modified glassy carbon electrodes with a NiAl LDH composite with chitosan, obtaining a good linear range (0.01–10 mM) for glucose detection [165]. Li et al. employed NiAl LDHs onto titanium electrodes, obtaining a detection limit of 5 μM and a linear range up to 10.0 mM [166]. More recently, the preparation of a glassy carbon electrode modified with a composite material based on gold nanoparticles decorated with NiAl LDHs and single-walled carbon nanotubes permitted to obtain a wide linear range from 10 μM to 6.1 mM [167]. The authors ascribed the good detection performances to the combined effects of enhanced electrical conductivity deriving from 3D network formed by carbon nanotubes, good accessibility to active reaction sites from NiAl LDH and more electron transfer passages provided by Au nanoparticles. Besides glucose, non-enzymatic LDHs-based sensors have also found applications in the detection of drugs, such as Terazosin hydrochloride, an Alpha-adrenergic Blocking Agent, by means of MgAl LDHs [168], or for the detection of nitrite ions from solution by NiFe LDHs fabricated onto carbon cloth substrates [169].

As another pivotal example, Asif et al. demonstrated the easy fabrication of hybrid LDH nanosheets with layers of reduced graphene oxide for electrochemical simultaneous determination of dopamine, uric acid and ascorbic acid [170]. They produced such 2D composite material by following a coprecipitation route in which the ZnNiAl LDH and GO precursors were added dropwise to a formamide solution with continuous stirring. In another synthesis procedure, they directly mixed ZnNiAl LDH and GO supernatant solutions and added hydrazine in order to obtain reduced GO. The latter synthesis approach allowed to self-assemble a periodic superlattice compound by integrating positively charged semiconductive sheets of a ZnNiAl LDH and negatively charged layers of reduced

graphene oxide. By operating at typical working potentials of −0.10 V, +0.13 V, and +0.27 V vs. saturated calomel electrode, the obtained lower detection limits for ascorbic acid, dopamine, and uric acid were 13.5 nM, 0.1 nM, and 0.9 nM, respectively. The authors also showed the possibility to track the dopamine released from human neuronal functioning neuroblastoma cell line SH-SY-5Y after being stimulated by highly K<sup>+</sup> buffer.

Along with electrochemical sensors, many efforts focused on the preparation of LDH-based sensors with fluorescence readout. Recently, Liu et al. demonstrated a fluorometric displacement assay for measuring the concentration of adenosine triphosphate (ATP) using layered cobalt(II) double hydroxide nanosheets [171]. In particular, they used a dye-labeled oligonucleotide adsorbed on the LDHs. The adsorption led to the complete and fast quenching of the green fluorescence of the label. In presence of ATP in the solution phase, the DNA oligonucleotide was rapidly detached from the LDH because of the stronger affinity of ATP for LDH, finally leading to the restoration of the green fluorescence signal. This remarkable effect was used by the authors to produce an assay showing a linear response in the 0.5–100 μM ATP concentration range and a 0.23 μM lower detection limit, allowing for the determination of ATP in spiked serum samples. In a similar approach, Abdolmohammad-Zadeh et al. showed a fluorescent sensor based on nanostructured MgAl LDH intercalated with salicylic acid (SA) for sensing the ferric ions in solution [172]. The calibration graph was linear in the concentration range of 0.07–100 μmol/L, along with a detection limit of 26 nmol/L. The authors demonstrated the intercalation of salicylic acid into the layers of the host Mg-Al LDH matrix by showing an increased interlayer spacing as measured by XRD analysis. The fluorescence intensity of salicylic acid was increased by its intercalation into LDHs, given the effect of confinement, which reduced the interaction between salicylic acid and the solvent. In presence of Fe3<sup>+</sup> ions, the fluorescence signal of MgAl LDHs intercalated with SA decreased with increasing of Fe3<sup>+</sup> ions concentration because of the formation of a stable complex with salicylic acid.

Among optical based techniques, chemiluminescence is also a powerful approach for biomolecular detection given its high sensitivity and low cost in comparison to fluorescence [173]. Many reports have shown the possibility to employ LDHs for the realization of chemiluminescent glucose sensors. For instance, Wang et al. demonstrated that MgAl LDHs can be supporting materials for immobilizing luminol reagent, by triggering luminol chemiluminescence in a moderately acid pH (5.8). In the presence of horseradish peroxidase, the luminol-LDH hybrid was able to produce chemiluminescence signal to glucose in the range of 0.005–1.0 mM, along with a detection limit for glucose equal to 0.1 μM [174]. In another paper, Pourfaraj et al. demonstrated that CoNi LDHs exhibit catalytic activities towards the luminol-H2O2 reaction. Under optimum conditions, the chemiluminescence intensity was linear in the range 0.1–12μM of H2O2 concentrations along with a detection limit (S/N=3) of 0.05 μM [175]. More recently, Pan et al. demonstrated the possibility to use the LDH–luminol–H2O2 system-based chemiluminescence platform for sensing carminic acid, a colorant used in food additives [176]. The detection principle consists of two steps: First, LDH adsorbs carminic acid onto the surface, then the carminic acid quenches the chemiluminescence of the LDH–luminol–H2O2 system by resonance energy transfer, reduction of reactive oxygen species, and occupation of the brucite-like layers. The authors demonstrate the possibility to obtain a linear response to the analyte in the concentration range from 0.5 to 10 μM, along with a limit of detection equal to 0.03 μM.

Telomeric DNA could also be a target analyte as recently shown by Haarone et al. [177], who produced a MgAl LDHs intercalated with DAPI (4- ,6-diamidino-2-phenylindole), which is a fluorescent molecule that strongly binds to adenine–thymine rich regions in DNA sequences. DAPI could assemble into the LDH nanosheets by blending with single strand DNA via the co-assembly method (see Figure 11). The DAPI containing LDH was able to detect long single strand DNA/telomere sequences simply by changing the single strand DNA sequence. The authors demonstrated a dynamic range within the 3–20 μg/mL range along with a detection limit of 20 μg/mL.

**Figure 11.** Scheme of the assembly of ssDNA blending into DAPI/LDH ultrathin nanosheets. Figure readapted from Ref. [177], with permission from Elsevier.

Finally, LDHs could be applied for guanine detection by means of silver nanoclusters (AgNCs) stabilized by nuclear fast red (NFR) sodium salt on Mg2Al LDH nanosheets [178]. Due to the confinement effects in 2D layered LDH nanosheets, the fluorescence intensity and photostability of the AgNCs-NFR/LDH compound were significantly improved if compared with those of the AgNCs-NFR based solution. By introducing Cu2<sup>+</sup> ions as a modulator, AgNCs-NFR/LDHs were successfully applied for determining guanine in the concentration range of 10 μM–100 μM, along with a detection limit equal to 1.85 μM. Recently, dopamine biosensors for real time detection of dopamine (detection limit down to 2 nM) from live cells (human neuronal functioning neuroblastoma cell line SH-SY 5Y) were realized in the form of composite materials produced by exfoliated charged nanosheets of LDHs and graphene [179]. Similarly, carbon nanotubes loaded onto a CuMn LDH nanohybrid allowed for a high-sensitivity electrochemical detection of H2S from live A375 cells (detection limit equal to 0.3 nM) [180].

#### **5. LDHs Applications in Nucleic Acids (DNA, RNA) Delivery**

Besides sensing molecules and nucleic acids in biological samples, LDHs can find important applications in life sciences given their ability to deliver biomolecules to biological systems. LDHs are efficient drug delivery systems, possess good biocompatibility, high drug-loading density, high drug-transportation efficiency, low toxicity to target cells, or organs, offering excellent protection to loaded molecules from undesired enzymatic degradation [160,181]. DNA is the carrier of the genetic information in living cells. The structure of DNA consists in two single strands of DNA, which can bind together by hydrogen bonding between complementary pairing bases (adenine to thymine and guanine to cytosine), thus forming the well-known DNA double helix structure. DNA delivery to cells represents one of the most investigated applications for the biological sciences. In this regard, the successful DNA delivery to cells strictly requires that the foreign DNA material must remain stable within the host cell by fusing into its genome or retaining the ability to integrate intracellularly replicate. This requires foreign DNA to be delivered by a suitable vector, which has the capability to enter the host cell and accurately deliver the DNA molecule to the cell's genome, being the vectors employed for gene delivery sorted into recombinant viruses and synthetic vectors. Similar to DNA, RNA is self-assembled as a chain of nucleotides, with the only difference that uracil is used instead of thymine. RNA has many important roles in biological organisms, such as conveying the genetic information and directing protein synthesis. In the last ten years, RNA molecules were being processed via the RNA interference (RNAi) pathway, which consists in silencing the expression of genes with complementary nucleotide sequences by degrading the mRNA after transcription from DNA, ultimately preventing translation. In this regard, the small interfering RNA (or silencing RNA, siRNA) therapy is hampered by the barriers for siRNA bioavailability to enter into cells cytoplasm and exert their gene silencing activity. In this scenario, LDHs have been demonstrated as an ideal synthetic vector for both DNA and RNA molecules, due to a well-elucidated adsorption mechanism, taking into account that the phosphate backbone of the DNA polymer coordinates with the metal cations of the LDH lattice via the ligand-exchange process [182,183].

The formation of LDH-DNA hybrids for DNA delivery can be obtained either through the incorporation of small DNA molecules and antisense oligonucleotides into the LDH matrix by a simple ion-exchange reaction [184], or by a more general coprecipitation route involving the in situ formation of LDH layers of various ionic compositions around intercalated DNA [185]. In particular, the feasibility of the latter approach was demonstrated by Desigaux et al. [185] that demonstrated the intercalation of DNA into the LDH matrix by the net increase of the interlayer distance, from ∼0.77 nm for all nitrate parent LDHs to ∼2.11 nm, ∼1.80 nm, and ∼1.96 nm for DNA molecules complexed with Mg2Al, Mg2Fe, and Mg2Ga, respectively (see Figure 12). Choy et al. were the first that showed the possibility to intercalate c-myc antisense oligonucleotide (As-myc) into MgAl LDH nanoparticles by anion exchange [186].

**Figure 12.** Powder X-ray diffraction patterns in the 2θ range 2◦–70◦ of LDH/DNA Hybrids. (**a**) Mg2Al/NO3 (spectrum a) and Mg2Al/DNA (spectrum b). (**b**) Mg2Fe/NO3 (spectrum a) and Mg2Fe/DNA (spectrum b). Reprinted with permission from Ref. [185]. Copyright (2006) American Chemical Society.

The results from cellular internalization experiments demonstrated a strong inhibition of the proliferation of HL-60 cancer cells exposed to As-myc–LDH hybrids, reaching 65% growth inhibition compared with untreated cells. In a more recent report, ZnAl LDH nanoparticles were loaded with the plasmid pCEP4 to permit the expression of the Cdk9 gene in C2C12 myoblasts cells, as confirmed by PCR and Western blotting results [187]. They were also loaded with valproate and methyldopa drugs, allowing for sustained pH-triggered drug delivery. With the aim to combine dissipative molecular-dynamics simulations and experimental work, Li et al. [188] demonstrated that delaminated LDH-DNA bioconjugates can penetrate the membrane walls of plant cells (BY-2 cells). The authors delaminated the LDH nanoparticles by intercalating lactate into the layers of LDH nanoparticles. They also demonstrated that only the DNA-LDH-DNA sandwich complex was efficiently taken by the cells, whereas LDH-DNA-LDH sandwich complex and the DNA-LDH complex were not compatible for intracellular delivery, mainly due to the fact that the hydrophobic sequences of DNA provided a driving force for penetration. For example, the DNA-LDH-DNA sandwich complex was gradually internalized into the membrane to minimize exposure of the DNA hydrophobic sequences in the hydrophilic solvent.

Along with the above reported methods, DNA oligonucleotides can be also immobilized by silanization of LDHs in aqueous suspension as demonstrated by Ádok-Sipiczki et al. [189]. In particular, the 3-aminopropyltriethoxysilane (APTES) was employed as linker. APTES covalently attached to the MgAl LDHs. In turn, the amino group of the APTES linker reacted with the activated 5' carboxylic functional group of the nucleic acid strand, for the activation of which 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride salt (EDC) and N-hydroxysuccinimide (NHS) were used. The EDC reacts with the carboxylic group of the nucleic acid to form the O-acylisourea mixed anhydride. The addition of a selected nucleophile such as NHS, which reacts faster than the competing acyl transfer, generates an intermediate compound being active enough to couple with the amino group, finally also preventing any possible side reactions. The authors conclude that the covalent linkage of the nucleic acids confers to this model nanoparticulate system promising properties and potential for applications as therapeutic agents, since the DNA could be taken into the cells allowing for intracellular delivery.

In a similar way to DNA, also RNA complexed with LDHs nanoparticles can be delivered to cells, being the application of siRNA the main driving force for developing this type of molecular delivery. For instance, Wong et al. [190] reported the delivery of siRNA to primary neuron cells resulting in the silencing of gene expression. This possibility has important applications for potential diseases such as the treatment of neurodegenerative conditions (e.g., Huntington's disease). Interestingly, authors found that it was possible to produce Mg2Al LDHs for simultaneously deliver anticancer drug 5-fluorouracil (5-FU) and Cell Death siRNA for effective cancer treatment, by loading the siRNA on the surface of LDH nanoparticles and the 5-FU into interlayer spacing [191]. Compared to treatment with only CD-siRNA or 5-FU, the combination of the two different molecular systems led to an enhanced cytotoxicity to three cancer cell lines, i.e. MCF-7, U2OS, and HCT-116. This interesting synergic effect was ascribed to a coordinated mitochondrial damage process. In another interesting approach, Park et al. [192] showed the possibility to obtain an efficient in vivo and in vitro delivery system for Survivin siRNA assembled with either passive MgAl LDHs, with a particle size of 100 nm, or active LDHs conjugated with a cancer overexpressing receptor targeting ligand, folic acid (LDHFA). These routes allowed targeting the tumor by either EPR-based clathrin-mediated or folate receptor-mediated endocytosis. The authors also showed the ability to induce potent gene silencing at mRNA and protein levels in vitro, and achieved a 3.0-fold higher suppression of tumor volume than LDH/Survivin in an in vivo tumor mouse model. Another interesting example of RNA intercalation in LDHs was provided by Acharya et al. for potential application in neurodegenerative diseases [193]. They employed short hairpin RNA (shRNA) intercalated in MgAl LDH nanoparticles at the mass ratio of (1:75), leading to the formation of shRNA-plasmid-LDH nanoconjugates, with an average size of 40–60 nm, which were efficiently transfected into mammalian neuroblastoma cells (SH-SY5Y), observing a maximum internalization of ∼26% at 24 h. This, in turn, led to a significant downregulation of the protein alpha TNF, demonstrating the efficient functionality of the delivered shRNA.

In the case of CaAl LDH nanoparticles, the pH at which these nanomaterials are prepared can affect the efficiency of RNA uptake [194] (see Figure 13). Rahaman et al. showed that the involvement of carbonate ion as impurity was critical during the preparation of CaAl LDHs (see Figure 13). The authors prepared CaAl LDHs at two different pHs, i.e., pH 8.5 vs. pH 12.5, finding that at the higher pH, more carbonate ions were intercalated into the CaAl LDH structure, leading to lower intercalated RNA, as demonstrated by FTIR and XRD characterizations. In particular, after intercalation of shRNA the *d* spacing of the basal plane (0 0 2) increased from 8.61 Å in the phase pure CaAl LDH to 20.30 Å in shRNA sample. Interestingly, these data were nicely correlated with cellular uptake using colon cancer cell line (HCT 116), since LDHs prepared at pH 8.5 led to a significantly higher uptake (9.34%) in comparison to LDHs prepared at pH 12.5 (3.54%).

**Figure 13.** Schematic representation of the mechanism of intercalation of anionic shRNA-plasmid, in presence of NO3 <sup>−</sup>/CO3 2–, in cationic layers of samples A (precipitated at pH 8.5) and B (precipitated at pH 12.5). Reprinted from Ref. [194], Copyright (2019), with permission from Elsevier.

In more recent reports, the researchers strived to the aim of improving the efficacy of LDH delivery of RNA in comparison with other nanomaterials. For example, Li et al. [195] demonstrated that siRNA could be more efficiently delivered to osteosarcoma (U2OS) cells by mannose-conjugated SiO2 coated LDH nanocomposites (Man-SiO2@LDH) compared to unmodified LDH NPs. The enhanced uptake was attributed to the active mannose-receptor interaction-mediated endocytosis, considering also the specific strong binding affinity towards lectin that is expressed on the cancer cell membrane.

Wu et al. [196] compared LDHs and lipid-coated calcium phosphate nanoparticles (LCPs) as effective vectors for siRNA delivery into suspended T lymphocytes (EL4) for silencing the target PD-1 gene. They found that LCPs showed a higher cellular uptake and higher PD-1 gene silence efficiency in mouse T cell line EL4 (about 70%) in comparison to LDHs (40%). They ascribed this difference to the smaller size of the LCPs with respect to LDHs (50 nm vs. 100–200 nm) and to the higher efficiency to get endosome escape when LCPs were dissolved in the endosomes, leading to more sustained RNA release in the cytosol. From this report, it appears that it is still necessary to obtain a better understanding of the optimal LDH:DNA ratio parameters for the delivery of functional siRNA using LDH nanoparticles. In this regard, Wu et al. [197] demonstrated that an optimal LDH/gene mass ratio was around 20:1 in terms of cellular uptake amount of gene segments, whereas the ratio was around 5:1 in terms of target gene silencing efficacy in MCF-7 cells. The authors ascribed these interesting results to a reasonable trade-off between DNA loading on the LDHs and dissolution rate. Cellular internalization of LDH NPs is basically driven by clathrin-mediated endocytosis. Once entered into the cells, LDH nanoparticles tend to be dissolved (at higher rate within the endosomes) and release the siRNA. The efficacy of RNA-induced silencing is therefore dependent upon the loading amount of dsDNA/ siRNA per LDH particle, the number of LDH particles, and the suitable release rate of dsDNA/siRNA. The 5:1 ratio seems to be a reasonable compromise between these different factors.

#### **6. LDHs Applications in Cell Biology: From Cellular Di**ff**erentiation to Cancer Therapy**

In general, 2D clay materials have been demonstrated as biocompatible [198,199], suitable systems for synthetic biology [200]. By employing micron sized colloidal objects these compounds have been effectively used for in vitro reconstituting cellular motility [201]. In the specific case of LDHs, these clays are starting to be more and more investigated as to their direct interaction with living cells [202,203].

In particular, the toxicity of LDHs to cells has been thoroughly investigated [204]. As expected from other types of clays, their toxicity potential is dose and time dependent with particle sizes, their shapes and surface charge being determinant features for cellular uptakes. The reticular endothelial system is able to sequestrate LDHs systems, especially those with sizes greater than 50 nm. Notably, LDHs with sizes between 50 and 200 nm show concentration dependent uptake, whereas sizes 350 nm and above are not concentration dependent [205]. LDHs enter cells through endocytotic pathway; however the hexagonal shaped LDH crystallite was found to be distributed within the nucleus of the cells [205]. These results suggested the promising drug delivery potential of LDH at the cellular level without damaging cell structure. In the biofluids, LDHs produce some tissue and cell friendlily by-products (H2O, Mg2<sup>+</sup>, Al3+, Zn2+) under physiological conditions [206]. In turn, this effect can mitigate the acidification tendencies in endosomes and lysosome of cells after the nanocomposite uptake, ultimately leading to a sustained and pH dependent drug release that is also beneficial for reducing drug toxicity [204].

#### *6.1. Cellular Di*ff*erentiation*

In a very recent work, LDHs have been reported as a promising material for bioengineering application, showing that LDH nanomaterials modulate cell adhesion, proliferation, and migration and demonstrating their suitability in the biomaterial field and in the specific context of tissue bioengineering [207]. In the following, LDH interaction with cells will be investigated focusing on the biocompatibility, the latest applications in cellular differentiation, and the applications in cancer therapy. A specific investigation regarding the LDH biocompatibility was recently carried out by Cunha et al. [208]. In their research, adult female Wistar rats were subjected to the intramuscular implantation of two types of LDHs of magnesium/ aluminum (Mg2Al-Cl) and zinc/ aluminum (Zn2Al-Cl). Interestingly, the LDHs did not lead to any sign of inflammatory reactions; on the contrary, they promoted collagen adsorption. More specifically, Mg2Al-Cl promoted multiple collagen invaginations (mostly collagen type-I), whereas Zn2Al-Cl induced collagen type–III. Li et al. reported on the fabrication of layered double hydroxide/poly-dopamine composite coating with surface heparinization onto Mg alloys for application in endothelialization and hemocompatibility [209]. The LDHs were directly grown onto the Mg sample by reported hydrothermal methods [210] in which Mg alloy plates are placed in a Teflon liner which contain an aluminum nitrate solution at 120 ◦ C for 12 h. The MgAl LDH was then coated with poly-dopamine and heparin. The resulting composite material was tested for its corrosion resistance and the ability to enhance, with respect to the bare Mg surface, the adherence, proliferation and migration rate of the human umbilical vein endothelial cells and inhibited the platelets adhesion.

The interaction between cells and nanomaterials can be employed for tuning differentiation switches of stem cells, according to precise biomechanical or biochemical triggers [211,212]. LDHs were demonstrated as biocompatible materials by Ramanathan et al. [213]. In their experiments, they prepared Poly(3-Hydroxybutyric acid)-Poly (N-vinylpyrrolidone) fibers loaded with MgAl LDHs and coated over the hydroxyapatite pellets for the realization of bone grafts; the resulting systems showed good compatibility towards MG63 human osteosarcoma 3AB-OS cancer stem cell lines. LDH nanocomposites with arginylglycylaspartic acid and kartogenin were incorporated into a gel together with tonsil-derived mesenchymal stem cells [214]. Interestingly, the cells were observed to aggregate in the nanocomposite system. By analysis of mRNA content, chondrogenic biomarkers of type II collagen and transcription factor SOX 9 significantly increased, ultimately facilitating a chondrogenic differentiation.

In a more in-depth investigation, Kang et al. [215] studied the effect of MgAl LDHs and ZnAl LDHs in promoting the dynamic expression of genes in osteogenic differentiation pathways. LDHs determined a cytoskeleton rearrangement on the pre-osteoblasts, dependent on the modulating of Cofilin and PP2A phosphorylations, and a significant upmodulation of the osteoblastic classical marker genes (Runt-related transcription factor 2, Osterix, and Osteocalcin), while requiring the activation of c-Jun N-terminal Kinase JNK and extracellular signal–regulated kinases genes. Very recently, a thorough investigation of the LDH-induced inflammatory landscape onto osteoblasts was carried out by da Silva Feltran et al. [216]. In their report, they demonstrated that MgAl LDHs and ZnAl LDHs challenged on the MC3T3-E1 pre-osteoblasts inducing a down-modulation of major pro-inflammatory genes related to cytokines, precisely the tnfα, nfkb, il18, il6, and il1ß genes. On the other hand, an up-modulation of anti-inflammatory cytokines il6, il10, and tgfß genes was observed (see Figure 14). The authors concluded that the effects observed by LDHs could be ascribed to the activation of the Sonic hedgehog signalling in the osteoblasts, permitting the obtaining of a better comprehension of the molecular mechanisms driving the later LDH-induced osteoblast differentiation.

**Figure 14.** Role of LDHs in inducing the anti-inflammatory Sonic hedgehog acute inflammatory molecular pathway. Reprinted from Ref. [216], Copyright (2019), with permission from Elsevier.

#### *6.2. Contrast Agents*

Manganese-based LDHs have been demonstrated as powerful magnetic contrast agents with outstanding pH response and high relaxivity. Indeed, Li et al. [217] reported that, in comparison with free Mn2<sup>+</sup> ions, the Mn-based LDH systems (prepared from isomorphic substitution from Mg3Al LDHs) showed a higher longitudinal relaxivity (9.48 mm−<sup>1</sup> s−<sup>1</sup> at pH 5.0, and 6.82 mm−<sup>1</sup> s−<sup>1</sup> at pH 7.0 vs. 1.16 mm−<sup>1</sup> s−<sup>1</sup> at pH 7.4 of free Mn2<sup>+</sup> ions). Intriguingly, they ascribed these results to the unique microstructural coordination environment surrounding Mn atoms in the Mn LDH framework, as demonstrated by EXAFS measurements. They also observed that the variation of Mn···(OH2)x coordination in Mn-LDHs in acidic buffer (pH 5.0) led to the excellent T1-weighted magnetic resonance imaging performance. Afterwards, they proved the intracellular uptake of the Mn-LDHs on the B16F10 cancer cells and the outstanding potentiality as in vivo tumor imaging systems (see Figure 15).

**Figure 15.** (**a**) Scheme of the Mg2.5Mn0.5Al LDH (Mn-LDH) from co-precipitation and isomorphic substitution. (**b**) Application of the Mn LDH as magnetic contrast agents showing an ultrasensitive pH response and high relaxivity. Reproduced from Ref. [217]. Copyright © 2014 by John Wiley and Sons, Inc. Reprinted with permission from John Wiley and Sons, Inc.

#### *6.3. Drug Delivery*

LDHs are reported as very efficient drug nanovehicles [218,219] since, in comparison to other inorganic nanovehicles, including silica and gold nanoparticles, quantum dots, and carbon nanotubes, they are featured with excellent biocompatibility [205], high drug loading capacity [220], and pH-responsive property [221], with biodegradability in the cellular cytoplasm [222]. Such outstanding properties make LDHs an efficient non-viral drug delivery vehicle, and also a reservoir for bioactive or bio-fragile molecules. Note that the intercalated drugs can be released either by deintercalation through anionic exchange with the surrounding anions (such as Cl− and phosphate), or through the acidic dissolution of LDH hydroxide layers. The predominant mechanism depends on the pH value and the nature of the drug. LDH internalization into cells is mainly carried out by clathrin-mediated endocytosis [223], in which the material to be internalized is surrounded by an area of cell membrane, which buds off inside the cell to form a vesicle containing the ingested material. However, following the reports from Bao et al. [224] for LDH nanosheets-mediated molecular delivery to plant cells, the typical inhibitors of endocytosis and low temperature incubation did not prevent LDH internalization, meaning that the penetration of the plasma membrane can also occur via non-endocytic pathways (see Figure 16).

Many reports have demonstrated the immense potentiality of these materials in drug loading and sustained release. For instance, model antioxidant drugs such as carnosine and gallic acid can be intercalated into MgAl LDHs by ion exchange and coprecipitation. The drugs are released in a pH = 7.4 phosphate buffered saline medium, following a gradual and biphasic release [225]. Drug release from LDHs can be triggered by three different mechanisms: ion exchange, desorption and weathering. Whereas ion exchange only depends on the anion nature, the desorption from LDHs is dependent on the pH of the medium. Taking ibuprofen as a model drug, anion exchange triggers ibuprofen release in

the intestinal medium, whereas surface reactions mediated by solid weathering determine the release in acid media [226]. Many recent examples report on the fabrication of drug-LDHs nanohybrids. Yasaei et al. [227] demonstrated the direct coprecipitation in the presence of simvastatin (a drug used for bone regeneration) and the ion exchange of nitrate and carbonate containing LDHs in a simvastatin solution. The authors found a higher drug loading and more sustained drug releases in the case of nitrate-based LDHs. Bouaziz et al. found similar efficient loading and sustained release of nisin (among the most widely used bacteriocin peptides used in food safety) onto zinc-aluminum LDHs [228]. Similarly, pH-sensitive bead systems composed of folic acid intercalated into LDHs and chitosan represent an ideal drug delivery system for folic acid release at simulated conditions similar to the gastrointestinal tract [229]. Among the possible investigated drug systems, alendronate represents a relevant example, being an anti-resorptive and bone-remodeling drug with poor intracellular permeability, which in turn results in low drug efficacy. In this regard, Piao et al. [230] demonstrated that MgAl LDHs were able to obtain a 10-fold enhancement of the cellular uptake efficacy of alendronate in MG63 cells with respect to the bare alendronate. Notably, LDH itself did not show any effect on proliferation and osteogenic differentiation of MG63 cells (see Figure 17).

**Figure 16.** Mechanisms of LDH-lactate internalization (clathrin-mediated endocytosis) for molecule delivery into plant cells. Figure reproduced from Ref. [224] under the terms of the Creative Commons Attribution Non-Commercial License.

**Figure 17.** Experimental results showing the biocompatibility of pristine MgAl LDHs (LDH), Alendronate (AL) and Alendronate-LDH nanohybrids (AL-LDH). Figure reproduced from Ref. [230] under the terms of the Creative Commons Attribution Non-Commercial License.

LDHs have also shown important applications in chemotherapy, since they can improve treatment of tumors by facilitating pH-triggered drug delivery. For instance, Khorsandi et al. [231] investigated upon the cellular responses to curcumin-LDH hybrid nanoparticles following a photodynamic treatment of MDA-MB-231 human breast cancer cells. For this purpose, the human breast cancer cells were treated with curcumin-LDH NPs and were then irradiated, demonstrating that the curcumin-LDH hybrid had a cytotoxic and antiprolifrative effect due to the generation of reactive oxygen species, which led to autophagy and apoptosis. Recently, Liu et al. [232] demonstrated a very intriguing pH-triggered hydrazone-carboxylate complex of doxorubicin, a model chemotherapy drug, which was encapsulated in MgAl LDH NPs via a ion-exchange processes. These nanohybrids were internalized by clathrin-dependent endocytosis and then shifted to lysosomes, where hydrazone-Dox complexes were released (due to the low pH of this compartment), ultimately resulting in free cytosolic doxorubicin which in turn facilitated the cell death (MCF-7 and HeLa cells) via cathepsin-mediated cell apoptosis. A more complex drug-nanohybrid was realized by Wen et al. [233] with the aim to produce a pH-triggered drug delivery system consisting of folic-acid (FA) functionalized tellurium nanodots (Te NDs) which were in-situ synthesized in paclitaxel (PTX)-loaded MgAl LDHs. The latter were, in turn, gated onto mesoporous silica nanoparticles (MSNs). The structural properties of the resulting nanosystem were characterized by SEM, XRD, XPS and FT-IR methods. Interestingly, these nanosystems combined the ability of Te nanodots as phototherapeutic agent and the pH-triggered release of paclitaxel to human cervical cancer cells HeLa and human embryonic kidney cells 293T (see Figure 18).

**Figure 18.** Experimental results showing the cytotoxicity of the hierarchical nanosystem consisting of Te NDs within Paclitaxel-Loaded MgAl LDH gated mesoporous silica NPs towards (**a**) HepG2 cells, and (**c**) HeLa cells for 48 h with or without irradiation. Cytotoxicity of MT@L-PTX@FA without irradiation and free Paclitaxel on (**b**) HL7702 cells, and (**d**) 293T cells for 48 h. Reprinted with permission from Ref. [233]. Copyright (2019) American Chemical Society.

CaAl LDHs have shown useful properties as a drug delivery system for methotrexate to MG-63 human osteosarcoma cell line [234]. Remarkably, CaAl LDHs alone have been recently demonstrated as an active anticancer agent, owing to the involvement of Ca2<sup>+</sup> ion with the CAMKIIα protein and associated SOD activity in cancer cell [235]. The results by Bhattacharjeeet al. exhibited significant down regulation of CAMKIIα and SOD gene by CaAl LDHs at cellular level, leading to apoptosis of the cancer cell. Exfoliated ultrathin MgAl LDHs prepared by a single-step procedure in presence of formamide [236] can be used as a confinement matrix of carbon nanodots (CDs), allowing for an enhancement of fluorescence lifetime of CDs. In turn, this leads to an improvement of their efficacy in photoacoustic imaging performance and tumor inhibition under the 808 nm irradiation [237] in Hela tumor-models consisting of male nude Balb/c mice.

LDHs have also been demonstrated as an effective drug delivery system of etoposide, a chemotherapeutic agent for the treatment of a number of diseases, including glioblastoma, one of the most common and lethal intrinsic brain tumors. Wang et al. [238] incorporated etoposide into LDH nanoparticles in order to overcome the inherent drug resistant problem, such as poor tumor selectivity, and low solubility in water, permitting to significantly facilitate uptake by glioblastoma cells and enhance apoptosis efficiency, self-renewal repression, and epithelial-mesenchymal transition program reversion. The authors conducted a profound transcriptomic analysis to find the role of different protein pathways involved in the delivery of etoposide into glioblastoma cells, including PI3K–AKt, downstream mTOR, Nuclear factor kappa B (NF-κB), mitochondrial Bcl-2, activated caspase, and Wnt-β-catenin signaling (see Figure 19).

**Figure 19.** Molecular mechanisms involved in etoposide LDH NPs reversing chemoresistance and eradicating human glioma stem cells. Republished with permission from the Royal Society of Chemistry, from Ref. [238]; permission conveyed through Copyright Clearance Center, Inc.

A different scenario has been elicited as it concerns antibiotics loading. Model antibiotics such as tetracycline and oxytetracyline [239] were shown not to be intercalated into LDHs, but more likely to be adsorbed on their external surface and released according to a Fickian diffusion model. The LDHs adsorbed antibiotics are still active towards *E. coli* and *S. epidermis*, showing a decrease of efficacy in comparison to the free molecules. Among the recent applications in anticancer drug delivery, Asiabi et al. [240] prepared a novel biocompatible pseudo-hexagonal NaCa-layered double metal hydroxides (see Figure 20), which allowed for a sustained pH-triggered release of dacarbazine. This system allowed for enhanced anticancer activity in malignant melanoma cells (malignant A-375 melanoma and breast cancer MCF-7 cell lines) at higher values in comparison to the free drug [240]. Similarly, Shahabadi et al. [241] demonstrated the suitability of Fe3O4@CaAl-LDHs as levodopa delivery systems for Mel-Rm Cells Melanoma (NCIt: C3224) cells, permitting improvement in the efficacy of the free drug, owing to a pH-triggered release mechanism.

**Figure 20.** Schematic illustration of the structure of NaCa LDH for dacarbazine drug loading. NaCa LDH has (**a**) pseudo-hexagonal (P-3m1) structure with (**b**) a trigonal crystal structure (R-3C) and (**c**) rhombohedral lattice system. (**d**) The M2+/M<sup>+</sup> ions are surrounded approximately octahedrally by hydroxide ions, (**e**) structure of NO3LDH, and (**f**) structure of the LDH intercalated with dacarbazine drug, showing an enlargement of the inter-layer spacing. Reprinted from Ref. [240], with permission from Elsevier.

#### **7. Conclusions**

The aim of this review is to provide the reader with a fresh view in the field of LDH applications in chemistry and biology. As for the chemistry-related applications, LDHs are a definitely promising platform for organics catalysis and photocatalysis. Their main advantages lie in the low cost, mild, eco-compatible, low temperature synthetic approaches in controlled reaction environments (i.e., the interlamellar space), along with the possibility to recycle the LDHs after their employment. However, a specific issue of these materials is the "trails and error" approach that typically characterizes the literature of these materials for chemistry-applications. In the future, a more predictive approach towards the understanding of the reaction mechanisms involving the metal centers forming the LDHs would highly benefit the development of this research field. As for the biology-related applications, LDHs have proved to be a suitable material for drug and nucleic acid delivery, due to their partially positive charge that greatly facilitates cellular uptake by electrostatic forces. We have focused on the novel still poor explored applications in DNA and siRNA delivery, where LDHs are able to play a key role given their biocompatibility and easy synthesis and loading approaches. Still, many new investigations are needed in the field of interactions with cells (especially stem cells and also cancer cells), with the aim to precisely understand the intracellular molecular pathways that can be activated by the interaction of LDHs with cells.

This overview on LDHs considers only a portion of the numerous organic reactions protocols which could benefit of LDHs catalytic properties as well as only some remarkable examples among the different applications of these materials in the field of drug delivery and advanced medical therapies. While this study does not pretend to detail all the literature actually available, the authors hope that the most important and recent achievements have been reviewed, with the ultimate goal to offer a panoramic vision of the importance and potential of this branch of chemistry in between material science, organic synthesis and biology.

**Author Contributions:** Methodology, G.P., A.O., A.M., G.A., and A.B.; writing—original draft preparation, G.A., A.B.; writing—review and editing, G.P. and E.M.; supervision, E.M., B.P. and P.G.M.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors acknowledge ATeN center (Unipa) for hospitality and support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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