2.3.2. Nanoparticles

Poly(lactic-co-glycolic acid) (PLGA) is a copolymer recently proposed for use in topical formulations to prevent and treat photodamage, one that is non-toxic and can be hydrolyzed in vivo into its biodegradable monomers, i.e., lactic and glycolic acids [53]. PLGA-AST nanoparticles (AST-PLGA NP) were developed by Hu et al. [47] in an optimization study, based on an experimental design, aiming to maximize encapsulation

efficiency (96.42 ± 0.73%) and drug-loading capacity (7.19 ± 0.12%), and to simultaneously minimize particle size (154.4 ± 0.35 nm). Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry and thermogravimetric analyses confirmed AST encapsulation within PLGA nanoparticles, while XRD analysis suggested its amorphous state, and scanning electron microscopy (SEM) and TEM evidenced globular nanoparticles without drug crystals. This would contribute to easier penetration compared to pure AST, and would afford good size distribution. To evaluate the capacity of cellular uptake, an in vitro model with a fluorescent probe was performed with HaCaT cells, an aneuploid immortal keratinocyte cell line. The results showed cellular uptake of the nanoparticles that increased in a time-dependent way. In vitro cytotoxicity, assessed by the MTT assay, showed no significant decrease in cell viability, while AST-PLGA NP increased cell viability and had stronger antioxidant activity, compared to pure AST. Protection by scavenging free radicals was performed on the same cell line, and pure AST and AST-PLGA NP displayed similar antioxidant properties.

Chitosan nanoparticles are promising as drug delivery systems, due to their biocompatibility, biodegradability, atoxicity, bioactivity, and large target options triggered by their cationic character [54]. Chitosan and natural DNA were used by Wang et al. [49] as oppositely charged biomaterials, to develop a nanoparticle system to deliver and release AST. Thus, AST-loaded DNA/chitosan (ADC) and empty nanocarrier (DNA/CS) were prepared by macromolecular co-assembly combined with the solvent evaporation method, and their antioxidant activities and cellular uptake were assessed. Both showed a positive surface charge, due to the predominant cationic characteristic of chitosan, and very close zeta potential (35.3 and 32.2 mV, respectively), which suggests that AST incorporation did not influence this parameter. The larger average particle size of ADC (211 ± 5 nm) compared to DNA/CS, and its PDI of 0.29 ± 0.01, confirmed its nano scale, good dispersity, and AST entrapment. The AST content in ADC was higher than in ethanol solution, confirming enhancement of its solubility. The MTT assay, performed to assess the cytoprotective effect, showed that ADC prevented the deleterious effect of H2O2 on cell viability more than vitamin C (positive control) and free AST, in spite of a vitamin C concentration that was three times higher than that of AST, likely due to the facilitated endocytosis of ADC nanoparticles. The ROS scavenging efficiency of ADC nanoparticles was twice that of free AST at the same bioactive concentration.

Liu et al. [50] prepared an innovative type of nanoparticle to improve solubility and stability, using chitosan oligosaccharides (COS) to coat PLGA AST nanoparticles (Ax-PLGA@COS NP). To prepare AST-loaded PLGA nanoparticles, a two-step process was used, which consisted of antisolvent precipitation, followed by coating nanoparticles with COS by electrostatic deposition. The formulation was characterized in terms of encapsulation efficiency, morphology, PDI, zeta potential and particle size, while the stability of nanoparticles was evaluated in terms of changes in their color and particle size during 72 h of storage at room temperature. Ax-PLGA@COS NP suspensions had a uniform orange color that did not change appreciably during storage. Furthermore, no particle aggregation or sedimentation was observed in these systems, and the formulation showed no cytotoxicity to Caco-2 cells.

Due to AST's susceptibility to heat, Tachaprutinun et al. [51] aimed to assess the resistance to thermal degradation of AST nanoparticles prepared by the solvent displacement process, using three different polymers, namely poly(ethylene oxide)-4-methoxy cinnamoyl phthaloyl-chitosan (PCPLC), poly(vinylalcohol-co-vinyl-4-methoxycinnamate) (PB4) and ethylcellulose (EC). While EC was inefficient and PB4 poorly efficient for encapsulating the bioactive compound, PCPLC allowed for an encapsulation efficiency of 98%, a loading of ~40%, and a 312 ± 5.83 nm particle size. In thermal degradation tests performed at 70 ◦C for two hours, most of the free AST was degraded, while the nanoparticle formulation was able to protect the molecule from degradation.

Solid lipid nanoparticles (SLN) are colloidal particles prepared from solid lipids, surfactants, the active ingredient and water. This method has shown several advantages, including the use of biocompatible lipids, high in vivo stability and a wide application spectrum. However, they have limitations, such as a low drug-carrying capacity and drug leakage during storage. Thus, nanostructured lipid carriers (NLC) emerge as secondgeneration lipid nanoparticles to overcome these deficiencies [55].

NLC are obtained using lipids (solids and liquids) and emulsifiers, forming a solid lipid matrix at body temperature that is capable of incorporating hydrophobic and hydrophilic molecules [25]. This matrix has promising potential for the pharmaceutical and cosmetics industry due to its beneficial effects, such as skin hydration, occlusion, greater bioavailability, and targeting application to the skin [55].

In this sense, Rodriguez-Ruiz et al. [48] developed an innovative possible solution with a green chemistry process formulation of AST-loaded nanostructured lipid carriers (NLC). The compounds used to synthesize NLC by the hot homogenization method were sunflower oil as the liquid lipid, glyceryl palmitostearate as the solid lipid, and Poloxamer 407 and Tween 80 as the surfactants. AST-loaded NLC and AST-free NLC were characterized by dynamic light scattering (DLS), atomic force microscopy (AFM) and SEM. DLS analysis showed that for the former particle size of 60 ± 7 nm, a polydispersity index (PDI) of around 0.33 ± 0.09 and zeta potential of −25.5 ± 0.7 mV were achieved, which is indicative of stability. The AFM and SEM analyses demonstrated nanoparticle spherical shape and nanoscale. To evaluate the stability, samples were stored for one month, protected from light, at low temperatures. Results showed no significant change in the size, PDI and zeta potential of both preparations. No less than 90 ± 5% of the starting AST content in NLC was observed after this time, and the additional effect of antioxidants present in sunflower oil seemed to protect the bioactive compound from oxidation. The antioxidant potential determined by the α-tocopherol equivalent antioxidant capacity assay demonstrated an inhibition curve slope that was almost double for AST-loaded NLC compared to the control, confirming the high antioxidant activity of AST [48].
