*2.2. Dendrimers*

The Greek phrase 'dendron', which means trees or branches, is the source for the word 'dendrimer'. Dendrimers are symmetrical, generation-dependent spherical polymers consisting of a core and dendrons (branches), possessing a hyperbranched, three-dimensional structure [13]. In 1941, Paul John Flory and colleagues (Nobel Prize in Chemistry 1974) introduced the theory of highly branched polymers [41,42], which can be synthesized through polycondensation of a monomer with one or more functional groups, avoiding the gelation process [43]. However, it was not stable and are without a cavity. Later, Vogtle and his team (1978) reported the formation of the first non-skid chain-like and cascadelike molecules with the topology of the molecular cavity, which is considered the earliest dendritic polymer form. The term "hyperbranched polymer" was first coined by Kim and Webster in 1988 in reference to the synthesis of soluble hyperbranched polyphenylene. This term was later used to describe the structure of dendrimers [44]. However, these particular types of polymers attract the academy's attention only with the work of Tomalia et al. (1985) [45] and Newkome et al. (1985) [46]. Further, Tomalia not only coined the term "dendrimer" as made a drastic breakthrough in dendrimers field by forming in a controlled manner using divergent synthesis, poly(amidoamine) (PAMAM) dendrimers with a hollow core in the center and outward branches of tendrils [47]. Currently, there are about 100 dendrimer families, which include beyond poly(amidoamine) (PAMAM) dendrimers, among others, polypropyleneimine (PPI), polyester-, polyamide-, phosphorus, and polyether-based dendrimers [12].

Dendrimers' molecular mass and size are specifically controlled during the polymerization process, which is not possible during linear polymer formation [48]. The unique molecular architecture of dendrimers results in improved physical and chemical properties compared to traditional linear polymers [49]. In general, dendrimers have a tightly packed spherical structure with excellent rheological properties and low viscosity than linear polymers [50,51]. It's worth mentioning that the intrinsic viscosity of a dendrimer reaches its peak at the fourth generation as its molecular mass increases [51,52]. The high solubility, miscibility, and reactivity of dendrimers can be attributed to the multiple chainends present in their structure [53]. Similarly, the solubility of the dendrimers depends on their surface group, where dendrimers with hydrophilic and hydrophobic terminations are soluble in both polar and nonpolar solvents, respectively [54]. Furthermore, the spherical shape and presence of internal cavities in dendrimers make them ideal for encapsulating desired molecules or drugs within the macromolecules [55]. These novel polymers are further sub-classified into cationic, neutral and anionic dendrimers, based on their surface charge [56]. It is worth noting that cationic dendrimers are cytotoxic and hemolytic, whereas dendrimers with carboxylate surfaces that are anionic are considered nontoxic for a broad range of concentrations [57,58]. However, the properties of dendrimers are significantly influenced by factors such as pH, solvent, precursor salt, and concentration [59]. Moreover, preparation of dendrimer in the nano-regime will further enhance their properties, due to their exceptional high surface-to-volume ratio and unique structure [60,61]. Figure 1 shows the structural aspects of Janus and dendrimer particles.

**Figure 1.** The general structure of (**A**) Janus, Adapted with permission from Honciuc Ref. [62]. Copyright 2019 Springer and (**B**) dendrimer particles, Adapted with permission from Araujo et al. Ref. [63]. Copyright 2018 MDPI.

### **3. Rational Design and Synthesis Approaches**

### *3.1. Janus Nanoparticles*

Janus nanoparticles are known as a new generation of smart building blocks for material evolution. They are innovative materials designed to have two or more physical and biochemical anisotropy as well as assembled layers to serve multiple functionalities. Their unique characteristics and structural designs make them superior to conventional particles. Their superior characteristics are such as the high interfacial activity; engineering of their interfacial activity via external stimulus (i.e., pH, temperature, or ionic strength); recyclable for sustainable usage attributed to the possibility of stimuli-induced separation; high surface-to-volume ratio; manipulation of mass transport across different interfacial layer; and their multi-functionalities in one Janus nanoparticle [64]. This allows them to be flexible as either a macro-phase separation or stable emulsion. Furthermore, many materials especially biodegradable or natural materials such as chitosan, alginates, and cellulose, have been widely used for the Janus nanoparticles fabrication [37,65–69]. Janus nanoparticles that respond to changes in pH have been reported for use in bio-imaging and anti-tumor drug delivery. These nanoparticles enable real-time monitoring and tracking of drug release [70,71].

Various synthetic techniques are available and optimized to fabricate Janus nanoparticles with the desired shape, size, and physical or biochemical functionality on their opposite side. Three widely recognized conservative fabrication methods are masking, phase separation, and self-assembly. Masking is the simplest technique used to fabricate Janus nanoparticles by covering up one side using either solid or liquid blocking surfaces while exposing the other side of the particles for chemical modifications. For instance, inorganic Janus particles were produced via the partial coating of calcium carbonate particles with platinum to create non-Brownian movement in the acidic environment of the tumor site [72]. This approach allows scientists to manipulate the chemical and structural features of Janus nanoparticles, resulting in exceptional design and functionality. However, the masking technique is only appropriate for small-scale laboratory production, making it useful for initial investigation and basic testing. The phase separation technique works by mixing two or more incompatible polymers or components before separating them into different compartments of a single Janus nanoparticle. The pH of the aqueous phase, the makeup of the dispersed phase, spreading coefficients, and interfacial tension can often impact phase separation and particle shape [73]. Self-assembly from di-block copolymer is considered one of the easy and simple methods to synthesize different polymeric Janus nanoparticles by adjusting the cross-linking degree and molecular weights or ratio of the blocks used [34].

Pickering emulsion on the other hand utilizes the interface of two immiscible fluids for both particle adsorption and unique surface modifications to synthesize the anisotropic particles. This technique enables the production of emulsions such as silica-based amphiphilic Janus particles with colloidal stability that lasts for more than 3 weeks [74]. Nonetheless, this technique comes with limitations including the tedious multiple steps that require strict monitoring; limited encapsulation and controlled release capacity; unsuitability for

biodegradable polymeric Janus particles due to the high temperature and specific solvents used to eliminate the masking layer [75].

Methods such as microfluidic assembly and a 'lab-on-a-chip' system have also been exploited to fabricate Janus-like dimer capsules. This technique comprises a series of steps such as fluidic assembly, cross-linkage, and droplet formation that occur on a microfluidic chip without manual control. Lu and team [67] have designed Janus-like dimers whereby the functionality and structure of each lobe have been precisely controlled to resume bowling pin and snowman structures. This method allows the formation of Janus-like dimer capsules by having two individual biopolymer mixtures with distinct compositions from two different channels meet at the junction and fuse into one stable formation with distinct compositions via controlled cross-linking and coalescence. There is also a study reported on the use of a microfluidic-mediated fabrication method to produce Janus nanoparticles in a microchannel whereby two adjacent fluid streams with a laminar flow are separated into discrete droplets via the immiscible phase and finally photo-polymerized into Janus nanoparticles [76]. However, this method comes with challenges such as low output efficiency, precise control, and limited polymer choices. Furthermore, there is a fluidic nanoprecipitation system developed to fabricate Janus nanoparticles made of PLGA. The results showed that these polymeric Janus nanoparticles effectively encapsulated two distinct drugs, Paclitaxel (a hydrophobic drug) and Doxorubicin Hydrochloride (a hydrophilic drug), resulting in varied drug release behavior [77]. This is a single-step method that utilizes dual inlets to deliver each half of the Janus compartment into the precipitation flow.

Another team of scientists [75] devised a straightforward, one-step solvent emulsion method for creating hybrid Janus particles. This approach enables the targeted encapsulation of both diagnostic and therapeutic agents into separate compartments of the Janus complex through drug-polymer interactions and the use of specific biopolymer ratios, as illustrated in Figure 2. This technique is capable to offer a more cost-effective, scalable, and seamless approach to synthesizing dual drug-encapsulated theranostic Janus particles with controlled and different release kinetics. With this method, both the immiscible biodegradable and non-biodegradable polymers are emulsified and segregated into two different compartments due to the interfacial tensions. This one synthesis setup technique could replace the single drug-encapsulated particle due to its simplicity and scalability.

**Figure 2.** A simple and single-step solvent emulsion technique used to fabricate polymeric Janus particles for theranostic applications. (**a**) Theranostic polymeric Janus particles made of both biodegradable and non-biodegradable polymers, (**b**) magnetic response of drug and SPIONs-encapsulated EVA/PLGA Janus particles, (**c**–**f**) a comparison of SPION encapsulation in different polymers i.e., PLLA, EVA, PLGA, and PMMA, (**g**–**i**) the formation of SPIONs-encapsulated Janus particles: EVA/PLLA, EVA/PLGA, and PMMA/PLGA. Reproduced with permission from Lim et al. Ref. [75]. Copyright 2019 Wiley.

### *3.2. Dendrimers*

Generally, dendrimers are synthesized using conventional methods, such as cascade reactions, and divergent as well as convergent approaches [78]. The cascade strategy involves the assembly of three sub-nano/picoscopic-sized repeated units via reaction with a divalent terminus to produce a branched cell with a branch juncture. The generation zero (0) of the dendrimer is formed by attaching the first branch to the reference point for yielding a labeled branch with a valency of four. Later, the first and second generation of dendrimers was formed by stepwise attachment of repeat units according to their incipient valency [79]. Even though the cascade strategy is initially used, convergent and divergent approaches are commonly utilized for the fabrication of dendrimers as shown in Figure 3. In a divergent approach, the dendrimers are synthesized from the core to the branches through repeated coupling and activation steps in an outward direction [45]. Hence, the cascade strategy is also considered a type of divergent approach for dendrimer formation [80]. Recently, Rauch et al. (2020) utilized an iterative divergent approach for the preparation of conjugated starburst borane dendrimers. In this study, the dendrimer was prepared in three steps: the functionalization of iridium-catalyzed carbon-hydrogen borylation and activation of fluorine-generated boronate ester with potassium bi-fluoride and expansion of trifluoroborate salts with aryl Grignard reagent reaction [81]. In a convergent approach, the branches (dendrons) of the dendrimer were prepared initially, which is finally coupled to the moiety in the core after the activation of their focal point [82]. It can be noted that the growth of the dendron is straightforwardly monitored via a convergent method, compared to a divergent approach [83]. Bondareva et al. (2020) prepared sulfonimidebased dendrimers and dendrons with the help of a convergent approach. In this study, both convergent and divergent methods were used to create sulfonimide-based dendrons and dendrimers with chlorosulfonic groups as the central focus. Suprisingly, the results indicated that the convergent method of creating dendrons reached its practical limit in the third generation, making a divergent approach necessary for producing sulfonimide-based dendrimers with more generations [84].

**Figure 3.** Schematic representation of (**A**) Divergent and (**B**) convergent approach, Concept adapted from Grayson et al. Ref. [85]. Copyright 2001 American Chemical Society.

Apart from the conventional divergent and convergent approach, revised traditional methods, such as the hyper monomer method, double-stage convergent growth, and double exponential growth were also used for the synthesis of dendrimers [86]. Monomers with higher functional group numbers were employed in the hyper monomer approach for dendrimer synthesis in fewer steps, compared to the traditional AB2 monomer [87]. Balaji and Lewis (2009) synthesized a novel aliphatic polyamide dendrimer by AB2 hyper monomer strategy and double exponential growth. The study showed that the yield of the dendrimer is about ~93%, which is reduced to about ~89% after 30 min of the purification process [88]. In a final step, low-generation dendrimers and dendrons are linked together through parallel synthesis in a double-stage convergent growth approach [89]. Recently, Agrahari et al. (2020) demonstrated the synthesis of novel glycol-dendrimers and dendrons coated with galactose was achieved through an efficient click approach and double-stage convergent method. The study emphasized the synthesis of a novel galactose coated 9peripheral glycodentromer of the zeroth generation and a galactose-coated 27-peripheral glycodentromer of the first generation [90]. Further, low-generation dendrons that were fully protected or deactivated were created, and then actively selected at their periphery or central focus, and joined to produce higher generations of fully protected dendrons through a double exponential growth method [91]. Hartwig et al. (2010) prepared A novel polyglutamate dendrimer of the fourth generation was produced through an iterative binomial exponential growth method. The research demonstrated that this synthesis technique can aid in incorporating either D-alt-L or all-(L) stereochemistry in the peptide backbone and is useful for post-functionalization of the dendritic core and periphery [92].

Additionally, orthogonal, chemoselective reactions (click chemistry), one-pot synthesis, and heterofunctional fabrication approaches were introduced for dendrimer formation. The click approach makes use of Diels-Alder cycloaddition and thiol-ene coupling, which are highly selective chemical reactions, and offer a reliable platform for the synthesis of complex macrostructures [93]. Ma et al. (2009) fabricated a novel polyester dendrimer via a sequential click coupling of asymmetrical monomers. In this study, mechanistic or kinetic chemoselectivity was combined with click reactions between the monomers for efficient dendrimer synthesis via simple sticking of generation by generation together [94]. When different monomers, such as Abx and CDy, instead of ABn monomer in divergent growth approach for dendrimer formation is termed as orthogonal growth method [95]. Liu et al. (2022) fabricated scaffold-modifiable dendrons via an orthogonal protection strategy. The study revealed that this approach can yield fourth-generation dendrons within 2 days without any significant defects in their structure [96]. Kothari et al. (2013) demonstrated an efficient and accelerated preparation of multifunctional dendrimers via nucleophilic substitution and orthogonal thiol-ene reactions. In this study, multifunctional dendrimers synthesized via an orthogonal approach did not possess the ability to carry out protectiondeprotection steps to apply for complex organic molecule construction [97]. Moreover, the heterofunctional method is employed to produce dendrimers with a precise and large number of unique functional groups while maintaining structural integrity within the framework [98]. Goodwin and colleagues synthesized a heterobifunctional, biodegradable dendrimer through the creation of a symmetric aliphatic ester dendrimer derived from 2, 2-bis(hydroxymethyl) propanoic acid. The dendrimer featured a cyclic carbonate periphery and functional amine at the opening of the carbonate moieties. This approach was deemed advantageous for the formation of a dendrimer with eight alkynes and eight protected aldehydes on its periphery, as the resulting dendrimer did not require purification through chromatography [99]. All the above-mentioned methods require purification approach to yield pure dendrimer, either one way or the other. Hence, one pot synthesis has been introduced for the formation of dendrimers, which does not require any purification steps [100]. Recently, Yan et al. (2022) utilized one pot periodate oxidation approach for the preparation of dialdehyde cellulose and are modified with polyamidoamine (PAMAM) dendrimer of zero generation via hyperbranched crosslinking synthesis to yield cellulosebased dialdehyde polymers. The study showed that the dendrimer was grafted onto the backbone of cellulose with a crystallinity of 46.5%, uniform long-strip structure and excellent particle size distribution [101].

In recent times, several novel synthesis approaches were introduced for the preparation of dendrimers. Mahdavijalal et al. (2023) prepared a PAMAM dendrimer that is anchored to tungsten disulfide (WS2) nano-sheets via a grafting approach. In this study, the stimulus-responsive polymer was prepared by suspending WS2, 2, 2 azobisisobutyronitrile (AIBN) as the radical initiator and poly (N-vinyl caprolactam) (PNVCL) in ethanol under a nitrogen atmosphere, stirring was conducted at 65 ◦C in a paraffin bath that was temperature-regulated, lasting for 7 h. The resultant surface-modified nano-sheets were utilized for the fabrication of three generations of PAMAM dendrimers via grafting, precipitation, and centrifugation technique. The electron micrograph results revealed that before the surface modification, the nano-sheets were 20–50 nm in size and had an inconsistent thickness. However, after the surface modification using PAMAM dendrimers, the size of the nano-sheets increased to 80–90 nm, with a more uniform thickness [102]. Further, Sohail et al. (2020) synthesized PAMAM dendrimers via a divergent approach with monomer coupling and monomer end-group transformation for the creation of reactive surface functionality as well as coupling of a novel monomer via solid-phase peptides or oligonucleotide synthesis. In this study, needle-like and spherical structures were produced during the synthesis of PAMAM dendrimers with ester-terminated half-generation and amino-terminated full-generation, respectively with ~827 nm of hydrodynamic size [103]. Furthermore, Bafrooee et al. (2020) utilized the chemical co-precipitation method for carboxyl-terminated hyperbranched PAMAM dendrimer formation, that is grafted with superparamagnetic iron oxide nanoparticles in a core-shell structure. The study revealed that the synthesis process resulted in the formation of a dendrimer with an average of 5.5 generations and an average pore diameter of 11.83 nm. The size of the dendrimer was observed to be in the range of 20–75 nm, with a spherical shape [104].

### **4. Bioavailability of Janus Nanoparticles**

Green chemistry aims to limit or reduce the use and/or creation of hazardous materials starting from the initial design process to production and finally to the application of a product. This promotes prevention over remediation for material sustainability and safety. The production of Janus nanoparticles is via green chemistry. This is because Janus nanoparticles are mostly made of biogenic materials, including both natural and synthetic biopolymers that are biodegradable and biocompatible. This leads to the development of bioavailable, sustainable, and eco-friendly materials, especially for biomedical applications, which can eventually help to address the toxicity issues of some available materials. Organic and soft materials are used to fabricate most biocompatible and biogenic Janus particles using e.g., single/double emulsion, microfluidic, co-jetting, solvent evaporation, polymerization [64]. In addition, many biocompatible synthetic biomaterials are used for Janus nanoparticles synthesis which includes the FDA-approved poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), and poly(lactic acid) (PLA) for their pharmaceutical delivery applications. They are often been utilized for time-programmed single/dual drug delivery and release at dual-site with simultaneous/stimuli-dependent release of two incompatible drugs due to their difference in biodegradability [73].

Some studies have reported the use of Janus nanoparticles with high bio-distribution for intercellular transportation and cellular uptake. For instance, Shao and their team have developed rod-like magnetic nanosized silica mesoporous particles via the modified sol-gel approach. The results indicated that a more targeted drug delivery efficacy has been achieved with higher retention and accumulation at the tumor sites via varying endocytic pathways, leading to higher intracellular internalization and bioavailability [105]. Another study by Shao, et al. [106] also demonstrated the bioavailability of doxorubicin-loaded Janus nanocomposites by concentrating the accumulation of the drug at the targeted site of the liver tumor with high tumor cell endocytosis whilst offering 'zero' release of doxorubicin to the other surrounding normal cells and blood circulatory system.

Many studies have investigated the cytotoxicity of Janus nanoparticles, indicating their excellent biocompatibility, especially for clinical applications. Cao and team have demonstrated the cytotoxicity of dual-drug loaded Janus nanoparticles in HeLa cells and MDA-MB-231 cancer cell line via the colorimetric cell viability assay [71]. Results revealed more than 90% of cell viability even with such a high concentration of Janus nanoparticles ~500 μg/mL. Moreover, it showed a lower IC50 as compared to free drugs. Furthermore, Janus nanoparticles are capable to offer a lowered systematic toxicity with significant tumor growth inhibition as compared to other core-shell nanoparticles [107]. Based on the cytotoxic Sulforhodamine B assessment performed under both normoxic and hypoxic environments, Janus nanoparticles can selectively eliminate the hypoxic liver cancer cells with dose-dependent cytotoxicity without inhibiting the normal surrounding cells for better and safer therapeutic outcomes. Besides, modifying the nanoparticles with silica could be crucial in enhancing their biocompatibility, specifically in the case of Janus nanoparticles.

Additionally, Zhao, et al. [108] have invented Janus hollow spheres with dose-dependent cytotoxicity. According to their research, the safety level of doxorubicin-loaded Janus hollow spheres was found to be favorable in the U87MG cell line when tested using the CCK-8 assay at both pH 6.4 and 7.4 with particle concentrations as high as 200 μg/mL. The results of both the cell viability tests and the pH-dependent toxicity evaluations indicate that Janus hollow spheres have the potential to be a secure and effective vehicle for targeted drug delivery. Another study has described the cytotoxicity evaluation of Janus nanoparticles after penetrating the blood-brain-barrier via the 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium-bromide (MTT) assay using the rat C6 glioma cell line, at different time intervals and pH. The study revealed that the optimal conditions for maintaining cell viability above 50% were a minimum pH of 6.2 and a maximum incubation time of 24 h. Also, the results indicated that fewer Janus nanoparticles were present in non-targeted normal cells, likely due to the extended blood circulation time of the Janus nanoparticles, as well as the strong covalent bond between the encapsulated doxorubicin and its Janus compartment. As a result, doxorubicin was only released when triggered by the acidic condition of cancerous cells via breakage of imine bond, indicating the safe usage of Janus nanoparticles for targeted drug therapies.

### **5. Bioavailability of Dendrimers**

It can be noted that dendrimers are biocompatible and less toxic to be used in biomedical applications, similar to Janus particles. Rehman et al. (2021) prepared a novel resorcin arene-based dendrimer vesicles of nano-size based benzyloxy macrocycle. In this study, 4-benzyloxy benzaldehyde and resorcinol were dissolved in acetic acid under constant stirring conditions. Later, their pH was altered using sulphuric acid under reflux conditions for 24 h at 50 ◦C. A brown precipitate was formed as a resultant sample, which was filtered, washed with water, and dried, where the recrystallization of brown precipitate in tetrahydrofuran or methanol mixture (2:8, *v*/*v*) with 88% of yield. This study demonstrated that the synthesized dendrimer was highly effective for encapsulating quercetin, which was found to exhibit low toxicity in the NIH-3 T3 cell lines. The lower toxicity of the dendrimer-encapsulated quercetin towards cells is identified to be due to the existence of four hydrophobic benzyl groups with appropriate lipophilicity of benzyloxy macrocycle and their slightly amphiphilic nature with structural saturation [109]. Further, Giorgadze et al. (2020) synthesized PAMAM dendrimers of four generations to be useful for the encapsulation of silver atoms. The results showed that the dendrimers were stable at ~5 nm in size with strong visible light absorption. The study emphasized that the silver atoms in dendrimers can able to overcome the cell nucleus and membrane of 8–9 nm of pore size, which can subsequently reduce their cytotoxicity and improve their biocompatibility [110]. Furthermore, Bhatt et al. (2019) developed a dendrimer via alpha-tocopheryl succinate (alpha-TOS) conjugation and coating polyethylene glycol (PEG) on the surface of PAMAM dendrimer (four generations) as shown in Figure 4. The resultant dendrimer was utilized to improve intracellular paclitaxel delivery, which is a chemotherapeutic drug that is poorly soluble in water. The hemolysis assay revealed that the resultant dendrimer was nontoxic to red blood cells with high biocompatibility. Later, the cellular uptake assay conducted on B16F10 monolayer cells and MDA MB231 3D spheroids showed that the conjugation of alpha-TOS significantly improved the time-dependent uptake of the nanoscale dendrimer [111].

Noorin et al. (2021) created a novel second-generation linear globular dendrimer made of Gadoterate Meglumine and of nanoscale size.The resultant dendrimer was identified to be almost nontoxic towards immortalized human embryonic HEK 293 kidney cells and human glioblastoma cell culture [112]. Similarly, Ahmed et al. (2021) prepared a novel surface modified four generation PAMAM dendrimer via 4-nitrophenyl chloroformate as an activator and 2 kDa of PEG with spherical or semi-spherical morphology for the controlled delivery of anti-tuberculosis rifampicin drug. The findings indicated that the surface of the dendrimer was coated with PEG at a level of 38–100%, leading to a slower release

rate of rifampicin compared to the non-PEGylated formulation and the unencapsulated drug. The study found that the fully functionalized dendrimer exhibited reduced toxicity towards raw 264.7 cell lines [113]. Likewise, Alfei et al. (2020) created a new biodegradable fifth-generation dendrimer based on polyester and featuring a free carboxylic group. The dendrimer was synthesized using dichloromethane, N, N'-dicyclohexylcarbodiimide (DCC), 4-(dimethylamino) pyridinium 4-toluene sulfonate and was protected with an acetonide group. Later, the dendrimer was used to encapsulate Etoposide (ETO), a substance derived from podophyllotoxin, the main chemical component found in the herbaceous plants named *Podophyllum hexandrum* and *P. peltatum*. The studies revealed that the dendrimer with ETO was 70 nm in size with nano-spherical morphology, 37% of drug loading, 53% of entrapment efficiency, and possess enhanced solubility in biocompatible solvents, such as ethanol and water. The cytotoxicity studies on neuroblastoma cells emphasized that the dendrimer and ETO possess cytotoxic and pro-oxidant properties and their bioactivity was synergistically improved after encapsulation due to the slow release of ETA [114].

**Figure 4.** Schematic representation of dendrimers with PEG and alpha-TOS along with their cell uptake, cell viability, growth inhibitions of 3D spheroids, tumor volume reduction, and terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate (UTP) nick end labeling (TUNEL) results. Reproduced with permission from Bhatt et al. Ref. [111]. Copyright 2019 ACS.

### **6. Janus Nanoparticles for Target-Specific Delivery Applications**

The advancement in nanotechnology has allowed Janus nanoparticles to serve as nanomedicines to regulate both the bio-distribution and tumor accumulation of administered medical drugs for better efficacy. Janus metallic mesoporous silica nanoparticles (JMMSNs) have been developed to be cancer-targeting for cancer theranostics with multifunctions such as magnetic resonance imaging as well as drug release and therapy [115]. This is attributed to JMMSNs that possess anisotropic compositions with stimuli-responsive

properties to provide both tumor diagnosis and therapy synergistically [115]. JMMSNs are capable to load both diagnostic and therapeutic agents in separate compartments and release them spatiotemporally in response to different stimuli for the cancer-targeted delivery. Janus magnetic nanoparticles possess magnetic properties that make them suitable for both enhancing MRI imaging as a contrast agent and delivering drugs when used in conjunction with hyperthermia [116]. Hence, Janus magnetic nanoparticles are widely recognized as safe and effective carriers for biological drugs such as genes, proteins, and other biological molecules due to their controlled and sustained drug release profile and their ability to efficiently load drugs through the use of an external magnetic field. Moreover, the size of nanoparticles allows them to penetrate targeted cells as well as the blood-brain-barrier whilst maintaining the retention effect [117]. Janus nanoparticles have been revealed as a highly promising delivery vehicle for pharmaceuticals due to their superior properties, and they can be decorated with various targeting, diagnostic, and therapeutic biological molecules further to advance the use of nanomedicine with multiple functions. Table 1 shows a summary of reported works that exploited Janus nanoparticles for target-specific delivery and therapy.

**Table 1.** A summary of reported studies on the targeted delivery applications of Janus nanoparticles.

