**7. Dendrimers for Target-Specific Delivery Applications**

Recently, numerous dendrimers were identified to be beneficial as potential targeted drug delivery systems for specific biomedical applications. Swanson et al. (2022) synthesized a novel fifth-generation PAMAM dendrimer, which was surface-modified and functionalized with folic acid. The remaining primary amines in the terminal dendrimer part were conjugated with the bifunctional 1, 4, 6, 10-tetraazacyclododecane-1, 4, 7, 10 tetraacetic acid, alpha-[5-isothiocyanato-2-methoxyphenyl]-, hydrochloride [9Cl] (NCS-DOTA) chelator, where stable gadolinium complexes were loaded into the dendrimer. The resultant dendrimer nanoparticle was about ~5 nm in size with the ability to be a potential target-specific magnetic resonance imaging (MRI) contrast agent with high affinity towards folate receptors in the liver and kidney for identifying human epithelial cancer cells in a murine model [120]. Similarly, Mbatha et al. (2019) prepared PAMAM dendrimer modified with folic acid and functionalized with nanosized gold particles for the efficient and targeted transport of exogenous small interfering ribose nucleic acid (siRNA). The study emphasized that the resultant dendrimer nanosized particles were spherical shaped with 65–128 nm of size and zeta potential above 25 mV, which indicates their high colloidal stability. Additionally, the dendrimer-based nanocomplex was less toxic to human embryo kidney (HEK-293) and HeLa-Tat-*Luc* cells (about 90% of cell viability). The study emphasized that the nanocomplex helped in the 75% increase in the induced transgene-silencing of siRNA and decrease the presence of excess folic acid for effective inhibition of hepatocellular carcinoma (HEPG2) and colon carcinoma (Caco-2) cells [121]. Likewise, Umeda et al. (2010) demonstrated the fabrication of polyethylene glycol (PEG) attached to a PAMAM dendrimer of four generations for the encapsulation of nanosized gold particles as shown in Figure 5. The results revealed that the nanosized gold particles were 2–3 nm in size with spherical morphology, which is engulfed by a single dendrimer particle, while the PEGylated dendrimer encapsulated gold nanoparticles were 15 nm in size. The study revealed that the dendrimers with gold nanoparticles possess enhanced toxicity against HeLa cells under the irradiation of visible light via photoinduced heat generation capability [122].

**Figure 5.** Growth of gold nanoparticles inside the PEGylated PAMAM dendrimers. Reproduced with permission from BUmeda et al. Ref. [122]. Copyright 2010 ACS.

Xiong et al. (2019) exhibited the formation of carboxybetaine acrylamide zwitterion functionalized fifth-generation PAMAM dendrimer and morphine, which is an agent to target lysosome and to entrap nanosized gold particles. The study showed that the nanosized gold particles entrapped by dendrimer are beneficial for the delivery of the H1C1 gene, enhanced by serum. The results showed that the dendrimer-entrapped nanosized gold particles were of size ~1.5 nm, where the vector system helped to carry the H1C1 protein for effective cancer cell migration and metastasis inhibition [123]. Further, Sharma et al. (2021) prepared novel hydroxyl terminated PAMAM dendrimers, that are modified with sugar moieties, such as alpha-D-mannose, beta-D-glucose, or beta-D-galactose via click chemistry approach. The resultant dendrimers were ~4 nm of average particle size with nearly neutral zeta potential and are utilized to target tumor-associated macrophages and microglia. The study showed that the glucose-modified dendrimer has increased brain penetration and cellular internalization, compared to other sugar moieties. It has been proposed that these dendrimers can be potential delivery vehicles for the treatment of glioblastoma and other types of cancers [124]. Furthermore, Zhang et al. (2020) utilized in situ growth approach

for the preparation of novel PAMAM dendrimers (0.5, 1.5, 2, and 2.5 generation) that are grafted on the persistent luminescence nanoparticles (PLNPs) surface. The aptamer AS1411 was coupled with the nanoparticle-dendrimer to specifically bind with the over-expressed nucleolin on the tumor cell membrane, thus increasing the intracellular accumulation of the nanoparticle. The PLNPs were spherical and 15.2 nm of average particle size. In addition, an anticancer drug named doxorubicin was also loaded in the dendrimer-nanoparticlebased delivery system via a pH-sensitive hydrazine, which was identified to be released in the targeted site of the intracellular acid environment. The new drug delivery system using dendrimers was found to have the capability of inducing apoptosis in HeLa tumor cells and inhibiting tumor growth [125]. The results of these studies suggest that dendrimers, particularly PAMAM dendrimers, have the potential to serve as effective drug delivery systems for treating specific diseases. Apart from drug delivery, dendrimers are also utilized as a potential gene delivery system. Ebrahimian et al. (2022) developed a novel lipo-polymeric PAMAM dendrimer-liposome, that are functionalized with transactivator of transcription (TAT) peptide and hyaluronic acid for targeted gene delivery system development. The study showed that the lipo-polymer possesses no significant toxicity with enhanced transfection efficiency in murine colon carcinoma cell line (C26), which will be beneficial for gene delivery applications [126]. Additionally, the incorporation or encapsulation of metals and metallocomplexes within the dendritic scaffold (metallodendrimers) holds the potential to yield novel metallodrugs or offer a new approach for the in situ delivery of metallodrugs. In this area, several works with in vitro and in vivo results were published by Rodrigues and coworkers, involving ruthenium compounds (an alternative metal for platinum-based anticancer resistant drugs) (Figure 6) [127,128], as well as platinum and platinum derivatives as anticancer drugs [128,129]. In any case, the research of the dendrimers is not exclusively restricted to the preparation of drug delivery systems. For instance, Rodrigues and coworkers developed new anionic poly (alkylideneamine) dendrimers until generation 3 with carboxylate and sulfonate terminal groups. These dendrimers have shown in vivo, to be very effective as microbicide against HIV-1 infection [130]. Thus, novel dendrimer structures were developed by the team, where their ability for targeted and controlled drug delivery is expected to be improved in the future.

**Figure 6.** In vivo studies in an MCF-7 xenograft mouse model of low-generation (0–2) nitrile poly(alkylidenamine)-based ruthenium dendrimers. Reproduced with permission from Maciel et al. [127] Copyright 2022 Royal Society of Chemistry.

### **8. Janus-Dendrimer Particles in Target-Specific Delivery Applications**

The enhanced targeted drug delivery ability of Janus structures and dendrimer nanoparticles has led to the emergence of novel Janus-dendrimer nanoparticles to improve their controlled delivery. Further, Janus-dendrimer possess exclusive properties and structures in combinations with different end-group types and distinct surfaces, which makes them a better candidate to form unique drug complexes and conjugates [131]. These Janusdendrimer particles were fabricated based on chemo-selective coupling, heterogenous double exponential growth, and mixed modular approach to be useful for stochastic and multiple drug conjugation-based combination therapy, solubility enhancement, antioxidant lyophilization, targeted delivery, and as fluorescent labels. In addition, the self-assembled Janus-dendrimer particles were identified to be beneficial for the vesicular delivery system, especially for pH-responsive delivery, targeted delivery, spatiotemporal delivery, and sitespecific drug delivery [132]. Pan et al. (2012) created a new Janus-dendrimer particle that was amphiphilic and had peripheral groups made up of acidic amino acids and naproxen molecules, designed for efficient drug delivery to bones. The second-generation dendrimers with >95% of binding rates towards hydroxyapatite in bones, 28-fold enhanced the solubility of naproxen for about 5.37 mg/mL of concentration, compared to the standalone drug. Moreover, the study also revealed that the Janus-dendrimers do not possess any significant cytotoxicity toward HEK293 cells [133]. Further, Iguarbe et al. (2019) created an effective liquid crystal Janus-dendrimer particle made up of mesogenic blocks based on two third-generation Percec-type dendrons with terminal dodecyloxy alkyl chains and one or two carbazole units serving as the electrically active component. The study reported that the carbazole dendrimers were prepared via electrodeposition to form semi-globular particles with electro-polymerizable units. Also, the resultant particles were able to retain the rigid or flexible characteristics of the linker, which eventually influences the size of the particle [134]. Furthermore, a link was established between hydrophobic paclitaxel and the Janus PEGylated peptide dendrimer by Li et al. (2017), through the use of an enzyme-sensitive glycylphenylalanylleucylglycine tetrapeptide as a connector, using an efficient click reaction as shown in Figure 7. The resultant Janus-dendrimer particle possesses the ability to encapsulate 21% of paclitaxel with an average hydrodynamic size of ~69 nm, a narrow polydispersity index of 0.23, the zeta potential of −16.9 mV and spherical morphology. The study showed that the Janus-dendrimer nanoparticle release paclitaxel via enzyme responsive feature and is identified to be highly cytotoxic towards 4T1 (murine breast) cancer cells without any toxicity against normal cells, such as 3T3 murine fibroblast and C2C12 murine myoblast cell lines [135].

Recently, Falanga et al. (2021) created a new type of Janus-like dendrimer that incorporates peptides derived from the glycoproteins (gH and gB) of Herpes Simplex Virus Type 1 (HSV1), aimed at inhibiting viral infection. This was achieved through the combination of copper-catalyzed bio-orthogonal 1,3-dipolar azide/alkyne cycloaddition and photoinitiated thiol-ene coupling, producing both monofunctional and bifunctional peptidodendrimer conjugates. The study revealed that the peptides released by the formulation possess enhanced antiviral activity by inhibiting the DNA replication of HSV1, compared to conventional antiviral drugs, such as foscarnet, acyclovir, and cifofovir [136]. Similarly, Najafi et al. (2020) prepared a novel poly (propyleneimine) (PPI) dendrimer of the fifth generation with a core of cystamine and a hydrophobic surface. Later, the structure scission approach was used to convert disulfide bonds to thiol group and hydrophilic PAMAM dendrons were formed with amine end groups. The study demonstrated that the Janus-like dendrimer, with an average hydrodynamic size of 4.2–28.2 nm, has the ability to enhance the solubility of hydrophobic drugs such as dexamethasone and tetracycline [137]. Likewise, Zhang et al. (2022) designed a hydrophobic multifunctional sequence-defined ionizable amphiphilic Janus-dendrimer region via dissimilar alkyl lengths. The research found that the Janus-dendrimer particles greatly enhanced the activity of the hydrophobic 3, 5-, 3, 4-, and 3,4, 5-substituted phenolic acids they encapsulated, by up to 90.2 times [138]. All these studies showed that the Janus-dendrimer nanoparticles possess enhanced drug delivery capacity, compared to standalone Janus particles and dendrimers.

**Figure 7.** Schematic representation of PEGylated Janus-dendrimer and their self-assembly for intravenous injection in murine models for enhanced stability and anticancer efficacy. Reproduced with permission from Li et al. Ref. [135]. Copyright 2017 ACS.

### **9. Limitations and Future Perspective**

Dendrimers, Janus particles, and Janus-dendrimer nanoparticles have been identified as potential candidates for improved drug delivery applications. However, there remain several limitations that must be overcome before they can be utilized in large-scale commercial applications. The synthesis of Janus particles is one of the major limitations, as it is a multi-step process that can be tedious and challenging to achieve sub-micrometer particle size [139]. The various synthesis approaches, including masking, phase separation, microfluidics, E-jetting, emulsion, and self-assembly, each have their own drawbacks which hinder their scalability. Additionally, general limitations of nanoparticles such as polydispersity, size-dependent toxicity, and the use of hazardous chemicals in synthesis are also applicable to Janus nanoparticles [140]. Dendrimers also face several limitations, particularly at generations higher than 3–4, including high cost of synthesis, a lack of understanding of their effects on biochemical pathways and toxicity, and difficulties in engineering multifunctional dendrimers, especially the tediousness in purification and the yields of the end product. The toxicity of dendrimers has been reported in various studies and can result from improper processing, leading to undesirable side effects, low tolerability, and inefficient drug delivery [141–143]. To address these limitations, the use of lower dendrimers generations with less toxic terminal groups and the use of computational software and machine learning approaches have been employed to optimize Janus, dendrimer, and Janus-dendrimer synthesis parameters. These computational approaches have the potential to reduce the number of experiments and the cost of production, while providing greater insight into the interaction of nanoparticles with biological environments. However, these computational approaches are still in the early stages of research, are highly dependent of the data availability, particularly with regards to the synthesis of nanoparticles and evaluation of their properties and interactions in biological environments [144,145]. In conclusion, the future of Janus and dendrimer nanoparticles for improved drug delivery will likely benefit from a combination of enhanced electron microscopes, synthesis techniques, computational approaches, and machine learning techniques. This will enable the

synthesis of novel and highly efficient nanoparticles with improved bioavailability for drug delivery applications.

### **10. Conclusions**

Recently, there has been an increasing interest in developing a novel drug delivery system that can enhance targeted and controlled drug delivery while reducing toxicity and increasing biocompatibility. Janus and dendrimer particles have been introduced as potential drug delivery systems due to their unique properties compared to conventional delivery systems. These particles have the ability to encapsulate and release drugs in a controlled manner, acts as a drug per se, which can improve the efficacy of drugs and reduce their side effects. However, there are limitations to the use of these materials in commercial pharmaceutics, including lack of scalability, high production cost, and the use of toxic chemicals in synthesis. The emergence of Janus-dendrimer particles holds promise for overcoming these limitations. The combination of Janus and dendrimer particles can result in enhanced drug delivery and improved biocompatibility. However, it is important to improve the stability and toxicity of these particles in the future. This can be achieved through simulation, modeling-based computational and machine-learning approaches, as well as by optimizing the synthesis process and reducing the use of toxic chemicals. With these advancements, Janus-dendrimer particles could become a key tool in advancing drug delivery technology and improving patient outcomes. Their utility extends to various applications within the healthcare field and beyond, opening up new possibilities for innovation.

**Author Contributions:** Writing—original draft preparation, J.J. and K.X.T.; writing—review and editing, J.R. and M.K.D.; supervision, J.R. and M.K.D. All authors have read and agreed to the published version of the manuscript.

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

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing not applicable. No new data were created or analyzed in this study. Data sharing is not applicable to this article.

**Acknowledgments:** The authors (J.J. and J.R.) acknowledge FCT-Fundação para a Ciência e a Tecnologia (Base Fund UIDB/00674/2020 and Programmatic Fund UIDP/00674/2020, Portuguese Government Funds). All the authors thank their respective department for the support during preparation of this article.

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