Chitosan Based MicroRNA Nanocarriers

Vectorization of microRNAs has shown to be a smart approach for their potential delivery to treat many diseases (i.e., cancer, osteopathy, vascular, and infectious diseases). However, there are barriers to genetic in vivo delivery regarding stability, targeting, specificity, and internalization. Polymeric nanoparticles can be very promising candidates to overcome these challenges. One of the most suitable polymers for this purpose is chitosan. Chitosan (CS), a biodegradable biocompatible natural polysaccharide, has always been of interest for drug and gene delivery. Being cationic, chitosan can easily form particles with anionic polymers to encapsulate microRNA or even complex readily forming polyplexes. However, fine tuning of chitosan characteristics is necessary for a successful formulation. In this review, we cover all chitosan miRNA formulations investigated in the last 10 years, to the best of our knowledge, so that we can distinguish their differences in terms of materials, formulation processes, and intended applications. The factors that make some optimized systems superior to their predecessors are also discussed to reach the highest potential of chitosan microRNA nanocarriers.


Introduction
Genetic material delivery has always been a dreadful challenge. In the last decade, non-viral vectors have shown great promise as safer alternatives to viral vectors for gene delivery. Non-viral vectors include lipidic and polymeric systems [1]. Cationic polymers were of special interest for their ability to complex genetic material readily. One of the most important cationic polymers known is chitosan since it is a natural, biocompatible, biodegradable, and generally recognized as safe (GRAS) by the FDA [2]. Chitosan is a unique polysaccharide copolymer of N-acetyl-D-glucosamine and D-glucosamine linked by a β-(1→4) glycosidic linkage ( Figure 1). It is readily derived from N-deacetylation of chitin which is abundant in the exoskeletons of crustaceans (shrimps, crabs, etc.) and the endoskeletons of cephalopods (squids) and is also found in many other species such as mollusks, insects, and fungi [3]. Depending on the duration and intensity of the deacetylation process, the amount of remaining N-acetyl-D-glucosamine moieties (called the degree of acetylation, DA) varies. This parameter, along with the degree of polymerization (DP) or in other words its molar mass (Mw), should be well controlled, as many physicochemical and even biological properties are impacted by the DA and Mw of chitosan [4]. Chitosan NPs have shown potential as highly effective and safe vectors for in vivo delivery of non-coding RNAs when formulae have been optimized [5]. A chitosan based commercial gene transfection reagent (Novafect) has also been developed by Novamatrix, Norway as a non-viral carrier for gene therapy [6].
Regulatory non-coding RNAs (ncRNAs) is a term commonly used for RNA not encoding a protein yet affecting gene regulation. Classified by their average size, regulatory ncRNAs are either small non-coding RNAs (sncRNAs) typically less than 200 nt long, or lncRNAs longer than 200 nt. SncRNAs are classified into microRNA (miRNA), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs) which are a class of animal  Both agents induce gene silencing through activation of RNA induced silencing complex (RISC). That leads to enzymatic cleavage of target mRNA in case of siRNA due to definite complementation of siRNA to its target. However, unlike siRNA, miRNA only achieves partial complementation to its target mRNA which makes it able to regulate expression of multiple targets ( Figure 2) [8,9]. MiRNAs have been proven to have a role in all physiological and pathophysiological mechanisms in our bodies, which makes them groundbreaking for many therapeutic applications [10].
As a result of their gene silencing mechanisms, miRNAs hold great potential for biomedical applications, but their lack of specificity remains a challenge. Moreover, using them solely as therapeutic agents faces extracellular and intracellular barriers [11]. The extracellular barrier would be the extracellular environment containing nucleases ready to cleave any RNA therapeutic and the reticuloendothelial system RES that will mark any foreign substance detected, deeming the half-life of a therapeutic ncRNA to about 10 min.
Then, if this agent was protected either by chemical modification or vectorization, it would face intracellular barriers of cellular uptake and intracellular trafficking. As RNA interference (RNAi) agents are negatively charged, they are repelled, when administered solely, by a counter negative charge of plasma membranes compromising their uptake. In addition, their diffusion through membranes will be impossible for their high molecular weight. To top it all, from a clinical perspective, administration of miRNA as such can induce an immune response and flu like symptoms [12,13]. Ideally, successful RNAi using a delivery system requires the nanocarrier material to be biocompatible, biodegradable, nonimmunogenic, and nontoxic and needs the nanocarrier to protect ncRNA molecules from enzymatic degradation, be of an optimum size (ideally < 200 nm), surface charge, and functionalization to avoid phagocytosis and get internalized by the cells, escape lysis in the endosome, and release ncRNA in cytoplasm [14].
Many vectorization materials, including lipidic, synthetic, and natural polymeric systems, have been studied to achieve this successful delivery of ncRNA. Among them, chitosan stands out as a suitably priced available natural biocompatible biodegradable safe polysaccharide with a positive charge readily available for interaction with negatively charged ncRNA forming nanoplexes. Moreover, chitosan's protonated amine groups enhance passage across cell membranes and endocytosis [15].
A lot of research has been carried out on the formulation of different forms of CS/siRNA nanoparticles and the importance of chitosan characteristics to achieving optimal (i) particle stability for ncRNA protection in biological media, (ii) colloidal stability maintaining the size of the nanoparticles avoiding aggregation, and (iii) subsequent transfection efficiency [16] but far less focus has been given to miRNA delivery using chitosan ( Figure 3). To date, in 2022, only 53 research papers, all of which are covered here, tackle NPs involving both chitosan and miRNA. Indeed, both siRNA and miRNA face similar challenges which are well reviewed in other articles concerning siRNA and include poor stability in vivo, delivery challenges, and off-target effects, and similar strategies may even be investigated to optimize their delivery [14]. However, their mechanisms of actions and biomedical applications are still distinct which demands more spotlight on miRNA.
In this article, we review all chitosan-based nano-formulations made for delivery of miRNA, which include:

•
Nanoparticles formulated by direct complexation between chitosan and miRNA (referred to as nano polyplexes = nanoplexes). Polyplexes are complexes made by electrostatic interaction between a cationic polymer (such as chitosan) and negatively charged genetic materials (such as miRNAs) [17]. • Nanoparticles based on the interaction between polycationic chitosan with a counter polyanion: • Nanoparticles made by polyelectrolyte complexation of polycationic chitosan with a counter polyanion (negatively charged polymer as hyaluronic acid (HA) or dextran sulphate (DS)), adsorbing or encapsulating miRNA (referred to as CS miRNA polyelectrolyte complexes (PECs)) [18][19][20]. • Nanoparticles made by ionic gelation of polycationic chitosan with a counter polyanionic small molecule such as tripolyphosphate (TPP), adsorbing or encapsulating miRNA (referred to as CS/TPP NPs) [21]. • More complex engineered systems.

Chitosan Molar Mass
Chitosan molar mass is an essential parameter in the formulation of miRNA nanoparticles with optimum size and stability. CS NPs need to be stable enough to protect miRNA until it is internalized, but able to release it in cytoplasm. As studied before with CS for nucleic acid delivery, the CS chain length's impact on NP morphological and physicochemical characteristics eventually affects their in vitro and in vivo activity [14,22].
In literature, some authors do not mention the molar mass of CS used in their studies or find it sufficient to just describe it as low or high. In this review, for uniformity, we use the same classification proposed by Ragelle et al. for CS/siRNA NPs; very high Mw would be (>300 kDa), high Mw (80-300 kDa), low Mw (10 to 80 kDa), and very low Mw CS (<10 kDa) [14].
As concluded from work on CS siRNA NPs, CS with a very short chain length cannot condense genetic material to form intact NPs. A minimum of 15 kDa molar mass was necessary for complete binding to siRNA and therefore protection against cleavage [14]. On the other hand, a very high Mw > 300 kDa may protect the payload too much, so that it faces a challenge in gene release and subsequently less gene silencing. Only CS with molar mass 5-10 times higher than siRNA's Mw can form compactly structured NPs of suitable size through electrostatic forces and interchain connections [23].
Most research conformed with these specifications set by experience gained with siRNA NPs. However, some exceptions can still be demonstrated. Rarely in literature has a CS of Mw more than 300 kDa been used for delivery of miRNA, however, Ning et al. reported formulation of galactosylated-CS-5-fluorouracil (GC-FU) NPs of 178.5 nm starting from 500 kDa chitosan [24]. These NPs were also loaded with liver-specific miRNA-122 to provide synergistic therapy for hepatocellular carcinoma.

Effect of Chitosan Molar Mass on (CS/Polyanion/MiRNA Nanoparticles)
Most researchers used a Mw around 100 kDa, which demonstrated NPs of less than 200 nm that were safely tolerated by cells in vitro and had a biological effect in vivo (when in vivo studies were performed). Yet, a systematic comparison of different CS molar masses has rarely been performed in the same study. Only Tekie et al. compared three different molar masses of CS (9, 18, and 45 kDa) in formulation of polyelectrolyte complexes (PECs) made with carboxymethyl dextran. PECs of 18 and 45 kDa chitosan had higher transfection efficiency, GFP expression, and reduction of cell proliferation by miR-145 when compared with PECs made of 9 kDA Mw CS [25,26]. CS/TPP NPs had a major share of interest of many scientists [9,[27][28][29][30], and all of them  [28,30,31]. For Deng et al., HA addition led to a synergistic effect with DOX enhancing its antitumor effect by repressing the expression of anti-apoptosis proto-oncogene [28]. On the other hand, Wu's NPs had a high transfection efficiency with significant enhancement of marrow mesenchymal stem cells osteogenesis [30]. Wang et al. had a similar effect of improving osteogenesis with their CS/HA system that did not include TPP at all using the same CS Mw of 100 kDa as in Wu's work [32].
Other polyanionic polymers, like dextran and chondroitin sulfates, were also studied. Tekie et al. used a double strategy to protect their miRNA-145 by conjugating it to dextran by a disulfide bond then further electrolyte complexation with CS of 18 kDa that led to particles of 47, 132, and 272nm depending on the dextran to chitosan molar ratios used (0.2, 1 and 5 respectively) [33]. Then an aptamer was introduced as a targeting agent to increase cellular uptake. Çelik et al. introduced both HA and chondroitin, which together with CS made a triple polysaccharide system capable of protection and delivery of the miRNA to decrease mRNA levels of its target [34].
Only Cosco et al. used CS with an extremely low molar mass (<10 kDa), but they introduced the nanoprecipitation method to form PLGA/CS miRNA NPs that were intact, and of optimal size [35]. This exception may be attributed to using a totally different formulation method (nanoprecipitation) than the ionic gelation method mentioned above. PLGA was dissolved in acetone then added to acidified water containing CS, poloxamer and miR-34a all homogenized together. As PLGA has hydrophobic properties and is negatively charged, its complexation with cationic chitosan could boost the encapsulation and delivery of negatively charged hydrophilic miRNA. These CS/PLGA nanospheres, shown in Figure 4, had a mean hydrodynamic diameter of 160nm, a positive zeta potential and a high level of entrapment efficiency up to 95%. The nanospheres structure protected miRNA from degradation and allowed high transfection efficiency and a significant antitumor effect in vitro against multiple myeloma cells [35].

Effect of Chitosan Molar Mass on Nanoplexes (Chitosan MiRNA Direct Polyplexes)
Following the same trend, all studies were conducted on molar masses of CS ranging from 20-200 kDa to form intact biologically active nanoplexes [24,25,[36][37][38][39][40][41][42][43]. Exceptionally, few teams compared different molar masses in the same study, which gives more standardized results. Tekie et al. studied their nanoplexes using 9, 18, and 45 kDa CS at different N/P ratios (2,5,10,20,30, and 100) [25]. N/P (amine/phosphate) ratio is the molar ratio of polymer's positively charged amine groups (N) to the negatively charged phosphate (P) groups of nucleic acids, and it is one of the most crucial physicochemical parameters for gene delivery, reviewed best in the work of Gary et al. and Alameh et al. [44,45]. As shown in Figure 5(B), a 9 kDa CS could not give NPs at N/P ratios of 5 and 10. Except for those two cases, all three molar masses used of CS gave nanoparticles at all used N/P ratios. However, only 9 kDa CS at N/P ratio 2 and 45 kDa CS at N/P ratio 10 gave a preferable polydispersity index PDI below 0.20. However, they indicated that their most efficient formulations were made of CS 18 and 45 kDA with N/P ratio of 40 and 100 although they showed delayed onset due to slow release [25].  Santos-Carballal et al. studied their CS nanoplexes using two ranges of low (1-2 kDa) and relatively higher molar masses (18-26 kDa) with varying degrees of acetylation (as shown in Table 2), all produced from the same parent CS (DA = 1.5%, Mw = 543 kDa) via nitrous depolymerization and subsequent reacetylation [36]. However, a variation in molar mass can be observed. Also, an identical degree of acetylation couldn't be achieved between comparative higher and lower molar mass couples, especially in the highest DA% couple (49% and 67%) [46,47]. These differences may have impacted the robustness of the comparison to study the effect of molar mass. All formulations led to polyplexes of average hydrodynamic diameters less than 190 nm rendering them available for cellular uptake ( Figure 6). They concluded that optimal formulations were observed using CS with a molar mass of~40 kDa, DA of 12%, and a (+/−) charge ratio of 1.5, resulting in a high transfection efficiency similar to the commercial reagents (DharmaFECT and Novafect) under a standard protocol recommended by DharmaFECT [36].  Louw et al. used three different molar masses to form CS miRNA polyplexes. A sizẽ 200 nm was achieved, and particles reduced activation of microglial cells in rat model showing a very exciting potential for spinal cord injury treatment by having a transfection efficiency that is even higher than viral vectors. Yet, the definite molar mass that gave these results was never mentioned. Indeed, undisclosed details can be noticed sometimes in literature for such a crucial parameter [40].

Degree of Acetylation (DA) of Chitosan/Deacetylation Degree (DD)
Chitosan can be re-acetylated in solution to a required tailored DA allowing the modulation of the density of primary amines on the polymer chains [48]. Upon protonation in weakly acidic media (i.e., at a pH lower than the pKa of chitosan around~6.4), the amine moieties provide the positive charges for complexation with the genetic material. Hence, the less DA chitosan has, the greater the density of positive charges it acquires. According to extensive research with siRNA, a high positive charge density is required to achieve strong enough electrostatic interactions with Chitosan, to obtain stable nanoparticles and to mediate endosomal escape [16]. Therefore, a DA of < 30% is required to complex siRNA and form efficient nanoplexes, have more efficient endosomal escape, and eliminate the need for a cross linker as TPP. The results of Alameh et al. in 2018 showed a significant effect of CS DA on the charge density of siRNA nanocomplexes, with optimal in vitro knock down rates using a 10 kDa CS with a 28% DA, and demonstrated a minor effect of the DP and N/P ratio on knock down efficiency [45].
Accordingly, almost all research on chitosan miRNA nanoplexes used CS with a DA ≤ 25%. However, in 2015 Santos-Carballal et al. had more efficient downregulation of the target gene using a higher DA of~30% and proved that larger complexes were obtained with higher DAs because of extra hydrophobic chains with no positive charge to interact with miRNA [36]. Additionally, miRNA would remain protected from degradation within these chitosan polyplexes due to their enhanced conformational flexibility. Moreover, the release of the RNA, subsequent to cell uptake, was favored due to the weak interactions between the carrier and its payload, an essential factor for the effectiveness of gene therapy, which led them to investigate the binding affinity between CS and miRNA using SPR spectroscopy.

Chemical Modifications to Chitosan Reported in MiRNA Formulations
CS is readily derived by partial deacetylation of chitin which is available from many natural sources (i.e., shrimp, crab, squids, insects, and fungi) [3]. Although the source should not be a factor affecting the physicochemical properties of nanoparticles, no study has been conducted to prove so. As a matter of fact, research teams usually keep using one source they are familiar with, which is acceptable as long as a validated purification process [49,50] is implemented to make sure there are no residual impurities.
The most essential reactions needed upon handling chitosan, unless used as commercialized, are reacetylation and depolymerization. Reacetylation of the primary amine group of CS is needed to tune its charge density, solubility and other physicochemical characteristics eventually affecting NPs behavior. The reaction is usually performed in a hydroalcoholic medium ensuring homogeneous N-acetylation reaction with acetic anhydride as described by Vachoud et al. [48].
Depolymerization is needed to obtain the exact lower molar mass needed to achieve desired NPs physicochemical characteristics. CS can be depolymerized chemically, enzymatically, or even by physical techniques reviewed by Younes et al. and Pandit et al. [51,52]. For the scope of our review, nitrous deamination reaction is the most well-known chemical technique to obtain CS of lower molar mass. Briefly, CS is dissolved in an acetic acid/ammonium acetate buffer of pH 4.5, then sodium nitrite is added and left stirring for a specific duration, as shown in Figure 7, needed to obtain the desired molar mass before quenching the reaction using ammonium hydroxide [36,46,47,49,53]. There are many possible modifications of CS to attain different characteristics (Figure 8), depending on the intended application, as it has three groups available for chemical alteration: a primary amine and two hydroxyl moieties ( Figure 2). Hence, one can investigate grafting macro/small molecules to these hydroxyl groups and/or the quaternization of the primary amine [14]. Moreover, surface functionalization of chitosan NPs became a valid option to investigate active targeting ensuring better uptake and higher transfection efficiency. Ligands such as REDV peptide in the works of Zhou et al. [39,54,55], RGD peptide [56], aptamers [33,57], folic acid [58,59], galactose [24,60], mannose [61], and glutathione [50] have all been investigated within the scope of CS/miR NPs. Most studies were concerning active targeting against cancer cells, however, Pereira et al. investigated the use of lactoferrin and stearic acid to enhance the brain targeting ability of their CS nanoplexes to treat Alzheimer's disease [62].
Many teams worked with quaternization of CS's primary amine, which increases the solubility of CS [39,54,63,64]. They worked on Trimethyl CS (TMC) (Figure 2), except for the Safari team that worked on Triethyl CS (TEC) and N-Diethyl N-methyl (DEMC), and all teams had promising results in vitro. Zhou's PEGylated TMC NPs enhanced endothelization in vivo when loaded in an electrospun bilayer scaffold.
PEGylation (i.e., attachment of polyethylene glycol) is a well-documented modification of drug nano-delivery systems. Sun et al. and Zhou et al. grafted PEG to their CS too to increase solubility and stability and avoid opsonization and subsequent rapid elimination in vivo [39,54,65,66]. PEG was usually used as a linker between CS and alkyl chains or ligands that can increase uptake of NPs by specific receptor mediated endocytosis. Sun et al. gave their CS amphiphilic properties by modifying it into PEGylated dioleoyl grafted carboxymethyl CS (DO-g-CMCS-mPEG), which was amphiphilic enough to self-assemble forming chitosomes (mimicking liposomes formed by amphiphilic lipids) (Figure 9) enabling them to co-load both hydrophobic docetaxel and hydrophilic anti-miR-21 in one NP formulation with an average particle size of 90nm [65].  Reprinted with permission from ref. [65]. Copyright 2020 Drug Deliv. Transl. Res.

Formulation Processes
The two main methods used in the literature for forming CS miRNA NPs are simple complexation and ionic gelation. Simple complexation is based on mixing miRNA with CS at definite N/P and weight ratios, both of which should be optimized with CS characteristics to form intact biologically active NPs [45]. Using this method, Santos-Carballal et al. showed that an N/P ratio of 1.5 was sufficient to achieve transfection efficiencies similar to commercial reagents (Novafect or DharmaFECT) without inducing toxicity ( Figure 6) [36].
Ionic gelation on the other hand, depends on ionic interaction between the anionic charges of miRNA and the cationic charges of CS, in the presence of a crosslinking agent like tripolyphosphate TPP stabilizing and strengthening the interactions between CS and miRNA [16,67]. Katas and his team reported CS-TPP NPs to be better gene delivery vectors compared to CS/siRNA polyplexes for higher binding and loading efficiencies. CS/SiRNA interaction strength was than with simple mixing that shows siRNA and CS easily dissociating from each other. This translated into 82% gene knock down using ionic gelation compared to 51% only with simple complexation, when tested with Chinese Hamster Ovary cells (CHO K1). However, lower transfection efficiency was observed when tested with HEK 293 human kidney cells, with ionic gelation still maintaining higher transfection efficiency than simple complexation [68].
Very similarly to these methods, polyelectrolyte complexation can provide a safe and green process for formulation of miRNA NPs. A green nanomedicine process would be quick, suitably priced, and would ensure the use of only nontoxic reagents [69]. Polyelectrolyte complexes (PECs) are safe vectors spontaneously formed by mixing two water solutions of oppositely charged polyelectrolytes with no need for organic solvents, surfactants or crosslinkers [18]. It is basically the same concept as the two methods above, but it uses polyanionic polymers instead of negatively charged miRNA only in simple mixing or TPP in ionic gelation. Moreover, the formulation process is so straightforward that it only requires the addition of one polyanion solution to another polycation solution or vice versa, forming NPs instantly under low shear rate at room temperature as in "one-shot addition method" investigated by Delair et al. or dripping as formulated by Carvalho et al. [19,20]. Using this method can both increase stability of NPs and convey beneficial characteristics of the polyanion used, as for the use of HA that enhances the biocompatibility of NPs [28,32,34,70] No study was published for systemic analysis of which process is better in terms of encapsulation, release, and transfection efficiency. Hence, the manner and order of adding miRNA, either before or after NPs formulation, remains unaddressed as there are various types, molar masses, and degrees of acetylation of CS and other external factors, such as pH, ionic strength, charge ratios, and even transfection medium used, that would make a consensus on that matter almost impossible. However, Katas et al. proved that, for their system of CS glutamate-TPP, lower gene silencing was observed with systems adsorbing siRNA onto NPs (added after formulation) than systems entrapping siRNA (added to TPP solution before mixing with CS) which was attributed to more exposure to cleavage by nucleases and/or weaker binding to CS when tested with gel retardation assay [68]. Eventually, genetic material can be either (i) adsorbed or surface-associated over the NPs, (ii) complexed with the polycation forming the NP, or (iii) encapsulated inside the NP [71].
Exceptionally, Çelik et al. made quite unique triple polysaccharide nanoparticles composed of CS, HA, and Chondritin Sulfate (ChoS) with TPP again as a crosslinker. Here, miRNA was added dropwise to preformulated NPs dispersion at an N/P ratio of 30. Another exceptional formulation was achieved by Tekie et al. who made glutathione responsive CS PECs composed of chitosan complexed with a miRNA145 conjugated thiolated dextran (miR-TD) for gene delivery. A disulfide bond connected miRNA and TD, enhancing the stability and efficacy of miRNA, and allowing for glutathione responsive cleavage and release in cancer cells. Above all, a targeting aptamer was decorated onto the formed PECs that greatly improved the uptake by cancer cells [33].
No consensus can be made claiming which system is best. The three options of (i) direct CS/miRNA polyplex with simple mixing, (ii) adding TPP for the formulation with ionic gelation, or (iii) adding a polyanion (such as HA or DS) with or replacing TPP completely for totally green polyelectrolyte complexation are all available and all made sound results in their respective in vitro and in vivo tests, when performed. However, CS should be tailored differently in each method. To mix directly with miRNA, we would need lower DA (up to 30% according to Santos). Adding TPP helps when higher DA CS are used and there is not enough charge density to form intact acceptable size NPs. Exchanging TPP with HA or other polyanions adds biocompatibility characteristics to the system and removes the need for a chemical cross linker, hence the choice of the system will depend mainly on the personalized intended use of CS NPs.

Therapeutic Applications of Chitosan MiRNA Delivery Systems
There are two main therapeutic approaches dealing with miRNA therapeutics: either restoring miRNA using a miRNA mimic in diseases in which this miR is downregulated like various cancer phenotypes [72] or inhibiting the function of miRNA through miRNA antagonists (antagomirs) in diseases showing miRNA overexpression [73]. Both strategies are included in Table 3 collectively as the distinction is already clear whether miRNA was used as a therapeutic agent or a therapeutic target. Enhancing osteogenesis of (hBMMSC) human bone marrow mesenchymal stem cell sheets Higher than 90% transfection efficacy after 24 h of treatment and more than 75% on the 14th day. Significant improvement in the in vitro osteogenic differentiation (through increased calcification-related gene expression, improved production of alkaline phosphatase, more secretion of collagen, and mineralized nodule formation.
As shown in the table, CS/miR nanoplexes made by simple complexation have all shown outstanding results tackling cancer with high transfection efficiencies in their respective studies. Ning and his team achieved suppressed tumor growth using their galactosylated CS with liver-specific miR 122 [24].
Besides nanoplexes, obtained by mere mixing of a chitosan solution and a miR solution, more sophisticated formulation processes were used, leading to chitosomes, lipopolyplexes, or nanobombs. Sun and their team achieved improved chemosensitivity of triple negative breast cancer cell line through synergy between anti-miR-21 and docetaxel in their "chitosomes" [65]. Furthermore, some research aimed at getting the best of polymer and lipids simultaneously, coming up with "lipopolyplexes" [96,97]. Recently, a near-infrared laser activated NP system, "Nano-bomb", was developed to overcome the limitations of both materials [86]. Wang and their team developed this three-phase water-in-oilin-water (w/o/w) structure encapsulating miR-34a indocyanine green, and ammonium bicarbonate in the inner hydrophilic phase dispersed in the polymeric hydrophobic phase of PLGA, Pluronic F127, and DPPC. The external hydrophilic phase consisted of HA and chitosan-modified-Pluronic F127. They called it a nano-bomb for the NIR laser photothermal response obtained by indocyanine green leading to the conversion of ammonium bicarbonate into gases allowing the system to escape endosome leading to efficient miR-34a delivery and gene therapy of prostate cancer stem-like cells (CSCs) [86].

Regenerative Medicine
Almost one third of literature on CS/miR NPs is for regenerative medicine applications. Naturally, CS has a great reputation in the regenerative medicine field as it stimulates tissue regeneration, alone or in combinations, and can convey valuable characteristics as antimicrobial and mucoadhesive properties to the systems incorporating it at definite parameters [98]. Moreover, CS has a chemical structural similarity to extracellular matrix (ECM) [99] that can be seen in the extensive research in CS/miRNA NPs for osteogenesis [30][31][32][83][84][85]87], chondrogenesis [34,89], neural tissue [76], and even blood vessels regeneration [39,54,55,64]. Jiang et al. formulated their chitosan antimir-133a/b NPs using the ionic gelation method with TPP, and hyaluronic acid then incorporated these NPs in a chitosan glycerophosphate gel making a sustained release formulation that was cytocompatible and promoted osteogenesis in vivo in a calvarial bone defect mouse model [31]. Li et al. also incorporated their CS/TPP/mir-222 NPs within silk nanofibrous scaffolds aiming at a transplantation therapy for neuronal regeneration. Their system showed good cytocompatibility and improved neural stem cells (NSCs) differentiation [76]. Zhou et al. also loaded their REDV functionalized PEGylated trimethyl chitosan miR-126 NPs into an electrospun bilayer vascular scaffold that accelerated vascular endothelial cells proliferation and could enhance reendothelialization in vivo [54].

Other Fields of Therapy
MiRNAs are involved in all physiological and pathophysiological mechanisms which make the opportunities for therapeutic applications endless [10]. Recently, they have received more attention for controlling inflammation [100]. Shamaeizadeh et al. investigated the use of a formulation of glutathione targeted tragacanthic acid modified CS polyplexes with miR-219a-5p for treatment of multiple sclerosis and they could prove decreased inflammation and brain cells regeneration when injecting their NPs into cuprizone MS mice model [50]. Louw et al. have also shown that their CS miR-124 polyplexes were able to reduce reactive oxygen species and TNF-α in vitro and reduce activation of microglial cells in rats in vivo aiming to treat spinal cord injury. Many more therapeutic applications are applicable depending on the miRNA chosen [40]. Cystic fibrosis [27], hepatic failure [82], and zoonotic echinococcosis [77] were also possible to investigate with CS/miR NPs

Conclusions and Prospects
Just recently in 2020, a huge surge of interest in nanomedicine has been noticed with the three phases of: waiting for a vaccine for COVID-19, the discovery of a vaccine based on nanomedicine and gene delivery, and the last phase of controversy gaining public trust with such a new system. This nanomedicine renaissance should be exploited to go further in therapeutic applications that have been overshadowed by the interest in cancer therapeutics. Fields like vaccinology, cardiovascular, hepatic, and renal diseases could use the benefits of nanomedicine. Among these nanomedicine candidates, CS NPs could provide a cheap, sustainable, stable, naturally available, and safer alternative to viral gene delivery. For it to reach the same efficiency of viral vectors, all it needs is a little push using the most recent, standardized, and consistent findings on making an efficient biologically active CS miRNA NP that is stable enough to protect miRNA from the extracellular environment, reach targeted cells, be uptaken and have weak enough binding to release miRNA in the cytoplasm for efficient gene silencing.
Achieving a systematic comparison between different factors (i.e., molar mass, degree of acetylation, N/P ratio, nanoparticle concentration, polyanions addition, and targeting moiety addition) in the same study is needed to reach a valid consensus. Recent Apliterature in the last 10 years showed more consistency and more promising results having more systemic literature-based modifications. Starting with using a molar mass of 10 kDa at least, a DA ≤ 30%, N/P ratio of 1.5-20, and PEGylation/ligand grafting when necessary is a good starting point for any future experiments. Depending on intended applications, the addition of other components to the formulations (as polyanions) can convey beneficial characteristics to the CS miRNA system. That offers a new perspective of CS and an opportunity for a theoretically old/practically new system to be exploited at its best for many therapeutic applications.