Among non‐viral vectors, chitosan and chitosan derivatives have also gained increasing interest as potential siRNA delivery systems because of their cationic charge, biodegradability and biocompatibility, as well as their mucoadhesive and permeability‐enhancing properties [72].
Chitosan is obtained by deacetylation of chitin, which is the structural element in the exoskeleton of crustaceans (crabs, shrimp, etc.) and cell walls of fungi. It is a biodegradable polysaccharide consisting of repeating D‐glucosamine and N‐acetyl‐ D‐glucosamine units, linked via (1–4) glycosidic bonds (Figure 6) [73].
Figure 6: Chemical structure of chitosan
Every deacetylated subunit of chitosan contains a primary amine group with a pKa value of about 6.5. Therefore, chitosan is soluble in acidic media and insoluble at neutral and alkaline pH values. In addition to pH, the solubility of chitosan is greatly influenced by the degree of deacetylation, molecular weight and ionic strength of the solution. In the physiological environment, chitosan can be digested by lysozymes, which can be produced by the normal flora in the human intestine [74]
or exist in the blood [75]. Therefore, it has been widely used in pharmaceutical research and in industry as a carrier for drug delivery. The potential use of chitosan as a siRNA delivery system is based on its cationic charge. At acidic pH, below the pKa, the primary amines in the chitosan backbone become positively charged. Interaction between the positively charged chitosan backbone and negatively charged DNA/siRNA leads to the spontaneous formation of nano‐size polyelectrolyte complexes (PECs) in aqueous media. At a sufficient nitrogen to phosphate (N:P) charge ratio, chitosan can condense siRNA to sizes compatible with cellular uptake while efficiently preventing nucleases from accessing the enclosed nucleic acid drugs by steric protection [76].
Several factors are to be considered when formulating chitosan for siRNA delivery [72]. Chitosan Mw has been shown to influence the physicochemical properties (size, zeta potential, morphology and complex stability) of the formed chitosan‐siRNA complex. Higher Mw chitosan was reported to offer higher degree of protection, however, dissociation of the complex at the target site is delayed, and vice versa. Thus, an appropriate balance needs to be achieved between protection and release for siRNA biological functionality with chitosan of the desired Mw. Another critical factor is the degree of deacetylation (DD), i.e. the percentage of deacetylated primary amine groups along the molecular chain, which subsequently determines the positive charge density when chitosan is dissolved at acidic conditions. A high DD (over 80%) has been identified as an important factor for increased binding capacity and efficient siRNA‐mediated knockdown. In the same context, it is important to mention that an optimized N/P ratio is of crucial importance (N/P ratio refers to the ratio of amine groups in chitosan to the phosphate groups in RNA backbone). It is now established that, as with all polycation‐based gene delivery systems, the most efficient N/P ratios for chitosan gene expression usually involve N/P ratios of at least 3–5 and sometimes of up to 30 [77, 78]. Also, the concentration of the polyion solution may affect PECs properties. A sufficiently diluted solution is required to ensure reproducibility of the coacervation process. Moreover, properties of the chitosan–siRNA nanoparticles are also influenced by chitosan salt form. Chitosan glutamate, which had a higher Mw than chitosan hydrochloride, produced smaller siRNA complexes with higher
loading capacity and a much better gene silencing effect in CHO K1 cells at 24 h post‐transfection compared to chitosan hydrochloride. Furthermore, based on a recent study [79] using atomic force microscopy (AFM), the interaction strengths between siRNA and chitosan were pH‐dependent and the adhesive interactions decreased as the pH was increased from 4.1 to 9.5. These findings were related to the decrease in charge density as well as solubility upon increasing pH, both of which leading to aggregation and poor stability of chitosan‐based formulations at physiological pH [77].
However, chitosan has some disadvantages that scientists had to tackle to improve its suitability for delivery of siRNA, in particular, poor stability in biological fluid, nonspecific cellular uptake and the low transfection ability of chitosan [72]. These improvements took place mainly in the form of chemical modifications. Selected examples are shown in Table 1. It is important to mention that we are here listing strategies aiming to improve intracellular uptake of chitosan complexes although some of these were not applied for siRNA delivery. Specific examples of the use of chitosan for delivery of siRNA will follow afterwards.
Being positively charged, chitosan complexes are subjected to electrostatic neutralization reaction with negatively charged serum albumins and other proteins leading to aggregation and fast clearance. To minimize these non‐specific interactions, hydrophilic polymers, such as PEG, can be conjugated to chitosan. The presence of PEG on the surface of polyplexes also provides stabilization by reducing inter‐particular aggregation [80]. In vivo studies showed that, compared with standard chitosan, transgene expression in the liver of rats transfected with PEGylated chitosan can be significantly increased one day after bile duct infusion and 3 days after portal vein infusion [81], which is hypothesized to be due to reduction of opsonization of the chitosan polyplex or prolongation of the blood circulation time of the complex after PEGylation. This hypothesis is supported by a recent study showing that PEGylation of chitosan can not only avoid clearance of the intracaudal‐venously infused polyplex by Kupffer cells in mice, but also increase the amount of the transfecting agent reaching the liver and tumor sites [82]. In addition, chitosan lacks cell specificity and thus cellular uptake of chitosan‐based nanoparticles mostly occurs via non‐specific adsorptive endocytosis, depending on the surface properties of the nanoparticles [83]. To improve cellular uptake efficiency and specificity toward the target cells, chitosan carriers are often modified by conjugating a cell‐specific ligand, which specifically recognizes and binds to membrane‐bound proteins of target cells. The specific ligand–receptor interaction leads to cellular uptake of the chitosan/siRNA polyplexes via receptor‐mediated endocytosis. To increase the cell specificity,
several ligands such as transferrin‐ [84, 85], folate‐ [86], mannose‐ [87] and galactose‐ [88]
conjugated chitosans have been designed and evaluated for receptor‐mediated endocytotic gene delivery, as summarized in Table 1. Moreover, chitosans were also modified to increase proton sponge capacity using arginine [89], histidine [90], urocanic acid [91], imidazole [92] or PEI [93] to facilitate endosomal escape. Furthermore, the sensitivity of the chitosan complex to pH change influences the stability of the complex and therefore the transfection efficiency. To improve the poor water solubility of chitosan at physiological pH and its low transfection efficiency, several chitosan derivatives have been synthesized in the last few years for different purposes. In a recent review, chitosan structure modification was described in detail [94]. To address the solubility issue, i.e. decrease of the sensitivity of the chitosan complexes to pH and enhance gene transfection, a series of hydrophilic chitosan derivatives were synthesized. Application of modified chitosan, such as trimethyl (quaternized) chitosan [95], PEGylated chitosan [81], PEGylated trimethyl chitosan [96]
and low Mw soluble chitosan [97], could potentially circumvent the known solubility issues.
Moreover, the addition of hydrophobic units to chitosan vectors are expected to increase transfection efficiency by modulating complex interactions with cells, such as adsorption on cell surfaces and cell uptake [98]. Hydrophobic units may also assist dissociation of siRNA from the complexes, to facilitate release of cargo that would otherwise be strongly bound through ionic interactions between cationic units and the phosphates of RNA. For these purposes, a series of hydrophobically modified chitosans, such as deoxycholic acid chitosan [99], N‐alkylated chitosan [100], stearic acid–chitosan [101], urocanic acid‐modified chitosan [91], and thiolate chitosan [102]
were studied, as listed in Table 1.
Table 1: Representative examples of chitosan derivatives “Adapted from reference [72]. Copyright 2010, with permission of Elsevier.”
Types of modification
Derivatives studied Underlying principles of transfection enhancement Ref
Hydrophilic modification
PEG–chitosan Improve stability, reduce the opsonization, elongate the plasma circulation time
[81]
Quaternized chitosan (TMC)
Make the chitosan soluble over a wide pH range, increased transfection efficiency
[95]
PEG‐g‐TMC Improve stability, reduce particle sizes of complexes [96]
Glycol chitosan Facilitate endocytic uptake of the complex and enhancing transfection efficiencies in vitro as well as in vivo
[109]
6‐Amino‐6‐deoxy‐chitosan Enhance water solubility in at neutral pH, showed transfection efficiency superior to chitosan
[110]
Arginine–chitosan Improve its water solubility and enhance its gene transfection efficiency
[111]
PEI–chitosan Enhance transfection efficiency with low cytotoxicity owing to induction of proton sponge effect by PEI.
[112]
Chitosan‐g‐L‐phenylalanine A higher degree of swelling and faster degradability [113]
Hydrophobic modification
Deoxycholic acid‐modified chitosan
Increase of cell membrane–carrier interactions and/or destabilization of cell membrane
[114]
Alkylated chitosan Easier unpacking of DNA from carriers, enhance perturbation of the model membrane system and increase the entry into cells
[100]
5b‐cholanic acid modified glycol chitosan (CGC)
Decrease particle size and improved stability against serum facilitate endocytic uptake
[109]
Stearic acid–chitosan Increase endosomal escape [101]
Thiolate chitosan Possess enhanced mucoadhesiveness and cell penetration properties
Enhance endosomal rupture through a proton sponge mechanism.
[91]
Hybrid chitosan/CD Promote cellular uptake, and decrease the cytotoxicity of the system
Efficiently transfer nucleic acid through a receptor‐mediated endocytosis mechanism
[84]
Folate–chitosan A ligand for targeting cell membrane and allows nanoparticles endocytosis via the folate receptor (FR)
[86]
Galactosylated chitosan Induce the receptor‐mediated endocytosis to bind asialoglycoproteins (ASGP) for hepatocyte‐targeting
[88]
Mannosylated chitosan Induce the receptor‐mediated endocytosis for targeting into antigen presenting cells (APCs).
[95]
Gal‐PEG‐CHI‐g‐PEI Improve hepatocyte specificity and transfection efficiency [118]
Galactosylated chitosan‐ KNOB protein conjugated
chitosan
Many groups have taken these approaches a step further by testing some of them in vivo. He et al.
[103] developed trimethyl chitosan‐cysteine (TMC‐Cyst) NPs for in vivo delivery of tumor necrosis factor α (TNF‐α) siRNA via oral gavage and intraperitoneal injection. TMC conjugates of different MWs were synthesized and siRNA loaded TMC‐Cyst NPs were prepared through ionic gelation with sodium tripolyphosphate (TPP). NPs had particle sizes below 160 nm and ζ potentials of around 30 mV. The siRNA encapsulation efficiencies were up to 84.7%. The presence of cysteine granted the delivery system a glutathione (GSH)‐responsive release profile, meaning that high release rates are only encountered intracellularly where GSH concentrations are elevated (10 mM), compared to lower concentrations in the extracellular compartments (4.5 µM). Lipopolysaccharide/D‐GalN‐
induced acute hepatic injury characterized by the overexpression of TNF‐α was adopted in a mouse model to evaluate the RNAi efficiency mediated by siRNA TMC‐Cyst NPs via single oral gavage or intraperitoneal injection. Orally delivered TMC‐Cyst NPs at a siRNA dose of 200 μg/kg significantly provided higher TNF‐α knockdown compared to intraperitoneal injection of the same dose (knockdown was up to 74% and 39%, respectively). Neither the naked TNF‐α siRNA nor TMC‐Cyst NPs containing scrambled siRNA evoked any RNAi effect. Another study by the same group [104]
utilized mannose‐modified trimethyl chitosan‐cysteine (MTC) conjugate nanoparticles to deliver TNF‐α siRNA orally. MTC NPs containing low amounts of TNF‐α siRNA (3.75 nM/kg) inhibited TNF‐α production in macrophages in vivo, which protected mice with acute hepatic injury from inflammation‐induced liver damage and lethality. Zhang et al. [105] investigated the in vivo biodistribution after oral administration of nanoparticles prepared by ionic gelation of chitosan, N‐
trimethyl chitosan (TMC), or thiolated trimethyl chitosan (TTMC) with TPP or hyaluronic acid (HA), encapsulating siRNA. TAMRA‐fluorescently labelled negative control siRNA (TAMRA‐NC siRNA) was used for in vivo siRNA quantification. Nanoparticles remarkably enhanced the intestinal absorption of siRNA as compared to the naked TAMRA‐NC siRNA solution, which was evidenced by 8 to 26‐fold increase in the TAMRA‐NC siRNA plasma concentration . Notably, the intestinal concentration of the TAMRA‐NC siRNA encapsulated into these nanoparticles decreased with time, while the distribution of TAMRA‐NC siRNA was increased in plasma, liver, and spleen within 6 h post oral administration. Moreover, TTMC/siRNA/TPP nanoparticles, rather than other nanoparticles examined, showed the highest distribution percentages in most organs and plasma in mice. This might be attributed to better stability and enhanced intestinal permeation results shown by these particles in earlier experiments.
Luo et al. [106] designed an inhalable delivery system for local targeted delivery of siRNA into the lungs. Chitosan was functionalized with a guanidinium group, reported to enhance cell transfection.
In addition, salbutamol, a β2‐adrenergic receptor agonist, was chemically coupled to the guanidinylated chitosan (GCS) to improve pulmonary targeting specificity of the siRNA carrier, and thereby remarkably enhanced the efficacy of gene silencing in the lung. Test formulations were sprayed directly into the lungs of mice via the endotracheal route using a MicroSprayer aerolizer.
Transgenic mice expressing green fluorescence protein (GFP) were used in these studies. Results observed by confocal laser scanning microscopy showed that a significant inhibition of GFP expression in the bronchial epithelial cells was evident in mice treated with Sul‐SGCS/GFP‐siRNA compared to GCS/GFP‐siRNA, and both showed a higher inhibition effect compared to the control mice or mice treated with CS/GFP‐siRNA complex.
Strategies combining siRNA and small molecule delivery have been proposed in an attempt to synergize individual effects. Wei et al. [107] designed a “two‐in‐one” nano‐complex to deliver mTERT siRNA and paclitaxel (PTX) by chitosan‐based nanoparticles. N‐((2‐hydroxy‐3‐
trimethylammonium) propyl) chitosan chloride (HTCC), a partially quaternized derivative of chitosan, has been used as a potential material for developing an oral delivery system because of its mucoadhesive properties and permeation‐enhancing effects. The tumor suppression effect was studied following oral administration of these nano‐complexes to xenograft mice models grafted with Lewis lung carcinoma (LLC) cells. Interestingly, NPs containing both siRNA and PTX showed significantly higher suppression effect on tumor growth compared to NPs delivering siRNA and PTX separately, at the same dose level. To explore possible reasons for the effects observed for separate NPs (siRNA/PTX) and the combinatorial delivery system NPs (siRNA)+NPs(PTX), the distribution of siRNA and PTX in tumor tissue cells was analyzed by flow cytometry and confocal microscopy. Results indicated that little siRNA could co‐localize with PTX in the combinational treatment group, suggesting that these two payloads were just separately internalized into different tumor cells, which would inevitably disperse their firepower against the target. On the contrary, siRNA could well co‐localize with PTX in the NPs (siRNA/PTX) group, demonstrating that this formulation could simultaneously deliver siRNA and PTX into the same tumor cells and thus concentrate their firepower.