Chitosan‐based self‐assembled nanomaterials: Their application in drug delivery

Nanoparticles have long been one of the most popular delivery systems for enhancing the stability of therapeutic agents, providing targeted drug transportation, sustaining drug concentration at sites of action, and promoting therapeutic efficacy. Chitosan‐based nanoparticles have been widely explored as drug delivery systems due to the unique biological properties, such as easy to chemical modifications, biocompatibility, mucoadhesive feature, and absorption enhancement. In this review, we outline the biological properties and stimuli‐sensitive strategies of chitosan‐based self‐assembled nanoparticles as well as the recent developments in various applications in drug delivery, including cancer treatment, oral administration, skin regeneration, nasal/ocular mucosal administration, and pulmonary drug delivery. Chitosan‐based nanoparticles exhibit excellent features as drug delivery systems due to the outstanding biological properties. As the suitable vehicles for drug administration, chitosan‐based nanoparticles facilitate and improve drug bioavailability in all cases.

due to the presence of functional amino and hydroxyl groups, which could broaden their application in physiological conditions. 4 Nowadays, plenty of nanocarriers prepared by chitosan or modified chitosan derivatives have been reported for delivery of various macromolecules or drugs. It is investigated that some chitosan-based nanocarriers possess outstanding physical and biological properties such as favorable hemocompatibility, lower cytotoxicity, enhanced antibacterial activity, and controlled/targeted drug release dynamics that is induced by specific external stimuli (eg, temperature, pH, enzyme, redox, etc). 5 With the favorable biological properties and

S C H E M E 1 Diagram of chitosan for utilizations in multiple domains
well-histocompatibility to tissue or organ, chitosan-based nanocarriers show tremendous potential in various fields such as cancer therapy, wound dressing preparation, and so on. This review aims at summarizing the preparation process, biological properties, and stimuli-responsive strategies of chitosan and chitosan-based nanomaterials, as well as illustrating their applications in different domains, especially in drug delivery field (Scheme 1).

Chemical modifications of chitosan
Chitosan, a natural polysaccharide, has an enormous potential for biomedical field. The presence of amino group at C 2 position and hydroxyl group at C 6 position of chitosan offers the opportunity of functionality using esterification reaction, etherification reaction, and amide reaction toward diverse biotechnological needs, especially in drug delivery system applications.

Hydrophilic modification
The biological applications of chitosan are limited due to its water-insoluble nature. To increase water solubility of chitosan, Amidi et al developed a N,N,N-trimethyl chitosan (TMC) that was prepared by N-methylation of N,N-dimethylchitosan, resulting in high degrees of quaternization. 11 Carboxymethyl chitosan, another watersoluble chitosan derivative, was successfully synthesized by the reaction between -OH or -NH 2 of chitosan and monochloroacetic acid under alkaline condition. 12 Moreover, hydroxybutyl chitosan was obtained by grafting of hydroxybutyl groups to the -OH at C 6 position and -NH 2 at C 2 position in the chitosan molecule to improve its hydrophobicity, leading to thermoresponsive behaviors. 13 These provide advantages of optimal hydrophilicity for chitosan and show promise in drug delivery applications.

Hydrophobic modification
Many studies on the chitosan derivatives focus on the hydrophobic modification, usually grafting a hydrophobic group (such as alkyl group, cholesterol) in the backbone of chitosan and reacting with fatty acid and enoic acid through amide reaction. Xing et al synthesized hydrophobically modified oleoyl-chitosan by conjugation of oleoyl and the -NH 2 at C 2 position, which formed nanoparticles and exhibited antibacterial activity. 14 Deoxycholic acid-modified chitosan was prepared by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction. 15 Li et al synthesized cholesterolmodified chitosan by covalently coupling cholesterol 3hemisuccinate to -NH 2 of the chitosan in the presence of EDC. 16 Hydrophobic modified chitosan can be used in the self-assembled nanoparticles, that is, nanoparticles encapsulated with hydrophobic drug in hydrophobic core.

Amphiphilic modification
By connecting a hydrophilic group (eg, N,N,N-trimethyl group, carboxymethyl group, and hydroxybutyl group) and a hydrophobic group (eg, cholesterol and deoxycholic acid) to the chitosan skeleton, many amphiphilic modified chitosans have been reported. For example, Sun et al made hydrophilic deoxycholate and hydrophobic cetirizine-grafted hydroxybutyl-chitosan as anti-allergic drug delivery vehicles. 17 These amphiphilic molecules can be widely used as carriers for small molecule drugs and macromolecular DNA and proteins by self-assembly to form valuable nanoparticles. These nanocarriers show a relatively simple way of the formation and excellent biocompatibility.

Methods of preparation of self-assembled nanoparticles
The obtained self-assembled nanoparticles by different preparation methods are different in particle size distribution and stability. The selection of the preparation method depends on many factors such as the swelling property and solubility of the chitosan derivative.

Ultrasonic method
The self-assembled nanoparticles can be prepared by ultrasonic method. Specifically, hydrophilic long chains of the chitosan derivative were broke through ultrasonic energy in aqueous solution; hydrophobic groups selfaggregated to form nanoparticles, which were mediated by intermolecular and intramolecular hydrophobic interaction. For instance, Jin et al prepared deoxycholic acidmodified carboxymethyl chitosan (DCMC) nanoparticles via probe-type sonication based on DCMC aqueous solution and illustrated the capacity of DCMC nanoparticles as hydrophobic drug DOX vector for cancer therapy. 18

Dialysis method
This method utilizes the replacement of solvent to prompt the hydrophobic group departure from aqueous media, and thus assembling into nanoparticles. For hydrophobically modified glycol chitosan (HGC), Son et al synthesized HGC nanoparticles encapsulating mono-lithocholic acid-conjugated exendin-4 at the Lys 27 residue (LAM1-Ex4) using a dialysis method, in which HGC (in DMSO) solutions and LAM1-Ex4 (in deionized water) were mixed and dialyzed using dialysis membrane against deionized water for 24 h at 4 • C. 19 HGC nanoparticles showed potential as a long-time sustained release drug delivery system.

Emulsification method
Emulsification method is frequently employed to prepare self-assembled nanoparticles. This method uses the drug solution and polymer solution to form an emulsion, and then disperses the emulsion in water or volatile solvent. After removing solvent, nanoparticles were formed. Li et al reported a magnetic resonance imaging probe that was synthesized by oleoyl-chitosans nanoparticle loaded with oleic acid-decorated iron oxide nanoparticles. In detail, the methylene chloride and O-chitosan acetic acid were mixed homogeneously, and then methylene chloride was removed under vacuum conditions and sodium tripolyphosphate was added as a cross-linking reagent. 20

Drug release
Chitosan nanoparticles have been utilized to deliver drugs, due to the ability of efficient payload and controlled release of drugs. Drug loading in chitosan nanoparticles can be done by two methods: incorporation (during the preparation of particles) and incubation (after the formation of particles). By incorporating the drug during the formation of nanoparticles, high drug loading can be achieved, and it may be affected by some factors such as the method of preparation, physicochemical properties of the drug, the presence of the additives, and so on. Drug release from chitosan nanoparticles depends upon the particle morphology, size, the extent of cross-linking, physicochemical properties of the drug, as well as the pH, polarity of the dissolution matrix, and the presence of enzymes. Three different mechanisms were involved in the release of drugs from chitosan nanoparticles: (a) release from the surfaces of the nanoparticles, in which drugs adsorbed or entrapped in the surface layer of particles instantaneously dissolve when incubated with the release matrix, resulting in a burst releasing effect; (b) diffusion through the swollen rubbery matrix, which involves three steps: first, water penetrates into the nanoparticles, which leads to the swelling of the carriers, the glassy particles transform into rubbery particles, and drugs diffuse from the swollen rubbery nanoparticles; and (c) release caused by the polymer erosion, the breakage, hydrolysis, or degradation of the nanoparticles backbone, leading to a long-time drug releasing. 21 Chitosan is degraded mainly by two pathways, chemical process and enzyme catalysis, and the latter is the major process in vivo. 21 In general, drug release follows more than one type of the mechanism. Rampino et al stated that the chitosan nanoparticles prepared by ionotropic gelation showed efficient protein loading efficiency of albumin from bovine serum (60% ± 6%), albumin from chicken egg white (76% ± 4%), and Zinc-free human insulin (55% ± 8%). 22 In addition, the chitosan nanoparticles exhibited loading capacity of hydrophobic cetirizine up to 14.82% ± 0.17% and showed controlled release of drug up to 80% in PBS (lysozyme, pH 7.4, 37 • C) for 72 h. 23

Hemocompatibility
One of the important evaluations for the application of medical biomaterials is that the materials should possess favorable hemocompatibility. The hemolysis test is commonly used to evaluate the hemocompatibility of the nanoparticles. 24 According to the standard of ASTM-F/756-08 (2000): hemolysis rate (HR) below 5% is considered to be nonhemolytic or slightly hemolytic ( Figure 1). 24 Chitosan-based nanoparticles have been proven to have good blood compatibility. Chitosan-grafted polylactic acid (CS-g-PLA) nanoparticles were proved to be nonhemolytic (HR% is 1.6-2.7%). 25 In addition, rejection reaction may occur when blood contacts with a foreign object because of the adsorption of foreign body by plasma proteins. Thus, plasma protein adsorption assay is necessary for investigating the hemocompatibility. For example, deoxycholatechitosan-hydroxybutyl (DAHBCs) nanoparticles exhibited favorable blood compatibility (HR% is 0.8-3.6%) and little serum protein adsorption (2.1-4.8%). 24

Cytotoxicity
Chitosan nanoparticles have been investigated as carriers for drugs delivery owing to its nontoxicity. 26 More than 80% relatively cell growth rate is considered to have ideal cell compatibility (ISO 10993-5:1999, IDT). The chitosan nanoparticles showed nontoxicity to L929 cells proliferation over a range of concentrations (62.5-1000 μg/mL) using MTT assay. 17 Liu et al investigated the toxicity of oleoyl-carboxymethyl-chitosan (OCMCS)/hyaluronic acid (HA) nanoparticles to Caco-2 cells and specified a safe range for the application of the nanoparticles. 27 The potential cytotoxicity of chitosan-based nanoparticles may be caused by the interactions of cationic particles with negatively charged cell membranes, and/or by cellular uptake and subsequent activation of intracellular signal transduction pathways. In general, the cytotoxicity of the nanoparticles increases with the increasing number of cationic groups (or charges). Thus, in vitro studies of the cytotoxicity on different cell lines and in-depth in vivo studies of OCMCS/HA nanoparticles are required to determine a safe range of concentration for clinical application.

Antibacterial activity
Chitosan nanoparticles have attracted considerable interest due to its unique wide-spectrum antibacterial activity. Different theories have been proposed to illuminate the antibacterial mechanism of chitosan. The most widely accepted mechanism involves the electrostatic interaction of the positively charged chitosan nanoparticles and the negatively charged components present on the bacterial surface ( Figure 2). 28 In addition, the quaternary

STIMULI-RESPONSIVE CHITOSAN NANOPARTICLES
The stimuli-responsive nanoparticles can undergo relatively violent transformations of physicochemical properties responding to minor changes of external environment factors. 32 The chain conformation of nanoparticles changes as they recognize and respond to specific external stimuli (including chemical stimuli and physical stimuli) as a signal. 33 Chemical stimuli, such as pH, ions, enzyme, chemical reagents, and so on, lead to altering of the interaction between the chains of the nanoparticles and solvents at a molecular level. Physical stimuli include temperature, mechanical stress, electric and magnetic field, and so on, which affect the various energy levels inside and outside the nanoparticles, resulting in the changes of intermolecular interaction at critical starting points. Stimuli-sensitive chitosan nanoparticles are widely utilized for triggered release of payloads as a consequence of exposure to stimuli and to obtain enhanced drug bioavailability.

Temperature
Temperature-responsive chitosan-based nanoparticles have gained much attention owing to their ability to sense and respond to the thermostimuli of specific body parts (eg, skin, gastrointestinal tract, and nasal cavity). The thermosensitive nanoparticles in aqueous solution undergo a transition from a molecularly dissolved state at low temperature to an insoluble state at temperature exceeding lower critical solution temperature (LCST). To understand the transformation of nanoparticles in aqueous solution, H-bonds between the nanoparticles and water molecules are significant. When the temperature is below the LCST, the nanoparticles' suspensions remain transparent. When temperature is above the LCST, the nanoparticles begin to aggregate and isolate from the solution because of the weakening intermolecular hydrogen bonding interactions between the nanoparticles and water molecules. Wang et al and Yang et al prepared a kind of thermosensitive DAHBCs nanoparticles by modification of chitosan with hydrophobic deoxycholate moieties and hydrophilic hydroxybutyl groups. The DAHBCs nanoparticles showed thermoresponsive changes of morphology and encapsulated drug releasing and might provide great potential as thermoresponsive nanovehicle for drugs delivery. 24,34

Enzyme
Chitosan, a natural polysaccharide, is composed of glucosamine and N-acetyl glucosamine units linked by β-(1-4) glycosidic bonds, which could be biodegraded by several enzymes, such as chitinases, chitosanases, and lysozyme. 35 Chitosan can be digested mainly by lysozyme in vivo, which exists in various human body fluids and tissues. 36 The rate of lysozyme degradation increased with decreasing molecular weight (MW) and degree of deacetylation (DD). 37 In the interaction between enzyme and polysaccharidic substrate, enzyme required plenty of binding subsites on glycosyl units in the substrate chain for reaction to produce oligosaccharides; chitosan with shorter chain could be hydrolyzed to oligosaccharides more easily. 38 Lysozyme contained hexameric binding sites, and hexasaccharide sequences containing more than three acetylated residues contributed mainly to the chain degradation of chitosan 39 ; lysozyme could not act on glucosamine segments and/or the segments with relatively small fractions of acetyl-glucosamine residues. 40 The lack of consecutive acetylated residues was responsible for the decreasing degradation rates of chitosan with increasing DD. Moreover, Nordtveit et al determined the effect of pH value (3-7) and ionic strength (0.1-1.1) on the rate of lysozyme degradation. It was observed that the degradation rate reached a broad optimum at pH 4 and ionic strength of 0.155 M. 41 Grafting of chitosan nanoparticles allows drugs to bind onto the chitosan backbone via covalent interaction and to form functional derivatives nanoparticles, which provide the potential for drug delivery and drug release by enzyme degradation. In the presence of lysozyme, the glycosidic bonds of the chitosan nanoparticles can be digested thus releasing fragments containing therapeutic agent into the matrix. The degradation of chitosan and its acetylated derivatives nanoparticles (NPs) meant that they were completely degraded into aminoglucose unit or into chitosan oligosaccharides with variable length. Chitosan and its acetylated derivatives NPs could be degradation to some extent both in vitro and in vivo. In vitro hydrolysis of chitosan (DD of 85%) NPs cross-linked with sodium tripolyphosphate in lysozyme solution (1 mg/mL, lysozyme concentration in human tears fluids) was investigated, and the size of the NPs was slightly reduced after incubating at 3 • C for 4 h due to a partial hydrolysis of chitosan molecules. 42 Mi et al had investigated the in vivo degradation of chitosan (MW of 70 KDa, DD of 85%) particles cross-linked with glutaraldehyde by injecting into the skeletal muscle of rats. The results indicated that the particles retrieved at 12 weeks postoperatively were already degraded into a loose and porous structure, and at 20 weeks postoperatively, the particles were severely degraded into fragments. 43 Yu et al provided evidence supporting this hypothesis by preparing functional amphiphilic cetirizinechitosan nanoparticles that showed two-step drug releasing property. The hydrolysis of glycosidic bonds leads to the cleavage of cetirizine molecules from saccharides fragments, thus prolonging release time and increasing cumulative drug release. 23 In a word, chitosan nanoparticles grafted with proper therapeutic agents as multistep nanodrug delivery carriers not only enhance the therapeutic effect but also extend the administration time, which was beneficial for the treatment of diseases.

pH
Chitosan nanoparticles delivery system with pHresponsive properties offers great promise to administer drugs, because the pKa value of the amino groups on chitosan is approximately 6.5. Hence, the amino groups on chitosan nanoparticles could act as tunable hydrophilic groups at low pH values. 44 Acidic condition in the gastric area has been characterized as a primary limiting factor for oral drug delivery system. 45 Chitosans are hydrophilic when they are protonated at acidic pH, whereas they are hydrophobic when they are deprotonated at neutral pH, which leads to a reversible hydrophobicityhydrophilicity transition resulting in the changes of aggregation states. Feng et al introduced a polyelectrolyte complex (chitosan/O-carboxymethyl chitosan-nanogels) as a pH-responsive carrier for oral drug delivery. Positively charged chitosan was able to form polyelectrolyte complex with negatively charged O-carboxymethyl chitosan (pKa = 2.0-4.0) via electrostatic interaction to overcome the water-insoluble limitation of chitosan. In gastrointestinal tract, the pH-responsive polyelectrolyte complex became unstable and disintegrated, resulting in a fairly fast release of drugs. 46 Thus, based on the ability of reducing the toxicities of the agents and controlled release of embedded drugs for chitosan, chitosan nanoparticles or chitosan derivatives nanocomplex with pH-sensitive properties represent promising approach as nanodrug delivery system for special sites.

Redox
The special microenvironment with high reductive glutathione in cytoplasm, such as of tumor sites, provides the possibility to develop redox-responsive nanoparticles for enhanced therapeutic efficiency. 47 Meanwhile, efficient intracellular delivery is needed for targeted delivery drugs to intracellular areas because the sites of chemotherapeutic agent action are located in the subcellular compartments. 48 Chitosan-based nanoparticles have turned out to be promising delivery systems for targeted delivery of drugs to intracellular areas to reduce the systemic toxicity and improve the treatment efficacy. The design of redox-responsive nanoparticles sparked an interest in attaining efficient intracellular delivery. Kinds of redox-sensitive nanoparticles based on thiol beard molecules have been designed, which could realize triggered drug release in cytoplasm or subcellular compartments by rapid cleavage of disulfide bonds under reductive potential. 49 Novel redox-sensitive intelligent nanoparticles (CMCS-TCS NPs) based on carboxymethyl-chitosan and thioglycolic acid-conjugated chitosan were established to provide precise spatiotemporal control for efficient intracellular delivery. Nanoparticles were prepared by covalently cross-linking carboxymethyl-chitosan and thioglycolic acid-conjugated chitosan via oxidation between thiol groups, which could achieve redox-sensitive disintegration in cytoplasm or subcellular compartments and release the payloads. 50

Cancer treatment
Anticancer drugs are available in plenty to treat cancer, and conventional agents have the disadvantages of drug toxicity and resistance induced by chemotherapy; therefore, cancer therapy thus remains a challenge in recent years. 51 Contrasting to small molecules therapeutic agents, nanotechnology in tumor therapy exhibits obvious superiorities, such as prolonged blood circulation time, increased accumulation at tumor sites via enhanced permeability and retention effect, and reduced systemic toxicity. 52 Chitosan nanoparticles have excellent properties of longer blood circulation time and led to increasing application as antitumor drugs or adjuvant drugs delivery systems. The therapeutic effect of anticancer agents depends on the dosages, thus demand-based targeted delivery to specific site is crucial for the transformation from experimental evaluation to clinical application ( Figure 3). With property of nonspecific electrostatic interaction with cell membranes and no specific receptors, Zhang et al developed DOX-loaded oleoyl-chitosan (OCH) self-assembled nanoparticles for cancer therapy. The OCH nanoparticles exhibited favorable anticancer potential, whereas insolubility in aqueous solution and low targeting to tumor sites limited their therapeutic effect. 53,54 To improve the antitumor effect, water-soluble carboxymethyl chitosan nanoparticles (CMCS-NPs) were exploited, with hydrophobic 5-fluorouracil (5-Fu) loaded by emulsion crosslinking. And the sustained drug release profiles at tumor sites were investigated. 55 Plenty of tumor-targeting nanodrug delivery systems have also been prepared to avoid drugs disintegration, decrease the toxicity, and enhance permeability and accumulation in tumor microenvironment. 56 To enhance the targeting ability of delivery system, chitosan nanoparticles with glucose conjugated were prepared based on the overexpression of glucose transporters (Gluts) by tumor cells. The anticancer agent DOX was embedded into glucoseconjugated chitosan nanoparticles (GCPs) and showed four-to fivefold killing activity of 4T1 cells than that of free DOX. Additionally, multidrug resistance (MDR) as hindering factor has gained great attention in the treatment of cancer chemotherapy. The phenomenon of MDR is due to the overexpression of ATP binding cassette (ABC) transporters, particularly P-glycoprotein (P-gp), which

F I G U R E 3 Confocal images of Hela cells incubated with TNGs (A) or TCNGs (B) at concentration of 250 μg/mL for 1 or 4 h, respectively.
Bar represents 10 μm. Neclues (Blue), lysosomes (Red), and TCS (green). Reprinted with permission. 60 Copyright 2017, Elsevier could lead to the outflow of drugs and consequently drug insensitivity. 58 The combination of nanocarriers and drugs exhibited potential for ABC transporter-mediated MDR treatment. Chitosan (CS) DOX and quercetin (QT; a P-gp inhibitor that could tackle transmembrane efflux of drugs) were used to constitute the QT-CS-DOX micelles. The micelles showed effectively increases of intracellular DOX concentration and inhibition of P-gp in drug-resistant Caco-2 cells. 59

Oral management
Oral route is a convenient drug delivery way, with main merit beings of needle-free systemic delivery, greatest safety, high patient compliance, and minimized side effects. 61 The main challenge of oral route is the poor bioavailability due to the peculiar physicochemical properties such as strong acid conditions of gastric and enzymatic intestinal and physiological barriers. 62 Nanoparticle-based oral drug delivery carriers pose potential advantages in conquering these shortcomings; the nanocarriers could avert the gastrointestinal degradation, promote drug absorption, and facilitate intestinal mucosa permeability. Effective drug absorption by intestinal epithelial cells via oral administration relies on transcellular or paracellular pathway. 63 Paracellular transport has mainly been the focus of the researchers, whereas drug absorbed by transcellular pathway is not easy to escape from the cells. The primary barrier of intestinal epithelial cells, also referred as tight junctions (TJs), could be disintegrated via degradation of TJ proteins. Chitosan-based nanocarriers with drug loaded have exhibited the potential to open the TJs on intestinal epithelial cells, thus promoting the paracellular transport of drugs. 64 Chitosan/carboxymethyl-chitosan (CS/CMCS) nanoparticles have been reported to have favorable safety and therapeutic effect in colorectal cancer therapy. The nanoparticles could improve oral bioavailability of DOX and enhance DOX absorption of small intestine via promotion by paracellular permeation. Amino groups of chitosan could be protonated in duodenum (pH < 7) and the TJs of intestinal epithelial cells were opened, leading to a promoted paracellular transport of DOX. Meanwhile, the carboxylic groups of CMCS supported Ca 2+ transferring adherens junctions (AJs) to CMCS and showed further promotion for the opening of TJs. 65 Liposomes coated with CMCS and quaternary ammonium chitosan (CMCS/TMC-LPs) were utilized to enhance the oral bioavailability of curcumin (CUR) and prolong the retention time of CUR in systemic circulation. 66,67 CMCS/TMC-LPs administrated via oral route could reach small intestine and increase paracellular transport of CUR markedly, which was attributed to the opening of TJs by -N + (CH 3 ) 3 and the chelating of Ca 2+ from AJs to -COO-. Insulin, a generally used agent for diabetes therapy, is limited by the inconvenience of repeated injections and complication. Oral route for delivery of insulin has the merits in reproducing the physiological Chitosan-based nanoparticles are capable for overcoming serial cellular and systemic barriers by oral administration of insulin, to obtain the maximum bioavailability and therapeutic effect. 68 To date, a number of strategies have been presented to enhance the nanoparticles permeability of healthy endothelium to achieve a valid blood concentration of oral drugs. The intestinal absorption ability of insulin depended on the surface charge of CMCS/CS-NPs. Compared to the positively charged CMCS/CS-NPs, the negative ones induced more drastic disintegration of TJs (Figure 4), which underwent stronger paracellular permeability, under synergetic effects of Ca 2+ deprivation and claudin-4 dephosphorylation. 69

Skin regeneration
As the primary barrier of the body, the skin holds crucial roles in continuously resisting pathogens, protecting the body from damage, and maintaining healthy. Repairing and rebuilding a big area of skin has become a hard problem. 70 Among them, chronic wounds refuse to heal for an extended period of time, which leads to severe complications or even death. To control the infection and acquire shorter healing time of chronic wounds, multifaceted therapeutic approaches have been designed. Wounds dressings with suitable mechanical properties, antimicrobial effect, and good histocompatibility have attracted broad attention. 71 Xia et al prepared composite hydrogel (CW/NPs/HBC-HG) wounds dressing consisting of chitin whisker (CW)/CMCS nanoparticles (CMCS NPs)/thermosensitive hydroxybutyl chi-tosan (HBC) ( Figure 5). The CW/NPs/HBC-HG composite hydrogel showed increased mechanical properties and prolonged cell proliferation activity for up to 5 days. 72 The efficacy of CW/NPs/HBC-HG composite hydrogel as a wound dressing was evaluated by in vivo chronic wound healing model, including significant wound healing, reepithelialization, collagen deposition, and angiogenesis. Wound infection is closely crucial in wound care management. Bacterial or fungal infection of wound constitutes one of the obstacles in the process of wound healing and may become a threat against people's health and life. Zhou et al developed pH-sensitive nanocarrier composed of quaternary ammonium chitosan (TMC) to combat pathogenic biofilms of bacteria or fungus. 28 Incubating with negatively charged biofilm, the positively charged nanocarrier could be absorbed. Chitosan/fucoidan nanoparticles have also been exploited for precise methotrexate (MTX) delivery to treat skin-related inflammatory diseases. MTX-loaded chitosan/fucoidan nanoparticles could obviously reduce production of pro-inflammatory cytokines and enhance skin permeation. 73

Nasal/ocular mucosal administration
Nasal administration has obvious advantages such as fast drug absorption due to the highly vascularized nasal mucosa and without the metabolism of first-pass effect, and thus offers a reliable and convenient route for the treatment of various diseases. 74 However, the physicochemical instability, the high metabolic activity, and low permeability of the nasal mucosal barriers of F I G U R E 5 TEM images of the CWs (A), CMCS NPs (B), and the HBC hydrogel containing CWs and CMCS NPs (C). Green arrows indicate CMCS NPs and red arrows indicate CWs. Reprinted with permission. 72 Copyright 2017, Royal Society of Chemistry macromolecules drugs via intranasal administration limit the bioavailability of the drugs. Chitosan is mucoadhesive, which provides a reliable opportunity of enhancing drug penetration capacity across nasal mucosal barriers. Zhang et al explored the potential of polyethylene glycol-grafted chitosan (PEG-g-chitosan) nanoparticles as nasal drug delivery systems to improve the systemic absorption of insulin. Compared to insulin-PEG-g-chitosan suspension and free insulin solution, PEG-g-chitosan nanoparticles showed significantly enhanced nasal absorption of insulin after intranasal administration. 75 Ocular administration of therapeutic agents commonly acts on anterior and posterior segments of the eye, whereas the absorption of the drugs is hampered by the special structure of the eye. When applied topically into the eyes, only 2-7% of the drug is available, due to the natural protective mechanism of eyes: TJs of corneal epitheliums limit the penetration of the drugs. Hence, it is essential to develop an ocular drug delivery system that could arrive at the anterior and posterior chambers and corneal stroma of eyes, and then achieve effective drug release. 76 Chitosan is expected to prolong the precorneal and ocular surface retention time of drugloaded nanocarriers through retaining and stabilizing tear fluids on the surface of eye. Dexamethasone-sodium phosphate-loaded mucoadhesive chitosan nanoparticles were prepared to enhance the intraocular drug penetration by binding with corneal epithelial cells, resulting in reversible loosening of the TJs of corneal epithelium. 77

Pulmonary drug delivery
Presently, there was an increased interest in nanocarriers for pulmonary delivery of therapeutic macromolecules (ie, drugs, peptides, proteins, and genes), not only for local, but also for systemic effect. Pulmonary route provided conspicuous advantages, for example, (a) large absorptive alveolar surface area, (b) thin blood-alveolar barrier, (c) relatively low metabolic activity, and (d) avoidance of gastrointestinal degradation and hepatic metabolism. 78 Compared with microparticles, nanocarriers showed the ability to diffuse through the mucus layer and translocate through the alveolar epithelium by endocytosis. Chitosan had long been extensively employed as nanocarrier due to its biodegradability, biocompatibility, low toxicity, and mucoadhesivity. Chitosan NPs could provide increased lung deposition and improved cellular absorption owing to their ability to enter intracellular compartments and escape macrophages phagocytosis, 79 which presented great potential as carriers for lung drug delivery. Microencapsulated insulin-loaded chitosan NPs were presented to investigate its potential to transport protein to the deep lung, where it was absorbed into systemic circulation. Microencapsulated chitosan NPs could be delivered into the deep lungs and showed more pronounced and prolonged hypoglycemic effect in hyperglycemia rats. 79 Bivas-Benita et al prepared chitosan-DNA NPs as pulmonary delivery carriers of a new DNA plasmid encoding eight HLA-A*0201-restricted T-cell epitopes from Mycobacterium tuberculosis. The release of the DNA vaccine from the NPs was able to stimulate dendritic cells (DCs) and induce the maturation of DCs in HLA-A2 transgenic mouse. Pulmonary delivery of DNA vaccines against tuberculosis showed increased immunogenicity by delivery of the DNA encapsulated in chitosan nanoparticles, which provided a more favorable delivery route. 80 Though presenting great potential in pulmonary drug delivery, the complete biodegradation of chitosan-based NPs in lung lacks comprehensive supporting data. 81 Islam et al had investigated the in vitro degradation of low-molecular-weight chitosan (MW of  kDa, DD of 92%) and its NPs cross-linked with glutaraldehyde in PBS (0.2 mg/mL lysozyme, pH 7.4, 37 • C) that mimic the lysozyme concentration in lung secretions to measure its potential for pulmonary drug delivery. The outcomes revealed that chitosan with a high DD and its NPs could not be degraded in lysozyme solution, thus unsuitable for pulmonary drug delivery. 82 Therefore, further studies were warranted to understand the in vitro/in vivo biodegradation of chitosan NPs with varying DD and MW to measure their potential in pulmonary drug delivery.

Gene therapy
Regenerative gene therapy and gene editing technologies are recently booming and have paved the way to cure refractory disease fundamentally. 83,84 To achieve desirable gene therapy, the carrier platform requires to be safe, efficient, and capable to overcome a battery of obstacles involving cellular barriers, extracellular barriers, enzymatic digestion barriers, immune barriers, and so on, which mainly refers to viral and nonviral delivery systems. 85,86 Chitosan, a promising nonviral gene delivery vector, has been recognized as a desirable candidate to deliver nucleic acids due to its availability, nontoxicity, mucosal-adhesion, low-immunogenicity, ease of administration and manufacture, as well as lack of DNA size limitation. 87-89 Liu et al presented the selfassembled nanoparticles (OCMCS-HA NPs) consisting of amphiphilic oleoyl-carboxymethyl-chitosan (OCMCS) and HA as novel potential vectors for gene delivery. When N/P ratio was 5 and OCMCS/HA weight ratio was 4, the smallest monodispersed NPs (∼165 nm) with positive charge (+14.2 mV) were obtained, presenting high plasmid DNA loading, and enhanced cellular transfection efficiency by Caco-2 cells. With the property of no significant cytotoxicity against Caco-2 cells, OCMCS-HA NPs exhibited great potential as safe and effective vehicles for targeted DNA delivery to gastrointestinal tract system, and further transferring to the intestinal epithelial cells. 90 Kamra et al synthesized water-soluble pyridine imine O-carboxymethyl chitosan ((py)CS(CH 2 COOH)) as vectors for co-delivery of gene and drug to achieve efficacious anticancer therapy. Obtained (py)CS(CH 2 COOH) and DNA can form nanosized positively charged polyelectrolyte complexes, with high gene binding rate, which were stable to DNase and physiological polyanions. Low toxicity and high transfection efficiency in vitro were observed, and meanwhile, co-delivery of functional gene (p53) and drug (doxorubicin) both in vitro and in vivo was accomplished, following by complete tumor regression with no recurrence and appreciable survivability, which proven the nanobiocomposite as a potential gene therapy option. 91

CONCLUSION AND PERSPECTIVE
Chitosan is widely used as targeted drug delivery system for protein, peptide, nucleic acid, and so on, due to the unique physicochemical and biological properties. Chitosan nanomaterials can be applied to prepare controlling agents, mucosal adsorbents, absorption permeation enhancers, drug carriers, disintegrating agents, coating material, and so on. Chitosan nanodrug delivery carriers endow the drugs with the prolonged contact time, improve drug absorption, enhance efficacy, and meanwhile avoid of side effects. Based on the researches in biomedical domains from 1994 to 2004, potential superiority of chitosan nanomaterials-based simple formulations has already aroused researchers' attention, compared to formulations of complicated preparation process. Positively charged chitosan is easy to bind with negatively charged tissues and cell surfaces, which leads to increased permeability of epithelial cells and activated immunoreaction. The phenomenon is affected by the relative molecular mass and the degree of acetylation of chitosan. The formulations of chitosan and its derivatives nanocarriers are optimizing with the development of technological means and the improvement of physical and chemical methods. Chitosan nanoparticles, as novel drug carriers, have many favorable properties such as low toxicity, wellbiocompatibility, biodegradability, enhanced drug stability, alterable administration route, and controlled drug release. Drug-loaded chitosan nanoparticles are prepared through various ways based on the structural features and natural properties. Chitosan nanoparticles are mainly used to encapsulate hydrophobic biomacromolecules to improve therapeutic efficacy by various routes, such as oral, nasal, and pulmonary administration. With the development of modern molecular designing field and synthetic technique, chitosan nanomaterials will open new avenue for nanotechnology and biomedicine in the future.

C O N F L I C T O F I N T E R E S T S
The authors declare no conflict interest.