Drug Delivery Applications of Chitosan Nanogels
The physicochemical properties of chitosan, in addition to their significant biological properties such as antimicrobial, hemostatic activity and wound healing capacity, differentiate this material from others, both synthetic and biomaterials commonly used as DDS. In 1996 Berthold etal.  reported the use of chitosan microspheres for oral delivery of corticosteroids. Since then, many researchers have reached nanometric size for developing DDSs. In this section, the most relevant publications of chitosan-based nanogels reported along the recent years are reviewed.
Drug Delivery Based on Mucoadhesion and Mucus Permeation
Several advantages have been associated to drug delivery via mucosal membranes, such as increased residence time, improved drug penetration or reduced administration frequency, among others. Chitosan-based nanoparticles could be highly suitable for the mucosal route due to their biological properties and their mucoadhesive capability. The cationic structure of chitosan will promote the bioadhesion with negatively charged mucous tissues; also, it could enhance the cellular absorption due to its capability to open the intercellular tight junctions, enhancing the cellular macromolecular absorption .
The defensive barriers present in the eye could make difficult the treatment of the ocular diseases. The protective physiological mechanisms present in the eye limit the efficacy of treatments. Corneal and conjunctival epithelia are sealed with intercellular tight junctions that limit the entrance of harmful substances or therapeutic drugs. On the other hand, the mucous tear removes particles and drugs through the lacrimal. Thus, the efficacy of drug delivery system depends on the interaction with the ocular mucosa [132-134].
Conventional ophthalmic formulations are rapidly eliminated by blink, nasolacrimal drainage and lacrimation, which lead to a poor bioavailability. Compared with traditional eye drops, nanoparticles can facilitate transport to different areas of the eye and provide the sustained drug release. These could increase the bioavailability of the drug, the contact time and, in consequence, the corneal penetration [135-137]. However, nanocarriers could be eliminated also from the eye, so in order to increase the residence time in precorneal area, mucoadhesive polymers are used . Chitosan-based nanogels have arisen as promising carriers due to their bioadhesion. In 2001 Alonso et al. reported the study of chitosan nanoparticles as ocular DDS . In that study, nanogels were obtained by ionotropic gelation using TPP as crosslinker. This approach has been often used to develop chitosan nanoparticles for encapsulating drugs such as Cyclosporin A, 5-fluorouracil, dorzolamide hydrochloride or ketorolac tromethamine, among others [139-142]. The ionotropic gelation by TPP has been used in nanogels formed with chitosan derivatives, such as galactosylated chitosan, for timolol delivery for the treatment of glaucoma .
The ionizable amino groups present in chitosan allow its complexation with other polymers; these interactions have been the driving force in the complex formation with other components like hyaluronic acid (HA) [144, 145] or lipids  as DDSs. Losa et al. reported that HA implication in the chitosan/HA complexed nanoparticles improves the cellular targeting in several ocular process. As suggested by de la Fuente et al., hyaluronic acid could induce corneal regeneration and conjunctival epithelial cells protection through an interaction with CD44 receptors .
Similarly, nanoparticles with prolonged mucoadhesive capacity and stability to lysozyme were reported by Chaiyasan et al. .
In their study, chitosan/dextran sulfate-based nanoparticles were prepared by polycomplex formation and they were ex vivo studied.
Other possible approach for nanoparticles fabrication was described by
Mahmoud et al. , who developed chitosan nanoparticles using sulfobutylether-B-cyclodextrin (SBE-B-CD) as polyanionic crosslinker agent.
Oral, Nasal and Pulmonary Drug Delivery
Oral drug delivery is the most common route due to the ease administration. However, oral DDS must overcome several challenges such as the presence of enzymes, the variation on the pH (highly acidic stomach), or the intestinal barrier to drug absorption. Chitosan nanoparticles could enhance the gastrointestinal absorption. Dube and co-workers studied the increase in the bioavailability and the stability of catechins, which are cardioprotective, neuroprotective and anticancer drug, by loading them in chitosan nanoparticles  The Fig. 8.14, illustrated an schema of the increase of the oral bioavailability of the chitosan nanoparticles in comparison with oral drug delivery . Another example of increased intestinal absorption was described for alginate-coated chitosan nanoparticles . These nanoparticles increased paracellular transport of the drug across intestinal epithelium of enoxaparin in rats; this enhanced permeation is attributed to chitosan's mucoadhesion capability. Several chitosan-based DDS have been developed as nanocarriers for different drugs such as gemcitabine , tolbutamide , tamoxifen citrate or insulin .
Other mucosal routes have been explored as delivery strategies, with nasal and pulmonary paths being the most studied after oral routes [154-156]. Nasal delivery is limited by the nasal mucous membrane absorption, as this absorption is conditioned by drugs' molecular weight, lipophilicity and charge. This limitation can be overcome by chitosan-based nanoparticles. The nasal absorption could take place in three ways: by transcellular pathway, by paracellular pathway or via trigeminal nerves . Many examples of chitosan-based DDS for nasal route have been developed. Ilium et al. described the enhanced absorption of several peptide drugs loaded in chitosan NPs across the nasal epithelium . In addition, sumatriptan succinate-loaded chitosan nanoparticles for migraine therapy have been also developed in order to reduce dosing frequency, toxicity and improving the analgesia . In another example, carbamazepine, a drug used in the epilepsy treatment, was successfully loaded in a carboxymethyl chitosan NPs. This nanocarrier presented an enhanced bioavailability and brain targeting capability .
Drug Delivery Applications of Chitosan Nanogels
Figure 8.14 Schema of in vivo oral delivery of drug and drug loaded chitosan nanoparticles from ref.  (CC BY 4.0).
In some examples of nasal DDS, chemical modification of chitosan has been employed to improve short retention time and nanoparticle solubilization at neutral pH values. Thiolated chitosan nanogels have been employed to deliver an antianxiety drug to the brain . In the recent years, similar approaches have been carried out by catechol-modified chitosan .
Finally, some chitosan nanocarriers have been developed for pulmonary delivery. Compared to other mucoadhesive paths, lung mucosal tissue presents as an advantage rapid and sustained drug delivery. Recently, Islam and Ferro reviewed many chitosan-based nanoparticles developed for pulmonary drug delivery . Until now, chitosan-based DDS have been reported for being capable of successfully delivering protein and peptide [164,165], antibacterial and antitubercular drugs [166, 167] or anti-asthma drugs [168, 169].
Intravesical Drug Delivery
There are diverse bladder diseases, such as cancer, inflammation, infection or incontinence, that require active pharmaceutical compounds for their treatment. Currently, the dosage forms used for these diseases are administered orally. However, only a small fraction of the drug reaches the target cells. Thus local application could be more effective for maximum drug delivery. This local application could be carried out by chitosan nanoparticles, Fig. 8.15, due to its known mucoadhesive capacity to the bladder mucosal tissues [170, 171].
Figure 8.15 Schematic representation of nanoparticles DOS in a bladder . Copyright 2017. Reproduced with kind permission from Elsevier Ltd.
Bilensoy et al.  reported the delivery of mitomycin C into bladder cancers by using nanoparticles of chitosan and chitosan- coated polycaprolactone (PCL). In this study, the successful use of chitosan as a coating material for bioadhesive intravesical nanosized DDS was demonstrated. Chitosan-thioglycolic acid (chitosan-TGA) nanoparticles (NP) and unmodified chitosan NPs have been also developed and loaded with trimethoprim during their formation. The addition of covalently attached thioglycolic acid improves the sustained release of TMP in comparison to unmodified chitosan NPs. The drug release for these systems took place over a period of 3 h in artificial urine at 372C . Recently, Lu and co-workers have developed chitosan-methacrylic acid nanocapsules capable of combining double effect therapies by including doxorubicin and peptide-modified cisplatin that could be an interesting approach for developing intravesical chemotherapy of non-muscle-invasive bladder cancers .
Gene therapy is a promising field for medicine which has attracted a lot of attention in the recent years for the development of new therapies to treat cancer, autoimmune diseases, viral and antibiotic- resistant bacterial infections, and genetic rare diseases, among others. The main goal of gene therapy is to introduce new genetic material (genes, pDNA, oligonucleotides, and small interfering RNA) into targeted cells in order to control and modulate the genomic expression leading to the direct production of proteins in the targeted cells . Effective gene therapy requires the protection of genes from degradation in the extracellular medium, the specific targeting into desired cells, and an enough cellular uptake of genetic material to produce a therapeutic effect . Therefore, the development of suitable vehicles for the efficient gene delivery has been intensively investigated in the last decades. These vehicles can be differentiated as viral and nonviral vectors. Since viral vectors can lead to mutational effects , although nonviral vectors usually show low transfection efficiency; as they are safer, cheap and easy to produce, they have become promising candidates for gene delivery . Ideal nonviral gene delivery vectors should present nanometric size to enable an adequate cellular uptake and properly protect the DNA until it reaches the target cell.
Cationic macromolecules, like chitosan, have become adequate carriers for gene therapy due to their capability to be complexed with the negatively charged phosphates groups from nucleotides in nucleic acids forming polypi exes that easily self-assemble .
In addition, it has been reported that the highest gene transfer has been obtained with cationic polymers with amine groups of pKa around physiological pH because these systems display the so-called "proton sponge" potential that induces endosomal disruption and protects nucleic acids from lysosomal degradation .
Chitosan-plasmid polyplexes were applied in vivo in the delivery of nucleic acids in the intestinal tissue , concluding that chitosan was an adequate plasmid delivery systems for the oral administration of nucleic acids vaccines.
However, chitosan usually leads to poor gene transfer efficiencies. This fact is ascribed to the low charge density of chitosan at physiological pH, which results in low colloidal stability of the nanoparticles. Thus, the effect of chitosan structure has been analyzed in the recent years to improve chitosan properties in gene delivery applications. Indeed, some specific parameters related to chitosan structure, such as the degree of acetylation, the molecular weight, or charged ratio, have shown to affect the particle size and stability of chitosan-based complexes for gene-delivery. Reported investigations suggest that low-molecular-weight chitosan or oligomers, high DA, small particle size (~100 nm) and moderate positive surface zeta potential increase transfection efficiency . Nevertheless, high molecular weights have been proved to entangle with DNA and RNA more readily leading to an efficient protection from enzymatic degradation and serum components .
In addition, some investigations have reported enhanced transfection when self-branched chitosan is employed comparatively with its linear counterpart. Nevertheless, no difference was observed regarding siRNA transfection efficiency [183,184].
In comparison with other cationic polymers, chitosan's nontoxicity and natural character make it a competitive candidate. Indeed, recently when it was compared with polyethylenimine, similar endosomal delivery was observed .
Chitosan-based nanocarriers for gene delivery can be prepared by simple complexation, ionic gelation or direct surface adsorption onto preformed chitosan nanoparticles (Fig. 8.16) . Self-assembling by complexation with polynucleotides is a rapid and simple method that involves mixing them in water. As a consequence, electrostatic attraction forces take place in solution and the nanoparticles are formed. Ionic gelation by the addition of polyanions like TPP in the presence of genetic material also leads to nano particles with lower size for gene delivery and increases the stability of the obtained complexes during incubation in biological fluids .
Recently, chitosan nanoparticles obtained by ionic gelation technique have been introduced by incubation within anionic liposomes showing better pDNA protection, reduced cytotoxicity and at least twofold higher transfection efficiency at physiological conditions, as well as, efficient delivery in vivo in the choroallantoic membrane model .
Chitosan nanoparticles have been also proposed to be applied combining chemotherapy (cisplatin) with gene therapy, leading to negligible toxic effects of the drug and significant improvement in lung tumor inhibition . Plenty of works with a great range of targeted diseases have been investigated about chitosan nanoparticles and their derivatives with gene delivery purposes in recent years, such as, different kinds of cancer [189, 190, 191], Alzheimer , HIV  or rheumatoid arthritis , among others.
Figure 8.16 Fluorescent chitosan nanoparticles interactionating with thiolated siRNA for gene delivery and diagnosis . Copyright 2016. Reproduced with kind permission from Elsevier Ltd.
Anticancer Drug Delivery
Cancer is one of the leading causes of morbidity and mortality worldwide and its incidence is expected to continue dramatically rising over the next decades. For this reason, huge effort has focused on acquiring greater knowledge about the causes and mechanisms of cancer, enabling enhanced diagnosis and treatments for this disease. New and future treatments and diagnostic methods have centered on the specific features of the different cancer types and in the development of targeted and personalized therapies.
The physicochemical properties of chitosan and its versatility in terms of functionalization and capability to develop nanostructured systems have promoted an intense investigation on chitosan nanogels and their derivatives as nanocarriers for passive and active tumor-targeted drug delivery .
Chitosan nanogels exhibit passive drug delivery capability leading to good therapeutic effect and increasing healthy cell survival rate. This is due to their prolonged circulation time in blood and their low uptake by the reticuloendothelial system (RES) as a consequence of their nanometric size and amphiphilic character. These conclusions are known since more than two decades. Mitra et al.  synthesized chitosan nanogels using the microemulsion method, which were loaded with dextran-doxorubicin for its passive delivery. This work proved the in vivo anticancer activity in Balb/c mice of chitosan nanogels, not only reducing side effects but also improving the therapeutic efficacy in the treatment of solid tumors. Recently, Yang et al.  synthesized pH-triggered chitosan nanogels via an ortho ester-based crosslinker for efficient drug loading of doxorubicin. These nanogels exhibited excellent pH-triggered drug release as a consequence of the degradation of ortho ester linkages in mildly acidic conditions characteristics of tumoral cells. Besides, in vitro and in vivo results demonstrated that the nanogels were successfully internalized by 2D cells and 3D-MCs increasing drug concentration in solid tumors, and leading to higher therapeutic efficacy.
However, one of the main limitations of passive tumor targeting is its difficulty to maintain the adequate drug concentration at the tumor site, which leads to reduced therapeutic efficacy and endorses adverse systemic effects [12,13]. To overcome these drawbacks and improve cancer specificity, active targeting of chitosan nanogels has been also intensively studied in the recent years. For this, chitosan and its derivatives have been functionalized with tumor-targeting ligands such as folic acid, antibodies, peptides, hyaluronic acid, biotin or avidin, which have the ability to recognize and link to specific receptors of some type of cancer cells.
Regarding folic acid, it is known that the internalization of folate conjugates by cells takes place via an endocytosis process in which pH approaches to 5.5-6.0 in endosomes and 4.5-5.0 in lysosomes. These pH values are markedly different from the physiological pH of 7.4 [197, 198]. According to this, Arteche et al. exploited the pH-sensitivity of chitosan-folate conjugates in form of nanogels covalently crosslinked with different biocompatible agent, to the targeted delivery of 5-fluoracil into potential cancer cells proving a higher concentration of free drug at the endosomic pH than at healthy physiological conditions [92,199].
It has been found that malignant cells with the highest metastatic potential show higher binding and internalization of hyaluronic acid. Indeed, isoforms of hyaluronic acid receptors such as CD44 and RHAMM are overexpressed in transformed human breast epithelial cells, colorectal carcinoma and other cancers . Hyaluronic acid was conjugated with chitosan in order to form nanoparticles that loaded oxaliplatinen for effective delivery to colon tumors after oral administration in Balb/c mice showing relatively high local drug concentration .
Biotin and avidin are targeting ligands capable of binding specifically with the receptors of hepatic carcinoma. As a result, chitosan nanogels functionalized with biotin or avidin were prepared as tumor-targeted nanocarriers and their physicochemical characteristics were examined .
PH and redox dual response was reported for nanogels prepared with chitosan derivatives, carboxymethyl chitosan and thiolated chitosan, respectively, which showed synergic effect for controlled delivery of doxorubicin .
A number of works have been reported for cancer drug delivery for generic tumor cells [185-188], and specific cancers, such as lung , liver [190,191], breast , brain , colon , among others.
Topical/transdermal path is the favored route for local drug delivery due to its convenience and affordability. The absorption limitations associated to the stratum corneum and the adverse side effects have given rise to the development of new DDS for topical/transdermal therapies . Chitosan-based nanocarriers have enhanced tissue penetration capability and biological properties [205-207]. Hasanovi et al. reported an improve skin permeation, residence time and a higher chemical stability of acyclovir loaded in chitosan/TPP nanoparticles. A similar synthetic approach, chitosan/TPP-based nanoparticles, was developed for skin gene delivery and antisense oligonucleotides by 6zba$-Turan and co-workers [208, 209]. These nanoparticles were successfully tested in rats, suggesting their potential for human skin treatments. In addition, hybrid skin targeting systems have also been reported by using chitosan nanoparticles crosslinked with TPP as a carrier for tacrolimus and nicotinamide for atopic dermatitis treatment . Designed DDS shown enhanced skin permeation in vitro and in vivo.
A different method was used by Shah et al. to develop a skin permeating nanogels of modified chitosan-poly-(lactide-co- glycolic acid) bilayered. These nanoparticles were designed as topical anti-inflammatory DDSs based on co-administration of two antiinflammatory drugs: spantide II (SP) and ketoprofen (KP) . Obtained results predict the use of these nanocarriers for the treatment of allergic contact dermatitis, psoriasis and other topical diseases.
Chitosan-based nanoparticles have been recently used for antibacterial wound dressing applications. In their study, Basha et al. developed a DDS based on in situ gel of cefadroxil loaded chitosan nanoparticles. This formulation could be a highly effective wound dressing and present antibacterial properties .
Poly(lactic acid)/chitosan nanoparticles have been studied as anti- human immunodeficiency virus (HIV) drug delivery system. Lamivudine, a potent and selective inhibitor of type 1 and type 2 HIV, was efficiently loaded in poly(lactic acid)/chitosan nanocarriers for controlled delivery of anti-HIV drugs .
Wilson et al. investigated a pre-clinical used of chitosan nanoparticles as DDS for tacrin, a drug with potential significance in Alzheimer's disease. These particles presented good drug-loading/ releasing capacity, the releasing mechanism being diffusion- controlled .