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=Chitosan-Based Nasal Drug Delivery Systems= Chitosan-based nasal drug delivery systems facilitate the delivery of therapeutically active molecules (e.g., vaccines, small molecule drugs, and biomolecules) across the nasal epithelium to produce local and/or systemic effects. These systems most commonly utilize nanoparticle or hydrogel formulations for the controlled release of therapeutic payloads with the ultimate goal of improving the efficacy associated with drug therapies. Chitosan-based nasal delivery systems can potentially mitigate the drawbacks of alternative parenteral delivery strategies, especially those aimed at brain-targeting and eliciting mucosal immunity, while also improving upon the efficacy issues associated with other nasal delivery systems.

Nasal Membrane Permeability and Absorption
Nasal delivery strategies are becoming of particular interest as they have the potential to increase both the bioavailability of drugs and efficacy of vaccines targeting mucosal immunity. However, the current application of nasal delivery strategies are limited by the inherent difficulty of transporting therapeutic molecules across the nasal epithelium. There are three main nasal barriers that therapeutics must encounter prior to exerting their physiological effect: the nasal mucosa, the nasal immune cells, and the nasal epithelium. The nasal mucosa contains an inherent clearance mechanism, known as mucociliary clearance, in which coordinated cilia movement removes foreign material from the nasal mucosa within 15-20 minutes. In addition to the nasal mucosa, microfold immune cells (M cells) of the nasal epithelium can uptake and clear therapeutic macromolecules (e.g., proteins) from the nose. The nasal epithelium is considered to be the most difficult barrier to cross because it contains intracellular junctions, known as tight junctions. The region where tight junctions are located is known as the zona occludens, which is located near the apical (i.e., most superficial) surface of nasal epithelial cells. At the molecular level, the zona occludens contains a series of complexed membrane and cytoplasmic proteins that create an exceedingly small paracellular transport pathway. In fact, it has been reported that the permeability of molecules larger than 1000 Da, which is smaller than most biological macromolecules, is very limited.

Mucoadhesion
Chitosan, a linear polysaccharide consisting of N-acetyl glucosamine and glucosamine units, has shown to be beneficial in nasal delivery strategies due to its mucoadhesiveness and permeability enhancing qualities. It is important to note that the ability of chitosan to be mucoadhesive and to promote enhanced membrane permeability are jointly responsible for the increased absorption across the nasal epithelium. Chitosan offers inherent mucoadhesive properties due to the amine functionality on its glucosamine units, which has a considerable amount of positively charged amines at nasal epithelium pH. These positively charged amine groups interact with negatively charged groups present in the nasal mucosa (e.g., sialic acid) which causes chitosan to adhere to the surface. Importantly, chitosan’s mucoadhesiveness is directly proportional to its size and flexibility.

While chitosan possesses excellent inherent mucoadhesive properties, it is often chemically modified to improve its mucoadhesiveness. One common way of improving the mucoadhesion of chitosan is accomplished by creating thiolated chitosan, which involves reacting chitosan’s primary amine group with molecules such as cysteine, thioglycolic acid, and 2-iminothiolane. In addition to their improved mucoadhesive properties, thiolated chitosan has been shown to have in situ gelling properties due to the formation of disulfide bonds, which also improves chitosan’s overall stability in the nasal mucosa. It is important to note that there are other ways in which chitosan is derivatized to improve mucoadhesiveness, such as creating quaternized chitosan derivatives like N-trimethyl chitosan (TMC) and N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC). In contrast to thiolated chitosan, both TMC and HTCC possess the ability to retain their charge, and thus mucoadhesiveness, across a wide pH range.

Enhancing Membrane Permeability
Regardless of chitosan being derivatized or not, the general mechanism in which chitosan enhances nasal epithelial membrane permeability is the same. That is, the positive charge of chitosan, either due to its amine group functionality or a positively charged derivative, is directly responsible for its permeability enhancing properties. More specifically, it has been reported that the general mechanism of action of chitosan is its ability to translocate tight junction proteins, specifically zona occludens 1 (ZO-1) and occludin, from the membrane into the internal cytoskeleton. Another study explored the influence of chitosan formulation on its permeability enhancement effects, specifically those related to changes in ZO-1 distributions. When comparing chitosan solution to chitosan nanoparticles, it was found that there is no difference in nasal epithelial membrane permeability. It is important to note that, while chitosan formulation does not appear to matter for its permeability enhancing effect, varying chitosan formulations have vastly different effects on drug release and bioavailability.

Promoting Mucosal Immunity
It has been widely proven that nasal administration of vaccines can confer effective humoral and cell-mediated responses, especially when combined with adjuvants and/or delivery systems. In general, nasally administered vaccines seek to target the nasal-associated lymphoid tissue (NALT) and produce IgG and secretory IgA (S-IgA) antibodies to a particular antigen. The NALT is a network of immune cells that consists of B-cell lymphoid follicles, T-cell interfollicular areas, and antigen-presenting cells (e.g., macrophages). There are two main avenues in delivering vaccines through the nasal epithelium into the NALT: paracellularly through the tight junctions and transcellularly via endocytosis. As mentioned in a previous section, there are limitations to utilizing the paracellular pathway for macromolecules due to their size. Therefore, when it comes to vaccines, most rely on transcellular transport via M cells, which act to phagocytose and transport vaccines across the nasal membrane to the NALT. In order for vaccines to make it to the NALT via transcellular transport and generate an antibody response, they must first evade mucociliary clearance.

An ideal nasal vaccine carrier would possess the ability to have sustained delivery of antigen, be biodegradable and generally recognized as safe (GRAS), and create a non-specific immunostimulatory effect. Oftentimes, the immunostimulatory effect of the carrier alone is insufficient, so adjuvants are added to improve the immune response to a given antigen. Chitosan offers a tremendous opportunity as a vaccine delivery system because it meets all of these criteria, as chitosan-based systems prevent antigen degradation, prolong the release of the antigen via mucoadhesion, are biodegradable, and stimulate both humoral and cell-mediated immune responses. In fact, it has been reported that chitosan-based influenza vaccines improve the mucosal antibody response in the entire respiratory tract as well as cell-mediated immune responses against many influenza strains compared to non-chitosan formulations. It is important to note that the immunostimulatory activity of chitosan is influenced by both the molecular weight and the degree of deacetylation. More specifically, it has been shown that higher molecular weights and higher degrees of deacetylation positively impact the immune response.

Design Parameters
Chitosan-based nanoparticle systems have been shown to provide enhanced protection of therapeutic agents against both chemical and enzymatic degradation, increased stability for volatile drugs, and increased overall residence time of proteins and protein antigens in the nasal mucosa. When synthesizing chitosan-based nanoparticles, the most common mechanism used involves blending the therapeutic molecule with the nanoparticle carrier during the manufacturing process. Several different methods utilize this strategy to load drug into and formulate the nanoparticles, such as ionic gelation, microemulsion, emulsification solvent diffusion, and other polyelectric interactions. Of these methods, the most common is ionic gelation, which utilizes ionic interactions between chitosan and an anionic crosslinker such as sodium sulfate or tripolyphosphate (TPP). It is important to note that ionic crosslinking strategies are far more preferable to chemical crosslinking strategies because they yield better therapeutic drug stability and do not involve toxic chemicals. In addition to the synthesis method, design parameters influencing the size distribution, shape, and cellular uptake of chitosan nanoparticles are dependent on the physical characteristics of chitosan used (e.g., degree of deacetylation and molecular weight).

Applications in Drug and Vaccine Delivery
One of the first reported applications of chitosan-based nanoparticles in nasal drug delivery was to deliver insulin systemically. It was found that high concentrations of insulin could be efficiently absorbed through the nasal epithelium and transported throughout the body. To this day, many other therapeutically active molecules have been incorporated into chitosan nanoparticles for brain-specific targeting, local drug delivery, and systemic drug delivery. One example of the utility of chitosan nanoparticles for brain targeting was a study done by Wang et al., which aimed to assess the nasal absorption and brain targeting ability of estradiol-loaded TMC nanoparticles and compared them to other estradiol-loaded formulations. More specifically, the paper assessed the distribution of estradiol in cerebral spinal fluid (CSF) after intranasal and intravenous administration. It was found that CSF levels of estradiol after intranasal administration were significantly greater compared to CSF levels from intravenous administration, which shows the promise nasally administered chitosan nanoparticles have for brain and/or CSF targeting applications.

In addition to using chitosan-based nanoparticles to deliver therapeutic drugs, extensive research has been performed to assess chitosan’s use as a nasal vaccine adjuvant and/or carrier. Across a wide array of studies, it has been proven that chitosan nanoparticle nasal vaccines can induce strong cellular and humoral immune responses. Two particular examples show the enhanced efficacy of nasal vaccines when utilizing chitosan nanoparticles. The first example is a study that utilized chitosan-TPP nanoparticles to nasally deliver a hemagglutinin-split influenza vaccine, in hopes of eliciting a stronger mucosal immune response compared to traditional intramuscular administration. When assessing the effectiveness of this vaccine formulation in mice, it was found that the nasally administered vaccine was 100% protective against challenge with a lethal influenza exposure, which meant that there was a sufficient level of mucosal immunity elicited by the vaccine. The second example is a study that assessed the efficacy of a mast cell activator compound combined with a chitosan nanoparticle-based anthrax vaccine. When comparing these nanoparticles to other carrier systems and mast cell activator alone, it was found that the chitosan-based nanoparticle formulation had the highest antibody titer and the most ideal Th1/Th2 (i.e., helper T cell) profile, indicating a balanced cellular and humoral immune response.

Design Parameters
Another interesting approach for chitosan-based nasal delivery systems involves the use of chitosan-based spray or drop formulations that form a mucoadhesive hydrogel in situ. Chitosan-based hydrogels are advantageous for nasal delivery as they significantly reduce mucociliary clearance and therapeutic agent degradation, allow for sustained release of drugs and vaccines, and have a moisturizing effect that reduces irritation in the nasal mucosa. When synthesizing in situ gelling chitosan-based hydrogels for nasal applications, the most common systems can be classified as either in situ covalent cross-linking systems or in situ phase separation systems. In situ covalent crosslinking systems take advantage of the cross-linkable functional groups on the chitosan chain (e.g., hydroxyl and amine groups), reacting them with a bifunctional cross-linking agent, the most popular of which is genipin. In situ phase separation systems take advantage of the changes in pH/temperature or rely on the elimination of the solvent once nasally administered. The most common in situ phase separation systems are those that utilize temperature to undergo a phase transition at nasal temperatures.

Applications in Drug and Vaccine Delivery
Many therapeutically active molecules, such as drugs or vaccines, have been incorporated into chitosan-based hydrogel nasal delivery systems for brain-specific targeting and local and/or systemic delivery. A great example of brain-specific targeting was a study that utilized a thiolated chitosan hydrogel loaded with liposomal donepezil HCl to treat Alzheimer’s disease. This study compared the mean brain content of donepezil HCl between the intranasal hydrogel and oral tablets, in which it was found that the intranasal hydrogel improved the average brain content of the drug by over two times that of the oral tablet, suggesting the advantages intranasally administered hydrogels have in brain targeting applications. It can also be seen that chitosan-based hydrogels offer significant utility in locally delivering drugs to the nose. One particular example is a study by Voros-Horvath et al., in which chemically cross-linked chitosan hydrogels were utilized to deliver ACE2 receptor inhibitors to reduce the risk of COVID-19 infection. In addition to local drug delivery, chitosan-based hydrogel vaccines have demonstrated the ability to confer both systemic and mucosal immunity. A great example of this is a chitosan-based hydrogel vaccine against influenza by Bedford et al., which demonstrated excellent in situ gelling properties and significantly prolonged the antigen residence time in the nasal epithelium. This combination of properties yielded a large repository of nasal tissue-resident memory T cells, indicating excellent immunity to influenza.