*3.2.3 Encapsulation of peptide into the nanocarrier*

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

There are two types of protease, including serine protease and Zn2+-dependent protease. Serine proteases, such as trypsin, chymotrypsin and elastase are pancreatic digestive enzymes. Meanwhile, Zn2+-dependent protease such as matrix metalloproteinase is an insulin-degrading enzyme [64]. Some enzyme inhibitors need chitosan to enhance their anti-protease activity and minimise peptide drug degradation [64]. For example, chymostatin is a protease inhibitor selectively to chymotrypsin-like serine proteases. This inhibitor will covalently be linked to the

The active site of matrix metalloproteinase, such as carboxypeptidase, contains Zn2+ binding motif. It requires Zn2+ to promote nucleophilic attack by water. This protease cleaves membrane-bound pre-proteins of the cell to release cytokine. The ethylenediaminetetraacetic acid (EDTA) is a complexing agent that is capable of forming a complex with Zn2+ and retard the nucleophilic attack of water on carboxypeptidase. To inhibit the Zn2+-dependent protease, the EDTA is covalently bound to the primary amino groups of the chitosan-inhibitor

Moreover, the study showed that the effect of trypsin inhibitors would be disrupted after the gastric phase. Therefore, the encapsulation of the peptide drug and trypsin inhibitor with chitosan-EDTA conjugates improve the controlled release of

Chitosan derivatives improve the permeation of water-soluble drug molecules due to their ability to adhere to the mucus [30]. Thiolated chitosan shows a greater effect in improving drug permeation through the cell membrane. With the formation of disulfide bridges, the thiol group of chitosan interacts with the cysteine-rich subdomains of mucus and allows greater mucoadhesion. Thus, the absorption of

It is worth noting that chitosan with a low degree of acetylation and high molecular weight leads to high charge density. The higher positive charge density of chitosan will bind to negatively charged tight junction channels. Ion displacement

The integrity and permeability of tight junctions can be illustrated with transepithelial electrical resistance (TER). The ability of TMC [66] and carboxymethyl chitosan [67] in decreasing the TER will increase the permeability of peptide drugs. TMC has been used to formulate buserelin, a synthetic peptide analogue for LHRH

Thiolated chitosan, such as glutathione, cysteine and N-acetylcysteine, have strong mucoadhesive properties due to covalent bonding with cysteine-rich subdomains of the mucus glycoprotein. For chitosan-glutathione, this derivative improves chitosan stability, enhanced mucoadhesion and permeation enhancing effect. This system has been applied to the oral delivery of immunostimulant drug,

Chitosan-cysteine shows similar mucoadhesive but improved cohesion as compared to unmodified chitosan. The cohesiveness of polymeric drug formulation is crucial to ensure the stability of the drug and will be released in a controlled manner. Furthermore, chitosan-N-acetylcysteine produces a longer retention time than unmodified chitosan. However, no drugs have been tested yet for these two

the peptide drug molecule increases with residence time [30].

occurs, leading to intracellular spaces loosening [16].

agonist, by the oral delivery system [68].

*3.2.1 Enzyme inhibition*

amino group of chitosan.

conjugate [27, 65].

the molecules.

*3.2.2 Chitosan as a mucoadhesive agent*

**62**

thymopentin [30].

chitosan derivatives [30].

By encapsulating peptides into a nanoparticulate system, enzymolysis and peptide aggregation can be avoided. This approach enhances the absorption of peptide drugs through the transmembrane of the small intestinal epithelium [69]. Nanoparticle will provide controlled-release properties in the presence of chitosan as a polymer. This condition will reduce repetitive dose administration and improve drug bioavailability [43]. In the presence of chitosan as a mucoadhesive agent, the retention time between formulation and absorption site will be maximised.

Cyclosporine is a cyclic peptide drug used to suppress the immune system, after organ transplantation. Cyclosporine with high molecular weight (1.32 kDa) shows poor bioavailability with low permeability through the biological barrier. Conventional oral cyclosporine has been shown to have an unpredictable low therapeutic concentration in the bloodstream.

Therefore, a nanoparticle drug delivery system is a promising strategy to improve the oral bioavailability of cyclosporine. Chitosan nanoparticles in the presence of tripolyphosphate, as a cross-linker, make it more convenient as compared to conventional ones. The bioavailability of the nanoparticulate cyclosporine increases by 73% [69]. Exendin-4 is a glucagon-like peptide-1 receptor agonist that has been approved to control type 2 diabetes mellitus. This peptide drug has high susceptible to enzymatic degradation [70]. Chitosan-tripolyphosphate conjugated nanoparticle was used to design oral suspension and enteric-coated capsules of exendin-4 to increase the bioavailability of exendin-4 slightly.

## *3.2.4 Efflux pump inhibition*

Efflux pump is a membrane protein located within the cytoplasmic membrane of a cell. It translocates a variety of substrates across extra- and intra-cellular membranes. Multidrug efflux pump can be one of the drug resistance mechanisms, as it pumps foreign substances (or drugs) out of cells. This active process is an ATP-dependent [71].

P-glycoprotein (P-gp) is a transmembrane glycoprotein and the best example of a multidrug efflux pump. It is expressed and located in the intestinal epithelium, liver cells and proximal tubule cells of the kidney. P-gp is also located within the blood–brain barrier (BBB), which provides an obstacle for drugs to enter the region. Therefore, it must be difficult for antipsychotic drugs to bypass BBB and exert their effect [57, 71, 72].

Chitosan may enhance drug permeation by opening of tight junctions which is highly related to CLDN4 [73]. Chitosan will modulate CLDN4 protein redistribution to the cytosol and disrupt tight junctions [62]. This phenomenon will enhance paracellular permeability and reduce TER. Thus, declining barrier function of epithelial cells to allow drugs to enter the cell [74].

Furthermore, the use of thiolated chitosan (thiomer) has shown to be useful in bypassing the P-gp. The thiol-moiety of thiolated chitosan may allow the formation of disulfide bonds between the cysteine groups of the P-gp [9]. The thiomers then enter the channels of the P-gp pump together with the therapeutic agent, which obstruct the function of the multidrug efflux pump [57].

For infectious disease by Gram-negative bacteria, chitosan plays an important role in facilitating effective delivery of antimicrobials to the infection site [15, 49]. Chitosan will encapsulate drugs and carry them into bacterial cells by attraction forces between polycationic chitosan and negatively charged bacteria [49]. This action avoids the efflux pump at the cell membrane of the bacterial cell.
