Study Of Protein Loaded N-trimethyl Chitosan Chloride Nanoparticles For Oral Drug Delivery

N-trimethyl chitosan chloride (TMC) is a quaternized chitosan derivative. Compared to chitosan, which is only soluble in water in an acidic pH range, TMC shows perfect solubility in water over a wide pH range. At physiological pH, chitosan loses its capacity to enhance drug permeability and absorption, which can only be achieved in its protonated form in acidic environments. However, TMC is still protonated in neutral and basic-pH environments, therefore has better absorption enhancing effect than chitosan.Nanoparticles can protect drugs from degradation in gastrointestinal tract, improve drug transmucosal transport and transcytosis by M cells, and provide controlled release properties for encapsulated drugs. These properties have made nanoparticles promising candidates for oral protein delivery. TMC nanoparticles (TMCNP) can be prepared by ionic crosslinking method, which is a mild preparation method and no organic solvent is required, therefore protects proteins and vaccines from inactivation during preparation.In this study, protein-loaded TMCNP were prepared using TMC as carrier material. Two model proteins-bovine serum albumin (BSA) and bovine hemoglobin (BHb), which have similar molecular weight but different pI, were selected as model drugs to investigate the drug loading, in vitro drug release properties of TMCNP. The influence of degree of quternization of TMC on these properties was also investigated. In vitro Caco-2 cell model was used to study the absorption enhancing properties of TMCNP. Urease, a vaccine protein for the treatment of Helicobacter pylori infection, was also used as a model vaccine to investigate the immunostimulating effect of TMCNP.Firstly, TMCs with different degrees of quaternization were synthesized from chitosan by altering reaction steps and time. The obtained TMCs showed perfect solubility over a wide pH range. TMCNP were prepared by ionic crosslinking of TMC with tripolyphosphate (TPP). The results showed that TMCNP can not be achieved with too high or too low TPP concentration. The pH value of TMCNP suspensions and TMC precipitation efficiency increased with the increase of TPP concentration and the decrease of degree of quaternization of TMC.Two model proteins with different pI values, BSA(pI=4.8) and BHb (pI=6.8), were used to investigate the loading and release features of the TMCNP. The results indicated that TMCNP prepared by method A had a high loading efficiency (95%) for BSA but a low one (30%) for BHb. The particle size and zeta potential were significantly affected by BSA concentration but not by BHb concentration. Nanoparticles of TMC with a lower degree of quaternization showed an increase in particle size, a decrease in zeta potential and a slower drug release profile. As for BSA-loaded TMCNP prepared using method B that nanoparticles were modified by alginate, a smaller size and lower zeta potential were observed and the burst release of BSA was reduced. TMCNP were also prepared by method C that BHb was firstly dissolved in TPP solution to achieve negative charges. Loading efficiency was largely improved and no influence was observed on drug release profile.The absorption enhancing properties of TMCNP were investigated on an in vitro model of GI epithelium (Caco-2 cells). Both alginate modified and non-modified TMCNP can improve drug permeation, open the tight junction and promote drug paracellular transport. Compared with non-modified TMCNP, alginate modified TMCNP had a stronger effect on enhancing drug transport through transcellular pathway but an equal effect on enhancing drug transport through paracellular pathway.The feasibility of applying TMCNP in oral vaccination was also studied using urease as a model vaccine. We successfully prepared urease-loaded TMCNP and primarily screened preparation conditions. Mice s.c. immunized with urease loaded TMCNP showed highest systematic immune response (IgG levels) but the lowest mucosal response (secretory IgA levels). In the contrast, mice i.g. immunized with urease loaded TMCNP showed much higher antibody titers of both IgG and secretory IgA than those immunized with urease solution or urease co-administrated with TMC solution. These results indicated that TMCNP are potential carriers for oral protein and vaccine delivery.