Studies On The Absorption Mechanism Of The Novel Norcantharidin-loaded Liver Targeting Chitosan Nanoparticles

Objective: In this paper, two novel live-targeting nanparticles, the norcantharidin- loaded chitosan nanoparticles (NCTD-CS-NPs) and the norcantharidin- associated galactosylated chitosan nanoparticles (NCTD-GC-NPs), were prepared by ionic cross-linkage process. The intestinal absorption mechanisms were studied with various methods including in situ circulation method, in vitro everted gut scas, franz chamber perfusion technique and Caco-2 cell model. The intestinal absorption characteristics of NCTD solution, NCTD-CS-NPs and NCTD-GC-NPs were investigated for further scientific research and development of new targeting drug delivery system (TDDS).Methods: (1) NCTD-NPs were prepared with ionic cross-linkage process. The particle size, size distribution, encapsulation efficiency, morphological characteristic, in vitro drug release were studied individually. (2) In situ circulation method was used to determin the absorption characteristic at different segments, and with different concentrations. (3) In vitro everted gut scas was used to investigate the absorption characteristic of NCTD solution and NCTD-NPs at the different segments and concentrations. The activity of intestinal cells in vitro were evaluated. Then, the effects of different concentrations of P-glycoprotein (P-gp) inhibitor on the intestinal absorption were studied. (4) Franz chamber perfusion technique was utilized in this investigation to explore the intestinal mucous membrane absorption of NCTD-NPs between Mucosa side to Serosa side (M-S) or vice versa. The effects of different intestinal segments, concentrations, preparations, absorption enhancers on intestinal mucous membrane absorption of NCTD were studied. (5) The Caco-2 cell model was established, validated, and was applied to study the absorption and transport of NCTD solution and NCTD-NPs. For the absorption study, the effects of absorption time, temperature, concentration and P-gp inhibitors were studied. For the transport study, the effects of concentration, inhibitors and enhancers were studied.Results: (1) The particle size of NCTD-CS-NPs and NCTD-GC-NPs were (131±11) nm and (119±9) nm, encapsulation efficiency were (45.12±0.33) % and (57.92±0.40) %, leading to final NCTD loading values (7.3±0.13)% and (10.38±0.06) %, respectively. The nanoparticles looked round and regular under transmission electron microscopy. And In vitro drug release followed Weibull equation. Compared to NCTD gelata, the nanoparticles were with the help of drug release. (2) On the experiment of drug absorption via small intestine in vivo, it was found that the absorption rate constants (Ka) of NCTD in different segments were duodenum>jejunum>ileum>colon. The concentration had no distinctive effect on the absorption kinetics, which suggested drug absorption is not dose-dependent. It was found that the Ka of nanoparticles was larger than NCTD solution, and t1/2a was smaller. (3) The absorption law in vitro was the same as in vivo. Compared to NCTD-CS-NPs, NCTD-GC-NPs are more conducive to drug absorption. The transport of NCTD was found to be inhibited by P-gp inhibitor, which indicated that NCTD might be the substrate of P-gp. (4) In franz chamber perfusion experiment, the apparent permeability (Papp) between M-S and S-M had no significant differences at duodenum and ileum, while Jejunum and colon were significantly different, indicating that these regions have some specific transporter to cause the drug transport in different directions. The order of the enhancers'effects were shown as follows: low molecular weight chitosan (CS-8000)> high molecular weight chitosan (CS-30000)> Poloxamer> SDS> SDCh. (5) The results of Caco-2 cell model show that: the uptake of NCTD is positively correlated to uptake time. Different concentrations were no significant differences. The drug uptake was the largest at 4℃. CyA and SDCh significantly enhanced the uptake amount, followed by NaN3, and Oxophenylarsine had little effect. The transport of NCTD cross Caco-2 cell monolyaers was directional. Papp of Basolateral to Apical was much more than that of Apieal toBasolateral.Conclusions: Four intestional absorption models in vivo/ in vitro were established. They are simple, easy, and effective for drug absorption studies. The intestinal absorption mechanisms of NCTD solution and NCTD-NPs were investigated. It is showed that NCTD-NPs significantly enhanced drug absorption, compared with NCTD solution. Intestinal absorption kinetics provided methods for profound study on the process in vivo and new oral drug development.