The Construction And Evaluation Of Oral Drug Delivery System Based On Functional Thiolated Polymeric Micelles

To date, chemotherapy is the major treatment for cancer therapy. Injection is the most widely used dosage form in clinical application, due to the inappropriate characteristics of anticancer drugs, including low solubility, poor membrane permeability, lack of selectivity in distribution, and easy degradation. However, severe side effects limit their application. Therefore, the development of the oral chemotherapy delivery system has important implications for tumor treatment.This research designed and established the functional thiolated polymeric micelle system, which can increase the solubility of chemotherapeutic drugs, inhibit P-gp protein, bioadhere, promote penetration and improve the oral absorption of hydrophobic chemotherapeutic drugs. Drug-loaded micelles were prepared, using paclitaxel (PTX) as model drug, synthetic functional amphiphilic copolymer as carrier material. The main contents of this study include synthesis, characterization and property study of the polymer, preparation drug-loaded micelles, and evaluation of bioadhesion, intestinal absorption, endocytosis mechanisms, in vivo pharmacokinetics. Moreover, mechanisms at celluar level of PTX-loaded micelles were also investigated.Three kinds of thiolated amphiphilic copolymer of chitosan-vitamin E succinate-N-acetylcysteine (CS-VES-NAC) was synthesized by using chitosan as hydrophilic backbone, vitamin E succinate as hydrophobic core, N-acetylcysteine as functional groups. The chemical structure of the copolymer was identified by IR,1H-NMR, TGA and EDS. Free thiol groups of CS-VES-NAC-1,-2,-3copolymers examined by Ellman’s method were182.6,275.5and451.5μmol/g, respectively, which render the copolymer has good swellaing behavior. Stability test results indicate that free sulfhydryl groups have good stability at the conditions of4℃, room temperature,37℃, and in pH1.2,5.4,6.8buffer solutions, which can meet the preparation requirements.The micelles were prepared by probe ultrasonication method. With the increase of sulfhydrylation substitution, mean diameters of blank CS-VES-NAC-1,-2,-3micelles were240.0,228.2and192.2nm, respectively. Zeta potential was+59.4,+58.4and+51.9mv, respectively. PTX was used as model drug to prepare the drug-loaded micelles. The effects of drug loading ratio on particle size and zeta potential were investigated. The particle size of drug-loaded micelles ranged from180nm to250nm, the encapsulation efficiency was about60%-80%, and the zeta potentials were all above+40mv. Moreover, sulfhydrylation substitution degree also has an effect on particle size and zeta potential. The images of drug-loaded micelles were spherical and round, with a narrow size distribution and a clear core-shell structure. Average particle size was about200nm, which was consistent with the measurement results of dynamic light scattering. DSC, X-ray results showed that PTX was in an amorphous form in the micellar core. Drug-loaded micelles showed a sustained release profile, which can maintain the stability of PTX and avoid drug leakage in physiological state.Mucin (type Ⅲ) was used as experimental model to study the biological adhesion. The results showed that CS-VES-NAC copolymer micelles could interact with mucin by covalent disulfide bonds, which can improve the adhesion significantly. Intestinal absorption of nanomicelles was investigated by in situ intestinal perfusion method. The results indicated that the absorption rate (Ka) of nanomicelles was higher than that of PTX solution, wherein CS-VES-NAC-1nanomicelles showed the most preferable absorption. The absorption rate was4.51times of PTX solution in duodenum segment. Besides, the apparent permeability (Papp) of CS-VES-NAC nanomicelles was also higher than that of PTX solution in duodenum, jejunum and ileum segments. The value of CS-VES-NAC-1nanomicelles was4.64times of pacllitaxel solution in duodenum segment. Intestinal absorption mechanisms showed that clathrin-dependent pathway, caveolin-dependent pathway, macropinocytosis, non-clathrin, non-caveolin-dependent pathway were all involved in the endocytosis process of CS-VES-NAC nanomicelles. Confocal laser scanning microscopy showed that after oral administration, thiolated micelles showed better permeability than that of free drug and CS-VES micelles.UPLC-MS/MS method was used to study the pharmacokinetics of PTX solution and PTX-loaded micelles. The experiment was conducted by duodenum administration, while PTX solution was intravenously administrated as control to obtain absolute bioavailability. The results showed that the relative bioavailability of PTX solution, CS-VES micelles, and CS-VES-NAC-1,-2,-3micelles was3.21%,3.39%,13.65%,13.33%and7.68%, respectively. AUC0-24h of CS-VES and CS-VES-NAC-1,-2,-3micelles was1.06,4.25,5.15and2.39fold higher than that of PTX solution, and the values of t1/2were1.33,4.31,1.78and1.25times higher than PTX solution. These indicated that thiolated polymeric micelles could increase the blood concentration of paclitaxel, increase bioavailability and prolong the systemic circulation.Human colon cancer cells (Caco-2) was applied to investigate the cellular biological mechanism of nanomicelles. Blank nanomicelles showed low cytotoxicity, suggesting good safety of the copolymer. Paclitaxel-loaded CS-VES-NAC nanomicelles could increase the cellular uptake due to bioadhesive interaction. Further, CS-VES-NAC-1nanomicelles could increase the permeation of PTX from apical side to basolateral side and decrease the efflux ratio of PTX by10-fold from the opposite direction (efflux ratio=2.13). The transcellular transport of micelles was energy-dependent, in which clathrin-dependent and caveolin-dependent pathway was both involved. Cellular uptake experiment also indicated that the P-gp inhibition effect of CS-VES and CS-VES-NAC-1copolymer could increase PTX cellular content. In addition, CS-VES-NAC-1copolymer could open the tight junctions between cells, increase paracellular transport, enhance the permeability of PTX solution by19.46-fold, and decrease the efflux ratio to0.75.