Preparation And In Vitro Evaluation Of Environment-responsive Nanoparticle Delivery System For Curcumin

Here, on the basis of the particularity of tumorous physiology, we established two environment-responsive nanoparticle drug delivery systems aiming at improving solubility and stability of curcumin. The content was divided into two parts.In the first part, we synthesized and evaluated the reduction-sensitive polymeric micelles of curcumin prodrug. The polymer was synthesized via three routes, from which the optimal route was determined. In the end, the polymer prodrug with disulfide bond was obtained and was named as MPEG-PLA-SS-CUR. The structure of synthesized polymers was confirmed by1H-NMR, FT-IR and UV-Vis methods.1H-NMR showed that the substitution rate of grafted CUR of the polymer was14%. UV-Vis methods confirmed the blue shift of the maximal absorption peaks. Pyrene fluorescence probe and DLS methods were employed to observe the self-assembled behavior in aqueous solutions of the polymer prodrug. The results showed that the CMC of MPEG-PLA-SS-CUR was1.25mg/L, with an average size of54.2nm.The polymer prodrug micelles were prepared by film dispersion method. To examine the drug release behavior of the polymer prodrug, we choosed the phosphate buffer of pH7.4with and without GSH10mM as the release medium. The outcome demonstrated that the reductive capability of the polymer in vitro. In the medium containing GSH, once the disulfide bond was cleaved by GSH, the release rate of curcumin grafed on the backbone of the polymer was relatively fast, exhibiting high accumulative weight. However, in the absence of GSH, the release rate was obviously more slowly.The MTT assay was adopted to study the effect of curcumin prodrug on the growth of human breast cancer cell line MCF-7and the resistant cell line MCF-7/ADR. The test showed that the polymer prodrug containing equal amount of curcumin exhibited enhanced cytotoxicity with lower IC50compared to the parent drug of curcumin. This indicated that the grafted curcumin on the polymeric prodrug released rapidly in reductive environment intracellularly and thus could inhibit the cell growth and revert the drug resistance of curcumin to MCF-7/ADR cells to some extent.In the second part, we established a pH-responsive amphiphilic polymer MPEG-PLA-PAE as polymeric micelles of curcumin based on polyethylene glycol-poly(lactic acid)-poly(amino esters). The synthetic route of MPEG-PLA-PAE was as follows. Firstly, the amphiphilic block copolymer MPEG-PLA that was primarily synthesized was reacted with acryloyl chloride to obtain the acrylated product of MPEG-PLA-A. The aimed product was obtained via Michael addition reaction with3-amino-l-propanol,1,4-butanediol diacrylate and MPEG-PLA-A.Curcumin-loaded pH-sensitive polymeric micelles were prepared by solvent evaporation method. To assess the stability of them, micelles with different drug-loading content were placed under various conditions including low temperature, room temperature and plasma. The results showed that all kinds of curcumin-loaded micelles of MPEG-PLA-PAE exhibited better stability under three conditions tested with encapsulation efficiency almost unchanged after24hours. However, the control group of MPEG-PLA drug loaded micelles showed greatly reduced stability. Only the group of5%loading content expressed good stability, while the encapsulation efficiency of high loading content group was remarkably reduced after24hours.We employed the phosphate buffer with different pH (7.4,6.5and5.5) as the release medium and examined the impact of pH values on the release behavior of curcumin-loaded MPEG-PLA-PAE micelles by dialysis method. The outcome showed that the drug-loaded micelles exhibited pH response in vitro. In a lower pH, the release of curcumin encapsulated in polymeric micelles was fast while the release was slowed down and the accumulative weight was reduced in a higher pH medium. We took it for granted that, in a lower pH, the structural change of micelles induced by protonated PAE blocks which was in the core of micelles accelerated release of curcumin.The flow cytometry and laser confocal microscopy (LSCM) were adopted to study the uptake of curcumin by human colon cancer cell line (HT-29). The results showed that pH-responsive curcumin-loaded micelles MPEG-PLA-PAE exhibited enhanced uptake compared to non-sensitive CUR-loaded micelles MPEG-PLA after two hours incubation with HT-29cells. The pH-responsive micelles which were charged positive slightly resulting from the weakly acid condition of tumorous physiology augmented the interaction between the drug-loaded miclles and cell membrane. As a result, the uptake of curcumin was increased by endocytosis pathway.Furthermore, we investigated the effect of drug-loaded micelles on the growth of human colon cancer HT-29cells. The results showed that the pH-responsive drug-loaded micelles significantly lowered the IC50compared to the normal drug-loaded micelles. This was in correspondence with the cellular uptake results.The researches performed above indicated that the synthesized two polymers exhibited reduction and pH-responsive characteristics, respectively. On the other hand, as nano-carriers, both polymers improved the solubility and stability of curcumin and enhanced the antitumor effect of curcumin.As a result, the two polymers provide great potential for increasing solubility, stability and enhancing therapeutic effects of hydrophobic drugs.