| Purpose:Nowadays, most anticancer drugs have poor selectivity and high toxicity, which limits their applications. The multi-drug resistance (MDR) is one of the major causes of unsuccessful therapy. Many kinds of reversing MDR agents have disadvantages of in vivo unstability, poor tumor targeting and high toxic side effects.Scientists all over the worlds have begun to scan some effective ingredients of low toxicity and revering MDR from Chinese drugs in recent years. Curcumin is a major effective ingredient of commonly-used traditional Chinese drug curcuma. It has many kinds of pharmacological activities and is described as one of an ideal antitumour agent. And acted as a Chinese herbal reversal agent, it has much more advantages than other chemical reversal agents. Despite great therapeutic potential of curcumin utilization for a variety of diseases, its clinical development has been hindered due to its poor water solubility, low bioavailability, fast metabolism and degradation.Biological nanocarriers can not only improve the solubility and bioavailability of drug, but also control the drug released and attenuate the toxic side effects.Based on the advantages of biological nanocarriers and the merits of curcumin, the purpose of present study was to produce CUR-PBCA-NPs by examining the anticancer activity and reversal mechanism in MCF-7/ADR, provide a basis of producing high effective, long-term, targeting and low toxicity curcumin and establish a new technological method for multi-drug resistance reversion.Methods:(1)Preparation and characterization of CUR-PBCA-NPs CUR-PBCA-NPs had been prepared by using anionic emulsion polymerization. The size, zeta potential and encapsulation ratio of the CUR-PBCA-NPs were evaluated to optimize the parameters of stabilizer, pH value, the concentration of?-BCA and curcumin.The size, morphology and Zeta potential were characterized by transmission electron microscopy (TEM) and Zetasizer Nano system. The encapsulation ratio and drug loading of curcumin were measured with UV-VIS spectrometer and HPLC at 420nm. The study of drug release behavior of CUR-PBCA-NPs with chitosan in vitro was performed by dialysis method.(2) Study of pharmacokinetics, anticancer activity in vitro and in vivoThe influences of growth inhibition of different concentrations of CUR-PBCA-NPs, PBCA-NPs and CUR on three kinds of hepatoma carcinoma cells like HepG2, Be17402 and Huh7 cell lines were investigated by MTT assay. The effect of CUR-PBCA-NPs, PBCA-NPs and CUR on cell cycles and apoptosis of HepG2 were studied with flow cytometry and fluorescent microscopy. The VEGF and COX-2 expression of HepG2 effected by CUR-PBCA-NPs, PBCA-NPs and CUR was measured by western blotting.The HCC nude mice models were established by injecting subcutaneously HepG2 cells into BALB/c-nu nude mice. The preparation of drugs like CUR-PBCA NPs, PBCA NPs and physiological saline were injected i.v. into the tail vein. The tumors were detected and the VEGF and COX-2 protein were measured by immunohistochemical method. The pharmacokinetics of CUR and CUR-PBCA NPs were studied in SD rats by determining the concentration of curcumin in serum.(3) Evaluation of overcoming multidrug resistance (MDR) effectThe MCF-7/ADR cells were selected as the model of resistant cell. The resistance index (RI) of CUR, CUR-PBCA-NPs and PBCA-NPs were studied by MTT assay. The effects of uptake of DOX and cell cycles of different concentration CUR-PBCA-NPs in MCF-7/ADR cells were investigated by flow cytometry. The expression of MDR1?MRP1?ABCG2 and Topo??proteins in MCF-7/ADR cells dealed with CUR, CUR-PBCA-NPs and PBCA-NPs were determined by western blotting, and the mechanism of reversing the MDR was studied.(4) Preparation of DOX-CUR-PBCA-NPs and study of drug resistanceThe PBCA nanoparticles co-encapsulated of DOX and CUR were prepared by emulsion polymerization. The size, zeta potential, drug loading and encapsulation ratio of the DOX-CUR-PBCA-NPs were evaluated to optimize the following parameters:the time intervals of added CUR from the initiation the polymerization reaction, the concentration of?-BCA, chitosan, doxorubicin and curcumin. DOX-CUR-PBCA-NPs were characterized by TEM, FTIR, GPC and DSC. The drug release behavior of DOX-CUR-PBCA-NPs in vitro was performed by dialysis method. Reversion efficiency of the formulations and various combination approaches were assessed using MTT assay and western blotting.Results:(1) When reaction volume was 10mL,the optimized emulsion polymerization to prepare CUR-PBCA NPs was as follows:pH=1.22, 100?L BCA monomer, 10mg chitosan, 1mg curcumin power, temperature 25?, stirring time 6h. In this condition, the CUR-PBCA NPs were spherical in shape and narrow in distribution. The average size of CUR-PBCA NPs was about 185±13.12nm in diameter, the drug loading and encapsulation ratio were 1.078±0.05% and 94.54±1.54%, respectively, and the zeta potential was +50.1±2.08mV. The drug release of CUR-PBCA NPs in vitro was according to the second phase release. Curcumin released from CUR-PBCA NPs was faster in the first 30min, then entered into release platform phase, and almost released completely after 7days. The obtained colloid solution were stabilized for at least half a year, there were no phase separation and deposition. The particle size and size distributions of had no significant change after half a year.(2) Both CUR-PBCA NPs and free CUR killed the HepG2, Be17402 and Huh7 cells were in a concentration and time dependent manner in vitro. While at the same concentration, CUR-PBCA NPs had more potent killing function than that of free CUR, which presented in different cell lines. IC50 of free CUR to HepG2 cells were 60 fold of CUR-PBCA NPs. According to flow cytometry and fluorescent microscopy, CUR-PBCA NPs induced HepG2 cells apoptosis at a time and dose dependent manner. When treated with CUR-PBCA NPs for 4h or longer time, HepG2 cells turned to circle, fell down from wall and proliferated slowly. Cell apoptosis percentage was gradually increased along with CUR-PBCA NPs concentration rising. The apoptosis rate at 10?g/mL,20?g/mL,40?g/mL,60?g/mL is about 4.68%, 14.25%,89.75%,99.95% respectively. After treatment with CUR-PBCA NPs, HepG2 cells were observed to block cell cycle in G2/M phase, which is similar to free CUR. The anticancer activity of CUR-PBCA NPs in vivo was studied in nude nice transplanted subcutaneously by HepG2 cell lines. The inhibitory effect of CUR-PBCA NPs on tumor growth 56.46% was stronger than that of normal saline group and PBCA NPs group (7.69%, P< 0.05).CUR-PBCA NPs significantly down-regulated protein level of VEGF and COX-2 in HepG2 cells and HepG2 xenografts. The levels of VEGF and COX-2 protein decreased significantly in the cells co-incubated with CUR-PBCA NPs at 20?g/mL for 48 h as compared with those treated with the same concentration of free CUR and in the control cells.(3) The drug concentration-time curves of CUR-PBCA NPs and CUR in mice blood were determined by HPLC, and the difference between the CUR-PBCA NPs and free CUR in vivo was observed. The pharmocokinetic parameters of CUR-PBCA NPs and free CUR were analyzed with DAS 2.1.1. The results showed that the area under the curve(AUC0-?)and the elimination half-life (t 1/2?) were 2 and 52 times bigger than that of free CUR, the mean residence time (MRT0-?) of CUR-PBCA NPs (63.787 h) was longer than that of free CUR (0.159h). There was a substantial increase in the volume of distribution (51-fold). This illustrated that the properties of particles (such as shape, size, charge, and hydrophilicity) can prolong the retention of CUR in the blood compartment. (4) The reversal index of CUR-PBCA NPs was almost two times of that of free CUR in MCF-7/ADR resistant cell lines. After co-administration of 10?g/mL DOX and 50?g/mL CUR-PBCA NPs, MCF-7/ADR cells were observed to arrest at G2/M phase. And blocked at S phase when co-administration of 40?g/mL DOX and 50?g/mL CUR-PBCA NPs. These differences are evidently remarkable compared with PBCA NPs and free DOX group. The amount of drug uptaken by MCF-7/ADR cells exposed to 50?g/mL CUR-PBCA NPs was significantly higher than that of free DOX solution (P< 0.05) at the same drug concentration.The CUR-PBCA NPs can cause down-regulated the expression of P-gp in MCF-7/ADR cell lines at a time dependent. After treatment with 50?g/mL CUR-PBCA NPs, the expression of P-gp began to decline on the 5th day and when the 7th day no P-gp could be measured. The MRP1 protein was decreased slower than that of MDR1, while there were no evident alternations of Topo Ila and ABCG2 protein. The results revealed that the mechanism of reversing the multi-drug resistance caused by CUR-PBCA NPs was likely to down-regulate the MDR expression but not to influence Topo??and ABCG2 expression obviously.(5) Co-encapsulated doxorubicin and curcumin in PBCA nanoparticles were prepared with emulsion polymerization. The mean particle size and the zeta potential of DOX-CUR-PBCA-NPs were 133±5nm in diameter and +32.23±4.56 mV, respectively. The entrapment efficiencies of DOX and CUR were 49.98±3.32% and 94.52±3.14%, respectively. The presence of associated DOX/CUR did not alter the release profile of CUR/DOX from the nanoparticles. Anticancer activities and reversal efficacy of the formulations and various combination approaches were assessed using MTT assay and western blotting. The results showed that the dual-agent loaded PBCA-NPs system had the similar cytotoxicity to co-administration of two single-agent loaded PBCA-NPs (DOX-PBCA-NPs + CUR-PBCA-NPs), which was slightly higher than that of the free drug combination (DOX-CUR) and one free drug/another agent loaded PBCA-NPs combination (DOX+CUR-PBCA-NPs or CUR+DOX-PBCA-NPs). The simultaneous administration of DOX and CUR could achieve the highest reversal efficacy, and down-regulate of P-gp in MCF-7/ADR cell lines. Multidrug-resistant can be enhanced by treated combination of encapsulated cytotoxic drugs and reversal agents.Conclusions:The novel cationic CUR-PBCA-NPs maintain and even enhance the anticancer activity and reversion P-gp mediated MDR in MCF-7/ADR of free curcumin. The solubility and bioavailability of curcumin have been improved. These colloidal delivery systems have been shown to increase the circulation time of curcumin in the serum. Co-encapsulation of anticancer drug DOX and reversal agent CUR can be more effective in reversing multi-drug resistance than the other formulations and might cause lower normal tissue drug toxicity and fewer drug-drug interactions. Nanodrugs have not only opened a new horizon to the treatment of disease, but also provided an idea for producing safe, reasonable and effective anticancer agents. It is anticipated that drug delivery system could contribute greatly to reversing MDR. Being a reliable and safe way to reverse MDR, drug delivery system represents a promising prospect both in research and application in recent years. |
PDF Full Text Request