| The serious side effects and MDR become the two major problems in cancer therapy. Doxorubicin hydrochloride (DOX) is one of the most widely used anthracycline antitumor drugs. However, the serious side effects such as cardiotoxicity limit the application of DOX on clinical greately. In addition, many cancer cells show MDR to DOX with the wide use of various drugs. MDR is mostly related to the over-expressed proteins on cell membranes, such as p-glycoprotein (P-gp). The over-expression of P-gp can pump P-gp substrate such as DOX out of the plasma membrane and lower the levels of drugs in the cytoplasm to decrease the anti-tumor effect. Verapamil hydrochloride (VER), a kind of P-gp inhibitor can prevent the pump-out of DOX by P-gp. Therefore, the combination of DOX and VER can improve the anticancer efficacy of DOX. However, the free VER administration can cause an additive cardiotoxicity. To reduce cardiotoxicity of both DOX and VER, drug delivery systems with high targeting ability are needed.The biodegradable polymers have become the main hot point in the field of drug delivery systems, such as PEG, PCLet al. The circulation time of drugs encapsulated in these polymers can be extended and the activities can be protected. With the development of polymer science, the polypeptides with secondary conformation have been widely studied. This is because that their physicochemical properties can be changed by varying the external environment such as pH and temperature. For example, the alanine and phenylalanine show high sensibility to temperature; the water solubility of glutamic acid and lysine can be tuned by varying the pH values. Therefore, the polypeptide-based polymers can be applied in the drug delivery systems with environmental-sensitivity.Folic acid (FA), a targeting ligand, can combine with folate receptor (FR) with high affinity to realize the highly active targeting. More importantly, it has been confirmed that the FR is only up-regulated in some cancer cells such as malignancies of the ovary and breast cells. Thus, the folate-modified drug delivery carriers could be internalized into cells via a specialized recognization with high efficiency.In this thesis, we have synthesized the pH-sensitive mPEG2K-PCL4K-PGAiK triblock copolymer to prepare co-delivery system poly(DOX+VER). The poly(DOX+VER) with sustained-release properties and pH-sensitivity could reverse the MDR efficiently. In a further step, the co-delivery system poly(DOX+VER) was modified by FA to form the active targeting co-delivery system FA-poly(DOX+VER). The thesis was divided into tree parts as below:1. Synthesis and characterization of mPEG2K-PCL4K-PGAiKThe mPEG2K-PCL4K-PGA1K was synthesized by ROP reaction in five steps:(1) The mPEG2K-PCL4K-OH was synthesized via ROP of ε-caprolactone and mPEG2K using stannous octoate as a catalyst; (2) The OH end-group of mPEG-PCL-OH was then reacted with Phe-NBOC under the catalysis of DCC and DMAP to synthesize mPEG2K-PCL4K-Phe-NBOC; (3) To obtain mPEG2K-PCL4K-NH2, the BOC group was removed by the acid hydrolysis of TFA; (4) The NCA-BLG was also reacted with the NH2 end-group of mPEG2K-PCL4K-NH2 through ROP reaction to form the mPEG2K-PCL4K-PBLG1K; (5) The benzyl group was removed and the desired product mPEG2K-PCL4K-PGA1K was obtained. The copolymer could assemble into polymersomes in aqueous solution and had low critical aggregation concentration (CAC) 0.0776 mg·L-1 (1.1086×105 mol·L-1). The low hemolysis ratio of mPEG2K-PCL4K-PGA1K confirmed that the copolymer show high biocompatibility for the application of intravenous injection.2. Studies on mPEG2K-PCL4K-PGA1K co-delivery systemThe co-delivery system of DOX and VER poly(DOX+VER) was prepared via dialysis method. We have test the size and drug-loading efficiency of poly(DOX+VER), as well as the influence of feed weight ratio of drugs on DL and EE. For high DL and EE for both drugs, the best feed weight ratio of DOX and VER was 1:1.5. And at this ratio, the DL and EE of DOX was (5.05±0.26)% and (59.89±3.40)% while that of VER was (10.72±1.08)%and (84.82±9.53)%, respectively. The release behavior of drug showed that the release rate of drugs from poly(DOX+VER) were much slower than that of free drugs. Compared with the release rate of drugs in pH5.0 PBS, the drugs released slower in pH7.4 PBS. All the results indicated that the poly(DOX+VER) showed sustained and pH-sensitive drug release behaviour. Therefore, the poly(DOX+VER) could accelerate the drug release and improve the accumulation of drugs in acidic tumor issue to enhance the anti-cancer effect. In addition, the addition of VER has no effect on the release of DOX which was proved by the almost same release plots for the polyDOX and poly(DOX+VER) in pH7.4 and pH5.0 PBS. To verify the MDR reversal of poly(DOX+VER), the cytotoxicity of DOX. polyDOX and poly(DOX+VER) on MCF-7 and MCF-7/ADR cells was tested via MTT method. Free DOX showed a little higher inhibition ratio to MCF-7 cells than that of polyDOX or poly(DOX+VER) sample due to the incomplete release of DOX from drug-loaded polymersomes. The polyDOX and poly(DOX+VER) exhibited similar inhibition ratio indicated that the VER had almost no cytotoxicity to cancer cells. However, for the MCF-7/ADR cells, due to the pump-out of DOX by P-gp, the inhibitory effect of free DOX or polyDOX was much weaker than that of poly(DOX+VER), indicating that the addition of P-gp inhibitor VER could significantly inhibit the activity of P-gp and improve the cytotoxicity of DOX to resistant cancer cells. In addition, the cellular uptake studies showed that the cellular uptake of DOX. polyDOX and poly(DOX+VER) by MCF-7 was almost similar. However, the cellular uptake of poly(DOX+VER) by MCF-7/ADR cells was stronger than that of DOX and poiyDOX. In a word, the cellular uptake studies also confirmed that poly(DOX+VER) could inhibit the activity of P-gp and prevent the pump-out of DOX.3. Studies on active targeted mPEG2K-PCL4K-PGA1K co-delivery systemThe folate-modified pH-sensitive mPEG2K-PCL4K-PGA1K was synthesized as follows:FA (folic acid) was firstly reacted with NHS (N-hydroxy succinimide) to form the active ester intermediate (NHS-folate) using EDC·HCl (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride) as a catalyst, the anhydride of NHS-folate was then reacted with the amine group of mPEG2K-PCL4K-PGA1K under alkaline conditions for 12 h. The folate-modified mPEG2K-PCL4K-PGA1K also had low critical aggregation concentration (CAC) 0.2969 mg·L-1 (3.9901×105 mol·L-1) and low hemolysis ratio, and the FA content was 3.19%. The folate-modified co-delivery system of DOX and VER FA-poly(DOX+VER) was also prepared by dialysis method. The experiment of drug release in vitro also confirmed that FA-poly(DOX+VER) exhibited sustained and pH-sensitive drug release behaviour. Comparing the inhibition ratio of FA-polyDOX and FA-poly(DOX+VER) on MCF-7 and MCF-7/ADR cells usisng MTT method, we could conclude that the addition of VER in FA-poly(DOX+VER) significantly improve the anticancer effect of DOX to MCF-7/ADR cells. Therefore, the FA-poly(DOX+VER) could reverse the MDR of resistant cancer cells efficiently. The targeting ability of folate-modified drug carrier was confirmed via comparing the cellular uptake of polyDOX and FA-polyDOX by MCF-7, and the cellular uptake of poly(DOX+VER) and FA-poly(DOX+VER) by MCF-7/ADR cells. It could be seen that both MCF-7 and MCF-7/ADR showed higher uptake of folate-modified drug delivery systems (FA-polyDOX and FA-poly(DOX+VER)). Therefore, it confirmed that the modification of FA could improve the uptake of drug delivery systems and improve the accumulation of drugs in cancer cells in a further step.In general, we have synthesized pH-sensitive mPEG2K-PCL4K-PGA1K and folate-modified mPEG2K-PCL4K-PGA1K which were then applied to co-deliver DOX and VER to form co-delivery systems poly(DOX+VER) and FA-poly(DOX+VER), respectively. The results of experiments indicated that the release of drug from poly(DOX+VER) and FA-poly(DOX+VER) had sustained and pH-sensitive behavior. In addition, they could also reverse the MDR of resistant cancer cells and improve the anticancer effect of DOX. Moreover, the modification of FA also increase the cellular uptake of FA-poly(DOX+VER) and thus enhancing the anticancer efficacy in a further step. Therefore, the study offered an intellectual rationale and had an important practical significance for the development of FA receptor-mediated active targeting drug delivery systems with high MDR reversal. |
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