| Nowadays, cancer is one of the most serious diseases to threaten the human life. According to information statistics, drug resistance results in failure for more than 90% cancer patients in clinical chemotherapy. Various respects lead to the tumor drug resistance, among them, due to the rapid growth of tumor, the microenvironment represents some abnormal phenomena such as high pressure, hypoxia, angiogenesis and acidic environment. The high pressure in the tumor interstitium directly results in the difficulty for delivering anticancer drugs into tumor tissues. The anoxic environment activates hypoxia-inducible factor(HIF-1) in making cancer cells resistant to the drugs. And the angiogenesis encourages thrombosis and impaired blood supply. The tumor-specific abnormalities in the microenvironment set up another hindrance for systems reaching the tumor tissues. Therefore, how to increase the drug internalization into drug-resistant tumors will be a great challenge.To address this challenge, we fabricate a rational site-specific drug-releasing delivery system with long-time circulation, which could not only unload the tumor vascular inhibitor specifically in the tumor vessels to temporarily ‘normalize’ the tumor vasculature to facilitate drug delivery, but also trigger in the tumor cells to increase effective drug concentration in drug-resistant tumor cells. With this concept, combretastatin A4(CA4), one of the most potent antivascular agents, was used in our system. Macromolecular prodrug(Pasp-DOX) was chosen as anticancer drugs. To construct our system, a biocompatible poly(ethylene glycol)-polyhistidine(PEG-Phis) polypeptide was chosen as the drug carrier to combination delivery of these two drugs elicit synergistic effect in antitumor activity. We mainly study the synthesis and identification of the polymer, preparation of the micelles, evaluation of the pharmaceutical pharmacy, cytotoxicity, cellular uptake, endocytosis mechanism, pharmacokinetic study and pharmacodynamics.(1) The PEG-Phis block polymers were prepared by ring-opening polymerization. To precisely tuning the p H-responsive range of the block polymer, a series of PEG-Phis(PEG-Phis20, PEG-Phis40, PEG-Phis60 and PEG-Phis80) was synthesized, and the degree of polymerization(DP) of the polyhistidine block was calculated by 1H NMR spectra. The p H-responsive macromolecular prodrug Pasp-DOX was prepared by conjugating the ketone group of DOX with the hydrazine group of polyaspartate to form a hydrazone linker. The structure was also confirmed by 1H NMR spectra.(2) The dual-p H responsive system(PEG-Phis/Pasp-DOX/CA4) was prepared by assembling block polymer PEG-Phis, macromolecular prodrug Pasp-DOX and small molecular inhibitor CA4 via a single-step nanoprecipitation method. Comparing with PEG-Phis40, PEG-Phis60 could form relatively smaller and uniform nanoparticles, and exhibited much better stability when incubated with 20% FBS. The loading efficiency of DOX and CA4 in this system determined by UV and HPLC analyses was 96.1% and 98.1%, respectively. Herein, PEG-Phis60/Pasp-DOX/CA4 was chosen as the optimal system in our following studies. To confirm if PEG-Phis60/Pasp-DOX/CA4 nanoparticles could gradually swell and sequential release of encapsulated CA4 and conjugated DOX in the corresponding sites to finally enhance the drug concentration in tumors, the sizes of the nanoparticles at acidic conditions were measured. The diameters of the nanoparticles sharply changed from p H 7.4 to p H 6.6, suggesting enormous volume swelling in respond to the acidic environment. The TEM also confirmed the morphology change as the sphere structures of the nanoparticles did not exist. The drug release amount of PEG-Phis60/Pasp-DOX/CA4 nanoparticles at different p H buffers was tested. The release of CA4 was very slow at p H 7.4, whereas exhibited a rapid process when the p H changed to 6.6, with 8.7% vs. 64.1% for 12 h. This suggested that the nanoparticles was stable in physiological condition, but could quickly respond to the tumor extracellular p H to set the CA4 free. The conjugated DOX had an obviously fast and continuous release when the p H changed from 7.4 to 5.8, 8.1% vs. 57.1% for two weeks, respectively. This demonstrates that the system could initiate the primary p H response in tumor microenvironment to enhance effective drug accumulation in drug-resistant tumors and then the hydrazone bonds facilitated to break when the p H less than 5.8 to release DOX to kill the tumor cells.(3) HUVECs was used as a model cell line of tumor endothelial cells, and MCF-7 and MCF-7/ADR cells as a model of tumor cells, the results evaluate the drug system could sequentially induce cell apoptosis in tumor endothelial cells and tumor cells. The in vitro antitumor activities, cellular uptake, endocytosis mechanism of systems against MCF-7 and MCF-7/ADR cells were all determined. In MTT assay, most formulations including DOX, Pasp-DOX, DOX+CA4, Pasp-DOX+CA4, PEG-Phis60/DOX/CA4, PEG-Phis60/Pasp-DOX/CA4 resulted in high cytotoxicity against MCF-7 cells. However, the formulations demonstrated different cytotoxicity against the drug-resistant cell lines. Among them, PEG-Phis60/Pasp-DOX/CA4 still demonstrated relatively high cytotoxicity against MCF-7/ADR cells. The cellular uptake of systems was visualized with a BD Calibur and confocal laser scanning microscopy(CLSM). The accumulations significantly increased when treated with PEG-Phis60/Pasp-DOX/CA4, confirming that drug delivery by nanoparticles through endocytosis could significantly enhance cellular uptake in drug-resistant tumor cells. The internalization mechanism was further examined that the two nano delivery system entered into MCF-7 and MCF-7/ADR cells by different endocytosis.(4) The in vivo pharmacokinetics of all the formulations were investigated and calculated by non-compartment pharmacokinetic model. PEG-Phis60/Pasp-DOX/CA4 exhibited the longest circulation time, which was needed for passive delivery via enhanced permeability and retention effect(EPR) and better accumulationin tumors. The inhibition effect of PEG-Phis60/Pasp-DOX/CA4 was 91.8% which showed that the system induced better inhibition of tumor growth. Due to the DOX self-fluorescene, the DOX distribution in tumor tissues and normal organs of the treated mice were evaluated. The strongest DOX fluorescence was observed in the tumor treated with PEG-Phis60/Pasp-DOX/CA4 and lower biodistribution of normal organs was also examined. Masson Trichrome assay showed that PEG-Phis60/Pasp-DOX/CA4 could significantly reduced the amount of collagen compared to PBS control, suggesting the regulation of tumor microenvironment. To further confirm the effect of the tumor vascular inhibitor to normalize the tumor microenvironment, blood vessels were stained by using an anti-CD31 antibody, the results suggested the same tendency to Masson Trichrome assay. The cell apoptosis in the tumor tissues after treatments was also analyzed using TUNEL assay and hematoxylin-eosin(H&E) staining. Compared with other formulations, PEG-Phis60/Pasp-DOX/CA4 markedly increased the number of TUNEL-positive tumor cells. The WBC results revealed that PEG-Phis60/Pasp-DOX/CA4 displayed no obvious toxicity, moreover, the histological examination also suggested that PEG-Phis60/Pasp-DOX/CA4 showed less cytotoxicity to normal tissues, including heart, kidney and liver, suggesting the better biocompatibility of our system. |
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