| DOX (doxorubicin) displays potent anti-tumor activity, but the disadvantages limite its application such as the lack of tumor targeting, strong side effects, causing MDR. Meanwhile, CA4P (combretastatin-A4 phosphate) has an antivascular effect at a tenth of the MTD, but it has also some drawbacks such as quick elimination and inhibiting tumor not completely in vivo. For these reasons, we designed the polymersomes for co-delivery of DOX and CA4P (Ps-DOX-CA4P) to passively target tumor via EPR effect and reduce adverse side effects. We will focus on investigating Ps-DOX-CA4P effect on improving tumor inhibition rate, reversing drug resistance and their mechanisms.This study includes the following three parts:First, a series of mPEG-PLA with different weight ratios of hydrophilic chain to hydrophobic chain were synthesized by ring-opening polymerization. mPEG114-PLA162 was optimized as drug carriers, which has suitable fEO, can self-assemble and become vesicles, and has potential capacity of loading drugs. Then, blank polymersomes and Ps-DOX-CA4P were prepared by solvent evaporation method. The resulting blank polymersomes have vesicles shape with uniform sizes of about 50 nm. The bilayer membrane is composed of hydrophilic coronas both on the inside and outside of the hydrophobic membrane. The membrane thickness was approximately 10 nm In contrast to blank polymersomes, Ps-DOX-CA4P have similar size and shape. The EE of DOX and C A4P in Ps-DOX-CA4P were more than 90% and 50%, respectively. The results of drugs release from Ps-DOX-CA4P indicated that the release of hydrophobic DOX was slightly slower than that of hydrophilic CA4P.Second, the cytotoxicity of Ps-DOX-CA4P toward KB cells was determined by the MTT assay. The results showed that the blank polymersomes exhibited low cytotoxicity. Compared with Ps-CA4P or Ps-DOX, Ps-DOX-CA4P evidently increased inhibiting tumor effects. The KB cells xenografts model in nude mice is established to evaluate the biodistribution of DOX and anti-tumor efficacy. The results indicated that the fluorescence intensity in tumor tissues was much higher after injection with polymersome formulations than with free drugs (p<0.05), suggesting that the polymersomes with small sizes tended to accumulate in solid tumors because of the enhanced permeability and retention (EPR) effect. Ps-CA4P can inhibit tumor growth quickly, but transitorily. By contrast, though Ps-DOX cause the effect slowly, they exhibit sustained inhibition tumor growth. Notably, Ps-DOX-CA4P (1:10) showed quick and sustained inhibition tumor growth, resulting to significant synergistic effects. The results of immunostaining of CD31 and Ki67 indicated that Ps-DOX-CA4P (1:10) have not only antiangio genic effects but also synergistic anti-tumor.Finally, The potential molecular mechanisms of reversing drug resistance are verified by cytotoxicity, cellular uptake of DOX, apoptosis, ATPase activity, depletion ATP, intracellular ROS, P-gp expression, and multicellular tumor spheroids. The results showed that Ps-DOX-CA4P could effectively reverse drug resistance by increasing cytotoxicity and apoptotic rate of DOX, promoting cellular uptake of DOX, stimulating ATPase activity, depleting ATP, raising intracellular ROS levels, suggesting that Ps-DOX-CA4P are able to inhibit P-gp function. Concurrently, Ps-DOX-CA4P can obviously facilitate DOX penetration into the multicellular tumor spheroids and inhibit their growth, suggesting that Ps-DOX-CA4P could reverse P-gp-mediated drug resistance and sensitize chemotherapy in vivo. |
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