Construction And Evaluation Of Drug-loaded Self-assemblies Based On Amphiphilic Polyphosphazenes Containing EAB Groups For Cancer Therapy

The rapid development of nanotechnology has greatly promoted the application of nano drug delivery systems in cancer therapy. In recent years, a novel class of nanosized self-assemblies called polymersomes has emerged and catched the eyes of researchers. Compared to the micelles with hydrophobic payload only, polymersomes exhibite structure similarity of liposomes, which have great potential for drug delivery application.With polyphosphazene as backbone, polyethylene glycol monomethyl ether (mPEG) and ethyl-p-aminobenzoate (EAB) were grafted to synthesize the amphiphilic PEP copolymer. A series of PEPs were obtained via the graft ratio adjustment of PEG and EAB. The final product was subjected to a series of experiments including nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR) and gel permeation chromatography (GPC). Self-assemblies of PEP could be prepared via common dialysis method. Besides, the correspondent structures of PEPs were characterized by transmission electron microscopy (TEM) and cryo-TEM that micelles were formed with PEG graft ratio (fPEG(g)) of 0.89, transition states were observed with fPEF(g) of 0.86 and fPEG(g) between 0.79 and 0.67 favored polymersomes formation.Either doxorubicine hydrochloride (DOX·HCl) or doxorubicin (DOX) was encapsulated in PEP via common dialysis method to investigate the encapsulation property of PEP. The enhanced EAB graft ratio facilitated efficient encapsulation of drugs, especially PEP-5. Both DOX·HCl and DOX loaded PEP-5 achieved high drug loading content (LC) with PEP-5-DH(16.23%) and PEP-5-D(15.55%), as well as over 90% encapsulation efficiency (EE) with PEP-5-DH (97.30%) and PEP-5-D (93.30%). 2D 1H-1H NOESY spectrum proved the existence of strong inter-molecular forces between carrier and the drug which facilited the drug loading process. Drug release profiles demonstrated the release rate of PEP-5-DH was faster than PEP-5-D and also dependent on pH conditions. Cytotoxicity study against MCF-7 breast cancer cells revealed that hardly any cytotoxicity was discovered by PEP-5 treatment. PEP-5-DH demonstrated similar cytotoxicity with free DOX·HCl against MCF-7 cells, while PEP-5-D displayed reduced cytotoxicity due to poor water-solubility of DOX and the resulted slow release behavior. PEGylated nanoparticles had been widely reported to have enhanced permeability and retention (EPR) effect associated with passive targeting by which the side effects of chemotherapeutics could be reduced. Then, MCF-7 tumor bearing nude mice were used to evaluate PEP drug delivery system in vivo. The results showed that the PEP carrier, water-solubility of drug and drug administration all affected the efficacy of tumor growth inhibition. Drug-loaded PEPs exhibited significant toxic reduction compared to free DOX·HCl. Although the dosage of 2 mg/kg (daily in 3 days) was lower than 5 mg/kg (per 3 days,3 times), the 2 mg/kg treated groups showed better efficacy. For 2 mg/kg treated groups, PEP-5-DH exhibited improved life safety without efficacy compromise compared with free DOX·HCl, which made PEP polymersome as an ideal candidate of drug delivery system.In addition, PEP was also used to codeliver DOX·HCl/DOX together with chloroquine (CQ). CQ was a traditional antimalarial drug, which had also been reported as anticancer drug sensitizer against multidrug resistance (MDR) and studied in clinical trials. The mechanism might be associated with inhibition of drug efflux proteins or the apoptosis of cancer stem cell (CSC). The cytotoxicity study discovered that DOX·HCl and CQ combination had synergism which was characterized by Chou-Talalay combination index, furthermore, the drug resistance reversal was increassed with the CQ ratio. In order to simulate the in vivo environment, tumor spheroids of adriamycin resistant MCF-7/Adr breast cancer cells were developed and the drug permeation related tumor inhibition effect were characterized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Free DOX-HCl was observed only around the surface of tumor spheroid, however, the combination therapy could penetrate deeper, especially PEP-DHC-1 with DOX-HCl and CQ ratio of 1:1 could permeate throughout the whole tumor spheroid, besides, after 7 days incubation, the tumor spheroid was severely destroyed. The flow cytometry (FC) also showed that PEP-DHC-1 treatment induced the highest apoptosis of cells in tumor spheroid. Hopefully, the formulation would reverse drug resistance in vivo and enhance the anti-cancer efficacy of free DOX·HCl.In breif, PEP polymersome as a drug delivery system with improved safety and uncompromised anti-cancer drug efficacy, as well as the drug resistance reversal ability is worthy of further study.