Folate-decorated Maleilated Pullulan-doxorubicin Polymeric Drug Delivery System: Preparation And Characterization

Despite the discovery and development of a spectrum of anticancer drugs for the treatment of cancers, their clinical outcomes have been disappointing due to severe side effects. The lack of tumor selectivity and recurrence of cancers with intrinsic or acquired drug resistance have also decreased the therapeutic efficacy of the drugs. To circumvent this problem, several drug delivery systems (DDS) have been developed. DDS based on polymer micelles, coated micro- and nanoparticles, liposomes and various prodrug systems including water-soluble polymer–drug conjugates and immunoconjugates have been prepared and extensively studied as novel drugs designed for cancer chemotherapy. To this regard, we have developed a FA-MP-DOX conjugate by three steps to enhance the therapeutic potential and reduce the systemic side effects of the drug. Pullulan was first modified with maleic anhydride to obtain maleilated pullulan (MP) which contained vinyl carboxylic acid groups. DOX was then chemically conjugated to MP via primary amide bonds; folic acid finally was bound to pendency hydroxyl groups of MP to produce FA-MP-DOX. The conjugation efficiency, stability and drug release of the conjugate were determined. And then f FA-MP-DOX conjugate nanoparticles were developed as carriers for doxorubicin for improved loading efficiency.1. Preparation of maleilated pullulan (MP)A certain amount of pullulan and maleic Anhydride were dissolved in a LiCl/DMF solvent system (10 wt %) under N2 gas. Triethylamine as catalyst (3 mol% to MA) was added to the mixed solution. Maleilated pullulan was obtained by this reaction, and the degree of substitution (DS) was 51 which was determained by 1H-NMR.2. Conjugation of doxorubicin to MP (MP-DOX)DOX was reacted with MP in the presence of EDC?HCL, NHS and TEA with the designed molar ratio in DMSO at 25℃under nitrogen atmosphere for 24h. The product was dialyzed for purification and saved after freeze-drying.3. Preparation of folic acid decorated maleilated pullulan- doxorubicin conjugate (FA-MP-DOX)Folic acid was first activated in anhydrous DMSO using EDC·HCl/NHSas catalysts for 24h under a nitrogen atmosphere at 25℃, and then MP-DOX and TEA were added in the above folic acid solution. Conjugation was allowed to take place for 24h under a nitrogen atmosphere at 25℃with stirring. FA-MP-DOX was obtained with the above Post-processing method. The polymer–drug conjugate was confirmed by FT-IR and1H-NMR analysis. The FA or DOX conjugation efficiency was determined by using microplate reader.4. In vitro release of DOX from the conjugateIn vitro drug release profiles were obtained by a dynamic dialysis method at various pH conditions. The released DOX was determined by fluorescence detection at 480 nm. The DOX release showed no dramatic initial burst and they are significantly pH-dependent. The lower the pH value was, the faster the drug released.5. Biological evaluation of FA-MP-DOX conjugateThe cellular uptake and in vitro cytotoxicity of the FA-MP-DOX conjugate were investigated by using ovarian cancer cell line A2780. Based on the IC50 values, the conjugate was found more effective with ovarian carcinoma A2780 cells than the parent drug after 48h culture. These results suggested that FA-MP-DOX conjugate could be a promising doxorubicin carrier for its targeted and intracellular delivery.5. Self-assembled nanoparticle from FA-MP-DOX conjugate and its charaterizationAs an extension of the previous work, we have developed FA-MP-DOX nanoparticles as carriers for DOX (abbreviated as FA-MP-DOX/DOX NPs) to provide an actively targetable drug delivery system for improved drug targeting of cancer cells with reduced side effects. And the DOX and FA content in the FA-MP-DOX conjugate were 7.5 wt%, 2.0 wt%, respectively. This proportion was confirmed the best for the formation of stable nanoparticles in our study. The resultant nanoparticles showed spherical geometry, with an average size of 150nm in diameter. The drug-release from nanoparticles were slow, pH-dependent sustained release, which did not show dramatic initial burst release.