Poly(β-malic Acid)-Hydroxycamptothecin Conjugate: Preparation, Characterization And Biological Evaluation

Many anticancer drugs used in the past decades have shown conspicuouscytotoxicity to tumor cells. Despite the discovery and development of aspectrum of anticancer drugs for the treatment of cancers, their clinicaloutcomes have been disappointing due to severe side effects. Non-selectivity ofchemotherapy drugs could result in lethal damages to the adjacent normalproliferating cells, leading to discontinuation of the therapy before all malignantcells are killed. To overcome these problems, several polymer drug deliverysystems (PDDS) have been developed.In recent years, polymer-based nanomedicine and polymer micelles orpolymer–drug conjugates bearing hydrophobic drugs, has received increasingattention for its ability to improve the efficacy of cancer therapeutics. Organicvesicular nanocarriers, such as liposomes and micelles, have the advantage of low toxicity and the versatility to carry diverse drugs and conjugate to targetingagents. Small size and excellent biocompatibility DDS can circulate in thebloodstream for long periods of time, allowing them to reach the target site.10-Hydroxycamptothecin, HCPT, blocks the rejoining step of thebreakage-rejoining reaction of mammalian DNA topoisomerase Ⅰ (Topo Ⅰ).HCPT has shown significant antitumor activity to lung, ovarian, breast, pancreas,and stomach cancers. HCPT, however, like a number of other potent anticanceragents such as paclitaxel, is extremely water insoluble. Furthermore,pharmacology studies have determined that prolonged schedules ofadministration given continuously are required. Thus, this insolubility hasrestricted its clinical application. For these reasons, a number of water-solubleanalogs have been synthesized and a number of different formulationapproaches have been investigated.Poly (beta-malic acid), PMLA, is a biodegradable, non-toxic,nonimmunogenic and absolute biodegradable polyester. After degradation, itstays trace scarcely, and the degradation products are non-toxic. Because of itsbiocompatibility, poly (beta-malic acid) has been designed to serve asnanoconjugate platforms in drug delivery. PMLA has pendant carboxyl groupsavailable for chemical conjugation of drug, targeting and auxiliary modules.A novel poly (beta-malic acid)-hydroxycamptothecin conjugate(abbreviated as PMLA-HCPT) for active tumor targeting was set up. Themodified HCPT and the PMLA platform are conjugated by an amino bond. Theacidic pH of extracellular fluid of solid tumors (tumor tissue pH6.8or lower,physiological pH7.4) has extensively been documented. The amino bond can bebroken up to release free drugs within the tumor tissue. 1. Synthesis of poly malic acid (PMLA)Racemic and optically active poly (β-malic acids)(PMLA) was synthesized byring opening polymerization (ROP) of β-substituted-β-lactone derived fromL-aspartic acid. All intermediate compounds and the resulting products wereconfirmed and characterized by FT-IR,1H-NMR.2. Chemical modification of HCPTA NH2must be introduced to HCPT, to make HCPT conjugate to the platformvia a disulfide bond. The conjugates used in this study were synthesized using atwo-step procedure modified from Refs. HCPT was reacts with t-boc-glycinate,then the t-Boc protection group was removed by dissolving in a mixture ofmethylene chloride and TFA.3. Preparation of PMLA-HCPT prodrugModified HCPT was reacted with poly malic acid in the presence of EDC HCl,and triethylamine with the designed molar ratio in DMF at25℃, under nitrogenatmosphere. The product was dialyzed for purification and saved afterfreeze-drying. The structure of this prodrug was confirmed by FT-IR and1H-NMR.4. In vitro release of HCPT from the PMLA-HCPT conjugateIn vitro drug release profiles were obtained by a dynamic dialysis method atthree different pH conditions of7.4,6.8,5.6. The released HCPT wasdetermined by HPLC fluorometric method. In vitro HCPT release fromPMLA-HCPT conjugate occurred at a faster rate at acidic pH compared withneutral pH (7.4). The released free HCPT after16h of incubation at pH5.6,6.8and7.4was76.8%,47.2%and18.1%, respectively. 5. Biological evaluation of PMLA-HCPT conjugateThe cytotoxicity in vitro of the PMLA-HCPT conjugate was investigated byusing colorectal neoplasms cell line SW480. These results suggested that thecytotoxicity of PMLA-HCPT conjugates against SW-480cells were lower thanfree HCPT at physiological pH.