Validated HPLC method for paclitaxel determination in PLGA submicron particles conjugated with α-fetoprotein third domain: Sample preparation case study.

OBJECTIVES: The goal of this study was to develop sample preparation method and validate the HPLC method for precise determination of paclitaxel (Ptx) in PLGA submicron particles conjugated with protein vector molecule. METHODS: Ptx loaded PLGA submicron particles were formulated by a single emulsification method. PLGA submicron particles were conjugated with alpha fetoprotein third domain (rAFP3d) via standard carbodiimide technique. The obtained conjugate was analyzed using 1525 binary pump and 2487 UV-VIS detector system (Waters, USA) and Reprosil ODS C-18 analytical column with the dimensions of 150mm×4.6mm ID×5µm (Dr. Maisch GmbH, Germany). Sample preparation method was developed utilizing guard cartridge with С18 stationary phase (Phenomenex, USA). HPLC method was validated according to the international conference on harmonization guidelines. RESULTS: Efficient sample preparation was achieved using 4% of DMSO pre-dissolution, following by 10min of centrifugation at 4500g. Ptx determination was performed using acetonitrile/0.1% phosphoric acid (50:50 v/v) mobile phase at a flow rate of 1.0mL/min, injection volume of 10µL, and at 227nm. The developed method showed linearity, accuracy and precision in the range from 0.03 to 360µg/mL, with LOD and LOQ values of 0.005 and 0.03µg/mL, respectively. The intra- and inter-day precisions presented RSD values of lower than 2%. CONCLUSION: The validated method was successfully applied to calculate Ptx encapsulation efficacy and drug loading in the developed formulation.

Type of pH sensitive linker reveals different time-dependent intracellular localization, in vitro and in vivo efficiency in alpha-fetoprotein receptor targeted doxorubicin conjugate.

Despite the presence of a variety of modern anticancer drugs at the market, doxorubicin (Dox) is still widely used in antineoplastic therapy, although its administration causes severe side effects. To enhance specific activity of such molecules, various approaches have been exploited: targeted moieties like monoclonal antibodies, onco-specific proteins and peptides are utilized as specific vector molecules; environment sensitive linkers are exploited to facilitate transported drug release at the target point etc. Acid-labile linkers are frequently used in synthesis due to the ability to be cleaved inside specific cellular compartments with acidic environment, avoiding possible recycling mechanisms. Two types of conjugates containing different acid-labile linkers have been synthesized. In vitro efficiency of doxorubicin conjugates with recombinant receptor-binding domain of human alpha-fetoprotein (3dAFPpG) synthesized with use of cis-aconitic anhydride (CAA) and linker based on succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 3-(2-pyridyldithio)propionic acid hydrazide (PDPH) was compared. The 3dAFPpG-SPDP-PDPH-Dox revealed a comparable with unmodified doxorubicin cytotoxic effect against the Dox sensitive MCF7 cell line and greater cytotoxicity against the anthracycline resistant MCF7Adr cells. Meanwhile the 3dAFPpG-CAA-Dox cytotoxic effect was significantly lower, although doxorubicin's pH-dependent release profiles and intracellular accumulation rates were similar. These differences in cytotoxic activity were arguably explained by the dissimilarities in intracellular doxorubicin localization, which may originate from thiol reductase activity in lysosomes and late endosomes.