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.

Determination of Carbon Chain Lengths of Fatty Acid Mixtures by Time Domain NMR.

Average carbon chain length is a key parameter that defines the quality of liquid biofuels. In this paper, a method for the determination of carbon chain lengths of fatty acid mixtures is presented. The approach is based on proton relaxation rates measured by time domain nuclear magnetic resonance. The spin-spin relaxation rates R2 were used for the estimation of the carbon chain lengths. The method was examined for the set of samples with different mean lengths of the main linear carbon chain. Samples were prepared using four different fatty acids and mixtures of two, three or four of these fatty acids. The correlation coefficient between the known and measured values was equal to 0.994. Based on the relaxation theory, a linear-like dependence between the relaxation rate and carbon chain length was briefly shown, which endorses the experimental results. The developed methodology for determining carbon chain lengths offers robustness and rapidity, which are significant advantages when it comes to online use of the method in real industrial environments.

Development of target delivery system based on actinomycin class drugs and recombinant alpha-fetoprotein.

A recombinant alpha-fetoprotein (rAFP) was obtained in the yeast P. pastoris system, and its functional activity was confirmed. A method for producing polymer particles loaded with dactinomycin was developed, and a conjugate of these nanoparticles with rAFP was synthesized. The efficiency of the obtained conjugate on the HeLa, SKOV3, and MG-63 tumor cells and the absence of toxicity on the normal cells was shown. Experiments in vivo demonstrated a significant increase in the antitumor efficacy of the conjugate at a lower general toxicity as compared to the commercially available dactinomycin.

[Accumulation of doxorubicin conjugates with dendritic polymer and vector protein in normal and tumor cells in vitro].

Accumulation of doxorubicin (Dox), its conjugates with the second generation dendritic polymer (G2-Dox) and vector pro- tein (recombinant third domain of alpha-fetoprotein - 3D-G2- Dox) in normal and tumor cells was studied in vitro within the framework of the development of selective transport system of anticancer drugs to the target cells. The objects of the study were cells of peripheral blood mononuclear fraction of healthy donors and cells of breast adenocarcinoma lines MCF-7 and MCF-7/MDR1, differing in chemosensitivity. G2-Dox and 3D-G2-Dox accumulated in tumor cells of the both lines better than free Dox (p<0,05). However removal of these drugs out of cells MCF-7 and MCF-7/MDR1 was significantly different: in the latter case all free Dox was excluded from the cells for 24 hours while Dox, accumulated in composition with dendrimers, still remained in the cells. It was important that 3D-G2-Dox (unlike the G2-Dox) accumulated in normal cells worse than free Dox (p<0.01). Thus, the results indicate that the use of 3D-G2-Dox is the most promising because it accumulates in tumor cells better and in normal cells worse than free Dox. Furthermore it can be assumed that the use of 3D-G2-Dox would be especially useful in cases of multi-drug resistance associated with the high expression of P-glycoprotein.

[The Combined Effects of Ionizing Radiation and Dendritic Polymers Loaded with Doxorubicin on the MCF-7 Breast Cancer Cell Line].

The dendritic polymers (dendrimers) are perspective nanocontainers for transportation of anticancer drugs into cells and a controlled release of the delivered substances. However, the combined effect of ionizing radiation and dendrimers loaded with anticancer drugs has been poorly studied and is the aim of this research. We used poliamidoamin (PAMAM) dendrimers of the second generation (G2) covalently conjugated with doxorubicin (Dox) via an acid labile linker, cis-aconitic anhydride. We compared the intracellular accumulation of Dox and growth rate of the MCF-7 cell culture under the single and combined action of ionizing radiation at a dose of 4 Gy, free Dox and G2-Dox. It was found that within 2 hours free Dox accumulated in cancer cells better than Dox connected with G2 dendrimers (p < 0.05 in the concentration range of 1-5 µmol/l). The intracellular accumulation of Dox was higher by 1.7 times for the free Dox than that connected with dendrimers (for concentration 0.5 µmol/l p = 0.02) after 26 hours of incubation. Like the intracellular accumulation of Dox, inhibition of the cell culture growth was more pronounced when using free Dox than G2-Dox in the case of both a single and combined action of these drugs. Subadditivity effects of the combined action of both drugs and ionizing radiation are shown in terms of reducing the number of tumor cells 24 hours after irradiation. The results indicate the need for further development of selective delivery systems for Doxin tumor cells, providing a more intense accumulation of anticancer drug in target cells.

Sensitivity and selectivity of electrochemical enzyme sensors for inhibitor determination.

The performance of electrochemical biosensors developed for the determination of inhibiting species is considered. The role of various factors affecting the analytical characteristics of biosensors, their selectivity toward inhibitors to be tested as well as operational characteristics is discussed. The choice of enzyme-inhibitor system, the influence of enzyme immobilization on the behaviour of a biosensor, the modes of the optimization of working conditions are discussed. Most conclusions are illustrated with the models of the application of biosensors for monitoring environmental pollutants.

Influence of surface-active compounds on the response and sensitivity of cholinesterase biosensors for inhibitor determination.

The influence of non-ionogenic surfactants, i.e., Tween-20, Triton X-100 and PEG-10,000, on the response of cholinesterase-based potentiometric biosensors and their sensitivity towards reversible and irreversible inhibitors were investigated. Acetyl- and butyrylcholinesterases were immobilized on nylon, cellulose nitrate films and tracing paper and were introduced into an assembly of potentiometric biosensors. The effect of surface-active compounds depends on the hydrophilic properties and porosity of the enzyme support material and the inhibition mechanism. In the range 0.002-0.3% m/v the surfactants show a reversible inhibiting effect on biosensor response. At lower concentrations (down to 10(-4)% m/v) the surfactants alter the analytical characteristics of reversible and irreversible inhibitor determination. The use of surface-active additives improves the biosensor selectivity in multi-component media.