THE SEARCH FOR AN EFFECTIVE METHOD OF INCORPORATING A FLUORESCENT LABEL INTO LIPOSOMES COMPOSITION

DOI: https://doi.org/10.29296/25877313-2018-04-09
Issue: 
4
Year: 
2018

O.A. Kulikov Ph.D. (Med.), Associate Professor, National Research Ogarev Mordovia State University (Saransk) E-mail: oleg-kulikov-84@mail.ru V.P. Ageev Post-graduate Student, National Research Ogarev Mordovia State University (Saransk) V.I. Inchina Dr.Sc. (Med.), Professor, National Research Ogarev Mordovia State University (Saransk)

Objective. Determine the most effective method of incorporating a fluorescent dye into the composition of liposomal vesicles. Materials. Cyanine dye (Cy-7); Dimethylsulfoxide; Lecithin (phosphatidylcholine); Cholesterol; Chloroform; Purified deionized water; Sodium chloride. Equipment. Rotary evaporator; Extruder; Nanoparticle size analyzer; Spectrophotometer; Dialysis chamber; Dialysis bag; Magnetic stirrer with heating; Vacuum oven. Method for obtaining a liposomal dispersion with a dye Cyanine-7. Liposomes were obtained by reversing the phases from lecithin and cholesterol. The dye was included in the composition of liposomal vesicles by two different methods. Method 1 was that the lipid film after drying was hydrated with a solution of the Cy-7 dye. The dye was dissolved in a mixture of dimethylsulfoxide and water in a ratio of 1:4. Method 2 was that before during the formation of the lipid film, a solution of the Cy-7 dye in chloroform was added, and then drying took place. After drying, the lipid film containing the dye was hydrated with physiological sodium chloride solution. The concentration of the dye in the solutions of both methods was 125 μg/ml. In both methods, the process for obtaining liposome vesicles was extrusion using a polycarbonate filter with a pore diameter of 400 nm. The size of the liposomes was determined using a nanoparticle analyzer. The average diameter of the liposomes was 342 ± 32 nm for method 1 and 313 ± 2 nm for method 2. Purification of liposomes from free dye was performed by dialysis through a membrane with a pore diameter of 12-14 kDa under a nitrogen pressure of 0.3 MPa. Spectrophotometry was used to quantify the content of Cy-7 in liposomes. The dialyzate obtained in the purification of liposomes was subjected to spectrophotometry. The optical density of the dialysate containing the dye not included in the liposomes was measured at λ = 760 nm. The concentration of the dye in the dialysate was found by the calibration schedule. To construct the calibration schedule, samples of solutions with different concentrations of the fluorescent dye were used. The effectiveness of the inclusion of the dye Cy-7 in liposomes using method 1 was 86%, using method 2-99%. The ratio of the dye to the lecithin of the purified liposomes was 0.003 and 0.0025, respectively. Conclusion. Thus, using the second method for preparing liposomes, the dye incorporation efficiency is higher and the dimensions of the liposomal vesicles are more uniform.

Keywords: 
cyanine dye (Сy-7)
fluorescence
spectrophotometry

References: 
  1. Ma P., Yu H., Zhang X. et al. Increased Active Tumor Targeting by An αvβ3-Targeting and Cell-Penetrating Bifunctional Peptide-Mediated Dendrimer-Based Conjugate // Pharm Res. 2017. V. 34. № 1.P. 121–135.
  2. Yu Y., Wang Zhao-H., Zhang L. et al. Mitochondrial targeting topotecan-loaded liposomes for treating drug-resistant breast cancer and inhibiting invasive metastases of melanoma // Biomaterials. 2012. № 33. P. 1808–1820.
  3. Kilic E., Lim Su-H., Kulikov O. et al. Formulation for Oral Delivery of Lactoferrin Based on Bovine Serum Albumin and Tannic Acid Multilayer Microcapsules // Scientific Reports. 7:44159. DOI: 10.1038/srep44159.
  4. Chena Liang-C., Changa Chih-H., Yua Chia-Y. et al. Pharmacokinetics, micro-SPECT/CT imaging and therapeutic
  5. efficacy of 188Re-DXR-liposome in C26 colon carcinoma ascites mice model // Nuclear Medicine and Biology. 2008. № 35. P. 883–93.
  6. Zou A., Chen Y., Huo M. In vivo Studies of OctreotideModified N-Octyl-O,N-Carboxymethyl Chitosan MicellesLoaded with Doxorubicin for Tumor-Targeted Delivery // J. of Pharmaceutical sciences. 2013. V. 102. № 1. P. 126–35.