The effect of the liposomal form of alteplase on the effectiveness of thrombolysis in coronary arteries in acute myocardial infarction

A liposomal (Lip) formulation of tissue plasminogen activator, alteplase (AlT), has been developed. The quantitative and qualitative composition of the liposomes, as well as their physicochemical properties and proteolytic activity, have been studied in relation to the thrombolytic liposomal form. It was determined that a formulation consisting of liposomes with a phosphatidylcholine/cholesterol ratio of 1.5 : 1, and lipids/alteplase ratio of 1 : 1, is optimal for treating acute myocardial infarction (AMI) in experimental models. At different component ratios, liposomes had a negative zeta potential value greater than 30 mV, indicating their aggregative stability, even after storage for two days at 20 degrees Celsius. Liposomes derived from soy phosphatidylcholine showed greater colloidal stability with a zeta potential of approximately –57 mV and a lower hydrodynamic diameter of approximately 140 nanometers, compared to liposomes derived from egg phosphatidylcholine, which had a zeta potential around –35.4 mV and a hydrodynamic diameter around 220 nanometers. The initial content of free AlT in the liposome supernatant from egg phosphatidylcholine (Lipeg) was 15.0 ± 4.0 %. During the incubation period of 4 days, the concentration of free AlT decreased to 9.0 ± 4.5 %. In contrast, in liposomes derived from soy phosphatidylcholine (LipS), the content of free AlT increased from 11.0 ± 4.5 % to 32.5 ± 6.0 % over the same incubation period. The value of the proteolytic activity of tissue plasminogen activator (tPA) in the compositions of Lipeg(AlT) and Lips (AlT) depends on the type of phosphatidylcholine. The initial tPA activity in Lipeg (AlT) was 36.0 %, and after 1 day, it increased to 45 %. In Lips (AlT), the initial activity was 61.0 % and increased to 66 % after 1 day. When using the liposomal form of alteplase for delivery into the coronary arteries of rats with acute myocardial infarction (AMI), a more complete fibrin lysis is noted compared to animals receiving the native form of the drug. The developed system of targeted delivery of alteplase using soy liposomes has been shown to significantly improve the degree of coronary artery lumen restoration by more than 15 %, compared to the use of a conventional drug (p < 0.05). 

1. Tarakhovskii Yu. S. Intelligent lipid nanocontainers in targeted drug delivery. Мoscow, Publishing house LKI, 2011. 280 p. (in Russian).

2. Greineder C. F., Howard M. D., Carnemolla R., Cines D., Muzykantov V. Advanced drug delivery systems for antithrombotic agents. Blood, 2013, vol. 122., no. 9, pp. 1565–1575. https://doi.org/10.1182/blood-2013-03-453498

3. Varna M., Juenet M., Bayles R., Mazighi M., Chauvierre C., Letourneur D. Nanomedicine as a strategy to fight thrombotic diseases. Future Science OA, 2015, vol. 1, no. 4. https://doi.org/10.4155/fso.15.46

4. Koudelka S., Mikulik R., Mašek J., Raška M., Turánek Knotigová P., Miller A. D., Turánek J. Liposomal nanocarriers for plasminogen activators. Journal of Controlled Release, 2016, vol. 227, no. 10, pp. 45–57. https://doi.org/10.1016/j.jconrel.2016.02.019

5. Maherani B., Arab-Tehrany E., Mozafari M. R., Gaiani C., Linder M. Liposomes: A Review of Manufacturing Techniques and Targeting Strategies. Current Nanoscience, 2011, vol. 7, iss. 3, pp. 436 – 452. https://doi.org/10.2174/157341311795542453

6. Crommelin D. J. A., Hoogevest P., Storm G. The role of liposomes in clinical nanomedicine development. What now? Now what? Journal of Controlled Release, 2020, vol. 318, pp. 256–263. https://doi.org/10.1016/j.jconrel.2019.12.023

7. Krasnopolsky Yu. M., Balabanyan V. Yu., Shobolov D. L., Shvets V. I. Prospective clinical applications of nanosized drugs. Russian Journal of General Chemistry, 2013, vol. 83, pp. 2524–2540. https://doi.org/10.1134/s1070363213120517

8. Dubatouka K. I., Lutsik I. L., Cherniavsky E. A., Bondarenko E. S., Adzerikho I. E., Agabekov V. E. Preparation of complex formulations based on liposomal streptokinase and their pharmacokinetic characteristics. Doklady Natsional’noi akademii nauk Belarusi = Doklady of the National Academy of Sciences of Belarus, 2017, vol. 61, no. 6, pp. 50–57 (in Russian).

9. Lutik I. L., Vladimirskaya T. E., Chernyavsky E. A., Dubatovka E. I., Adzerikho I. E. Experimental study of the physicochemical, pharmacokinetic properties and degree of safety of a complex preparation of streptokinase based on fibrin-specific liposomes. Cardiology in Belarus, 2019, vol. 11, no. 5, pp. 729–743 (in Russian).

10. Vaidya B., Nayak M. K, Dash D., Agrawal G. P., Vyas S. P. Development and characterization of site specific target sensitive liposomes for the delivery of thrombolytic agents. International Journal of Pharmaceutics, 2011, vol. 403, no. 1–2, pp. 254–261. https://doi.org/10.1016/j.ijpharm.2010.10.028

11. Adzerikho I. E., Vladimirskaya T. E., Lutsik I. L., Dubatouka K. I., Agabekov V. E., Branovitskaya E. S., Chernyavsky E. A., Lugovska N. Fibrinspecific liposomes as a potential method of delivery of the thrombolytic preparation streptokinase. Journal of Thrombosis and Thrombolysis, 2022, vol. 53, no. 2, pp. 313–320. https://doi.org/10.1007/s11239-021-02614-0

12. Dubatouka K. I., Agabekov V. E., Lutsik I. L., Yatsevich V. N., Adzerikho I. E. Effect of liposomal streptokinase on D-dimers formation. Doklady Natsional’noi akademii nauk Belarusi = Doklady of the National Academy of Sciences of Belarus, 2016, vol. 60, no. 6, pp. 54–58 (in Russian).

13. Bradford M. M. A rapid and sensitive method for the estimation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976, vol. 72, no. 1-2, pp. 248–254. https://doi.org/10.1006/abio.1976.9999

14. Сompton S. J., Jones, G. G. Mechanism of dye response and interference in the Bradford protein assay, Analytical Biochemistry, 1985, vol. 151, no. 2, pp. 369–374. https://doi.org/10.1016/0003-2697(85)90190-3

15. Adzerikho I., Vladimirskaya T., Lutsik I., Dubatouka K., Agabekov V. Efficiency of targeted delivery of streptokinase based on fibrin-specific liposomes in the in vivo experiment. Drug Delivery and Translational Research, 2023, vol. 13, iss. 3, pp. 811–821. https://doi.org/10.1007/s13346-022-01242-2

16. Sgouris J. T., Inman J. K., McCall K. B., Hyndman L. A., Anderson H. D. The preparation of human fibrinolysin (plasmin). Vox Sanguinis, 1960, vol. 5, no. 4, pp. 357–376. https://doi.org/10.1111/j.1423-0410.1960.tb03750.x

17. Saxena V., Johnson C. G., Negussie A. H., Karun V. S., Dreher M. R., Wood B. J. Temperature-sensitive liposome-mediated delivery of thrombolytic agents. International Journal of Hyperthermia, 2015, vol. 31, no. 1, pp. 67–73. https://doi.org/10.3109/02656736.2014.991428

18. Kim J.-Y., Kim J.-K., Park J.-S., Byun Y., Kim C.-K. Biomaterials The use of PEGylated liposomes to prolong circulation lifetimes of tissue plasminogen activator. Biomaterials, 2009, vol. 30, no. 29, pp. 5751–5756. https://doi.org/10.1016/j.biomaterials.2009.07.021

19. Wang Y. J., Pearlman R. Stability and Characterization of Protein and Peptide Drugs. Pharmaceutical biotechnology. New York ; London, Plenum press Publ, 1993. 353 p. https://doi.org/10.1007/978-1-4899-1236-7

20. Shalimov S.A., Radzihovskij A.P., Kejsevich L.V. Manual of experimental surgery. Мoscow: Meditsina Publ, 1989. 144 p. (in Russian).

21. Absar S., Nahar K., Y. Kwon M., Ahsan F. Thrombus-targeted nanocarrier attenuates bleeding complications associated with conventional thrombolytic therapy. Pharmaceutical Research, 2013, vol. 30, pp. 1663–1676. https://doi.org/ 10.1007/s11095-013-1011-x