Liposomal factor VIII as an efficient pharmaceutical system for the treatment of hemophilia

Document Type : Original Article


1 Department of Pharmaceutics and Pharmaceutical Nanotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

3 Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

4 Protein Technology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

5 Department of Pharmaceutical Nanotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran


Objective(s): Currently, the most important treatment approach for hemophilia type A is recombinant Factor VIII. However, due to its low retention time in the blood, the patients usually need successive injections. In addition, neutralization of injected proteins by antibodies complicates treatment. We examined the prolongation of the persistence time of injectable FVIII in the blood and the potential effects on survival using promising PEGylated liposomes (PEGLip) utilizing hydrogenated soy phosphatidylcholine (HSPC, Tm= 54.5 ºC) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC, Tm= – 2 ºC).
Materials and Methods: Nanoliposomes with different percentages of PEG (3% and 5%) were obtained via the thin film hydration procedure and extrusion. Liposomal FVIII formulation was prepared and characterization was done.
Results: The results revealed that the formulations are in the 80–120 nm range with uniform dispersion, which was confirmed using transmission electron microscopy (TEM) imaging. The phase transition temperature (Tm) of the liposomes was obtained by differential scanning calorimetry (DSC). With an attachment efficacy of approximately 87%, proteins bind non-covalently yet with a strong affinity to the exterior of PEGLip. The final formulations underwent additional examination. No significant change was observed in size, charge, and PDI between the FVIII-conjugated liposomal formulations and their liposomal nanoparticles. The selected formulations were injected into BALB/c mice. The circulation time and potential clotting effectiveness of PEGLip-FVIII are vastly improved over free protein, in non-hemophilic mice. 
Conclusion: The obtained results showed that using phospholipids with high Tm (HSPC) can improve the hemostatic efficiency of liposomes more than phospholipids with low Tm (POPC).


Main Subjects

1. Sanchez-Lara PA, Valentino LA. Genetics of hemophilia A and B.  Advances in Hemophilia Treatment: From Genetics to Joint Health: Springer; 2022. p. 11-17.
2. Veysel G, Ekrem Ü. Comprehensive approach to hemophilia. J Health Sci Med 2022; 5:1199-1206.
3. Cormier MC. Characterization of natural anti-FVIII antibodies and the regulatory role of the gut microbiota in FVIII immunogenicity: Doctoral dissertation, Queen’s University (Canada); 2023.
4. Hazendonk HCAM, van Moort I, Mathôt RAA, Fijnvandraat K, Leebeek FWG, Collins PW, et al. Setting the stage for individualized therapy in hemophilia: What role can pharmacokinetics play? Blood Rev 2018; 32:265-271.
5. Srivastava A, Brewer AK, Mauser-Bunschoten EP, Key NS, Kitchen S, Llinas A, et al. Guidelines for the management of hemophilia. haemophilia 2013; 19:e1-47.
6. Dias MM, Mesquita Camelo R, de Magalhães LP, Lemos Jardim L, Gonçalves de Oliveira A, Ribeiro RdA, et al. Changing recombinant factor VIII to plasma-derived factor VIII during immune tolerance induction. J Pediatr Hematol Oncol 2024; 41:74-80.
7.Chavin SI, Fay PJ. The Purification, Structure, and Function of Factor VIII.  Factor VIII—von Willebrand Factor: CRC Press; 2021. p. 1-23.
8. Furukawa S, Nogami K, Ogiwara K, Shima M. Potential role of activated factor VIII (FVIIIa) in FVIIa/tissue factor-dependent FXa generation in initiation phase of blood coagulation. Int J Hematol 2019; 109:390-401.
9. Tiede A. Half‐life extended factor VIII for the treatment of hemophilia A. J Thromb Haemost 2015; 13:S176-S179.
10. Di Minno MND, Di Minno A, Calcaterra I, Cimino E, Dell’Aquila F, Franchini M, editors. Enhanced half-life recombinant factor VIII concentrates for hemophilia A: insights from pivotal and extension studies. Seminars in Thrombosis and Hemostasis; 2021: Thieme Medical Publishers, Inc.
11. Wu Y, Sun SX, Fan T. Comparison of real-world dose and consumption for two extended half-life recombinant factor VIII products for the treatment of hemophilia A in the United States. J Blood Med 2022; 24:517-524.
12. Manco-Johnson M, Kempton C, Reding M, Lissitchkov T, Goranov S, Gercheva L, et al. Randomized, controlled, parallel-group trial of routine prophylaxis vs. on-demand treatment with sucrose-formulated recombinant factor VIII in adults with severe hemophilia A (SPINART). J Thromb Haemost 2013; 11:1119-1127.
13. Tieu P, Chan A, Matino D. Molecular mechanisms of inhibitor development in hemophilia. Mediterr J Hematol Infect Dis 2020; 12:e2020001.
14. Schep SJ, Schutgens REG, Fischer K, Boes ML. Review of immune tolerance induction in hemophilia A. Blood Rev 2018; 32:326-338.
15. Saenko EL, Pipe SW. Strategies towards a longer acting factor VIII. Haemophilia 2006; 12:42-51.
16. Croteau SE, Wang M, Wheeler AP. 2021 clinical trials update: Innovations in hemophilia therapy. Am J Hematol 2021; 96:128-144.
17. Verma SP, Tripathi AK, Sharma GS, Kumar N, Kushwaha R. Low dose long-acting factor VIII prophylaxis in pediatric and young adult patients with hemophilia A: Short-term single-center experience from a developing country. J Hematol Allied Sci 2021; 1:75-80.
18. Nakhaei P, Margiana R, Bokov DO, Abdelbasset WK, Jadidi Kouhbanani MA, Varma RS, et al. Liposomes: Structure, biomedical applications, and stability parameters with emphasis on cholesterol. Front bioeng biotechnol 2021; 9:705886.
19. Dos Santos Rodrigues B, Banerjee A, Kanekiyo T, Singh J. Functionalized liposomal nanoparticles for efficient gene delivery system to neuronal cell transfection. Int J Pharm 2019; 566:717-730.
20. Nsairat H, Khater D, Sayed U, Odeh F, Al Bawab A, Alshaer W. Liposomes: structure, composition, types, and clinical applications. Heliyon 2022; 8:e09394.
21. Kesharwani R, Jaiswal P, Patel DK, Yadav PK. Lipid-based drug delivery system (LBDDS): An emerging paradigm to enhance oral bioavailability of poorly soluble drugs.     Biomed Mater Diagn Devices 2022:1:648-663.
22. De Leo V, Milano F, Agostiano A, Catucci L. Recent advancements in polymer/liposome assembly for drug delivery: From surface modifications to hybrid vesicles. Polymers 2021; 13:1027.
23. Rao BLN, Krishnan SP, Reddy CB. Vesicular and stealth vesicular drug delivery–A review. J Pharm Res Int 2021; 33:76-88.
24.Gopi S, Balakrishnan P. Liposomal nanostructures: Properties and applications.  Nanoscale processing: Elsevier; 2021. p. 163-179.
25. Baru M, Carmel-Goren L, Barenholz Y, Dayan I, Ostropolets S, Slepoy I, et al. Factor VIII efficient and specific non-covalent binding to PEGylated liposomes enables prolongation of its circulation time and haemostatic efficacy. Thromb Haemost 2005; 93:1061-1068.
26. Powell JS, Nugent DJ, Harrison JA, Soni A, Luk A, Stass H, Gorina E. Safety and pharmacokinetics of a recombinant factor VIII with pegylated liposomes in severe hemophilia A. J Thromb Haemost 2008; 6:277-283.
27. Spira J, Plyushch OP, Andreeva TA, Khametova RN. Evaluation of liposomal dose in recombinant factor VIII reconstituted with pegylated liposomes for the treatment of patients with severe haemophilia A. Thromb Haemost 2008; 100:429-434.
28. Pan J, Liu T, Kim JY, Zhu D, Patel C, Cui ZH, et al. Enhanced efficacy of recombinant FVIII in noncovalent complex with PEGylated liposome in hemophilia A mice. Blood 2009; 114:2802-2811.
29. Spira J, Plyushch OP, Andreeva TA, Andreev Y. Prolonged bleeding-free period following prophylactic infusion of recombinant factor VIII reconstituted with pegylated liposomes. Blood 2006; 108:3668-3673.
30. Dayan I, Robinson M, Baru M. Enhancement of haemostatic efficacy of plasma-derived FVIII by formulation with PEGylated liposomes. Haemophilia 2009; 15:1006-1013.
31. Yatuv R, Dayan I, Carmel-Goren L, Robinson M, Aviv I, Goldenberg-Furmanov M, et al. Enhancement of factor VIIa haemostatic efficacy by formulation with PEGylated liposomes. Haemophilia 2008; 14:476-483.
32. Yazdi JR, Tafaghodi M, Sadri K, Mashreghi M, Nikpoor AR, Nikoofal-Sahlabadi S, et al. Folate targeted PEGylated liposomes for the oral delivery of insulin: In vitro and in vivo studies. Colloids Surf B Biointerfaces 2020; 194:111203.
33. Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem 1959; 234:466-468.
34. Matbou Riahi M, Sahebkar A, Sadri K, Nikoofal-Sahlabadi S, Jaafari MR. Stable and sustained release liposomal formulations of celecoxib: In vitro and in vivo anti-tumor evaluation. Int J Pharm 2018; 540:89-97.
35. Chountoulesi M, Naziris N, Pippa N, Pispas S, Demetzos C. Differential scanning calorimetry (DSC): An invaluable tool for the thermal evaluation of advanced chimeric liposomal drug delivery nanosystems. Thermodynamics and Biophysics of Biomedical Nanosystems: Applications and Practical Considerations 2019:297-337.
36. Heath TD, Macher BA, Papahadjopoulos D. Covalent attachment of immunoglobulins to liposomes via glycosphingolipids. Biochim Biophys Acta 1981; 640:66-81.
37. Powell JS. Liposomal approach towards the development of a longer-acting factor VIII. Haemophilia 2007; 13:23-28.
38. Yatuv R, Robinson M, Dayan-Tarshish I, Baru M. The use of PEGylated liposomes in the development of drug delivery applications for the treatment of hemophilia. Int J Nanomedicine 2010; 5:581-591.
39. Zadeh Mehrizi T, Mousavi Hosseini K. An overview on the investigation of nanomaterials’ effect on plasma components: immunoglobulins and coagulation factor VIII, 2010–2020 review. Nanoscale Adv 2021; 3:3730-3745.
40. High KA. The leak stops here: platelets as delivery vehicles for coagulation factors. J Clin Invest 2006; 116:1840-1842.
41. Girish A, Jolly K, Alsaadi N, de la Fuente M, Recchione A, An R, et al. Platelet-inspired intravenous nanomedicine for injury-targeted direct delivery of thrombin to augment hemostasis in coagulopathies. ACS Nano 2022; 16:16292-16313.