Development and characterization of lipidic cochleate containing recombinant factor VIII |
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Authors: | Razvan D. Miclea Prashant R. Varma Sathy V. Balu-Iyer |
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Affiliation: | a Department of Molecular and Cellular Biophysics and Biochemistry, Roswell Park Cancer Institute, Buffalo, New York 14263, USA b Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14260, USA |
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Abstract: | Hemophilia A, a life-threatening bleeding disorder, is caused by deficiency of factor VIII (FVIII). Replacement therapy using rFVIII is the first line therapy for hemophilia A. However, 15-30% of patients develop neutralizing antibody, mainly against the C2, A3 and A2 domains. It has been reported that PS-FVIII complex reduced total and neutralizing anti-rFVIII antibody titers in hemophilia A murine models. Here, we developed FVIII-containing cochleate cylinders, utilizing PS-Ca2+ interactions and characterized these particles for optimal in vivo properties using biophysical and biochemical techniques. Approximately 75% of the protein was associated with cochleate cylinders. Sandwich ELISA, acrylamide quenching and enzymatic digestion studies established that rFVIII was shielded from the bulk aqueous phase by the lipidic structures, possibly leading to improved in vivo stability. Freeze-thawing and rate-limiting diffusion studies revealed that small cochleate cylinders with a particle size of 500 nm or less could be generated. The release kinetics and in vivo experiments suggested that there is slow and sustained release of FVIII from the complex upon systemic exposure. In vivo studies using tail clip method indicated that FVIII-cochleate complex is effective and protects hemophilic mice from bleeding. Based on these studies, we speculate that the molecular interaction between FVIII and PS may provide a basis for the design of novel FVIII lipidic structures for delivery applications. |
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Keywords: | aPTT, activated partial thromboplastin time FVIII, B domain deleted recombinant factor VIII BPS, brain phosphatidylserine BSA, bovine serum albumin ELISA, enzyme-linked immunosorbent assay FVIII, factor VIII PB, phosphate buffer PBA, phosphate buffer with 1% bovine serum albumin PBT, Tween 20 containing phosphate buffer PS, phosphatidylserine rFVIII, recombinant human factor VIII SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis |
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