Synthesis and characterization of recombinant factor VIIa‐conjugated magnetic iron oxide nanoparticles for hemophilia treatment

Abstract

Maghemite (gamma-Fe(2)O(3)) nanoparticles of 15.0 +/- 2.1 nm in diameter were prepared by nucleation, followed by controlled growth of magnetic iron oxide thin films onto gelatin nuclei. Functionalization of these magnetic nanoparticles with activated double bonds was accomplished by interacting divinyl sulfone with the gelatin coating of the gamma-Fe(2)O(3) nanoparticles. The activated double bonds were then used for covalent binding, via Michael addition reaction, of recombinant factor VIIa and human serum albumin to the surface of these nanoparticles. Recombinant factor VIIa was also physically bound to the magnetic nanoparticles by interacting this factor with the human serum albumin conjugated gamma-Fe(2)O(3) nanoparticles. The influence of factor VIIa concentration on the immobilization yield has been elucidated. Leakage of the bound factor VIIa into PBS containing 4% albumin was insignificant. The coagulant activity of the physically adsorbed recombinant factor VIIa was similar to that of the free one and was significantly better than that of the covalently bound. The blood half-life of free factor VIIa is short, about 2-3 h, because of digestion by proteolytic enzymes and inhibitory effects. Stabilization of factor VIIa against trypsin (a model proteolytic enzyme) and chloromethyl ketone-type inhibitor was accomplished by conjugation of the factor to the gamma-Fe(2)O(3) nanoparticles. This stabilization may extend the blood half-life of factor VIIa. Therefore, IV injection of factor VIIa conjugated gamma-Fe(2)O(3) nanoparticles instead of free factor may avoid the frequent dosing and reduce the cost of hemophilia treatment.

Novel magnetic fibrin hydrogel scaffolds containing thrombin and growth factors conjugated iron oxide nanoparticles for tissue engineering

“…A similar stabilization effect was reported previously by us for thrombin, 9,42 glial cell-derived neurotrophic factors (GDNF), 55 Factor VII, 46 and methotrexate 56 conjugated to the γ-Fe 2 O 3 nanoparticles. For a quantitative determination of the effect of free/ conjugated bFGF on the kinetics of the migration of the NOM cells from the MCs aggregates to the magnetic fibrin hydrogel scaffolds, the total number of cells in the fibrin scaffolds was counted using the ImageJ software.…”

“…The physical conjugation of these bioactive molecules onto the albumin-coated layer is based on the fact that albumin is a carrier protein with a high affinity to various exogenous and endogenous compounds. 45,46 The binding yield of albumin precipitated on the surface of the γ-Fe 2 O 3 nanoparticles following addition of 5 mg albumin to 10 mg nanoparticles dispersed in PBS is 99.4% (496.7 µg albumin/mg nanoparticles), as determined by Bradford assay. The concentrations and the conjugation yields of the thrombin and the bFGF bound to the γ-Fe 2 O 3 nanoparticles are shown in Table 1.…”

Antibody-conjugated, dual-modal, near-infrared fluorescent iron oxide nanoparticles for antiamyloidgenic activity and specific detection of amyloid-β fibrils

“…This observation may be a result of the fact that the immobilization of BAM10 to the nanoparticles increases its stability during the Aβ 40 fibrillation process, and thus prolongs its activity compared with that of the free antibody. A similar stabilization effect was reported previously by us for thrombin, 60 glial cell-derived neurotrophic factor, 61 basic fibroblast growth factor, 62 tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), 63 factor VIIa, 64 and methotrexate 65 conjugated to γ-Fe 2 O 3 nanoparticles. Long-term stability of thrombin, TRAIL, and methotrexate conjugated to the γ-Fe 2 O 3 nanoparticles compared with the free ones was also previously demonstrated.…”

Magnetic Scaffolds Enriched with Bioactive Nanoparticles for Tissue Engineering

“…The magnetic scaffolds were produced by the interaction of thrombin conjugated iron oxide (maghemite, γ‐Fe 2 O 3 ) nanoparticles of 23 ± 4.7 nm diameter and fibrinogen. These γ‐Fe 2 O 3 nanoparticles are of particular importance due to their high surface area to volume ratio, magnetic properties, biocompatibility, relative non‐toxicity and biodegradability 8–11. The fibrin viscous solution, before gelation, was subcutaneously injected into adult rats’ thighs, and the formed scaffolds were then visualized by MRI.…”

“…The main disadvantage of the free (non‐conjugated) growth factors is their short in vivo half‐life time due to rapid enzymatic degradation, leading to loss of biological activity and functions 12. In order to stabilize the growth factors, the magnetic scaffolds were enriched with growth factors, e.g., basic fibroblast growth factor (bFGF), beta nerve growth factor (βNGF) or glial cell‐derived neurotrophic factor (GDNF) by covalent conjugation to fluorescent rhodamine γ‐Fe 2 O 3 nanoparticles (R‐γ‐Fe 2 O 3 ) 8–11. These scaffolds enriched with the bioactive nanoparticles provided a supporting 3D environment for massive proliferation of various cells, e.g., nasal olfactory mucosa (NOM) and dorsal root ganglia (DRG) cells.…”