Novel platelet substitutes: disk-shaped biodegradable nanosheets and their enhanced effects on platelet aggregation.

We have studied biocompatible spherical carriers carrying a dodecapeptide, HHLGGAKQAGDV (H12), on their surface as platelet substitutes. This peptide is a fibrinogen γ-chain carboxy-terminal sequence (γ400-411) and specifically recognizes the active form of glycoprotein IIb/IIIa on activated platelets. Our purpose is to assess the possibility of making a novel platelet substitute consisting of disk-shaped nanosheets having a large contact area for the targeting site, rather than conventional small contact area spherical carriers. The H12 peptide was conjugated to the surface of the free-standing nanosheets made of biodegradable poly(d,l-lactide-co-glycolide) (PLGA). These H12-PLGA nanosheets were fabricated onto 3 µm disk-shaped patterned hydrophobic octadecyl regions on a SiO(2) substrate. By way of comparison, spherical H12-PLGA microparticles with the same surface area and conjugation number of H12 were also prepared. The resulting H12-PLGA nanosheets specifically interacted with the activated platelets adhered on the collagen surface at twice the rate of the H12-PLGA microparticles under flow conditions, and showed platelet thrombus formation in a two-dimensional spreading manner. Thus, H12-PLGA nanosheets might be a suitable candidate novel platelet alternative substitute for infused human platelet concentrates for the treatment of bleeding in patients with severe thrombocytopenia.

Fabrication of free-standing albumin-nanosheets having heterosurfaces.

Sheet-shaped carriers, having both obverse and reverse surfaces and thus a large contact area for targeting a site, have several advantages over spherical-shaped carriers, which have an extremely small contact area for targeting sites. Here, we proposed a novel method to prepare a free-standing ultrathin and biocompatible nanosheet having heterosurfaces, by a combination of four processes: (1) specific adsorption of recombinant human serum albumin (rHSA) molecules onto a patterned octadecyltrimethoxysilane self-assembled monolayer region (ODS-SAM), (2) preparation of nanosheets of rHSA molecules bearing thiol groups (SH-rHSA) via two-dimensionally disulfide crosslinking, (3) surface modification of the resulting nanosheet, and (4) preparation of the free-standing nanosheet by detachment from the ODS-SAM. The SH-rHSA molecules at pH 5.0 and a concentration of 1 microg/mL were specifically adsorbed on the patterned ODS-SAM regions by hydrophobic interaction, and were two-dimensionally crosslinked in the presence of copper ion as an oxidant. The rHSA-nanosheets were then simply detached from the ODS-SAM by treatment with surfactant. We succeeded in the preparation of rectangular (10 microm x 30 microm) and ultrathin (4.5 +/- 1.0 nm) rHSA-nanosheets on a patterned ODS-SAM, and could also obtain free-standing rHSA-nanosheets having heterosurfaces by surface modification with fluorescent latex beads. Thus, the rHSA-nanosheets having heterosurfaces could be regarded as a new biomaterial for drug carriers, hemostatic reagents, wound dressing for burn injury, and so forth.