The effect of surface functionalization of PLGA nanoparticles by heparin- or chitosan-conjugated Pluronic on tumor targeting.

The poly (lactide-co-glycolide) (PLGA)-based nanoparticles, coated by the heparin- or chitosan-Pluronic conjugate, were used to improve a relatively low tumor-targeting efficiency of the bare PLGA nanoparticles. The prepared nanoparticles were in the size range of 100-150nm, and the surface exposure of the functional moiety (heparin or chitosan) was confirmed by negatively or positively increased zeta potential values, respectively. The viability tests for both normal and tumor cells displayed minimal cytotoxicity of the nanoparticles. The stable surface coating, which was evident from no change in the size distribution profiles in spite of the surface charge changes in serum environment, effectively provided the desired functionalized surface that clearly enhanced the in vitro cellular uptake of the nanoparticles for both heparin and chitosan functionalization. The in vivo tumor model study, which was carried out in SCC7 tumor-bearing athymic mice, demonstrated that there was a limited, but positive effect of surface functionalization, more effective for chitosan functionalization. The accumulation of chitosan-functionalized PLGA nanoparticles in tumor was 2.4 folds higher than that of the control, PLGA nanoparticles coated with bare Pluronic, and the accumulation in liver was lower than the control. In the case of heparin functionalization, the mean value was 2.2 folds higher than that of the control, but the accumulation in liver was similar to that of the control. Therefore, the surface-functionalization by the chitosan- or heparin-conjugated Pluronic may be an effective approach for the hydrophobic nanoparticle systems aiming for the enhanced tumor imaging and therapy.

Silk fibroin nanoparticles for cellular uptake and control release.

Silk nanoparticles were prepared from silk fibroin solutions of domesticated Bombyx mori and tropical tasar silkworm Antheraea mylitta and investigated in respect to its particle size, surface charge, stability and morphology along with its cellular uptake and release of growth factors. The nanoparticles were stable, spherical, negatively charged, 150-170nm in average diameter and exhibited mostly Silk II (beta-sheet) structure and did not impose any overt toxicity. Cellular uptake studies showed the accumulation of fluorescence isothiocyanate conjugated silk nanoparticles in the cytosol of murine squamous cell carcinoma cells. In vitro VEGF release from the nanoparticles showed a significantly sustained release over 3 weeks, signifying the potential application as a growth factor delivery system.

Efficient revascularization by VEGF administration via heparin-functionalized nanoparticle-fibrin complex.

We investigated the angiogenic bioactivity and therapeutic angiogenic effect of vascular endothelial growth factor (VEGF) administration by the heparin-functionalized nanoparticle-fibrin gel complex. The markedly increased bioactivity was observed by the VEGF-loaded nanoparticle-fibrin gel complex, compared to the VEGF-loaded fibrin gel, the nanoparticle-fibrin gel complex without VEGF, or fibrin gel (control) in terms of the capillary density in a mouse subcutaneous implantation model. Furthermore, the VEGF-loaded nanoparticle-fibrin gel complex significantly enhanced the therapeutic angiogenic effect in a rabbit ischemic hind limb model: the noticeable increase in the recovered calf blood pressure, the angiographic score, and the density of collaterals, as well as the stable maintenance of the organized collaterals, compared to the VEGF-loaded fibrin gel. These results show the enhanced angiogenic potential of VEGF administration by the proposed heparin-functionalized nanoparticle-fibrin gel complex.

In situ chondrogenic differentiation of human adipose tissue-derived stem cells in a TGF-beta1 loaded fibrin-poly(lactide-caprolactone) nanoparticulate complex.

When conducting cartilage tissue engineering with stem cells, it is well known that chemical and physical stimulations are very important for the induction and maintenance of chondrogenesis. In this study, we induced chondrogenic differentiation of human adipose tissue-derived stem cells (hASCs) in situ by effective stimulation via the continuous controlled release of TGF-beta1 from a heparin-functionalized nanoparticle-fibrin-poly(lactide-co-caprolactone) (PLCL) complex. PLCL scaffolds were fabricated with 85% porosity and 300-500 microm pore size by a gel-pressing method. Heparin-functionalized nanoparticles were prepared by a solvent-diffusion method, composed of poly(lactide-co-glycolide) (PLGA), Pluronic F-127, and heparin, and then TGF-beta1 was loaded to the nanoparticles. A mixture of hASCs, fibrin gels and TGF-beta1 loaded nanoparticles was then seeded onto PLCL scaffolds and cultured in vitro, after which they were subcutaneously implanted into nude mice for up to five weeks. The results of in vitro and in vivo studies revealed that chondrogenic differentiation of the hASCs on the complex was induced and sustained by continuous stimulation by TGF-beta1 from the heparin-functionalized nanoparticles. In addition, there was no significant difference between the predifferentiation condition prior to incubation in chondrogenic medium and the proliferation condition, which suggests that in situ chondrogenic differentiation of hASCs was induced by the TGF-beta1 loaded nanoparticles. Consequently, the hybridization of fibrin and PLCL scaffolds for three-dimensional spatial organization of cells and the effective delivery of TGF-beta1 using heparin-functionalized nanoparticles can induce hASCs to differentiate to a chondrogenic lineage and maintain their phenotypes.

Preparation of liposomes containing oleanolic acid via micelle-to-vesicle transition.

Micelle-to-vesicle transition method was used to make liposomes containing oleanolic acid. First, the solubilization of potassium salt of oleanolic acid at basic condition by micelle formation was confirmed. Using the soluble state of oleanolic acid at basic condition, liposomes containing oleanolic acid was prepared by adjusting pH. After making homogeneous aqueous mixture of potassium salt of oleanolic acid and lecithin in basic condition, the solution was neutralized to produce the lecithin-based liposomes that contain oleanolic acid inside the lipid bilayers. The optimal loading of oleanolic acid to lecithin (about 25 mole%) was found to exist to produce liposomal suspension of small size without homogenization step. Electron microscopy and dynamic light scattering studies showed that the narrowly distributed, reconstituted oleanolic acid-containing liposomes were prepared without severe mechanical treatment.

Enhanced bone regeneration with BMP-2 loaded functional nanoparticle-hydrogel complex.

As an efficient sustained release system of BMP-2, a functional nanoparticle-hydrogel complex, composed of heparin-functionalized nanoparticles and fibrin gel, was developed and used as a bone graft. In vivo bone formation was evaluated by soft X-ray, histology, alkaline phosphatase (ALP) activity, immunostaining, and mineral content analysis, based on the rat calvarial critical size defect model. Significantly improved and effective bone regeneration was achieved with the recombinant BMP-2 (4 mug) loaded nanoparticle-fibrin gel complex, as compared to bare fibrin gel, the nanoparticle-fibrin gel complex without BMP-2, or even the BMP-2 loaded fibrin gel. These improvements included areas such as radiodensity, the bone-specific ALP activity, the osteocalcin immunoreactivity, and the ratios of calcium and phosphate contents with respect to normal bone in the regenerated bone area. The remodeling process of new bone developed with BMP-2 was significantly enhanced, and more mature and highly-mineralized bone was obtained by utilizing the functional nanoparticle-hydrogel complex. These results indicate that the nanoparticle-fibrin gel complex can be a promising candidate for a new bone defect replacement matrix, and an enhanced BMP-2 carrier.

A facile method to prepare heparin-functionalized nanoparticles for controlled release of growth factors.

A new, facile method to prepare the heparin-functionalized PLGA nanoparticle (HEP-PLGA NP) for the controlled release of growth factors is developed. This system is composed of PLGA as a hydrophobic core, Pluronic F-127 as a hydrophilic surface layer, and heparin as the functional moiety. HEP-PLGA NPs were prepared by a solvent-diffusion method without chemical modification of the components. The entrapment of heparin molecules was confirmed by a negatively increased zeta potential value and the specific binding affinity to antithrombin III. The average diameter and the surface charge of the nanoparticles were ranged from 139+/-2 to 188+/-4 nm and from -26.0+/-1.1 to -44.1+/-1.3 mV by increasing the amount of heparin during the nanoparticle preparation. Accordingly, the amount of heparin on the nanoparticle increased from 0% to 4.7%. As a model in vitro release experiment, lysozyme was loaded into HEP-PLGA NPs, and a sustained release profile over 2 weeks was obtained with maintaining its bioactivity. The release of rhVEGF, one of the heparin-binding growth factors, showed a more sustained and prolonged profile than that of lysozyme over one month.

(S)-selective dynamic kinetic resolution of secondary alcohols by the combination of subtilisin and an aminocyclopentadienylruthenium complex as the catalysts.

A new procedure for the dynamic kinetic resolution (DKR) of racemic alcohols into single enantiomers is described. This procedure employs surfactant-treated subtilisin as an (S)-selective resolving catalyst and an aminocyclopentadienylruthenium complex as a racemizing catalyst. The DKR is performed best in the presence of an acyl donor such as trifluoroethyl butyrate in THF at room temperature. Eight simple secondary alcohols have been efficiently resolved with high optical purities and good yields. The subtilisin-based DKR is complementary in stereoselectivity to its lipase-based counterpart. For an acyl-carrying alcohol, both subtilisin- and lipase-based DKRs have proceeded equally well to give a pair of enantiomeric products (>99.5% ee each) with opposite optical rotations in high yields (94-95%).