The expression of indoleamine 2,3-dioxygenase (IDO), which metabolizes tryptophan, an essential amino acid, into kynurenine, has been identified as having a key role in the prevention of the immune rejection of the semi-allogeneic fetus during pregnancy. We have previously demonstrated that IDO expressed in fibroblasts causes bystander CD4(+) T cell damage as well as THP-1 cell damage by apoptosis. As T cells are primarily responsible for graft rejection, here, we asked the question of whether engraftment of IDO-expressing xenogeneic fibroblasts populated in a collagen matrix can be immuno-protected in an animal model. The results show a significant reduction in the number of infiltrated CD3(+) T lymphocytes on days 14 and 28 post-transplantation in the wounds receiving IDO-expressing fibroblasts relative to controls. IDO-expressing human fibroblasts embedded in bovine collagen on wounds in a rat model accelerates wound healing by promoting neovascularization during the early stages and providing protection of the xenograft fibroblasts. Using a co-culture system, we further confirm that IDO can induce angiogenesis through the depletion of tryptophan. These findings suggest that IDO may have an application in promoting the engraftment of skin substitutes and other transplanted organs.
Nanoparticle (NP)-based delivery has gained importance for improving the potency of therapeutic agents. The bovine serum albumin (BSA) NPs, obtained by a coacervation process, was modified by electrostatic adsorption of cationic polyethylenimine (PEI) to NP surfaces for delivery of bone-inducing growth factor, bone morphogenetic protein-2 (BMP-2). Different concentrations of PEI were utilized for coating BSA NPs to stabilize the colloidal system and to control the release of BMP-2. The NPs were characterized by size and zeta potential measurements, as well as by Scanning Electron Microscopy and Atomic Force Microscopy. The encapsulation efficiency was typically [90% in all NP preparations. In vitro release kinetics showed that the PEI concentration used for coating the NPs efficiently controlled the release of BMP-2, demonstrating a gradual slowing, sustained release pattern during a 10-day study period. The bioactivity of the encapsulated BMP-2 and the toxicity of the NPs were examined by the alkaline phosphatase (ALP) induction assay and the MTT assay, respectively, using C2C12 cells. The results indicated that PEI was the primary determinant of NP toxicities, and BSA NPs coated with 0.1 mg/mL PEI demonstrated tolerable toxicity, retained the bioactivity of BMP-2, and efficiently slowed the release rate of BMP-2. We conclude that BMP-2 encapsulated in BSA NPs might be an efficient way to deliver the protein for in vivo bone induction.
Palmitic acid conjugates of poly-L-lysine (PLL-PA) were prepared, and their ability to deliver plasmid DNA into human skin fibroblasts was evaluated in vitro. The conjugates were capable of condensing a 4.7 kb plasmid DNA into 50-200 nm particles (mean +/- SD = 112 +/- 34 nm), which were slightly smaller than the particles formed by PLL (mean +/- SD = 126 +/- 51 nm). Both PLL and PLL-PA were readily taken up by the cells, but PLL-PA delivered the plasmid DNA into a higher proportion of cells. DNA delivery was found to be reduced by endocytosis inhibitor Brefeldin A, suggesting an active mechanism of particle uptake. Using enhanced green fluorescent protein (EGFP) as a reporter gene, PLL-PA was found to give the highest number of EGFP-positive cells among several carriers tested, including polyethyleneimine, Lipofectamine-2000, and an adenovirus. Although some carriers gave a higher percentage of EGFP-positive cells than PLL-PA, they were also associated with higher toxicities. We conclude that PLL-PA is a promising gene carrier for non-viral modification of human fibroblasts.
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