Lysine-based peptide-functionalized PLGA foams for controlled DNA delivery.

Due to its hydrophobicity and negatively charged surfaces, PLGA-based scaffolds have encountered problems in controlled-release and tissue engineering applications. The effects of charge modification of PLGA micro-porous foams on DNA delivery and DNA transfection are investigated herein. Tailor-designed l-lysine peptides (K4 and K20) were employed to modify the surface charge of PLGA foams using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide cross linkers and the effects of charge modification of PLGA were examined in three main aspects: DNA adsorption, DNA release properties and DNA transfection. Successful conjugation of peptide and DNA adsorption were verified by X-ray photoelectron spectroscopy. A plasmid encoding bone morphogenetic protein-2 (BMP2) was used throughout the current study and the results indicate that adsorption capacity and release behavior of DNA were highly dependent on the charge properties of the foam surfaces. The release rates of DNA from the K4- and K20-functionalized foams are more sustainable as compared to the blank foam. As a result, the sustained release of DNA from modified foams led to negligible cytotoxicity and sustained expression of DNA which is favorable for DNA delivery and tissue engineering application. Furthermore, the ease of fabrication and modification of PLGA foams makes it a promising DNA delivery device.

Enzymatically crosslinked collagen-mimetic dendrimers that promote integrin-targeted cell adhesion.

Collagen is made up of a diverse family of the extracellular matrices, most of which are generally found crosslinked in vivo. To more closely mimic the biological function of collagen, this work focuses on establishing a molecular strategy to engineer a functional biomimetic collagen that exhibits stable collagen-like triple-helical conformation with cell-binding activity, in addition to an enzyme-mediated crosslinking by tissue transglutaminase (tTGase). A novel sequence spanning residues 2800-2807 of human fibrillin-1 (EDGFFKI) was first identified as an amine donor substrate for tTGase, using a previously characterized APQQEA derived from human osteonectin as an amine acceptor probe. Subsequently, collagen-mimetic peptides (CMPs) supplemented with a cell-binding sequence (GFOGER) and the identified EDGFFKI and APQQEA substrate sequences were conjugated onto a generation 2 poly(amidoamine) dendrimer, resulting in a crosslinkable collagen-mimetic dendrimer, denoted as CMD-K and CMD-Q, respectively. Both CMD-K and CMD-Q exhibited enhanced triple-helical stability and supported cell adhesion in an integrin-specific manner. Finally, tTGase-mediated crosslinking between CMD-K and CMD-Q resulted in a supramolecular structure that exhibited stable collagen-like triple-helical conformation and improved cellular recognition. The results show that the triple-helical structure is important in preserving the GFOGER cell-binding site while the tTGase-mediated protein crosslinking may also be crucial for the recognition by cell surface integrin receptors.

Template-assembled triple-helical peptide molecules: mimicry of collagen by molecular architecture and integrin-specific cell adhesion.

Most proteins fold into specific structures to exert their biological functions, and therefore the creation of protein-like molecular architecture is a fundamental prerequisite toward realizing a novel biologically active protein-like biomaterial. To do this with an artificial collagen, we have engineered a peptide template characterized by its collagen-like primary structure composed of Gly-Phe-Gly-Glu-Glu-Gly sequence to assemble (Pro-Hyp-Gly)n (n = 3 and 5) into triple-helical conformations that resemble the native structure of collagen. The peptide template has three carboxyl groups connected to the N-termini of three collagen peptides. The coupling was accomplished by a simple and direct branching protocol without complex strategies. A series of biophysical studies, including melting curve analyses and CD and NMR spectroscopy, demonstrated the presence of stable triple-helical conformation in the template-assembled (Pro-Hyp-Gly)3 and (Pro-Hyp-Gly)5 solution. Conversely, nontemplated peptides showed no evidence of assembly of triple-helical structure. A cell binding sequence (Gly-Phe-Hyp-Gly-Glu-Arg) derived from the collagen alpha1(I) chain was incorporated to mimic the integrin-specific cell adhesion of collagen. Cell adhesion and inhibition assays and immunofluorescence staining revealed a correlation of triple-helical conformation with cellular recognition of collagen mimetics in an integrin-specific way. This study offers a robust strategy for engineering native-like peptide-based biomaterials, fully composed of only amino acids, by maintaining protein conformation integrity and biological activity.

The specific recognition of a cell binding sequence derived from type I collagen by Hep3B and L929 cells.

In this study, the affinity of two different cell types toward a specific cell binding sequence (Gly-Phe-Hyp-Gly-Glu-Arg or GFOGER) derived from type I collagen using peptide template (PT)-assembled collagen peptides of different triple helicity as a model for natural collagen is examined. A series of biophysical studies, including melting curve analysis and circular dichroism spectroscopy, demonstrated the presence of stable triple-helical conformation in the PT-assembled (GPO)3-GFOGER-(GPO)3, (GPO)-GFOGER-(GPO), and (Pro-Hyp-Gly)5 solution. Conversely, non-templated peptides, except (GPO)3-GFOGER-(GPO)3, showed no evidence of assembly into triple-helical structure. Biological assays, including cell adhesion, competitive inhibition, and immunofluorescence staining, revealed a correlation of triple-helical conformation with the cellular recognition of GFOGER in an integrin-specific manner. The triple helix was shown to be important, but not crucial for cell adhesion to native collagen. Hep3B and L929 cells displayed significant differences in the recognition of GFOGER, mainly because of the differences in their expression of specific integrin receptors for collagen. For example, PT-assembled (GPO)3-GFOGER-(GPO)3 was shown to perform comparably to collagen for L929, but not Hep3B, cell adhesion. The result showed that a specific cell binding motif may not fully mimic the extracellular matrix (ECM) microenvironment, suggesting the need to use a combination of two or more cell binding sequences for targeting a wide range of integrin receptors expressed by a specific cell type to better mimic the ECM.

Characterization of triple-helical conformations and melting analyses of synthetic collagen-like peptides by reversed-phase HPLC.

There is a confusion in the application of circular dichroism (CD) spectroscopy in analyzing collagen's structure for the overlapping of the spectral shapes and positions of the collagen triple helix and poly(proline-II)-like structure. The unique repetitive sequence of the collagen triple helix is susceptible to misalignment during the spontaneous assembly. Such misaligned structures are usually difficult to be characterized by CD or NMR spectroscopy. Here, RP-HPLC was developed as a conformational characterization technique for synthetic collagen-like peptides based on the different hydrophobicities exhibited by the triple-helical and unassembled peptides. RP-HPLC was also used to study thermal transitions and to measure melting point temperatures (Tm) of the collagen-like peptides.

An integrin-specific collagen-mimetic peptide approach for optimizing Hep3B liver cell adhesion, proliferation, and cellular functions.

This study focused on mimicking collagen structurally and biologically using various peptide sequences toward realizing an artificial collagen-like biomaterial. Collagen-mimetic peptides (CMPs) incorporating integrin-specific glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) sequence from residues 502 to 507 of collagen alpha(1)(I) were used as a bioadhesive matrix and grafted onto poly(3-hydroxybutyrate-co3-hydroxyvalerate) microspheres to optimize cell adhesion, proliferation, and functions. Cell recognition of these biomolecules appeared to be conformation dependent, with the CMP1 of higher triple helix stability being preferred. Absence of the GFOGER hexapeptide in the CMP1' and CMP2' caused an adverse effect on the level of cell adhesion (<10%). The GFOGER-containing triple-helical CMPs effectively inhibited cell adhesion to collagen in a competition assay. The cell-adhesion activity of the CMP1 was approximately 50% of that of collagen. The cell spreading on the CMP1 was comparable with that observed on collagen. The presence of the CMP1 promoted cell attachment and spreading on the microspheres and extensive cell proliferation and bridging. Slower cell proliferation was observed on the blank microspheres. Live-dead assay showed that most cells are viable after 10-day culture. The presence of CMP1 on the microspheres maintained the albumin secretion and P-450 activity levels of the liver cells for up to 14 days. Our results established the potential of CMP1 to create a collagen-like microenvironment for optimizing cellular responses for liver tissue engineering.