Establishment of Collagen: Hydroxyapatite/BMP-2 Mimetic Peptide Composites.

Extensive efforts were undertaken to develop suitable biomaterials for tissue engineering (TE) applications. To facilitate clinical approval processes and ensure the success of TE applications, bioinspired concepts are currently focused on. Working on bone tissue engineering, we describe in the present study a method for biofunctionalization of collagen/hydroxyapatite composites with BMP-2 mimetic peptides. This approach is expected to be fundamentally transferable to other tissue engineering fields. A modified BMP-2 mimetic peptide containing a negatively charged poly-glutamic acid residue (E7 BMP-2 peptide) was used to bind positively charged hydroxyapatite (HA) particles by electrostatic attraction. Binding efficiency was biochemically detected to be on average 85% compared to 30% of BMP-2 peptide without E7 residue. By quartz crystal microbalance (QCM) analysis, we could demonstrate the time-dependent dissociation of the BMP-2 mimetic peptides and the stable binding of the E7 BMP-2 peptides on HA-coated quartz crystals. As shown by immunofluorescence staining, alkaline phosphatase expression is similar to that detected in jaw periosteal cells (JPCs) stimulated with the whole BMP-2 protein. Mineralization potential of JPCs in the presence of BMP-2 mimetic peptides was also shown to be at least similar or significantly higher when low peptide concentrations were used, as compared to JPCs cultured in the presence of recombinant BMP-2 controls. In the following, collagen/hydroxyapatite composite materials were prepared. By proliferation analysis, we detected a decrease in cell viability with increasing HA ratios. Therefore, we chose a collagen/hydroxyapatite ratio of 1:2, similar to the natural composition of bone. The following inclusion of E7 BMP-2 peptides within the composite material resulted in significantly elevated long-term JPC proliferation under osteogenic conditions. We conclude that our advanced approach for fast and cost-effective scaffold preparation and biofunctionalization is suitable for improved and prolonged JPC proliferation. Further studies should prove the functionality of composite scaffolds in vivo.

Fine-tuned PEGylation of chitosan to maintain optimal siRNA-nanoplex bioactivity.

Polyethylene glycol (PEG) is a widely used modification for drug delivery systems. It reduces undesired interaction with biological components, aggregation of complexes and serves as a hydrophilic linker of ligands for targeted drug delivery. However, PEGylation can also lead to undesired changes in physicochemical characteristics of chitosan/siRNA nanoplexes and hamper gene silencing. To address this conflicting issue, PEG-chitosan copolymers were synthesized with stepwise increasing degrees of PEG substitution (1.5% to 8.0%). Subsequently formed PEG-chitosan/siRNA nanoplexes were characterized physicochemically and biologically. The results showed that small ratios of chitosan PEGylation did not affect nanoplex stability and density. However, higher PEGylation ratios reduced nanoplex size and charge, as well as cell uptake and final siRNA knockdown efficiency. Therefore, we recommend fine-tuning of PEGylation ratios to generate PEG-chitosan/siRNA delivery systems with maximum bioactivity. The degree of PEGylation for chitosan/siRNA nanoplexes should be kept low in order to maintain optimal nanoplex efficiency.

Immunoliposomes for Targeted Delivery of an Antifibrotic Drug.

Excessive extracellular matrix formation in organs and tissues arises from an imbalance between the synthesis and degradation of matrix proteins, especially collagen. This condition interferes with proper wound healing and regeneration, and to date, no specific treatment is available. In the present study, we propose a targeted drug delivery system consisting of cell-specific immunoliposomes (ILs) loaded with deferoxamine (DFO) as an antifibrotic drug. ILs were functionalized with polyethylene glycol (PEG) to improve the steric stability and prolong their half-life. In addition, a single-chain Fv (scFv) antibody fragment that specifically targets fibroblast activation protein (FAP) was incorporated. An in vitro fibrosis model was employed to test this construct. This model consisted of highly activated pro-fibrotic fibroblasts with 2- to 6-fold induction of selected fibrosis markers: cell/matrix deposited collagen I, total soluble collagen, and α smooth muscle actin. The activation was accompanied by a significant and cell-specific elevation of FAP expression and activity, thereby confirming that FAP is an adequate target for antifibrotic drug delivery. Purified anti-FAP scFv was shown to bind specifically to these cells without influencing the FAP enzymatic activity. DFO was demonstrated to have a dose-dependent antifibrotic activity as quantified by collagen deposition. Specific binding and intracellular uptake of DiI-labeled ILs into the activated fibroblasts were shown by flow cytometry and microscopy. Finally, DFO-loaded ILs targeted to FAP caused a significant reduction in the collagen deposition, whereas no effect was observed using liposomes that lacked the targeting antibody fragment. These results suggest that the FAP-specific scFv-conjugated liposomes have considerable potential for cell-specific targeting applicable as a therapy for excessive collagen deposition during fibrosis. In general, through liposome encapsulation, bioactive molecules, such as DFO, that have broad effects and poor cell penetration can be converted into cell-specific composites for targeted drug delivery.