Development of bioactive peptide amphiphiles for therapeutic cell delivery

Abstract

There is great clinical interest in cell-based therapies for ischemic tissue repair in cardiovascular disease. However, the regenerative potential of these therapies is limited due to poor cell viability and minimal retention following application. We report here the development of bioactive peptide amphiphile nanofibers displaying the fibronectin-derived RGDS cell adhesion epitope as a scaffold for therapeutic delivery of bone marrow derived stem and progenitor cells. When grown on flat substrates, a binary peptide amphiphile system consisting of 10 wt.% RGDS-containing molecules and 90wt.% negatively charged diluent molecules was found to promote optimal cell adhesion. This binary system enhanced adhesion 1.4-fold relative to substrates composed of only the non-bioactive diluent. Additionally, no enhancement was found upon scrambling the epitope and adhesion was no longer enhanced upon adding soluble RGDS to the cell media, indicating RGDS-specific adhesion. When encapsulated within self-assembled scaffolds of the binary RGDS nanofibers in vitro, cells were found to be viable and proliferative, increasing in number by 5.5 times after only 5 days, an effect again lost upon adding soluble RGDS. Cells encapsulated within a non-bioactive scaffold and those within a binary scaffold with scrambled epitope showed minimal viability and no proliferation. Cells encapsulated within this RGDS nanofiber gel also increase in endothelial character, evident by a decrease in the expression of CD34 paired with an increase in the expression of endothelial-specific markers VE-Cadherin, VEGFR2 and eNOS after 5 days. In an in vivo study, nanofibers and luciferase-expressing cells were co-injected subcutaneously in a mouse model. The binary RGDS material supported these cells in vivo, evident by a 3.2-fold increase in bioluminescent signal attributable to viable cells; this suggests the material has an anti-apoptotic and/or proliferative effect on the transplanted bone marrow cells. We conclude that the binary RGDS-presenting nanofibers developed here demonstrate enhanced viability, proliferation and adhesion of associated bone marrow derived stem and progenitor cells. This study suggests potential for this material as a scaffold to overcome current limitations of stem cell therapies for ischemic diseases.

Figure 1

Chemical structure of the E₃ diluent PA (A), RGDS PA (B), and DGSR scrambled PA (C) along with molecular graphics representations of binary PA fibers assembled from 90% E₃ diluent and 10% RGDS PA accented in yellow (D) or 10% scrambled PA accented in green (E).

Figure 2

Viability of BMNCs when cultured for 1 day on surfaces coated with E₃ diluent PA (A), K₃ diluent PA (B), and R₃ diluent PA (C) with red indicating dead cells and green indicating live cells. Viability was quantified after 1, 3, and 6 days (E). n=5 for each group. The scale bar for all images is 500 μm. *** p<0.001

Figure 3

(A) Optimization of BMNC adhesion to PA coated surfaces by altering the content of RGDS PA using E₃ PA. n=8 for each group. *** p<0.001, displayed for 10% RGDS relative to other RGDS compositions. (B) Quantitative viability of BMNCs cultured on PA coated surfaces of the same RGDS content as those used in (A).

Figure 4

BMNC adhesion to surfaces coated with 10% RGDS PA compared to surfaces coated with E₃ diluent only and 10% scrambled PA only, in the presence and absence of soluble RGDS. n=12 for each group. *** p<0.001

Figure 5

BMNCs cultured on surfaces coated with 10% RGDS PA, exhibiting extensive process formation (A, B, C) in contact with the PA coated surface at higher magnification. (D) Scale bars are 20 μm (A), 5 μm (B), 2 μm (C), and 500nm (D).

Figure 6

Viable BMNCs encapsulated within E₃ PA (A) and 10% RGDS PA (B). Viable cells are noted by green fluorescence. The red channel is not shown due to background fluorescence from interaction of the PA with EthD-1. The scale bar for all images is 500 μm. Also, proliferation after 5 days for BMNCs encapsulated within E₃ diluent, binary mixtures of 10% RGDS or 10% scrambled PAs, expressed as a percent of the day 0 cell signal, in the presence and absence of soluble RGDS (C). *** p<0.001. n=5 for each group.

Figure 7

Gene expression of BMNCs encapsulated within the binary RGDS scaffold for 0 and 5 days, examining hematopoietic stem cell (CD34) and endothelial cell (VE-Cadherin, VEGFR2, and eNOS) marker expression by RT-PCR. Data are normalized to 18s expression and displayed relative to day 0 values. n=5 for each group. * p<0.05, ** p<0.01, *** p<0.001

Figure 8

Quantification from in vivo bioluminescent imaging of transplanted luciferase-expressing BMNCs (A) injected subcutaneously, encapsulated within the binary RGDS system (B, n=15) and E₃ diluent PA (C, n=11), along with a saline control (D, n=13). ** p<0.01