Avidity-controlled hydrogels for injectable co-delivery of induced pluripotent stem cell-derived endothelial cells and growth factors

Abstract

To translate recent advances in induced pluripotent stem cell biology to clinical regenerative medicine therapies, new strategies to control the co-delivery of cells and growth factors are needed. Building on our previous work designing Mixing-Induced Two-Component Hydrogels (MITCHs) from engineered proteins, here we develop protein-polyethylene glycol (PEG) hybrid hydrogels, MITCH-PEG, which form physical gels upon mixing for cell and growth factor co-delivery. MITCH-PEG is a mixture of C7, which is a linear, engineered protein containing seven repeats of the CC43 WW peptide domain (C), and 8-arm star-shaped PEG conjugated with either one or two repeats of a proline-rich peptide to each arm (P1 or P2, respectively). Both 20kDa and 40kDa star-shaped PEG variants were investigated, and all four PEG-peptide variants were able to undergo a sol-gel phase transition when mixed with the linear C7 protein at constant physiological conditions due to noncovalent hetero-dimerization between the C and P domains. Due to the dynamic nature of the C-P physical crosslinks, all four gels were observed to be reversibly shear-thinning and self-healing. The P2 variants exhibited higher storage moduli than the P1 variants, demonstrating the ability to tune the hydrogel bulk properties through a biomimetic peptide-avidity strategy. The 20kDa PEG variants exhibited slower release of encapsulated vascular endothelial growth factor (VEGF), due to a decrease in hydrogel mesh size relative to the 40kDa variants. Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) adopted a well-spread morphology within three-dimensional MITCH-PEG cultures, and MITCH-PEG provided significant protection from cell damage during ejection through a fine-gauge syringe needle. In a mouse hindlimb ischemia model of peripheral arterial disease, MITCH-PEG co-delivery of hiPSC-ECs and VEGF was found to reduce inflammation and promote muscle tissue regeneration compared to a saline control.

Keywords: Endothelial cell; Hydrogel; Protein engineering; VEGF; iPSC.

Fig. 1

Schematic of MITCH-PEG hydrogel formation. Component 1 is a recombinant protein copolymer bearing CC43 WW domains (denoted as C) and RGD cell-binding domains. Component 2 is an 8-arm PEG-peptide conjugate bearing complementary proline-rich peptide domains (denoted as P). Simple mixing of the two components results in hydrogel network formation. Inset shows variants of the 8-arm PEG-peptide conjugate, created by varying domain repeat (P1 for one domain or P2 for two domains) and the PEG molecular weight (20 kD or 40 kD).

Fig. 2

Characterization of peptide conjugation to PEG. (a) Conjugation reaction scheme by Michael addition. (b) ¹H NMR spectra of the 8-arm PEG-VS precursors and the 8-arm PEG-peptide conjugate products. The disappearance of the vinyl (CH₂=CH-) peaks from the precursors and the appearance of tyrosine doublets in the products (inset) confirm the conjugation reaction. Conjugation efficiency was calculated from the tyrosine:PEG backbone peak ratio. (c) Polyacrylamide gel electropherogram of the precursor peptides and the purified 8-arm PEG-peptide conjugates. Only one band is detected for each sample at various molecular weights, indicating purity and success of conjugation. Due to non-denaturing conditions, bands appear at positions different from those expected from the ladder standards.

Fig. 3

Dependence of hydrogel shear moduli and thixotropy on crosslinking strength and molecular weight between crosslinks. (a) Binding isotherms obtained from isothermal titration calorimetry (ITC) injections of C1 peptide into P1 or P2. Table shows binding enthalpy and stoichiometry derived from fitting binding isotherms to an independent site model. (b) Storage moduli (G’, closed symbols) and loss moduli (G”, open symbols) of MITCH-PEG variants as a function of frequency (10% w/v gels, 5% strain, 37 °C). (c) Linear shear viscosity measured under alternating shear rates, showing shear-thinning and self-healing behavior (10% w/v gels, 30-sec durations of 0.1 and 10 s⁻¹ shear rate, 37 °C).

Fig. 4

FRAP characterization of dextran diffusivity within MITCH-PEG variants. (a) Representative time-lapse fluorescence images of a photobleached area (upper left panel) and the corresponding fluorescence recovery curves obtained from 40 kD dextran (bottom). (b) Diffusivity constants derived from fluorescence recovery curves are plotted on a semi-logarithmic graph and reported in the table (c). Values obtained from encapsulation in common biomatrices (collagen and Matrigel) and PBS are included for comparison.

Fig. 5

Kinetics of hydrogel erosion and dextran and VEGF release. (a) Cumulative release curves of 20 kD dextran from MITCH-PEG into bulk PBS medium. Solid lines represent curve-fits for Fick's 2^nd law of diffusion from thin slabs. (b) Cumulative fraction of hydrogel material eroded into bulk PBS medium. (c) Quantification of VEGF₁₆₅ cumulative release by ELISA.

Fig. 6

(a) Bioluminescence imaging (BLI) quantification of acute hiPSC-ES viability following in vitro injection. Pre-encapsulation of hiPSC-ECs (Fluc+) in MITCH improved post-injection viability relative to injected cells suspended in PBS,* p < 0.05. (b) Three-dimensional (3D) culture of hiPSC-ECs in 8P2-20k variant of MITCH-PEG. Visualization of cell morphology at day 4 of encapsulation by confocal immunofluorescence. Cell nuclei shown by DAPI staining in blue, F-actin cytoskeleton by phalloidin staining in green, and CD31 endothelial cell marker in red. (c) BLI quantification of hiPSC-ECs within four different variants of MITCH-PEG at days 0 and 4 post-injection, * p < 0.05.

Fig. 7

Histology scores of (a) inflammation, (b) necrosis, and (c) regeneration for a mouse hindlimb ischemia injury model. Scores were evaluated according to the criteria listed in the Methods. Different lowercase letters denote statistically different outcomes, as assessed by ANOVA with Tukey post-hoc test, p < 0.05, (n.s. = not significant). (d-h) Photomicrographs of cryosectioned samples of skeletal muscles 14 days after femoral artery ligation-induced ischemia. Hematoxylin and eosin (H&E) stained images of muscle tissue explants for (d) control tissue with no ischemia and ischemic tissue injected with (e) PBS, (f) 8P2-20k, (g) hiPSC-ECs and VEGF in PBS, and (h) hiPSC-ECs and VEGF in 8P2-20k. Inflammation, characterized by expansion of the connective tissues between myofibers, is marked with a blask asterisk in panels (e) and (f). Inflammatory cells in the connective tissues is marked with a white asterisk in panel (g).