Controlling hydrogelation kinetics by peptide design for three-dimensional encapsulation and injectable delivery of cells

Abstract

A peptide-based hydrogelation strategy has been developed that allows homogenous encapsulation and subsequent delivery of C3H10t1/2 mesenchymal stem cells. Structure-based peptide design afforded MAX8, a 20-residue peptide that folds and self-assembles in response to DMEM resulting in mechanically rigid hydrogels. The folding and self-assembly kinetics of MAX8 have been tuned so that when hydrogelation is triggered in the presence of cells, the cells become homogeneously impregnated within the gel. A unique characteristic of these gel-cell constructs is that when an appropriate shear stress is applied, the hydrogel will shear-thin resulting in a low-viscosity gel. However, after the application of shear has stopped, the gel quickly resets and recovers its initial mechanical rigidity in a near quantitative fashion. This property allows gel/cell constructs to be delivered via syringe with precision to target sites. Homogenous cellular distribution and cell viability are unaffected by the shear thinning process and gel/cell constructs stay fixed at the point of introduction, suggesting that these gels may be useful for the delivery of cells to target biological sites in tissue regeneration efforts.

Fig. 1.

Self-assembly, shear-thinning, and self-healing mechanism allowing rapid formation of hydrogels that can be subsequently syringe-delivered. (a) Addition of DMEM (pH 7.4, 37°C) to a buffered solution (25 mM Hepes, pH 7.4) of unfolded peptide induces formation of a β-hairpin structure that undergoes lateral and facial self-assembly affording a rigid hydrogel with a fibrillar supramolecular structure. Subsequent application of shear stress disrupts the noncovalently stabilized network, leading to the conversion of hydrogel to a low-viscosity gel. Upon cessation of shear stress, the network structure recovers converting the liquid back to a rigid hydrogel. (b) Peptide sequences of MAX8 and MAX1.

Fig. 2.

Encapsulation of mesenchymal C3H10t1/2 stem cells in 0.5 wt%MAX1 and MAX8 hydrogels. Shown are LSCM z-stack images (viewed along the y axis) showing the incorporation of cells into a MAX1 gel leading to cell sedimentation (a) and into a MAX8 gel resulting in cellular homogeneity (b). Cells are prelabeled with cell tracker green to aid visualization. (Scale bars: 100 μm.)

Fig. 3.

CD spectroscopy and oscillatory rheology of MAX1 and MAX8 hydrogels. (a) Kinetics of β-sheet formation for MAX1 (squares) and MAX8 (triangles). The evolution of β-sheet is monitored during the solution-hydrogel phase transition by recording [θ]₂₁₆ as a function of time for a 0.5 wt% peptide solution at 37°C after folding and self-assembly is initiated by the addition of DMEM (pH 7.4). Inset shows CD wavelength spectra characteristic of β-sheet structure for MAX1 and MAX8 hydrogels after the kinetics measurements. (b) DTS measurements of MAX1 (squares) and MAX8 (triangles) monitoring the evolution of storage modulus (G′) as a function of time for 0.5 wt% hydrogel at 37°C in DMEM (pH 7.4); frequency = 6 rad·sec⁻¹, strain = 0.2%.

Fig. 4.

TEM micrographs of MAX1 and MAX8 hydrogels showing the nanostructure of 0.5 wt% gel network prepared at 37°C with DMEM for MAX1 (a) and MAX8 (b) negatively stained with uranyl acetate. (Scale bars: 100 nm.) (Insets) Magnification of MAX1 and MAX8 fibrils that are ≈3 nm in width. (Scale bars: 20 nm.)

Fig. 5.

Gel recovery kinetics, distribution of cells, and cell viability after shear-thinning. (a) Gel recovery assessed by monitoring G′ as a function of time after shear-thinning a 0.5 wt% MAX8 gel initially prepared from DMEM (pH 7.4). Region I shows onset of gelation as a function of time for the initial gelation event at 0.2% stain; II shows shear-thinning of the resulting hydrogel on application of 1,000% strain; and III shows recovery of hydrogel rigidity after reduction of strain to 0.2%; frequency = 6 rad·sec⁻¹ for all measurements. (b) LSCM z-stack image (viewed along the y axis) showing the distribution of cells in a 0.5 wt% MAX8 hydrogel. The gel–cell construct was initially prepared in a syringe as described in Fig. 2a and shear-thinned into a confocal plate for imaging. Inset shows the syringe loaded with gel–cell construct before shear-thinning. Cells were prelabeled with cell tracker green for visualization. (Scale bar: 100 μm.) (c) LSCM z-stack image (viewed along the z axis) showing a Live-Dead assay of cells after being shear-thin delivered at T = 3 h after delivery. Red, dead cells; green, alive cells. (Scale bar: 100 μm.)

Fig. 6.

Photographs of 0.5 wt% MAX8 hydrogels prepared in a syringe and shear-thinned to tissue culture treated polystyrene plate (a) and applied to a vertical borosilicate surface (b).