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. 2007 May 8;104(19):7791-6.
doi: 10.1073/pnas.0701980104. Epub 2007 Apr 30.

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

Lisa Haines-Butterick  1 , Karthikan Rajagopal, Monica Branco, Daphne Salick, Ronak Rughani, Matthew Pilarz, Matthew S Lamm, Darrin J Pochan, Joel P Schneider
Affiliations

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

Lisa Haines-Butterick et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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.
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.
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.
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.
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.
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).
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