Elastin-like protein hydrogels with controllable stress relaxation rate and stiffness modulate endothelial cell function.

Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell-applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin-like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine-modified ELP (ELP-HYD) and aldehyde/benzaldehyde-modified polyethylene glycol (PEG-ALD/PEG-BZA). The reversible DCC crosslinks in ELP-PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast-relaxing or slow-relaxing hydrogels with a range of stiffness (500-3300 Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two-dimensional substrates, on which ECs exhibited greater cell spreading on fast-relaxing hydrogels up through 3 days, compared with slow-relaxing hydrogels at the same stiffness. In three-dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast-relaxing, low-stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast-relaxing, low-stiffness hydrogel produced significantly more vascularization compared with the slow-relaxing, low-stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast-relaxing, low-stiffness hydrogels supported the highest capillary density in vivo.

Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids.

Human intestinal organoids from primary human tissues have the potential to revolutionize personalized medicine and preclinical gastrointestinal disease models. A tunable, fully defined, designer matrix, termed hyaluronan elastin-like protein (HELP) is reported, which enables the formation, differentiation, and passaging of adult primary tissue-derived, epithelial-only intestinal organoids. HELP enables the encapsulation of dissociated patient-derived cells, which then undergo proliferation and formation of enteroids, spherical structures with polarized internal lumens. After 12 rounds of passaging, enteroid growth in HELP materials is found to be statistically similar to that in animal-derived matrices. HELP materials also support the differentiation of human enteroids into mature intestinal cell subtypes. HELP matrices allow stiffness, stress relaxation rate, and integrin-ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid-matrix interactions and potential patient-specific optimization. Organoid formation in HELP materials is most robust in gels with stiffer moduli (G' ≈ 1 kPa), slower stress relaxation rate (t1/2 ≈ 18 h), and higher integrin ligand concentration (0.5 × 10-3-1 × 10-3 m RGD peptide). This material provides a promising in vitro model for further understanding intestinal development and disease in humans and a reproducible, biodegradable, minimal matrix with no animal-derived products or synthetic polyethylene glycol for potential clinical translation.

Protein-engineered hydrogels enhance the survival of induced pluripotent stem cell-derived endothelial cells for treatment of peripheral arterial disease.

A key feature of peripheral arterial disease (PAD) is damage to endothelial cells (ECs), resulting in lower limb pain and restricted blood flow. Recent preclinical studies demonstrate that the transplantation of ECs via direct injection into the affected limb can result in significantly improved blood circulation. Unfortunately, the clinical application of this therapy has been limited by low cell viability and poor cell function. To address these limitations we have developed an injectable, recombinant hydrogel, termed SHIELD (Shear-thinning Hydrogel for Injectable Encapsulation and Long-term Delivery) for cell transplantation. SHIELD provides mechanical protection from cell membrane damage during syringe flow. Additionally, secondary in situ crosslinking provides a reinforcing network to improve cell retention, thereby augmenting the therapeutic benefit of cell therapy. In this study, we demonstrate the improved acute viability of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) following syringe injection delivery in SHIELD, compared to saline. Using a murine hind limb ischemia model of PAD, we demonstrate enhanced iPSC-EC retention in vivo and improved neovascularization of the ischemic limb based on arteriogenesis following transplantation of iPSC-ECs delivered in SHIELD.

The Diverse Roles of Hydrogel Mechanics in Injectable Stem Cell Transplantation.

Stem cell delivery by local injection has tremendous potential as a regenerative therapy but has seen limited clinical success. Several mechanical challenges hinder therapeutic efficacy throughout all stages of cell transplantation, including mechanical forces during injection and loss of mechanical support post-injection. Recent studies have begun exploring the use of biomaterials, in particular hydrogels, to enhance stem cell transplantation by addressing the often-conflicting mechanical requirements associated with each stage of the transplantation process. This review explores recent biomaterial approaches to improve the therapeutic efficacy of stem cells delivered through local injection, with a focus on strategies that specifically address the mechanical challenges that result in cell death and/or limit therapeutic function throughout the stages of transplantation.

A novel protein-engineered hepatocyte growth factor analog released via a shear-thinning injectable hydrogel enhances post-infarction ventricular function.

In the last decade, numerous growth factors and biomaterials have been explored for the treatment of myocardial infarction (MI). While pre-clinical studies have demonstrated promising results, clinical trials have been disappointing and inconsistent, likely due to poor translatability. In the present study, we investigate a potential myocardial regenerative therapy consisting of a protein-engineered dimeric fragment of hepatocyte growth factor (HGFdf) encapsulated in a shear-thinning, self-healing, bioengineered hydrogel (SHIELD). We hypothesized that SHIELD would facilitate targeted, sustained intramyocardial delivery of HGFdf thereby attenuating myocardial injury and post-infarction remodeling. Adult male Wistar rats (n = 45) underwent sham surgery or induction of MI followed by injection of phosphate buffered saline (PBS), 10 µg HGFdf alone, SHIELD alone, or SHIELD encapsulating 10 µg HGFdf. Ventricular function, infarct size, and angiogenic response were assessed 4 weeks post-infarction. Treatment with SHIELD + HGFdf significantly reduced infarct size and increased both ejection fraction and borderzone arteriole density compared to the controls. Thus, sustained delivery of HGFdf via SHIELD limits post-infarction adverse ventricular remodeling by increasing angiogenesis and reducing fibrosis. Encapsulation of HGFdf in SHIELD improves clinical translatability by enabling minimally-invasive delivery and subsequent retention and sustained administration of this novel, potent angiogenic protein analog. Biotechnol. Bioeng. 2017;114: 2379-2389. © 2017 Wiley Periodicals, Inc.

Fluorescent dye incorporation causes weakened gene association and intracellular aggregate formation in nonviral carriers.

BACKGROUND: The successful application of nonviral gene transfer technologies requires both improved understanding and control with respect to intracellular trafficking and release. However, the intracellular space is highly complex and hence well-defined, stable structures are necessary to probe the stages of the delivery pathway. Fluorescent labeling is a regularly used approach to monitor nonviral delivery and release, yet few studies investigate the effects of label incorporation on the structure and activity of gene-containing vehicles. METHODS: In the present study, the impacts of label incorporation on the assembly and gene transfer capacity of DNA polyplexes were determined through the utilization of a model DNA-polyethylenimine (PEI) delivery system. PEI was fluorescently labeled with the Oregon Green® dye prior to polyplex formation and delivery to CHO-K1 cells. RESULTS: The present study provides evidence showing that routine labeling strategies for polyplexes weakened DNA binding affinity, produced large quantities of extracellular structures and significantly increased intracellular polyplex aggregation. Additionally, cellular internalization studies showed that increased labeling fractions led to reductions in polyplex uptake as a result of weakened complexation. CONCLUSIONS: These results not only provide insight into the assembly of these structures, but also help to identify labeling strategies sufficient to preserve activity at the same time as enabling detailed studies of trafficking and disassembly.

Light-mediated activation of siRNA Release in diblock copolymer assemblies for controlled gene silencing.

Controllable release is particularly important for the delivery of small interfering RNA (siRNA), as siRNAs have a high susceptibility to enzymatic degradation if release is premature, yet lack silencing activity if they remain inaccessible within the cytoplasm. To overcome these hurdles, novel and tailorable mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) diblock copolymers containing light-sensitive o-nitrobenzyl moieties and pendant amines are employed to provide both efficient siRNA binding, via electrostatic and hydrophobic interactions, as well as triggered charge reversal and nucleic acid release. In particular, siRNA/mPEG-b-P(APNBMA)23.6 polyplexes show minimal aggregation in physiological salt and serum, and enhanced resistance to polyanion-induced unpackaging compared to polyethylenimine preparations. Cellular delivery of siRNA/mPEG-b-P(APNBMA)23.6 polyplexes reveals greater than 80% cellular transfection, as well as rapid and widespread cytoplasmic distribution. Additionally, UV irradiation indicates ≈70% reduction in targeted gene expression following siRNA/mPEG-b-P(APNBMA)23.6 polyplex treatment, as compared to 0% reduction in polyplex-treated cells without UV irradiation, and only ≈30% reduction for Lipofectamine-treated cells. The results here highlight the potential of these light-sensitive copolymers with a well-defined on/off switch for applications including cellular patterning for guided cell growth and extension, and cellular microarrays for exploring protein and drug interactions that require enhanced spatiotemporal control of gene activation.

Catch and Release: Photocleavable Cationic Diblock Copolymers as a Potential Platform for Nucleic Acid Delivery.

Binding interactions between DNA and cationic carriers must be sufficiently strong to prevent nuclease-mediated degradation, yet weak enough to permit transcription. We demonstrate cationic diblock copolymers containing PEG and o-nitrobenzyl moieties that facilitated tailorable DNA complexation and light-activated release. This design unlocks a new approach to advance non-viral gene packaging.