Temporally Controlled Photouncaged Epidermal Growth Factor Influences Cell Fate in Hydrogels.

Hydrogels are powerful materials that more accurately mimic the cellular microenvironment over static two-dimensional culture. Photochemical strategies enable dynamic complexity to be achieved within hydrogels to better mimic the extracellular matrix; however, many photochemical systems to pattern proteins within hydrogels are complicated by long reaction times to immobilize these proteins wherein the protein can lose activity. As proof-of-concept, we demonstrate an elegant method where photocaged proteins are immobilized in hydrogels and then directly photoactivated. Specifically, we immobilized streptavidin-ortho-nitrobenzyl-modified epidermal growth factor (EGF) to cross-linked hyaluronan hydrogels and cultured two EGF-responsive cancer cells of breast and lung therein. We used light to temporally uncage and control EGF activation, thereby inducing cell death in breast cancer cells and proliferation in lung cancer cells. These results show how temporal, photochemical, protein activation influences cellular response and lays the foundation for further advances in manipulating the in vitro environment to control cell fate.

Stable oxime-crosslinked hyaluronan-based hydrogel as a biomimetic vitreous substitute.

Vitreous substitutes are clinically used to maintain retinal apposition and preserve retinal function; yet the most used substitutes are gases and oils which have disadvantages including strict face-down positioning post-surgery and the need for subsequent surgical removal, respectively. We have engineered a vitreous substitute comprised of a novel hyaluronan-oxime crosslinked hydrogel. Hyaluronan, which is naturally abundant in the vitreous of the eye, is chemically modified to crosslink with poly(ethylene glycol)-tetraoxyamine via oxime chemistry to produce a vitreous substitute that has similar physical properties to the native vitreous including refractive index, density and transparency. The oxime hydrogel is cytocompatible in vitro with photoreceptors from mouse retinal explants and biocompatible in rabbit eyes as determined by histology of the inner nuclear layer and photoreceptors in the outer nuclear layer. The ocular pressure in the rabbit eyes was consistent over 56 d, demonstrating limited to no swelling. Our vitreous substitute was stable in vivo over 28 d after which it began to degrade, with approximately 50% loss by day 56. We confirmed that the implanted hydrogel did not impact retina function using electroretinography over 90 days versus eyes injected with balanced saline solution. This new oxime hydrogel provides a significant improvement over the status quo as a vitreous substitute.

Benchmarking to the Gold Standard: Hyaluronan-Oxime Hydrogels Recapitulate Xenograft Models with In Vitro Breast Cancer Spheroid Culture.

Many 3D in vitro models induce breast cancer spheroid formation; however, this alone does not recapitulate the complex in vivo phenotype. To effectively screen therapeutics, it is urgently needed to validate in vitro cancer spheroid models against the gold standard of xenografts. A new oxime-crosslinked hyaluronan (HA) hydrogel is designed, manipulating gelation rate and mechanical properties to grow breast cancer spheroids in 3D. This HA-oxime breast cancer model maintains the gene expression profile most similar to that of tumor xenografts based on a pan-cancer gene expression profile (comprising 730 genes) of three different human breast cancer subtypes compared to Matrigel or conventional 2D culture. Differences in gene expression between breast cancer cultures in HA-oxime versus Matrigel or 2D are confirmed for 12 canonical pathways by gene set variation analysis. Importantly, drug response is dependent on the culture method. Breast cancer cells respond better to the Rac inhibitor (EHT-1864) and the PI3K inhibitor (AZD6482) when cultured in HA-oxime versus Matrigel. This study demonstrates the superiority of an HA-based hydrogel as a platform for in vitro breast cancer culture of both primary, patient-derived cells and cell lines, and provides a hydrogel culture model that closely matches that in vivo.

Rationally Designed 3D Hydrogels Model Invasive Lung Diseases Enabling High-Content Drug Screening.

Cell behavior is highly dependent upon microenvironment. Thus, to identify drugs targeting metastatic cancer, screens need to be performed in tissue mimetic substrates that allow cell invasion and matrix remodeling. A novel biomimetic 3D hydrogel platform that enables quantitative analysis of cell invasion and viability at the individual cell level is developed using automated data acquisition methods with an invasive lung disease (lymphangioleiomyomatosis, LAM) characterized by hyperactive mammalian target of rapamycin complex 1 (mTORC1) signaling as a model. To test the lung-mimetic hydrogel platform, a kinase inhibitor screen is performed using tuberous sclerosis complex 2 (TSC2) hypomorphic cells, identifying Cdk2 inhibition as a putative LAM therapeutic. The 3D hydrogels mimic the native niche, enable multiple modes of invasion, and delineate phenotypic differences between healthy and diseased cells, all of which are critical to effective drug screens of highly invasive diseases including lung cancer.

Diels-Alder Click-Cross-Linked Hydrogels with Increased Reactivity Enable 3D Cell Encapsulation.

Engineered hydrogels have been extensively used to direct cell function in 3D cell culture models, which are more representative of the native cellular microenvironment than conventional 2D cell culture. Previously, hyaluronan-furan and bis-maleimide polyethylene glycol hydrogels were synthesized via Diels-Alder chemistry at acidic pH, which did not allow encapsulation of viable cells. In order to enable gelation at physiological pH, the reaction kinetics were accelerated by replacing the hyaluronan-furan with the more electron-rich hyaluronan-methylfuran. These new click-cross-linked hydrogels gel faster and at physiological pH, enabling encapsulation of viable cells, as demonstrated with 3D culture of 5 different cancer cell lines. The methylfuran accelerates Diels-Alder cycloaddition yet also increases the retro Diels-Alder reaction. Using computational analysis, we gain insight into the mechanism of the increased Diels-Alder reactivity and uncover that transition state geometry and an unexpected hydrogen-bonding interaction are important contributors to the observed rate enhancement. This cross-linking strategy serves as a platform for bioconjugation and hydrogel synthesis for use in 3D cell culture and tissue engineering.

Independently Tuning the Biochemical and Mechanical Properties of 3D Hyaluronan-Based Hydrogels with Oxime and Diels-Alder Chemistry to Culture Breast Cancer Spheroids.

For native breast cancer cell growth to be mimicked in vitro as spheroids, a well-defined matrix that mimics the tumor microenvironment is required. Finding a biomimetic material for 3D cell culture other than Matrigel has challenged the field. Because hyaluronan is naturally abundant in the tumor microenvironment and can be chemically modified, we synthesized a hyaluronan (HA) hydrogel with independently tunable mechanical and chemical properties for 3D culture of breast cancer cells. By modifying HA with distinct bioorthogonal functional groups, its mechanical properties are controlled by chemical cross-linking via oxime ligation, and its biochemical properties are controlled by grafting bioactive peptides via Diels-Alder chemistry. A series of hydrogels were screened in terms of stiffness and peptide composition for cancer spheroid formation. In the optimal hydrogel formulation, the 3D breast cancer spheroids showed decreased drug diffusion into their core and upregulation of cellular multidrug-resistant efflux pumps similar to what is observed in drug-resistant tumors. Our results highlight the potential of these tunable and well-defined gels in drug screening assays.

Designing Peptide and Protein Modified Hydrogels: Selecting the Optimal Conjugation Strategy.

Hydrogels are used in a wide variety of biomedical applications including tissue engineering, biomolecule delivery, cell delivery, and cell culture. These hydrogels are often designed with a specific biological function in mind, requiring the chemical incorporation of bioactive factors to either mimic extracellular matrix or to deliver a payload to diseased tissue. Appropriate synthetic techniques to ligate bioactive factors, such as peptides and proteins, onto hydrogels are critical in designing materials with biological function. Here, we outline strategies for peptide and protein immobilization. We specifically focus on click chemistry, enzymatic ligation, and affinity binding for transient immobilization. Protein modification strategies have shifted toward site-specific modification using unnatural amino acids and engineered site-selective amino acid sequences to preserve both activity and structure. The selection of appropriate protein immobilization strategies is vital to engineering functional hydrogels. We provide insight into chemistry that balances the need for facile reactions while maintaining protein bioactivity or desired release.

Synthesis and biological evaluation of prodigiosene conjugates of porphyrin, estrone and 4-hydroxytamoxifen.

To generate the first series of prodigiosene conjugates, the tripyrrolic skeleton was appended to estrone, tamoxifen and porphyrin frameworks by way of ester linkers and various hydrocarbon chain lengths. The ability of the conjugates to inhibit various types of cancer cells was evaluated in vitro. The porphyrin conjugates did not exhibit significant activity. The estrone conjugates exhibited modest activity, for the most part. However, significantly greater growth inhibition activity against certain breast, colon, lung, leukemia, melanoma and prostate cell lines was noted. This unusual effect for this first generation model class of compound warrants further investigation and comparison to cases where estrogens are linked to prodigiosenes via connection points that do not feature in estrogen receptor binding. The 4-hydroxytamoxifen conjugates exhibit nanomolar range activity against the MCF-7 breast cancer cell line, paving the way to expand the scope and connectivity of prodigiosene-tamoxifen conjugates.