Viscoelastic Supramolecular Hyaluronan-Peptide Cross-Linked Hydrogels.

Viscoelasticity plays a key role in hydrogel design. We designed a physically cross-linked hydrogel with tunable viscoelasticity, comprising supramolecular-assembled peptides coupled to hyaluronan (HA), a native extracellular matrix component. We then explored the structural and molecular mechanisms underlying the mechanical properties of a series of these HA-peptide hydrogels. By modifying the peptide sequence, we modulated both long- and short-time stress relaxation rates as a way to target viscoelasticity with limited impact on stiffness, leading to gels that relax up to 60% of stress in 10 min. Gels with the highest viscoelasticity exhibited large mesh sizes and ß-sheet secondary structures. The stiffness of the gel correlated with hydrogen bonding between the peptide chains. These gels are cytocompatible: highly viscoelastic gels that mimic the native skin microenvironment promote dermal fibroblast cell spreading. Moreover, HA-peptide gels enabled cell encapsulation, as shown with primary human T cells. Overall, these physically-cross-linked hydrogels enable tunable viscoelasticity that can be used to modulate cell morphology.

Synthesis of an Enzyme-Mediated Reversible Cross-linked Hydrogel for Cell Culture.

Detachment of fragile cell types cultured on two-dimensional (2D) surfaces has been shown to be detrimental to their viability. For example, detachment of induced pluripotent stem cell (iPSC)-derived neurons grown in vitro in 2D typically results in loss of neuronal connections and/or cell death. Avoiding cell detachment altogether by changing the properties of the substrate on which the cells are grown is a compelling strategy to maintain cell viability. Here, we present the synthesis of a reversible cross-linked hydrogel that is sufficiently stable for cell culture and differentiation and is cleaved by an external stimulus, facilitating injection. Specifically, hyaluronan (HA) and methylcellulose (MC) were modified with ketone and aldehyde groups, respectively, and a TEV protease-degradable peptide was synthesized via solid-state synthesis and modified at both termini with oxyamine groups to cross-link HA-ketone and MC-aldehyde to produce oxime-cross-linked HA × MC. The HA × MC hydrogel demonstrated good stability, enzyme-sensitive degradation, and cytocompatibility with iPSC-derived neural progenitor cells, laying the framework for broad applicability.

Modeling of Photoreceptor Donor-Host Interaction Following Transplantation Reveals a Role for Crx, Müller Glia, and Rho/ROCK Signaling in Neurite Outgrowth.

The goal of photoreceptor transplantation is to establish functional synaptic connectivity between donor cells and second-order neurons in the host retina. There is, however, limited evidence of donor-host photoreceptor connectivity post-transplant. In this report, we investigated the effect of the host retinal environment on donor photoreceptor neurite outgrowth in vivo and identified a neurite outgrowth-promoting effect of host Crx(-/-) retinas following transplantation of purified photoreceptors expressing green fluorescent protein (GFP). To investigate the noncell autonomous factors that influence donor cell neurite outgrowth in vitro, we established a donor-host coculture system using postnatal retinal aggregates. Retinal cell aggregation is sensitive to several factors, including plate coating substrate, cell density, and the presence of Müller glia. Donor photoreceptors exhibit motility in aggregate cultures and can engraft into established aggregate structures. The neurite outgrowth-promoting phenotype observed in Crx(-/-) recipients in vivo is recapitulated in donor-host aggregate cocultures, demonstrating the utility of this surrogate in vitro approach. The removal of Müller glia from host aggregates reduced donor cell neurite outgrowth, identifying a role for this cell type in donor-host signaling. Although disruption of chondroitin sulfate proteoglycans in aggregates had no effect on the neurite outgrowth of donor photoreceptors, disruption of Rho/ROCK signaling enhanced outgrowth. Collectively, these data show a novel role of Crx, Müller glia, and Rho/ROCK signaling in controlling neurite outgrowth and provide an accessible in vitro model that can be used to screen for factors that regulate donor-host connectivity. Stem Cells 2019;37:529-541.

Inverse Electron-Demand Diels-Alder Methylcellulose Hydrogels Enable the Co-delivery of Chondroitinase ABC and Neural Progenitor Cells.

A hydrogel that can deliver both proteins and cells enables the local microenvironment of transplanted cells to be manipulated with a single injection. Toward this goal, we designed a hydrogel suitable for the co-delivery of neural stem cells and chondroitinase ABC (ChABC), a potent enzyme that degrades the glial scar that forms after central nervous system (CNS) injury. We leveraged the inverse electron-demand Diels-Alder reaction between norbornene and methylphenyltetrazine to form rapidly gelling (<15 min) crosslinked methylcellulose (MC) hydrogels at physiological temperature and pH, with Young's modulus similar to that of brain tissue (1-3 kPa), and degradable, disulfide-containing crosslinkers. To achieve tunable, affinity-controlled release of a ChABC-Src homology 3 (SH3) fusion protein, we immobilized norbornene-functionalized SH3-binding peptides onto MC-methylphenyltetrazine and observed release of bioactive ChABC-SH3 over 4 days. We confirmed cytocompatibility by evaluating neural progenitor cell survival and proliferation. The combined encapsulation of neural stem cells and chondroitinase ABC from one hydrogel lays the framework for future in vivo studies to treat CNS injuries.

An Injectable Hyaluronan-Methylcellulose (HAMC) Hydrogel Combined with Wharton's Jelly-Derived Mesenchymal Stromal Cells (WJ-MSCs) Promotes Degenerative Disc Repair.

Intervertebral disc (IVD) degeneration is one of the predominant causes of chronic low back pain (LBP), which is a leading cause of disability worldwide. Despite substantial progress in cell therapy for the treatment of IVD degeneration, significant challenges remain for clinical application. Here, we investigated the effectiveness of hyaluronan-methylcellulose (HAMC) hydrogels loaded with Wharton's Jelly-derived mesenchymal stromal cell (WJ-MSCs) in vitro and in a rat coccygeal IVD degeneration model. Following induction of injury-induced IVD degeneration, female Sprague-Dawley rats were randomized into four groups to undergo a single intradiscal injection of the following: (1) phosphate buffered saline (PBS) vehicle, (2) HAMC, (3) WJ-MSCs (2 × 104 cells), and (4) WJ-MSCs-loaded HAMC (WJ-MSCs/HAMC) (n = 10/each group). Coccygeal discs were removed following sacrifice 6 weeks after implantation for radiologic and histologic analysis. We confirmed previous findings that encapsulation in HAMC increases the viability of WJ-MSCs for disc repair. The HAMC gel maintained significant cell viability in vitro. In addition, combined implantation of WJ-MSCs and HAMC significantly promoted degenerative disc repair compared to WJ-MSCs alone, presumably by improving nucleus pulposus cells viability and decreasing extracellular matrix degradation. Our results suggest that WJ-MSCs-loaded HAMC promotes IVD repair more effectively than cell injection alone and supports the potential clinical use of HAMC for cell delivery to arrest IVD degeneration or to promote IVD regeneration.

Induction of Rod and Cone Photoreceptor-Specific Progenitors from Stem Cells.

Retinal degeneration includes a variety of diseases for which there is no regenerative therapy. Cellular transplantation is one potential approach for future therapy for retinal degeneration, and stem cells have emerged as a promising source for future cell therapeutics. One major barrier to therapy is the ability to specify individual photoreceptor lineages from a variety of stem cell sources. In this review, we focus on photoreceptor genesis from progenitor populations in the developing embryo and how this understanding has given us the tools to manipulate cultures to specific unique rod and cone lineages from adult stem cell populations. We discuss experiments and evidence uncovering the lineage mechanisms at play in the establishment of fate-specific rod and cone photoreceptor progenitors. This may lead to an improved understanding of retinal development in vivo, as well as new cell sources for transplantation.

Engineering Cellular Microenvironments with Photo- and Enzymatically Responsive Hydrogels: Toward Biomimetic 3D Cell Culture Models.

Conventional cell culture techniques using 2D polystyrene or glass have provided great insight into key biochemical mechanisms responsible for cellular events such as cell proliferation, differentiation, and cell-cell interactions. However, the physical and chemical properties of 2D culture in vitro are dramatically different than those found in the native cellular microenvironment in vivo. Cells grown on 2D substrates differ significantly from those grown in vivo, and this explains, in part, why many promising drug candidates discovered through in vitro drug screening assays fail when they are translated to in vivo animal or human models. To overcome this obstacle, 3D cell culture using biomimetic hydrogels has emerged as an alternative strategy to recapitulate native cell growth in vitro. Hydrogels, which are water-swollen polymers, can be synthetic or naturally derived. Many methods have been developed to control the physical and chemical properties of the hydrogels to match those found in specific tissues. Compared to 2D culture, cells cultured in 3D gels with the appropriate physicochemical cues can behave more like they naturally do in vivo. While conventional hydrogels involve modifications to the bulk material to mimic the static aspects of the cellular microenvironment, recent progress has focused on using more dynamic hydrogels, the chemical and physical properties of which can be altered with external stimuli to better mimic the dynamics of the native cellular microenvironment found in vivo. In this Account, we describe our progress in designing stimuli-responsive, optically transparent hydrogels that can be used as biomimetic extracellular matrices (ECMs) to study cell differentiation and migration in the context of modeling the nervous system and cancer. Specifically, we developed photosensitive agarose and hyaluronic acid hydrogels that are activated by single or two-photon irradiation for biomolecule immobilization at specific volumes within the 3D hydrogel. By controlling the spatial location of protein immobilization, we created 3D patterns and protein concentration gradients within these gels. We used the latter to study the effect of VEGF-165 concentration gradients on the interactions between endothelial cells and retinal stem cells. Hyaluronic acid (HA) is particularly compelling as it is naturally found in the ECM of many tissues and the tumor microenvironment. We used Diels-Alder click chemistry and cryogelation to alter the chemical and physical properties of these hydrogels. We also designed HA hydrogels to study the invasion of breast cancer cells. HA gels were chemically cross-linked with matrix metalloproteinase (MMP)-degradable peptides that degrade in the presence of cancer cell-secreted MMPs, thus allowing cells to remodel their local microenvironment and invade into HA/MMP-degradable gels.

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.

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.

Leveraging Colloidal Aggregation for Drug-Rich Nanoparticle Formulations.

While limited drug loading continues to be problematic for chemotherapeutics formulated in nanoparticles, we found that we could take advantage of colloidal drug aggregation to achieve high loading when combined with polymeric excipients. We demonstrate this approach with two drugs, fulvestrant and pentyl-PABC doxazolidine (PPD; a prodrug of doxazolidine, which is a DNA cross-linking anthracycline), and two polymers, polysorbate 80 (UP80) and poly(d,l-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-graft-poly(ethylene glycol) (PLAC-PEG; a custom-synthesized, self-assembling amphiphilic polymer). In both systems, drug-loaded nanoparticles had diameters < 200 nm and were stable for up to two days in buffered saline solution and for up to 24 h in serum-containing media at 37 °C. While colloidal drug aggregates alone are typically unstable in saline and serum-containing media, we attribute the colloid stability observed herein to the polymeric excipients and consequent decreased protein adsorption. We expect this strategy of polymer-stabilized colloidal drug aggregates to be broadly applicable in delivery formulations.

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.

Hydrogel for Simultaneous Tunable Growth Factor Delivery and Enhanced Viability of Encapsulated Cells in Vitro.

Poor cell survival in vitro and in vivo is one of the key challenges in tissue engineering. Prosurvival therapeutic proteins, such as insulin-like growth factor-1 (IGF-1), can promote cell viability but require controlled delivery systems due to their short half-lives and rapid clearance. Biocompatible materials are commonly used for drug delivery platforms or to encapsulate cells for increased viability, but few materials have been used for both applications simultaneously. In this work, we present a dual-use platform. A blend of hyaluronan and methylcellulose, known to promote cell survival, was covalently modified with Src homology 3 (SH3)-binding peptides and demonstrated tunable, affinity-based release of the prosurvival fusion protein SH3-IGF-1. The material also significantly increased the viability of retinal pigment epithelium cells under anchorage-independent conditions. This novel platform is applicable to a broad range of cells and protein therapeutics and is a promising drug delivery/cell transplantation strategy to increase the viability of both exogenous and endogenous cells in tissue engineering applications.

6-Bromo-7-hydroxy-3-methylcoumarin (mBhc) is an efficient multi-photon labile protecting group for thiol caging and three-dimensional chemical patterning.

The photochemical release of chemical reagents and bioactive molecules provides a useful tool for spatio-temporal control of biological processes. However, achieving this goal requires the development of highly efficient one- and two-photon sensitive photo-cleavable protecting groups. Thiol-containing compounds play critical roles in biological systems and bioengineering applications. While potentially useful for sulfhydryl protection, the 6-bromo-7-hydroxy coumarin-4-ylmethyl (Bhc) group can undergo an undesired photoisomerization reaction upon irradiation that limits its uncaging efficiency. To address this issue, here we describe the development of 6-bromo-7-hydroxy-3-methylcoumarin-4-ylmethyl (mBhc) as an improved group for thiol-protection. One- and two-photon photolysis reactions demonstrate that a peptide containing a mBhc-caged thiol undergoes clean and efficient photo-cleavage upon irradiation without detectable photoisomer production. To test its utility for biological studies, a K-Ras-derived peptide containing an mBhc-protected thiol was prepared by solid phase peptide synthesis using Fmoc-Cys(mBhc)-OH for the introduction of the caged thiol. Irradiation of that peptide using either UV or near IR light in presence of protein farnesyltransferase (PFTase), resulted in generation of the free peptide which was then recognized by the enzyme and became farnesylated. To show the utility of this caging group in biomaterial applications, we covalently modified hydrogels with mBhc-protected cysteamine. Using multi-photon confocal microscopy, highly defined volumes of free thiols were generated inside the hydrogels and visualized via reaction with a sulfhydryl-reactive fluorophore. The simple synthesis of mBhc and its efficient removal by one- and two-photon processes make it an attractive protecting group for thiol caging in a variety of applications.

A novel polymeric drug delivery system for localized and sustained release of tacrolimus (FK506).

Despite substantial improvement in microsurgical techniques for nerve repair, recovery after peripheral nerve injury is usually incomplete. FK506, an FDA approved immunosuppressant, improves functional recovery and reinnervation following peripheral nerve injury in animal models. However, systemically delivered FK506 causes undesirable global immunosuppression. We have, therefore, engineered a biodegradable local delivery system for FK506 using fibrin gel as a drug reservoir that could be placed at a site of nerve injury. FK506 was incorporated into fibrin gel in solubilized, particulated, and poly(lactic-co-glycolic) acid (PLGA) microspheres-encapsulated forms. A tunable release of FK506 in the fibrin gel from days to weeks was observed with the rate of release being most rapid for the solubilized form and then the particulate form. The most prolonged period of release was seen with the PLGA microsphere-encapsulated form. As analyzed by in vitro dorsal root ganglion (DRG) neurite extension assay, PLGA microsphere encapsulation of FK506 did not alter the drug's properties and the released FK506 maintained its bioactivity over the entire period of release. This study suggests that local delivery of FK506 with fibrin hydrogel could be used to enhance peripheral nerve regeneration.

Affinity-based drug delivery systems for tissue repair and regeneration.

Affinity-based release systems use transient interactions to sustain and control the release of a therapeutic from a polymeric matrix. The most common affinity-based systems use heparin-based scaffolds to sustain the release of heparin-binding proteins, such as fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF). However, novel affinity-based systems based on, for example, protein-protein or DNA-protein interactions, are emerging to control the release of an expanding repertoire of therapeutics. Mathematical models of affinity-based systems have provided a thorough understanding of which parameters affect release rate from these systems, and how these release rates can be tuned. In this review, recent affinity-based release systems will be described, including an overview of the various types of affinity interactions used to modulate release, the mechanisms by which release from these systems is tuned, and the time scales of sustained release. This advanced drug delivery paradigm provides tunable and predictable release rates and has expanded the scope of deliverable therapeutics for tissue repair and regeneration.

Long-Acting Ocular Injectables: Are We Looking In The Right Direction?

The complex anatomy and physiological barriers of the eye make delivering ocular therapeutics challenging. Generally, effective drug delivery to the eye is hindered by rapid clearance and limited drug bioavailability. Biomaterial-based approaches have emerged to enhance drug delivery to ocular tissues and overcome existing limitations. In this review, some of the most promising long-acting injectables (LAIs) in ocular drug delivery are explored, focusing on novel design strategies to improve therapeutic outcomes. LAIs are designed to enable sustained therapeutic effects, thereby extending local drug residence time and facilitating controlled and targeted drug delivery. Moreover, LAIs can be engineered to enhance drug targeting and penetration across ocular physiological barriers.

Engineering Biomaterials to Model Immune-Tumor Interactions In Vitro.

Engineered biomaterial scaffolds are becoming more prominent in research laboratories to study drug efficacy for oncological applications in vitro, but do they have a place in pharmaceutical drug screening pipelines? The low efficacy of cancer drugs in phase II/III clinical trials suggests that there are critical mechanisms not properly accounted for in the pre-clinical evaluation of drug candidates. Immune cells associated with the tumor may account for some of these failures given recent successes with cancer immunotherapies; however, there are few representative platforms to study immune cells in the context of cancer as traditional 2D culture is typically monocultures and humanized animal models have a weakened immune composition. Biomaterials that replicate tumor microenvironmental cues may provide a more relevant model with greater in vitro complexity. In this review, the authors explore the pertinent microenvironmental cues that drive tumor progression in the context of the immune system, discuss how these cues can be incorporated into hydrogel design to culture immune cells, and describe progress toward precision oncological drug screening with engineered tissues.

Hyaluronan improves photoreceptor differentiation and maturation in human retinal organoids.

Human stem cell-derived organoids enable both disease modeling and serve as a source of cells for transplantation. Human retinal organoids are particularly important as a source of human photoreceptors; however, the long differentiation period required and lack of vascularization in the organoid often results in a necrotic core and death of inner retinal cells before photoreceptors are fully mature. Manipulating the in vitro environment of differentiating retinal organoids through the incorporation of extracellular matrix components could influence retinal development. We investigated the addition of hyaluronan (HA), a component of the interphotoreceptor matrix, as an additive to promote long-term organoid survival and enhance retinal maturation. HA treatment had a significant reduction in the proportion of proliferating (Ki67+) cells and increase in the proportion of photoreceptors (CRX+), suggesting that HA accelerated photoreceptor commitment in vitro. HA significantly upregulated genes specific to photoreceptor maturation and outer segment development. Interestingly, prolonged HA-treatment significantly decreased the length of the brush border layer compared to those in control retinal organoids, where the photoreceptor outer segments reside; however, HA-treated organoids also had more mature outer segments with organized discs structures, as revealed by transmission electron microscopy. The brush border layer length was inversely proportional to the molar mass and viscosity of the hyaluronan added. This is the first study to investigate the role of exogenous HA, viscosity, and polymer molar mass on photoreceptor maturation, emphasizing the importance of material properties on organoid culture. STATEMENT OF SIGNIFICANCE: Retinal organoids are a powerful tool to study retinal development in vitro, though like many other organoid systems, can be highly variable. In this work, Shoichet and colleagues investigated the use of hyaluronan (HA), a native component of the interphotoreceptor matrix, to improve photoreceptor maturation in developing human retinal organoids. HA promoted human photoreceptor differentiation leading to mature outer segments with disc formation and more uniform and healthy retinal organoids. These findings highlight the importance of adding components native to the developing retina to generate more physiologically relevant photoreceptors for cell therapy and in vitro models to drive drug discovery and uncover novel disease mechanisms.

Transplanted human photoreceptors transfer cytoplasmic material but not to the recipient mouse retina.

BACKGROUND: The discovery of material transfer between transplanted and host mouse photoreceptors has expanded the possibilities for utilizing transplanted photoreceptors as potential vehicles for delivering therapeutic cargo. However, previous research has not directly explored the capacity for human photoreceptors to engage in material transfer, as human photoreceptor transplantation has primarily been investigated in rodent models of late-stage retinal disease, which lack host photoreceptors. METHODS: In this study, we transplanted human stem-cell derived photoreceptors purified from human retinal organoids at different ontological ages (weeks 10, 14, or 20) into mouse models with intact photoreceptors and assessed transfer of human proteins and organelles to mouse photoreceptors. RESULTS: Unexpectedly, regardless of donor age or mouse recipient background, human photoreceptors did not transfer material in the mouse retina, though a rare subset of donor cells (< 5%) integrated into the mouse photoreceptor cell layer. To investigate the possibility that a species barrier impeded transfer, we used a flow cytometric assay to examine material transfer in vitro. Interestingly, dissociated human photoreceptors transferred fluorescent protein with each other in vitro, yet no transfer was detected in co-cultures of human and mouse photoreceptors, suggesting that material transfer is species specific. CONCLUSIONS: While xenograft models are not a tractable system to study material transfer of human photoreceptors, these findings demonstrate that human retinal organoid-derived photoreceptors are competent donors for material transfer and thus may be useful to treat retinal degenerative disease.