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.

Ischemic stroke results in a loss of neurons for which there are no available clinical strategies to stimulate regeneration. While preclinical studies have demonstrated that functional recovery can be obtained by transplanting an exogenous source of neural progenitors into the brain, it remains unknown at which stage of neuronal maturity cells will provide the most benefit. We investigated the role of neuronal maturity on cell survival, differentiation, and long-term sensorimotor recovery in stroke-injured rats using a population of human cortically-specified neuroepithelial progenitor cells (cNEPs) delivered in a biocompatible, bioresorbable hyaluronan/methylcellulose hydrogel. We demonstrate that transplantation of immature cNEPs result in the greatest tissue and functional repair, relative to transplantation of more mature neurons. The transplantation process itself resulted in the least cell death and phenotypic changes in the immature cNEPs, and the greatest acute cell death in the mature cells. The latter negatively impacted host tissue and negated any potential positive effects associated with cell maturity and the hydrogel vehicle, which itself showed some functional and tissue benefit. Moreover, we show that more mature cell populations are drastically altered during the transplantation process itself. The phenotype of the cells before and after transplantation had an enormous impact on their survival and the consequent tissue and behavioral response, emphasizing the importance of characterizing injected cells in transplantation studies more broadly.

Therapeutic delivery to the brain is limited by the blood-brain barrier and is exacerbated by off-target effects associated with systemic delivery, thereby precluding many potential therapies from even being tested. Given the systemic side effects of cyclosporine and erythropoietin, systemic administration would be precluded in the context of stroke, leaving only the possibility of local delivery. We wondered if direct delivery to the brain would allow new reparative therapeutics, such as these, to be identified for stroke. Using a rodent model of stroke, we employed an injectable drug delivery hydrogel strategy to circumvent the blood-brain barrier and thereby achieved, for the first time, local and sustained co-release to the brain of cyclosporine and erythropoietin. Both drugs diffused to the sub-cortical neural stem and progenitor cell (NSPC) niche and were present in the brain for at least 32 days post-stroke. Each drug had a different outcome on brain tissue: cyclosporine increased plasticity in the striatum while erythropoietin stimulated endogenous NSPCs. Only their co-delivery, but not either drug alone, accelerated functional recovery and improved tissue repair. This platform opens avenues for hitherto untested therapeutic combinations to promote regeneration and repair after stroke.

The regenerative capacity of injured adult central nervous system (CNS) tissue is very limited. Specifically, traumatic spinal cord injury (SCI) leads to permanent loss of motor and sensory functions below the site of injury, as well as other detrimental complications. A potential regenerative strategy is stem cell transplantation; however, cell survival is typically less than 1%. To improve cell survival, stem cells can be delivered in a biomaterial matrix that provides an environment conducive to survival after transplantation. One major challenge in this approach is to define the biomaterial and cell strategies in vitro. To this end, we investigated both peptide-modification of gellan gum and olfactory ensheathing glia (OEG) on neural stem/progenitor cell (NSPC) fate. To enhance cell adhesion, the gellan gum (GG) was modified using Diels-Alder click chemistry with a fibronectin-derived synthetic peptide (GRGDS). Amino acid analysis demonstrated that approximately 300 nmol of GRGDS was immobilized to each mg of GG. The GG-GRGDS had a profound effect on NSPC morphology and proliferation, distinct from that of NSPCs in GG alone, demonstrating the importance of GRGDS for cell-GG interaction. To further enhance NSPC survival and outgrowth, they were cultured with OEG. Here NSPCs interacted extensively with OEG, demonstrating significantly greater survival and proliferation relative to monocultures of NSPCs. These results suggest that this co-culture strategy of NSPCs with OEG may have therapeutic benefit for SCI repair.

Traumatic injury to the spinal cord causes cell death, demyelination, axonal degeneration, and cavitation resulting in functional motor and sensory loss. Stem cell therapy is a promising approach for spinal cord injury (SCI); however, this strategy is currently limited by the poor survival and uncontrolled differentiation of transplanted stem cells. In an attempt to achieve greater survival and integration with the host tissue, we examined the survival and efficacy of adult brain-derived neural stem/progenitor cells (NSPCs) injected within a hydrogel blend of hyaluronan and methyl cellulose (HAMC) into a subacute, clinically relevant model of rat SCI. Prior to use, HAMC was covalently modified with recombinant rat platelet-derived growth factor-A (rPDGF-A) to promote oligodendrocytic differentiation. SCI rats transplanted with NSPCs in HAMC-rPDGF-A showed improved behavioral recovery compared to rats transplanted with NSPCs in media. Rats with NSPC/HAMC-rPDGF-A transplants had a significant reduction in cavitation, improved graft survival, increased oligodendrocytic differentiation, and sparing of perilesional host oligodendrocytes and neurons. These data suggest that HAMC-rPDGF-A is a promising vehicle for cell delivery to the injured spinal cord.

We have previously shown that a novel synthetic hydrogel channel composed of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (pHEMA-MMA) is biocompatible and supports axonal regeneration after spinal cord injury. Our goal was to improve the number and type of regenerated axons within the spinal cord through the addition of different matrices and growth factors incorporated within the lumen of the channel. After complete spinal cord transection at T8, pHEMA-MMA channels, having an elastic modulus of 263+/-13 kPa were implanted into adult Sprague Dawley rats. The channels were then filled with one of the following matrices: collagen, fibrin, Matrigel, methylcellulose, or smaller pHEMA-MMA tubes placed within a larger pHEMA-MMA channel (called tubes within channels, TWC). We also supplemented selected matrices (collagen and fibrin) with neurotrophic factors, fibroblast growth factor-1 (FGF-1) and neurotrophin-3 (NT-3). After channel implantation, fibrin glue was applied to the cord-channel interface, and a duraplasty was performed with an expanded polytetrafluoroethylene (ePTFE) membrane. Controls included animals that had either complete spinal cord transection and implantation of unfilled pHEMA-MMA channels or complete spinal cord transection. Regeneration was assessed by retrograde axonal tracing with Fluoro-Gold, and immunohistochemistry with NF-200 (for total axon counts) and calcitonin gene related peptide (CGRP, for sensory axon counts) after 8 weeks survival. Fibrin, Matrigel, methylcellulose, collagen with FGF-1, collagen with NT-3, fibrin with FGF-1, and fibrin with NT-3 increased the total axon density within the channel (ANOVA, p<0.05) compared to unfilled channel controls. Only fibrin with FGF-1 decreased the sensory axon density compared to unfilled channel controls (ANOVA, p<0.05). Fibrin promoted the greatest axonal regeneration from reticular neurons, and methylcellulose promoted the greatest regeneration from vestibular and red nucleus neurons. With Matrigel, there was no axonal regeneration from brainstem motor neurons. The addition of FGF-1 increased the axonal regeneration of vestibular neurons, and the addition of NT-3 decreased the total number of axons regenerating from brainstem neurons. The fibrin and TWC showed a consistent improvement in locomotor function at both 7 and 8 weeks. Thus, the present study shows that the presence and type of matrix contained within synthetic hydrogel guidance channels affects the quantity and origin of axons that regenerate after complete spinal cord transection, and can improve functional recovery. Determining the optimum matrices and growth factors for insertion into these guidance channels will improve regeneration of the injured spinal cord.

Drug delivery to solid tumours remains a challenge because both tumour physiology and drug solubility are unfavourable. Engineered materials can provide the basis for drug reformulation, incorporating active compounds and modulating their pharmacokinetic and biodistribution behaviour. To this end, we encapsulated docetaxel, a poorly soluble taxane drug, in a self-assembled polymeric nanoparticle micelle of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol) (poly(TMCC-co-LA)-g-PEG). This formulation was compared with its conventional ethanolic polysorbate 80 formulation in terms of plasma circulation and biodistribution in an orthotopic mouse model of breast cancer. Notably, the polymeric nanoparticle formulation achieved greater tumour retention, resulting in prolonged exposure of cancer cells to the active drug. This behaviour was unique to the tumour tissue. The active drug was eliminated at equal or greater rates in all other tissues assayed when delivered in the polymeric nanoparticles vs. the free drug formulation. Thus, these polymeric nanoparticles are promising vehicles for solid tumour drug delivery applications, offering greater tumour exposure while eliminating the need for toxic solvents and surfactants in the dosing formulation.

Breast cancer cell invasion is influenced by growth factor concentration gradients in the tumor microenvironment. However, studying the influence of growth factor gradients on breast cancer cell invasion is challenging due to both the complexities of in vivo models and the difficulties in recapitulating the tumor microenvironment with defined gradients using in vitro models. A defined hyaluronic acid (HA)-based hydrogel crosslinked with matrix metalloproteinase (MMP) cleavable peptides and modified with multiphoton labile nitrodibenzofuran (NDBF) was synthesized to photochemically immobilize epidermal growth factor (EGF) gradients. We demonstrate that EGF gradients can differentially influence breast cancer cell invasion and drug response in cell lines with different EGF receptor (EGFR) expression levels. Photopatterned EGF gradients increase the invasion of moderate EGFR expressing MDA-MB-231 cells, reduce invasion of high EGFR expressing MDA-MB-468 cells, and have no effect on invasion of low EGFR-expressing MCF-7 cells. We evaluate MDA-MB-231 and MDA-MB-468 cell response to the clinically tested EGFR inhibitor, cetuximab. Interestingly, the cellular response to cetuximab is completely different on the EGF gradient hydrogels: cetuximab decreases MDA-MB-231 cell invasion but increases MDA-MB-468 cell invasion and cell number, thus demonstrating the importance of including cell-microenvironment interactions when evaluating drug targets.

We developed a novel taxane-binding peptide (TBP) modified, biodegradable polymeric micelle that overcomes limitations of drug loading and poor serum stability typically seen with particle delivery, leading to enhanced pharmacokinetics and tumor distribution of docetaxel (DTX). The use of the taxane-binding peptide to increase docetaxel loading is particularly compelling as it takes advantage of a known intracellular binding mechanism in a new way. Docetaxel is a potent chemotherapeutic with a therapeutic index often limited by the toxicity of the excipients that are necessary to enhance its solubility for intravenous delivery. Our polymeric micelle has terminal furan groups that enable facile antibody Fab conjugation by Diels-Alder chemistry for targeted delivery. Compared to the conventional ethanolic polysorbate 80 formulation (Free DTX), our nanoparticle (NP DTX) formulation exhibited a two-fold increase in exposure and tumor accumulation. Notably, the reduced toxicity of the NP DTX formulation increased the therapeutic index and allowed for higher dosing regimens, with a maximum tolerated dose (MTD) 1.6-fold higher than that of the Free DTX formulation, which is significant and similar to enhancements observed in clinical products for docetaxel and other drugs. These improved properties led to enhanced mouse survival in an orthotopic model of breast cancer; however, the targeted formulation of Fab-NP DTX did not further improve efficacy. Together, these results clearly demonstrate the benefits of the TBP-modified polymeric micelles as promising carriers for docetaxel.

We demonstrate a novel approach to reverse advanced stages of blindness using hydrogel-mediated delivery of retinal pigmented epithelium (RPE) and photoreceptors directly to the degenerated retina of blind mice. With sodium iodate (NaIO3) injections in mice, both RPE and photoreceptors degenerate, resulting in complete blindness and recapitulating the advanced retinal degeneration that is often observed in humans. We observed vision restoration only with co-transplantation of RPE and photoreceptors in a hyaluronic acid-based hydrogel, and not with transplantation of each cell type alone as determined with optokinetic head tracking and light avoidance assays. Both RPE and photoreceptors survived significantly better when co-transplanted than in their respective single cell type controls. While others have pursued transplantation of one of either RPE or photoreceptors, we demonstrate the importance of transplanting both cell types with a minimally-invasive hydrogel for vision repair in a degenerative disease model of the retina.

Adult skeletal muscle tissue harbors the capacity for self-repair due to the presence of tissue resident muscle stem cells (MuSCs). Advances in the area of prospective MuSC isolation demonstrated the potential of cell transplantation therapy as a regenerative medicine strategy to restore strength and long-term regenerative capacity to aged, injured, or diseased skeletal muscle tissue. However, cell loss during ejection, limits to post-injection proliferation, and poor donor cell dispersion distal to the injection site are amongst hurdles to overcome to maximize MuSC transplant impact. Here, we assess a physical blend of hyaluronan and methylcellulose (HAMC) as a bioactive, shear thinning hydrogel cell delivery system to improve MuSC transplantation efficiency. Using in vivo transplantation studies, we found that the HAMC delivery system results in a >45% increase in the number of donor-derived fibers as compared to saline delivery. We demonstrate that increases in donor-derived fibers when using HAMC are attributed to increased MuSC proliferation via a CD44-independent mechanism, preventing injected cell active clearance, and supporting in vivo expansion by delaying differentiation. Furthermore, we observed a significant improvement in donor fiber dispersion when MuSCs were delivered in HAMC. Our study results suggest that HAMC is a promising muscle stem cell delivery vehicle.