Hydrogel Delivery Device for the In Vitro and In Vivo Sustained Release of Active rhGALNS Enzyme.

Morquio A disease is a genetic disorder resulting in N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, and patients are currently treated with enzyme replacement therapy via weekly intravenous enzyme infusions. A means of sustained enzyme delivery could improve patient quality of life by reducing the administration time, frequency of hospital visits, and treatment cost. In this study, we investigated poly(ethylene-glycol) (PEG) hydrogels as a tunable, hydrolytically degradable drug delivery system for the encapsulation and sustained release of recombinant human GALNS (rhGALNS). We evaluated hydrogel formulations that optimized hydrogel gelation and degradation time while retaining rhGALNS activity and sustaining rhGALNS release. We observed the release of active rhGALNS for up to 28 days in vitro from the optimized formulation. rhGALNS activity was preserved in the hydrogel relative to buffer over the release window, and encapsulation was found to have no impact on the rhGALNS structure when measured by intrinsic fluorescence, circular dichroism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In vivo, we monitored the retention of fluorescently labeled rhGALNS in C57BL/6 albino mice when administered via subcutaneous injection and observed rhGALNS present for up to 20 days when delivered in a hydrogel versus 7 days in the buffer control. These results indicate that PEG hydrogels are suitable for the encapsulation, preservation, and sustained release of recombinant enzymes and may present an alternative method of delivering enzyme replacement therapies that improve patient quality of life.

Microfluidic Chip Device for In Situ Mixing and Fabrication of Hydrogel Microspheres via Michael-Type Addition.

Hydrogel microspheres are sought for a variety of biomedical applications, including therapeutic and cellular delivery, sensors, and lubricants. Robust fabrication of hydrogel microspheres with uniform sizes and properties can be achieved using microfluidic systems that rely on droplet formation and subsequent gelation to form microspheres. Such systems work well when gelation is initiated after droplet formation but are not practical for timed gelation systems where gelation is initiated prior to droplet formation; premature gelation can lead to device blockage, variable microsphere diameter due to viscosity changes in the precursor solution, and limited numbers of microspheres produced in a single run. To enable microfluidic fabrication of microspheres from timed gelation hydrogel systems, an in situ mixing region is needed so that various hydrogel precursor components can be added separately. Here, we designed and evaluated three mixing devices for their effectiveness at mixing hydrogel precursor solutions prior to droplet formation and subsequent gelation. The serpentine geometry was found to be the most effective and was further improved with the inclusion of a pillar array to increase agitation. The optimized device was shown to fully mix precursor solutions and enable the fabrication of monodisperse polyethylene glycol microspheres, offering great potential for use with timed gelation hydrogel systems.

Biodegradable polyethylene glycol hydrogels for sustained release and enhanced stability of rhGALNS enzyme.

Mucopolysaccharidosis IVA (Morquio A disease) is a genetic disorder caused by deficiency of N-acetylgalactosamine-6-sulfate-sulfatase (GALNS), leading to accumulation of keratan sulfate and chondroitin-6-sulfate in lysosomes. Many patients become wheelchair-dependent as teens, and their life span is 20-30 years. Currently, enzyme replacement therapy (ERT) is the treatment of choice. Although it alleviates some symptoms, replacing GALNS enzyme poses several challenges including very fast clearance from circulation and instability at 37 °C. These constraints affect frequency and cost of enzyme infusion and ability to reach all tissues. In this study, we developed injectable and biodegradable polyethylene glycol (PEG) hydrogels, loaded with recombinant human GALNS (rhGALNS) to improve enzyme stability and bioavailability, and to sustain release. We established the enzyme's release profile via bulk release experiments and determined diffusivity using fluorescence correlation spectroscopy. We observed that PEG hydrogels preserved enzyme activity during sustained release for 7 days. In the hydrogel, rhGALNS diffused almost four times slower than in buffer. We further confirmed that the enzyme was active when released from the hydrogels, by measuring its uptake in patient fibroblasts. The developed hydrogel delivery device could overcome current limits of rhGALNS replacement and improve quality of life for Morquio A patients. Encapsulated GALNS enzyme in a polyethylene glycol hydrogel improves GALNS stability by preserving its activity, and provides sustained release for a period of at least 7 days.

Predicting Drug Release From Degradable Hydrogels Using Fluorescence Correlation Spectroscopy and Mathematical Modeling.

Predicting release from degradable hydrogels is challenging but highly valuable in a multitude of applications such as drug delivery and tissue engineering. In this study, we developed a simple mathematical and computational model that accounts for time-varying diffusivity and geometry to predict solute release profiles from degradable hydrogels. Our approach was to use time snapshots of diffusivity and hydrogel geometry data measured experimentally as inputs to a computational model which predicts release profile. We used two model proteins of varying molecular weights: bovine serum albumin (BSA; 66 kDa) and immunoglobulin G (IgG; 150 kDa). We used fluorescence correlation spectroscopy (FCS) to determine protein diffusivity as a function of hydrogel degradation. We tracked changes in gel geometry over the same time period. Curve fits to the diffusivity and geometry data were used as inputs to the computational model to predict the protein release profiles from the degradable hydrogels. We validated the model using conventional bulk release experiments. Because we approached the hydrogel as a black box, the model is particularly valuable for hydrogel systems whose degradation mechanisms are not known or cannot be accurately modeled.

Whispering gallery mode resonator sensor for in situ measurements of hydrogel gelation.

Whispering gallery mode (WGM) resonators are compact and ultrasensitive devices, which enable label-free sensing at the single-molecule level. Despite their high sensitivity, WGM resonators have not been thoroughly investigated for use in dynamic biochemical processes including molecular diffusion and polymerization. In this work, the first report of using WGM sensors to continuously monitor a chemical reaction (i.e. gelation) in situ in a hydrogel is described. Specifically, we monitor and quantify the gelation dynamics of polyacrylamide hydrogels using WGM resonators and compare the results to an established measurement method based on rheology. Rheology measures changes in viscoelasticity, while WGM resonators measure changes in refractive index. Different gelation conditions were studied by varying the total monomer concentration and crosslinker concentration of the hydrogel precursor solution, and the resulting similarities and differences in the signal from the WGM resonator and rheology are elucidated. This work demonstrates that WGM alone or in combination with rheology can be used to investigate the gelation dynamics of hydrogels to provide insights into their gelation mechanisms.

Intrinsic Response Towards Physiologic Stiffness is Cell-Type Dependent.

In the continuous search for better tissue engineering scaffolds it has become increasingly clear that the substrate properties dramatically affect cell responses. Here we compared cells from a physiologically stiff tissue, melanoma, to cells isolated from a physiologically soft tissue, brain. We measured the cell line responses to laminin immobilized onto glass or polyacrylamide hydrogels tuned to have a Young's modulus ranging from 1 to 390 kPa. Single cells were analyzed for spreading area, shape, total actin content, actin-based morphological features and modification of immobilized laminin. Both cell types exhibited stiffness- and laminin concentration-dependent responses on polyacrylamide and glass. Melanoma cells exhibited very little spreading and were rounded on soft (1, 5, and 15 kPa) hydrogels while cells on stiff (40, 100, and 390 kPa) hydrogels were spread and had a polarized cell shape with large lamellipodia. On rigid glass surfaces, spreading and actin-based morphological features were not observed until laminin concentration was much higher. Similarly, increased microglia cell spreading and presence of actin-based structures were observed on stiff hydrogels. However, responses on rigid glass surfaces were much different. Microglia cells had large spreading areas and elongated shapes on glass compared to hydrogels even when immobilized laminin density was consistent on all gels. While cell spreading and shape varied with Young's modulus of the hydrogel, the concentration of f-actin was constant. A decrease in laminin immunofluorescence was associated with melanoma and microglia cell spreading on glass with high coating concentration of laminin, indicating modification of immobilized laminin triggered by supraphysiologic stiffness and high ligand density. These results suggest that some cell lines are more sensitive to mechanical properties matching their native tissue environment while other cell lines may require stiffness and extracellular ligand density well above physiologic tissue before saturation in cell spreading, elongation and cytoskeletal re-organization are reached.

Sustained release of multicomponent platelet-rich plasma proteins from hydrolytically degradable PEG hydrogels.

Platelet-rich plasma (PRP), an autologous blood derived product is a concentrated mix of multiple growth factors and cytokines. Direct injections of PRP are clinically used for treatment of various musculoskeletal disorders and in wound healing. However, PRP therapy has met with limited clinical success possibly due to unpredictable and premature bolus delivery of PRP growth factors. The objective of this study was to predictably control the bioavailability of PRP growth factors using a hydrolytically degradable polyethylene glycol (PEG) hydrogel. We used a step-growth polymerization based on a Michael-type addition reaction between a 6-arm PEG-acrylate and a dithiol crosslinker, which led to the formation of a homogenous hydrogel network under mild, physiologically relevant conditions. Specifically, to model the release of multicomponent PRP through PEG hydrogels, we examined bulk diffusion of PRP as well as model proteins in a size range corresponding to that of growth factors found in PRP. Our results indicated that protein size and hydrogel degradation controlled diffusion of all proteins and that secondary structure of proteins encapsulated during gelation remained unaffected post-release. Analysis of specific PRP proteins released from the hydrogel showed sustained release until complete hydrogel degradation. PRP released from hydrogels promoted proliferation of human dermal fibroblast, indicating retained bioactivity upon encapsulation and release. The versatile hydrogel system holds clinical potential as a therapeutic drug delivery depot of multicomponent mixtures like PRP. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3304-3314, 2017.

Polymeric particle-mediated molecular therapies to treat spinal cord injury.

Spinal cord injury (SCI) is a physically and psychologically debilitating condition that mainly affects young, healthy males who are at the peak of their personal and professional development. SCI damages axons and disrupts myelination, which interrupts sensory and motor neuronal function. Current treatments are mostly palliative, aimed at reducing further damage and pain, but do not provide a cure. Polymeric particles have shown tremendous promise to provide patients with effective treatments that can bring partial or full functional recovery. Their unique properties can facilitate delivery of therapeutic agents to the injury site, provide protection from the host immunity or provide platforms to stimulate the regeneration of damaged axons. This review highlights the current benefits and challenges of the use of polymeric particles to control the release of molecular therapeutics as potential strategies for SCI treatment.

Mechanical factors direct mouse aortic remodelling during early maturation.

Numerous diseases have been linked to genetic mutations that lead to reduced amounts or disorganization of arterial elastic fibres. Previous work has shown that mice with reduced amounts of elastin (Eln+/-) are able to live a normal lifespan through cardiovascular adaptations, including changes in haemodynamic stresses, arterial geometry and arterial wall mechanics. It is not known if the timeline and presence of these adaptations are consistent in other mouse models of elastic fibre disease, such as those caused by the absence of fibulin-5 expression (Fbln5-/-). Adult Fbln5-/- mice have disorganized elastic fibres, decreased arterial compliance and high blood pressure. We examined mechanical behaviour of the aorta in Fbln5-/- mice through early maturation when the elastic fibres are being assembled. We found that the physiologic circumferential stretch, stress and modulus of Fbln5-/- aorta are maintained near wild-type levels. Constitutive modelling suggests that elastin contributions to the total stress are decreased, whereas collagen contributions are increased. Understanding how collagen fibre structure and mechanics compensate for defective elastic fibres to meet the mechanical requirements of the maturing aorta may help to better understand arterial remodelling in human elastinopathies.