Waffle-inspired hydrogel-based macrodevice for spatially controlled distribution of encapsulated therapeutic microtissues and pro-angiogenic endothelial cells.

Macro-encapsulation systems for delivery of cellular therapeutics in diabetes treatment offer major advantages such as device retrievability and high cell packing density. However, microtissue aggregation and absence of vasculature have been implicated in the inadequate transfer of nutrients and oxygen to the transplanted cellular grafts. Herein, we develop a hydrogel-based macrodevice to encapsulate therapeutic microtissues positioned in homogeneous spatial distribution to mitigate their aggregation while concurrently supporting an organized intra-device network of vascular-inductive cells. Termed Waffle-inspired Interlocking Macro-encapsulation (WIM) device, this platform comprises two modules with complementary topography features that fit together in a lock-and-key configuration. The waffle-inspired grid-like micropattern of the "lock" component effectively entraps insulin-secreting microtissues in controlled locations while the interlocking design places them in a co-planar spatial arrangement with close proximity to vascular-inductive cells. The WIM device co-laden with INS-1E microtissues and human umbilical vascular endothelial cells (HUVECs) maintains desirable cellular viability in vitro with the encapsulated microtissues retaining their glucose-responsive insulin secretion while embedded HUVECs express pro-angiogenic markers. Furthermore, a subcutaneously implanted alginate-coated WIM device encapsulating primary rat islets achieves blood glucose control for 2 weeks in chemically induced diabetic mice. Overall, this macrodevice design lays foundation for a cell delivery platform, which has the potential to facilitate nutrients and oxygen transport to therapeutic grafts and thereby might lead to improved disease management outcome.

Cerium Oxide Nanoparticles with Entrapped Gadolinium for High T 1 Relaxivity and ROS-Scavenging Purposes.

Gadolinium chelates are employed worldwide today as clinical contrast agents for magnetic resonance imaging. Until now, the commonly used linear contrast agents based on the rare-earth element gadolinium have been considered safe and well-tolerated. Recently, concerns regarding this type of contrast agent have been reported, which is why there is an urgent need to develop the next generation of stable contrast agents with enhanced spin-lattice relaxation, as measured by improved T 1 relaxivity at lower doses. Here, we show that by the integration of gadolinium ions in cerium oxide nanoparticles, a stable crystalline 5 nm sized nanoparticulate system with a homogeneous gadolinium ion distribution is obtained. These cerium oxide nanoparticles with entrapped gadolinium deliver strong T 1 relaxivity per gadolinium ion (T 1 relaxivity, r 1 = 12.0 mM-1 s-1) with the potential to act as scavengers of reactive oxygen species (ROS). The presence of Ce3+ sites and oxygen vacancies at the surface plays a critical role in providing the antioxidant properties. The characterization of radial distribution of Ce3+ and Ce4+ oxidation states indicated a higher concentration of Ce3+ at the nanoparticle surfaces. Additionally, we investigated the ROS-scavenging capabilities of pure gadolinium-containing cerium oxide nanoparticles by bioluminescent imaging in vivo, where inhibitory effects on ROS activity are shown.

Immuno-Modulatory Effects of Microparticles Formulated from Degradable Polystyrene Analogue.

Environmental accumulation of non-degradable polystyrene (PS) microparticles from plastic waste poses potential adverse impact on marine life and human health. Herein, microparticles from a degradable PS analogue (dePS) are formulated and their immuno-modulatory characteristics are comprehensively evaluated. Both dePS copolymer and microparticles are chemically degradable under accelerated hydrolytic condition. In vitro studies show that dePS microparticles are non-toxic to three immortalized cell lines. While dePS microparticles do not induce macrophage polarization in vitro, dePS microparticles induce in vivo upregulation of both pro-inflammatory and anti-inflammatory biomarkers in immuno-competent mice, suggesting the coexistence of mixed phenotypes of macrophages in the host immune response to these microparticles. Interestingly, on day 7 following subcutaneous in mice, dePS microparticles induce a lower level of several immuno-modulatory biomarkers (matrix metallo-proteinases (MMPs), tumor necrosis factor (TNF-α), and arginase activity) compared to that of reference poly(lactic-co-glycolic acid) microparticles. Remarkably, compared to PS microparticles, dePS microparticles exhibit similar in vitro and in vivo bioactivity while acquiring additional chemical degradability. Overall, this study gains new insights into the host immune response to dePS microparticles and suggests that this dePS analogue might be explored as an alternative material choice for biomedical and consumer care applications.

Modular design of a hybrid hydrogel for protease-triggered enhancement of drug delivery to regulate TNF-α production by pro-inflammatory macrophages.

Systemic drug administration has conventionally been prescribed to alleviate persistent local inflammation which is prevalent in chronic diseases. However, this approach is associated with drug-induced toxicity, particularly when the dosage exceeds that necessitated by pathological conditions of diseased tissues. Herein, we developed a modular hybrid hydrogel which could be triggered to release an anti-inflammatory drug upon exposure to elevated protease activity associated with inflammatory diseases. Modular design of the hybrid hydrogel enabled independent optimization of its protease-cleavable and drug-loaded subdomains to facilitate hydrogel formation, cleavability by matrix-metalloprotease-9 (MMP-9), and tuning drug release rate. In vitro study demonstrated the protease-triggered enhancement of drug release from the hybrid hydrogel system for effective inhibition of TNF-α production by pro-inflammatory macrophages and suggested its potential to mitigate drug-induced cytotoxicity. Using non-invasive imaging to monitor the activity of reactive oxygen species in biomaterial-induced host response, we confirmed that the hybrid hydrogel and its constituent materials did not induce adverse immune response after 5 days following their subcutaneous injection in immuno-competent mice. We subsequently incorporated this hybrid hydrogel onto a commercial wound dressing which could release the drug upon exposure to MMP-9. Together, our findings suggested that this hybrid hydrogel might be a versatile platform for on-demand drug delivery via either injectable or topical application to modulate inflammation in chronic diseases.

Microencapsulated islet-like microtissues with toroid geometry for enhanced cellular viability.

Transplantation of immuno-isolated islets is a promising strategy to restore insulin-secreting function in patients with Type 1 diabetes. However, the clinical translation of this treatment approach remains elusive due to the loss of islet viability resulting from hypoxia at the avascular transplantation site. To address this challenge, we designed non-spherical islet-like microtissues and investigated the effect of their geometries on cellular viability. Insulin-secreting microtissues with different shapes were fabricated by assembly of monodispersed rat insulinoma beta cells on micromolded nonadhesive hydrogels. Our study quantitatively demonstrated that toroid microtissues exhibited enhanced cellular viability and metabolic activity compared to rod and spheroid microtissues with the same volume. At a similar level of cellular viability, toroid geometry facilitated efficient packing of more cells into each microtissue than rod and spheroid geometries. In addition, toroid microtissues maintained the characteristic glucose-responsive insulin secretion of rat insulinoma beta cells. Furthermore, toroid microtissues preserved their geometry and structural integrity following their microencapsulation in immuno-isolatory alginate hydrogel. Our study suggests that adopting toroid geometry in designing therapeutic microtissues potentially reduces mass loss of cellular grafts and thereby may improve the performance of transplanted islets towards a clinically viable cure for Type 1 diabetes. STATEMENT OF SIGNIFICANCE: Transplantation of therapeutic cells is a promising strategy for the treatment of a wide range of hormone or protein-deficiency diseases. However, the clinical application of this approach is hindered by the loss of cell viability and function at the avascular transplantation site. To address this challenge, we fabricated hydrogel-encapsulated islet-like microtissues with non-spheroidal geometry and optimal surface-to-volume ratio. This study demonstrated that the viability of therapeutic cells can be significantly increased solely by redesigning the microtissue configuration without requiring any additional biochemical or operational accessories. This study suggests that the adoption of toroid geometry provides a possible avenue to improve the long-term survival of transplanted therapeutic cells and expedite the translation of cell-based therapy towards clinical application.

Cell-laden microengineered and mechanically tunable hybrid hydrogels of gelatin and graphene oxide.

Incorporating graphene oxide inside GelMA hydrogels enhances their mechanical properties and reduces UV-induced cell damage while preserving their favorable characteristics for 3D cell encapsulation. NIH-3T3 fibroblasts encapsulated in GO-GelMA microgels demonstrate excellent cellular viability, proliferation, spreading, and alignment. GO reinforcement combined with a multi-stacking approach offers a facile engineering strategy for the construction of complex artificial tissues.

Glucose-responsive microgels integrated with enzyme nanocapsules for closed-loop insulin delivery.

A glucose-responsive closed-loop insulin delivery system represents the ideal treatment of type 1 diabetes mellitus. In this study, we develop uniform injectable microgels for controlled glucose-responsive release of insulin. Monodisperse microgels (256 ± 18 µm), consisting of a pH-responsive chitosan matrix, enzyme nanocapsules, and recombinant human insulin, were fabricated through a one-step electrospray procedure. Glucose-specific enzymes were covalently encapsulated into the nanocapsules to improve enzymatic stability by protecting from denaturation and immunogenicity as well as to minimize loss due to diffusion from the matrix. The microgel system swelled when subjected to hyperglycemic conditions, as a result of the enzymatic conversion of glucose into gluconic acid and protonation of the chitosan network. Acting as a self-regulating valve system, microgels were adjusted to release insulin at basal release rates under normoglycemic conditions and at higher rates under hyperglycemic conditions. Finally, we demonstrated that these microgels with enzyme nanocapsules facilitate insulin release and result in a reduction of blood glucose levels in a mouse model of type 1 diabetes.

Enhanced function of immuno-isolated islets in diabetes therapy by co-encapsulation with an anti-inflammatory drug.

Immuno-isolation of islets has the potential to enable the replacement of pancreatic function in diabetic patients. However, host response to the encapsulated islets frequently leads to fibrotic overgrowth with subsequent impairment of the transplanted grafts. Here, we identified and incorporated anti-inflammatory agents into islet-containing microcapsules to address this challenge. In vivo subcutaneous screening of 16 small molecule anti-inflammatory drugs was performed to identify promising compounds that could minimize the formation of fibrotic cell layers. Using parallel non-invasive fluorescent and bioluminescent imaging, we identified dexamethasone and curcumin as the most effective drugs in inhibiting the activities of inflammatory proteases and reactive oxygen species in the host response to subcutaneously injected biomaterials. Next, we demonstrated that co-encapsulating curcumin with pancreatic rat islets in alginate microcapsules reduced fibrotic overgrowth and improved glycemic control in a mouse model of chemically-induced type I diabetes. These results showed that localized administration of anti-inflammatory drug can improve the longevity of encapsulated islets and may facilitate the translation of this technology toward a long-term cure for type I diabetes.

Injectable nano-network for glucose-mediated insulin delivery.

Diabetes mellitus, a disorder of glucose regulation, is a global burden affecting 366 million people across the world. An artificial "closed-loop" system able to mimic pancreas activity and release insulin in response to glucose level changes has the potential to improve patient compliance and health. Herein we develop a glucose-mediated release strategy for the self-regulated delivery of insulin using an injectable and acid-degradable polymeric network. Formed by electrostatic interaction between oppositely charged dextran nanoparticles loaded with insulin and glucose-specific enzymes, the nanocomposite-based porous architecture can be dissociated and subsequently release insulin in a hyperglycemic state through the catalytic conversion of glucose into gluconic acid. In vitro insulin release can be modulated in a pulsatile profile in response to glucose concentrations. In vivo studies validated that these formulations provided improved glucose control in type 1 diabetic mice subcutaneously administered with a degradable nano-network. A single injection of the developed nano-network facilitated stabilization of the blood glucose levels in the normoglycemic state (<200 mg/dL) for up to 10 days.

Painting blood vessels and atherosclerotic plaques with an adhesive drug depot.

The treatment of diseased vasculature remains challenging, in part because of the difficulty in implanting drug-eluting devices without subjecting vessels to damaging mechanical forces. Implanting materials using adhesive forces could overcome this challenge, but materials have previously not been shown to durably adhere to intact endothelium under blood flow. Marine mussels secrete strong underwater adhesives that have been mimicked in synthetic systems. Here we develop a drug-eluting bioadhesive gel that can be locally and durably glued onto the inside surface of blood vessels. In a mouse model of atherosclerosis, inflamed plaques treated with steroid-eluting adhesive gels had reduced macrophage content and developed protective fibrous caps covering the plaque core. Treatment also lowered plasma cytokine levels and biomarkers of inflammation in the plaque. The drug-eluting devices developed here provide a general strategy for implanting therapeutics in the vasculature using adhesive forces and could potentially be used to stabilize rupture-prone plaques.

Localized delivery of dexamethasone from electrospun fibers reduces the foreign body response.

Synthetic scaffolds are crucial to applications in regenerative medicine; however, the foreign body response can impede regeneration and may lead to failure of the implant. Herein we report the development of a tissue engineering scaffold that allows attachment and proliferation of regenerating cells while reducing the foreign body response by localized delivery of an anti-inflammatory agent. Electrospun fibers composed of poly(l-lactic) acid (PLLA) and poly(ε-caprolactone) (PCL) were prepared with and without the steroid anti-inflammatory drug, dexamethasone. Analysis of subcutaneous implants demonstrated that the PLLA fibers encapsulating dexamethasone evoked a less severe inflammatory response than the other fibers examined. They also displayed a controlled release of dexamethasone over a period of time conducive to tissue regeneration and allowed human mesenchymal stem cells to adhere to and proliferate on them in vitro. These observations demonstrate their potential as a building block for tissue engineering scaffolds.

Spatiotemporal effects of a controlled-release anti-inflammatory drug on the cellular dynamics of host response.

In general, biomaterials induce a non-specific host response when implanted in the body. This reaction has the potential to interfere with the function of the implanted materials. One method for controlling the host response is through local, controlled-release of anti-inflammatory agents. Herein, we investigate the spatial and temporal effects of an anti-inflammatory drug on the cellular dynamics of the innate immune response to subcutaneously implanted poly(lactic-co-glycolic) microparticles. Noninvasive fluorescence imaging was used to investigate the influence of dexamethasone drug loading and release kinetics on the local and systemic inhibition of inflammatory cellular activities. Temporal monitoring of host response showed that inhibition of inflammatory proteases in the early phase was correlated with decreased cellular infiltration in the later phase of the foreign body response. We believe that using controlled-release anti-inflammatory platforms to modulate early cellular dynamics will be useful in reducing the foreign body response to implanted biomaterials and medical devices.

Real-time in vivo detection of biomaterial-induced reactive oxygen species.

The non-specific host response to implanted biomaterials is often a key challenge of medical device design. To evaluate biocompatibility, measuring the release of reactive oxygen species (ROS) produced by inflammatory cells in response to biomaterial surfaces is a well-established method. However, the detection of ROS in response to materials implanted in vivo has not yet been demonstrated. Here, we develop a bioluminescence whole animal imaging approach to observe ROS released in response to subcutaneously-implanted materials in live animals. We compared the real-time generation of ROS in response to two representative materials, polystyrene and alginate, over the course of 28 days. High levels of ROS were observed near polystyrene, but not alginate implants, and persisted throughout the course of 28 days. Histological analysis revealed that high levels of ROS correlated not only with the presence of phagocytic cells at early timepoints, but also fibrosis at later timepoints, suggesting that ROS may be involved in both the acute and chronic phase of the foreign body response. These data are the first in vivo demonstration of ROS generation in response to implanted materials, and describe a novel technique to evaluate the host response.

Rapid biocompatibility analysis of materials via in vivo fluorescence imaging of mouse models.

BACKGROUND: Many materials are unsuitable for medical use because of poor biocompatibility. Recently, advances in the high throughput synthesis of biomaterials has significantly increased the number of potential biomaterials, however current biocompatibility analysis methods are slow and require histological analysis. METHODOLOGY/PRINCIPAL FINDINGS: Here we develop rapid, non-invasive methods for in vivo quantification of the inflammatory response to implanted biomaterials. Materials were placed subcutaneously in an array format and monitored for host responses as per ISO 10993-6: 2001. Host cell activity in response to these materials was imaged kinetically, in vivo using fluorescent whole animal imaging. Data captured using whole animal imaging displayed similar temporal trends in cellular recruitment of phagocytes to the biomaterials compared to histological analysis. CONCLUSIONS/SIGNIFICANCE: Histological analysis similarity validates this technique as a novel, rapid approach for screening biocompatibility of implanted materials. Through this technique there exists the possibility to rapidly screen large libraries of polymers in vivo.

Microfabrication of homogenous, asymmetric cell-laden hydrogel capsules.

Cell encapsulation has been broadly investigated as a technology to provide immunoprotection for transplanted endocrine cells. Here we develop a new fabrication method that allows for rapid, homogenous microencapsulation of insulin-secreting cells with varying microscale geometries and asymmetrically modified surfaces. Micromolding systems were developed using polypropylene mesh, and the material/surface properties associated with efficient encapsulation were identified. Cells encapsulated using these methods maintain desirable viability and preserve their ability to proliferate and secrete insulin in a glucose-responsive manner. This new cell encapsulation approach enables a practical route to an inexpensive and convenient process for the generation of cell-laden microcapsules without requiring any specialized equipment or microfabrication process.

Preparation of monodisperse biodegradable polymer microparticles using a microfluidic flow-focusing device for controlled drug delivery.

Degradable microparticles have broad utility as vehicles for drug delivery and form the basis of several therapies approved by the US Food and Drug Administration. Conventional emulsion-based methods of manufacturing produce particles with a wide range of diameters (and thus kinetics of release) in each batch. This paper describes the fabrication of monodisperse, drug-loaded microparticles from biodegradable polymers using the microfluidic flow-focusing (FF) devices and the drug-delivery properties of those particles. Particles are engineered with defined sizes, ranging from 10 microm to 50 microm. These particles are nearly monodisperse (polydispersity index = 3.9%). A model amphiphilic drug (bupivacaine) is incorporated within the biodegradable matrix of the particles. Kinetic analysis shows that the release of the drug from these monodisperse particles is slower than that from conventional methods of the same average size but a broader distribution of sizes and, most importantly, exhibit a significantly lower initial burst than that observed with conventional particles. The difference in the initial kinetics of drug release is attributed to the uniform distribution of the drug inside the particles generated using the microfluidic methods. These results demonstrate the utility of microfluidic FF for the generation of homogenous systems of particles for the delivery of drugs.