Reversal of pulmonary veno-occlusive disease phenotypes by inhibition of the integrated stress response.

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension arising from EIF2AK4 gene mutations or mitomycin C (MMC) administration. The lack of effective PVOD therapies is compounded by a limited understanding of the mechanisms driving the vascular remodeling in PVOD. We show that the administration of MMC in rats mediates the activation of protein kinase R (PKR) and the integrated stress response (ISR), which lead to the release of the endothelial adhesion molecule VE-Cadherin in the complex with Rad51 to the circulation, disruption of endothelial barrier, and vascular remodeling. Pharmacological inhibition of PKR or ISR attenuates the depletion of VE-Cadherin, elevation of vascular permeability, and vascular remodeling instigated by MMC, suggesting potential clinical intervention for PVOD. Finally, the severity of PVOD phenotypes was increased by a heterozygous BMPR2 mutation that truncates the carboxyl tail of BMPR2, underscoring the role of deregulated BMP signal in the development of PVOD.

Injectable Devices for Delivery of Liquid or Solid Protein Formulations.

Sustained delivery of protein therapeutics remains a largely unsolved problem across anatomic locations. Miniaturized devices that can provide sustained delivery of protein formulations have the potential to address this challenge via minimally invasive administration. In particular, methodologies that can optimize protein formulation independent of device manufacture have the greatest potential to provide a platform suitable for wide applications. The techniques developed here demonstrate the fabrication of tubular devices for sustained release of protein therapeutics. Utilizing a dip-casting process, fine-scale tubes can be reliably produced with wall thickness down to 30 µm. Techniques were developed that enabled effective loading of either solid or liquid formulations, while maintaining a cylindrical form-factor compatible with placement in a 22-gauge needle. Further, highly compacted protein pellets that approach the expected density of the raw materials were produced with a diameter (∼300 µm) suitable for miniaturized devices. Release from a solid-loaded device was capable of sustaining release of a model protein in excess of 400 days. Given significant interest in ocular applications, intravitreal injection was demonstrated in a rabbit model with these devices. In addition, to simulate repeated injections in ocular applications, serial intravitreal injection of two devices in a rabbit model demonstrated acceptable ocular safety without significant intraocular inflammation from clinical exam and histology.

Local decorin delivery via hyaluronic acid microrods improves cardiac performance, ventricular remodeling after myocardial infarction.

Heart failure (HF) remains a global public health burden and often results following myocardial infarction (MI). Following injury, cardiac fibrosis forms in the myocardium which greatly hinders cellular function, survival, and recruitment, thus severely limits tissue regeneration. Here, we leverage biophysical microstructural cues made of hyaluronic acid (HA) loaded with the anti-fibrotic proteoglycan decorin to more robustly attenuate cardiac fibrosis after acute myocardial injury. Microrods showed decorin incorporation throughout the entirety of the hydrogel structures and exhibited first-order release kinetics in vitro. Intramyocardial injections of saline (n = 5), microrods (n = 7), decorin microrods (n = 10), and free decorin (n = 4) were performed in male rat models of ischemia-reperfusion MI to evaluate therapeutic effects on cardiac remodeling and function. Echocardiographic analysis demonstrated that rats treated with decorin microrods (5.21% ± 4.29%) exhibited significantly increased change in ejection fraction (EF) at 8 weeks post-MI compared to rats treated with saline (-4.18% ± 2.78%, p < 0.001) and free decorin (-3.42% ± 1.86%, p < 0.01). Trends in reduced end diastolic volume were also identified in decorin microrod-treated groups compared to those treated with saline, microrods, and free decorin, indicating favorable ventricular remodeling. Quantitative analysis of histology and immunofluorescence staining showed that treatment with decorin microrods reduced cardiac fibrosis (p < 0.05) and cardiomyocyte hypertrophy (p < 0.05) at 8 weeks post-MI compared to saline control. Together, this work aims to contribute important knowledge to guide rationally designed biomaterial development that may be used to successfully treat cardiovascular diseases.

Replenishable prevascularized cell encapsulation devices increase graft survival and function in the subcutaneous space.

Beta cell replacement therapy (BCRT) for patients with type 1 diabetes (T1D) improves blood glucose regulation by replenishing the endogenous beta cells destroyed by autoimmune attack. Several limitations, including immune isolation, prevent this therapy from reaching its full potential. Cell encapsulation devices used for BCRT provide a protective physical barrier for insulin-producing beta cells, thereby protecting transplanted cells from immune attack. However, poor device engraftment posttransplantation leads to nutrient deprivation and hypoxia, causing metabolic strain on transplanted beta cells. Prevascularization of encapsulation devices at the transplantation site can help establish a host vascular network around the implant, increasing solute transport to the encapsulated cells. Here, we present a replenishable prevascularized implantation methodology (RPVIM) that allows for the vascular integration of replenishable encapsulation devices in the subcutaneous space. Empty encapsulation devices were vascularized for 14 days, after which insulin-producing cells were inserted without disrupting the surrounding vasculature. The RPVIM devices were compared with nonprevascularized devices (Standard Implantation Methodology [SIM]) and previously established prevascularized devices (Standard Prevascularization Implantation Methodology [SPVIM]). Results show that over 75% of RPVIM devices containing stem cell-derived insulin-producing beta cell clusters showed a signal after 28 days of implantation in subcutaneous space. Notably, not only was the percent of RPVIM devices showing signal significantly greater than SIM and SPVIM devices, but the intraperitoneal glucose tolerance tests and histological analyses showed that encapsulated stem-cell derived insulin-producing beta cell clusters retained their function in the RPVIM devices, which is crucial for the successful management of T1D.

Encapsulation of ß-NGF in injectable microrods for localized delivery accelerates endochondral fracture repair.

Introduction: Currently, there are no non-surgical FDA-approved biological approaches to accelerate fracture repair. Injectable therapies designed to stimulate bone healing represent an exciting alternative to surgically implanted biologics, however, the translation of effective osteoinductive therapies remains challenging due to the need for safe and effective drug delivery. Hydrogel-based microparticle platforms may be a clinically relevant solution to create controlled and localized drug delivery to treat bone fractures. Here, we describe poly (ethylene glycol) dimethacrylate (PEGDMA)-based microparticles, in the shape of microrods, loaded with beta nerve growth factor (ß-NGF) for the purpose of promoting fracture repair. Methods: Herein, PEGDMA microrods were fabricated through photolithography. PEGDMA microrods were loaded with ß-NGF and in vitro release was examined. Subsequently, bioactivity assays were evaluated in vitro using the TF-1 tyrosine receptor kinase A (Trk-A) expressing cell line. Finally, in vivo studies using our well-established murine tibia fracture model were performed and a single injection of the ß-NGF loaded PEGDMA microrods, non-loaded PEGDMA microrods, or soluble ß-NGF was administered to assess the extent of fracture healing using Micro-computed tomography (µCT) and histomorphometry. Results: In vitro release studies showed there is significant retention of protein within the polymer matrix over 168 hours through physiochemical interactions. Bioactivity of protein post-loading was confirmed with the TF-1 cell line. In vivo studies using our murine tibia fracture model show that PEGDMA microrods injected at the site of fracture remained adjacent to the callus for over 7 days. Importantly, a single injection of ß-NGF loaded PEGDMA microrods resulted in improved fracture healing as indicated by a significant increase in the percent bone in the fracture callus, trabecular connective density, and bone mineral density relative to soluble ß-NGF control indicating improved drug retention within the tissue. The concomitant decrease in cartilage fraction supports our prior work showing that ß-NGF promotes endochondral conversion of cartilage to bone to accelerate healing. Discussion: We demonstrate a novel and translational method wherein ß-NGF can be encapsulated within PEGDMA microrods for local delivery and that ß-NGF bioactivity is maintained resulting in improved bone fracture repair.

Codelivery of synergistic antimicrobials with polyelectrolyte nanocomplexes to treat bacterial biofilms and lung infections.

Bacterial biofilm infections, particularly those of Pseudomonas aeruginosa (PA), have high rates of antimicrobial tolerance and are commonly found in chronic wound and cystic fibrosis lung infections. Combination therapeutics that act synergistically can overcome antimicrobial tolerance; however, the delivery of multiple therapeutics at relevant dosages remains a challenge. We therefore developed a nanoscale drug carrier for antimicrobial codelivery by combining approaches from polyelectrolyte nanocomplex (NC) formation and layer-by-layer electrostatic self-assembly. This strategy led to NC drug carriers loaded with tobramycin antibiotics and antimicrobial silver nanoparticles (AgTob-NCs). AgTob-NCs displayed synergistic enhancements in antimicrobial activity against both planktonic and biofilm PA cultures, with positively charged NCs outperforming negatively charged formulations. NCs were evaluated in mouse models of lung infection, leading to reduced bacterial burden and improved survival outcomes. This approach therefore shows promise for nanoscale therapeutic codelivery to treat recalcitrant bacterial infections.

Biomaterials to enhance stem cell transplantation.

The successful transplantation of stem cells has the potential to transform regenerative medicine approaches and open promising avenues to repair, replace, and regenerate diseased, damaged, or aged tissues. However, pre-/post-transplantation issues of poor cell survival, retention, cell fate regulation, and insufficient integration with host tissues constitute significant challenges. The success of stem cell transplantation depends upon the coordinated sequence of stem cell renewal, specific lineage differentiation, assembly, and maintenance of long-term function. Advances in biomaterials can improve pre-/post-transplantation outcomes by integrating biophysiochemical cues and emulating tissue microenvironments. This review highlights leading biomaterials-based approaches for enhancing stem cell transplantation.

Lyophilized yeast powder for adjuvant free thermostable vaccine delivery.

Thermolabile nature of commercially available vaccines necessitates their storage, transportation, and dissemination under refrigerated condition. Maintenance of continuous cold chain at every step increases the final cost of vaccines. Any breach in the cold chain even for a short duration results in the need to discard the vaccines. As a result, there is a pressing need for the development of thermostable vaccines. In this proof-of-concept study, we showed that E. coli curli-green fluorescent fusion protein remains stable in freeze-dried yeast powder for more than 18 and 12 months when stored at 30 °C and 37 °C respectively. Stability of the heterologous protein remains unaffected during the process of heat-inactivation and lyophilization. The mass of lyophilized yeast powder remains almost unchanged during the entire period of storage and expressed protein remains intact even after two cycles of freeze and thaws. The protease-deficient strain appears ideal for the development of whole recombinant yeast-based vaccines. The cellular abundance of expressed antigen in dry powder after a year was comparable to freshly lyophilized cells. Scanning electron microscopy showed the intact nature of cells in powdered form even after a year of storage at 30 °C. Observation made in this study showed that freeze-dry yeast powder can play a vital role in the development of thermostable vaccines.Key Points• Yeast-based vaccines can overcome problem of cold chain associated with conventional vaccines• Lyophilized yeast powder can be a simple way for long-term storage of immunogen(s)• Protease deficient strain is important for whole recombinant yeast-based vaccines.

Modulating the foreign body response of implants for diabetes treatment.

Diabetes Mellitus is a group of diseases characterized by high blood glucose levels due to patients' inability to produce sufficient insulin. Current interventions often require implants that can detect and correct high blood glucose levels with minimal patient intervention. However, these implantable technologies have not reached their full potential in vivo due to the foreign body response and subsequent development of fibrosis. Therefore, for long-term function of implants, modulating the initial immune response is crucial in preventing the activation and progression of the immune cascade. This review discusses the different molecular mechanisms and cellular interactions involved in the activation and progression of foreign body response (FBR) and fibrosis, specifically for implants used in diabetes. We also highlight the various strategies and techniques that have been used for immunomodulation and prevention of fibrosis. We investigate how these general strategies have been applied to implants used for the treatment of diabetes, offering insights on how these devices can be further modified to circumvent FBR and fibrosis.

Theranostic biomaterials for tissue engineering.

Tissue engineering strategies, notably biomaterials, can be modularly designed and tuned to match specific patient needs. Although many challenges within tissue engineering remain, the incorporation of diagnostic strategies to create theranostic (combined therapy and diagnostic) biomaterials presents a unique platform to provide dual monitoring and treatment capabilities and advance the field toward personalized technologies. In this review, we summarize recent developments in this young field of regenerative theranostics and discuss the clinical potential and outlook of these systems from a tissue engineering perspective. As the need for precision and personalized medicines continues to increase to address diseases in all tissues in a patient-specific manner, we envision that such theranostic platforms can serve these needs.