Abstract Allogeneic islet transplantation is an effective treatment for type 1 diabetes (T1D). Graft rejection is controlled by immunosuppressive drugs that have adverse effects. An imbalance between Teffs and Tregs is the primary cause of T1D and graft rejection. Activated Teff cells express Fas and are sensitive to FasL-mediated apoptosis. Treg cells are relatively refractive to apoptosis and expand in response to IL-2. Thus, a combination of Fas and IL-2R agonists has the potential to modulate alloreactive responses for sustained graft survival. Given the off-target effects of systemic delivery of biologics, we established a lipocoacervate formulation consisting of a biodegradable polycation, PEAD, and polyanion heparin to form coacervate for controlled delivery of a novel Fas agonist, SA-FasL, and IL-2. Release kinetics and activities of proteins were assessed in vitro. The immunomodulatory efficacy of protein-loaded lipocoacervate was assessed in vivo. IL-2 and SA-FasL proteins showed a steady release over 30 days in vitro. IL-2 released on day 14 had minimal activity loss, whereas FasL showed ~20% activity loss on day 9. Treatment with SA-FasL/IL-2-loaded lipocoacervate modulated in vivo alloreactive T cell responses, resulting in an increased Treg/Teff cell ratio. In conclusion, lipocoacervate is an effective platform for controlled and sustained release of SA-FasL and IL-2 biologics to modulate allo and autoreactive T cells with significant therapeutic potential for T1D.

Carbic anhydride is an underappreciated starting material for 3D-printable, non-hydrogel photopolymers. Compared with other norbornene precursors, carbic anhydride is cheaper and reactive via aminolysis. As a result, the generalized and efficient functionalization with carbic anhydride can increase the utilization of thiol-norbornene photopolymers. Here, we report carbic anhydride's catalyst-free condensation with two commodity polymers: amine-functionalized polypropylene glycol and polydimethylsiloxane. The reaction completes in 1 h, produces water as the only byproduct, and does not require purification. It is therefore affordable, facile, and green. Mixing the product with thiol cross-linkers and the appropriate photoadditives produces photopolymers that are printable via Digital Light Processing. The photopolymers exhibit tunable tensile properties and a functional surface by varying the polymer backbone and thiol stoichiometry. Moreover, the photopolymers are 3D-printed into true-to-scale human aorta models and porous scaffolds with high resolution. The simple yet versatile platform will benefit additive manufacturing of soft materials and beyond.

Although the male epididymal fat pad is an effective site for islet transplantation, females lack this tissue. Here, we present a protocol to assess the parametrial fat pad (PFP) adjacent to the uterine horn in females as an alternative site for islet transplantation. We describe steps for islet isolation from the pancreas, counting, transplantation into PFP, and monitoring for engraftment. Transplantation into PFP is minimally invasive, time efficient, and supports long-term engraftment of syngeneic islets and rejection of allogeneic islets. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1

Controlled delivery of proteins and other biologics is a growing medium of therapy for diseases previously untreatable. Here we report a self-assembling, tunable vesicle for the controlled delivery of growth factors and cytokines. Coacervate made of heparin and a biocompatible polycation, PEAD, forms the core of the vesicle; lipids form the membrane of the vesicle. We call this vesicle lipocoacervate (LipCo), which has a high affinity for growth factors and cytokines due to heparin. LipCo is a tunable protein delivery vehicle. The vesicle size is controlled through polymer and salt concentrations. Membrane functionalization enables potential for targeting capabilities with long-term storage through lyophilization. Importantly, the controlled delivery of therapeutics also avoids high toxicity to treated cells in vitro. Here we report on these key principles of LipCo assembly and design.

Transdermal delivery is an attractive delivery method that increases bioavailability, is suitable for a wide variety of therapeutics, and offers stable delivery outcomes. However, many therapeutics are unable to readily cross the stratum corneum. Microneedles mechanically disrupt the cutaneous barrier to deliver small molecules, proteins, and vaccines. To date, microneedles have not been used in conjunction with coacervate, a liquid-liquid phase separation that protects unstable proteins. A three-layer microneedle for the controlled release of three different molecules is designed. Through micromolding, microneedles are efficiently generated, which benefits product scalability. The microneedles have good mechanical integrity and effectively penetrate porcine skin ex vivo. The three layers, in the microneedles, release the cargo in a three-phase manner. The released protein maintains its structure well. Moreover, layer thickness can be controlled by varying fabrication parameters. The microneedles can incorporate both small molecule drugs and protein therapeutics, thus promising uses in multi-drug therapies through a single treatment.

Nonunion following bone fracture and segmental bone defects are challenging clinical conditions. To combat this clinical dilemma, development of new bone tissue engineering therapies using biocompatible materials to deliver bone growth factors is desirable. This aim of this study is to use a heparin/polycation coacervate sustained-release platform to compare 5 bone morphogenetic proteins (BMPs) for promoting bone defect healing in a critical sized calvarial defect model. The in vitro 3D osteogenic pellet cultures assays demonstrated that BMPs 2, 4, 6, 7 and 9 all enhanced mineralization in vitro compared to the control group. BMP2 resulted in higher mineralized volume than BMP4 and BMP6. All BMPs and the control group activated the pSMAD5 signaling pathway and expressed osterix (OSX). The binding of BMP2 with coacervate significantly increased the coacervate average particle size. BMP2, 4, 6, & 7 bound to coacervate significantly increased the Zeta potential of the coacervate while BMP9 binding showed insignificant increase. Furthermore, using a monolayer culture osteogenic assay, it was found that hMDSCs cultured in the coacervate BMP2 osteogenic medium expressed higher levels of RUNX2, OSX, ALP and COX-2 compared to the control and BMPs 4, 6, 7 & 9. Additionally, the coacervate complex can be loaded with up to 2 μg of BMP proteins for sustained release. In vivo, when BMPs were delivered using the coacervate sustained release system, BMP2 was identified to be the most potent BMP promoting bone regeneration and regenerated 10 times of new bone than BMPs 4, 6 & 9. BMP7 also stimulated robust bone regeneration when compared to BMPs 4, 6 & 9. The quality of the newly regenerated bone by all BMPs delivered by coacervate is equivalent to the host bone consisting of bone matrix and bone marrow with normal bone architecture. Although the defect was not completely healed at 6 weeks, coacervate sustain release BMPs, particularly BMP2 and BMP7, could represent a new strategy for treatment of bone defects and non-unions.