3D bioprinted mesenchymal stem cell laden scaffold enhances subcutaneous vascularization for delivery of cell therapy.

Subcutaneous delivery of cell therapy is an appealing minimally-invasive strategy for the treatment of various diseases. However, the subdermal site is poorly vascularized making it inadequate for supporting engraftment, viability, and function of exogenous cells. In this study, we developed a 3D bioprinted scaffold composed of alginate/gelatin (Alg/Gel) embedded with mesenchymal stem cells (MSCs) to enhance vascularization and tissue ingrowth in a subcutaneous microenvironment. We identified bio-ink crosslinking conditions that optimally recapitulated the mechanical properties of subcutaneous tissue. We achieved controlled degradation of the Alg/Gel scaffold synchronous with host tissue ingrowth and remodeling. Further, in a rat model, the Alg/Gel scaffold was superior to MSC-embedded Pluronic hydrogel in supporting tissue development and vascularization of a subcutaneous site. While the scaffold alone promoted vascular tissue formation, the inclusion of MSCs in the bio-ink further enhanced angiogenesis. Our findings highlight the use of simple cell-laden degradable bioprinted structures to generate a supportive microenvironment for cell delivery.

Modeling of a Bioengineered Immunomodulating Microenvironment for Cell Therapy.

Cell delivery and encapsulation platforms are under development for the treatment of Type 1 Diabetes among other diseases. For effective cell engraftment, these platforms require establishing an immune-protected microenvironment as well as adequate vascularization and oxygen supply to meet the metabolic demands of the therapeutic cells. Current platforms rely on 1) immune isolating barriers and indirect vascularization or 2) direct vascularization with local or systemic delivery of immune modulatory molecules. Supported by experimental data, here a broadly applicable predictive computational model capable of recapitulating both encapsulation strategies is developed. The model is employed to comparatively study the oxygen concentration at different levels of vascularization, transplanted cell density, and spatial distribution, as well as with codelivered adjuvant cells. The model is then validated to be predictive of experimental results of oxygen pressure and local and systemic drug biodistribution in a direct vascularization device with local immunosuppressant delivery. The model highlights that dense vascularization can minimize cell hypoxia while allowing for high cell loading density. In contrast, lower levels of vascularization allow for better drug localization reducing systemic dissemination. Overall, it is shown that this model can serve as a valuable tool for the development and optimization of platform technologies for cell encapsulation.

Antiviral potency of long-acting islatravir subdermal implant in SHIV-infected macaques.

Treatment nonadherence is a pressing issue in people living with HIV (PLWH), as they require lifelong therapy to maintain viral suppression. Poor adherence leads to antiretroviral (ARV) resistance, transmission to others, AIDS progression, and increased morbidity and mortality. Long-acting (LA) ARV therapy is a promising strategy to combat the clinical drawback of user-dependent dosing. Islatravir (ISL) is a promising candidate for HIV treatment given its long half-life and high potency. Here we show constant ISL release from a subdermal LA nanofluidic implant achieves viral load reduction in SHIV-infected macaques. Specifically, a mean delivery dosage of 0.21 ± 0.07 mg/kg/day yielded a mean viral load reduction of -2.30 ± 0.53 log10 copies/mL at week 2, compared to baseline. The antiviral potency of the ISL delivered from the nanofluidic implant was higher than oral ISL dosed either daily or weekly. At week 3, viral resistance to ISL emerged in 2 out of 8 macaques, attributable to M184V mutation, supporting the need of combining ISL with other ARV for HIV treatment. The ISL implant produced moderate reactivity in the surrounding tissue, indicating tolerability. Overall, we present the ISL subdermal implant as a promising approach for LA ARV treatment in PLWH.

Intratumoral nanofluidic system enhanced tumor biodistribution of PD-L1 antibody in triple-negative breast cancer.

Immune checkpoint inhibitors (ICI), pembrolizumab and atezolizumab, were recently approved for treatment-refractory triple-negative breast cancer (TNBC), where those with Programmed death-ligand 1 (PD-L1) positive early-stage disease had improved responses. ICIs are administered systemically in the clinic, however, reaching effective therapeutic dosing is challenging due to severe off-tumor toxicities. As such, intratumoral (IT) injection is increasingly investigated as an alternative delivery approach. However, repeated administration, which sometimes is invasive, is required due to rapid drug clearance from the tumor caused by increased interstitial fluid pressure. To minimize off-target drug biodistribution, we developed the nanofluidic drug-eluting seed (NDES) platform for sustained intratumoral release of therapeutic via molecular diffusion. Here we compared drug biodistribution between the NDES, intraperitoneal (IP) and intratumoral (IT) injection using fluorescently labeled PD-L1 monoclonal antibody (αPD-L1). We used two syngeneic TNBC murine models, EMT6 and 4T1, that differ in PD-L1 expression, immunogenicity, and transport phenotype. We investigated on-target (tumor) and off-target distribution using different treatment approaches. As radiotherapy is increasingly used in combination with immunotherapy, we sought to investigate its effect on αPD-L1 tumor accumulation and systemic distribution. The NDES-treated cohort displayed sustained levels of αPD-L1 in the tumor over the study period of 14 days with significantly lower off-target organ distribution, compared to the IP or IT injection. However, we observed differences in the biodistribution of αPD-L1 across tumor models and with radiation pretreatment. Thus, we sought to extensively characterize the tumor properties via histological analysis, diffusion evaluation and nanoparticles contrast-enhanced CT. Overall, we demonstrate that ICI delivery via NDES is an effective method for sustained on-target tumor delivery across tumor models and combination treatments.

Long-acting refillable nanofluidic implant confers protection against SHIV infection in nonhuman primates.

The impact of pre-exposure prophylaxis (PrEP) on slowing the global HIV epidemic hinges on effective drugs and delivery platforms. Oral drug regimens are the pillar of HIV PrEP, but variable adherence has spurred development of long-acting delivery systems with the aim of increasing PrEP access, uptake, and persistence. We have developed a long-acting subcutaneous nanofluidic implant that can be refilled transcutaneously for sustained release of the HIV drug islatravir, a nucleoside reverse transcriptase translocation inhibitor that is used for HIV PrEP. In rhesus macaques, the islatravir-eluting implants achieved constant concentrations of islatravir in plasma (median 3.14 nM) and islatravir triphosphate in peripheral blood mononuclear cells (median 0.16 picomole per 106 cells) for more than 20 months. These drug concentrations were above the established PrEP protection threshold. In two unblinded, placebo-controlled studies, islatravir-eluting implants conferred 100% protection against infection with SHIVSF162P3 after repeated low-dose rectal or vaginal challenge in male or female rhesus macaques, respectively, compared to placebo control groups. The islatravir-eluting implants were well tolerated with mild local tissue inflammation and no signs of systemic toxicity over the 20-month study period. This refillable islatravir-eluting implant has potential as a long-acting drug delivery system for HIV PrEP.

Changes in local tissue microenvironment in response to subcutaneous long-acting delivery of tenofovir alafenamide in rats and non-human primates.

Several implantable long-acting (LA) delivery systems have been developed for sustained subcutaneous administration of tenofovir alafenamide (TAF), a potent and effective nucleotide reverse transcriptase inhibitor used for HIV pre-exposure prophylaxis (PrEP). LA platforms aim to address the lack of adherence to oral regimens, which has impaired PrEP efficacy. Despite extensive investigations in this field, tissue response to sustained subcutaneous TAF delivery remains to be elucidated as contrasting preclinical results have been reported in the literature. To this end, here we studied the local foreign body response (FBR) to sustained subdermal delivery of three forms of TAF, namely TAF free base (TAFfb), TAF fumarate salt (TAFfs), and TAFfb with urocanic acid (TAF-UA). Sustained constant drug release was achieved via titanium-silicon carbide nanofluidic implants previously shown to be bioinert. The analysis was conducted in both Sprague-Dawley (SD) rats and rhesus macaques over 1.5 and 3 months, respectively. While visual observation did not reveal abnormal adverse tissue reaction at the implantation site, histopathology and Imaging Mass Cytometry (IMC) analyses exposed a local chronic inflammatory response to TAF. In rats, UA mitigated foreign body response to TAF in a concentration-dependent manner. This was not observed in macaques where TAFfb was better tolerated than TAFfs and TAF-UA. Notably, the level of FBR was tightly correlated with local TAF tissue concentration. Further, regardless of the degree of FBR, the fibrotic capsule (FC) surrounding the implants did not interfere with drug diffusion and systemic delivery, as evidenced by TAF PK results and fluorescence recovery after photobleaching (FRAP).

Implantable niche with local immunosuppression for islet allotransplantation achieves type 1 diabetes reversal in rats.

Pancreatic islet transplantation efficacy for type 1 diabetes (T1D) management is limited by hypoxia-related graft attrition and need for systemic immunosuppression. To overcome these challenges, we developed the Neovascularized Implantable Cell Homing and Encapsulation (NICHE) device, which integrates direct vascularization for facile mass transfer and localized immunosuppressant delivery for islet rejection prophylaxis. Here, we investigated NICHE efficacy for allogeneic islet transplantation and long-term diabetes reversal in an immunocompetent, male rat model. We demonstrated that allogeneic islets transplanted within pre-vascularized NICHE were engrafted, revascularized, and functional, reverting diabetes in rats for over 150 days. Notably, we confirmed that localized immunosuppression prevented islet rejection without inducing toxicity or systemic immunosuppression. Moreover, for translatability efforts, we showed NICHE biocompatibility and feasibility of deployment as well as short-term allogeneic islet engraftment in an MHC-mismatched nonhuman primate model. In sum, the NICHE holds promise as a viable approach for safe and effective islet transplantation and long-term T1D management.

Advanced strategies to thwart foreign body response to implantable devices.

Mitigating the foreign body response (FBR) to implantable medical devices (IMDs) is critical for successful long-term clinical deployment. The FBR is an inevitable immunological reaction to IMDs, resulting in inflammation and subsequent fibrotic encapsulation. Excessive fibrosis may impair IMDs function, eventually necessitating retrieval or replacement for continued therapy. Therefore, understanding the implant design parameters and their degree of influence on FBR is pivotal to effective and long lasting IMDs. This review gives an overview of FBR as well as anti-FBR strategies. Furthermore, we highlight recent advances in biomimetic approaches to resist FBR, focusing on their characteristics and potential biomedical applications.

Localization of drug biodistribution in a 3D-bioengineered subcutaneous neovascularized microenvironment.

Local immunomodulation has shown the potential to control the immune response in a site-specific manner for wound healing, cancer, allergy, and cell transplantation, thus abrogating adverse effects associated with systemic administration of immunotherapeutics. Localized immunomodulation requires confining the biodistribution of immunotherapeutics on-site for maximal immune control and minimal systemic drug exposure. To this end, we developed a 3D-printed subcutaneous implant termed 'NICHE', consisting of a bioengineered vascularized microenvironment enabled by sustained drug delivery on-site. The NICHE was designed as a platform technology for investigating local immunomodulation in the context of cell therapeutics and cancer vaccines. Here we studied the ability of the NICHE to localize the PK and biodistribution of different model immunomodulatory agents in vivo. For this, we first performed a mechanistic evaluation of the microenvironment generated within and surrounding the NICHE, with emphasis on the parameters related to molecular transport. Second, we longitudinally studied the biodistribution of ovalbumin, cytotoxic T lymphocyte-associated antigen-4-Ig (CTLA4Ig), and IgG delivered locally via NICHE over 30 days. Third, we used our findings to develop a physiologically-based pharmacokinetic (PBPK) model. Despite dense and mature vascularization within and surrounding the NICHE, we showed sustained orders of magnitude higher molecular drug concentrations within its microenvironment as compared to systemic circulation and major organs. Further, the PBPK model was able to recapitulate the biodistribution of the 3 molecules with high accuracy (r â€‹> â€‹0.98). Overall, the NICHE and the PBPK model represent an adaptable platform for the investigation of local immunomodulation strategies for a wide range of biomedical applications.

Engineered implantable vaccine platform for continuous antigen-specific immunomodulation.

Cancer vaccines harness the host immune system to generate antigen-specific antitumor immunity for long-term tumor elimination with durable immunomodulation. Commonly investigated strategies reintroduce ex vivo autologous dendritic cells (DCs) but have limited clinical adoption due to difficulty in manufacturing, delivery and low clinical efficacy. To combat this, we designed the "NanoLymph", an implantable subcutaneous device for antigen-specific antitumor immunomodulation. The NanoLymph consists of a dual-reservoir platform for sustained release of immune stimulants via a nanoporous membrane and hydrogel-encapsulated antigens for local immune cell recruitment and activation, respectively. Here, we present the development and characterization of the NanoLymph as well as efficacy validation for immunomodulation in an immunocompetent murine model. Specifically, we established the NanoLymph biocompatibility and mechanical stability. Further, we demonstrated minimally invasive transcutaneous refilling of the drug reservoir in vivo for prolonging drug release duration. Importantly, our study demonstrated that local elution of two drugs (GMCSF and Resiquimod) generates an immune stimulatory microenvironment capable of local DC recruitment and activation and generation of antigen-specific T lymphocytes within 14 days. In summary, the NanoLymph approach can achieve in situ immunomodulation, presenting a viable strategy for therapeutic cancer vaccines.

Enhanced In Vivo Vascularization of 3D-Printed Cell Encapsulation Device Using Platelet-Rich Plasma and Mesenchymal Stem Cells.

The current standard for cell encapsulation platforms is enveloping cells in semipermeable membranes that physically isolate transplanted cells from the host while allowing for oxygen and nutrient diffusion. However, long-term viability and function of encapsulated cells are compromised by insufficient oxygen and nutrient supply to the graft. To address this need, a strategy to achieve enhanced vascularization of a 3D-printed, polymeric cell encapsulation platform using platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) is investigated. The study is conducted in rats and, for clinical translation relevance, in nonhuman primates (NHP). Devices filled with PRP, MSCs, or vehicle hydrogel are subcutaneously implanted in rats and NHP and the amount and maturity of penetrating blood vessels assessed via histopathological analysis. In rats, MSCs drive the strongest angiogenic response at early time points, with the highest vessel density and endothelial nitric oxide synthase (eNOS) expression. In NHP, PRP and MSCs result in similar vessel densities but incorporation of PRP ensues higher levels of eNOS expression. Overall, enrichment with PRP and MSCs yields extensive, mature vascularization of subcutaneous cell encapsulation devices. It is postulated that the individual properties of PRP and MSCs can be leveraged in a synergistic approach for maximal vascularization of cell encapsulation platforms.

Neovascularized implantable cell homing encapsulation platform with tunable local immunosuppressant delivery for allogeneic cell transplantation.

Cell encapsulation is an attractive transplantation strategy to treat endocrine disorders. Transplanted cells offer a dynamic and stimulus-responsive system that secretes therapeutics based on patient need. Despite significant advancements, a challenge in allogeneic cell encapsulation is maintaining sufficient oxygen and nutrient exchange, while providing protection from the host immune system. To this end, we developed a subcutaneously implantable dual-reservoir encapsulation system integrating in situ prevascularization and local immunosuppressant delivery, termed NICHE. NICHE structure is 3D-printed in biocompatible polyamide 2200 and comprises of independent cell and drug reservoirs separated by a nanoporous membrane for sustained local release of immunosuppressant. Here we present the development and characterization of NICHE, as well as efficacy validation for allogeneic cell transplantation in an immunocompetent rat model. We established biocompatibility and mechanical stability of NICHE. Further, NICHE vascularization was achieved with the aid of mesenchymal stem cells. Our study demonstrated sustained local elution of immunosuppressant (CTLA4Ig) into the cell reservoir protected transcutaneously-transplanted allogeneic Leydig cells from host immune destruction during a 31-day study, and reduced systemic drug exposure by 12-fold. In summary, NICHE is the first encapsulation platform achieving both in situ vascularization and immunosuppressant delivery, presenting a viable strategy for allogeneic cell transplantation.