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.

Advances in Skin-on-a-Chip Technologies for Dermatological Disease Modeling.

Skin-on-a-chip (SoC) technologies are emerging as a paradigm shift in dermatology research by replicating human physiology in a dynamic manner not achievable by current animal models. Although animal models have contributed to successful clinical trials, their ability to predict human outcomes remains questionable, owing to inherent differences in skin anatomy and immune response. Covering areas including infectious diseases, autoimmune skin conditions, wound healing, drug toxicity, aging, and antiaging, SoC aims to circumvent the inherent disparities created by traditional models. In this paper, we review current SoC technologies, highlighting their potential as an alternative to animal models for a deeper understanding of complex skin conditions.

Engineering platforms for localized long-acting immune modulation.

Systemic immunotherapeutics have been a clinical staple in the treatment of cancer, infectious diseases, organ and cell transplantation, autoimmunity, and allergies. Although their utility remains unquestioned, systemic administration of these drugs is associated with limited efficacy, significant adverse off-target effects, transient activity, and the requirement for frequent repeated dosing. To this end, recent technological advancements have provided novel means for sustained drug delivery to specific tissues and targeted localized approaches for immunotherapeutics. In this article, we present various cutting-edge platform technologies, including implants, multireservoir systems, and scaffolds encapsulating immunomodulatory agents for local administration. Examples of their application in cancer, cell transplantation, allergy, and infectious diseases are discussed, highlighting the potential of such systems for innovative immunomodulatory intervention.

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.

Robotic self-modulation enhances implantable long-acting drug delivery devices.

Integrating fibrotic capsule sensing with soft robotics may boost long-term performance of implantable drug delivery devices.

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).

Sustained Intratumoral Administration of Agonist CD40 Antibody Overcomes Immunosuppressive Tumor Microenvironment in Pancreatic Cancer.

Agonist CD40 monoclonal antibodies (mAb) is a promising immunotherapeutic agent for cold-to-hot tumor immune microenvironment (TIME) conversion. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer known as an immune desert, and therefore urgently needs more effective treatment. Conventional systemic treatment fails to effectively penetrate the characteristic dense tumor stroma. Here, it is shown that sustained low-dose intratumoral delivery of CD40 mAb via the nanofluidic drug-eluting seed (NDES) can modulate the TIME to reduce tumor burden in murine models. NDES achieves tumor reduction at a fourfold lower dosage than systemic treatment while avoiding treatment-related adverse events. Further, abscopal responses are shown where intratumoral treatment yields growth inhibition in distant untreated tumors. Overall, the NDES is presented as a viable approach to penetrate the PDAC immune barrier in a minimally invasive and effective manner, for the overarching goal of transforming treatment.

Emerging immunomodulatory strategies for cell therapeutics.

Cellular therapies are poised to transform the field of medicine by restoring dysfunctional tissues and treating various diseases in a dynamic manner not achievable by conventional pharmaceutics. Spanning various therapeutic areas inclusive of cancer, regenerative medicine, and immune disorders, cellular therapies comprise stem or non-stem cells derived from various sources. Despite numerous clinical approvals or trials underway, the host immune response presents a critical impediment to the widespread adoption and success of cellular therapies. Here, we review current research and clinical advances in immunomodulatory strategies to mitigate immune rejection or promote immune tolerance to cellular therapies. We discuss the potential of these immunomodulatory interventions to accelerate translation or maximize the prospects of improving therapeutic outcomes of cellular therapies for clinical success.

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.

Zonal Intratumoral Delivery of Nanoparticles Guided by Surface Functionalization.

Delivery of small molecules and anticancer agents to malignant cells or specific regions within a tumor is limited by penetration depth and poor spatial drug distribution, hindering anticancer efficacy. Herein, we demonstrate control over gold nanoparticle (GNP) penetration and spatial distribution across solid tumors by administering GNPs with different surface chemistries at a constant injection rate via syringe pump. A key finding in this study is the discovery of different zone-specific accumulation patterns of intratumorally injected nanoparticles dependent on surface functionalization. Computed tomography (CT) imaging performed in vivo of C57BL/6 mice harboring Lewis lung carcinoma (LLC) tumors on their flank and gross visualization of excised tumors consistently revealed that intratumorally administered citrate-GNPs accumulate in particle clusters in central areas of the tumor, while GNPs functionalized with thiolated phosphothioethanol (PTE-GNPs) and thiolated polyethylene glycol (PEG-GNPs) regularly accumulate in the tumor periphery. Further, PEG functionalization resulted in larger tumoral surface coverage than PTE, reaching beyond the outer zone of the tumor mass and into the surrounding stroma. To understand the dissimilarities in spatiotemporal evolution across the different GNP surface chemistries, we modeled their intratumoral transport with reaction-diffusion equations. Our results suggest that GNP surface passivation affects nanoparticle reactivity with the tumor microenvironment, leading to differential transport behavior across tumor zones. The present study provides a mechanistic understanding of the factors affecting spatiotemporal distribution of nanoparticles in the tumor. Our proof of concept of zonal delivery within the tumor may prove useful for directing anticancer therapies to regions of biomarker overexpression.

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.

Extending drug release from implants via transcutaneous refilling with solid therapeutics.

Long-acting (LA) implantable drug delivery systems (IDDS) offer an effective approach for the management or prevention of chronic conditions by sustained parenteral therapeutic administration. LA IDDS can and improve adherence to treatment regimens by minimizing dosing frequency. However, their clinical deployment is challenged by factors such as poor drug loading capacity, which limit their lifespan and require repeated surgical replacement for continued therapy. To address these challenges, and by leveraging previous work on nanofluidic systems, a reservoir-based IDDS that enables transcutaneous refilling of solid drug formulations through minimally invasive needle injection is presented. With thousand-fold higher drug loading efficiency, the implant affords minimal volume and aspect ratio suitable for discrete subcutaneous deployment. Key parameters for clinical acceptability, namely implant safety, access port robustness, and refilling method were systematically evaluated. The implant and refilling procedure are studied in rats and nonhuman primates with therapeutics used clinically for type 2 diabetes and human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP). The ability to extend drug release and maintain equivalent pharmacokinetics (PK) profiles pre- and post-drug refilling is demonstrated. This technology presents a clinically viable LA approach to prolong drug release for lifelong prevention or management of chronic conditions.

Comprehensive Analysis of Electrostatic Gating in Nanofluidic Systems.

Molecular transport in nanofluidic systems exhibits properties that are unique to the nanoscale. Here, the electrostatic and steric interactions between particle and surfaces become dominant in determining particle transport. At the solid-liquid interface of charged surfaces an electric double layer (EDL) forms due to electrostatic interactions between surfaces and charged particles. In these systems, tunable charge-selective nanochannels can be generated by manipulating electrostatic gating via co-ions exclusion and counterions enrichment of the EDL at the solid-liquid interface. In this context, electrostatic gating has been used to modulate the selectivity of nanofluidic membranes for drug delivery, nanofluidic transistors, and FlowFET, among other applications. While an extensive body of literature investigating nanofluidic systems exists, there is a lack of a comprehensive analysis accounting for all major parameters involved in these systems. Here we performed an all-encompassing modeling investigation corroborated by experimental analysis to assess the influence of nanochannel size, electrolyte properties, surface chemistry, gate voltage, dielectric properties, and molecular charge and size on the exclusion and enrichment of charged analytes in nanochannels. We found that the leakage current in electrostatic gating, often overlooked, plays a dominant role in molecular exclusion. Importantly, by independently considering all ionic species, we found that counterions compete for EDL formation at the surface proximity, resulting in concentration distributions that are nearly impossible to predict with analytical models. Achieving a deeper understanding of these nanofluidic phenomena will help the development of innovative miniaturized systems for both medical and industrial applications.

Emerging local immunomodulatory strategies to circumvent systemic immunosuppression in cell transplantation.

INTRODUCTION: Cell transplantation is a promising curative therapeutic strategy whereby impaired organ function can be restored without the need for whole-organ transplantation. A key challenge in allotransplantation is the requirement for life-long systemic immunosuppression to prevent rejection, which is associated with serious adverse effects such as increased risk of opportunistic infections and the development of neoplasms. This challenge underscores the urgent need for novel strategies to prevent graft rejection while abrogating toxicity-associated adverse events. AREAS COVERED: We review recent advances in immunoengineering strategies for localized immunomodulation that aim to support allograft function and provide immune tolerance in a safe and effective manner. EXPERT OPINION: Immunoengineering strategies are tailored approaches for achieving immunomodulation of the transplant microenvironment. Biomaterials can be adapted for localized and controlled release of immunomodulatory agents, decreasing the effective dose threshold and frequency of administration. The future of transplant rejection management lies in the shift from systemic to local immunomodulation with suppression of effector and activation of regulatory T cells, to promote immune tolerance.

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.

Emerging strategies for beta cell transplantation to treat diabetes.

Beta cell replacement has emerged as an attractive therapeutic alternative to traditional exogenous insulin administration for management of type 1 diabetes (T1D). Beta cells deliver insulin dynamically based on individual glycometabolic requirements, providing glycemic control while significantly reducing patient burden. Although transplantation into the portal circulation is clinically available, poor engraftment, low cell survival, and immune rejection have sparked investigation of alternative strategies for beta cell transplantation. In this review, we focus on current micro- and macroencapsulation technologies for beta cell transplantation and evaluate their advantages and challenges. Specifically, we comment on recent methods to ameliorate graft hypoxia including enhanced vascularization, reduction of pericapsular fibrotic overgrowth (PFO), and oxygen supplementation. We also discuss emerging beta cell-sourcing strategies to overcome donor shortage and provide insight into potential approaches to address outstanding challenges in the field.