Nanoparticle-Conjugated Toll-Like Receptor 9 Agonists Improve the Potency, Durability, and Breadth of COVID-19 Vaccines.

Development of effective vaccines for infectious diseases has been one of the most successful global health interventions in history. Though, while ideal subunit vaccines strongly rely on antigen and adjuvant(s) selection, the mode and time scale of exposure to the immune system has often been overlooked. Unfortunately, poor control over the delivery of many adjuvants, which play a key role in enhancing the quality and potency of immune responses, can limit their efficacy and cause off-target toxicities. There is a critical need for improved adjuvant delivery technologies to enhance their efficacy and boost vaccine performance. Nanoparticles have been shown to be ideal carriers for improving antigen delivery due to their shape and size, which mimic viral structures but have been generally less explored for adjuvant delivery. Here, we describe the design of self-assembled poly(ethylene glycol)-b-poly(lactic acid) nanoparticles decorated with CpG, a potent TLR9 agonist, to increase adjuvanticity in COVID-19 vaccines. By controlling the surface density of CpG, we show that intermediate valency is a key factor for TLR9 activation of immune cells. When delivered with the SARS-CoV-2 spike protein, CpG nanoparticle (CpG-NP) adjuvant greatly improves the magnitude and duration of antibody responses when compared to soluble CpG, and results in overall greater breadth of immunity against variants of concern. Moreover, encapsulation of CpG-NP into injectable polymeric-nanoparticle (PNP) hydrogels enhances the spatiotemporal control over codelivery of CpG-NP adjuvant and spike protein antigen such that a single immunization of hydrogel-based vaccines generates humoral responses comparable to those of a typical prime-boost regimen of soluble vaccines. These delivery technologies can potentially reduce the costs and burden of clinical vaccination, both of which are key elements in fighting a pandemic.

Injectable Nanoparticle-Based Hydrogels Enable the Safe and Effective Deployment of Immunostimulatory CD40 Agonist Antibodies.

When properly deployed, the immune system can eliminate deadly pathogens, eradicate metastatic cancers, and provide long-lasting protection from diverse diseases. Unfortunately, realizing these remarkable capabilities is inherently risky as disruption to immune homeostasis can elicit dangerous complications or autoimmune disorders. While current research is continuously expanding the arsenal of potent immunotherapeutics, there is a technological gap when it comes to controlling when, where, and how long these drugs act on the body. Here, this study explored the ability of a slow-releasing injectable hydrogel depot to reduce dose-limiting toxicities of immunostimulatory CD40 agonist (CD40a) while maintaining its potent anticancer efficacy. A previously described polymer-nanoparticle (PNP) hydrogel system is leveraged that exhibits shear-thinning and yield-stress properties that are hypothesized to improve locoregional delivery of CD40a immunotherapy. Using positron emission tomography, it is demonstrated that prolonged hydrogel-based delivery redistributes CD40a exposure to the tumor and the tumor draining lymph node (TdLN), thereby reducing weight loss, hepatotoxicity, and cytokine storm associated with standard treatment. Moreover, CD40a-loaded hydrogels mediate improved local cytokine induction in the TdLN and improve treatment efficacy in the B16F10 melanoma model. PNP hydrogels, therefore, represent a facile, drug-agnostic method to ameliorate immune-related adverse effects and explore locoregional delivery of immunostimulatory drugs.

Delivery of CAR-T cells in a transient injectable stimulatory hydrogel niche improves treatment of solid tumors.

Adoptive cell therapy (ACT) has proven to be highly effective in treating blood cancers, but traditional approaches to ACT are poorly effective in treating solid tumors observed clinically. Novel delivery methods for therapeutic cells have shown promise for treatment of solid tumors when compared with standard intravenous administration methods, but the few reported approaches leverage biomaterials that are complex to manufacture and have primarily demonstrated applicability following tumor resection or in immune-privileged tissues. Here, we engineer simple-to-implement injectable hydrogels for the controlled co-delivery of CAR-T cells and stimulatory cytokines that improve treatment of solid tumors. The unique architecture of this material simultaneously inhibits passive diffusion of entrapped cytokines and permits active motility of entrapped cells to enable long-term retention, viability, and activation of CAR-T cells. The generation of a transient inflammatory niche following administration affords sustained exposure of CAR-T cells, induces a tumor-reactive CAR-T phenotype, and improves efficacy of treatment.

Hydrogel-Based Slow Release of a Receptor-Binding Domain Subunit Vaccine Elicits Neutralizing Antibody Responses Against SARS-CoV-2.

The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID-19. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, clinically-relevant adjuvants Alum, AddaVax, and CpG/Alum are found unable to elicit neutralizing responses following a prime-boost immunization. Here, it has been shown that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer-nanoparticle (PNP) hydrogel elicited potent anti-RBD and anti-spike antibody titers, providing broader protection against SARS-CoV-2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically-relevant adjuvant systems. Notably, a SARS-CoV-2 spike-pseudotyped lentivirus neutralization assay revealed that hydrogel-based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.

Consistent tumorigenesis with self-assembled hydrogels enables high-powered murine cancer studies.

Preclinical cancer research is heavily dependent on allograft and xenograft models, but current approaches to tumor inoculation yield inconsistent tumor formation and growth, ultimately wasting valuable resources (e.g., animals, time, and money) and limiting experimental progress. Here we demonstrate a method for tumor inoculation using self-assembled hydrogels to reliably generate tumors with low variance in growth. The observed reduction in model variance enables smaller animal cohorts, improved effect observation and higher powered studies.

Modulation of injectable hydrogel properties for slow co-delivery of influenza subunit vaccine components enhance the potency of humoral immunity.

Vaccines are critical for combating infectious diseases across the globe. Influenza, for example, kills roughly 500,000 people annually worldwide, despite annual vaccination campaigns. Efficacious vaccines must elicit a robust and durable antibody response, and poor efficacy often arises from inappropriate temporal control over antigen and adjuvant presentation to the immune system. In this work, we sought to exploit the immune system's natural response to extended pathogen exposure during infection by designing an easily administered slow-delivery influenza vaccine platform. We utilized an injectable and self-healing polymer-nanoparticle (PNP) hydrogel platform to prolong the co-delivery of vaccine components to the immune system. We demonstrated that these hydrogels exhibit unique dynamic physical characteristics whereby physicochemically distinct influenza hemagglutinin antigen and a toll-like receptor 7/8 agonist adjuvant could be co-delivered over prolonged timeframes that were tunable through simple alteration of the gel formulation. We show a relationship between hydrogel physical properties and the resulting immune response to immunization. When administered in mice, hydrogel-based vaccines demonstrated enhancements in the magnitude and duration of humoral immune responses compared to alum, a widely used clinical adjuvant system. We found stiffer hydrogel formulations exhibited slower release and resulted in the greatest improvements to the antibody response while also enabling significant adjuvant dose sparing. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of influenza subunit vaccines.

Full closed loop open-source algorithm performance comparison in pigs with diabetes.

Understanding how automated insulin delivery (AID) algorithm features impact glucose control under full closed loop delivery represents a critical step toward reducing patient burden by eliminating the need for carbohydrate entries at mealtimes. Here, we use a pig model of diabetes to compare AndroidAPS and Loop open-source AID systems without meal announcements. Overall time-in-range (70-180 mg/dl) for AndroidAPS was 58% ± 5%, while time-in-range for Loop was 35% ± 5%. The effect of the algorithms on time-in-range differed between meals and overnight. During the overnight monitoring period, pigs had an average time-in-range of 90% ± 7% when on AndroidAPS compared to 22% ± 8% on Loop. Time-in-hypoglycemia also differed significantly during the lunch meal, whereby pigs running AndroidAPS spent an average of 1.4% (+0.4/-0.8)% in hypoglycemia compared to 10% (+3/-6)% for those using Loop. As algorithm design for closed loop systems continues to develop, the strategies employed in the OpenAPS algorithm (known as oref1) as implemented in AndroidAPS for unannounced meals may result in a better overall control for full closed loop systems.

Hydrogel-based slow release of a receptor-binding domain subunit vaccine elicits neutralizing antibody responses against SARS-CoV-2.

The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID-19. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, we found that clinically-relevant adjuvants Alum, AddaVax, and CpG/Alum were unable to elicit neutralizing responses following a prime-boost immunization. Here we show that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer-nanoparticle (PNP) hydrogel elicited potent anti-RBD and anti-spike antibody titers, providing broader protection against SARS-CoV-2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically-relevant adjuvant systems. Notably, a SARS-CoV-2 spike-pseudotyped lentivirus neutralization assay revealed that hydrogel-based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.

Prolonged Codelivery of Hemagglutinin and a TLR7/8 Agonist in a Supramolecular Polymer-Nanoparticle Hydrogel Enhances Potency and Breadth of Influenza Vaccination.

The sustained release of vaccine cargo has been shown to improve humoral immune responses to challenging pathogens such as influenza. Extended codelivery of antigen and adjuvant prolongs germinal center reactions, thus improving antibody affinity maturation and the ability to neutralize the target pathogen. Here, we develop an injectable, physically cross-linked polymer-nanoparticle (PNP) hydrogel system to prolong the local codelivery of hemagglutinin and a toll-like receptor 7/8 agonist (TLR7/8a) adjuvant. By tethering the TLR7/8a to a NP motif within the hydrogels (TLR7/8a-NP), the dynamic mesh of the PNP hydrogels enables codiffusion of the adjuvant and protein antigen (hemagglutinin), therefore enabling sustained codelivery of these two physicochemically distinct molecules. We show that subcutaneous delivery of PNP hydrogels carrying hemagglutinin and TLR7/8a-NP in mice improves the magnitude and duration of antibody titers in response to a single injection vaccination compared to clinically used adjuvants. Furthermore, the PNP gel-based slow delivery of influenza vaccines led to increased breadth of antibody responses against future influenza variants, including a future pandemic variant, compared to clinical adjuvants. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of influenza subunit vaccines.

Injectable Hydrogels for Sustained Codelivery of Subunit Vaccines Enhance Humoral Immunity.

Vaccines aim to elicit a robust, yet targeted, immune response. Failure of a vaccine to elicit such a response arises in part from inappropriate temporal control over antigen and adjuvant presentation to the immune system. In this work, we sought to exploit the immune system's natural response to extended pathogen exposure during infection by designing an easily administered slow-delivery vaccine platform. We utilized an injectable and self-healing polymer-nanoparticle (PNP) hydrogel platform to prolong the codelivery of vaccine components to the immune system. We demonstrated that these hydrogels exhibit unique delivery characteristics, whereby physicochemically distinct compounds (such as antigen and adjuvant) could be codelivered over the course of weeks. When administered in mice, hydrogel-based sustained vaccine exposure enhanced the magnitude, duration, and quality of the humoral immune response compared to standard PBS bolus administration of the same model vaccine. We report that the creation of a local inflammatory niche within the hydrogel, coupled with sustained exposure of vaccine cargo, enhanced the magnitude and duration of germinal center responses in the lymph nodes. This strengthened germinal center response promoted greater antibody affinity maturation, resulting in a more than 1000-fold increase in antigen-specific antibody affinity in comparison to bolus immunization. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of subunit vaccines.

Nanoparticles Presenting Potent TLR7/8 Agonists Enhance Anti-PD-L1 Immunotherapy in Cancer Treatment.

Cancer immunotherapy can be augmented with toll-like receptor agonist (TLRa) adjuvants, which interact with immune cells to elicit potent immune activation. Despite their potential, use of many TLRa compounds has been limited clinically due to their extreme potency and lack of pharmacokinetic control, causing systemic toxicity from unregulated systemic cytokine release. Herein, we overcome these shortcomings by generating poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) nanoparticles (NPs) presenting potent TLR7/8a moieties on their surface. The NP platform allows precise control of TLR7/8a valency and resulting surface presentation through self-assembly using nanoprecipitation. We hypothesize that the pharmacokinetic profile of the NPs minimizes systemic toxicity, localizing TLR7/8a presentation to the tumor bed and tumor-draining lymph nodes. In conjunction with antiprogrammed death-ligand 1 (anti-PD-L1) checkpoint blockade, peritumoral injection of TLR7/8a NPs slows tumor growth, extends survival, and decreases systemic toxicity in comparison to the free TLR7/8a in a murine colon adenocarcinoma model. These NPs constitute a modular platform for controlling pharmacokinetics of immunostimulatory molecules, resulting in increased potency and decreased toxicity.

An ultrafast insulin formulation enabled by high-throughput screening of engineered polymeric excipients.

Insulin has been used to treat diabetes for almost 100 years; yet, current rapid-acting insulin formulations do not have sufficiently fast pharmacokinetics to maintain tight glycemic control at mealtimes. Dissociation of the insulin hexamer, the primary association state of insulin in rapid-acting formulations, is the rate-limiting step that leads to delayed onset and extended duration of action. A formulation of insulin monomers would more closely mimic endogenous postprandial insulin secretion, but monomeric insulin is unstable in solution using present formulation strategies and rapidly aggregates into amyloid fibrils. Here, we implement high-throughput-controlled radical polymerization techniques to generate a large library of acrylamide carrier/dopant copolymer (AC/DC) excipients designed to reduce insulin aggregation. Our top-performing AC/DC excipient candidate enabled the development of an ultrafast-absorbing insulin lispro (UFAL) formulation, which remains stable under stressed aging conditions for 25 ± 1 hours compared to 5 ± 2 hours for commercial fast-acting insulin lispro formulations (Humalog). In a porcine model of insulin-deficient diabetes, UFAL exhibited peak action at 9 ± 4 min, whereas commercial Humalog exhibited peak action at 25 ± 10 min. These ultrafast kinetics make UFAL a promising candidate for improving glucose control and reducing burden for patients with diabetes.

A co-formulation of supramolecularly stabilized insulin and pramlintide enhances mealtime glucagon suppression in diabetic pigs.

Treatment of patients with diabetes with insulin and pramlintide (an amylin analogue) is more effective than treatment with insulin only. However, because mixtures of insulin and pramlintide are unstable and have to be injected separately, amylin analogues are only used by 1.5% of people with diabetes needing rapid-acting insulin. Here, we show that the supramolecular modification of insulin and pramlintide with cucurbit[7]uril-conjugated polyethylene glycol improves the pharmacokinetics of the dual-hormone therapy and enhances postprandial glucagon suppression in diabetic pigs. The co-formulation is stable for over 100 h at 37 °C under continuous agitation, whereas commercial formulations of insulin analogues aggregate after 10 h under similar conditions. In diabetic rats, the administration of the stabilized co-formulation increased the area-of-overlap ratio of the pharmacokinetic curves of pramlintide and insulin from 0.4 ± 0.2 to 0.7 ± 0.1 (mean ± s.d.) for the separate administration of the hormones. The co-administration of supramolecularly stabilized insulin and pramlintide better mimics the endogenous kinetics of co-secreted insulin and amylin, and holds promise as a dual-hormone replacement therapy.

Injectable supramolecular polymer-nanoparticle hydrogels enhance human mesenchymal stem cell delivery.

Stem cell therapies have emerged as promising treatments for injuries and diseases in regenerative medicine. Yet, delivering stem cells therapeutically can be complicated by invasive administration techniques, heterogeneity in the injection media, and/or poor cell retention at the injection site. Despite these issues, traditional administration protocols using bolus injections in a saline solution or surgical implants of cell-laden hydrogels have highlighted the promise of cell administration as a treatment strategy. To address these limitations, we have designed an injectable polymer-nanoparticle (PNP) hydrogel platform exploiting multivalent, noncovalent interactions between modified biopolymers and biodegradable nanoparticles for encapsulation and delivery of human mesenchymal stem cells (hMSCs). hMSC-based therapies have shown promise due to their broad differentiation capacities and production of therapeutic paracrine signaling molecules. In this work, the fundamental hydrogel mechanical properties that enhance hMSC delivery processes are elucidated using basic in vitro models. Further, in vivo studies in immunocompetent mice reveal that PNP hydrogels enhance hMSC retention at the injection site and retain administered hMSCs locally for upwards of 2 weeks. Through both in vitro and in vivo experiments, we demonstrate a novel scalable, synthetic, and biodegradable hydrogel system that overcomes current limitations and enables effective cell delivery.