a-translational-surgical-porcine-model-for-postoperative-intra

A Translational Surgical Porcine Model for Postoperative Intra-Abdominal Adhesion Formation

A porcine model of postoperative intra-abdominal adhesion formation was established using Yucatan mini pigs. The protocol combines midline laparotomy, ...

Stanford University

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.

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

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.

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

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving the desired rheological behavior as well as tunable  and  payload release kinetics. However, quantitation of hydrogel composition is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG--PLA nanoparticles, and a therapeutic compound, bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (, proteins) and was suitable for composition analysis as well as for evaluating  release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with (1) a multi-angle light scattering detector (RPLC-MALS) or (2) high resolution mass spectrometry (RPLC-MS) and a Fourier-transform based deconvolution algorithm. We envision that this analytical strategy could be generalized to characterize critical quality attributes of other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

Label-Free Composition Analysis of Supramolecular Polymer-Nanoparticle Hydrogels by Reversed-Phase Liquid Chromatography Coupled with a Charged Aerosol Detector.

Over the past few decades, the development of potent and safe immune-activating adjuvant technologies has become the heart of intensive research in the constant fight against highly mutative and immune evasive viruses such as influenza, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and human immunodeficiency virus (HIV). Herein, we developed a highly modular saponin-based nanoparticle platform incorporating Toll-like receptor agonists (TLRas) including TLR1/2a, TLR4a, and TLR7/8a adjuvants and their mixtures. These various TLRa-saponin nanoparticle adjuvant constructs induce unique acute cytokine and immune-signaling profiles, leading to specific T helper responses that could be of interest depending on the target disease for prevention. In a murine vaccine study, the adjuvants greatly improved the potency, durability, breadth, and neutralization of both COVID-19 and HIV vaccine candidates, suggesting the potential broad application of these adjuvant constructs to a range of different antigens. Overall, this work demonstrates a modular TLRa-SNP adjuvant platform that could improve the design of vaccines and affect modern vaccine development.

Saponin nanoparticle adjuvants incorporating Toll-like receptor agonists drive distinct immune signatures and potent vaccine responses.

Optical methods for studying the brain offer powerful approaches for understanding how neural activity underlies complex behavior. These methods typically rely on genetically encoded sensors and actuators to monitor and control neural activity. For microendoscopic calcium imaging, injection of a virus followed by implantation of a lens probe is required to express a calcium sensor and enable optical access to the target brain region. This two-step process poses several challenges, chief among them being the risks associated with mistargeting and/or misalignment between virus expression zone, lens probe and target brain region. Here, an adeno-associated virus (AAV)-eluting polymer coating is engineered for gradient refractive index (GRIN) lenses enabling the expression of a genetically encoded calcium indicator (GCaMP) directly within the brain region of interest upon implantation of the lens. This approach requires only one surgical step and guarantees alignment between GCaMP expression and lens in the brain. Additionally, the slow virus release from these coatings increases the working time for surgical implantation, expanding the brain regions and species amenable to this approach. These enhanced capabilities should accelerate neuroscience research utilizing optical methods and advance the understanding of the neural circuit mechanisms underlying brain function and behavior in health and disease.

Viral Vector Eluting Lenses for Single-Step Targeted Expression of Genetically-Encoded Activity Sensors for in Vivo Microendoscopic Calcium Imaging.

The inflammatory process resulting in the fibrotic encapsulation of implants has been well studied. However, how acellular dermal matrix (ADM) used in breast reconstruction elicits an attenuated foreign-body response (FBR) remains unclear. Here, by leveraging single-cell RNA-sequencing and proteomic data from pairs of fibrotically encapsulated specimens (bare silicone and silicone wrapped with ADM) collected from individuals undergoing breast reconstruction, we show that high levels of the extracellular-matrix protein osteopontin are associated with the use of ADM as a silicone wrapping. In mice with osteopontin knocked out, FBR attenuation by ADM-coated implants was abrogated. In wild-type mice, the sustained release of recombinant osteopontin from a hydrogel placed adjacent to a silicone implant attenuated the FBR in the absence of ADM. Our findings suggest strategies for the further minimization of the FBR.

Osteopontin attenuates the foreign-body response to silicone implants.

Messenger RNA (mRNA) delivered in lipid nanoparticles (LNPs) rose to the forefront of vaccine candidates during the COVID-19 pandemic due to scalability, adaptability, and potency. Yet, there remain critical areas for improvements of these vaccines in durability and breadth of humoral responses. In this work, we explore a modular strategy to target mRNA/LNPs to antigen-presenting cells with an injectable polymer-nanoparticle (PNP) hydrogel technology, which recruits key immune cells and forms an immunological niche in vivo. We characterize this niche on a single-cell level and find it is highly tunable through incorporation of adjuvants like MPLAs and 3M-052. Delivering commercially available severe acute respiratory syndrome coronavirus 2 mRNA vaccines in PNP hydrogels improves the durability and quality of germinal center reactions, and the magnitude, breadth, and durability of humoral responses. The tunable immune niche formed within PNP hydrogels effectively skews immune responses based on encapsulated adjuvants, creating opportunities to precisely modulate mRNA/LNP vaccines for various indications from infectious diseases to cancers.

Generation of an inflammatory niche in a hydrogel depot through recruitment of key immune cells improves efficacy of mRNA vaccines.

The threat of future coronavirus pandemics requires developing effective vaccine technologies that provide broad and long-lasting protection against circulating and emerging strains. Here we report a multivalent liposomal hydrogel vaccine technology comprising the receptor binding domain (RBD) of up to four SARS and MERS coronavirus strains non-covalently displayed on the surface of the liposomes within the hydrogel structure. The multivalent presentation and sustained exposure of RBD antigens improved the potency, neutralizing activity, durability, and consistency of antibody responses across homologous and heterologous coronavirus strains in a naïve murine model. When administrated in animals pre-exposed to wild-type SARS-CoV-2 antigens, liposomal hydrogels elicited durable antibody responses against the homologous SARS and MERS strains for over six months and elicited neutralizing activity against the immune-evasive SARS-CoV-2 variant Omicron BA.4/BA.5. Overall, the tunable liposomal hydrogel platform we report here generates robust responses against diverse coronaviruses, supporting global efforts to respond to future viral outbreaks.

Sustained exposure to multivalent antigen-decorated nanoparticles generates broad anti-coronavirus responses.

Sustained serum levels of broadly neutralizing antibodies (bnAbs) are crucial for effective passive immunization against infectious diseases as protection persists only while these bnAbs remain at adequate concentrations within the body. Current obstacles, such as poor pharmacokinetics (PK) and burdensome administration, must be overcome to make bnAbs a viable option for pre- and post-exposure prophylaxis. In this work, we explore how a polymer-nanoparticle (PNP) hydrogel depot technology can be engineered to prolong protein delivery and enable drug exposure on the order of weeks to months. In-vivo studies in mice and rats demonstrate extended protein release compared to bolus administration, and modeling efforts predict the impact of both the elimination half-life of the active pharmaceutical ingredient and hydrogel depot volume on overall pharmacokinetics. Moreover, flow cytometry characterization reveals that immune cell infiltration into the hydrogel depot can result in faster-than-expected release of antibody cargo on account of active transport via cellular uptake. We then demonstrate that co-formulation of antibodies with an anti-inflammatory agent reduces cellular infiltration and resulting active transport, further extending delivery and pharmacokinetics. Finally, multicompartmental modeling predicts the human PK profiles of clinically relevant HIV bnAbs delivered via subcutaneous hydrogel injection. These findings aid in the development of next generation hydrogel materials that stabilize and slowly release bnAbs for long-term pre-exposure immunoprophylaxis.

Article	Year
Hydrogel-Based Slow Release of a Receptor-Binding Domain Subunit Vaccine Elicits Neutralizing Antibody Responses Against SARS-CoV-2. Advanced materials (Deerfield Beach, Fla.)\| PubMed ID: 34651342	2021
Injectable Nanoparticle-Based Hydrogels Enable the Safe and Effective Deployment of Immunostimulatory CD40 Agonist Antibodies. Advanced science (Weinheim, Baden-Wurttemberg, Germany)\| PubMed ID: 35975424	2022
Label-Free Composition Analysis of Supramolecular Polymer-Nanoparticle Hydrogels by Reversed-Phase Liquid Chromatography Coupled with a Charged Aerosol Detector. Analytical chemistry\| PubMed ID: 38567987	2024
Saponin nanoparticle adjuvants incorporating Toll-like receptor agonists drive distinct immune signatures and potent vaccine responses. Science advances\| PubMed ID: 39110802	2024
Viral Vector Eluting Lenses for Single-Step Targeted Expression of Genetically-Encoded Activity Sensors for in Vivo Microendoscopic Calcium Imaging. Macromolecular bioscience\| PubMed ID: 39283817	2025
Osteopontin attenuates the foreign-body response to silicone implants. Nature biomedical engineering\| PubMed ID: 40128393	2025
Generation of an inflammatory niche in a hydrogel depot through recruitment of key immune cells improves efficacy of mRNA vaccines. Science advances\| PubMed ID: 40215318	2025
Sustained exposure to multivalent antigen-decorated nanoparticles generates broad anti-coronavirus responses. Matter\| PubMed ID: 40821443	2025
Hydrogel formulations for sustained-release of broadly neutralizing antibodies. Journal of controlled release : official journal of the Controlled Release Society\| PubMed ID: 41177463	2025

Emily L. Meany

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