Screening hydrogels for antifibrotic properties by implanting cellularly barcoded alginates in mice and a non-human primate.

Screening implantable biomaterials for antifibrotic properties is constrained by the need for in vivo testing. Here we show that the throughput of in vivo screening can be increased by cellularly barcoding a chemically modified combinatorial library of hydrogel formulations. The method involves the implantation of a mixture of alginate formulations, each barcoded with human umbilical vein endothelial cells from different donors, and the association of the identity and performance of each formulation by genotyping single nucleotide polymorphisms of the cells via next-generation sequencing. We used the method to screen 20 alginate formulations in a single mouse and 100 alginate formulations in a single non-human primate, and identified three lead hydrogel formulations with antifibrotic properties. Encapsulating human islets with one of the formulations led to long-term glycaemic control in a mouse model of diabetes, and coating medical-grade catheters with the other two formulations prevented fibrotic overgrowth. High-throughput screening of barcoded biomaterials in vivo may help identify formulations that enhance the long-term performance of medical devices and of biomaterial-encapsulated therapeutic cells.

Lipid Deposition Profiles Influence Foreign Body Responses.

Fibrosis remains a significant cause of failure in implanted biomedical devices and early absorption of proteins on implant surfaces has been shown to be a key instigating factor. However, lipids can also regulate immune activity and their presence may also contribute to biomaterial-induced foreign body responses (FBR) and fibrosis. Here it is demonstrated that the surface presentation of lipids on implant affects FBR by influencing reactions of immune cells to materials as well as their resultant inflammatory/suppressive polarization. Time-of-flight secondary ion mass spectroscopy (ToF-SIMS) is employed to characterize lipid deposition on implants that are surface-modified chemically with immunomodulatory small molecules. Multiple immunosuppressive phospholipids (phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin) are all found to deposit preferentially on implants with anti-FBR surface modifications in mice. Significantly, a set of 11 fatty acids is enriched on unmodified implanted devices that failed in both mice and humans, highlighting relevance across species. Phospholipid deposition is also found to upregulate the transcription of anti-inflammatory genes in murine macrophages, while fatty acid deposition stimulated the expression of pro-inflammatory genes. These results provide further insights into how to improve the design of biomaterials and medical devices to mitigate biomaterial material-induced FBR and fibrosis.

Immune profiling of adeno-associated virus response identifies B cell-specific targets that enable vector re-administration in mice.

Adeno-associated virus (AAV) vector-based gene therapies can be applied to a wide range of diseases. AAV expression can last for months to years, but vector re-administration may be necessary to achieve life-long treatment. Unfortunately, immune responses against these vectors are potentiated after the first administration, preventing the clinical use of repeated administration of AAVs. Reducing the immune response against AAVs while minimizing broad immunosuppression would improve gene delivery efficiency and long-term safety. In this study, we quantified the contributions of multiple immune system components of the anti-AAV response in mice. We identified B-cell-mediated immunity as a critical component preventing vector re-administration. Additionally, we found that IgG depletion alone was insufficient to enable re-administration, suggesting IgM antibodies play an important role in the immune response against AAV. Further, we found that AAV-mediated transduction is improved in µMT mice that lack functional IgM heavy chains and cannot form mature B-cells relative to wild-type mice. Combined, our results suggest that B-cells, including non-class switched B-cells, are a potential target for therapeutics enabling AAV re-administration. Our results also suggest that the µMT mice are a potentially useful experimental model for gene delivery studies since they allow repeated dosing for more efficient gene delivery from AAVs.

Clinically translatable cytokine delivery platform for eradication of intraperitoneal tumors.

Proinflammatory cytokines have been approved by the Food and Drug Administration for the treatment of metastatic melanoma and renal carcinoma. However, effective cytokine therapy requires high-dose infusions that can result in antidrug antibodies and/or systemic side effects that limit long-term benefits. To overcome these limitations, we developed a clinically translatable cytokine delivery platform composed of polymer-encapsulated human ARPE-19 (RPE) cells that produce natural cytokines. Tumor-adjacent administration of these capsules demonstrated predictable dose modulation with spatial and temporal control and enabled peritoneal cancer immunotherapy without systemic toxicities. Interleukin-2 (IL2)-producing cytokine factory treatment eradicated peritoneal tumors in ovarian and colorectal mouse models. Furthermore, computational pharmacokinetic modeling predicts clinical translatability to humans. Notably, this platform elicited T cell responses in NHPs, consistent with reported biomarkers of treatment efficacy without toxicity. Combined, our findings demonstrate the safety and efficacy of IL2 cytokine factories in preclinical animal models and provide rationale for future clinical testing in humans.

Immune-modulatory alginate protects mesenchymal stem cells for sustained delivery of reparative factors to ischemic myocardium.

Paracrine factors secreted by mesenchymal stem cells (MSCs) have been previously shown to improve cardiac function following acute myocardial infarction (MI). However, cell therapy activates the innate immune response, leading to the rapid elimination of transplanted cells and only short-term therapeutic delivery. Herein, we describe a new strategy to deliver sustained paracrine-mediated MSC therapy to ischemic myocardium. Using an immune evasive, small molecule modified alginate, we encapsulated rat MSC cells in a core-shell hydrogel capsule and implanted them in the pericardial sac of post-MI rats. Encapsulated cells allowed diffusion of reparative paracrine factors at levels similar to non-encapsulated cells in vitro. Encapsulation enabled sustained cell survival with localization over the heart for 2 weeks. The effect of the experimental group on ventricular function and fibrosis was compared with blank (cell free) capsules and unencapsulated MSCs injected into infarcted myocardium. MSC capsules improved post-MI ventricular function ∼2.5× greater than MSC injection. After 4 weeks, post-MI fibrosis was reduced ∼2/3 with MSC capsules, but unchanged with MSC injection. MSC encapsulation with alginate core-shell capsules sustains cell survival and potentiates efficacy of therapy.

Nanocrystals: A perspective on translational research and clinical studies.

Poorly soluble small molecules typically pose translational hurdles owing to their low solubility, low bioavailability, and formulation challenges. Nanocrystallization is a versatile method for salvaging poorly soluble drugs with the added benefit of a carrier-free delivery system. In this review, we provide a comprehensive analysis of nanocrystals with emphasis on their clinical translation. Additionally, the review sheds light on clinically approved nanocrystal drug products as well as those in development.

Detachment of ligands from nanoparticle surface under flow and endothelial cell contact: Assessment using microfluidic devices.

Surface modification of nanoparticles is a well-established methodology to alter their properties to enhance circulation half-life. While literature studies using conventional, in vitro characterization are routinely used to evaluate the biocompatibility of such modifications, relatively little attention has been paid to assess the stability of such surface modifications in physiologically relevant conditions. Here, microfluidic devices were used to study the effect of factors that adversely impact surface modifications including vascular flow and endothelial cell interactions. Camptothecin nanoparticles coated with polyethylene glycol (PEG) and/or folic acid were analyzed using linear channels and microvascular networks. Detachment of PEG was observed in cell-free conditions and was attributed to interplay between the flow and method of PEG attachment. The flow and cells also impacted the surface charge of nanoparticles. Presence of endothelial cells further increased PEG shedding. The results demonstrate that endothelial cell contact, and vascular flow parameters modify surface ligands on nanoparticle surfaces.

Microfluidic co-culture devices to assess penetration of nanoparticles into cancer cell mass.

In vitro and in vivo assessment of safety and efficacy are the essential first steps in developing nanoparticle-based therapeutic systems. However, it is often challenging to use the knowledge gained from in vitro studies to predict the outcome of in vivo studies since the complexity of the in vivo environment, including the existence of flow and a multicellular environment, is often lacking in traditional in vitro models. Here, we describe a microfluidic co-culture model comprising 4T1 breast cancer cells and EA.hy926 endothelial cells under physiological flow conditions and its utilization to assess the penetration of therapeutic nanoparticles from the vascular compartment into a cancerous cell mass. Camptothecin nanocrystals (∼310 nm in length), surface-functionalized with PEG or folic acid, were used as a test nanocarrier. Camptothecin nanocrystals exhibited only superficial penetration into the cancerous cell mass under fluidic conditions, but exhibited cytotoxicity throughout the cancerous cell mass. This likely suggests that superficially penetrated nanocrystals dissolve at the periphery and lead to diffusion of molecular camptothecin deep into the cancerous cell mass. The results indicate the potential of microfluidic co-culture devices to assess nanoparticle-cancerous cell interactions, which are otherwise difficult to study using standard in vitro cultures.

DAFODIL: A novel liposome-encapsulated synergistic combination of doxorubicin and 5FU for low dose chemotherapy.

PEGylated liposomes have transformed chemotherapeutic use of doxorubicin by reducing its cardiotoxicity; however, it remains unclear whether liposomal doxorubicin is therapeutically superior to free doxorubicin. Here, we demonstrate a novel PEGylated liposome system, named DAFODIL (Doxorubicin And 5-Flurouracil Optimally Delivered In a Liposome) that inarguably offers superior therapeutic efficacies compared to free drug administrations. Delivery of synergistic ratios of this drug pair led to greater than 90% reduction in tumor growth of murine 4T1 mammary carcinoma in vivo. By exploiting synergistic ratios, the effect was achieved at remarkably low doses, far below the maximum tolerable drug doses. Our approach re-invents the use of liposomes for multi-drug delivery by providing a chemotherapy vehicle which can both reduce toxicity and improve therapeutic efficacy. This methodology is made feasible by the extension of the ammonium-sulfate gradient encapsulation method to nucleobase analogues, a liposomal entrapment method once conceived useful only for anthracyclines. Therefore, our strategy can be utilized to efficiently evaluate various chemotherapy combinations in an effort to translate more effective combinations into the clinic.

Mejoras en el acceso a la información de los ensayos clínicos / Improved access to clinical assay information

El Centro de Inmunología Molecular ha venido desarrollando biomoléculas para el tratamiento del cáncer. En la medida que se avanza en el desarrollo de estos productos, se avanza en las fases I, II, III y IV de los ensayos clínicos, lo que trae aparejado un incremento en el número de pacientes a tratar y en el número de hospitales involucrados. Se cuenta con diez productos diferentes, implicados en más de 50 ensayos clínicos, nacionales y multinacionales, con un pronóstico de inclusión de 2 500 pacientes nuevos por año. En este proceso están incluidos alrededor de 30 hospitales en 13 provincias del país. Para mejorar el acceso a la información de ensayos clínicos por parte de los investigadores comprometidos, se creó un sitio Web formado por cuatro secciones fundamentales, relacionadas con los productos y sus indicaciones, buenas prácticas clínicas, la entrada remota de datos y la gerencia de los ensayos clínicos. Todo lo anterior mejora fundamentalmente la calidad de la información brindada a los investigadores clínicos involucrados y redunda en una mayor organización de la compleja actividad de los ensayos clínicos en Cuba(AU)

The Molecular Immunology Center has been developing biomolecules for cancer treatment. As the development of such products continues, phases I, II, III and IV of clinical assays also advance, which leads to an increase of the number of patients to be treated and the number of involved hospitals. There are ten different products involved in over 50 national and multinational clinical assays in which 2 500 patients are predicted to be included every year. Approximately 30 hospitals from 13 provinces are included in this process. For the purpose of improving the access of committed researchers to the clinical assay information, a new Website with four sections was devised. Those sections are related to products and their indications, good clinical practice, remote data entry and clinical assay management. All the above-mentioned improves the quality of the information given to involved clinical researchers and results in better organization of the complex activity of clinical assays in Cuba(AU)

Mejoras en el acceso a la información de los ensayos clínicos / Improved access to clinical assay information

El Centro de Inmunología Molecular ha venido desarrollando biomoléculas para el tratamiento del cáncer. En la medida que se avanza en el desarrollo de estos productos, se avanza en las fases I, II, III y IV de los ensayos clínicos, lo que trae aparejado un incremento en el número de pacientes a tratar y en el número de hospitales involucrados. Se cuenta con diez productos diferentes, implicados en más de 50 ensayos clínicos, nacionales y multinacionales, con un pronóstico de inclusión de 2 500 pacientes nuevos por año. En este proceso están incluidos alrededor de 30 hospitales en 13 provincias del país. Para mejorar el acceso a la información de ensayos clínicos por parte de los investigadores comprometidos, se creó un sitio Web formado por cuatro secciones fundamentales, relacionadas con los productos y sus indicaciones, buenas prácticas clínicas, la entrada remota de datos y la gerencia de los ensayos clínicos. Todo lo anterior mejora fundamentalmente la calidad de la información brindada a los investigadores clínicos involucrados y redunda en una mayor organización de la compleja actividad de los ensayos clínicos en Cuba

The Molecular Immunology Center has been developing biomolecules for cancer treatment. As the development of such products continues, phases I, II, III and IV of clinical assays also advance, which leads to an increase of the number of patients to be treated and the number of involved hospitals. There are ten different products involved in over 50 national and multinational clinical assays in which 2 500 patients are predicted to be included every year. Approximately 30 hospitals from 13 provinces are included in this process. For the purpose of improving the access of committed researchers to the clinical assay information, a new Website with four sections was devised. Those sections are related to products and their indications, good clinical practice, remote data entry and clinical assay management. All the above-mentioned improves the quality of the information given to involved clinical researchers and results in better organization of the complex activity of clinical assays in Cuba