Steven R. Little

Ischemic heart disease is a highly prevalent and debilitating condition throughout the world. Following an ischemic injury, the immune response can become overly active and spread damage to nearby healthy tissue. Because the heart lacks regenerative capacity, preventing excessive damage is critical. Existing immunosuppressive therapeutics provide indiscriminate suppression of inflammatory and repair functions of infiltrating immune cells. Regulatory T cells (Treg) are the body's highly sophisticated system for regulating immunologic activity. These cells can provide powerful immune suppression while amplifying repair pathways through direct stimulation of local cells. Our research group has previously developed a polymeric delivery system comprised of poly(lactic‐co‐glycolic acid) (PLGA) degradable microparticles (MP) loaded with the Treg‐attracting protein, CCL22. In our previous work, we demonstrated CCL22MP attracts Treg to the injection site and can suppress disadvantageous inflammation. Here we combine our Treg‐attracting MP technology with H ydrogels to E nhance or A ttract R egulatory T cells ( HEART ). Topically applied HEART significantly reduced infarct size and improved ejection fraction after permanent‐ligation MI, and in a cardiac transplant ischemia model reduced graft fibrosis while significantly increasing intragraft Foxp3 ⁺ Treg at 30 days. Efficacy tracked with injury severity, identifying severe ischemic injury as a setting where this therapeutic strategy may be most beneficial.

Controlling the molecular transport of nutrients through the gut is an attractive strategy to modulate host metabolism. Herein, a technique of stress‐based evolution of an individual's own microbiota to enhance lipid metabolism is presented, which is based on sequential culture of these bacteria in higher concentrations of lipids. Using this technique, a probiotic formulation of bacterial colonies that exhibit increased lipid metabolism was generated from oral microbiota samples from mice, canine, and human sources. Mice fed a high‐fat diet (HFD) and administered lipid stress evolved (LSE) probiotics excreted increased lipids in stool and reduced triglyceride transport into the blood by three‐fold till 3 h post‐oral gavage of soybean oil, as compared to controls. In addition, these enhanced probiotics prevented weight gain in mice fed a HFD five‐fold better than controls and induced weight loss in mice with diet change three‐fold faster than diet change alone. In these mice, there was a marked change in appearance with a more healthy, less oily coat. Controlled metabolic cage experiments demonstrated that the total movement, food intake, and water intake were not significantly different between mice receiving LSE probiotic versus a control probiotic formulation, suggesting that important health measures are unchanged with LSE probiotic administration. Overall, this facile stress‐based culture technique can be utilized to modulate bacterial metabolism and applied to different industrial processes of probiotic generation and to affect different disease outcomes such as obesity.

Liposomes are microscale lipid vesicles used in pharmaceuticals, food products, and most recently, agriculture. Several studies have shown that liposomes can deliver nutrients to plant leaves, often more efficiently than traditional forms. However, the delivery of plant nutrients to soil via liposomes remains understudied. Interactions between liposomes and soil microbes, including metabolism of the lipid carbon (C) and assimilation of liposome-encapsulated nutrients into soil microbial biomass, could alter the availability of nutrients within the soil. We assessed the impact of lecithin liposomes with nitrogen (N) cargo on C and N cycling during a 7-day incubation experiment. We quantified changes in concentrations of carbon dioxide, nitrous oxide, oxygen, and soil inorganic N pools including soil extractable nitrate (NO₃ ^–-N) and ammonium (NH₄ ⁺-N). Liposome additions increased microbial respiration and resulted in rapid soil NO₃ ^–-N immobilization, suggesting that liposomes may be a tool to immobilize N and reduce agricultural N losses.

Poly(lactic-co-glycolic acid) terminated with charged terminal groups physical property assessment, quantification of formed microparticle degradation and uptake by phagocytes, and measurement of biomolecule release.

Background/Objectives: Controlled release systems, such as polymeric microparticles (MPs), have emerged as a promising solution to extend the bioavailability and reduce dosing frequency for biologic drugs; however, the formulation of these systems to encapsulate highly sensitive, hydrophilic biologic drugs within hydrophobic polymers remains a nontrivial task. Although scalable manufacturing and FDA approval of single emulsion processes encapsulating small molecules has been achieved, scaling more complex double emulsion processes to encapsulate hydrophilic biologics remains more challenging. Methods: Here, we demonstrate that two hydrophilic, low-molecular-weight, recombinant chemokines, CCL22 and CCL2, can be encapsulated in poly(lactic-co-glycolic acid) (PLGA) MPs using a single emulsion method where the proteins are dissolved in an organic solvent during formulation. Results: As expected, we observed some differences in release kinetics from single emulsion MPs compared to double emulsion MPs, which traditionally have been used to encapsulate proteins. Single emulsion MPs exhibited a substantially reduced initial burst. Importantly, protein released from single emulsion CCL22-MPs also retained biological activity, as determined by a cell-based functional assay. Decreasing particle size or changing the polymer end group from PLGA-COOH to PLGA-OH increased the initial burst from single emulsion MPs, demonstrating tunability of release kinetics for protein-loaded, single emulsion MPs. Finally, to improve scalability and enable more precise control over MP formulations, the single emulsion process was adapted to a microfluidic, continuous manufacturing system, and the resulting MPs were evaluated similarly. Conclusions: Altogether, this study demonstrates the feasibility of using a single emulsion encapsulation method for at least some protein biologics.

The use of inorganic nutrient fertilizers in crop agriculture is often inefficient due to rapid leaching of nutrients with percolating water in soil. To address this, a liposomal carrier was developed to slow transport and reduce leaching of inorganic nutrient loads within agricultural soils. Liposomes, spherical lipid bilayers, have been widely used in medicine but remain under-characterized for agricultural applications. Their biocompatibility, high loading capacity, and stability under certain compositions and environmental conditions suggest they could effectively deliver agrochemicals to crops. We present soil column experiments to evaluate the ability of a liposomal carrier to reduce the transport of an ionic tracer load, sodium bromide, under varying soil and water saturation conditions. Results from saturated column experiments demonstrate that encapsulation in liposomes slowed tracer transport and reduced leachate concentrations in sand and silty clay loam soils. Reduction in tracer leachate was also observed for unsaturated column experiments in silty clay loam soil. Subsequent experiments suggested a combination of processes (i.e., attachment, aggregation, physical exclusion, and biogenic immobilization) were responsible for the observed behavior of liposome encapsulated tracer in the soil column experiments. These findings support the use of liposomes as an effective carrier of inorganic nutrients to reduce leaching.

Allergic contact dermatitis (ACD), a T-cell mediated inflammatory skin condition, is prompted by multiple, subsequent exposures to contact allergens (e.g., nickel). Current treatment approaches for ACD include repeated topical application or systemic delivery of immunosuppressants. These treatment strategies have many limitations, including non-specific mechanism of actions and the occurrence of side effects due to their delivery method. For this reason, we developed a novel therapeutic approach that is based upon adenosine (Ado) receptor signaling, a known anti-inflammatory pathway. Specifically, we developed a polymer microparticle-based controlled release system capable of presenting IBMECA (IBMECA-MPs), an Ado receptor agonist, to the local environment. In this study, we first sought to study the immunosuppressive effects of IBMECA on immune cells implicated in the pathogenesis of ACD (e.g., dendritic cells) in vitro. Subsequently, we examined the effects of enhancing adenosine signaling in contact hypersensitivity (CHS), an in vivo model of ACD, through local administration of IBMECA-MPs. We observed that IBMECA-MPs were capable of reducing the inflammatory response associated with CHS by reducing maturation markers of antigen-presenting cells, altering cytokine secretion, and reducing relative frequencies of effector T cell populations. To our knowledge, this is the first demonstration of therapeutic efficacy of IBMECA in CHS, as well as the first proof-of-principle demonstration of IBMECA application in the context of a local drug delivery system. Ultimately, this delivery system has the potential to be adapted for use in other T-cell mediated inflammatory conditions (e.g., transplant rejection), suggesting broader implications of this study. Graphical abstract

Background/Objectives: Microparticle-based drug delivery systems offer several advantages for protein-based drug formulations, enhancing patient compliance and therapeutic efficiency through the sustained delivery of the active pharmaceutical ingredient. Over the past few decades, the microfluidics method has emerged as a continuous manufacturing process for preparing drug-encapsulating microparticles, mainly for small molecule drugs. However, comparative assessments for the conventional batch method vs. the microfluidics method for protein-based drug formulations have been lacking. The main objective of this study was to generate immunomodulatory protein drug-loaded injectable formulations using both conventional batch and microfluidics methods. Methods: Therefore, rhCCL22-loaded poly(lactic-co-glycolic) acid (PLGA) microparticles were prepared by conventional homogenization and microfluidics methods. Results: The resulting microparticles were analyzed comparatively, focusing on critical quality attributes such as microparticle size, size distribution, morphology, drug encapsulation efficiency, release kinetics, and batch-to-batch variations in relation to the manufacturing method. Our results demonstrated that the conventional method resulted in microparticles with denser surface porosity and wider size distribution as opposed to microparticles prepared by the microfluidics method, which could contribute to a significant difference in the drug-release kinetics. Additionally, our findings indicated minimal variation within batches for the microparticles prepared by the microfluidics method. Conclusion: Overall, this study highlights the comparative assessment of several critical quality attributes and batch variations associated with the manufacturing methods of protein-loaded microparticles which is crucial for ensuring consistency in efficacy, regulatory compliance, and quality control in the drug formulation manufacturing process.

Coronary artery disease leads to over 360,000 deaths annually in the United States, and off-the-shelf bypass graft options are currently limited and/or have high failure rates. Tissue-engineered vascular grafts (TEVGs) present an attractive option, though the promising mesenchymal stem cell (MSC)-based implants face uncertain regulatory pathways. In this study, “artificial MSCs” (ArtMSCs) were fabricated by encapsulating MSC-conditioned media (CM) in poly(lactic-co-glycolic acid) microparticles. ArtMSCs and control microparticles (Blank-MPs) were incubated over 7 days to assess the release of total protein and the vascular endothelial growth factor (VEGF-A); releasates were also assessed for cytotoxicity and promotion of smooth muscle cell (SMC) proliferation. Each MP type was loaded in previously published “lyogel” silk scaffolds and implanted as interposition grafts in Lewis rats for 1 or 8 weeks. Explanted grafts were assessed for patency and cell content. ArtMSCs had a burst release of protein and VEGF-A. CM increased proliferation in SMCs, but not after encapsulation. TEVG explants after 1 week had significantly higher patency rates with ArtMSCs compared to Blank-MPs, but similar to unseeded lyogel grafts. ArtMSC explants had lower numbers of infiltrating macrophages compared to Blank-MP explants, suggesting a modulation of inflammatory response by the ArtMSCs. TEVG explants after 8 weeks showed no significant difference in patency among the three groups. The ArtMSC explants showed higher numbers of SMCs and endothelial cells within the neotissue layer of the graft compared to Blank-MP explants. In sum, while the ArtMSCs had positive effects acutely, efficacy was lost in the longer term; therefore, further optimization is needed.

Many chronic inflammatory diseases are attributed to disturbances in host–microbe interactions, which drive immune-mediated tissue damage. Depending on the anatomic setting, a chronic inflammatory disease can exert unique local and systemic influences, which provide an exceptional opportunity for understanding disease mechanism and testing therapeutic interventions. The oral cavity is an easily accessible environment that allows for protective interventions aiming at modulating the immune response to control disease processes driven by a breakdown of host–microbe homeostasis. Periodontal disease (PD) is a prevalent condition in which quantitative and qualitative changes of the oral microbiota (dysbiosis) trigger nonresolving chronic inflammation, progressive bone loss, and ultimately tooth loss. Here, we demonstrate the therapeutic benefit of local sustained delivery of the myeloid-recruiting chemokine (C-C motif) ligand 2 (CCL2) in murine ligature-induced PD using clinically relevant models as a preventive, interventional, or reparative therapy. Local delivery of CCL2 into the periodontium inhibited bone loss and accelerated bone gain that could be ascribed to reduced osteoclasts numbers. CCL2 treatment up-regulated M2-macrophage and downregulated proinflammatory and pro-osteoclastic markers. Furthermore, single-cell ribonucleic acid (RNA) sequencing indicated that CCL2 therapy reversed disease-associated transcriptomic profiles of murine gingival macrophages via inhibiting the triggering receptor expressed on myeloid cells-1 (TREM-1) signaling in classically activated macrophages and inducing protein kinase A (PKA) signaling in infiltrating macrophages. Finally, 16S ribosomal ribonucleic acid (rRNA) sequencing showed mitigation of microbial dysbiosis in the periodontium that correlated with a reduction in microbial load in CCL2-treated mice. This study reveals a novel protective effect of CCL2 local delivery in PD as a model for chronic inflammatory diseases caused by a disturbance in host–microbe homeostasis.