Inflammation is a key homeostatic process involved in the body’s response to a multitude of disease states including infection, autoimmune disorders, cancer, and other chronic conditions. When the initiating event is poorly controlled, severe inflammation and globally dysregulated immune responses can occur. To address the lack of therapies that efficaciously address the multiple aspects of the dysregulated immune response, we developed cargo-less immunomodulatory nanoparticles (iNPs) comprised of poly(lactic acid) (PLA) with either poly(vinyl alcohol) (PVA) or poly(ethylene-alt-maleic acid) (PEMA) as stabilizing surfactants and investigated the mechanisms by which they exert their inherent anti-inflammatory effects. We identified that iNPs leverage a multimodal mechanism of action by physically interfering with the interactions between pathogen-associated molecular patterns (PAMPs) and bone marrow-derived macrophages (BMMΦs). Additionally, we showed that iNPs mitigate proinflammatory cytokine secretions induced by LPS via a time- and composition-dependent abrogation of NF-κB p65 and p38 MAPK activation. Lastly, inhibition studies were performed to establish the role of a pH-sensing G-protein-coupled receptor, GPR68, on contributing to the activity of iNPs. These data provide evidence for the multimodal mechanism of action of iNPs and establish their potential use as a novel therapeutic for the treatment of severe inflammation.

Histone deacetylase inhibitors (HDACi) induce potent anti-inflammatory responses when used to treat inflammatory diseases. Suberoylanilide hydroxamic acid (SAHA), a pan-HDACi, decreases pro-inflammatory cytokine levels and attenuates cytokine storm in sepsis; however, its toxicity profile toward immune cells has limited its use as a sepsis therapeutic. Here, we developed a modification to SAHA by para-hydroxymethylating the capping group to generate SAHA-OH. We discovered that SAHA-OH provides a favorable improvement to the toxicity profile compared to SAHA. SAHA-OH significantly reduced primary macrophage apoptosis and splenic B cell death as well as mitigated organ damage using a lipopolysaccharide (LPS)-induced endotoxemia mouse model. Furthermore, SAHA-OH retained antiinflammatory responses similar to SAHA as measured by reductions in LPS-induced proinflammatory cytokine secretions in vitro and in vivo. These effects were attributed to a decreased selectivity of HDAC1, 2, 3, 8 and an increased selectivity for HDAC6 for SAHA-OH as determined by IC 50 values. Our results support the potential for SAHA-OH to modulate acute proinflammatory responses while mitigating SAHA-associated drug toxicity for use in the treatment of inflammation-associated diseases and conditions.

Nanoparticles (NPs) have emerged as a highly useful and clinically translatable drug delivery platform for vast therapeutic payloads. Through the precise tuning of their physicochemical properties, NPs can be engineered to exhibit controlled drug release properties, enhanced circulation times, improved cellular uptake and targeting, and reduced toxicity profiles. Conventional bulk methods for the production of polymeric NPs suffer from the ability to control their size and polydispersity, batch-to-batch variability, significant preparation times, and low recovery. Here, we describe the development and optimization of a high-throughput microfluidic method to produce cargo-less immunomodulatory nanoparticles (iNPs) and their formulation-dependent anti-inflammatory properties for the modulation of lipopolysaccharide (LPS)-induced macrophage responses. Using poly(lactic acid) (PLA) as the core-forming polymer, a rapid and tunable microfluidic hydrodynamic flow-focusing method was developed and optimized to systematically evaluate the role of polymer and surfactant concentration, surfactant chemistry, and flow rate ratio (FRR) on the formation of iNPs. A set of iNPs with 6 different surface chemistries and 2 FRRs was then prepared to evaluate their inherent anti-inflammatory effects using bone marrow-derived macrophages stimulated with the Toll-like receptor 4 agonist, LPS. Finally, a lyophilization study was performed using various cryoprotectants and combinations to identify preferable conditions for iNP storage. Overall, we demonstrate a highly controlled and reproducible method for the formulation of iNPs using microfluidics and their formulation-dependent inherent anti-inflammatory immunomodulatory properties, which represents a potentially promising strategy for the management of inflammation.

Extrahepatic nucleic acid delivery using polymers typically requires the synthesis and purification of custom monomers, post-synthetic modifications, and incorporation of additional excipients to augment their stability, endosomal escape, and in vivo effectiveness. Here, we report the development of a single-component and excipient-free, polyester-based nucleic acid delivery nanoparticle platform comprising ionizable N-methyldiethanolamine (MDET) and various hydrophobic alkyl diols (Cp) that achieves lung-selective nucleic acid transfection in vivo. PolyMDET and polyMDET-Cp polyplexes displayed high serum and enzymatic stability, while delivering pDNA or mRNA to "hard-to-transfect" innate immune cells. PolyMDET-C4 and polyMDET-C6 mediated high protein expression in lung alveolar macrophages and dendritic cells without inducing tissue damage or systemic inflammatory responses. Improved strategies using readily available starting materials to produce a simple, excipient-free, non-viral nucleic acid delivery platform with lung-selective and innate immune cell tropism has the potential to expedite clinical deployment of polymer-based genetic medicines.

Genetic engineering of innate and adaptive immune cells represents a potential solution to treat numerous immune-mediated pathologies. Current immune engineering methods to introduce nucleic acids into cells with high efficiency rely on physical mechanisms such as electroporation, viral vectors, or other chemical methods. Gene delivery using non-viral nanoparticles offers significant flexibility in biomaterial design to tune critical parameters such as nano-bio interactions, transfection efficiency, and toxicity profiles. However, their clinical utility has been limited due to complex synthetic procedures, high toxicity at increased polymer (nitrogen, N) to DNA ratios (phosphate, P) (N/P ratios), poor transfection efficiency and nanoparticle stability in the presence of serum, and short-term gene expression. Here, we describe the development of a simple, polymer-based non-viral gene delivery platform based on simple modifications of polyethylenimine (PEI) that displays potent and serum-independent transfection of innate and adaptive immune cells. Cationic acetylated PEI (Ac-PEI) was synthesized and complexed with plasmid DNA (pDNA) followed by enveloping with an anionic polyelectrolyte layer of poly(ethylene-alt-maleic acid) (PEMA) to form immunoplexes (IPs). Cellular interactions and gene expression could be precisely controlled in murine RAW 264.7 macrophages, murine DC2.4 dendritic cells, and human Jurkat T cells by altering the levels of PEMA envelopment, thus providing a strategy to engineer specific cell targeting into the IP platform. Optimally formulated IPs for immune cell transfection in the presence of serum utilized high N/P ratios to enable high