**1. Introduction**

Severe inflammation is a complex and global multi-step physiological process implicated in the development of a systemic dysregulated immune environment. Using sepsis as an example of severe inflammation, epidemiological data suggests that one-in-five of all global deaths is due to sepsis or sepsis-related causes [1]. However, the standard of care for sepsis has failed to move far beyond antibiotics and supportive care, thus leaving much room for the development of new treatment strategies to improve outcomes. To date, over 100 clinical trials have been conducted for potential therapies, but curative strategies remain elusive [2,3]. Previous attempts to address and manage severe inflammation and sepsis have focused on the development of single molecular agents targeted against specific molecules or aspects of molecular pathways implicated in the development of the

**Citation:** Lasola, J.J.M.; Cottingham, A.L.; Scotland, B.L.; Truong, N.; Hong, C.C.; Shapiro, P.; Pearson, R.M. Immunomodulatory Nanoparticles Mitigate Macrophage Inflammation via Inhibition of PAMP Interactions and Lactate-Mediated Functional Reprogramming of NF-κB and p38 MAPK. *Pharmaceutics* **2021**, *13*, 1841. https://doi.org/10.3390/ pharmaceutics13111841

Academic Editors: João Paulo Longo and Luís Alexandre Muehlmann

Received: 3 September 2021 Accepted: 28 October 2021 Published: 2 November 2021

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severe inflammatory response. Despite these methods often demonstrating outstanding preclinical success, translating these results to viable therapeutics for critically ill patients has been unrealized [4]. It has been hypothesized that these attempts have failed because of the profound clinical heterogeneity of sepsis, the lack of fundamental understandings of the different endotypes of sepsis, and treatments that have been targeted towards only a single molecular pathway, leaving redundant pathways associated with immune activation and a multifaceted immune dysfunction unaddressed [5,6]. Therefore, a significant need exists to develop multimodal therapeutics to address the complexity of immune responses present in severe inflammation and sepsis.

Modulation of the innate immune system using nanoparticles serves as the basis for many new and promising therapies for some of the most prevalent and/or severe diseases [7–9]. We recently reviewed the various strategies of nanoparticle-mediated immunomodulation for the treatment of severe inflammation and sepsis [10]. Three mechanisms were proposed by which nanoparticles can be utilized to offset the negative immune mediators of severe inflammation: (1) sequestration of activating pathogen-associated molecular patterns (PAMPs) or proinflammatory cytokines; (2) functional reprogramming of inflammatory immune cell phenotypes; and (3) redirection of inflammatory immune cell trafficking from sites of inflammation.

Our group [11,12] and others [13] have developed cargo-less immunomodulatory nanoparticles (iNPs) that lacked incorporation of small molecules, proteins, or other immunomodulating agents and showed that the physicochemical properties of the nanoparticles were major contributors to the observed therapeutic effects. In our previous studies, antigen presenting cells treated with cargo-less poly(lactic-co-glycolic acid) (PLGA)- and poly(D,L-lactic acid) (PLA)-based iNPs prepared with highly negative zeta potentials could mitigate proinflammatory cytokine secretions such as IL-6 and TNF-α when stimulated with extracellular and intracellular PAMPs, namely Toll-like receptor 4 (TLR4)-targeted lipopolysaccharide (LPS) and TLR9-targeted unmethylated CpG oligodeoxynucleotides (CpG ODN). Furthermore, their immunomodulatory properties translated into a survival benefit in lethal murine LPS-induced endotoxemia models [11]. Initial analysis hinted at a potential role for modulation of NF-κB, IRF1, and STAT1; however, the mechanisms by which iNPs elicit their favorable therapeutic effects remains poorly understood. For these nanoparticle-based strategies to move forward, a greater understanding of the biological effects of these materials and mechanisms by which they exert their immunomodulatory effects is warranted.

Nanoparticles are complex systems and can function through multiple mechanisms where each component involved in its production (i.e., stabilizing surfactant and polymer composition) can potentially alter cellular and inflammatory mediator interactions including rate of uptake, trafficking, rate of degradation and degradation products, etc. Stabilizing surfactants such as poly(vinyl alcohol) (PVA) and poly(ethylene-alt-maleic acid) (PEMA) are ideal for testing the impact of surface characteristics on nano-bio interactions given the variability in zeta potentials and surface chemistry while allowing for control of iNP size. PLA is ideal for understanding the role of the polymer composition and further use in nanoparticle development due to its Food and Drug Administration (FDA) approved status for internal use in humans. Its degradation occurs via autocatalytic cleavage of the ester bonds through hydrolysis into oligomers and monomers of lactic acid, which are substrates of the Krebs cycle [14]. For this reason, minimal toxicity is usually observed due to its biodegradable and biocompatible properties. Although not toxic, there has been a growing appreciation in immunology of the effects of metabolic byproducts in driving observed immune phenotypes [15–19]. Specifically, lactate has been implicated in modifying inflammatory macrophage responses, although controversy remains as to how lactate acts to do this and whether its role is protective or detrimental [20,21]. Additionally, although PLA is a widely used biomaterial in nanoparticle formulation, its effects following degradation are not well characterized in comparison to other commonly used polymeric materials.

In this study, we assess the physical and biological mechanisms that affect iNPmediated modulation of macrophage activation by TLR agonists. We hypothesize that the anti-inflammatory effects of iNPs are multimodal, such that the choice of surfactants elicits differences in the nano-bio interactions, while the choice of nanoparticle composition and its degradation products abrogate the activation of proinflammatory cell signaling pathways. Two formulations of iNPs were prepared using PLA with either PVA or PEMA as surfactants to evaluate the role of surface chemistry and charge on inducing antiinflammatory immune responses. We first evaluated the ability for iNPs to directly interact with PAMPs and the impact of iNP-cell interactions on PAMP-cellular interactions. Next, we assessed the time course-dependent effects of PLA-based iNPs on modulation of NF-κB and p38 mitogen-activated protein kinase (MAPK) signaling. The composition-dependent effects of iNPs on NF-κB and p38 MAPK signaling were subsequently investigated by comparing PLA-based iNPs with commonly utilized commercially available nanoparticles. Lastly, we established a potential role for the pH-sensing G protein-coupled receptor (GPR) 68 on the anti-inflammatory activity of iNPs. Taken together, our study provides evidence for the multimodal mechanisms by which iNPs exert their inherent anti-inflammatory immunomodulatory effects. This work serves as a foundation for further investigation of the inherent immunomodulatory properties of biomaterials and how their specific design features can be tuned to elicit predictable immunological responses through novel strategies and systematic testing with the potential of opening new avenues of research to treat a variety of immune-mediated diseases.
