**5. Conclusions**

Taken together, this work establishes that iNPs take advantage of multiple mechanisms to mitigate severe inflammatory responses and suggests a novel multimodal approach to improve prospects for patients with sepsis and other inflammation-mediated diseases. Polymer-based nanoparticles show promise in serving as drug carriers for controlled delivery of active chemotherapeutic agents; however, the inherent immunomodulatory nature of the materials themselves remains not well characterized. We have described the nano-bio interactions for PLA-based iNPs with varying surface charge and applied these formulations to modulating BMMΦ activity in response to diverse inflammatory agents. We showed that iNPs modify proinflammatory cytokine secretions and also establish that the mechanisms by which this occurs are broad and rely on both physical interactions and reprogramming of BMMΦs. Physical interaction of the BMMΦs with iNPs limit uptake of LPS and CpG ODN interaction. Furthermore, iNPs elicit intrinsic changes in the BMMΦs through metabolic alterations such that NF-κB and p38 MAPK activity is downregulated in response to LPS stimulation. Future studies aim to address applications of iNPs to improve clinical outcomes in murine models of severe inflammation and sepsis and to further characterize nano-bio interactions of iNPs with other key players of the innate immune response, particularly those regulating immunometabolism.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/pharmaceutics13111841/s1, Figure S1: Physicochemical characterization of the iNPs with their fluorophore-conjugated formulations, Figure S2: Confocal microscopy showing extent of iNP uptake in RAW 264.7 cells and subsequent colocalization with FITC-CpG ODN, Figure S3: Flow cytometry reveals iNPs trigger downregulation of CD14 and TLR4 surface expression in bone marrow-derived dendritic cells, Figure S4: Control immunoblots showing p38 inhibition with BIRB 796 and lack of NF-κB p65 and p38 MAPK activation with iNPs alone, Figure S5: LPS-stimulated RAW 264.7 IL-6 and TNF-α secretion using the same experimental design as in Figure 4, Figure S6: Physicochemical characterization of commercially available particles, Figure S7: LPS-stimulated RAW 264.7 IL-6 and TNF-α secretion using the same experimental design as described previously with the inclusion of commercially available particles as described in Figure 5, Figure S8: LPS-stimulated RAW 264.7 IL-6 and TNF-α secretion using the same experimental design as described previously with the inclusion of GPR68 and GPR81 inhibitors described in Figure 6.

**Author Contributions:** Conceptualization, J.J.M.L. and R.M.P.; methodology, J.J.M.L., P.S. and R.M.P.; investigation, J.J.M.L., A.L.C., B.L.S. and N.T.; formal analysis, J.J.M.L. and A.L.C.; resources, C.C.H., P.S. and R.M.P.; writing—original draft preparation, J.J.M.L.; writing—review and editing, P.S., R.M.P. and C.C.H.; visualization, J.J.M.L., A.L.C. and R.M.P.; funding acquisition, R.M.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by startup funds from the University of Maryland School of Pharmacy, the New Investigator Award from the American Association of Colleges of Pharmacy (AACP), and the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM142752 awarded to R.M.P. Additional support was provided by the NIH/NIAID Signaling Pathways in Innate Immunity Training Program (NIH T32AI095190) and NIH/NHLBI Interdisciplinary Training Program in Cardiovascular Disease (NIH T32HL007698) to support J.J.M.L. The authors declare no competing conflicts of interest.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the University of Maryland, Baltimore Office of Animal Welfare Assurance #D16-00125 (A3200-01). The experiments described were approved by the University of Maryland, Baltimore Institutional Animal Care and Use Committee (IACUC) protocol #0721010.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available upon request from the corresponding author, R.M.P., upon request.

**Acknowledgments:** The authors wish to acknowledge the support of the University of Maryland School of Medicine Center for Innovative Biomedical Resources, Flow Cytometry Core—Baltimore, Maryland for technical assistance and experimental design support for this study.

**Conflicts of Interest:** The authors declare no conflict of interest.
