Maca-derived nanoparticles and methods of use

Abstract

Isolation and characterization of MNDP, lipid nanoparticles derived from Lepidium meyenii Walp (maca), are disclosed. MDNP sequestered and neutralized multiple pro-inflammatory cytokines and APPs in its protein corona to reveal a highly effective therapeutic strategy for managing severe inflammatory responses. Lipid nanoparticle compositions based on the lipidomic analysis of MNDP are disclosed. Methods for the prevention and treatment for inflammatory diseases such as sepsis are disclosed.

Description

MACA-DERIVED NANOPARTICLES AND METHODS OF USE STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0001] This invention was made with government support under Grant Number GM142752 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0002] This invention relates to plant-derived lipid nanoparticle compositions. The invention further relates to methods for treating inflammatory disease. BACKGROUND OF INVENTION

[0003] Edible plant-derived nanoparticles (PDNPs) are nanostructured and membrane- enveloped vesicles secreted by plant cells that serve as carriers of various endogenous bioactive substances. Although previously perceived as cellular debris, PDNPs are recognized as crucial entities that regulate cell-cell communication and immune responses against pathogens1. The evolving understanding of the biogenesis of PDNPs has been described in previous literature2,3. Briefly, the process initiates with the formation of a trans-Golgi network or early endosome. Plant cells then generate the matured multivesicular endosome that integrates and fuses with the plasmalemma, releasing PDNPs through the inward budding of multivesicular endosomes4. There are several studies pointing to the potential therapeutic benefits of PDNPs, which in some cases demonstrate anti-inflammatory5, antioxidant6, and anti-cancer activity7. This emerging class of naturally derived nanoparticles offers highly advantageous features for use as nanomedicines, exhibiting negligible toxicity or immunogenicity, efficient cellular uptake, and having capacity to deliver a variety of therapeutic agents through re-engineering8.

[0004] Cytokine secretions and the acute phase response (APR) are early, systemic immune reactions that develop within minutes to hours as part of the body’s early defense mechanism to injury, infection, or immune challenge9,10. The APR is triggered by the release of pro- inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-a), interleukin-1b (IL-1b), and others by macrophages, neutrophils, and various immune cells at the site of inflammation11. This response triggers the production of acute phase proteins (APPs), which are typically undetectable under healthy conditions but increased 10- to 1000-fold duringinflammation12. These proteins play roles in modulating inflammation, enhancing pathogen clearance, and promoting tissue repair13. The APR is generally recognized as beneficial for restoring homeostasis disturbed by such injuries, however, APPs have been described to elicit a range of functions to induce pro- or anti-inflammatory responses. During severe inflammatory responses like sepsis, a dysregulated APR can potentiate the inflammatory response leading to coagulopathy, organ failure, and even death14–16.

[0005] Several groups have developed strategies to mitigate the inflammatory response by sequestering pro-inflammatory cytokines17. Multiple studies highlight that the use of membrane- coated nanoparticles that mimic biological cell membranes to trap and neutralize these inflammatory cytokines18. By acting as macrophage decoys, these nanoparticles can be developed to bind and neutralize endotoxins and pro-inflammatory cytokines19. Similarly, neutrophil membrane-coated nanoparticles are produced using neutrophils isolated from mouse bone marrow after LPS stimulation20. The process involves membrane extraction, incubation, and extrusion or sonication with polymeric nanoparticle cores. These membrane-camouflaged nanoparticles reduce inflammation by binding and neutralizing endotoxins and cytokines such as IL-6, and TNF-a19,21. Despite these advancements, the complex synthesis and formulation required for these designs to sequester pro-inflammatory mediators presents a significant challenge to clinical translation. As a result, there is critical need to develop simpler anti- inflammatory strategies that can sequester multiple inflammatory mediators to mitigate pro- inflammatory responses to augment sepsis survival.

[0006] Lepidium meyenii Walp, also referred to as maca, is a biennial root plant indigenous to the Peruvian Andes. Maca possesses high contents of fiber, amino acids, fatty acids, and other essential nutrients, while also containing various bioactive compounds that are involved in integral cellular activities within plants22. Several studies have shown that maca alleviates fatigue23, oxidative stress24, tumor formation25, and inflammation26. However, studies have focused on the raw material or crude extract, which contains mixture of different active ingredients, to evaluate in disease models27,28. Despite these known advantages, maca remains relatively underexplored.BRIEF SUMMARY OF INVENTION

[0007] The present invention is generally directed to maca-derived lipid nanoparticles (MDNP), nanoparticles comprising lipids identified from MNDPs and methods of treatment of inflammatory diseases with said nanoparticles.

[0008] In more detail, the present inventors isolated, characterized, and developed therapeutics based on PDNPs isolated from maca root, termed maca-derived lipid nanoparticles (MDNP), for the treatment of severe inflammation as exemplified using in vitro and in vivo models of lipopolysaccharide (LPS)-induced endotoxemia and polymicrobial sepsis. MDNP were isolated and characterized for physicochemical properties, as well as lipid composition using lipidomic analysis. The toxicity profile of MDNP was then established prior to determining their uptake profile and anti-inflammatory properties. In vitro, MDNP efficiently sequestered multiple pro-inflammatory cytokines, which led to comprehensive in vivo assessment of their biodistribution and therapeutic activity. Therapeutic administration of MDNP to LPS-challenged mice led to significant reductions in plasma pro-inflammatory cytokines, reductions in inflammation-induced organ damage, and improved survival.

[0009] When nanoparticles are introduced into a biological fluid, they are rapidly covered by a layer of biomolecules known as the biomolecular corona (or protein corona)29,30. To identify the mechanism by which MDNP elicited its anti-inflammatory effects, untargeted proteomic analysis of the MDNP protein corona was performed to uncover the inflammatory mediators sequestered by MDNP and corresponding pathways and upstream regulators modulated. MDNP were found to sequester and neutralize a variety of APPs, which promote the propagation of pro- inflammatory immune responses, in addition to pro-inflammatory cytokines. Lastly, the therapeutic efficacy of MDNP was assessed using a clinically relevant mouse model of polymicrobial sepsis and found to significantly increase survival. These results demonstrate the potential of MDNP as an abundant, cost effective, naturally derived therapeutic agent for use as a multimodal intervention for a variety of inflammatory diseases due to their ability to sequester a broad spectrum of inflammatory mediators.

[0010] The present invention is directed to isolated maca-derived nanoparticles (MDNP), wherein the nanoparticles are isolated from Lepidium meyenii Walp, also known as maca. In one embodiment, MDNP may be isolated by a method comprising performing sucrose gradientultracentrifugation on a maca extract. In one embodiment, the invention is directed to lipid nanoparticle compositions comprising isolated MDNP.

[0011] The present invention is also directed to lipid nanoparticle compositions comprising a lipid component. In one embodiment, the lipid component includes some or all of the lipid component of isolated MDNP. In a non-limiting example, the lipid nanoparticle composition comprises a lipid component which includes a triglyceride in an amount of about 77.4 % to about 62.0% by weight of the lipid component; and a ceramide in an amount of about 25.0% to about 11.8%, by weight of the lipid component. In some embodiments the triglyceride is a phyto- triglyceride and / or the ceramide is a phytoceramide. In certain embodiments the lipid component may include additional lipids, such as phytohexosylceramide, free fatty acid, digalactosyldiacylglycerol, phosphatidylcholine, phosphatidylinositol, lysophosphatidylcholine, cholesterol, or a combination thereof. The phytohexosylceramide, if present, may be in an amount of about 2.70 % to about 2.16%, by weight of the lipid component; the free fatty acid, if present, may be in an amount of about 1.95% to about 1.56%, by weight of the lipid component; the digalactosyldiacylglycerol, if present, may be in an amount of about 0.720% to about 0.576%, by weight of the lipid component; the phosphatidylcholine, if present, may be in an amount of about 0.960% to about 0.768%, by weight of the lipid component; the phosphatidylinositol, if present, may be in an amount of about 0.850% to about 0.680%, by weight of the lipid component; the lysophosphatidylcholine, if present, may be in an amount of about 0.130% to about 0.104%, by weight of the lipid component; and the cholesterol, if present, may be in an amount of about 20.0% to about 5.0%, by weight of the lipid component. In one embodiment, the triglyceride is in an amount of about 75% by weight of the lipid component, and the ceramide is in an amount of about 25% by weight of the lipid component. In another embodiment, the triglyceride is in an amount of about 77.4% by weight of the lipid component; the ceramide is in an amount of about 14.8% by weight of the lipid component; the phytohexosylceramide in an amount of about 2.70% by weight of the lipid component; the free fatty acid is in an amount of about 1.95% by weight of the lipid component; the digalactosyldiacylglycerol is in an amount of about 0.720% by weight of the lipid component; the phosphatidylcholine is in an amount of about 0.960% by weight of the lipid component; the phosphatidylinositol is in an amount of about 0.850% by weight of the lipid component; and the lysophosphatidylcholine is in an amount of about 0.130% by weight of the lipid component.

[0012] The present invention is also directed to the lipid nanoparticle compositions of the invention further containing therapeutic agents. In some embodiments, the lipid nanoparticle composition of any of the above embodiments further contains a therapeutic agent. In other embodiments, the lipid nanoparticle composition includes a lipid composition which includes some or all of the lipid component of isolated MDNP, and a therapeutic agent. In certain embodiments the therapeutic agent is dexamethasone.

[0013] The present invention is also directed to the lipid nanoparticle compositions of the invention further containing a polymer core. In some embodiments, the lipid nanoparticle composition of any of the above embodiments further contains a polymer core. In other embodiments, the lipid nanoparticle composition includes a lipid composition which includes some or all of the lipid component of isolated MDNP, and a polymer core. In certain embodiments, the polymer core is a poly (lactic acid) core.

[0014] The present invention is also directed to methods for preventing or treating an inflammatory disease in a subject, said method comprising, administering a therapeutically effective amount of the lipid nanoparticle composition of any of the above embodiments, to a subject having inflammatory disease. In certain embodiments, the inflammatory disease is selected from rheumatoid arthritis, inflammatory bowel diseases, COVID-19 cytokine storms, and sepsis. In some embodiments, a bacterium causes the sepsis. In certain embodiments, the lipid nanoparticle composition sequesters an acute phase protein or a pro-inflammatory cytokine. In certain embodiments the pro-inflammatory cytokine is selected from IL-6 and TNF-α. In some embodiments the therapeutically effective amount is 0.01 to 1 g / kg body weight.

[0015] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described herein, which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that any conception and specific embodiment disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be betterunderstood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that any description, figure, example, etc. is provided for the purpose of illustration and description only and is by no means intended to define the limits of the invention. BRIEF DESCRIPTION OF DRAWINGS

[0016] FIG.1 shows the isolation and characterization of MDNP. (A) Schematic representation of the isolation process for maca-derived lipid nanoparticles (MDNP). Created in BioRender. Sung, J. (2025) https: / / BioRender.com / h22o988. (B) Before and after images of MDNP isolation. (C) MDNP recovery from pure maca powder. (D) A representative TEM image of MDNP. (E) Room temperature measurement of size (174.5 nm), zeta potential (-9.6 mV), and PDI (0.231) of freshly isolated MDNP. (F) Storage stability test of MDNP under various storage conditions up to day 6. (G) Heat flow curve of MDNP measured by differential scanning calorimetry showing the summit of melting peak at 74.3ºC. (H) Lipid extraction and component analysis by liquid chromatography coupled to high resolution LC-MS / MS. Bar graph shows abundance of lipids. (I) Pie graph shows the whole ratio of each component, and the vertical slice magnifies the smaller portion of composition excluding TG. N=3 / group.

[0017] FIG.2 shows the in vitro cytotoxicity, cellular internalization, immunomodulation, and therapeutic effects against LPS-induced inflammation. (A) PI / Annexin V assay by flow cytometry using 1 mg / mL of MDNP in BMDM. Quantitative analysis of live, apoptotic, and dead cells after MDNP treatment. (B) Uptake of Cy5.5 labeled MDNP by BMDM after 4 and 8 hrs. Cells were stained with DAPI and FITC-PI to visualize the nucleus and F-actin, respectively. (C-F) Immunomodulatory activity of MDNP in LPS stimulated BMDM using flow cytometry of CD80, CD86, MHCII, and CD206 markers. (G) A timeline for prophylactic treatment of MDNP. ELISA cytokine measurements of (H) IL-6 and (I) TNF-a. Created in BioRender. Sung, J. (2025) https: / / BioRender.com / x40h804. (J) A timeline for therapeutic treatment. ELISA cytokine measurements of (K) IL-6 and (L) TNF-a. Data are expressed as mean ± SD (n=5). Data are expressed as mean ± SD (n=3). *P<0.05, **p<0.05 versus LPS control group.

[0018] FIG.3 shows the in vitro sequestration of pro-inflammatory cytokines from LPS- induced bone marrow derived macrophages. (A) Time course reduction of LPS-stimulated pro- inflammatory cytokine by treatment of MDNP. Created in BioRender. Sung, J. (2025)https: / / BioRender.com / n91r920. ELISA cytokine measurements of (B) IL-6 and (C) TNF-a. (D) Schematic of IL-6 cytokine sequestration study. Created in BioRender. Sung, J. (2025) https: / / BioRender.com / n10c223. (E) Concentration-dependent sequestration of IL-6 protein by MDNP. (F) Schematic of NF-^^B activity assay using RAW-Blue 264.7 cells. Created in BioRender. Sung, J. (2025) https: / / BioRender.com / y73h708. (G) NF-^^B activity after treatment with MDNP in LPS stimulated cells. PLGA was used as a control. All data are expressed as means ± SD (n=5). **p<0.05, ***p<0.005, and ****p<0.0005 versus LPS or NT control group.

[0019] FIG.4 shows the route of administration-dependent biodistribution and anti- inflammatory effects of MDNP. (A) Timeline of MDNP treatments following LPS challenge. The schematic illustrates the IP and IV injection performed on group of C57BL / 6J mice (LPS only, LPS+IP injection, and LPS+IV injection, n=3 per group). Created in BioRender. Sung, J. (2025) https: / / BioRender.com / n92o995. Created in BioRender. Sung, J. (2025) https: / / BioRender.com / v94b216. (B) IVIS image of biodistribution of fluorescently labeled MDNP in mouse organs (liver, kidney, spleen, lung, and heart). (C) Fluorescence intensity measurement of MDNP biodistribution. (D) Heatmap of cytokine expression including: (E) IL-6, (F) TNF-α, (G) MCP-1, (H) IL-10, (I) IL-1b, (J) IFN-b, and (K) GROα from mice plasma samples. Data are expressed as mean ± SD (n=3). *P<0.05, **p<0.05, ***p<0.005, and ****p<0.0005 versus PBS control group.

[0020] FIG.5 shows representative images of organ recovery and survival effect by treatment of MDNP during LPS-induced sepsis. (A) Hematoxylin and eosin (H&E) staining of liver, kidney, spleen, lung, and heart. The LPS treated group images show presence of neutrophil infiltration (yellow arrow) in liver, tubular necrosis (white arrow) in the tubules in kidney, focal necrosis and dysregulation in white pulp of spleen, appearance of extensive clots (red arrow) in the alveoli in lungs, while no noticeable damage in heart. On the contrary, mice organs administered with IP and IV displayed notably reduced organ damage similar to the level of the non-treated control group. (B-F) Quantification of H&E staining. Levels of injury scores were calculated. Data are expressed as mean ± SD (n=3). *P<0.05, **p<0.05 versus LPS control group. (G) Schematic of survival study of LPS-treated and LPS+MDNP-treated C57BL / 6J mice (n=10 / group). Created in BioRender. Sung, J. (2025) https: / / BioRender.com / l63d302. (H) The survival analysis graph displays a significant difference between LPS only and the MDNP treatment group. Mice were challenged with 20 mg / kg LPS intraperitoneally followed by twoinjections of MDNP. Kaplan-Meier curves and a log-rank test were performed to compare the survival rates. ***p<0.0005

[0021] FIG.6 shows the mapping and identification of protein corona adsorbed onto the surface of MDNP. (A) Schematic showing the process of evaluating the MDNP protein corona. Created in BioRender. Sung, J. (2025) https: / / BioRender.com / j58w712. (B) Venn diagram illustrates a total of 296 protein corona including unique (1 in healthy and 26 in LPS) and 270 shared proteins in the corona. (C) Heatmap shows abundance of 49 upregulated and 100 downregulated proteins in the LPS compared to healthy MDNP corona groups. (D) Volcano plot of significantly upregulated and downregulated protein corona. Hp, Serpina3n and Saa1 (red dots) are categorized as pro-inflammatory and acute phase response signaling proteins. (E) Top 10 canonical pathways associated with the significantly enriched corona proteins were identified, with acute phase response signaling being the most prominent pathway. (F) A network representation of acute phase response signaling proteins and major upstream regulators. (G) Ex vivo sequestration of pro-inflammatory cytokines by incubating LPS plasma with MDNP. Both IL-6 and TNF-α was significantly reduced following 8 hr incubation with MDNP. Created in BioRender. Sung, J. (2025) https: / / BioRender.com / f87s608. (H) A graphical depiction illustrates the proposed mechanism of how the MDNP sequesters and deactivates pro-inflammatory cytokines and acute phase proteins to reduce overall inflammation to enhance sepsis survival. Created in BioRender. Sung, J. (2025) https: / / BioRender.com / p54m386.

[0022] FIG.7 shows survival effects of MDNP treatment using the severe CLP model of polymicrobial sepsis. (A) Schematic timeline of survival study of cecal ligation and puncture (CLP). C57BL / 6J mice underwent CLP surgery to induce severe inflammation and polymicrobial infection and three separate doses of MDNP were administered intraperitoneally (0.5, 2, 24 hr timepoint, 2 mg / injection) to analyze survival (n=5 / group). Created in BioRender. Sung, J. (2025) https: / / BioRender.com / q03l127. (B) Ex vivo cytokine sequestration assay using CLP plasma following incubation with MDNP. Kaplan-Meier curves and a log-rank test were performed to compare the survival rates. Created in BioRender. Sung, J. (2025) https: / / BioRender.com / t95w854. (C) The survival test displayed a significant improvement (40%) by the treatment of MDNP. **p< 0.005. Data are expressed as mean ± SD (n=4). ****p<0.0005 versus PBS control group.

[0023] FIG.8 shows stability test for MDNP in culture medium and mouse plasma. MDNP was centrifuged followed by incubation in (A) culture medium and (B) mouse plasma at RT and 37ºC, and its size and zeta potential were monitored up to 24 hrs. No significant fluctuation was observed, indicating stable dispersion behavior under both conditions.

[0024] FIG.9 shows stability test for lyophilized MDNP with and without cryoprotectant. (A) Size was stable after reconstituting lyophilized MDNP. (B) Zeta potential of lyophilized MDNP displayed stable measurement in both cryoprotectant-free and cryoprotectant added samples.

[0025] FIG.10 shows chemical structures of lipid components in four distinctive lipid groups (sphingolipids, galactolipids, neutral, and phospholipids).

[0026] FIG 11 shows quantitative measure of mean fluorescence intensity (MFI) from the uptake of Cy5.5-labeled MDNP at 0, 4, 8 hr time point in Figure 2B. A significant uptake was detected by confocal microscopy over time. Measurement originates from in vitro therapeutic treatment of MDNP.

[0027] FIG.12 shows the therapeutic effect of MDNP on pro-inflammatory cytokines. Unlike IL-6 and TNF-a, there was no significant reduction in IL-1b and IFN-g.

[0028] FIG.13 shows the incapability of anti-inflammatory effect in crude extract after MDNP isolation. (A) Crude extract in yellow rectangles is the remaining solution after MDNP isolation through sucrose gradient separation. Result does not display significant removal effect on pro-inflammatory cytokines (B) IL-6 and (C) TNF-a by treatment of crude extract from maca juice.

[0029] FIG.14 shows a concentration dependent therapeutic test demonstrating that 100 ug / mL exhibited the most significant reduction at 8 hr time point of (A) IL-6 and (B) TNF-a.

[0030] FIG.15 shows that restructured MDNP (reMDNP) retains its ability to sequester cytokines whereas disrupted MDNP by PEG and cholesterol exhibited partial or complete loss of function to remove pro-inflammatory cytokines, (A) IL-6 and (B) TNF-a.

[0031] FIG.16 shows the blood biochemistry analysis of LPS challenged and MDNP administered mice plasma (A) Albumin (B) Alanine Transaminase (C) Creatine (D) Aspartate transferase (E) Globulin (F) Total protein (G) Blood Urea Nitrogen. All data is expressed as mean ± SD (n=3 / group).

[0032] FIG.17 shows the top 25 proteins that are upregulated and downregulated in each healthy and LPS plasma groups incubated with MDNP. N=3 / group.

[0033] FIG.18 shows that LPS-induced mouse plasma coated MDNP was tested to show there was no induction of pro-inflammatory cytokines production in BMDM. All data are expressed as means ± SD (n=3). ****p<0.0005 versus LPS only group.

[0034] FIG.19 shows a strategy for synergetic mitigation of inflammation by dexamethasone encapsulated in TCNP.

[0035] FIG.20 shows the preparation and characterization of TCNP. (A) A novel triglyceride (TG):ceramide (Cer) (TCNP) LNP was synthesized based on the reported lipid composition of maca-derived lipid nanoparticle (MDNP), as visualized by TEM. (B-C) TCNP were formulated at various TG-Cer ratios and characterized for particle size and zeta potential following extrusion at 40C and 75C. The 75:25 ratio exhibited the most stable physicochemical properties, closely resembling those of original MDNP. (D) Stability of optimized TCNP formulation was confirmed at both RT and 4C up to 10 days, demonstrating high structural integrity and consistency.

[0036] FIG.21 shows that in vitro sequestration test demonstrates that 75:25 TCNP possesses ability to reduce IL-6 and TNF-a similar to MDNP.

[0037] FIG.22 shows encapsulation of dexamethasone (Dex) within TCNP. (A) A graphical summary illustrating the formulation of Dex-TCNP drug delivery system. (B) Dex was encapsulated in TCNP at 0.01, 0.1, 0.5 and 1 µg / mL. Encapsulation slightly increased particle sizes and reduced zeta potential compared to empty TCNP. All formulations displayed low PDI, indicating uniform particle distribution. (C) Encapsulation efficiency of Dex in TCNP was approximately 77.33%. (D) The release profile showed rapid release within first 7 hrs, reaching a plateau after 12 hrs.

[0038] FIG.23 shows cytotoxicity of Dex-TCNP. Dose dependent cytotoxicity of Dex- loaded TCNP was evaluated in BMDM. Treatment of Dex-TCNP showed no significant cytotoxicity. Free Dex also exhibited minimal toxicity, indicating good cellular tolerance.

[0039] FIG.24 shows a schematic depicting the coating of PLA nanoparticles with MDNP- derived lipids.

[0040] FIG.25 shows the characterization of MDNP-PLA and cytokine sequestration ability assessment.DETAILED DESCRIPTION OF THE INVENTION I. Definitions

[0041] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technical references.

[0042] As used herein, “a” or “an” may mean one or more. As used herein when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms include pluralities, and plural terms include the singular.

[0043] As used herein, “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., + / - 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure. II. The Present Invention

[0044] Disclosed herein is the isolation, characterization, and therapeutic development of PDNPs isolated from maca root, termed maca-derived lipid nanoparticles (MDNP), for the treatment of inflammation as exemplified using in vitro and in vivo models of lipopolysaccharide (LPS)-induced endotoxemia and polymicrobial sepsis.

[0045] MDNP were isolated and characterized for physicochemical properties, as well as lipid composition using lipidomic analysis. The toxicity profile of MDNP was then established prior to determining their uptake profile and anti-inflammatory properties. In vitro, MDNP efficiently sequestered multiple pro-inflammatory cytokines, which led to comprehensive in vivoassessment of their biodistribution and therapeutic activity. Therapeutic administration of MDNP to LPS-challenged mice led to significant reductions in plasma pro-inflammatory cytokines, reductions in inflammation-induced organ damage, and improved survival. When nanoparticles are introduced into a biological fluid, they are rapidly covered by a layer of biomolecules known as the biomolecular corona (or protein corona)29,30.To identify the mechanism by which MDNP elicited its anti-inflammatory effects, untargeted proteomics analysis of the MDNP protein corona was performed to uncover the inflammatory mediators sequestered by MDNP and corresponding pathways and upstream regulators modulated. MDNP were found to sequester and neutralize a variety of APPs, which promote the propagation of pro-inflammatory immune responses, in addition to pro-inflammatory cytokines. Additionally, the efficacy of MDNP was assessed using a clinically relevant mouse model of polymicrobial sepsis and found to significantly increase survival. These results demonstrate the potential of MDNP as an abundant, cost effective, naturally derived therapeutic agent for use as a multimodal intervention for a variety of inflammatory diseases due to their ability to sequester a broad spectrum of inflammatory mediators. Isolated MDNP

[0046] In one aspect, the present invention is directed to isolated maca-derived nanoparticles (MDNP), wherein the nanoparticles are isolated from Lepidium meyenii Walp, also known as maca. In one embodiment, MDNP is isolated by a method comprising performing sucrose gradient ultracentrifugation on a maca extract. “Isolated MDNP” is used herein to mean that the MDNP have at least been removed from other components of the maca. The isolated MDNP has anti-inflammatory activity.

[0047] In one embodiment, maca powder is dissolved to make maca juice, which is then centrifuged to remove fibrous content. The supernatant is collected and ultracentrifuged, and the pellets are suspended and dispersed, followed by transfer to a sucrose gradient. The MDNP appear as a cloudy band between certain percentages of the sucrose gradient. The MDNP are collected and analyzed.

[0048] In certain embodiments, isolated MDNP are dissolved, and the lipid components are reassembled to form restructured MDNP (reMDNP). In some embodiments, isolated MDNP aredissolved in organic solvents such as chloroform:methanol and subsequently reassembled using a thin-film rehydration method. In one embodiment, the MDNP is a reMDNP.

[0049] In another aspect, the present invention is directed to lipid nanoparticle compositions comprising MDNP. In one embodiment, lipid nanoparticles of the invention comprise MDNP. For example, the lipid nanoparticle composition comprises MDNP and a carrier, diluent, or an excipient. In a similar embodiment, the invention is directed to lipid nanoparticle compositions comprising reMDNP and a carrier, diluent, or excipient. In some embodiments, other lipids, therapeutic agents, or polymers are added, resulting in different lipid nanoparticle compositions. In one embodiment, cholesterol is added. In some embodiments, the lipid component of MDNP is used to coat polymer cores. In some embodiments a therapeutic agent is added, resulting in a lipid nanoparticle composition with the therapeutic agent encapsulated within the lipid nanoparticle.

[0050] The MDNP of the invention have an average particle size ranging between 100 and 1000 nm. Suitable particle sizes also include a range of between 100 and 900 nm, 100 and 800 nm, 100 and 700 nm, 200 and 1000 nm, 200 and 900 nm, 200 and 800 nm, 200 and 700 nm, 300 and 1000 nm, 300 and 900 nm, 300 and 800 nm, 300 and 700 nm, 300 and 650 nm, 300 and 600 nm, 300 and 550 nm, 300 and 500 nm, 300 and 450 nm, 300 and 400 nm, 350 and 700 nm, 350 and 650 nm, 350 and 600 nm, 350 and 550 nm, 350 and 500 nm, 350 and 450 nm, 350 and 400 nm, 400 and 700 nm, 400 and 650 nm, 400 and 600 nm, 400 and 550 nm, 400 and 500 nm, 400 and 450 nm, 450 and 700 nm, 450 and 650 nm, 450 and 600 nm, 450 and 550 nm, 450 and 500 nm, 500 and 700 nm, 500 and 650 nm, 500 and 600 nm, 500 and 550 nm, 550 and 700 nm, 550 and 650 nm, 550 and 600 nm, 600 and 700 nm, 600 and 650 nm, and 650 and 700 nm. In certain embodiments of the invention, MDNP of the invention have an average particle size of 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 650, 700, 750, 800, 850, 900, 950 or 1000 nm.

[0051] The MDNP of the invention have a negative zeta potential of between 0 mV and -20. Suitable zeta potentials also include about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, - 8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, - 19 mV, -20 mV. Suitable zeta potentials further include about -0.5 mV, -1.5 mV, -2.5 mV, -3.5 mV, -4.5 mV, -5.5 mV, -6.5 mV, -7.5mV, -8.5 mV, -9.5 mV, -10.5 mV, -11.5 mV, -12.5 mV, - 13.5 mV, -14.5 mV, -15.5 mV, -16.5 mV, -17.5 mV, -18.5 mV, and -19.5 mV.Lipid components of lipid nanoparticle compositions

[0052] In one aspect, the present invention is related to lipid nanoparticle compositions containing: a lipid component; and optionally, a cargo, which may be a therapeutic agent or polymer core. Therapeutic agents may be selected from small molecules, peptides, nucleic acids such as mRNA or siRNA, proteins, or plasmid DNA. The lipid nanoparticle compositions include sphingolipids, galactolipids, triglycerides, ceramides, neutral lipids, and phospholipids as lipid components. “Lipid nanoparticle” refers to, without limiting the meaning, a particle that comprises a plurality of (i.e., more than one) lipid components physically associated with each other by intermolecular forces.

[0053] In one embodiment, the lipid nanoparticle composition comprises a lipid component which includes a triglyceride in an amount of about 80% to about 60%, or about 77.4% to about 62.0% by weight of the lipid component; and a ceramide in an amount of about 30% to about 10% or about 25.0% to about 11.8%, by weight of the lipid component. Suitable amounts of triglyceride include about 77.4% to 62.0%, 75.0% to 64.0%, 72.0% to 66.0%, and 70.0% to 68.0% by weight of the lipid component. Suitable further amounts of triglycerides include about 77.4%, 74.4%, 71.3%, 68.2%, 65.1%, and 62.0% by weight of the lipid component. Suitable amounts of ceramide include about 25.0% to 11.8%, 23.0% to 13.8%, 21.0% to 15.8%, 19.0% to 17.8% by weight of the lipid component. Suitable further amounts of ceramides include about 25.0%, 24.4%, 23.8%, 23.2%, 22.6%, 22.0%, 21.4%, 20.8%, 20.2%, 19.6%, 19.0%, 18.4%, 17.8%, 17.2%, 16.6%, 16.0%, 15.4%, 14.8%, 14.2%, 13.6%, 13.0%, 12.4%, and 11.8% by weight of the lipid component.

[0054] The triglyceride in the lipid component may be a combination of one or more individual triglycerides. In some embodiments the triglyceride is a phyto-triglyceride. In certain embodiments the triglyceride is a medium chain triglyceride. In some embodiments, triglycerides in the lipid component contain from 21 to 70 carbons. Suitable triglycerides (TG) for the lipid component include TG 43:0, TG 47:0, TG 47:1, TG 51:5, TG 49:3, TG 50:3, TG 51:1, TG 51:4, TG 56:0, TG 52:4, TG 52:5, TG 54:6, TG 52:6, TG 58:0, TG 48:3, TG 51:2, TG 56:1, TG 56:2, TG 53:11, TG 53:2, TG 53:5, TG 53:6, TG 54:1, TG 54:5, TG 62:0, TG 54:3, TG 54:5, TG 54:7, TG 55:5, TG 56:4, TG 58:4, TG 54:8, TG 56:5, TG 54:9, TG 44:0, TG 45:0, TG 47:0, TG 47:1, TG 47:2, TG 49:1, TG 50:0, TG 50:1, TG 50:1, TG 52:0, TG 52:2, TG 54:3, TG 54:4, TG 54:7, TG 54:8, TG 54:9, TG 56:3.

[0055] In certain embodiments, the ceramide in the lipid component is a combination of one or more individual ceramides. In some embodiments the ceramide is a phyto-ceramide. In some embodiments, ceramides in the lipid component contain from 40 to 44 carbons. Suitable ceramides for the lipid component include Cer_ADS d42:0, Cer_AP t42:2, Cer_AP t40:1, Cer_AP t41:1, Cer_AP t42:2, Cer_AP t42:1, Cer_AP t42:0, Cer_AP t43:2, Cer_AP t43:1, Cer_AP t44:2, Cer_AP t44:1, Cer_AS d42:1.

[0056] In certain embodiments the lipid component contains additional lipids, including phytohexosylceramide, free fatty acid, digalactosyldiacylglycerol, phosphatidylcholine, phosphatidylinositol, lysophosphatidylcholine, cholesterol, or a combination thereof.

[0057] In some embodiments, phytohexosylceramide is present in an amount of about 3% to about 2% or about 2.70 % to about 2.16%, by weight of the lipid component. Suitable amounts of phytohexosylceramides include about 2.70%, 2.66%, 2.56%, 2.46%, 2.36%, 2.26%, and 2.16%, by weight of the lipid component. The phytohexosylceramide, in certain embodiments, is a combination of one or more individual phytohexosylceramides. Suitable phytohexosylceramides for the lipid component include HexCer_AP t42:2, HexCer_AP t42:1, HexCer_AP t43:2.

[0058] In some embodiments, the free fatty acid, is present in an amount of about 2% to about 1.5 %, or about 1.95% to about 1.56%, by weight of the lipid component. Suitable amounts of free fatty acid include about 1.95%, 1.88%, 1.80%, 1.72%, 1.64%, and 1.56%. The free fatty acid, in certain embodiments, is a combination of one or more individual fatty acids. Suitable free fatty acids for the lipid component include FA 16:0, FA 18:2, FA 18:0.

[0059] In some embodiments, the digalactosyldiacylglycerol is present in an amount of about 0.8 to about 0.5% or about 0.720% to about 0.576%, by weight of the lipid component. Suitable amounts of digalactosyldiacylglycerol include 0.720%, 0.692%, 0.663%, 0.634%, 0.605%, and 0.576 by weight of the lipid component. The digalactosyldiacylglycerol, in certain embodiments, is a combination of one or more individual digalactosyldiacylglycerols. Suitable digalactosyldiacylglycerols include DGDG 34:2, DGDG 36:4, DGDG 36:3.

[0060] In some embodiments, the phosphatidylcholine is present in an amount of about 1% to about 0.75% or about 0.960% to about 0.768%, by weight of the lipid component. Suitable amounts of phosphatidylcholine include about 0.960%, 0.922%, 0.883%, 0.845%, 0.806%, and 0.768% by weight of the lipid component. The phosphatidylcholine, in certain embodiments, isa combination of one or more individual phosphatidylcholines. Suitable phosphatidylcholines include PC 34:3, PC 36:5, PC 36:6.

[0061] In some embodiments, the phosphatidylinositol is present in an amount of about 1% to about 0.5 % or about 0.850% to about 0.680%, by weight of the lipid component. Suitable amounts of phosphatidylinositol include about 0.850%, 0.816%, 0.782%, 0.748%, 0.714%, and 0.680%, by weight of the lipid component. The phosphatidylinositol, in some embodiments, is a combination of one or more individual phosphatidylinositols. Suitable phosphatidylinositols include PI 34:3, PI 34:2.

[0062] In some embodiments, lysophosphatidylcholine is present in an amount of about 0.15% to about 0.1%, or about 0.130% to about 0.104%, by weight of the lipid component. Suitable amounts of lysophosphatidylcholine include about 0.130%, 0.125%, 0.119%, 0.114%, 0.109%, and 0.104%, by weight of the lipid component. The lysophosphatidylcholine, in some embodiments, is a combination of one or more individual lysophosphatidylcholines. In one embodiment, the lysophosphatidylcholine is an LPC comprising 16 carbon atoms.

[0063] In some embodiments, cholesterol is present in an amount of about 20.0% to about 5.0%, by weight of the lipid component. Suitable amounts of cholesterol include 20.0%, 19.0%, 18.0%, 17.0%, 16.0%, 15.0%, 14.0%, 13.0%, 12.0%, 11.0%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, and 5.0%, by weight of the lipid component.

[0064] In one embodiment, in the lipid component of the lipid nanoparticle composition contains triglyceride in an amount of about 75% by weight of the lipid component, and ceramide in an amount of about 25% by weight of the lipid component.

[0065] In a preferred embodiment, the lipid component of the lipid nanoparticle composition contains triglyceride in an amount of about 77.4% by weight of the lipid component; ceramide in an amount of about 14.8% by weight of the lipid component; phytohexosylceramide in an amount of about 2.70% by weight of the lipid component; free fatty acid in an amount of about 1.95% by weight of the lipid component; digalactosyldiacylglycerol in an amount of about 0.720% by weight of the lipid component; phosphatidylcholine in an amount of about 0.960% by weight of the lipid component; phosphatidylinositol in an amount of about 0.850% by weight of the lipid component; and lysophosphatidylcholine in an amount of about 0.130% by weight of the lipid component.Lipid nanoparticle compositions with therapeutic agents

[0066] In another aspect, the present invention is related to lipid nanoparticle compositions containing therapeutic agents. In some embodiments, the lipid nanoparticle composition of any of the above embodiments further contains a therapeutic agent. In other embodiments, the lipid nanoparticle composition includes a lipid composition which includes the lipid component of isolated MDNP, and a therapeutic agent. In one embodiment, the lipid nanoparticle composition comprises triglyceride in an amount of about 75% by weight of the lipid component, and ceramide in an amount of about 25% by weight of the lipid component, and a therapeutic agent. In another embodiment, the lipid nanoparticle composition contains triglyceride in an amount of about 77.4% by weight of the lipid component; ceramide in an amount of about 14.8% by weight of the lipid component; phytohexosylceramide in an amount of about 2.70 % by weight of the lipid component; free fatty acid in an amount of about 1.95% by weight of the lipid component; digalactosyldiacylglycerol in an amount of about 0.720% by weight of the lipid component; phosphatidylcholine in an amount of about 0.960% by weight of the lipid component; phosphatidylinositol in an amount of about 0.850% by weight of the lipid component; and lysophosphatidylcholine in an amount of about 0.130% by weight of the lipid component, and a therapeutic agent.

[0067] The therapeutic agent may be a single therapeutic agent or a combination of different therapeutic agents. As understood in one embodiment, a therapeutic agent, i.e., a drug, includes, but is not limited to, small organic molecules, inorganic molecules, therapeutic peptides and proteins, antibodies, radioisotopes, siRNA and nucleic acids for gene therapy, and toxins that are functional in intracellular compartments, and that can be used to treat, diagnose, inhibit, or prevent the progression of a disease, i.e., an abnormal condition affecting the body.

[0068] Examples of classes of therapeutic agents include anti-inflammatory agents, neurological therapies, pulmonary therapies, gene and mRNA therapies, oncology (cancer therapies), autoimmune and rheumatologic therapies, cardiovascular therapies, metabolic disorder treatments, infectious disease therapies, inflammatory bowel disease therapies, psychiatric and mood disorder treatments, dermatological therapies.

[0069] Examples of suitable therapeutic agents in the lipid nanoparticle compositions of the invention include: levodopa, carbidopa, memantine, donepezil, rivastigmine, interferon beta-1a, glatiramer acetate, edaravone, riluzole, salbutamol, budesonide, fluticasone, formoterol,salmeterol, roflumilast, ivacaftor, lumacaftor, tobramycin, isoniazid, rifampin, azithromycin, amoxicillin, ciprofloxacin, doxycycline, vancomycin, ceftriaxone, remdesivir, oseltamivir, efavirenz, dolutegravir, acyclovir, valganciclovir, Paxlovid (nirmatrelvir / ritonavir), fluconazole, amphotericin B, itraconazole, dexamethasone, hydrocortisone, tocilizumab, baricitinib, anakinra, canakinumab, mRNA-1273, BNT162b2, patisiran, givosiran, inclisiran, pembrolizumab, nivolumab, ipilimumab, trastuzumab, paclitaxel, doxorubicin, cisplatin, 5-fluorouracil, methotrexate, infliximab, adalimumab, etanercept, ustekinumab, vedolizumab, abatacept, hydroxychloroquine, colchicine, atorvastatin, simvastatin, rosuvastatin, ezetimibe, bempedoic acid, metformin, pioglitazone, semaglutide, liraglutide, tirzepatide, phentermine / topiramate, orlistat, naltrexone / bupropion, dapagliflozin, empagliflozin, sitagliptin, linagliptin, insulin, obeticholic acid, vitamin E, resmetirom, aramchol, lanifibranor, fluoxetine, sertraline, escitalopram, bupropion, lithium, risperidone, aripiprazole, clozapine, betamethasone, tretinoin, benzoyl peroxide, imiquimod, vismodegib, mesalamine, sulfasalazine, olsalazine, balsalazide, prednisone, budesonide (IBD), hydrocortisone (IBD), azathioprine, 6-mercaptopurine, cyclosporine, certolizumab pegol, golimumab, tofacitinib, upadacitinib, ozanimod, interleukin-4 (IL-4), interleukin-13 (IL-13), IL-10, TGF-β, PPAR-γ agonists (pioglitazone, rosiglitazone, 15d- PGJ2), histone deacetylase inhibitors (TSA, valproic acid), statins (atorvastatin, simvastatin), glucocorticoids (dexamethasone), resveratrol, curcumin, vitamin D, metformin, omega-3 fatty acids (EPA, DHA), lactoferrin, ginsenosides.

[0070] In certain embodiments the therapeutic agent is dexamethasone.

[0071] The therapeutically-effective amount of the one or more therapeutic agents and the amount sufficient to achieve the stated goals of the methods of treatment disclosed herein in each dosage will vary, for example, in view of the physical characteristics of the subject, the severity of the subject’s symptoms, the form of the infection, the identity of the therapeutic agent, the formulation and the means used to administer the therapeutic agent, and the method being practiced. The specific dose for a given subject is usually set by the judgment of the attending physician.

[0072] The lipid nanoparticle compositions of the invention containing therapeutic agents have an average particle size ranging between 100 and 1000 nm. Suitable particle sizes also include a range of between 100 and 900 nm, 100 and 800 nm, 100 and 700 nm, 200 and 1000 nm, 200 and 900 nm, 200 and 800 nm, 200 and 700 nm, 300 and 1000 nm, 300 and 900 nm, 300and 800 nm, 300 and 700 nm, 300 and 650 nm, 300 and 600 nm, 300 and 550 nm, 300 and 500 nm, 300 and 450 nm, 300 and 400 nm, 350 and 700 nm, 350 and 650 nm, 350 and 600 nm, 350 and 550 nm, 350 and 500 nm, 350 and 450 nm, 350 and 400 nm, 400 and 700 nm, 400 and 650 nm, 400 and 600 nm, 400 and 550 nm, 400 and 500 nm, 400 and 450 nm, 450 and 700 nm, 450 and 650 nm, 450 and 600 nm, 450 and 550 nm, 450 and 500 nm, 500 and 700 nm, 500 and 650 nm, 500 and 600 nm, 500 and 550 nm, 550 and 700 nm, 550 and 650 nm, 550 and 600 nm, 600 and 700 nm, 600 and 650 nm, and 650 and 700 nm. In certain embodiments of the invention, MDNP of the invention have an average particle size of 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 650, 700, 750, 800, 850, 900, 950 or 1000 nm.

[0073] The lipid nanoparticle compositions of the invention containing therapeutic agents have a negative zeta potential of between 0 mV and -20. Suitable zeta potentials also include about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV. Suitable zeta potentials further include about -0.5 mV, -1.5 mV, -2.5 mV, -3.5 mV, -4.5 mV, -5.5 mV, -6.5 mV, -7.5mV, -8.5 mV, -9.5 mV, -10.5 mV, -11.5 mV, -12.5 mV, -13.5 mV, -14.5 mV, -15.5 mV, -16.5 mV, -17.5 mV, -18.5 mV, and -19.5 mV. Lipid nanoparticle compositions with polymer cores

[0074] In yet another aspect, the present invention is related to lipid nanoparticle compositions containing a polymer core. In some embodiments, the lipid nanoparticle composition of any of the above embodiments further contains a polymer core. In other embodiments, the lipid nanoparticle composition includes a lipid composition which includes the lipid component of isolated MDNP, and a polymer core. In one embodiment, the lipid nanoparticle composition comprises triglyceride in an amount of about 75% by weight of the lipid component, and ceramide in an amount of about 25% by weight of the lipid component, and a polymer core. In another embodiment, the lipid nanoparticle composition contains triglyceride in an amount of about 77.4% by weight of the lipid component; ceramide in an amount of about 14.8% by weight of the lipid component; phytohexosylceramide in an amount of about 2.70 % by weight of the lipid component; free fatty acid in an amount of about 1.95% by weight of the lipid component; digalactosyldiacylglycerol in an amount of about 0.720% by weight of the lipid component; phosphatidylcholine in an amount of about 0.960% by weight of the lipidcomponent; phosphatidylinositol in an amount of about 0.850% by weight of the lipid component; and lysophosphatidylcholine in an amount of about 0.130% by weight of the lipid component, and a polymer core.

[0075] The polymer cores may be poly(lactic acid) (PLA) cores, poly(lactic-co-glycolic acid) cores, poly(itaconic acid) cores, poly(mesaconic acid) cores, poly(citraconic acid) cores, polystyrene cores, poly(methyl methacrylate) cores, or poly(alpha-keto glutarate) cores.

[0076] In a preferred embodied, the cores are prepared from poly(lactic acid) (PLA). PLA is thermoplastic polyester with backbone formula [-C(CH3)HC(=O)O–]n. It is biodegradable, biocompatible and renewable, mainly derived from corn starch.

[0077] PLA cores of the invention may be obtained commercially or prepared by ring opening polymerization of lactide or through polycondensation.

[0078] In certain embodiments, the polymer core is a poly(lactic acid) core.

[0079] The lipid nanoparticle compositions of the invention containing polymer cores have an average particle size ranging between 100 and 1000 nm. Suitable particle sizes also include a range of between 100 and 900 nm, 100 and 800 nm, 100 and 700 nm, 200 and 1000 nm, 200 and 900 nm, 200 and 800 nm, 200 and 700 nm, 300 and 1000 nm, 300 and 900 nm, 300 and 800 nm, 300 and 700 nm, 300 and 650 nm, 300 and 600 nm, 300 and 550 nm, 300 and 500 nm, 300 and 450 nm, 300 and 400 nm, 350 and 700 nm, 350 and 650 nm, 350 and 600 nm, 350 and 550 nm, 350 and 500 nm, 350 and 450 nm, 350 and 400 nm, 400 and 700 nm, 400 and 650 nm, 400 and 600 nm, 400 and 550 nm, 400 and 500 nm, 400 and 450 nm, 450 and 700 nm, 450 and 650 nm, 450 and 600 nm, 450 and 550 nm, 450 and 500 nm, 500 and 700 nm, 500 and 650 nm, 500 and 600 nm, 500 and 550 nm, 550 and 700 nm, 550 and 650 nm, 550 and 600 nm, 600 and 700 nm, 600 and 650 nm, and 650 and 700 nm. In certain embodiments of the invention, MDNP of the invention have an average particle size of 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 650, 700, 750, 800, 850, 900, 950 or 1000 nm.

[0080] The lipid nanoparticle compositions of the invention containing polymer cores have a negative zeta potential of between 0 mV and -20. Suitable zeta potentials also include about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV. Suitable zeta potentials further include about -0.5 mV, -1.5 mV, -2.5 mV, -3.5 mV, -4.5 mV, -5.5 mV, -6.5 mV, -7.5mV,-8.5 mV, -9.5 mV, -10.5 mV, -11.5 mV, -12.5 mV, -13.5 mV, -14.5 mV, -15.5 mV, -16.5 mV, - 17.5 mV, -18.5 mV, and -19.5 mV. Methods of using lipid nanoparticle compositions

[0081] The present invention also relates to methods for preventing or treating an inflammatory disease in a subject, said method comprising, administering a therapeutically effective amount of the lipid nanoparticle composition of any of the above embodiments, to a subject having inflammatory disease. In methods of the invention, lipid nanoparticle compositions of the invention as described above, including: lipid nanoparticle compositions comprising MDNP and / or reMDNP as identified above; lipid nanoparticle compositions identified above with a lipid component comprising a triglyceride in an amount of about 77.4 % to about 62.0% by weight of the lipid component and a ceramide in an amount of about 25.0% to about 11.8%, by weight of the lipid component; lipid nanoparticle compositions further comprising therapeutic agents as identified above; and lipid nanoparticle compositions further comprising polymer cores, as identified above are administered.

[0082] As suggested above, the methods of the present invention are based on experimental evidence demonstrating lipid nanoparticle compositions of the invention have an anti- inflammatory effect in subjects.

[0083] Examples of inflammatory disease of the present disclosure include but not limited to Alzheimer’s, multiple sclerosis, ankylosing spondylitis, arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis), asthma, atherosclerosis, Crohn’s disease, ulcerative colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, inflammatory bowel diseases, systemic lupus erythematous (SLE), nephritis, Parkinson’s disease, ulcerative colitis, inflammation due to cytokine release syndrome, inflammation due to traumatic injury or infections such as sepsis or cytokine storms. In certain embodiments, the inflammatory disease is selected from rheumatoid arthritis, inflammatory bowel diseases, COVID-19-related cytokine storms, CAR-T cell cytokine release syndrome, and sepsis.

[0084] As used herein, the term “sepsis” has its ordinary and customary meaning. Generally, sepsis is a medical condition resulting from the presence of a microorganism in the blood and / or tissues of a subject, and an uncontrolled inflammatory response by the immune system of the subject to their presence, characterized by harmful levels of inflammation in the subject,potentially leading to malfunctioning of various organs, shock and death of the subject. Hallmark features of sepsis include acute proinflammatory cytokine responses, and the activation and recruitment of inflammatory immune cells. Symptoms include fever, difficulty breathing, low blood pressure, fast heart rate, and mental confusion.

[0085] In the context of the embodiments of the invention provided herein, sepsis may be caused by bacteria, viruses, or fungi. Sepsis may also be characterized as ‘sterile sepsis’ as in the case of systemic inflammatory response syndrome (SIRS) caused by injuries or conditions where the body’s response is similar to that of an infection, but no actual infection is present. Examples of injuries or conditions that can result in sterile sepsis include severe burns, pancreatitis, trauma, ischemia / reperfusion injury, severe allergic reactions, severe organ damage (like liver failure or NASH), extensive tissue damage (like muscle crush injury), or hemorrhagic shock.

[0086] With respect to bacterial sepsis, the causative agent may be gram-negative or gram- positive bacteria. Bacterial sepsis may be caused, for example and without limitation, by Staphylococcus aureus, Streptococcus pyogenes, Klebsiella spp., Escherichia coli, Pseudomonas aeruginosa, Enterococcus, Streptococcus pneumoniae, and Neisseria meningitidis.

[0087] Cytokine storms are a pathological immune response where immune system overreacts by releasing too many inflammatory signaling molecules. These storms can be triggered by various factors, including infections (like influenza or COVID-19), autoimmune diseases, certain medications, and even certain cancers.

[0088] In certain embodiments, the lipid nanoparticle composition of the invention sequesters an acute phase protein or a pro-inflammatory cytokine. The lipid nanoparticle compositions of the invention, when introduced into a biological fluid like blood plasma bind to proteins, forming a protein corona. Sequestration, in this context, means the proteins are trapped or bound to the nanoparticle surface.

[0089] Acute phase proteins are proteins in the blood that change in response to inflammation. Examples include C-reactive protein (CRP), haptoglobin, serum amyloid A (SAA), fibrinogen, alpha-1 acid glycoprotein (AGP), ceruloplasmin, complement C3 and C4, and pro-inflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). In certain embodiments the pro-inflammatory cytokine is selected from IL-6 and TNF-α.

[0090] As used herein, the terms “treating” and “treatment” mean at least the mitigation of a symptom associated with inflammatory disease, in a subject having inflammatory disease. The terms “treating” and “treatment” include curing, healing, inhibiting, relieving from, improving and / or alleviating, in whole or in part, inflammatory disease or an associated condition or symptom. The mitigation of a symptom associated with sepsis may be about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% in the subject, versus a subject to which lipid nanoparticle compositions have not been administered.

[0091] The therapeutically-effective amount of lipid nanoparticle compositions of the invention and the amount sufficient to achieve the stated goals of the methods of treatment disclosed herein in each dosage will vary, for example, in view of the physical characteristics of the subject, the severity of the subject’s symptoms, the form of the infection, the identity of the microorganism, the formulation and the means used to administer the lipid nanoparticle compositions, and the method being practiced. The specific dose for a given subject is usually set by the judgment of the attending physician. However, in each dose a therapeutically effective amount of lipid nanoparticle compositions is typically between about 0.01 to 1 g / kg body weight. Suitable ranges of therapeutically effective amounts of the lipid nanoparticle compositions also include about 0.01 to 1 g / kg, 0.01 to 0.1 g / kg, 0.1 to 1 g / kg, and 0.01 to 0.1 g / kg body weight. In a specific embodiment, the therapeutically effective amount of the lipid nanoparticle compositions is about 0.150 g / kg body weight.

[0092] The methods of the invention may further comprise administering a therapeutically effective amount of one or more therapeutic agents to the subject. Thus, the subject having an inflammatory disease would receive both lipid nanoparticle compositions and one or more therapeutic agents.

[0093] The lipid nanoparticle compositions and one or more therapeutic agent may be administered separately or together; concurrently or separated in time; and in any order. Modes of Administration

[0094] The lipid nanoparticle compositions of the present invention may be formulated, for example, for oral, enteral, sublingual, intranasal, intraocular, rectal, intravaginal, transdermal, mucosal, topical or parenteral administration. Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (i.m.), intravenous(i.v.), intraperitoneal (i.p.), intra-arterial, intramedullary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). Any known device useful for parenteral injection or infusion of the lipid nanoparticle compositions can be used to effect such administration. In certain aspects of each of the embodiments of the invention, the lipid nanoparticle compositions are administered to the subject via intraperitoneal (i.p.) injection or intravenously (i.v.).

[0095] Formulations for parenteral administration can be in the form of aqueous or non- aqueous isotonic sterile solutions, suspensions or fat emulsions. The unit dosage of these solutions or suspensions can be in a concentrated liquid, powder or granular form for ex tempore reconstitution in the appropriate pharmaceutically acceptable carrier, such as sterile water, at the time of delivery. In addition to the above-mentioned excipients, powder forms optionally include bulking agents (e.g. mannitol, glycine, lactose, sucrose, trehalose, dextran, hydroxyethyl starch, ficoll and gelatin), and cryo or lyoprotectants. In an alternative embodiment, the parenteral unit dosage form of lipid nanoparticle compositions can be a ready-to-use solution of the lipid nanoparticle compositions in a suitable carrier in sterile, hermetically sealed ampoules or in sterile pre-loaded syringes.

[0096] Carriers optionally included in a formulation comprising the lipid nanoparticle compositions include excipients. Suitable excipients include, without limitation, sterile water, water-for-injection, stabilizing agents (e.g. carbohydrates, amino acids and polysorbates, such as 5% dextrose), solubilizing agents (e.g. cetrimide, sodium docusate, glyceryl monooleate, polyvinylpyrolidone (PVP) and polyethylene glycol (PEG)), surfactants (e.g. polysorbates, tocopherol PEG succinate, poloxamer and Cremophor™), buffers (e.g. acetates, citrates, phosphates, tartrates, lactates, succinates, amino acids and the like), antioxidants and preservatives (e.g. BHA, BHT, gentisic acids, vitamin E, ascorbic acid, sodium ascorbate and sulfur containing agents such as sulfites, bisulfites, metabisulfites, thioglycerols, thioglycolates and the like), tonicity agents (for adjusting physiological compatibility), suspending or viscosity agents, antibacterials (e.g. thimersol, benzethonium chloride, benzalkonium chloride, phenol, cresol and chlorobutanol), chelating agents, and administration aids (e.g. local anesthetics, anti- inflammatory agents, anti-clotting agents, vaso-constrictors for prolongation and agents that increase tissue permeability), and combinations thereof.

[0097] Administration frequencies for the lipid nanoparticle compositions of the invention will vary based on the method being practiced, the physical characteristics of the subject, the severity of the subject’s symptoms, the form of the infection, the identity of the microorganism, and the formulation and the means used to administer the lipid nanoparticle compositions. However, administration frequencies will generally include 4, 3, 2 or once daily, every other day, every third day, every fourth day, every fifth day, every sixth day, once weekly, every eight days, every nine days, every ten days, bi-weekly, monthly and bi-monthly. The duration of treatment will be based on the condition being treated and will be best determined by the attending physician. Under some conditions, treatment will be continued for a number of days or weeks. Under other conditions, complete treatment will be achieved through administering one, two or three dose of the lipid nanoparticle compositions over the entire course of treatment.

[0098] As used herein, a “subject” means an animal, such as a mammal, including humans, other higher primates, lower primates, and animals of veterinary importance, such as dogs, cats, horses, sheep, goats, and cattle and the like. III. Examples Materials and methods Isolation, purification, and characterization of maca-derived lipid nanoparticles (MDNP)

[0099] A fine ground organic powder of 100% Lepidium meyenii Walp (maca) (Family: Brassicaceae, Order: Brassicas) was purchased from an organic producer, Happy Andes (https: / / www.happyandes-usa.com / ). Maca powder (30 g) was mixed in 400 mL of autoclaved deionized water and stirred for 12 hrs at room temperature to make maca juice. The obtained maca juice was transferred to 50 mL tubes and centrifuged at 3,000 x g for 20 min at 20°C then the supernatant was centrifuged again in new 50 mL tubes at 10,000 x g for 2 hrs at 20°C to remove small and large fibers. Next, the clear light brown colored supernatant was collected in 70 mL polycarbonate ultracentrifuge bottles (Beckman Coulter, Brea, CA) and was ultracentrifuged with using a 45 Ti rotor (Beckman Coulter, Brea, CA) at 150,000 x g for 2 hrs at 4^C. Then, the collected pellets were suspended in 2 mL of phosphate-buffered saline (PBS) through dispersion with an ultrasonic processor. PBS-suspended maca pellets were transferred to a sucrose gradient (8%, 20%, 35% [g / v]) and ultracentrifuged at 150,000 x g for 2 hrs at 4^C, then the cloudy band between 8% and 20% sucrose was collected as previously described31. Theconcentration of the MDNP was measured using a Bio-Rad quantification assay. The quantified MDNP were stored at 4^C. The size and zeta potential of the MDNP was measured by dynamic light scattering (DLS) using a Zetasizer Nano ZSP (Malvern, UK) and Nanoparticle Tracking Analysis NS300 (NTA) (Malvern, UK). MDNP stability was evaluated under various storage temperatures (room temperature (RT), 4°C, -20°C, and -80°C) in PBS. In addition, stability of the nanoparticles was tested in cell culture medium and mouse plasma at both RT and 37^C to assess behavior under biologically relevant conditions. Transmission electron microscopy (TEM) images of MDNP were acquired using a formvar-coated copper grid and staining using 1% uranyl acetate. Differential Scanning Calorimeter (DSC) 2500 (TA Instruments, New Castle, DE) was used to analyze the temperature and heat flow associated with thermal transition in the MDNP. 5 µL of sample in liquid form was added into a hermetic aluminum pan and sealed with metal lid (DSC Consumables, Austin, MN) with Tzero sample press kit (TA Instruments, New Castle, DE). The following thermal procedure was used to scan: ramp 2°C / min from 25°C to 80°C. The heat flow transition was analyzed on a plot by TA analysis software Trios (Version 5.4.0.300). For lyophilization, frozen MDNP in 1.5 mL tubes with holes on top of screw caps were placed in a benchtop freeze-dryer (Labconco, Kansas City, MO) overnight. Completely dried samples were collected and reconstituted in PBS for stability testing. Lipid extraction and composition analysis

[0100] A total lipid extract was prepared using a modified methyl-tert-butyl ether (MTBE) lipid extraction protocol60. Briefly, 400 µL of cold methanol and 10 µL of internal standard mixture (EquiSPLASH lipidomix) were added to each sample followed by incubation at 4°C, 650 RPM shaking for 15 min. Next, 500 µL of cold MTBE was added followed by incubation at 4°C for 1 hr with 650 RPM shaking.500 µL of cold water was added slowly and resulting extract was maintained 4°C, 650 rpm shaking for 15 min. Phase separation was completed by centrifugation at 8,000 RPM for 8 min at 4°C. The upper, organic phase was removed and set aside on ice. The bottom, aqueous phase was re-extracted with 200 µL of MTBE followed by 15 min of incubation at 4°C with 650 RPM shaking. Phase separation was completed by centrifugation at 8,000 RPM for 8 min at 4°C. The organic extract was dried under a steady stream of nitrogen at 30°C. The recovered lipids were reconstituted in 200 µL ofchloroform:methanol (1:1, v / v) containing 200 µM of butylated hydroxytoluene. Prior to analysis, samples were further diluted with acetonitrile:isopropanol:water (1:2:1, v / v / v). The total lipid extract was analyzed by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-MS / MS). The LC-MS / MS analyses were performed on an Agilent 1290 Infinity LC coupled to an Agilent 6560 Quadrupole Time-of-Flight (Q-TOF) mass spectrometer. The separation was achieved using a C18 CSH (1.7 µm; 2.1 x 100 mm) column (Waters, Milford, MA). Mobile phase A was 10 mM ammonium formate with 0.1% formic acid in water / acetonitrile (40:60, v / v) and mobile phase B was 10 mM ammonium formate with 0.1% formic acid in acetonitrile / isopropanol (10:90, v / v). The gradient was ramped from 40% to 43% B in 1 min, ramped to 50% in 0.1 min, ramped to 54% B in 4.9 min, ramped to 70% in 0.1 min, and ramped to 99% B in 2.9 min. The gradient was returned to initial conditions in 0.5 min and held for 1.6 min for column equilibration. The flow rate was 0.4 mL / min. The column was maintained at 55°C and the auto-sampler was kept at 5°C. A 2 µL injection was used for all samples. LC-MS data from the iterative MS / MS workflow was analyzed for lipid identification via Agilent’s Lipid Annotator (version 1.0). Positive and negative ion mode adducts included [M+H]+, [M+Na]+, [M+NH4]+, [M-H]-, and [M+CH3CO2]-, respectively. The LC-MS data from the MS1workflow were processed using Agilent’s MassHunter Profinder (version 10.0). Characterization of MDNP protein composition and protein corona fingerprints in healthy and LPS plasma

[0101] To form the MDNP protein corona, purified MDNP were incubated with plasma isolated from healthy and LPS-treated C57BL / 6J mice for 4 hrs at 37^C. As described in a previous publication, MDNP bound to proteins were washed and centrifuged 3 times at 13,000 xg, 4°C with 1x cold PBS62. Prepared samples were lysed in a lysis buffer containing 5% sodium dodecyl sulfate (Sigma, St. Louis, MO), 50 mM triethylammonium bicarbonate (1M, pH 8.0) (Sigma, 7408). Proteins were extracted and digested using S-trap micro columns (ProtiFi, Farmingdale, NY). The eluted peptides from the S-trap column were dried, and peptide concentration was determined using a BCA assay kit (Thermo Fisher Scientific, 23275), after reconstitution in 0.1% formic acid. All tryptic peptides were separated on a nanoACQUITY Ultra-Performance Liquid Chromatography analytical column (BEH130 C18, 1.7 µm, 75 µm x 200 mm; Waters Corporation, Milford, MA, USA) over a 185-min linear acetonitrile gradientwith 0.1% formic acid on a nanoACQUITY Ultra-Performance Liquid Chromatography system (Waters Corp, Milford, MA) and analyzed on a coupled Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific, San Jose, CA). Full scans were acquired at 240,000 resolutions, and precursors were fragmented by high-energy collisional dissociation at 35% for up to 3 sec. MS / MS raw files were processed using Thermo Proteome Discoverer (PD, version 3.0.0.757) with the Sequest HT search engine against the plant metagenome database and UniProt mouse reference proteome. Trypsin was used with a maximum of two missed cleavages, and peptide lengths were restricted to 6-144 amino acids. Label-free quantification was performed using the Minora feature detector. Protein identification was filtered at a 1% false discovery rate across PSM, peptide, and protein levels using the Percolator algorithm in PD. Exported protein abundances were analyzed with Perseus software (version 1.6.14.0), with further filtering to include only proteins identified without missing values across all samples. The quantitative protein data were log2 transformed and further normalized using median centering. Two-tailed student’s t-test (adjusted p-value < 0.05) was applied to determine the differentially expressed proteins (DEPs). Plant data was analyzed with library from Uniprot and the Plant Proteome Database (Lepidium meyenii: taxonomy ID 153348, entry: 92 sequences; Plant metagenome: taxonomy ID 1297885, entry: 27312 sequences; Zingiberales: taxonomy ID 4618, entry: 357900) Ingenuity Pathway Analysis (Qiagen) was utilized to identify canonical pathways, biological function, upstream regulators and disease association. Cell culture

[0102] The generation of bone marrow-derived macrophages (BMDM) followed a previously published method61. As established, the tibia and femur from a C57BL / 6J mouse was isolated by removing bulk muscles and connective tissues. RPMI media (supplemented with L- glutamine, penicillin (100 units / mL), streptomycin (100 µg / mL), 10% heat-inactivated FBS, and 20% L929 cell-conditioned media) drawn needles were inserted into bones to flush the marrow into a 10 cm petri dish. The collected bone marrow was filtered to grow in uncoated 10 cm cell culture plates. BMDM were cultured in RPMI media conditioned with L929 at 37^C, 5% CO2. The media was replaced every 3 days. On Day 8-10, BMDM were lifted using Versene (Gibco, Grand Island, NY), to be used for subsequent experiments. Trypan blue solution was used todetermine the cell number and viability with an EVETMAutomated Cell Counter (NanoEntek, Waltham, MA). RAW-blue cells were also cultured to confluency in 75 cm2flasks in identical incubation condition with Dulbecco’s Eagle Medium (DMEM) supplemented with penicillin (100 U / mL), streptomycin (100 U / mL), and heat inactivated fetal bovine serum (10%). Mice

[0103] Male C57BL / 6 (6 to 8-week-old) purchased from the Jackson Laboratory (Bar Harbor, ME) were maintained in cages at ambient temperature, 55% relative humidity, and under a 12 hr dark / light cycle. LPS-induced endotoxemia model: Mice were challenged with 5 mg / kg LPS intraperitoneally (IP) prior to treatment with two doses of 2 mg MDNP at 30 min and 2 hr via both IP and intravenous (IV) injection. Cecal ligation and punction (CLP) model: Mice were lightly anesthetized with a mixture of ketamine (75 mg / kg) and xylazine (15 mg / kg) at 1:1 ratio. After abdominal fur was removed, a small incision was made to expose the cecum. The cecum was then ligated with a silk suture and perforated with 19-gauge needle. A small amount of feces was extruded by gently squeezing the cecum, the cecum was replaced, and the abdomen was sutured after the bowel was repositioned. Experimental groups were IP injected with three doses of 2 mg MDNP at 30 min, 2 hr, and 24 hr timepoint. The selection of the appropriate dose was based on well-established tolerability for multiples nanoparticle administration discussed in literatures63,64. All experiments were performed in compliance with the protocol by the University of Maryland, Baltimore Institutional Animal Care and Use Committee (IACUC) as well as the ARRIVE guidelines. Cytotoxicity assay

[0104] To measure the cytotoxicity of MDNP, a PI / Annexin V-FITC apoptosis assay was conducted using BMDM. The assay utilizes propidium iodide (PI) staining to distinguish between early and late-stage apoptotic cells. FITC labeling allows for the visualization of Annexin V-bound cells using flow cytometry. Cultured BMDM at a density of 1.0 x 105per well were treated with MDNP at 1 mg / mL for 8 hrs, then the cells were harvested with Versene after washing with PBS. Collected cells were centrifuged at 500 x g for 5 min and cell pellets were resuspended in 1x binding buffer.100 μL of cell suspension in flow cytometry tubes were addedwith Annexin V-FITC and PI to be analyzed by flow cytometry. The data was analyzed by De Novo software FCS Express 7 (Dotmatics, Boston, MA). In vitro cellular internalization

[0105] BMDM were seeded overnight in Falcon Culture slides at a density of 0.5 x 105per well. Cy5.5 was incubated with MDNP under shaking for 30 min in the dark to label the MDNP and excessive Cy5.5 dye was removed by 20 min centrifugation at 13,000 x g prior to use. Then, 50 μL of Cy5.5-labled MDNP were added to 0.5 mL of culture medium and incubated with cells for 0, 4, 8 hrs. Negative control, untreated cells were used to establish the 0 hr timepoint. At the determined timepoints, cells were washed with PBS three times, fixed with 4% paraformaldehyde (PFA) for 10 min, and dehydrated with acetone for 5 min at -20^C. After blocking the culture with 1% BSA in 1x PBS 30 min, the cells were washed again with 1x PBS and treated with 100 µL of fluorescein isothiocyanate (FITC)-labeled phalloidin (1:50 dilution in PBS) for 30 min to stain F-actin. The cells were then washed two times with 1x PBS and dried in the dark condition and glass cover slips were mounted with mounting medium containing 4’,6- diamidino-2-phenylindole (DAPI) after carefully removing the plastic chambers. The final fluorescence images were captured using an Olympus fluorescence microscope (Tokyo, Japan) equipped with Hamamatsu Digital Camera ORCA-03G and Nikon software was used to analyze the image data. Flow cytometric phenotyping of MDNP-treated macrophages

[0106] BMDM were seeded at a density of 1.0 x 105per well were in a sterile 24 well plate. Cells were treated with 100 ng / mL of LPS in complete media for 24 hrs to induce inflammation. Then, 100 μg / mL and 200 μg / mL MDNP were treated for 8 hrs. Cells were resuspended in MACS buffer (PBS pH 7.2 supplemented with 1% FBS and 0.4% 0.5 M EDTA, Quality Biological, Gaithersburg, MD) and transferred to the flow cytometry tubes. FcR blocking (CD16 / 32, BioLegend, San Diego, CA) was performed and cells were stained with following surface marker antibodies: Live / Dead fixable green, F4 / 80, CD11b, MHCII, CD206, CD80, and CD86 (BioLegend, San Diego, CA). Samples were analyzed using Cytek Aurora 3 (Fremont,CA). FCS Expression 7 Flow Cytometry De Novo Software was used for flow data processing. Mean fluorescence intensity (MFI) was generated by GraphPad Prism Software 10.1.1. In vitro pro-inflammatory cytokine sequestration and effects on NF-kB activity

[0107] Anti-inflammatory properties of MDNP were investigated in two ways; prophylactic and therapeutic. For prophylactic treatment, BMDM at a density of 1.0 x 105per well were treated with 100 μg / mL MDNP for 8 hrs and 100 ng / mL of LPS in complete media in 24 well plates (Corning, Corning, NY) were incubated for 4 hrs. For therapeutic study, BMDM in identical culture condition as above were treated with 100 ng / mL of LPS for 4 hrs and 100 µg / mL of MDNP were subsequently added and incubated for 8 hrs. Supernatants were then collected to measure the reduction of pro-inflammatory cytokines including IL-6, TNF-a, IL-1b, and IFN-g by enzyme-linked immunosorbent assay (ELISA) (BioLegend, San Diego, CA). The ability of MDNP to modulate cytokine levels in the absence of cells was also assessed. The supernatants from another set of LPS-treated BMDM were collected and incubated with 100 µg / mL of MDNP in a time dependent manner (0, 0.5, 1, 4, 8, 12, and 24 hrs). To evaluate whether disrupting MDNP structure affects its ability to sequester pro-inflammatory cytokines, MDNP were reformulated using the thin-film hydration method with additional components such as DSPE-PEG600 and cholesterol. MDNP were first dissolved in a chloroform:methanol mixture (2:1 v / v) and subjected to rotary evaporation (Model I-300, Buchi, Switzerland) at 37°C, 100^mBar, and 280^rpm for 1 hour. DSPE-PEG600 or cholesterol was then incorporated at varying concentrations (5%, 10%, and 20%) to generate distinct restructured lipid nanoparticles. Following rehydration, the nanoparticles were extruded through a 0.4^µm polycarbonate membrane using an Avanti Mini Extruder (Birmingham, AL). Lastly, the direct ability of MDNP to sequester IL-6 was assessed by incubating 5 ng / mL of IL-6 recombinant protein in DMEM with 100 µg / mL of MDNP for 8 hrs. Remaining IL-6 levels were measured using ELISA.

[0108] An NF-^^B reporter cell line, RAW-Blue, was used to determine the ability of MDNP to alter NF-^^B activity due to LPS stimulation. The cells were plated at a density of 1.0 x 105per well and were stimulated with LPS for 4 hrs and treated with 100 µg / mL MDNP for 8 hrs. Then, QUANTI-Blue was added to the collected supernatant in flat bottom 96 well plate. It was incubated at 37ºC for 1 hr, then secreted embryonic alkaline phosphatase (SEAP) reporter wasdetected using a spectrophotometer at 620-655 nm. Poly(lactic-co-glycolic acid) (PLGA) particles of similar size as MDNP were used as a control and fabricated using a microfluidics device as previously described65. The size and zeta potential of the PLGA was measured 163.8 nm and -39.3 mV, respectively. Ex vivo cytokine sequestration assay

[0109] To evaluate the cytokine sequestration ability of MDNP in mouse plasma ex vivo, 100 µg / mL of MDNP in PBS was prepared and added to 100 µL of diluted LPS-treated or CLP mouse plasma to ultracentrifuge tubes. The mixture tubes were incubated at 37ºC for 8 hrs. After incubation, samples were centrifuged at 2,000 x g for 10 min to pellet the cytokine bound MDNP. Then the supernatant was collected to measure IL-6 and TNF-a levels using ELISA (BioLegend, San Diego, CA). In vivo biodistribution and anti-inflammatory effect following LPS challenge

[0110] The in vivo biodistribution study was performed by intraperitoneal (IP) or intravenous (IV) injection of MDNP that were labeled with the near-infrared fluorescent Cy5.5 dye. Mice were administered 2 mg of Cy5.5-MDNP twice (0.5 and 2 hr time point)-post LPS challenge (5 mg / kg) and euthanized after 4 hrs to collect blood by cardiac puncture and organs including liver, lung, spleen, heart, and kidneys. The fluorescence of the organs was imaged using an in vivo imaging system (IVIS) with emission (720 nm) upon laser excitation (675 nm). Blood samples were centrifuged at 1,000 x g for 20 min at 4^C in microtainer capillary blood collection plasma tubes. The plasma samples were used to measure the alteration of systematic cytokines levels using a Luminex MAGPIX System (Luminex, Austin, TX). The 7-plex panel included murine IL-6, TNF-a, MCP-1, IL-10, IL-1b, IFN-b, and IFN-γ. Blood biochemistry was also measured for alanine aminotransferase (ATL / SGPT), aspartate aminotransferase (AST / SGOT), creatine (CREA), blood urea nitrogen (BUN), total protein (TP), and globulin, and albumin (VRL Animal Health Diagnostics, Gaithersburg, MD). The collected organs were fixed in 4% formalin immediately after euthanasia and paraffin-embedded for sectioning. Hematoxylin & eosin (H&E) staining was performed using standard procedures by the Pathology Biorepository Shared Services Core at the University of Maryland, Baltimore. The level of injury scores wascalculated based on the scoring criteria: score 0, no damage; score 1, >10% slight inflammation; score 2, 10-25% mild; score 3, 26-50% moderate; score 4, 51-75% severe; score 5, >75% necrosis. In vivo sepsis survival study

[0111] The in vivo survival study was carried out using two representative sepsis models: a lethal LPS-induced endotoxemia and CLP polymicrobial mouse model. For endotoxemia, C57BL / 6J mice were challenged with 20 mg / kg LPS and two doses MDNP (2 mg) administered via IP injection. For the CLP model, the cecum of anesthetized C57BL / 6J mice was perforated to release fecal materials into peritoneal cavity to generate an exacerbated polymicrobial infection. Three doses of MDNP (2 mg / injection) were administered via IP route. Survival for each model was evaluated over the course of 5 days. Body temperature was also recorded every day for CLP mice. Preparation and Characterization of TG-Ceramide Lipid Nanoparticles (TCNP)

[0112] Two distinct lipids—triglyceride (#M4200, Spectrum Chemical, New Brunswick, NJ) and ceramide (#62510, Cayman Chemical, Ann Arbor, MI)—were dissolved in 100% ethanol at 40°C at varying ratios (75:25, 50:50, and 25:75) and transferred to a round-bottom flask. The solvent was evaporated under reduced pressure using a rotary evaporator (Buchi, Switzerland) at 40°C and 100 mbar for 1 hour to form a thin lipid film. The lipid film was hydrated in phosphate-buffered saline (PBS, pH 7.4) and dispersed using a bath sonicator for 10 minutes to minimize aggregation and reduce particle size. To further homogenize the formulation, the resulting TG-ceramide nanoparticles (TCNP) were extruded through a 0.8 µm Nuclepore track- etch membrane (#10417301, Cytiva, Marlborough, MA) and a 10 mm diameter PE drain disc (#230300, Cytiva, Marlborough, MA) using a mini-extruder (#610000, Avanti Polar Lipids, Alabaster, AL) at 45°C and 75°C, with 10 passes per membrane. The final nanoparticle formulation was characterized for hydrodynamic diameter, polydispersity index (PDI), and zeta potential using dynamic light scattering (Zetasizer Nano ZSP, Malvern Instruments, UK). Data acquisition was performed using Zetasizer software (version 7.12). Stability of TCNP was further assessed under various storage conditions, including room temperature (RT) and 4°C.Transmission electron microscopy (TEM) (Tecnai T12, FEI, Hillsboro, OR) images of TCNP were obtained using a formvar-coated copper grid and stained with 1% uranyl acetate. Engineering and evaluation of dexamethasone encapsulated TCNP (Dex-TCNP)

[0113] Dexamethasone (Dex) was encapsulated within TCNP by adding the drug into the lipid mixture during the thin-film formation process. Briefly, varying concentration of Dex (0.01, 0.1, 0.5, 1 µg / mL) was added to prepared TG-Cer thin film using rotatory evaporation and hydrated in PBS, followed by sonication and membrane extrusion as described above. The resulting formulations were characterized for particle size, PDI, and zeta potential using DLS. The encapsulation efficiency (EE) was quantified by centrifugation. Dex-loaded TCNP was centrifuged at 12,000 x g for 15 min to pellet the nanoparticles. The supernatant containing unencapsulated free Dex was collected and quantified using Ultraviolet-visible spectroscopy (UV-Vis) detection method at 242 nm. Dex release profiles were further evaluated up to 24 hr period. In vitro sequestration of TCNP

[0114] The in vitro anti-inflammatory efficacy of TCNP was assessed in a therapeutic setting. Bone marrow–derived macrophages (BMDM) were seeded at a density of 1.0 × 10^5 cells per well in 24-well plates (Corning, Corning, NY) and maintained in complete growth medium. Cells were first stimulated with 100 ng / mL lipopolysaccharide (LPS) for 4 h to induce inflammation, then treated with 100 μg / mL TCNP for an additional 8 h. Following treatment, culture supernatants were harvested and the concentrations of the pro-inflammatory cytokines IL-6 and TNF-α were quantified by ELISA (BioLegend, San Diego, CA). Cytotoxicity assay with TCNP

[0115] Cellular cytotoxicity was assessed by quantifying lactate dehydrogenase (LDH) release using a commercially available LDH Cytotoxicity Detection kit (#426401, BioLegend, San Diego, CA), according to the manufacturer’s instructions. BMDM were seeded in 96 well plates in serum-free Dulbecco’s Modified Eagle Medium (DMEM) and treated with TCNP, Dex- TCNP formulations or controls for 4 hrs. Triton-X-100 (MFCD00128254, Sigma Aldrich, Burlington, MA) was used as a negative control. Following treatment, cell culture supernatants were collected and transferred to a new plate. An equal volume of LDH reaction mixture wasadded to each well and incubated at RT, protected from light for 30 min. Absorbance was measured at 490 nm using a microplate reader with background subtraction at 680 nm. Percent cytotoxicity was calculated by comparing LDH released from treated cells to the total LDH released from fully lysed cells, using this formula. ^^^^^^^^^^^^ − ^^^^^^ ^^^^^^^^^^^^^^% ^^^^^^^^^^^^^^^^^^^^^^^^ = [^^^^^^ℎ ^^^^^^^^^^^^^^ − ^^^^^^ ^^^^^^^^^^^^^^] × 100Synthesis and

[0116] PLA nanoparticles were fabricated using a nanoprecipitation approach facilitated by a microfluidic system (Dolomite, Royson, UK). Prior to use, all solutions including the surfactant (0.1% PVA), polymer (1% PLA dissolved in acetone), and acetone were passed through 0.2 µm syringe filters to eliminate undissolved particulates that could potentially obstruct the microfluidic tubing or chip. The organic polymer solution was further degassed using a bath sonicator. For nanoprecipitation, the flow rates of both the PLA-PVA solutions were set to 1000 µL / min, establishing a flow rate ratio (FRR) of 1. FRR was defined as the ratio of the aqueous (surfactant) phase to the organic (polymer) phase. Laminar flow at the chip junction was continuously visualized using a Meros digital microscope to ensure consistent particle formation. The resulting PLA nanoparticles were collected and left at room temperature overnight to allow complete evaporation of acetone. On the following day, particles were filtered through 40 µm cell strainers and subsequently characterized for their physicochemical properties including size, zeta potential, and polydispersity index (PDI) using dynamic light scattering (Zetasizer Nano, Malvern Instruments, UK). Preparation of hybrid MDNP-coated PLA nanoparticles (MDNP-PLA)

[0117] MDNP were prepared as previously described. To generate the lipid coating, MDNP were dissolved in 100% ethanol and transferred into round-bottom flask. The solvent was evaporated under reduced pressure using a rotary evaporator to form a uniform thin lipid film. After complete drying, pre-formed PLA nanoparticles were added to the flask and hydrated with PBS with gentle agitation to facilitate lipid self-assembling onto the PLA surface. Then, the mixture was sonicated briefly to promote uniform coating. The resulting MDNP-coated PLA nanoparticles were collected and characterized for size, polydispersity index (PDI), and zeta potential using DLS.In vitro sequestration of MDNP-PLA nanoparticles

[0118] The anti-inflammatory effects of MDNP-PLA nanoparticles were evaluated using therapeutic approach. Bone marrow-derived macrophages (BMDM) were plated to a density of 1.0 x 105cells per wells in 24 well plates (Corning, Corning, NY) and maintained in complete growth medium. Cells were initially stimulated with 100 ng / mL of lipopolysaccharide (LPS) for 4 hrs to induce inflammation, after which 100 ug / mL of MDNP-PLA were administered and incubated for additional 8 hrs. Following treatment, culture supernatants were harvested to assess levels of pro-inflammatory cytokines (IL-6 and TNF-a) using enzyme-linked immunosorbent assay (ELISA) kits (BioLegend, San Diego, CA) Statistical analysis

[0119] Data evaluation was performed using GraphPad Prism Software 10.1.1 (San Diego, CA). One-way ANOVA and t-test for unpaired data were employed to evaluate statistical significance along with Tukey’s multiple comparison test. Kaplan-Meier survival curve and statistical significance of mouse survival were determined with a long-rank (Mantel-Cox) X2test. Significant differences were indicated as *p<0.05, **p<0.005, ***p<0.0005 related to control unless otherwise stated. Example 1: Isolation and characterization of maca-derived lipid nanoparticles (MDNP)

[0120] MDNP were isolated from maca juice using differential ultracentrifugation and density gradient sucrose gradient centrifugation (FIG.1A)31. MDNP accumulated at the interface of 8% / 20% / 35% sucrose gradient, and the recovery was approximately 4 mg per 30 g of maca powder (FIG.1B, C). TEM revealed that MDNP display a spherical morphology, and their core structure was consistent with that of solid lipid nanoparticles (FIG.1D). Size and zeta potential of MDNP measured an average of 174.5 nm and -9.6 mV, respectively (FIG.1E). Next, the stability of MDNP at multiple temperatures in PBS was assessed. MDNP could tolerate a freeze and thaw cycle and were stable at 4^C for up to 6 days, the maximum length of time tested. All different temperature storage conditions kept MDNP stable except for room temperature (RT), where after 3 days the size was significantly increased (FIG.1F). The stability of MDNP were also tested in cell culture medium and mouse plasma at RT and 37^C.There were minimal changes in size and zeta potential (FIG.8). The sizes and zeta potential of MDNP were also stable upon lyophilization (FIG.9). Differential scanning calorimetry (DSC) analysis determined the summit of melting peak of MDNP, which was measured as 74.3^C (FIG.1G).

[0121] To determine the lipid composition of MDNP, untargeted high throughput lipidomics was performed. A bar graph (FIG.1H) and a pie / vertical slice chart (FIG.1I) shows the total lipid abundance and ratio in percentage of each component in MDNP. The lipidomics identified a total of eight individual lipids in four categories including sphingolipids, galactolipids, neutral lipids, and phospholipids (FIG.10). The most prevalent species within the MDNP was the triglyceride (TG) subclass with 51 identifications (77.45%), followed by phytoceramide (Cer (Phyto); 14.77%), phytohexosylceramide (HexCer (Phyto)) (2.7%), free fatty acids (FFAs) (1.95%), digalactosyldiacylglycerol (DGDG) (0.72%), phosphatidylcholine (PC) (0.96%), phosphatidylinositol (PI) (0.85%), and lysophosphatidylcholine (LPC) (0.13%) (Table 1). Table 1 RT mz Abund Polarity Lipid Class Mass Ion Species Theo mz ppm Sum Comp ance Error14.582 728.6394 17580 Negative Ceramide 683.6428 (M+CHO2) 728.6410 -2.2 Cer_AP alpha-hydroxy - t42:0 fatty acid- h t in in A A A15.91 754.6958 13230 Positive Triacylglycerol 736.6581 (M+H4N)+ 754.6919 5.1 TG 43:0 16.54 810.7590 103704 Positive Triacylglycerol 792.7207 (M+H4N)+ 810.7545 5.6 TG 47:0 1 27 742 22 P iti Ti l l l 7 7 M+H4N+ 7 4 T 47174 16.47 905.7569 11530 Positive Triacylglycerol 882.7676 (M+Na)+ 905.7569 0.1 TG 54:4 75 15.64 899.7120 64915 Positive Triacylglycerol 876.7207 (M+Na)+ 899.7099 2.4 TG 54:7 7 1 7 4 4 P iti T i l l l 747 M+N + 7 4 2 T 4revealed undetectable levels of proteins that could be linked to plant databases from Uniprot and the Plant Proteome Database. This aligns with the lipidomics results indicating that MDNP are primarily composed of lipids, with minimal or no associated protein content. Overall, these data demonstrate the successful isolation, purification, and characterization of MDNP from bulk maca extract. Example 2: In vitro cytotoxicity, internalization, and therapeutic effect of MDNP

[0123] To analyze potential toxicity, BMDM were treated with MDNP (1 mg / mL) for 8 hours prior to flow cytometry analysis to measure viability and apoptosis (FIG.2A). MDNP treatment did not affect the cell viability or induce apoptosis. Furthermore, a time-dependent cellular uptake of Cy5.5-labeled MDNP was observed using BMDM (FIG.2B). The quantitative measurement of mean fluorescence intensity (MFI), a commonly used method in confocal microscopy analysis32, can be found in FIG.11. These findings demonstrate the MDNP are non- toxic and efficiently internalized by BMDM.

[0124] BMDM were treated with LPS followed by MDNP to assess their ability to mitigate immune activation. The observed decrease in CD80 (reduced but not significant), CD86, and MHC II expression following MDNP treatment suggests that MDNP reduced macrophage activation, contributing to a decreased pro-inflammatory response (FIG.2C-E). Modulation of macrophage polarization is one of the strategies to modulate inflammation, support tissue regeneration and reestablish disrupted homeostasis33. A reduction in CD206 as a marker of anti- inflammatory M2-like macrophages was also measured, suggesting MDNP induced a shift in macrophage phenotype towards a more undifferentiated or M0-like state (FIG.2F). These results indicate that MDNP can impact the activation status of macrophages, suggesting that they aid in shifting their profile towards a more balanced and neutral immune response.

[0125] To assess the anti-inflammatory effects of MDNP, prophylactic and therapeutic activity were investigated by treating MDNP on LPS-challenged BMDM. In the prophylacticsetting, MDNP were treated for 8 hr prior to 4 hr LPS exposure (FIG.2G). However, treatment did not significantly attenuate the release of pro-inflammatory cytokines (IL-6 and TNF-a) (FIG. 2H, I) suggesting that it was unlikely that MDNP treatment directly modulated pro-inflammatory signaling pathways in macrophages responsible for cytokine secretion. In the therapeutic setting, BMDM were challenged with LPS for 4 hrs prior to the treatment of MDNP for 8 hr (FIG.2J). The result shows that pro-inflammatory cytokines including IL-6, and TNF-a induced by LPS were significantly reduced, whereas IL-1b, and IFN-g were only slightly reduced (FIG.12). Furthermore, additional testing was conducted on the crude extract after MDNP isolation to determine if it possessed any anti-inflammatory efficacy; however, no cytokine reduction was detected (FIG.13). These results suggested that MDNP can reduce pro-inflammatory responses, and the anti-inflammatory component of maca is the purified MDNP. Example 3: Sequestration of cytokines and mitigation of NF-^^B activation

[0126] Interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) are well known to be involved in pathogenesis of chronic inflammation, autoimmune diseases, and cancer34,35. Utilizing data from the in vitro anti-inflammatory assay, an investigation was conducted to confirm whether MDNP possessed the capability to sequester pro-inflammatory cytokines in the absence of cells. In a time-dependent experiment, BMDM were treated with 100 ng / mL LPS for 4 hrs, then the supernatant rich in inflammatory cytokines was incubated with 100 µg / mL MDNP for up to 24 hrs (FIG.3A). ELISA analysis of these supernatants indicated a gradual decrease in IL-6 levels, with a maximum of 48% reduction after 8 hrs (FIG.3B). A similar trend was observed for TNF-a levels, with a 28% reduction over the same timeframe (FIG.3C). Optimization of MDNP concentration revealed concentration-dependent cytokine sequestration properties of MDNP and 100 µg / mL MDNP exhibited the most significant sequestration of IL-6 and TNF-a, whereas PLGA nanoparticles (as control) did not display any cytokine sequestration properties (FIG.14). The ability of MDNP to sequester recombinant IL-6 was confirmed to exclude the possibility of other proteins contributing to cytokine removal in vitro (FIG.3D, E). Lastly, to assess whether the cytokine-sequestering ability of MDNP is dependent on its structural integrity, MDNP were dissolved in a chloroform:methanol mixture and reassembled using the thin-film rehydration method, with or without PEGylated lipid or cholesterol (FIG. 15). The restructured MDNP retained its cytokine-binding activity, suggesting that its coreproperties are essential for function. However, the inclusion of PEGylated lipid abolished cytokine sequestration, while the addition of up to 20% cholesterol impaired this ability, although cytokine levels remained significantly lower than in the LPS control. These results indicate that MDNP can effectively sequester pro-inflammatory cytokines to mitigate inflammation, and that structural modifications can diminish this function, underscoring the importance of the native MDNP composition.

[0127] The innate immune responses that are triggered by LPS are mediated by Toll-like receptor (TLR)-4 and subsequent activation of the transcription factors nuclear factor kappa B (NF-^^B) and activator protein 1 (AP-1)36. Upon encountering various PAMPs and DAMPs, macrophages undergo rapid activation and secrete a diverse range of cytokines and chemokines including IL-6 and TNF-a37. In this experiment, RAW-Blue cells, an NF-^^B macrophage reporter cell line that releases secreted alkaline phosphatase (SEAP) after activation, were used to gauge MDNP-mediated effects following LPS stimulation (FIG.3F). Following treatment with 100 µg / mL MDNP for 1 hr, a significant decrease in NF-^^B activity was measured, confirming that MDNP possessed the capacity to mitigate pro-inflammatory cell signaling and reduce macrophage activation (FIG.3G). PLGA was used as a control, which was unable to reduce NF-^^B activation. Example 4: In vivo biodistribution and anti-inflammatory effects of MDNP

[0128] For the in vivo biodistribution study, MDNP were labeled with Cy5.5 and administered intraperitoneally (IP) or intravenously (IV) into LPS-challenged mice (5 mg / kg LPS dose) twice at an MDNP dose of 2 mg / injection at 0.5 and 2 hr (FIG.4A). At 4 hr, mice were euthanized to image the biodistribution of Cy5.5-MDNP in different organs including liver, spleen, heart, kidney, and lung with an in vivo imaging system (IVIS). Most of the Cy5.5-labeled MDNP were delivered to the liver and kidney by both IP and IV injection, and very low amounts were detected in the spleen, heart, and lung (FIG.4B, C).

[0129] The route of administration (IP versus IV) was also investigated. Mice were challenged with 5 mg / kg LPS IP followed by treatment with 2 mg MDNP at 0.5 hr and 2 hr post- LPS treatment. After 4 hrs, mice were euthanized to collect organs samples and plasma (FIG. 4A). Plasma samples were processed using a MAGPIX Luminex multiplexing immunoassay system. This system was employed to analyze the expression level of a 7-plex panel of cytokineswhich included IL-6, TNF-a, MCP-1, IL-10, IL-1β, IFN-β, and GROa. The heatmap demonstrates overall reduction of pro-inflammatory cytokines for both routes (FIG.4D). The analysis revealed a significant reduction in IL-6 level following both administration routes (FIG. 4E), and a notable reduction in TNF-a, MCP-1, and GROa for IP administered MDNP (FIG.4F- K). IV administration of MDNP also reduced systemic cytokine levels, however it was slightly less effective. Blood biochemistry of albumin, alanine transaminase, creatine, aspartate transferase, globulin, total protein, and blood urea nitrogen was further evaluated. Although minor reductions were observed for MDNP-treated mice, there was no significant alteration in levels of blood biochemistry parameters compared to LPS-treated controls (FIG.16). The non- significant reductions in blood biochemistry following MDNP-treatment could be due to the pre- established severe inflammatory response, which could not be completely reversed by the endpoint of the study. Example 5: Treatment with MDNP accelerated organ recovery and improved survival

[0130] Histological analysis was used to evaluate the level of inflammation in tissues isolated from experiments conducted in FIG.4. H&E staining of five different organs including liver, kidney, spleen, lung, and heart are shown in FIG.5. LPS-treated liver showed severe immune cell infiltration, tubular necrosis was detected in the kidney, focal necrosis, and dysregulation in white pulp of spleen was observed, and extensive clots in the alveoli of lung appeared, but no significance of heart injury was measured. On the contrary, the IV and IP administered MDNP groups displayed notable improvements in histology scores when compared to the LPS group (FIG.5B-F). Based on the overall safety profile and reductions in pro- inflammatory cytokines, MDNP were used to further evaluate survival in vivo. Mice were challenged with lethal dose of 20 mg / kg LPS in sterile PBS intraperitoneally. Then, mice were monitored after two doses of 2 mg MDNP (FIG.5G). As a result, 60% of MDNP treated mice survived, whereas 0% of the LPS treated (control) mice survived (FIG.5H). Example 6: Proteomic profiling of MDNP protein corona

[0131] The ability of MDNP to reduce systemic cytokines and promote LPS mouse survival prompted an exploration of the composition and types of proteins sequestered by the MDNP. Adsorbed proteins were characterized by forming a protein corona on MDNP ex vivo byincubating with either healthy or LPS-treated mouse plasma (MDNP-H or MDNP-LPS, respectively) (FIG.6A). Proteomics analysis of these coronas identified 297 total proteins (Tables 2A and 2B), with 270 common between the two conditions, 1 distinctive for healthy, and 26 for LPS-treated (FIG.6B). Table 3 presents a list of uniquely adsorbed proteins from each group. Among these, CD14, LCN2 (lipocalin-2), APCS (Serum Amyloid P component), FABP4 (Fatty Acid Binding Protein 4), NGP (Neutrophilic Granule Protein), and HSP90AA (Heat Shock Protein 90 alpha) are known to play roles in inflammation and categorized as APPs excluding NGP. The heatmap in FIG.6C shows the differential expression of total 297 detected proteins. Comparing MDNP-H to MDNP-LPS, highlighted significant changes in protein abundance, with 49 proteins being significantly upregulated and 100 proteins downregulated (FIG.6D). Haptoglobin (Hp), which is known for its pro-inflammatory properties38, was found to be the most differentially abundant protein in the MDNP-LPS corona. The top 25 proteins identified in the MDNP-LPS group are shown in FIG.17. Among these, Hp, Seprina3n, and Saa1 are well-known for their pro-inflammatory roles in regulating inflammatory process and promoting the release of inflammation cytokines. Their levels can increase dramatically during inflammation39. FIG.6E lists the canonical pathways of MDNP-LPS coronas from ingenuity pathway analysis (IPA). APR signaling was most highly associated with MDNP-LPS corona. The network diagram in FIG.6F illustrates the complex but close interaction of APPs and these upstream pro-inflammatory regulators within the APR signaling pathway. The highlighted connections between APR shows its direct interaction with the APPs sequestered on MDNP-LPS coronas (Hp, Saa1, SerpinA3N) and a subset of upstream regulators (IL-6, TNF, IL1B, AGT, HNF1A). Importantly, IL-6, TNF and IL1B play pivotal roles in either indirectly or directly activating these APPs. FIG.6G illustrates the result of ex vivo cytokine sequestration assays using MDNP. It demonstrates that MDNP retains the ability to bind and remove pro- inflammatory cytokines, such as IL-6 and TNF-a, from the LPS plasma. It is also important to point out that protein corona formed on the surface of MDNP did not activate any pro- inflammatory responses when treated on BMDM, supporting that the formation of the corona effectively neutralizes the pro-inflammatory responses of these various mediators (FIG.18). FIG.6H summarizes the proposed mechanism of MDNP by forming a multimodal protein corona comprised of pro-inflammatory cytokines and APPs and neutralization. This inhibits the key inflammatory pathways, blocking the production of pro-inflammatory upstream regulators,ultimately reducing pro-inflammatory cytokines, improving organ function, and increasing survival.

[0132] A list of unique protein corona adsorbed onto the surface of MDNP from LPS and healthy plasma is provided in Table 3. A total of 26 proteins are derived from the LPS plasma protein corona, while only one protein is found in healthy plasma corona. Acute phase proteins are highlighted with circles to underscore the potential presence of pro-inflammatory proteins associated with MDNP. Table 2A Accession Description Gene Abundancies (Normalized) Abundancies (Normalized) Symbol MDNP-H MDNP-LPS P02088 Hemoglobin subunit beta-1 Hbb-b1 928003409.2 3714237272P32261 Antithrombin-III Serpinc1 132951486.3 114955170.1 P13020 Gelsolin Gsn 122925696.2 112717714.4P34928 Apolipoprotein C-I Apoc1 25204072.49 22481305.91 P06330 Ig heavy chain V region AC38 205.12 15878369.91 21571691.03Q5FW60 Major urinary protein 20 Mup20 2352747.672 6801889.379 Q61704 Inter-alpha-trypsin inhibitor heavy chain H3 Itih3 6604642.255 6787648.943Q61508 Extracellular matrix protein 1 Ecm1 2409400.968 2826543.008 Q9Z2K1 Keratin, type I cytoskeletal 16 Krt16 385143.2046 2819454.191P01844 Ig lambda-2 chain C region Iglc2 933514.4961 1199107.648 P11352 Glutathione peroxidase 1 Gpx1 448819.0804 1156240.245P35700 Peroxiredoxin-1 Prdx1 117636.8253 478144.7418 P14430 H-2 class I histocompatibility antigen, Q8 alpha chain H2-Q8 239335.4636 469309.8116Q8BK48 Pyrethroid hydrolase Ces2e Ces2e 138648.6251 141534.1001 Q9DA19 Corepressor interacting with RBPJ 1 Cir1 143355.989 131390.7276P12246 Serum amyloid P-component Apcs 307144.909 P28666 Murinoglobulin-2 Mug2Abundance Abundance Abundance Abundance Abundance Abundance s s s s s s ze PS 09 09 09 09 09 48 11 09 92 39 87 01 29 75 84 40 47 05 90 92Alpha-2-HS-glycoprotein P29699 [OS=Mus musculus] Ahsg 230117007 201801577 319822702 256420472 203711974 254513355 li i 19 69 41 08 36 99 26 13 96 46 12 84 95 07 63 60 53 83 94 92 33 17Complement C5 [OS=Mus 42195860. 29841220. P06684 musculus] C5 5 35253339 43226301 33339312 6 31351350 h i i 44 57 61 80 17 75 18 16 72 39 84 95 46 15 27 40 70 21 04 55 70Major urinary protein 17 17308177. 31477651. B5X0G2 [OS=Mus musculus] Mup17 3 18463893 17261350 30772036 1 33966760 l hi i 85 65 08 38 61 14 60 83 9.6 11 92 45 1.9 10 16 3.5 92 72 7.5 51 6.1 6.1Carboxypeptidase N catalytic chain [OS=Mus 6459556.9 Q9JJN5 musculus] Cpn1 7376015.2 8100394.6 5789547.9 6289510.3 9 6085393.1 1.7 00 6.6 6.3 4.9 8.9 09 55 4.6 47 6.3 1.4 7.2 0.3 4.9 4.4 4.2 7.1 6.6 2.5 8.9Keratin, type II cytoskeletal 2 epidermal [OS=Mus 963460.88 7929576.3 Q3TTY5 musculus] Krt2 3 4551184.7 504160.43 2316333.5 5 1682918.7 6.9 9.6 2.2 3.7 84 76 39 1.1 5.1 1.9 5.4 80 9.4 0.8 3.2 4.2 1.6 6.8 9.6 64 4.2 2.8Ig kappa chain V-III region PC 7132 [OS=Mus 2638372.1 4148828.7 P01655 musculus] 5 2173667.3 2530767.3 4126911.7 3 4587525 00 56 2.1 7.1 1.4 2.8 11 0.1 11 8.9 0.2 91 8.3 2.2 46 91 04 0.1 0.8 0.1EGF-containing fibulin-like extracellular matrix protein 936621.53 Q8BPB5 1 [OS=Mus musculus] Efemp1 8 957110.26 1381219.4 1277030.1 1051273.4 1113724.6 6.9 62 3.8 39 9.3 9.2 09 63 81 87 81 25 60 12 59 0.8 2.8 99 4.3Regulator of G-protein signaling 4 [OS=Mus 637679.25 499218.12 O08899 musculus] Rgs4 7 508507.63 658493.75 601239.08 6 611611.55 6.5 79 8.1 91 34 7.2 2.7 48 42 3.1 7.1 95 53 13 24 41 1.4 64 1.3 98 66Ig kappa chain V-II region 292064.62 220323.32 P01630 7S34.1 [OS=Mus musculus] 9 280837.73 283650.91 254268.87 5 191531.78 29 44 06 59 3.4 59 45 68 38 9.7 41 0.5 28 13 8.4 38 85 11 97 56Contactin-2 [OS=Mus 74217.781 289166.31 Q61330 musculus] Cntn2 8 134956.12 132676.42 5 248870.57 i i 24 88 3.3 7.5 44 52 5.1 45 5.5 45 29 6.6 0.1 49 85 01 58 82 18 02L-selectin [OS=Mus 70289.683 66985.259 P18337 musculus] Sell 7 58937.108 116049.97 82181.208 3 77968.279 i l h 67 62 97 05 86 89 11 6.1 88 49 15 0.1 25 13 56 73 86 86 8.4 56Low molecular weight phosphotyrosine protein h h t [OS=M 74136966 18616920 5.6 85 31 3.7 17 68 7.8 4.1 47 09 17 91 6.7 71Rho GDP-dissociation inhibitor 1 [OS=Mus 185554.68 Q99PT1 musculus] Arhgdia 139601.89 3 178621.27 36 16 26 91 05 28 21 22 89 11Extracellular superoxide dismutase [Cu-Zn] [OS=Mus O09164 musculus] Sod3E3 ubiquitin-protein ligase Q4U2R1 HERC2 [OS=Mus musculus] Herc2 6.9Metalloproteinase inhibitor P39876 3 [OS=Mus musculus] Timp3TABLE 3 Protein Accession Descri Gene Acute Corona ption Symbol Function PhaseP39876 Metalloproteinase inhibitor 3 Timp3 Regulates hematopoietic stem cell proliferation, differentiation, and trafficking P02089 Hemoglobin subunit beta-2 Hbb-b2 Oxygen, CO transport and pH regulation P62806 Hi t H4 H4 Central role in transcription regulation, DNA repair

[0133] The survival benefit of MDNP was also tested in a clinically relevant severe polymicrobial cecal ligation and puncture (CLP) sepsis model (FIG.7A). Mice underwent the CLP procedure and were treated with three doses of 2 mg MDNP, followed by monitoring for 5 days. To confirm MDNP could sequester pro-inflammatory cytokines in CLP plasma, MDNP were first incubated with plasma isolated at 3 hr post-CLP and an almost complete reduction in IL-6 and TNF-a (FIG.7B) was observed. In the survival study, MDNP treated mice showed significant improvement in survival (40%) compared to control group (0%) (FIG.7C). Taken together, the ability of MDNP to increase the survival of septic mice in the absence of antibiotics highlights their translational potential for treating inflammatory diseases, including sepsis. Example 8: Physicochemical characterization and optimization of TCNP formulates

[0134] Based on lipidomic analysis of MDNP, triglycerides and ceramides were identified as the major lipid constituents. Utilizing these two components, a synthetic lipid nanoparticle, termed TCNP (Triglyceride–Ceramide Nanoparticle) was synthesized by employing a thin film hydration method, as a bioinspired drug delivery platform (FIG.19). Transmission electron microscopy (TEM) images of the original MDNP and TCNP (FIG.20A) demonstrated similarly uniform morphology. TCNP was formulated at three triglyceride-to-ceramide ratios (75:25, 50:50, and 25:75) to identify the optimal composition that most closely mimics the physicochemical properties of MDNP. Following extrusion at 40°C and 75°C, particle size and zeta potential were measured. At the 75:25 ratio, particle sizes were 137.9 nm (40°C) and 361.3 nm (75°C), with zeta potentials of -28.3 mV and -31.2 mV, respectively. The 50:50 formulation yielded sizes of 196.5 nm (40°C) and 269.4 nm (75°C), and zeta potentials of -21.5 mV and - 22.0 mV. The 25:75 formulation showed a significant increase in particle size of 872.1 nm (40°C) and 1384 nm (75°C) and decreased surface charge (-16.8 mV and -13.6 mV) (FIG. 20B,C). Among the three formulations, the 75:25 ratio exhibited the most comparable physicochemical characteristics to native MDNP. Additionally, stability testing of TCNP stored at room temperature (RT) and 4°C for up to 10 days showed no significant changes in particle size or zeta potential, indicating high colloidal stability under both storage conditions (FIG. 20D). Example 9: In vitro evaluation of anti-inflammatory effects and pro-inflammatory cytokine sequestration of TCNP nanoparticles

[0135] Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) are critical mediators in the progression of chronic inflammation. To assess the cytokine-binding and inflammation- reducing potential of TCNPs, an in vitro assay was performed. ELISA results showed that TCNP significantly decreased the concentrations of both IL-6 and TNF-α (FIG.21). While control MDNP exhibited a strong cytokine-reducing effect, the reduction achieved by TCNP was not as efficient but produced a significant reduction. Example 10: Characterization of dexamethasone encapsulated TCNP

[0136] Dexamethasone (Dex) was encapsulated into the optimized TCNP formulation at four different concentrations (0.01, 0.1, 0.5, and 1.0^μg / mL) using the thin film hydration method toassess its impact on particle characteristics and encapsulation efficiency (FIG.22A). The average particle size and zeta potential for empty TCNPs were 151.32^nm and –31.21^mV, respectively. With Dex loading, slight increases in size and reductions in surface charge were observed: 157.29^nm, -29.71^mV for 0.01^μg / mL, 160.10^nm, -28.56^mV for 0.1^μg / mL, 156.78^nm, -28.20^mV for 0.5^μg / mL, and 171.39^nm, -26.11^mV for 1.0^μg / mL. All formulations exhibited low polydispersity index (PDI), indicating uniform particle distribution (FIG.22B). The average encapsulation efficiency was approximately 77.33% (FIG.22C). The drug release kinetics showed rapid Dex release within the first 7 hours, reaching a plateau after 12 hours (FIG.22D). Example 11: Cytotoxicity measurement of Dex-TCNP

[0137] Dose-dependent cytotoxicity was assessed using the LDH assay, which provides a direct and reliable measure of membrane damage through LDH enzyme release (FIG.23). Bone marrow-derived macrophages (BMDM) were treated with Dex-loaded TCNP at concentrations of 0.01, 0.1, 0.2, and 0.5^μg / mL. Across all doses, no significant increase in LDH release was observed compared to the Triton X-100 positive control, which caused substantial cell lysis. Moreover, treatment with free Dex (non-encapsulated) at equivalent concentrations did not induce cytotoxicity, indicating that both free and encapsulated Dex were well tolerated by BMDM. Example 12: Characterization of MDNP-PLA nanoparticles

[0138] PLA NP cores were coated with MDNP-associated lipids as shown in FIG.24. Precisely formulated hybrid MDNP-coated PLA (MDNP-PLA) was characterized for particle size and surface charge (FIG.25A). Uncoated PLA nanoparticles exhibited an average diameter of 197.3^nm and a zeta potential of –26.3^mV. Following MDNP coating, the particle size increased to 258.76^nm, while the zeta potential remained consistent at –26.3^mV. The increase in particle size is attributed to the formation of an outer lipid layer derived from MDNP, confirming successful surface coating. Example 13: In vitro evaluation of anti-inflammatory effect of MDNP-PLA nanoparticles

[0139] Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a) are key mediators implicated in the development of chronic inflammation. MDNP-PLA nanoparticles were used to explore their ability to bind and reduce pro-inflammatory cytokine levels in vitro. ELISA analysis revealed that MDNP-PLA significantly reduced both IL-6 and TNF-a (FIG.25B). Although native MDNP demonstrated notable cytokine reduction, the effect observed with MDNP-PLA was slightly tempered compared to that of the MDNP treatment. Discussion

[0140] A tremendous amount of research has been implemented to enhance the clinical management of inflammatory diseases caused by excessive stress40, infections41, autoimmune disorders42, or secondary diseases43. Despite many clinical trials aimed at blocking specific inflammatory mediators, most have been unsuccessful due to the complexity and multifactorial nature of inflammatory diseases. This highlights the need for multimodal approaches that target multiple inflammatory mediators simultaneously to improve treatment outcomes. The core pathophysiology of sepsis is known to involve the disruption of the finely tuned balance between inflammatory and anti-inflammatory immune responses44. Recognition of pathogen- or damage- associated molecular patterns (PAMPs / DAMPs) by innate immune cells functions as the trigger to initiate inflammatory cell signaling and subsequent cytokine release, leading to multiorgan damage, and death. Notably, studies have shown that increased plasma IL-6 and TNF-a levels in sepsis patients are correlated with mortality45–47. MDNP described herein efficiently reduced systemic pro-inflammatory cytokines including IL-6, TNF-α, among others (FIG.4E, 6G, 7B), while simultaneously neutralizing additional pro-inflammatory mediators (FIG.6D). Strategies for reducing pro-inflammatory mediators include conventional adsorption resins like Cytosorb®, or more sophisticated membrane-coated nanoparticles19, telodendrimer nanotraps48, or abiotic histone-capturing hydrogel nanoparticles49. Although these approaches have shown success in certain cases, the complex structural composition and manufacturing processes may hinder rapid development. In contrast, MDNP were developed, isolated from maca root, as a novel approach to mitigate broad pro-inflammatory responses from abundant and readily available plant material, facilitating convenient access, and long-term storage potential (FIG.1). Their ability to reduce organ damage and improve survival was further demonstrated as reflected in tworepresentative and lethal sepsis models (FIG.5A, 5H, 7C) through the formation of a multimodal protein corona (FIG.6).

[0141] Sepsis endotypes include hyperinflammatory (early) and immunosuppressive (late) states50. A clinical study indicated that early-identified sepsis is associated clinically with higher mortality than late-identified sepsis51. Thus, achieving control over the cytokine storm holds great potential to improve patient survival. Modes of cytokine sequestration have generally relied on charge-based interactions, where most pro-inflammatory cytokines are negatively charged, and anti-inflammatory cytokines are positively charged at neutral pH52. Interestingly, the zeta potential of MDNP was -9.6 mV (FIG.1E), yet cytokines like IL-6 (pI 6.96) and TNF-a (pI 5.01) were strongly sequestered but IL-1b (pI 4.96) and IFN-g (pI 8.25) were not (FIG.2K, L, 13). These differences suggest the possibility of a combination of forces driving the binding of cytokines to the surface of MDNP. Our lipidomics analysis of MDNP (FIG.1H, I) showed the presence of multiple charged or ionizable lipid species like Cer (Phyto), HexCer (Phyto), FFA, LPC, and PC (FIG.10). Therefore, it is possible that a combination of local charge and hydrophobic interactions are contributing to the apparently selective sequestering of specific pro- inflammatory cytokines. Although it has not been elaborated how MDNP facilitate a selective binding interaction for certain cytokines, which is a limitation of the present study, the results disclosed herein provide the basis for future studies to investigate the role of specific lipid components and their ability to alter binding interactions. Also, while the present disclosure demonstrates promising safety and therapeutic effects with the 2 mg / dose administration, future studies that investigate the impact of MDNP dose level and frequency will yield valuable information regarding the potential therapeutic enhancements or long-term safety implications, which could be particularly relevant for chronic conditions requiring sustained therapeutic intervention.

[0142] Upon exposure to biological fluids, nanoparticles dynamically adsorb biomolecules onto their surface, forming a protein corona that dictates how nanoparticles interact with host cells. These interactions can alter both drug delivery efficiency53and biological function54. The analysis of MDNP protein coronas revealed significant differences between MDNP-H and MDNP-LPS groups, highlighting that unique protein fingerprints are associated with MDNP under inflammatory conditions. This is consistent with the concept of the ‘personalized protein corona’, which demonstrates that unique protein fingerprints are formed onthe surface of nanoparticles as a function of disease state55,56. The ability of MDNP to bind cytokines in LPS- or CLP-treated mouse plasma confirmed its ability to directly modulate inflammatory responses in ongoing disease settings (FIG.6G, 7B). An unexpected finding from the proteomics analysis was the identification of multiple pro-inflammatory APPs being upregulated in the coronas of MDNP-LPS like Hp, SerpinA3N, and Saa1 (FIG.6D). Hp activates immune cells, promotes secretion of pro-inflammatory cytokines, and interacts with complement system, potentially intensifying inflammatory responses57. SerpinA3N is involved in regulating inflammatory responses by controlling protease activity to prevent excessive tissue damage, but it is upregulated under inflammatory conditions58. Saa1 plays direct role in recruiting immune cells and modulating pro-inflammatory cytokines59. Supporting that MDNP neutralized the pro-inflammatory functions of corona constituents, MDNP-LPS was cultured with BMDM, and no significant immune activation was measured when evaluating IL-6 or TNF- a secretions (FIG.18). These results highlighted that MDNP formed multimodal protein corona under inflammatory conditions that could mitigate aberrant immune activation caused by individual components, representing a novel approach to the treatment of inflammatory diseases.

[0143] Here, it is demonstrated that MDNP sequestered and neutralized multiple pro- inflammatory cytokines and APPs in its protein corona to reveal a highly effective anti- inflammatory PDNP-based therapeutic strategy for managing severe inflammatory responses. The data indicates that MDNP exhibit negligible toxicity, are efficiently internalized by macrophages, display immunomodulatory activities, and demonstrate significant therapeutic efficacy. Administration of MDNP in vivo effectively reduced pro-inflammatory cytokines and promoted improved survival in two representative sepsis mouse models, LPS-induced endotoxemia and CLP polymicrobial sepsis. This research underscores the broad therapeutic potential of the MDNP, which leverages a unique and never previously examined mechanism of action through the formation of a multimodal protein corona that effectively binds and neutralizes pro-inflammatory cytokines and APPs from propagating systemic inflammation. * * * *

[0144] While the invention has been described with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention. The scope of the appended claims is not to be limited to the specific embodiments described.REFERENCES

[0145] All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which the invention pertains. Each cited patent and publication is incorporated herein by reference in its entirety. All of the following references have been cited in this application: 1. Shao, H., Im, H., Castro, C.M., Breakefield, X., Weissleder, R., and Lee, H. (2018). New Technologies for Analysis of Extracellular Vesicles. Chem Rev 118, 1917–1950. https: / / doi.org / 10.1021 / acs.chemrev.7b00534. 2. Chafe, S.C. (1969). The fine structure of the collenchyma cell wall. Planta 90, 12–21. https: / / doi.org / 10.1007 / BF00389292. 3. Robards, A.W. and Kidwai, P. (1969). Vesicular involvement in differentiating plant vascular cells. New Phytologist 68, 343–349. https: / / doi.org / https: / / doi.org / 10.1111 / j.1469-8137.1969.tb06446.x. 4. Li, X., Bao, H., Wang, Z., Wang, M., Fan, B., Zhu, C., and Chen, Z. (2018). Biogenesis and function of multivesicular bodies in plant immunity. Front Plant Sci 9. https: / / doi.org / 10.3389 / fpls.2018.00979. 5. Yang, C., Long, D., Sung, J., Alghoul, Z., and Merlin, D. (2021). Orally Administered Natural Lipid Nanoparticle-Loaded 6-Shogaol Shapes the Anti-Inflammatory Microbiota and Metabolome. Pharmaceutics 13. https: / / doi.org / 10.3390 / pharmaceutics13091355. 6. Perut, F., Roncuzzi, L., Avnet, S., Massa, A., Zini, N., Sabbadini, S., Giampieri, F., Mezzetti, B., and Baldini, N. (2021). Strawberry-Derived Exosome-Like Nanoparticles Prevent Oxidative Stress in Human Mesenchymal Stromal Cells. Biomolecules 11. https: / / doi.org / 10.3390 / biom11010087. 7. Wang, Q., Zhuang, X., Mu, J., Deng, Z.-B., Jiang, H., Zhang, L., Xiang, X., Wang, B., Yan, J., Miller, D., et al. (2013). Delivery of therapeutic agents by nanoparticles made of grapefruit-derived lipids. Nat Commun 4, 1867. https: / / doi.org / 10.1038 / ncomms2886. 8. Fang, X., Feng, J., Zhu, X., Feng, D., and Zheng, L. (2024). Plant-derived vesicle-like nanoparticles: A new tool for inflammatory bowel disease and colitis-associated cancer treatment. Molecular Therapy 32, 890–909. https: / / doi.org / https: / / doi.org / 10.1016 / j.ymthe.2024.02.021.Markanday, A. (2015). Acute Phase Reactants in Infections: Evidence-Based Review and a Guide for Clinicians. Open Forum Infect Dis 2, ofv098. https: / / doi.org / 10.1093 / ofid / ofv098. Chen, L., Deng, H., Cui, H., Fang, J., Zuo, Z., Deng, J., Li, Y., Wang, X., and Zhao, L. (2018). Inflammatory responses and inflammation-associated diseases in organs. Oncotarget 9, 7204–7218. https: / / doi.org / 10.18632 / oncotarget.23208. Tanaka, T., Narazaki, M., and Kishimoto, T. (2014). IL-6 in Inflammation, Immunity, and Disease. Cold Spring Harb Perspect Biol 6, a016295–a016295. https: / / doi.org / 10.1101 / cshperspect.a016295. Ansar, W., and Ghosh, S. (2016). Inflammation and Inflammatory Diseases, Markers, and Mediators: Role of CRP in Some Inflammatory Diseases. In Biology of C Reactive Protein in Health and Disease, W. Ansar and S. Ghosh, eds. (Springer India), pp.67–107. https: / / doi.org / 10.1007 / 978-81-322-2680-2_4. Ehlting, C., Wolf, S.D., and Bode, J.G. (2021). Acute-phase protein synthesis: a key feature of innate immune functions of the liver.402, 1129–1145. https: / / doi.org / doi:10.1515 / hsz-2021-0209. Markanday, A. (2015). Acute Phase Reactants in Infections: Evidence-Based Review and a Guide for Clinicians. Open Forum Infect Dis 2, ofv098. https: / / doi.org / 10.1093 / ofid / ofv098. Castellheim, A., Brekke, O.-L., Espevik, T., Harboe, M., and Mollnes, T.E. (2009). Innate Immune Responses to Danger Signals in Systemic Inflammatory Response Syndrome and Sepsis. Scand J Immunol 69, 479–491. https: / / doi.org / https: / / doi.org / 10.1111 / j.1365-3083.2009.02255.x. Zhang, Y., and Ning, B. (2021). Signaling pathways and intervention therapies in sepsis. Signal Transduct Target Ther 6, 407. https: / / doi.org / 10.1038 / s41392-021-00816-9. Lasola, J.J.M., Kamdem, H., McDaniel, M.W., and Pearson, R.M. (2020). Biomaterial- Driven Immunomodulation: Cell Biology-Based Strategies to Mitigate Severe Inflammation and Sepsis. Front Immunol 11. Khatoon, N., Zhang, Z., Zhou, C., and Chu, M. (2022). Macrophage membrane coated nanoparticles: a biomimetic approach for enhanced and targeted delivery. Biomater Sci 10, 1193–1208. https: / / doi.org / 10.1039 / D1BM01664D.Thamphiwatana, S., Angsantikul, P., Escajadillo, T., Zhang, Q., Olson, J., Luk, B.T., Zhang, S., Fang, R.H., Gao, W., Nizet, V., et al. (2017). Macrophage-like nanoparticles concurrently absorbing endotoxins and proinflammatory cytokines for sepsis management. Proceedings of the National Academy of Sciences 114, 11488–11493. https: / / doi.org / 10.1073 / pnas.1714267114. Zhang, Q., Hu, C., Feng, J., Long, H., Wang, Y., Wang, P., Hu, C., Yue, Y., Zhang, C., Liu, Z., et al. (2024). Anti-inflammatory mechanisms of neutrophil membrane-coated nanoparticles without drug loading. Journal of Controlled Release 369, 12–24. https: / / doi.org / https: / / doi.org / 10.1016 / j.jconrel.2024.03.030. Shen, S., Han, F., Yuan, A., Wu, L., Cao, J., Qian, J., Qi, X., Yan, Y., and Ge, Y. (2019). Engineered nanoparticles disguised as macrophages for trapping lipopolysaccharide and preventing endotoxemia. Biomaterials 189, 60–68. https: / / doi.org / https: / / doi.org / 10.1016 / j.biomaterials.2018.10.029. Wang, S., and Zhu, F. (2019). Chemical composition and health effects of maca (Lepidium meyenii). Food Chem 288, 422–443. https: / / doi.org / https: / / doi.org / 10.1016 / j.foodchem.2019.02.071. Zhu, H., Wang, R., Hua, H., Qian, H., and Du, P. (2022). Deciphering the potential role of Maca compounds prescription influencing gut microbiota in the management of exercise-induced fatigue by integrative genomic analysis. Front Nutr 9. Fei, W., Zhang, J., Yu, S., Yue, N., Ye, D., Zhu, Y., Tao, R., Chen, Y., Chen, Y., Li, A., et al. (2022). Antioxidative and Energy Metabolism-Improving Effects of Maca Polysaccharide on Cyclophosphamide-Induced Hepatotoxicity Mice via Metabolomic Analysis and Keap1-Nrf2 Pathway. Nutrients 14. https: / / doi.org / 10.3390 / nu14204264. Cao, F., Zhang, H., Yan, Y., Chang, Y., and Ma, J. (2023). Extraction of polysaccharides from Maca enhances the treatment effect of 5-FU by regulating CD4+T cells. Heliyon 9, e16495. https: / / doi.org / https: / / doi.org / 10.1016 / j.heliyon.2023.e16495. Yang, J., Cho, H., Gil, M., and Kim, K.E. (2023). Anti-Inflammation and Anti- Melanogenic Effects of Maca Root Extracts Fermented Using Lactobacillus Strains. Antioxidants 12. https: / / doi.org / 10.3390 / antiox12040798.Fei, W., Hou, Y., Yue, N., Zhou, X., Wang, Y., Wang, L., Li, A., and Zhang, J. (2020). The effects of aqueous extract of Maca on energy metabolism and immunoregulation. Eur J Med Res 25, 24. https: / / doi.org / 10.1186 / s40001-020-00420-7. Tenci, B., Di Cesare Mannelli, L., Maresca, M., Micheli, L., Pieraccini, G., Mulinacci, N., and Ghelardini, C. (2017). Effects of a water extract of Lepidium meyenii root in different models of persistent pain in rats.72, 449–457. https: / / doi.org / doi:10.1515 / znc- 2016-0251. Shaw, J., and Pearson, R.M. (2022). Nanoparticle personalized biomolecular corona: implications of pre-existing conditions for immunomodulation and cancer. Biomater Sci 10, 2540–2549. https: / / doi.org / 10.1039 / D2BM00315E. Pearson, R.M., Juettner, V. V, and Hong, S. (2014). Biomolecular corona on nanoparticles: a survey of recent literature and its implications in targeted drug delivery. Front Chem 2. Sung, J., Yang, C., Viennois, E., Zhang, M., and Merlin, D. (2019). Isolation, Purification, and Characterization of Ginger-derived Nanoparticles (GDNPs) from Ginger, Rhizome of Zingiber officinale. Bio Protoc 9, e3390. https: / / doi.org / 10.21769 / bioprotoc.3390. Mandal, S., Zhou, Y., Shibata, A., and Destache, C.J. (2015). Confocal fluorescence microscopy: An ultra-sensitive tool used to evaluate intracellular antiretroviral nano-drug delivery in HeLa cells. AIP Adv 5, 084803. https: / / doi.org / 10.1063 / 1.4926584. Parisi, L., Gini, E., Baci, D., Tremolati, M., Fanuli, M., Bassani, B., Farronato, G., Bruno, A., and Mortara, L. (2018). Macrophage Polarization in Chronic Inflammatory Diseases: Killers or Builders? J Immunol Res 2018, 8917804. https: / / doi.org / https: / / doi.org / 10.1155 / 2018 / 8917804. Popa, C., Netea, M.G., van Riel, P.L.C.M., van der Meer, J.W.M., and Stalenhoef, A.F.H. (2007). The role of TNF-α in chronic inflammatory conditions, intermediary metabolism, and cardiovascular risk. J Lipid Res 48, 751–762. https: / / doi.org / https: / / doi.org / 10.1194 / jlr.R600021-JLR200. Hirano, T. (2021). IL-6 in inflammation, autoimmunity and cancer. Int Immunol 33, 127– 148. https: / / doi.org / 10.1093 / intimm / dxaa078.Nyati, K.K., Masuda, K., Zaman, M.M.-U., Dubey, P.K., Millrine, D., Chalise, J.P., Higa, M., Li, S., Standley, D.M., Saito, K., et al. (2017). TLR4-induced NF-κB and MAPK signaling regulate the IL-6 mRNA stabilizing protein Arid5a. Nucleic Acids Res 45, 2687–2703. https: / / doi.org / 10.1093 / nar / gkx064. Liu, T., Zhang, L., Joo, D., and Sun, S.-C. (2017). NF-κB signaling in inflammation. Signal Transduct Target Ther 2, 17023. https: / / doi.org / 10.1038 / sigtrans.2017.23. Shen, H., Colangelo, C., Wu, T., Maffei, M., and Goldstein, D.R. (2011).235 Haptoglobin: A Novel Innate Ligand that Induces Inflammation after Tissue Necrosis. The Journal of Heart and Lung Transplantation 30, S84. https: / / doi.org / 10.1016 / j.healun.2011.01.242. Massart, I.S., Paulissen, G., Loumaye, A., Lause, P., Pötgens, S.A., Thibaut, M.M., Balan, E., Deldicque, L., Atfi, A., Louis, E., et al. (2020). Marked Increased Production of Acute Phase Reactants by Skeletal Muscle during Cancer Cachexia. Cancers (Basel) 12. https: / / doi.org / 10.3390 / cancers12113221. Miller, A.H., and Raison, C.L. (2016). The role of inflammation in depression: from evolutionary imperative to modern treatment target. Nat Rev Immunol 16, 22–34. https: / / doi.org / 10.1038 / nri.2015.5. Ikoba, U., Peng, H., Li, H., Miller, C., Yu, C., and Wang, Q. (2015). Nanocarriers in therapy of infectious and inflammatory diseases. Nanoscale 7, 4291–4305. https: / / doi.org / 10.1039 / C4NR07682F. Miller, S.D., Turley, D.M., and Podojil, J.R. (2007). Antigen-specific tolerance strategies for the prevention and treatment of autoimmune disease. Nat Rev Immunol 7, 665–677. https: / / doi.org / 10.1038 / nri2153. Dufour, J.-F., Anstee, Q.M., Bugianesi, E., Harrison, S., Loomba, R., Paradis, V., Tilg, H., Wong, V.W.-S., and Zelber-sagi, S. (2022). Current therapies and new developments in NASH. Gut 71, 2123. https: / / doi.org / 10.1136 / gutjnl-2021-326874. Jarczak, D., Kluge, S., and Nierhaus, A. (2021). Sepsis—Pathophysiology and Therapeutic Concepts. Front Med (Lausanne) 8. Mera, S., Tatulescu, D., Cismaru, C., Bondor, C., Slavcovici, A., Zanc, V., Carstina, D., and Oltean, M. (2011). Multiplex cytokine profiling in patients with sepsis. APMIS 119, 155–163. https: / / doi.org / https: / / doi.org / 10.1111 / j.1600-0463.2010.02705.x.Unsinger, J., McGlynn, M., Kasten, K.R., Hoekzema, A.S., Watanabe, E., Muenzer, J.T., McDonough, J.S., Tschoep, J., Ferguson, T.A., McDunn, J.E., et al. (2010). IL-7 Promotes T Cell Viability, Trafficking, and Functionality and Improves Survival in Sepsis. The Journal of Immunology 184, 3768–3779. https: / / doi.org / 10.4049 / jimmunol.0903151. Wu, H.-P., Chen, C.-K., Chung, K., Tseng, J.-C., Hua, C.-C., Liu, Y.-C., Chuang, D.-Y., and Yang, C.-H. (2009). Serial cytokine levels in patients with severe sepsis. Inflammation Research 58, 385–393. https: / / doi.org / 10.1007 / s00011-009-0003-0. Shi, C., Wang, X., Wang, L., Meng, Q., Guo, D., Chen, L., Dai, M., Wang, G., Cooney, R., and Luo, J. (2020). A nanotrap improves survival in severe sepsis by attenuating hyperinflammation. Nat Commun 11, 3384. https: / / doi.org / 10.1038 / s41467-020-17153-0. Koide, H., Okishima, A., Hoshino, Y., Kamon, Y., Yoshimatsu, K., Saito, K., Yamauchi, I., Ariizumi, S., Zhou, Y., Xiao, T.-H., et al. (2021). Synthetic hydrogel nanoparticles for sepsis therapy. Nat Commun 12, 5552. https: / / doi.org / 10.1038 / s41467-021-25847-2. Hotchkiss, R.S., Monneret, G., and Payen, D. (2013). Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy. Nat Rev Immunol 13, 862–874. https: / / doi.org / 10.1038 / nri3552. Jee, W., Jo, S., Lee, J.B., Jin, Y., Jeong, T., Yoon, J.C., and Park, B. (2020). Mortality difference between early-identified sepsis and late-identified sepsis. Clin Exp Emerg Med 7, 150–160. https: / / doi.org / 10.15441 / ceem.19.009. Messina, J.M., Luo, M., Hossan, M.S., Gadelrab, H.A., Yang, X., John, A., Wilmore, J.R., and Luo, J. (2024). Unveiling cytokine charge disparity as a potential mechanism for immune regulation. Cytokine Growth Factor Rev 77, 1–14. https: / / doi.org / https: / / doi.org / 10.1016 / j.cytogfr.2023.12.002. Corbo, C., Molinaro, R., Parodi, A., Toledano Furman, N.E., Salvatore, F., and Tasciotti, E. (2016). The Impact of Nanoparticle Protein Corona on Cytotoxicity, Immunotoxicity and Target Drug Delivery. Nanomedicine 11, 81–100. https: / / doi.org / 10.2217 / nnm.15.188. Cedervall, T., Lynch, I., Lindman, S., Berggård, T., Thulin, E., Nilsson, H., Dawson, K.A., and Linse, S. (2007). Understanding the nanoparticle–protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proceedings of theNational Academy of Sciences 104, 2050–2055. https: / / doi.org / 10.1073 / pnas.0608582104. Ju, Y., Kelly, H.G., Dagley, L.F., Reynaldi, A., Schlub, T.E., Spall, S.K., Bell, C.A., Cui, J., Mitchell, A.J., Lin, Z., et al. (2020). Person-Specific Biomolecular Coronas Modulate Nanoparticle Interactions with Immune Cells in Human Blood. ACS Nano 14, 15723– 15737. https: / / doi.org / 10.1021 / acsnano.0c06679. Shaw, J., and Pearson, R.M. (2022). Nanoparticle personalized biomolecular corona: implications of pre-existing conditions for immunomodulation and cancer. Biomater Sci 10, 2540–2549. https: / / doi.org / 10.1039 / D2BM00315E. Torres, A., Vivanco, S., Lavín, F., Pereda, C., Chernobrovkin, A., Gleisner, A., Alcota, M., Larrondo, M., López, M.N., Salazar-Onfray, F., et al. (2022). Haptoglobin Induces a Specific Proteomic Profile and a Mature-Associated Phenotype on Primary Human Monocyte-Derived Dendritic Cells. Int J Mol Sci 23. https: / / doi.org / 10.3390 / ijms23136882. Liu, C., Zhao, X.-M., Wang, Q., Du, T.-T., Zhang, M.-X., Wang, H.-Z., Li, R.-P., Liang, K., Gao, Y., Zhou, S.-Y., et al. (2023). Astrocyte-derived SerpinA3N promotes neuroinflammation and epileptic seizures by activating the NF-κB signaling pathway in mice with temporal lobe epilepsy. J Neuroinflammation 20, 161. https: / / doi.org / 10.1186 / s12974-023-02840-8. Ye, R.D., and Sun, L. (2015). Emerging functions of serum amyloid A in inflammation. J Leukoc Biol 98, 923–929. https: / / doi.org / 10.1189 / jlb.3VMR0315-080R. Matyash, V., Liebisch, G., Kurzchalia, T. V, Shevchenko, A., and Schwudke, D. (2008).Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics*s ⃞. J Lipid Res49, 1137–1146. https: / / doi.org / https: / / doi.org / 10.1194 / jlr.D700041-JLR200. Truong, N., Cottingham, A.L., Dharmaraj, S., Shaw, J.R., Lasola, J.J.M., Goodis, C.C., Fletcher, S., and Pearson, R.M. (2024). Multimodal nanoparticle-containing modified suberoylanilide hydroxamic acid polymer conjugates to mitigate immune dysfunction in severe inflammation. Bioeng Transl Med 9, e10611. https: / / doi.org / https: / / doi.org / 10.1002 / btm2.10611.Shaw, J., and Pearson, R.M. (2022). Nanoparticle personalized biomolecular corona: implications of pre-existing conditions for immunomodulation and cancer. Biomater Sci 10, 2540–2549. https: / / doi.org / 10.1039 / D2BM00315E. Ju, S., Mu, J., Dokland, T., Zhuang, X., Wang, Q., Jiang, H., Xiang, X., Deng, Z.-B., Wang, B., Zhang, L., et al. (2013). Grape Exosome-like Nanoparticles Induce Intestinal Stem Cells and Protect Mice From DSS-Induced Colitis. Molecular Therapy 21, 1345– 1357. https: / / doi.org / https: / / doi.org / 10.1038 / mt.2013.64. Suzuki, Y., Hyodo, K., Tanaka, Y., and Ishihara, H. (2015). siRNA-lipid nanoparticles with long-term storage stability facilitate potent gene-silencing in vivo. Journal of Controlled Release 220, 44–50. https: / / doi.org / https: / / doi.org / 10.1016 / j.jconrel.2015.10.024. Truong, N., Black, S.K., Shaw, J., Scotland, B.L., and Pearson, R.M. (2021). Microfluidic-Generated Immunomodulatory Nanoparticles and Formulation-Dependent Effects on Lipopolysaccharide-Induced Macrophage Inflammation. AAPS J 24, 6. https: / / doi.org / 10.1208 / s12248-021-00645-2.

Claims

WHAT IS CLAIMED IS:

1. Isolated maca-derived nanoparticles (MDNP), wherein the nanoparticles are isolated from Lepidium meyenii Walp.

2. The isolated MDNP of claim 1, wherein the MDNP are isolated by a method comprising performing sucrose gradient ultracentrifugation on a maca extract.

3. A lipid nanoparticle composition comprising isolated MDNP of claim 1 or 2.

4. A lipid nanoparticle composition comprising a lipid component, wherein the lipid component comprises a) a triglyceride in an amount of about 77.4 % to about 62.0% by weight of the lipid component; and b) a ceramide in an amount of about 25.0% to about 11.8%, by weight of the lipid component.

5. The lipid nanoparticle composition of claim 4, wherein the triglyceride is a phyto- triglyceride.

6. The lipid nanoparticle composition of claim 4 or 5, wherein the ceramide is a phytoceramide.

7. The lipid nanoparticle composition of any one of claims 4 to 6, wherein the lipid component further comprises phytohexosylceramide, free fatty acid, digalactosyldiacylglycerol, phosphatidylcholine, phosphatidylinositol, lysophosphatidylcholine, cholesterol, or a combination thereof.

8. The lipid nanoparticle composition of claim 7, wherein: c) the phytohexosylceramide, if present, is in an amount of about 2.70 % to about 2.16%, by weight of the lipid component; d) the free fatty acid, if present, is in an amount of about 1.95% to about 1.56%, by weight of the lipid component; e) the digalactosyldiacylglycerol, if present, is in an amount of about 0.720% to about 0.576%, by weight of the lipid component; f) the phosphatidylcholine, if present, is in an amount of about 0.960% to about 0.768%, by weight of the lipid component; g) the phosphatidylinositol, if present, is in an amount of about 0.850% to about 0.680%, by weight of the lipid component;h) the lysophosphatidylcholine, if present, is in an amount of about 0.130% to about 0.104%, by weight of the lipid component; and i) the cholesterol, if present, is in an amount of about 20.0% to about 5.0%, by weight of the lipid component; 9. The lipid nanoparticle composition of any one of claims 4 to 6, wherein the triglyceride is in an amount of about 75% by weight of the lipid component, and the ceramide is in an amount of about 25% by weight of the lipid component.

10. The lipid nanoparticle composition of any one of claims 4 to 8, wherein the lipid component comprises: a) the triglyceride in an amount of about 77.4% by weight of the lipid component; b) the ceramide in an amount of about 14.8% by weight of the lipid component; c) the phytohexosylceramide in an amount of about 2.70 % by weight of the lipid component; d) the free fatty acid in an amount of about 1.95% by weight of the lipid component; e) the digalactosyldiacylglycerol in an amount of about 0.720% by weight of the lipid component; f) the phosphatidylcholine in an amount of about 0.960% by weight of the lipid component; g) the phosphatidylinositol in an amount of about 0.850% by weight of the lipid component; and h) the lysophosphatidylcholine in an amount of about 0.130% by weight of the lipid component.

11. A lipid composition comprising the lipid component of the isolated MDNP of claims 1 or 2.

12. The lipid nanoparticle composition of any one of claims 4 to 10, or a lipid nanoparticle composition wherein the lipid component comprises the lipid composition of claim 11, further comprising a therapeutic agent.

13. The lipid nanoparticle composition of claim 12, wherein the therapeutic agent is dexamethasone.

14. A lipid nanoparticle composition of any one of claims 4 to 10, or a lipid nanoparticle composition wherein the lipid component comprises the lipid composition of claim 11, further comprising a polymer core.

15. The lipid nanoparticle composition of claim 14, wherein the polymer core is a poly(lactic acid) core.

16. A method of preventing or treating an inflammatory disease in a subject, said method comprising, administering a therapeutically effective amount of the lipid nanoparticle composition of any one of claims 3-10 and 12-15, to a subject having inflammatory disease.

17. The method of claim 16, wherein the inflammatory disease is selected from, rheumatoid arthritis, inflammatory bowel diseases, COVID-19-related cytokine storms, CAR-T cell cytokine release syndrome, and sepsis.

18. The method of claim 16 or 17, wherein the lipid nanoparticle composition sequesters an acute phase protein or a pro-inflammatory cytokine.

19. The method of claim 18, wherein the pro-inflammatory cytokine is selected from IL-6 and TNF-α.

20. The method of any one of claims 16 to 19, wherein the inflammatory disease is sepsis.

21. The method of claim 20, wherein the sepsis is caused by a bacterium.

22. The method of any one of claims 16-21, wherein the therapeutically effective amount is 0.01 to 1 g / kg body weight.

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