Compositions and methods for reducing nanoparticle-associated coagulation

Abstract

Composition and methods for preventing or reducing coagulation or thrombosis resulting from nanoparticle administration to a subject are provided.

Description

[0001]COMPOSITIONS AND METHODS FOR REDUCING NANOPARTICLE- ASSOCIATED COAGULATION STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under HL153510, HL160694, AI166778, and HL164594 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION Since the first Food and Drug Administration (FDA) approval of a solid LNP in 2018, LNPs have rapidly become the preferred drug delivery platform of the biopharma industry, with the trend accelerating markedly after the development of COVID-19 LNP vaccines. Preferable delivery of LNPs to specific organs or cell types is desirable for many applications and is generally accomplished by one of two methods, both of which have certain advantages and disadvantages. The older method, still very useful research, is conjugation of LNPs to affinity moieties (e.g., antibodies) that bind to known epitopes on a target cell. At the present time, this approach presents major challenges for scale-up manufacturability and immunogenicity, partially contributing to it never having been applied in an FDA-approved targeted LNP or liposome. However, conjugation of LNPs to affinity moieties does allow for the selection of target molecules favorable for a given utility. For example, knowing localization of these molecules in organs and tissues provides opportunities to accumulate the drug in the desired sites, and knowing sub- cellular trafficking and distribution helps control intracellular uptake. The second and newer method for LNP targeting is to screen large numbers of LNP formulations for those that have fortuitous “physicochemical tropism” to a target organ. In physicochemical tropism / targeting, some (usually unknown) physical or chemical features of the LNP cause it to accumulate in a given organ or tissue, and to bind and enter particular cells. Over the last decade, the physicochemical approach has become the dominant method of achieving targeting in both academia and industry because of the ease of in vivo screening and highly efficient manufacturing processes. As physicochemical tropism has been able to target many different organs and cell types, it has the potential to meet LNPs’ promise of being able to modulate any protein in any cell type or organ. However, knowledge of contributors to the localization of a given type of LNP is still lacking. As such, investigations and development of tropism-based drug delivery systems, in terms of medical utility, has been based solely on localization of the carrier. Pursuing useful tropisms hangs on the hope that biological mechanisms, activity, and effects will eventually fit specific therapeutic needs. In this context, the prospects of identifying a drug delivery system with attractive tropism is marred with potential toxicities, adversities, or other side effects that are often only revealed after significant investment is made in a formulation. There remains a need in the art for improved nanoparticle compositions and methods of effectively targeting nanoparticles to various target organs. SUMMARY OF THE INVENTION In one aspect, provided herein is a method of delivering an agent to a target organ of a subject, the method comprising administering to the subject an anticoagulant and / or a platelet inhibitor and a nanoparticle comprising the agent, wherein coagulation or thrombosis resulting from administration of the nanoparticle is reduced or prevented. In certain embodiments, the nanoparticle is a lipid nanoparticle (LNP) or liposome. In certain embodiments, the nanoparticle comprises an outer surface layer comprising a cationic lipid. In certain embodiments, the cationic lipid is 1,2-dioleoyl-3-trimethylammonium- propane (DOTAP), 1,2-Dioleoyl-3-trimethylammonium propane (DOTMA), DLin-KC2- DMA, or D-Lin-MC3-DMA. In certain embodiments, the nanoparticle comprises an outer surface layer comprising an ionizable lipid. In certain embodiments, the ionizable lipid is 306-N16B, lipid 29d, or 514O6,10. In certain embodiments, the nanoparticle has a zeta potential greater than 0 mV, at least about 5 mV, at least about 10 mV, or at least about 15 mV, optionally wherein the zeta potential is measured in a solution at a pH of about 7.0 or about 7.0 to 7.5. In certain embodiments, the nanoparticle comprises a direct thrombin inhibitor or a coagulation cascade protease inhibitor conjugated to a lipid component of the nanoparticle’s outer surface. In certain embodiments, the direct thrombin inhibitor is D-phenylalyl-L-prolyl-L-arginyl-chloromethyl ketone (PPACK). In certain embodiments, the direct thrombin inhibitor is hirudin, bivalirudin, desirudin, lepirudin, dabigatran, argatroban, inogatran, or melagatran. In certain embodiments, the anticoagulant is a direct thrombin inhibitor or a coagulation cascade protease inhibitor. In certain embodiments, the direct thrombin inhibitor is hirudin, bivalirudin, desirudin, lepirudin, dabigatran, argatroban, inogatran, or melagatran. In certain embodiments, the platelet inhibitor is acetylsalicylic acid, clopidogrel, prasugrel, ticagrelor, dipyridamole, eptifibatide, tirofiban, abciximab, cilostazol,or vorapaxar. In certain embodiments, the nanoparticle is capable of binding fibrinogen present in the subject’s plasma following administration to the subject. In certain embodiments, the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension. In certain embodiments, the nanoparticle is not capable of binding fibrinogen or has reduced binding to fibrinogen following administration to the subject. In certain embodiments, the nanoparticle is administered intravenously or intra-arterially. In certain embodiments, the nanoparticle and anticoagulant and / or the platelet inhibitor are administered to the subject via the same route. In certain embodiments, the nanoparticle and anticoagulant and / or the platelet inhibitor are administered sequentially. In certain embodiments, the agent is a therapeutic, diagnostic, or imaging agent. In certain embodiments, the nanoparticle is administered intravenously and clotting in the lung is reduced or prevented. In certain embodiments, administration of the anticoagulant and / or the platelet inhibitor does not alter localization of the nanocarrier when compared to delivery of the nanoparticle without administering the anticoagulant and / or the platelet inhibitor. In certain embodiments, the coagulation resulting from administration of the therapeutic nanoparticles is nanoparticle-induced clotting (thrombosis) in a target organ of the subject. In certain embodiments, the target organ is the lung. In certain embodiments, the nanoparticle comprises RNA and administration of the anticoagulant and / or the platelet inhibitor does not alter expression of the RNA when compared to delivery of the nanoparticle without administering the anticoagulant. In certain embodiments, the reduction or prevention of coagulation is determined by measuring platelet activation, platelet aggregation, partial thromboplastin time (PTT), activated partial thromboplastin time (aPTT), and / or thrombin–antithrombin (TAT) complex in a biological sample obtained from the subject, optionally wherein the biological sample is blood, serum, or plasma. In another aspect, provided herein is a nanoparticle comprising an agent and a direct thrombin inhibitor or a coagulation cascade protease inhibitor conjugated to a lipid component of the nanoparticle’s outer surface. In certain embodiments, the nanoparticle is a lipid nanoparticle (LNP) or liposome. In certain embodiments, the direct thrombin inhibitor is D-phenylalyl-L-prolyl-L-arginyl-chloromethyl ketone (PPACK). In certain embodiments, the direct thrombin inhibitor is hirudin, bivalirudin, desirudin, lepirudin, dabigatran, argatroban, inogatran, or melagatran. In certain embodiments, the nanoparticle comprises an outer surface layer comprising a cationic lipid. In certain embodiments, the cationic lipid is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-Dioleoyl-3- trimethylammonium propane (DOTMA), DLin-KC2-DMA, or D-Lin-MC3-DMA. In certain embodiments, the nanoparticle comprises an outer surface layer comprising a ionizable lipid. In certain embodiments, the ionizable lipid is 306-N16B, lipid 29d, or 514O6,10. In certain embodiments, the nanoparticle has a zeta potential greater than 0 mV, at least about 5 mV, at least about 10 mV, or at least about 15 mV, optionally wherein the zeta potential is measured in a solution at a pH of about 7.0 or about 7.0 to 7.5. In certain embodiments, the nanoparticle is capable of binding plasma fibrinogen. In certain embodiments, the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension. In certain embodiments, the nanoparticle is not capable of binding fibrinogen or has reduced binding to fibrinogen following administration to the subject. In certain embodiments, the agent is a therapeutic, diagnostic, or imaging agent. In yet another aspect, provided herein is a method of delivering an agent to a target organ of a subject, the method comprising administering to the subject an anticoagulant and / or a platelet inhibitor and a nanoparticle comprising the agent, wherein coagulation or thrombosis resulting from administration of the nanoparticle is reduced or prevented. In certain embodiments, the anticoagulant is a direct thrombin inhibitor or a coagulation cascade protease inhibitor. In certain embodiments, the direct thrombin inhibitor is hirudin, bivalirudin, desirudin, lepirudin, dabigatran, argatroban, inogatran, or melagatran. In certain embodiments, the platelet inhibitor is acetylsalicylic acid, clopidogrel, prasugrel, ticagrelor, dipyridamole, eptifibatide, tirofiban, abciximab, cilostazol,or vorapaxar. In certain embodiments, the nanoparticle is administered intravenously or intra-arterially. In certain embodiments, the nanoparticle and the anticoagulant and / or the platelet inhibitor are administered to the subject via the same route. In certain embodiments, the nanoparticle and the anticoagulant and / or the platelet inhibitor are administered sequentially. In certain embodiments, the nanoparticle is administered intravenously and clotting in the lung is reduced or prevented. In certain embodiments, administration of the anticoagulant and / or the platelet inhibitor does not alter localization of the nanocarrier when compared to delivery of the nanoparticle without administering the anticoagulant. In certain embodiments, the coagulation resulting from administration of the therapeutic nanoparticle includes clotting (thrombosis) in a target organ in the subject. In certain embodiments, the target organ is the lung. In certain embodiments, the nanoparticle comprises RNA and administration of the anticoagulant and / or the platelet inhibitor does not alter expression of the RNA when compared to delivery of the nanoparticle without administering the anticoagulant. In certain embodiments, the reduction or prevention of coagulation is determined by measuring platelet activation, platelet aggregation, partial thromboplastin time (PTT), activated partial thromboplastin time (aPTT), and / or thrombin–antithrombin (TAT) complex in a biological sample obtained from the subject, optionally wherein the biological sample is blood, serum, or plasma. In certain embodiments, the reduction or prevention of coagulation is determined by measuring platelet activation, platelet aggregation, partial thromboplastin time (PTT), activated partial thromboplastin time (aPTT), and / or thrombin–antithrombin (TAT) complex in a biological sample obtained from the subject, optionally wherein the biological sample is blood, serum, or plasma. In yet another aspect, provided herein is a pharmaceutical composition comprising a nanoparticle described herein and a pharmaceutically acceptable carrier, excipient, and / or preservative. Still other aspects and advantages of these compositions and methods are described further in the following detailed description of the preferred embodiments thereof. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1A – FIG.1G show cationic LNPs with physicochemical tropism to the lungs induce acute, severe side effects in mice. (FIG.1A) Formulation of −DOTAP and +DOTAP LNPs. (FIG.1B) Size distribution of −DOTAP and +DOTAP LNPs determined by dynamic light scattering (DLS) shows no significant difference in size (99.22 ± 4.27 nm for −DOTAP LNPs and 102.47 ± 0.48 nm for +DOTAP LNPs). (FIG.1C) Zeta potential graphs of −DOTAP LNP (black) versus +DOTAP LNP (pink), show that −DOTAP LNPs are slightly negatively charged at −4.05 mV and +DOTAP LNPs have a surface charge of 14.34 mV. (FIG.1D) Gross anatomical comparison of naïve mice intravenously (IV) injected with either −DOTAP LNPs or +DOTAP LNPs reveals severe lung hepatization (liver-like appearance) with +DOTAP LNP injected mice. LNPs were allowed to circulate for 30 min. (FIG.1E) Comparison of cumulative distance traveled for 1 h after injection of +DOTAP LNPs assessed by AI software, DeepLabCut, compared to healthy, uninjected mice reveals a significant reduction in total distance traveled, indicating lethargy in these mice. (FIG.1F) Dose-response of the effect of +DOTAP LNP dose on protein concentration in bronchoalveolar lavage (BAL) fluid shows a dose- dependent increase in BAL protein, a measure of endothelial barrier dysfunction. (FIG. 1G) This is further exacerbated in mice with pre-existing lung inflammation (nebulized LPS mice; note the 5-fold y-axis augmentation between FIG.1F and FIG.1G). Statistics: n = 3–5 and data shown represents mean ± SEM; For FIG.1E, comparisons between groups were made using an unpaired t-test with Welch's correction. For all other graphs, comparisons between groups were made using one-way ANOVA with Tukey's post-hoc test. * = p < 0.05, ** * = p < 0.001, **** = p < 0.0001. FIG.2 shows the effect of +DOTAP LNP dose on cell count in bronchoalveolar lavage (BAL) fluid in naïve mice is dose-dependent. +DOTAP LNPs were injected into naïve mice and 24h later, BAL fluid was extracted and the number of cells was measured. FIG.3 shows that +DOTAP LNPs do not increase the plasma concentration of the complement protein C3a 30 minutes after IV injection into naive mice. C3a concentration was measured by conducting a mouse C3a ELISA on plasma extracted from injected mice. FIG.4A – FIG.4I show coagulation is activated by positively charged LNPs and other nanoparticles, across diverse formulations, both in vivo and in ex vivo whole blood. (FIG.4A) +DOTAP LNPs induce large clots (black arrows) in the pulmonary arteries (scale bar = 100 µm). −DOTAP or +DOTAP LNPs were injected into healthy mice at a dose of 10 µg of mRNA and 30 min after, lungs were harvested and prepared for histology (Masson's trichrome). −DOTAP LNPs do not show such clots. (FIG.4B) In vivo, +DOTAP LNPs increase plasma levels of thrombin-antithrombin (TAT), a marker of recent clotting. The TAT increase is > 2-fold in naïve mice, with an additional > 1.5-fold increase in mice with pre-existing lung inflammation (nebulized-LPS). +DOTAP LNPs were injected into naïve mice and 30 min post-injection, plasma was collected for TAT assay. (FIG.4C) 306-N16B LNPs increase TAT plasma levels by ≈2.5-fold compared to naive mice while control −DOTAP LNPs do not.2E11 LNPs per mouse were injected and TAT plasma levels were measured after 30 min. (FIG.4D) Schematic of the two major coagulation pathways – intrinsic and extrinsic – which both converge into the common pathway. The extrinsic pathway is triggered by damage that occurs outside the blood vessel, exposing tissue factors. The intrinsic pathway is initiated by factors within the blood vessel lumen. Both the intrinsic and extrinsic pathways converge to the common pathway which involves the conversion of prothrombin to thrombin – an enzyme that converts fibrinogen to fibrin. Fibrin forms the mesh-like framework of clots. The prothrombin time (PT) and activated partial thromboplastin time (APTT) are laboratory tests used to measure the time to clot through the extrinsic and intrinsic coagulation pathways, respectively. (FIG.4E) +DOTAP LNPs do not change PT time in vitro, showing that they do not induce coagulation through the extrinsic coagulation pathway. (FIG.4F) Timeline and schematic of in vitro APTT measurements. (FIG.4G) +DOTAP LNPs increase APTT, showing that they induce coagulation through the extrinsic pathway. (FIG.4H) +DOTAP LNPs increase APTT through the intrinsic pathway regardless of ionizable lipid identity, as ALC-0315, SM-102, and C12-200 DOTAP LNPs also show elevated APTT. (FIG.4I) Positively charged, non-LNP nanoparticles, here liposomes containing DOTAP, also increase APTT time. Statistics: n = 3–6 and the data shown represents mean ± SEM; For FIG.4I, comparisons between groups were made using an unpaired t-test with Welch's correction. For all other graphs, comparisons between groups were made using one-way ANOVA with Tukey's post-hoc test. * = p < 0.05, *** = p < 0.001, **** = p < 0.0001. FIG.5A – FIG.5C show that -DOTAP and +DOTAP LNPs do not cause clotting in the (FIG.5A) liver and (FIG.5B) spleen of naïve mice when injected intravenously (scale bars = 200µm). LNPs were injected into healthy mice at a dose of 10ug of mRNA and 30 minutes after, lungs were harvested and prepared for histology (Masson’s Trichrome). (FIG.5C) +DOTAP LNPs cause visible indications of clotting in the brain when injected through the carotid artery after a circulation time of 30 minutes. FIG.6 shows lipid formulations and molar percentages of DOTAP LNPs fabricated with the ionizable lipids SM-102, ALC-0315, and C12-200 as well as DOTAP liposomes, 306-N16B LNPs, and DOTMA LNPs. FIG.7 shows size, PDI and zeta potential measurements of nanoparticle formulations. FIG.8A – FIG.8C show LNPs formulated with the ionizable lipid 306-N16B induce clotting. (FIG.8A) Structure of 306-N16B. LNP formulations and characterizations are described in FIG.6 and FIG.7. (FIG.8B) Gross lung anatomical comparison of naïve mice or mice intravenously (IV) injected with 306-N16B LNPs reveals severe lung hepatization (liver-like appearance) with 306-N16B LNP injected mice. In these experiments, 2E11 LNPs injected into mice and allowed to circulate for 30 minutes and whole-body perfusion through the left ventricle was carried out with PBS. (FIG.8C) Left - 306-N16B LNPs significantly increase clotting time in vivo indicating a pro-thrombotic state due to depletion of coagulation factors.30 minutes post-injection of 306-N16B LNPs into naïve mice, plasma was isolated and immediately after recalcification, the time to clot was measured. Right - 306-N16B LNPs significantly increase APTT time in vivo showing that they activate the intrinsic coagulation pathway. APTT reagent was added prior to calcification for an incubation period of 4 minutes and then the time to clot was measured. FIG.9A – FIG.9E show in vivo dose-dependence of +DOTAP LNPs on platelet count and time to clot achieved by varying either the number of LNPs injected while keeping the mol fraction of DOTAP constant at 50% (FIG.9A, FIG.9B) or titrating the mol fraction of DOTAP and keeping the number of LNPs injected constant at 8E11 particles per mouse (FIG.9C, FIG.9D). Increasing either the number of particles injected or the mol fraction of DOTAP in the LNP formulation leads to an increase in platelet count and time to clot. In these studies, LNPs were injected for a circulation time of 30 minutes. Complete blood counts were taken to measure platelet numbers. Plasma was then isolated and after recalcification, the time to clot was measured. (FIG.9E) In vitro dose dependence of +DOTAP LNP particle dose on APTT. As the number +DOTAP LNPs administered increases, the APTT increases. FIG.10 shows LNPs formulated with the cationic lipid DOTMA also increase APTT in vitro. FIG.11A – FIG.11C show DOTAP liposomes localization. (FIG.11A) DOTAP liposomes localize primarily to the lungs when injected into naïve mice. DOTAP liposomes were radiolabeled and intravenously injected for a circulation period of 30 minutes. Negatively charged LNPs formulated with the anionic lipid DOPS do not change (FIG.11B) APTT or (FIG.11C) PT in vitro. LNP formulations and characterizations are described in FIG.6 and FIG.7. FIG.12A – FIG.12K show DOTAP LNPs cause platelet activation. (FIG.12A) +DOTAP LNPs significantly decrease platelet count in naive mice and even more so in mice with pre-existing inflammation (nebulized LPS). LNPs were IV-injected and platelet count was measured from whole blood 30 min later. (FIG.12B) 306-N16B LNPs decrease platelet count in naïve mice while control −DOTAP LNPs do not. (FIG.12C) Schematic depicting what different flow cytometry metrics describe platelet physiology. (FIG.12D) Schematic of protocol used for platelet flow cytometry. After isolation of platelet-rich plasma (PRP), samples were either left untreated or treated with −DOTAP or +DOTAP LNPs for an incubation period of 30 min at 37 °C. (FIG.12E) Representative FSC-A versus SSC-A graphs of unstimulated platelets (left) and platelets treated with +DOTAP (right) or −DOTAP LNPs (center), showing a clear change in the scatterplot with +DOTAP LNPs. There are significant increases in (FIG.12F) FSC and (FIG.12G) SSC upon treatment with +DOTAP LNPs suggesting larger and more complex platelet aggregates. +DOTAP LNPs cause upregulation of (FIG.12H) CD42d and (FIG.12I) P- selectin, based on mean fluorescence intensity (MFI). (FIG.12J) Platelets were assayed for whether they were physically associated with LNPs, as measured by the fraction of platelets that were positive for a fluorescent lipid that had been incorporated into the LNPs during synthesis. While 50% of platelets were associated with −DOTAP LNPs, nearly 100% of platelets were physically associated with +DOTAP LNPs. (FIG.12K) Staining with P-selectin shows that while only 30% of P-selectin positive platelets were associated with −DOTAP LNPs, almost 100% of P-selectin positive platelets were LNP positive as well, showing preference for an activated state. Statistics: n = 3–6 and data shown represents mean ± SEM; For (FIG.12J, FIG.12K), Welch's t-test was performed. For all other graphs, Brown-Forsythe and Welch's ANOVA was performed with the Dunnett T3 post-hoc test. * = p < 0.05, *** = p < 0.001, **** = p < 0.0001. FIG.13A – FIG.13E show that LNPs targeted to the lung using antibodies against platelet endothelial cell adhesion molecule (PECAM) do not have the same effect on platelets. (FIG.13A) Complete blood counts (CBCs) from naïve or nebulized-LPS mice injected with +DOTAP LNPs showing counts of white blood cells (WBC), lymphocytes (LYM), monocytes (MON), and neutrophils (NEU). (FIG.13B) CBC showing counts of red blood cells (RBC), hemoglobin (HGB) and hematocrit (HCT) in naïve or nebulized- LPS mice injected with +DOTAP LNPs. (FIG.13C) -DOTAP LNPs conjugated to antibodies against Platelet Endothelial Cell Adhesion Molecule (PECAM; a lung targeting moiety) do not affect platelet count in naive and nebulized-LPS mice 30 minutes post-LNP injection. (FIG.13D) PECAM LNPs maintain APTT at naïve levels in vitro. (FIG.13E) Control -DOTAP LNPs with the formulation 75 / 18.5 / 5 / 1.5, cKK- E12 / Cholesterol / DOPE / PEG-DMG2k do not change APTT in vitro. FIG.14A – FIG.14C show flow cytometry gating for platelet flow cytometry and platelet neutrophil aggregates. (FIG.14A) Flow cytometry gating for platelet flow cytometry using a dual confirmation method, gating by both size of platelets as positivity for CD42d, a unique surface marker for platelets. (FIG.14B) Flow cytometry MFI for CD41 expression on the surface of platelets. For FIG.14A and FIG.1B, left column in each set of columns is DOTAP LNP - Naïve and right column in each set of columns is DOTAP LNP - LPS. (FIG.14C) Flow cytometry plots showing that about 5% of neutrophils also stain positive for CD41 compared to fluorescence minus one (FMO) control. Similarly, about 30% of neutrophils also stain positive for p-selectin compared to FMO controls, showing the effects of thrombo-inflammation in the blood. FIG.15A – FIG.15K show fibrinogen binds to +DOTAP LNPs, is required for LNP-induced activation of platelets, and is likely the first step in LNP-induced clotting. (FIG.15A) To measure LNP binding to fibrinogen, we fluorescently labeled fibrinogen, mixed it with plasma and LNPs, and used nanoparticle tracking analysis (NTA) to identify individual nanoparticles that had become bound to fluorescent fibrinogen. (FIG.15B) Representative NTA images of fluorescent fibrinogen on −DOTAP or +DOTAP LNPs, showing visible binding of fibrinogen to +DOTAP LNPs but no visible binding to −DOTAP LNPs, or vehicle control. (FIG.15C) NTA data was turned into particle size versus concentration histograms, and here we plot fibrinogen-positive particles only. +DOTAP LNPs bind strongly to fluorescent fibrinogen and this leads to LNPs of many different sizes, while −DOTAP LNPs barely have any fibrinogen-positive signal at any particle size. Inset: Size versus concentration histogram ranging from 100–500 nm. (FIG.15D) Number of detected fibrinogen-bound particles from FIG.15C showing that +DOTAP LNPs generate ≈37.7× more fibrinogen-bound particles than −DOTAP LNPs. (FIG.15E) Next, we IV-injected radiolabeled fibrinogen into mice, followed by LNPs 2 min later. Organs were harvested 30 min after LNP injection and the radioactivity in each organ was measured. (FIG.15F) Amount of radiolabeled fibrinogen in the blood and lungs after the protocol of FIG.15E, shown as the % of injected dose per gram of tissue (%ID g−1). +DOTAP LNPs lead to a ≈2× increase in fibrinogen lung uptake, while −DOTAP LNPs do not alter fibrinogen biodistribution compared to control. (FIG.15G) Localization ratios of fibrinogen from FIG.15F calculated as %ID g−1of tissue in the lung divided by that in the blood. +DOTAP LNPs cause a 2.4-fold higher localization ratio of fibrinogen compared to −DOTAP LNPs and vehicles. Confocal microscopy images of lung sections from mice injected with fluorescent fibrinogen followed by fluorescent LNPs was obtained, showing visible fibrinogen aggregates in the lungs of +DOTAP LNP injected mice (n = 3 biologically independent animals). (FIG.15H) From the microscopy images, Mean fluorescence intensities (MFIs) in the lung sections from FIG.15H of − or +DOTAP LNPs and fibrinogen showing significantly higher MFIs of LNPs and fibrinogen in +DOTAP LNP injected mice compared to −DOTAP LNP mice. (FIG.15I) From the microscopy images, the percentage of fibrinogen signal overlap with that of LNPs or percentage of LNP signal overlap with fibrinogen signal in the presence of − or +DOTAP LNPs shows a strong co-localization of fibrinogen with +DOTAP LNPs. (FIG.15J) Circular dichroism of fibrinogen (1 mg ml−1) in the presence of −DOTAP or +DOTAP LNPs (4E11 LNPs per ml) reveals that +DOTAP LNPs alter the secondary structure of fibrinogen while −DOTAP LNPs do not. (FIG.15K) To determine if fibrinogen is necessary for LNP-induced activation of platelets, we added tPA to plasma to deplete the fibrinogen. Fibrinogen depletion nearly completely prevented LNP-induced activation of platelets. Statistics: n = 4–12 for FIG.15H and FIG.15I and n = 3–4 for all other graphs. Data shown represents mean ± SEM; For FIG.15F, FIG.15J, and FIG.15I, comparisons between groups were made using two-way ANOVA with Tukey's post-hoc test. For all other graphs, comparisons between groups were made using one-way ANOVA with Tukey's post-hoc test. * = p < 0.05, *** = p < 0.001, **** = p < 0.0001. FIG.16 show DLS size distributions of plasma alone or plasma treated with + or - DOTAP LNPs show aggregation with +DOTAP LNPs. Naïve mouse plasma was collected and incubated with LNPs at a dose of at 37℃ for 30 minutes. After a 100-fold dilution with PBS, the samples were read with a Zetasizer. FIG.17A – FIG.17B show that +DOTAP LNPs aggregate significantly with fibrinogen after incubation with fibrinogen alone. (FIG.17A) - or + DOTAP LNPs do not precipitate significantly in PBS. (FIG.17B) When incubated with citrated plasma, recalcified citrated plasma, or fibrinogen alone, +DOTAP LNPs (right column in each set) show significant aggregation and precipitation while -DOTAP LNPs (left column in each set) do not. In these experiments, radioactively labeled LNPs were incubated in PBS, plasma, or fibrinogen for 30 minutes at 37℃. After centrifugation for 10 minutes at 100 x g, the radioactivity in the pellet and the supernatant were quantified. The fraction of LNPs in the pellet can be interpreted as the fraction of LNPs that are aggregated. FIG.18A – FIG.18F show biodistributions of fibrinogen in (FIG.18A) naïve and (FIG.18B) nebulized-LPS injured mice injected with fibrinogen alone or fibrinogen and + or - DOTAP LNPs for an LNP circulation time of 30 minutes. Biodistribution of fibrinogen in naïve mice injected with fibrinogen alone or + DOTAP LNPs after (FIG. 18C) 4 hours and (FIG.18D) 24 hours of circulation. While the increase in fibrinogen uptake in the lung persists after 4 hours, it is attenuated after 24 hours indicating fibrinolysis. (FIG.18E and FIG.18F) Localization ratios of fibrinogen from (FIG.18C and FIG.18D) calculated as %ID / g of tissue in the lung divided by that in the blood. For FIG.18C, FIG.18D, FIG.18E, and FIG.18F, left column in each set of columns is vehicle, and right column in each set of columns is + DOTAP LNPs. FIG.19A – FIG.19D show anticoagulation ameliorates the side effects of DOTAP LNPs, but only select anticoagulants still allow LNP targeting to the lungs. (FIG.19A) Histology showing clots in the lungs of mice given +DOTAP LNPs (top row), but not in mice that were given +DOTAP LNPs preceded by heparin (middle row) or bivalirudin (bottom row; scale bar = 200 µm). (FIG.19B) Schematics showing the protocol for biodistribution studies (using In-111-labeled LNPs) and luciferase expression studies. (FIG.19C) 30-min biodistribution in naïve mice of +DOTAP LNPs with no treatment or pretreated with heparin or bivalirudin. Pretreatment with heparin ablates localization of +DOTAP LNPs to the lung. Interestingly, pretreatment with bivalirudin maintains lung localization. (FIG.19D) Additionally, bivalirudin further preserves luciferase expression of +DOTAP LNPs whereas heparin pretreatment attenuates this expression capacity. Statistics: n = 3 and the data shown represents mean ± SEM. For FIG.19C and FIG.19D, comparisons between groups were made using a two-way ANOVA with Tukey's post-hoc test. * = p < 0.05, *** = p < 0.001, **** = p < 0.0001. FIG.20A – FIG.20B show biodistributions of fibrinogen after pre-treatment with the anticoagulants (FIG.20A) heparin or (FIG.20B) bivalirudin followed by +DOTAP LNP treatment. Heparin pre-treatment completely reverses fibrinogen lung uptake to vehicle levels while bivalirudin does not alter fibrinogen biodistribution compared to injection with +DOTAP LNPs alone. FIG.21A – FIG.21C show the effect of pre-treatment with the direct thrombin inhibitor dabigatran on luciferase expression of +DOTAP LNPs. (FIG.21A) Dabigatran attenuates +DOTAP luciferase expression in the lung, liver, and spleen. Dabigatran pre- treatment does not alter (FIG.21B) fibrinogen biodistribution and (FIG.21C) localization ratio compared to +DOTAP LNPs alone. In all experiments, dabigatran was administered to naïve mice 5 minutes before +DOTAP LNP injection (4-hour LNP circulation time for FIG.21A and 30-minute LNP circulation time for FIG.21B and FIG.21C). Left column in each set of columns is +DOTAP LNPs, and right column in each set of columns is + +DOTAP LNPs + Dabigatran. FIG.22A – FIG.22E show results from administration of +DOTAP LNPs. (FIG. 22A) The direct thrombin inhibitor PPACK was surface conjugated onto +DOTAP LNPs using azide-DBCO click chemistry. (FIG.22B) A standard curve was generated to measure the inhibition of thrombin with PPACK at different concentrations using a Chromozymassay. PPACK was incubated with thrombin followed by the addition of the chromophoricthrombin substrate Chromozym. The change in absorbance at 405nm has a negative correlation with thrombin inhibition. (FIG.22C) At undiluted concentrations, PPACK-conjugated +DOTAP LNPs have ~100% inhibition of thrombin. (FIG.22D) Lung histological samples from mice injected with PPACK-DOTAP LNPs show a significantly reduced appearance of clots. Unfortunately, these PPACK-DOTAP LNPs were found to cause an increase in mouse mortality via an unknown mechanism, with our hypothesis being hemorrhage. Therefore, further engineering is needed to make anticoagulant conjugated LNPs safe and effective. (scale bar = 200µm). (FIG.22E) Size, PDI and zeta potential of PPACK-conjugated DOTAP LNPs. FIG.23A – FIG.23J show decreasing the size of DOTAP LNPs prevents fibrinogen binding and the resulting coagulation and platelet activation. (FIG.23A) +DOTAP LNP size has a positive correlation with coagulation as measured by APTT. 180 nm +DOTAP LNPs lead to a ≈1.6-fold increase in APTT compared to standard 100 nm +DOTAP LNPs while 80 nm +DOTAP LNPs maintain APTT at naive levels. (FIG.23B) 80 nm +DOTAP LNPs do not cause significant platelet activation as measured by P-Selectin MFI. (FIG.23C) Particle size versus concentration histograms from the NTA fluorescent fibrinogen binding assay comparing the number of detected fibrinogen bound particles with 80 or 100 nm +DOTAP LNPs showing that 80 nm +DOTAP LNPs bind to significantly less fibrinogen compared to 100 nm +DOTAP LNPs. Inset: Size versus concentration histogram ranging from 100–500 nm. (FIG.23D) Number of detected fibrinogen-bound particles from FIG.23C showing that 80 nm +DOTAP LNPs generate ≈79× fewer fibrinogen-bound particles than 100 nm +DOTAP LNPs. The number of fibrinogen-bound particles detected with 80 nm +DOTAP LNPs is not significantly different from the vehicle. (FIG.23E) Amount of fibrinogen in the blood and lungs with injection of radiolabeled fibrinogen alone (vehicle) or radiolabeled fibrinogen followed by 80 or 100 nm +DOTAP LNPs.80 nm +DOTAP LNPs do not alter fibrinogen biodistribution compared to control. (FIG.23F) 80 nm +DOTAP LNPs do not alter the localization ratio of fibrinogen calculated from FIG.23E (%ID g−1of tissue in the lung divided by that in the blood). (FIG.23G) Hypothesized mechanism behind the abrogation of clotting side effects below a size threshold: At a large radius of curvature (≥ 100 nm), +DOTAP LNPs present a flat enough surface to bind fibrinogen lengthwise, which then activates platelets. (FIG.23H) Lung histological samples reveal that 80 nm +DOTAP LNPs do not generate visible clots as is the case with 100 nm +DOTAP LNPs. (FIG.23I) 80 nm +DOTAP LNPs maintain their biodistribution and lung localization (10 µg mRNA per mouse, 30 min; scale bar = 200 µm) and (FIG.23J) express mRNA primarily in the lung measured by luciferase expression (10 µg mRNA per mouse, 4 h). Statistics: n = 3 and the data shown represents mean ± SEM. For FIG.23E, comparisons between groups were made using a two-way ANOVA with Tukey's post-hoc test. For all other graphs, comparisons between groups were made using one-way ANOVA with Tukey's post-hoc test. * = p < 0.05, *** = p < 0.001, **** = p < 0.0001. DETAILED DESCRIPTION Provided herein are nanoparticle compositions and methods that reduce or prevent pathological coagulation and thrombosis in a subject. As described herein, toxicity associated with delivery of nanoparticles, particularly in the lungs, was not previously reported. The present inventors developed several approaches to avoid deleterious effects, including clotting, resulting from nanoparticle delivery to a target organ. Nanoparticles, including lipid nanoparticles (LNPs), have become the dominant drug delivery technology in industry, particularly to improve delivery of RNA to up- or down-regulate any protein of interest. This usually requires targeting to specific cell types or organs, which is most commonly done by physicochemical targeting, in which a nanoparticle’s lipid composition is adjusted to find a mixture with the desired tropism. Physicochemical targeting is frequently accomplished by adjusting surface charge, such as negative charge localizing to the spleen, and positive charge to the lungs. As described herein, positively charged LNPs induce massive thrombosis. This finding was quite general, as it held for LNPs with different permanently positively charged lipids, regardless of their co-loaded ionizable lipid, and not just LNPs, but also liposomes. Perhaps most surprisingly, we also observed thrombosis caused by LNPs that lacked a permanently cationic lipid (one possessing a quaternary amine) but instead contained an ionizable lipid (possessing a tertiary amine) that was found via screening to confer lung tropism. The lung-tropic LNPs induced profound pulmonary embolism when administered intravenously, induced stroke-like effects when administered via the carotid artery, and modified clotting processes in ex vivo blood. Detailed mechanisms of LNP- induced clotting were investigated, showing that lung-tropic LNPs bind to the core clotting protein, fibrinogen, alter its secondary structure, and cause aggregation and activation of platelets. Elucidating these detailed mechanisms of clotting enabled led to the identification of solutions that can permit positively charged LNPs to maintain their lung- targeting property while ameliorating dangerous clotting. In contrast to specific targeting based on the affinity binding to characterized ligands, the nature and mechanisms of tropisms of nanoparticles are not well understood. Therefore, it is not possible reengineer tropism-driven particles in a cognizant manner, thereby reducing undesirable effects while improving desirable features. Notwithstanding these challenges, it was found that altering the size of nanoparticles yields high levels of anti-thrombotic protection along with other approaches including treatment with anticoagulants (including direct thrombin inhibitors), treatment with platelet inhibitors, and conjugation of nanoparticles to direct thrombin inhibitors. These solutions, without introducing any limitations on the disclosed invention, are summarized as follows: 1) treatment with anticoagulants - Treatment of a subject with direct thrombin inhibitors, such as Bivalirudin, prevents coagulation but also sustain expression and localization of nanocarriers. While heparin prevented coagulation induced by DOTAP LNPs, RNA expression was completely attenuated and localization of LNPs was shifted from the lung to the liver and spleen. This effect could be attributed to the high negative charge of heparin which could interact with cationic nanocarriers and affect their functionality; 2) treatment with platelet inhibitors – Delivery of nanoparticles can result in aggregation and activation of platelets that can be ameliorated by administration of a platelet inhibitor; 3) surface conjugating anticoagulation agents to nanoparticles - Having determined that direct thrombin inhibitors are effective anticoagulants, all-in-one nanoparticles having surface conjugated with the direct thrombin inhibitor PPACK were engineered; and 4) altering nanoparticle size – It was found that as the size of nanoparticles increases above 100nm, they bind more fibrinogen units leading to an increased amount of fibrinogen taken up by the lungs and exacerbated clotting side effects. However, nanoparticles with sizes of or below 80nm do not cause fibrinogen uptake in the lung and also do not induce coagulation. Notably, reducing nanoparticle size is easily applied and does not impose the potential for complications that other approaches for reducing coagulation or thrombosis may have. It is described herein that in addition to the inflammatory side effects previously outlined, LNP delivery induces acute side effects in mice including lethargy, lung hepatization, and capillary leak, which is due to the activation of coagulation. After screening multiple formulations, it was found that these effects occur regardless of ionizable lipid and even with liposomes formulated with cationic lipids. Given that these potentially toxic LNPs have entered into clinical trials, it is important to identify overcome their side effects to ensure the safe translatability of this technology to the clinic. As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” nanoparticle can mean a single nanoparticle or a multiplicity of nanoparticles. Various embodiments in the specification are presented using “comprising” language, which is inclusive of other components or method steps. When “comprising” is used, it is to be understood that related embodiments include descriptions using the “consisting of” terminology, which excludes other components or method steps, and “consisting essentially of” terminology, which excludes any components or method steps that substantially change the nature of the embodiment or invention. All scientific and technical terms used herein have their known and normal meaning to a person of skill in the fields of biology, biotechnology and molecular biology and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. However, for clarity, certain terms are defined as provided herein. As used herein, the term “about” refers to a variant of ±10% from the reference integer and values therebetween. For example, “about” 100 nanometers (nm), includes ±10 nm (i.e., 90 – 110 nm), which includes the integers 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110). The term “about” is inclusive of all values within the range including both integers and fractions. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. By “nanoparticle” or “NP” (also referred to as “nanocarrier” or “NC”) as used herein is meant a particle that is typically between about 1 to about 500 nm across the largest dimension of the structure. Nanoparticles include nanogels, solid colloidal nanoparticles, magnetic nanoparticles, noble metal nanoparticles, semiconductor nanoparticles, multimodal nanoparticles, composite nanoparticles, and other nanoparticles typically used for biomedical applications (see, e.g., Blanco-Andujar et al. Annu. Rep. Prog. Chem., Sect. A, 2010, 106, 553-568, incorporated by reference herein). A nanoparticle may be spherical, but is not required to be spherical. Regardless of the shape of the nanoparticle, the exterior of the nanoparticle should be capable of comprising at least one agent, therapeutic, diagnostic, imaging, or otherwise. Inclusive in the definition of nanoparticle are particles that are at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, at least 280, or at least 300 nm across their largest dimension. In certain embodiments, also included are particles of at least 320, at least 340, at least 360, at least 380, at least 400, at least 420, at least 440, at least 460, at least 480, at least 500, at least 520, at least 540, at least 560, at least 580, at least 600, at least 620, at least 640, at least 660, at least 680, or at least 700nm. In yet other embodiment, also included are particles having diameters of at least 720, at least 740, at least 760, at least 780, at least 800, at least 820, at least 840, at least 860, at least 880, at least 900, at least 920, at least 940, at least 960, at least 980, or up to about 1000 nm across their largest dimension. In yet further embodiments, the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension. All numbers and fractions between any two of these numbers are also included. The size of a nanoparticle can be represented, for example, by the z- average as determined by dynamic light scattering. In certain embodiments, the nanoparticle is a liposome. By “liposome” as used herein is meant a material a microscopic spherical particle formed by a lipid bilayer enclosing an aqueous compartment. In certain embodiments, the nanoparticle is a hydrogel nanoparticle (also referred to as a nanogel or NG). In certain embodiments, the nanoparticle is a dendrimer. In yet other embodiments, the nanoparticle is a polymersome, a hybrid carrier combining artificial and natural components, or a protein multimolecular composition (e.g., ferritin or albumin aggregates). By “hydrogel nanoparticle” or “nanogel” as used herein is mean a polymeric material having a hydrophilic structure which renders it capable of holding amounts of selected drug compounds in their three-dimensional networks, the resulting particle having nanoparticle dimensions. Macroscopic dextran hydrogels have shown Young’s moduli of ~10-50kPa in the literature. See, e.g., Hwang MR, Kim JO, Lee JH, Kim YI, Kim JH, Chang SW, Jin SG, Kim JA, Lyoo WS, Han SS, Ku SK,Yong CS, Choi HG. Gentamicin- Loaded Wound Dressing with Polyvinyl Alcohol / Dextran Hydrogel: Gel Characterization and In vivo Healing Evaluation. AAPS PharmSciTech 2010;11(3):1092-1103. In certain embodiments, the NG is a lysozyme-dextran nanogel (also referred to as LDNG). In certain embodiments, the LDNG is a synthetic construct of lysozyme and dextran. In other embodiments, the nanogel is made of chitosan or chitin, pullulan, hyaluronic acid, PEG, pluronics (e.g. F127), poly(acrylic acid) or poly(acrylate), poly(oligo(ethylene glycol)methyl ether methacrylate), poly(ethylene oxide), polyethylenimine, poly(caprolactone), and poly(N-isopropylacrylamide), among other options encompassing a wide range of hydrophilic polymers capable of chemical modifications enabling incorporation in a nanoparticle. See, e.g., Eckmann DM, Composto RJ, Tsourkas A, Muzykantov VR. Nanogel Carrier Design for Targeted Drug Delivery. J Mater Chem B Mater Biol Med 2015;2(46):8085-8097; and Ahmed EM, Mar.2015, “Hydrogel: Preparation, Characterization, and Applications: A Review”, J. Adv. Res., 6(2):105-121, among other publications in the art. In certain embodiments described herein, nanogel particles containing a drug and associated with an RBC behave best in this form of drug delivery. In certain embodiments, the nanoparticle employed herein is a lipid nanoparticle (“LNP”). LNPs useful herein are known in the art. LNPs are formulated with components that include cholesterol (aids in stability and promotes membrane fusion), a phospholipid (which provides structure to the LNP bilayer and also may aid in endosomal escape), a polyethylene glycol (PEG) derivative (which reduces LNP aggregation and “shields” the LNP from non-specific endocytosis by immune cells), and an ionizable lipid (complexes negatively charged RNA and enhances endosomal escape), which form the LNP-forming composition. Fenton et al, Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent in vivo mRNA Delivery, Adv Mater.2016 Apr 20; 28(15): 2939–2943, which is incorporated herein by reference. The various components of an LNP-forming composition may be selected based on the desired target, cargo, size, etc. For example, previous studies have shown that polymeric nanoparticles made of low molecular weight polyamines and lipids can deliver nucleic acids to endothelial cells with high efficiency. Dahlman, et al, In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight, Nat Nanotechnol.2014 Aug; 9(8): 648–655, which is incorporated herein by reference in its entirety. LNP-forming composition may include one or more ionizable lipids or lipid-like material. In certain embodiments, the ionizable lipid confers lung tropism. In certain embodiments, the ionizable lipid is 306-N16B. In certain embodiments, the ionizable lipid is Lipid 29d. In certain embodiments, the ionizable lipid is 514O6,10. In certain embodiments, the ionizable lipid is SM-102. In certain embodiments, the ionizable lipid is ALC-0315. In certain embodiments, the ionizable lipid is C12-200. In In certain embodiments, the ionizable lipid the ionizable lipid is CKK-E12. In certain embodiments, the ionizable lipid is 5A2-SC8. In certain embodiments, the ionizable lipid is BAMEA‐ O16B. In certain embodiments, the ionizable lipid is 306O10. In certain embodiments, the ionizable lipid is 7C1. See, Love et al, Lipid-like materials for low-dose, in vivo gene silencing, Proceedings of the National Academy of Sciences Feb 2010, 107 (5) 1864- 1869; Dong et al, Lipopeptides and selective siRNA delivery, Proceedings of the National Academy of Sciences Mar 2014, 111 (11) 3955-3960; Cheng et al, Dendrimer‐Based Lipid Nanoparticles Deliver Therapeutic FAH mRNA to Normalize Liver Function and Extend Survival in a Mouse Model of Hepatorenal Tyrosinemia Type I, Advanced Materials, 30(52) (Dec 2018); Liu et al, Fast and Efficient CRISPR / Cas9 Genome Editing In Vivo Enabled by Bioreducible Lipid and Messenger RNA Nanoparticles, Advanced Materials, 31(33), Aug 2019; and Hajj et al, Branched‐Tail Lipid Nanoparticles Potently Deliver mRNA In Vivo due to Enhanced Ionization at Endosomal pH, Small, 15(6) (Feb 2019), each of which are incorporated herein by reference. Other ionizable lipids are known in the art and are useful herein. In certain embodiments, the compositions and methods utilize LNP formulations that are lung-tropic and include ionizable lipids. See, e.g., Xue et al., Nat Commun.2024 Feb 29;15(1):1884; Qiu et al. Proc Natl Acad Sci U S A.2022 Feb 22;119(8):e2116271119; Radmand et al. Proc Natl Acad Sci U S A.2024 Mar 12;121(11):e2307801120; Zeng et al. ACS Appl Mater Interfaces.2024 May 22;16(20):25698-25709; Eygeris et al. Proc Natl Acad Sci U S A.2024 Mar 12;121(11):e2307813120; Petersen et al., Adv Healthc Mater.2024 Jun 18:e2400225; and Han et al.. Fast and facile synthesis of amidine-incorporated degradable lipids for versatile mRNA delivery in vivo. Nat Chem.2024 Jul 9. PMID: 38982196, each of which is incorporated herein by reference. Nanoparticles may be prepared using any method known in the art. For example, particulate formulations can be formed by methods as nanoprecipitation, flow focusing using fluidic channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsion procedures, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, and other methods well known to those of ordinary skill in the art. Alternatively or additionally, aqueous and organic solvent syntheses for monodisperse semiconductor, conductive, magnetic, organic, and other nanoparticles have been described. In certain embodiments, nanoparticles are prepared by the nanoprecipitation process or spray drying. Conditions used in preparing particles may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology, “stickiness,” shape, etc.). The method of preparing the particle and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may depend on the therapeutic agent to be delivered and / or the composition of the polymer matrix. Methods for making nanoparticles for delivery of encapsulated agents are described in the literature (see, e.g., Doubrow, Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; all of which are incorporated herein by reference). Zeta potential is a measurement of surface potential of a nanoparticle. In certain embodiments, the nanoparticle has a positive zeta potential. In certain embodiments, the nanoparticle has a zeta potential ranging from about 0 mV to about +50 mV. In certain embodiments, the nanoparticle has a zeta potential ranging from about 0 mV to about +25 mV. In certain embodiments, the nanoparticle has a zeta potential ranging from about 0 mV to about +10 mV. In certain embodiments, the nanoparticle has a zeta potential ranging from about +5 mV to about +10 mV. In certain embodiments, the nanoparticle has a zeta potential ranging from about 0 mV to about +15 mV. In certain embodiments, the nanoparticle has a zeta potential ranging from about 0 mV to about +20 mV. In certain embodiments, the nanoparticle has a zeta potential ranging from about 0 mV to about +25 mV. In certain embodiments, the nanoparticle has a zeta potential ranging from about 0 mV to about +30 mV. In certain embodiments, the nanoparticle has a zeta potential ranging from about 0 mV to about +35 mV. In certain embodiments, the nanoparticle has a zeta potential ranging from about 0 mV to about +40 mV. In certain embodiments, the nanoparticle has a zeta potential ranging from about 0 mV to about +50 mV. In certain embodiments, the nanoparticle has a zeta potential ranging from about +5 mV to about +50 mV. In certain embodiments, the nanoparticle has a zeta potential greater than 0 mV, at least about 5 mV, at least about 10 mV, or at least about 15 mV. In certain embodiments, the zeta potential of a nanoparticle is measure in a solution at a pH of about 7.0. In further emboidments, the zeta potential of a nanoparticle is measure in a solution at a pH of about 7.0 to 7.5. The term “anticoagulant” as used herein, refers to a substance that prevents the clotting of blood (coagulation). Anticoagulants are commonly administered to subjects to prevent or treat thrombosis. Generally, anticoagulants are administered to treat or prevent deep vein thrombosis, pulmonary embolism, myocardial infarction, stroke, and mechanical prosthetic heart valves. Various types of anticoagulants with different mechanisms of action are available including anticoagulants that inhibit the effect of vitamin K (such as coumadin) or thrombin directly (referred to herein as a “direct thrombin inhibitor”, such as argatroban, lepirudin, bivalirudin, or ximelagatran) or that activate antithrombin II that in turn blocks thrombin from clotting blood (such as heparin and derivative substances thereof). In certain embodiments, the anticoagulant is a direct thrombin inhibitor. In certain embodiments, the anticoagulant is a direct thrombin inhibitor that is one of hirudin, bivalirudin, desirudin, lepirudin, dabigatran, argatroban, inogatran, or melagatran. In certain embodiments, the direct thrombin inhibitor is D-phenylalyl-L-prolyl-L-arginyl- chloromethyl ketone (PPACK). In certain embodiments, the anticoagulant is a direct inhibitor or a coagulation cascade protease, including but not limited to factor Xa, factor XIa, or factor XIIa. In certain embodiments, the anticoagulant is apixaban, edoxaban, fondaparinux, or rivaroxaban. The term “platelet inhibitor” or “platelet aggregration inhibitor” as used herein, refers to a substance that interferes with the clotting cascase to prevent platelet adhesion and clot formation. The most commonly used platelet inhibiotro is aspirin, but other include: adenosine diphosphate (ADP) receptor inhibitors (clopidogrel, ticagrelor, ticlopidine, prasugrel; adenosine reuptake inhibitors (dipyridamole); glycoprotein platelet inhibitors (abciximab, eptifibatide, tirofiban); phosphodiesterase inhibitors (cilostazol); and protease-activated receptor (PAR-1) antagonists (vorapaxar). In certain embodiments, the platelet inhibitor is aspirin (acetylsalicylic acid), clopidogrel (Plavix), prasugrel (Effient), ticagrelor (Brilinta), dipyridamole (Persantine), Eptifibatide (Integrilin), Tirofiban (Aggrastat), Abciximab (ReoPro), Cilostazol (Pletal), or Vorapaxar (Zontivity). In certain embodiments, the nanoparticle is associated with one or more targeting moieties. By the term “targeting moiety” as used herein, is meant a molecule, including an antibody, a fragment thereof or an antibody fusion protein, which is capable of specifically binding to another molecule. A targeting moiety may be an antibody, an aptamer, a nucleic acid, a peptide, a carbohydrate (sugar), a lipid, a vitamin, a toxin, a component of a microorganism, a hormone, a receptor ligand, and or any derivative thereof. If the targeting moiety is an antibody, it may be a monoclonal antibody, a humanized antibody, a synthetic antibody, a heavy chain antibody, and a biologically active fragment of an antibody, wherein the biologically active fragment is a Fab fragment, a F(ab')2fragment, and a Fv fragment. By the term “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. As used herein, antibody or antibody molecule contemplates intact immunoglobulin molecules, immunologically active portions of an immunoglobulin molecule, and fusions of immunologically active portions of an immunoglobulin molecule. The antibody may be a naturally occurring antibody or may be a synthetic or modified antibody (e.g., a recombinantly generated antibody; a chimeric antibody; a bispecific antibody; a humanized antibody; a camelid antibody; and the like). The antibody may comprise at least one purification tag. In a particular embodiment, the framework antibody is an antibody fragment. The term “antibody fragment” includes a portion of an antibody that is an antigen binding fragment or single chains thereof. An antibody fragment can be a synthetically or genetically engineered polypeptide. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those in the art, and the fragments can be screened for utility in the same manner as whole antibodies. Antibody fragments include, without limitation, immunoglobulin fragments including, without limitation: single domain (Dab; e.g., single variable light or heavy chain domain), Fab, Fab', F(ab')2, and F(v); and fusions (e.g., via a linker) of these immunoglobulin fragments including, without limitation: scFv, scFv2, scFv-Fc, minibody, diabody, triabody, and tetrabody. The antibody may also be a protein (e.g., a fusion protein) comprising at least one antibody or antibody fragment. As used herein, “specifically binding,” “binds specifically to,” “specific binding” refer, for example, to an antibody selectively or preferentially binding to an antigen. For example, with respect to a targeting moiety (such as an antibody), specifically binding refers to preferential binding refers to the ability of the antibody to bind one or more epitopes of an antigen or binding partner of interest without substantially recognizing and binding other molecules in a sample or environment containing a mixed population of antigens. Specific binding interactions are mediated by one or, typically, more noncovalent bonds between the binding molecules or binding partners. The antibodies utilized herein may be further modified. For example, the antibodies may be humanized. In a particular embodiment, the antibodies (or a portion thereof) are inserted into the backbone of an antibody or antibody fragment construct. For example, the variable light domain and / or variable heavy domain of the antibodies of the instant invention may be inserted into another antibody construct. Methods for recombinantly producing antibodies are well-known in the art. Indeed, commercial vectors for certain antibody and antibody fragment constructs are available. In certain embodiments, the nanoparticle compositions provided herein include a second targeting moiety that is selected for delivery to, e.g, a target organ or tissue, such as antibody that bind to the endothelium (e.g., antibodies targeting endothelial proteins including PECAM-1, ICAM-1, VCAM, ACE, transferrin receptor, tissue factor / platelet tissue factor / factor III, and others) or antibodies that bind to other targeted cells. The targeting moiety may bind to at least one of a cell surface protein, carbohydrate, or lipid. In some embodiments, the targeting moiety binds to a cell adhesion molecule (CAM). The CAM may be intercellular adhesion molecule (ICAM), platelet-endothelial cell adhesion molecule (PECAM), activated leukocyte cell adhesion molecule (ALCAM), B- lymphocyte cell adhesion molecule (BL-CAM), vascular cell adhesion molecule (VCAM), mucosal vascular addressin cell adhesion molecule (MAdCAM), CD44, LFA-2, LFA-3, P- selectin, and basigin. In certain embodiments, the targeting moiety binds specifically to a CAM selected from ICAM, PECAM, or VECAM. In certain embodiments, the nanoparticles include a second targeting moiety specific for ICAM-1 and are targeted to pulmonary endothelial cells and white blood cells in the lungs (since ICAM-1 is expressed on both cell types). In yet other embodiments, the targeting moiety binds specifically to ACE, transferrin receptor, or a suitable endothelial target known in the art (See e.g., Simone et al. Cell Tissue Res.2009 Jan; 335(1): 283–300, which is incorporated herein by reference). In other embodiments, the targeting moiety binds to a cell surface molecule associated with classical endocytosis. Preferably, the cell surface molecule associated with classical endocytosis is one of mannose-6-phosphate receptor and transferrin receptor. In certain embodiments, the nanoparticle is pre-loaded with an agent (also referred to as a “cargo” or a “drug”). In certain embodiments, the agent is a drug. The drug may be encapsulated in the inner volume and / or bound to the surface of nanoparticles. In one embodiment, the NP loading is high capacity, e.g., the mass of the drug is >5% the mass of the NP. In one embodiment the selected nanoparticle contains a single drug component. In another embodiment, the selected nanoparticle is loaded with multiple drug components. The term “drug” as used herein is meant any therapeutic, prophylactic, or diagnostic compound or reagent that is contained within the flexible nanoparticles described herein. In one embodiment, the drug is a water-miscible compound. In another embodiment, the drug is an anti-rejection drug. Anti-rejection drugs include agents such as alemtuzumab, tacrolimus, and other drugs currently delivered systemically post-transplant to prevent rejection. In another embodiment, the drug is an anti-inflammatory agent. Anti- inflammatory agents include corticosteroids, methotrexate, mycophenolate mofetil, azathioprine and other agents intended to limit inflammation. In another embodiment, the drug is an anti-edema agent, e.g., albuterol. In still another embodiment the drug is a compound that prevent ischemia-reperfusion injury, such as N-acetylcysteine, allopurinol, L-arginine, among known agents. In certain embodiments, the drug includes combinations of agents that act on multiple cell types, such as albuterol (acting on epithelial ENac to pump out alveolar fluid), dexamethasone (enhances endothelial barrier function and decreases neutrophil activity), and palifermin (enhances repair and regeneration of alveoli). In certain embodiments, the nanoparticle includes an agent that is an RNA molecule. The RNA molecule may express, for example, a therapeutic protein or an antigen (i.e. for inducing a protective immune response) in the case of nanoparticle-based vaccine. As described herein, administration of an anticoagulant in combination with a nanoparticle administration may affect delivery of a nanoparticle for a target organ and / or expression of an RNA molecule delivered by a nanoparticle. The present inventors have shown, for example, that administration of a direct thrombin inhibitor (such as bivalirudin) can preserve or restore the tropism of a nanoparticle composition and expression of an RNA in a target organ. In certain embodiments, the administration of a direct thrombin inhibitor preserves or restores the tropism of a nanoparticle composition and expression of an RNA in a target organ, while reducing or preventing coagulation. In still another embodiment, the agent is an imaging agent. An “imaging agent” as used herein is a compound that has one or more properties that permit its presence and / or location to be detected directly or indirectly. Examples of such imaging agents include proteins and small molecule compounds incorporating a labeled entity that permits detection. Among suitable imaging agents are molecules containing radionuclides that are amenable to SPECT or PET imaging (e.g., Indium-111 for SPECT imaging); molecules containing moieties that provide contrast for CT imaging (e.g., gold nanoparticles or iodinated contrast agents); molecules containing moieties that provide contrast for MRI imaging (e.g., gadolinium); nano- or micro-scale complexes that provide contrast for ultrasound imaging (e.g., microbubbles filled with gas). A “detectable label” is a marker used for detection or imaging. Examples of such labels include: a radiolabel, a fluorophore, a chromophore, or an affinity tag. In one embodiment, the label is a radiolabel used for medical imaging, for example tc99m or iodine-123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen- 15, oxygen- 17, gadolinium, manganese, iron, etc. In another aspect, a suitable nanoparticle for use in the methods herein is miscible with endothelial glycocalyx (e.g., carbohydrates on nanogel interdigitate with endothelial cells). In certain embodiments, the nanoparticle of the invention includes other components, such as one or more anti-platelet agents, glycoprotein inhibitors, fibrinolytic agents, thrombolytic agents, antibodies, or small molecule drugs. Non-limiting examples may include streptokinase, urokinase, or tissue plasminogen. The terms “associated with,” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more entities, means that the entities are physically associated or connected with one another, either directly or via one or more additional entities that serves as a linking agent, to form a structure that is sufficiently stable so that the entities remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An “association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization-based connectivity sufficiently stable such that the “associated” entities remain physically associated. As used herein, the terms “exterior” or “outer surface layer” with reference to a nanoparticle refers to the surface of the nanoparticle that contacts blood plasma when the nanoparticle is administered to a subject. The term “expression” is used herein in its broadest meaning and comprises the production of RNA, of protein, or of both RNA and protein. With respect to RNA, the term “expression” or “translation” relates in particular to the production of peptides or proteins. The term “antibody fragment” as used herein for the described methods and compositions refers to less than an intact antibody structure having antigen-binding ability. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as e.g. single chain Fab, scFv, and multispecific antibodies formed from antibody fragments. The “single chain Fab” format is described, e.g., in Hust M. et al. BMC Biotechnol.2007 Mar 8;7:14. scFvV constructs include complementary scFvs produced as a single chain (tandem scFvs) or bispecific tandem scFvs. As used herein, “specifically binding,” “binds specifically to,” “specific binding” refer, for example, to an antibody selectively or preferentially binding to an antigen. For example, with respect to a targeting moiety (such as an antibody), specifically binding refers to preferential binding refers to the ability of the antibody to bind one or more epitopes of an antigen or binding partner of interest without substantially recognizing and binding other molecules in a sample or environment containing a mixed population of antigens. Specific binding interactions are mediated by one or, typically, more noncovalent bonds between the binding molecules or binding partners. “Patient” or “subject” or “individual” as used herein means a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research. In certain embodiments, the subject is a human. The term “therapeutically effective amount” or “effective amount” refers to an amount agent that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject is effective to prevent or ameliorate a condition or disease. Compositions Provided herein are nanoparticle compositions that are modified to reduce or prevent coagulation following their delivery to a subject. In certain embodiments, the nanoparticle is associated with an anticoagulant on its exterior surface. The term “lipid nanoparticle”, also referred to as LNP, refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids). In certain embodiments, the lipid nanoparticle comprises a cationic lipid. In certain embodiments, the lipid nanoparticle comprises a cationic lipid and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid). In certain embodiments, an mRNA, or a portion thereof, is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells. In some embodiments, the mRNA or a portion thereof is associated with the lipid nanoparticles. In the context of the present disclosure, lipid nanoparticles are not restricted to any particular morphology, and should be interpreted as to include any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g., in an aqueous environment and / or in the presence of a nucleic acid compound. For example, a liposome, a lipid complex, a lipoplex and the like are within the scope of a lipid nanoparticle. A described herein, the nanoparticle binding to fibrinogen is associated with nanoparticle-induced toxicity. In certain embodiments, the provided nanoparticle composition is not capable of binding to fibrinogen, thereby preventing or reducing side effects associated with nanoparticle delivery. In certain embodiments, the co- administration of an anticoagulant prevents or reduced binding of fibrinogen to a nanoparticle following its administration to a subject. An LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids. In certain embodiments, the LNP comprises one or more cationic lipids as described herein, cholesterol, a helper phospholipid, and a polyethylene glycol-modified lipid. As mentioned, the LNP may be formulated to include a cationic lipid. The cationic lipid may be ionizable, i.e., it becomes protonated as the pH is lowered below the pKa of the ionizable group of the lipid, but is progressively more neutral at higher pH values. When positively charged, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease. The LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. In certain embodiments, the LNP comprises a cationic or ionizable lipid, i.e., any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N- dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3- (N—(N′,N′dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1-(2,3- dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoyl- 3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). Other useful lipids include, without limitation, 98N12-5, C12-200, PLGA, PEG, PEG-DMG, PEGylated lipids, amino alcohol lipids, and KL22. Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2-dioleoyl-sn- 3phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1- (2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2- dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA). In certain embodiments, the further cationic lipid is an amino lipid. Suitable amino lipids useful in the invention include those described in WO2012 / 016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy- 3morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy- 3dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3- (N,Ndilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 4- (dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13- nonadecadien-1-yl ester (DLin-MC3-DMA), N,N-dimethyl-2,2-di-(9Z,12Z)-9,12- octadecadien-1-yl-1,3-dioxolane-4-ethanamine (DLin-KC2-DMA). See also, e.g., WO2014 / 089486, US 2018 / 0353616A1, and US 8,853,377B2, which are incorporated by reference. In certain embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, or at least about 60% of the lipids of the LNP are a cationic or ionizable lipid. In certain embodiments, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 30% to about 95%, about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, or about 40% to about 50% of the lipids of the LNP are a cationic or ionizable lipid. In certain embodiments, LNP formulation is performed using routine procedures comprising cholesterol, ionizable lipid, helper lipid, PEG-lipid and polymer forming a lipid bilayer around encapsulated mRNA (Kowalski et al., 2019, Mol. Ther.27(4):710- 728). In some embodiments, LNP comprises a cationic lipid (i.e. N-[1-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), or 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP)) with helper lipid DOPE. In some embodiments, LNP comprises an ionizable lipid Dlin-MC3-DMA ionizable lipids, or diketopiperazine- based ionizable lipids (cKK-E12). In some embodiments, polymer comprises a polyethyleneimine (PEI), or a poly(β-amino)esters (PBAEs). In some embodiments, the LNP comprises C14-4 / DOPE / Chol / PEG-lipid. See, Rybakova Y., Kowalski P. S., Huang Y., Gonzalez J. T., Heartlein M. W., DeRosa F., et al.. (2019). mRNA delivery for therapeutic anti-HER2 antibody expression in vivo. Mol. Ther.27, 1415–1423 which is incorporated by reference. See also, e.g., WO2014 / 089486, US 2018 / 0353616A1, US2013 / 0037977A1, WO2015 / 074085A1, US9670152B2, and US 8,853,377B2, which are incorporated by reference. Certain LNPs useful herein include those that are described in WO 2021 / 077066 and WO 2021 / 055892, each of which is incorporated herein by reference in its entirety. LNP formulations may be varied to enhance delivery. For example, the type and ionizable lipid:mRNA ratio, molar ratio of ionizable lipid, phosopholipid, cholesterol, and PEG- lipid, etc. may be varied. In one embodiment, the LNP is one described by Kauffman, K. J.; Dorkin, J. R.; Yang, J. H.; Heartlein, M. W.; DeRosa, F.; Mir, F. F.; Fenton, O. S.; Anderson, D. G., Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano letters 2015, 15 (11), 7300-7306, which is incorporated herein by reference. In certain embodiments, the LNPs are designed with ionizable lipid: mRNA weight ratios varying between 5:1 to 25:1. In certain embodiments, the ionizable lipid: mRNA weight ratio is 5:1, 10:1, 12.5:1, 15:1, 20:1, or 25:1. Other LNPs have been described and are useful herein. See, e.g., WO 2016 / 118724, US 10,413,618B2, US 10,723,692B2, and US8754062B2, each of which is incorporated herein by reference. In certain embodiments, the LNP comprises one or more additional lipids which stabilize the formation of particles during their formation. Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- lcarboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2- oleoylphosphatidyethanol amine (SOPE), and 1,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl- sn-glycero-3phosphocholine (DSPC). In certain embodiments, the LNP includes a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2:1 to about 8:1. In various embodiments, the LNP includes a steroid or steroid analogue. In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid to cholesterol ranges from about 5:1 to 1:1. The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, Ndodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, Nglutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids. In certain embodiments, the LNP includes glycolipids (e.g., monosialoganglioside GM1). In certain embodiments, the LNP includes a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethoxy- polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s-DMG) and the like. In certain embodiments, the LNP includes an additional, stabilizing-lipid which is a polyethylene glycol-lipid (pegylated lipid). Suitable polyethylene glycollipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG- modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy- polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as ω-methoxy(polyethoxy)ethyl-N- (2,3di(tetradeca noxy)propyl)carba mate or 2,3-di(tetradecanoxy)propyl-N-(w- methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100:1 to about 25:1. Other exemplary LNPs and their manufacture are described in the art, for example in U.S. Patent Application Publication No. U520120276209, Semple et al., 2010, Nat Biotechnol., 28(2):172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1: e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids.2, e139; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which are incorporated by reference in their entirety. Pharmaceutical Compositions In another aspect, a pharmaceutical composition is provided that contains a nanoparticle. The pharmaceutical composition further contains a carrier, excipient, and / or preservative. As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host. In certain embodiments, the composition includes a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition may be transported as a concentrate which is diluted for administration to a subject. In other embodiments, the composition may be lyophilized and reconstituted at the time of administration. Methods and agents well known in the art for making formulations are described, for example, in “Remington's Pharmaceutical Sciences,” Mack Publishing Company, Easton, Pa. Formulations may, for example, contain excipients, carriers, stabilizers, or diluents such as sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes, preservatives (such as octadecyldimethylbenzyl, ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, and dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are nontoxic. In one embodiment, a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Pluronic® F68 [BASF], also known as Poloxamer 188, which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are commonly named with the letter “P” (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content. In one embodiment Poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension. These above compositions may be administered in a variety of volumes of carrier, excipient or buffer formulations, ranging from about 25 to about 1000 microliters, or higher volumes, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. Any suitable route of administration may be selected. Accordingly, pharmaceutical compositions may be formulated for any appropriate route of administration, for example, in the form of liquid solutions or suspensions (as, for example, for intravenous or intra- arterial administration). Methods The compositions provided herein are useful for reducing or preventing coagulation resulting from administration of a nanoparticle composition. In certain embodiments, a method of delivering an agent to a target organ of a subject is provided, wherein the method comprises administering to the subject an anticoagulant and / or and a nanoparticle comprising the agent. In certain embodiments, the methods decrease coagulation (thrombosis) in a target organ. In yet another embodiment, the methods increase the time needed for clotting (thrombosis) to occur in a target organ. Methods of assessing coagulation (thombosis) in a subject are known in the art. In certain embodiments, the methods comprise assessing coagulation (thombosis) in a subject by measuring platelet activation (e.g., P-selectin expression levels), platelet aggregation, partial thromboplastin time (PTT) or activated partial thromboplastin time (aPTT), and / or thrombin–antithrombin (TAT) complex in a biological sample obtain from the subject. In certain embodiments, the biological sample is whole blood, serum, or plasma. By “administering” or “route of administration” is meant delivery of composition described herein, with or without a pharmaceutical carrier, excipient, and / or preservative, to the subject. Routes of administration may be combined, if desired. In certain embodiments, the method includes intravenous delivery of a composition described herein. In certain the method includes intraarterial delivery of a composition described herein. In certain embodiments, the nanoparticles are used to treat acute respiratory distress syndrome (ARDS). ARDS is an acute, diffuse, inflammatory lung injury with a variety of causes, most commonly pneumonia and sepsis. ARDS causes the lungs’ air sacs, called alveoli, to fill up with proteinaceous liquid, preventing the lungs from oxygenating the blood. The impact of ARDS is enormous, with 190,000 US cases per year, and a mortality rate of 35%. Decades of research have yielded myriad drug targets, but after the failure of more than a dozen large clinical trials, there are still no FDA approved drugs that improve survival in ARDS. From a pharmacology perspective, there are three reasons why many rationally chosen drugs have failed in ARDS. Firstly, ARDS patients are too fragile to tolerate drug side effects. These patients have multi-organ dysfunction, and thus cannot tolerate even mild side effects. Secondly, the inhalational route of delivery, useful for so many pulmonary problems, has limited benefit in ARDS, as the flooded alveoli (those filled with liquid) are covered by a column of fluid, which means that topical delivery to the alveoli is not possible via the inhaled route. Finally, ARDS is a very heterogeneous disease, so targeting a single pathway is unlikely to be sufficient. In another aspect, provided herein are methods of treating inflammation or treating an inflamed tissue. The method includes administering nanoparticles to a subject in need thereof. The inflammation may be attributable, e.g., to an inflammatory disorder and / or infection in the subject. In certain embodiments, the inflamed tissue is lung. In certain embodiments, the method includes administration of a nanoparticle intravenously. Optionally, routes other than intravenous administration may be used, such as, e.g., oral, intranasal, intratracheal, intraarterial, intraocular, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Routes of administration may be combined, if desired. In certain embodiments, administration of the compositions described herein can be intravenous (iv) for delivery of the drug to the lungs. For example, where the disease is ARDS, pneumonia, interstitial lung disease, idiopathic pulmonary fibrosis, post- pulmonary embolism, pulmonary capilliaritis syndrome, emphysema, or a viral infection (such as SARS, influenza, or COVID), the composition may be administered intravenously. In certain embodiments, the methods employ injecting intravenously the compositions described herein carrying one or more of albuterol, dexamethasone, and palifermin for the treatment of ARDS. In certain embodiments, where the disease involves any other selected mammalian organ, the composition is administered in vivo intra- arterially immediately upstream of the target organ for delivery of an agent. Additionally, for treatment of disease involving a selected mammalian organ designated for transplantation (other than the lung), the composition is administered ex vivo via feeding arterial opening into the organ prior to reperfusion and transplantation. In certain embodiments, the composition is administered via an arterial conduit of a selected organ using an arterial catheter. In any intra-arterial administration, the composition can be administered via an intra-arterial catheter. Such organs include, without limitation, the heart, brain, eyes, thyroid, kidney, liver, pancreas, spleen, intestines, or prostate. In certain embodiments, the method further includes administration of a therapeutic agent in addition to an nanoparticle composition. In certain embodiments, the therapeutic agent is an anticoagulant. In certain embodiments, the nanoparticle composition and additional agent are delivered essentially simultaneously via the same route of administration. In certain embodiments, the nanoparticle composition is delivered subsequent to the additional agent(s). In other embodiments, the nanoparticle composition is delivered first. As used herein, the term “administered in combination” or “combined administration” means that two or more compositions (e.g., a nanoparticle and an anti- coagulant) are administered to a subject at the same time or within an interval such that there can be an overlap of an effect of each composition on the patient. In certain embodiments, they are administered within about 60 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute of one another. In certain embodiments, the administrations of the compositions are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved. In some embodiments, the administration in combination can be concurrent (i.e., all the nanoparticles are administered as part of a single formulation, or different nanoparticles in different formulation are administered simultaneous), or consecutive (e.g., several nanoparticles in several formulations are administered consecutively). In still a further aspect, the compositions and methods can also be employed for imaging, such as to map capillary structure or pathology, wherein the drug is an imaging reagent. In one embodiment, a method of imaging a mammalian organ comprises injecting intravenously or intraarterially in vivo or ex vivo a composition comprising dual-targeted nanoparticles described herein, wherein the nanoparticles contain an imaging drug, and wherein the nanoparticles are transferred from the dual-targeted nanoparticles to the vascular bed of a target organ of the subject and release the imaging drug to the selected target organ or vascular tissue. EXAMPLES The following examples disclose specific embodiments of preparation of compositions of this invention, their characteristics, and methods of use thereof. These examples should be construed to encompass any and all variations that become evident as a result of the teachings provided herein. Example 1: Materials and Methods DOTAP, 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE), cholesterol, 1,2- dimyristoyl-rac-glycero-3methoxypolyethylene glycol-2000 (DMG-PEG 2000), dipalmitoyl phosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3phosphoethanolamine- N-(TopFluor AF594) (18:1 PE TopFluor AF 594, ammonium salt), 1,2-distearoylsn- glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (18:0 PE-DTPA, ammonium salt), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS, sodium salt), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (DSPE-PEG2000-azide, ammonium salt) were purchased from Avanti Polar Lipids. Ionizable lipids cKK-E12, 306-N16B, SM-102, ALC-0315, and C12-200 were purchased from Echelon Biosciences. Anti-mouse BV421 CD41, PE-Cy7 CD62P AF700 Ly6G, and APC CD42d were purchased from Biolegend. Indium-111 chloride (In-111) was purchased from BWXT Medical. A modified Lowry assay kit (DC Protein Assay) was purchased from Bio-Rad Laboratories. tPA was purchased from Millipore. Animals All animal studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD), and all animal protocols were approved by the University of Pennsylvania Institutional Animal Care and Use Committee. All animal experiments were carried out using male, 6–8 week old C57BL / 6 mice (23–25 g) (The Jackson Laboratory, Bar Harbor, ME). The mice were maintained at 22–26 °C and adhered to a 12 / 12 h dark / light cycle with food and water ad libitum. For all experiments, mice were anesthetized in a chamber with 4% isofluorane in 100% oxygen. Nanoparticle Formulation LNPs were formulated using the microfluidic mixing method. An organic phase containing a mixture of lipids dissolved in ethanol at a designated molar ratio (FIG. xx) was mixed with an aqueous phase (50 mM citrate buffer, pH 4) containing luciferase mRNA that was either purchased by TriLink (most experiments) or made in-house via in vitro transcription (IVT) (see Parhiz, H. et al., J. Controlled Release 2022, 344, 50). at a flow rate ratio of 1:3 and at a total lipid / mRNA weight ratio of 40:1 in a microfluidic mixing device (NanoAssemblr Ignite, Precision Nanosystems). LNPs were dialyzed against 1 × PBS in a 10 kDa molecular weight cut-off cassette for 2 h, sterilized through a 0.22 µm filter, and stored at 4 °C. To manufacture LNPs of 180 nm, the flow rate of the NanoAssemblr Ignite varied, from 6 mL min−1(for 100 nm) to 1 mL min−1(180 nm). The authors were unable to produce uniform LNPs < 100 nm with TriLink mRNA, but with in-house IVT mRNA, LNPs of 80 nm were made at a flow rate of 6 ml min−1. Liposomes were synthesized using the thin film hydration method. Lipids were dissolved in chloroform and combined in a borosilicate glass tube. Chloroform and ethanol were evaporated by blowing nitrogen over the solution until visibly dry (≈15 min) and then placed in the tube under vacuum for > 1 h. Dried lipid films were hydrated with 1 × PBS, pH 7.4 to a total lipid concentration of 20 mM. The rehydrated lipid solution was vortexed and sonicated in a bath sonicator until visually homogeneous (≈1 min each of vortexing and sonication). The solution was then extruded 21 times through a 0.2 µm polycarbonate filter. Nanoparticle Characterization Dynamic light scattering measurements of hydrodynamic nanoparticle size, distribution, polydispersity index, and zeta potential were made using a Zetasizer Pro ZS (Malvern Panalytical). LNP RNA encapsulation efficiencies and concentrations were measured using a Quant-iT RiboGreen RNA assay (Invitrogen). Nebulized LPS Model Mice were exposed to nebulized LPS in a “whole-body” exposure chamber, with separate compartments for each mouse (MPC-3 AERO; Braintree Scientific, Inc.; Braintree MA). To maintain adequate hydration, mice were injected with 1 mL of sterile saline, 37 °C, intraperitoneally, immediately before exposure to LPS. LPS (L2630- 100 mg, Sigma Aldrich) was reconstituted in PBS to 10 mg mL−1and stored at −80 °C until use. Immediately before nebulization, LPS was thawed and diluted to 5 mg mL−1with PBS. LPS was aerosolized via a mesh nebulizer (Aerogen, Kent Scientific) connected to the exposure chamber (NEB-MED H, Braintree Scientific, Inc.). 5 mL of 5 mg mL−1LPS was used to induce the injury. Nebulization was performed until all liquid was nebulized (≈20 min). TAT and Blood Count Measurements LNPs were retro-orbitally injected into naïve or nebulized LPS-injured mice for a circulation time of 30 min. Blood was collected from mice into tubes containing EDTA. Blood cells were analyzed using an Abaxis VetScan HM5 Hematology Analyzer for complete blood counts. Blood was then centrifuged at 1500 × g for 10 min and plasma was collected and analyzed with a TAT ELISA kit (Abcam) according to the manufacturer's instructions. Lung Histology LNPs were retro-orbitally injected into naïve mice for a circulation time of 30 min. After exsanguination and perfusion via the right ventricle with ≈5 mL of phosphate- buffered saline (PBS) at a constant pressure of 25 cm H2O, whole lungs were inflated and fixed with neutral buffered 10% formalin. Paraffin-embedded 5 µm lung sections were stained and imaged with Masson's trichrome by the Pathology Core Laboratory of Children's Hospital of Philadelphia. In Vitro and In Vivo PT, APTT, and Time to Clot Assays PT and APTT measurements were carried out with a STart Analyzer (Stago) and Pacific Hemostasis reagents at a temperature of 37 °C. In these studies, an oscillating stainless steel ball was placed in the sample and after the formation of a clot, this ball was attenuated and the time interval for clot formation was automatically recorded. Blood was first collected from naïve mice and anticoagulated with 3.2% sodium citrate at a ratio of 10:1, blood:citrate, vol:vol. Blood samples were then centrifuged at 1500 × g for 10 min and the plasma was collected.50 µl of plasma was incubated with 0.5 µg of mRNA in LNPs or a specified number of particles for 5 min. For the PT assay, 50 µl of LNP-treated plasma was diluted up to 100 µl with PBS and 200 uµl of PT reagent (Pacific Hemostasis) was added and the time to clot was automatically measured. For the APTT assay, 50 µl of LNP-treated plasma was diluted up to 100 µl of PBS and 100 µl of APTT-XL reagent (Pacific Hemostasis) was added for an incubation time of 4 min.100 µl of 0.02 M calcium chloride was then added and the time to clot was automatically measured. For in vivo measurements, blood was collected from treated mice and then centrifuged at 1500 × g for 10 min to isolate the plasma. Plasma was then analyzed as described. For time-to-clot assays, 50 µl of plasma was placed in the STart Analyzer with an oscillating stainless steel ball. After recalcification with 100 µl of 0.02 M calcium chloride, the time to clot was immediately measured based on the attenuation of the ball. NTA Assay for Fibrinogen Aggregation with LNPs To prepare fluorophore-labeled fibrinogen, mouse fibrinogen (Innovative Research) was incubated with NHS ester Alexa Fluor 488 (ThermoFisher) at 1:10 mol:mol ratio in PBS at 4 °C for 16 h. Afterward, excess fluorophore was removed from fibrinogen by a 3-fold passage against a 10 kDa molecular weight cut-off centrifugal filter (Amicon) with PBS washing between passages. After fibrinogen recovery from the centrifugal filter, spectrophotometer measurement of optical density at 280 nm (Nanodrop) determined fluorescent fibrinogen concentration and optical density measurement at 488 nm determined the number of fluorophores per fibrinogen. Immediately before experiments, LNP concentrations were determined by NTA (Nanosight, Malvern). In a total reaction volume of 40 µL, 4E10 LNPs were combined with 20 µL of heparinized mouse serum (a pooled sample obtained from n = 3 mice) and fluorescent fibrinogen was doped into the solution at a final physiologically relevant concentration of 3 mg mL−1. Fluorescent fibrinogen, serum, and LNPs were incubated in the dark at room temperature for 10 min. Fluorescent fibrinogen was also added to serum solutions at identical concentrations, without LNPs, verifying that fluorescent fibrinogen did not detectably adhere to endogenous serum components. The fibrinogen-serum-LNP reactions were terminated by 1:250 dilution in PBS and the diluted suspensions were used for nanoparticle tracking analysis. NTA was conducted with a 488 nm excitation laser and a 500 nm long pass filter to image and track the Alexa Fluor 488 signal from fluorescent fibrinogen on LNPs. Automated analysis of fluorescence nanoparticle tracking data in Malvern Nanosight software used a uniform detection threshold of 5 for all samples. For both fluorescence data and scattering data, three to five technical replicates were obtained for each sample and an average of those replicates was taken as representative of the size- concentration profile for each sample. Circular Dichroism Measurements were recorded using a 1500 circular dichroism spectrometer (Jasco) with a 1 mm path length quartz cell over a wavelength range of 190–260 nm. Data were collected at a temperature of 37 °C with a bandwidth of 1 nm at 50 nm min−1and a CD scale of 200mdeg / 0.1dOD. Fibrinogen was diluted in PBS at a concentration of 1 mg ml−1and for readings with LNPs, 4E11 particles were incubated with fibrinogen for 5 min before reading. Radiolabeling and Biodistributions For biodistribution studies, nanoparticles were traced with In-111 as previously described (Marcos-Contreras, O. A., et al., Proc. Natl. Acad. Sci. U. S. A.2020, 117, 3405). Nanoparticles were produced as described above with 0.1 mol% of 18:0 PE-DTPA (a chelator containing lipid) using metal-free buffers. Trace metals were removed from the buffers using a Chelex 100 resin, per the manufacturer's instructions, to prevent unwanted occupancy of the chelator. In-111 chloride was added to the nanocarriers at a specific activity of 1 µCi of In-111 per 1 µmol of lipid. The mixture was incubated at room temperature for 30 min. Then, unincorporated In-111 was removed using a Zeba Spin desalting column. The removal of unincorporated In-111 was verified using thin film chromatography (TLC). A 1 µL sample of nanoparticles was applied to the stationary phase (silica gel strip). The strip was placed in the mobile phase of 10 mM EDTA until the solvent front was 1 cm from the end of the strip (≈10 min). The strip was cut 1 cm above the initial sample location. In-111 chelated to the nanoparticles stayed at the origin, while unchelated In-111 traveled with the solvent front. The activity in each section was measured using a gamma counter. The percent of In-111 chelated to the nanoparticles was calculated as the activity in the origin strip divided by the total activity in both strips. For all experiments, > 95% of In-111 was chelated to the nanoparticles. For biodistributions of fibrinogen, the protein was radiolabeled with iodine-125 using the Iodogen method. Tubes coated with 100 µg of Iodogen reagent were incubated with fibrinogen (2 mg mL−1) and Na125I (115µCi per µg protein) for 5 min on ice. Unincorporated I-125 was removed with a Zeba column followed by TLC analysis as described above. For nanoparticle biodistributions, In-111 labeled nanoparticles were injected into mice for a circulation time of 30 min. For fibrinogen biodistributions, I-125 fibrinogen was allowed to circulate for 2 min before the injection of nanoparticles for 30 min before harvest. The radioactivity in each organ was then read with a gamma counter (Wizard2, PerkinElmer). Confocal Microscopy Healthy mice were injected with 150 µg of fibrinogen fluorescently labeled with AF647-NHS-Ester. Two minutes later, mice were injected with fluorescent + or −DOTAP LNPs formulated with 0.3 mol % of 18:1 PE TopFluor AF 594. After a 30-min circulation the animals were sacrificed and perfused with 5 ml of cold PBS and lungs were harvested and freshly frozen.10 µm lung slices were cryosectioned for imaging using a Leica TCS SP8 confocal microscope. Luciferase Delivery Luciferase mRNA LNPs fabricated as described above were injected into mice for a circulation time of 4 h. Select organs were then flash-frozen until the day of analysis or homogenized immediately. Samples were suspended in 900 µL of homogenization buffer (5 mM EDTA, 10 mM EDTA, 1:100 diluted stock protease inhibitor [Sigma], and 1 × PBS, samples were then loaded with a steel bead [Qiagen], then placed in a tissue homogenizer [Powerlyzer 24, Qiagen] using the following settings: Speed (S) 2000 rpm, 2 cycles (C), time (T) 45 s, and pause for 30 s). After this, 100 µL of lysis buffer (10% Triton-X 100 and PBS) was added into each tube and then allowed to incubate for 1 h at 4 °C. After this, they were immediately transferred into fresh tubes, and sonicated, using a point sonicator to remove in excess DNA, using an amplitude of 30%, five cycles of 3 s on / off. After this, samples were then centrifuged at 16 000 × g for 10 min. The resultant lysate was either frozen or prepared for luminometry analysis. For luciferase expression, 20 µL of undiluted sample was loaded onto a black 96 well-plate and then 100 uL luciferin solution (Promega) was added immediately before reading on a luminometer (Wallac). Last, a Lowry assay (Bio-Rad) was performed according to manufacturer's specifications using diluted samples, specifically a 1:40 dilution for lung and spleen tissues and a 1:80 dilution for liver tissues. Final luminescence readings were then normalized based on the total protein concentration obtained from the Lowry assay. Platelet Flow Cytometry Platelet-rich plasma (PRP) was collected by first adding 15 µL of 10 mg mL−1bivalirudin as well as flushing 23G collection needles with the same solution bivalirudin, collecting between 500–1000 uL of blood per mouse. After the blood draw, samples were then centrifuged at room temperature (RT) at 200 × g for 10 min. After centrifugation, PRP was collected, taking care not to disturb the resultant pellet. After collection, samples were immediately read on a hematology analyzer (Vetscan HM2, Abaxis) to obtain platelet count. Resultant platelets were then aliquoted to achieve a total of 400 000 platelets per analysis with Tyrode's buffer. These platelet samples were then incubated with 0.5µg mRNA dose of AlexFluor 594 labeled −DOTAP or +DOTAP LNPs for 30 min at 37 °C. After incubation with nanoparticles, samples were stained with flow cytometry antibodies against CD41, CD42d, and P-selectin and allowed to incubate at RT for 10 min. Samples were then immediately diluted with 350 µL of Tyrode's buffer and read on LSR Fortessa (BD Bioscience). Analysis was then completed using FloJo and the gating strategy employed is found in FIG. xx, Supporting Information. For fibrinogen- depleted plasma, 0.278 mg of tPA was added per mL of platelet-free plasma, followed by 3 h incubation at 37 °C. Statistics All results were expressed as mean ± SEM unless specified otherwise. Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software) * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001. Example 2: Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting Here, we show that lung tropic, positively charged, LNPs induce massive thrombosis. This finding was quite general, as it held for LNPs with different permanently positively charged lipids, regardless of their co-loaded ionizable lipid, and not just LNPs, but also liposomes. Perhaps most surprisingly, we also observed thrombosis caused by LNPs that lacked a permanently cationic lipid (one possessing a quaternary amine) but instead contained an ionizable lipid (possessing a tertiary amine) that was found via screening to confer lung tropism. The lung-tropic LNPs induced profound pulmonary embolism when administered intravenously, induced stroke-like effects when administered via the carotid artery, and modified clotting processes in ex vivo blood. We investigated detailed mechanisms of LNP-induced clotting, showing that lung-tropic LNPs bind to the core clotting protein, fibrinogen, alter its secondary structure, and cause aggregation and activation of platelets. Elucidating these detailed mechanisms of clotting enabled us to develop three solutions that may permit positively charged LNPs to maintain their lung- targeting property while ameliorating dangerous clotting: Pre-treatment with anticoagulants (direct thrombin inhibitors, but not the seemingly obvious choice of heparin), conjugation of the LNPs to direct thrombin inhibitors, or reduction of the LNP size. Lung-Tropic LNPs with Cationic Lipids Induce Gross Side Effects In Vivo We fabricated physicochemically-targeted, lung-tropic LNPs by the previously published method of adding in a cationic lipid, 1,2-dioleoyl-3-trimethylammonium- propane (DOTAP, chloride salt). We initially synthesized these LNPs with the ionizable lipid cKK-E12 (herein referred to as +DOTAP LNPs, unless otherwise stated). This base +DOTAP LNP formulation consisted of the lipids DOTAP, cKK-E12, 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), cholesterol, 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG 2000) (50 / 25 / 5 / 18.5 / 1.5, mol / mol) and mRNA (lipid / mRNA ratio = 40 / 1, wt / wt). Control, non-lung tropic LNPs were synthesized without DOTAP (FIG.1A). There was no significant difference in the sizes of these LNPs (99.22 ± 4.27 nm for −DOTAP LNPs and 102.47 ± 0.48 nm for +DOTAP LNPs) (FIG. 1B). However, +DOTAP LNPs had a significantly higher zeta potential than −DOTAP LNPs (14.34 mV vs −4.05 mV) due to the addition of the cationic lipid (FIG.1C). We next intravenously (IV) injected the above LNPs into naive mice at a dose of 10 µg of mRNA per mouse and sacrificed 30 min later for gross anatomical inspection. We observed that in mice that received +DOTAP LNPs, the lungs were strikingly red and firm, resembling “hepatization” (liver-like appearance) described by pathologists for human acute respiratory distress syndrome (ARDS) (FIG.1D). We observed clear lethargy and sluggishness in +DOTAP LNP mice. Using the software DeepLabCut, we found that after a recording time interval of 1 h post-LNP injection, +DOTAP LNP mice walked a total distance that was > 17-fold less than naïve mice (FIG.1E). We further assessed lung- specific toxicity by measuring the contents of the bronchoalveolar lavage (BAL) fluid 20 h after IV +DOTAP LNP injection, which assesses lung capillary leak. In naive mice, there was a dose-dependent increase in both BAL protein and leukocyte count from 5 to 10 µg of mRNA (FIG.1F and FIG.2). Because lung-targeted LNPs will generally be given to patients with lung pathology, we next investigated whether +DOTAP LNPs caused even more severe injury in mice with pre-existing lung inflammation. We utilized a mouse model of acute lung inflammation achieved by administering nebulized lipopolysaccharides (LPS). +DOTAP LNPs were IV injected 4 h after LPS exposure and mice were sacrificed 20 h later. As in naïve mice, we saw a dose-dependent increase in BAL protein concentration from 5 to 10 µg of mRNA (FIG.1G). However, in nebulized LPS mice, there was an exacerbation of BAL edema at each dose tested. At the 10 µg dose of mRNA, nebulized LPS mice have 6-fold higher BAL edema compared to naïve mice injected with +DOTAP LNPs. This indicates that the toxicity of +DOTAP LNPs is amplified under pre-existing inflammatory conditions. Based on these data, a dose of 10 µg of mRNA in LNPs was used for further in vivo studies unless otherwise stated. This is a therapeutically relevant dose for mRNA- LNP delivery and enabled us to adequately assess the mechanisms behind DOTAP LNP toxicity. Coagulation is Triggered by LNPs and Other Nanoparticles with Cationic Lipids, across Diverse Formulations Given the very acute nature of the side effects of +DOTAP LNPs, seen within a minute on the AI-measured distance-walked test, we hypothesized that the nanoparticles were activating a protein-based defense system in the blood. The three major plasma protein defense systems are the complement cascade, immunoglobulins, and the coagulation cascade. Since complement has been long implicated in nanoparticle defense, and since immunoglobulins’ acute effects also lead to complement activation (via IgG-mediated activation of the classical pathway of complement), we first investigated if these LNPs activate the complement system. Surprisingly, we discovered that +DOTAP LNPs do not activate complement, as we detected no increase in the plasma concentration of C3a 30 min after +DOTAP LNP IV injection into naive mice (FIG.3), noting that we have extensively shown C3a production induced by other nanoparticles. Without C3a production, it is highly unlikely that complement activation, or acute immunoglobulin effects, mediate +DOTAP LNPs’ acute toxicity. Therefore, we investigated whether the toxicity was produced by the coagulation cascade. To investigate this, we stained lung sections with Masson's trichrome to look for evidence of clots. Lung histological samples show large clots both at the large vessel and capillary levels in naïve mice IV injected with +DOTAP LNPs, compared to mice injected with −DOTAP LNPs where there are no observable clots (FIG.4A). This was a clear indication of thrombosis (clotting) due to +DOTAP LNPs. In these mice, visible clots were not observed in the liver or the spleen (FIG.5A and FIG.5B). However, this effect is not restricted to the lungs since we also observed evidence of clotting in the brain when +DOTAP LNPs were injected via the carotid artery (FIG.5C). These results indicate the incidence of clots in the first capillary bed downstream of injection (lungs after IV injection; brain after intra-arterial injection). To quantify the extent of clotting, we measured thrombin-antithrombin (TAT) plasma levels in both naïve and nebulized-LPS injured mice. TAT is a stable complex formed after thrombin activation that serves as a marker of coagulation. We found that +DOTAP LNPs increase TAT levels by > 2-fold in naive mice and by an additional > 1.5-fold in nebulized-LPS injured mice (FIG.4B). This confirmed that +DOTAP LNPs activate thrombin (the common pathway of coagulation), and this is exacerbated in pre-existing inflammatory conditions, probably as a result of thrombo-inflammation. Furthermore, we show that in addition to permanently cationic lipids like DOTAP, ionizable cationic lipids that are used to induce lung tropism also induce clotting. An example of such an ionizable lipid is 306-N16B, which was shown in a recent PNAS paper to produce excellent lung tropism and is also cationic at neutral pH (FIG.6 and FIG.7). However, we found that 306-N16B LNPs also increase TAT plasma levels by ≈2.5-fold in naïve mice (FIG.4C) and demonstrate other indications of coagulation (FIG.8A – FIG.8C). This finding therefore generalizes this clotting phenomenon to a wide variety of physicochemically-targeted LNPs that rely on positive charge for lung localization. We sought to determine which molecular coagulation pathways underlie LNP- induced thrombosis. Coagulation can be divided into the intrinsic and extrinsic pathways (FIG.4D). The extrinsic pathway is activated by trauma that damages the endothelium, leading to exposure of tissue factor which activates the coagulation protein factor VII. The intrinsic pathway is initiated by factors within the blood vessel lumen and has been shown to be triggered by charged surfaces (such as foreign particles) or collagen, which trigger the activation of the coagulation protein factor XII. The extrinsic and intrinsic pathways converge to the common pathway which involves the conversion of prothrombin to thrombin – the enzyme that converts fibrinogen to fibrin. Fibrin forms the mesh-like framework of clots. The prothrombin time (PT) and activated partial thromboplastin time (APTT) are laboratory tests used to measure the time to clot through either the extrinsic or intrinsic pathways, respectively. To determine if +DOTAP LNP-activated coagulation employs the intrinsic or extrinsic pathway, we measured PT and APTT after doping LNPs into plasma in vitro. While +DOTAP LNPs did not change the PT (FIG.4E), they led to a significant increase in APTT (FIG.4F and FIG.4G). This indicates that +DOTAP LNPs activate the intrinsic coagulation pathway, thus depleting intrinsic pathway proteins and prolonging the APTT. Furthermore, the effects of +DOTAP LNPs on coagulation are dependent on both the number of LNPs injected and the mole fraction of DOTAP in the formulation (FIG.9B, FIG.9D, and FIG.9E). We formulated liposomes and other LNPs with DOTAP to see if the coagulation side effects were a generalizable result of co-formulation with cationic lipids. We formulated LNPs with various ionizable lipids (C12-200, ALC-0315, SM-102; FIG.6 and FIG.7) and found that +DOTAP LNPs increase APTT, regardless of the ionizable lipid type (FIG.4H). We used DOTMA, rather than DOTAP, as our cationic lipid and observed the same effect on APTT (FIG.10). Adding DOTAP to liposomes, rather than LNPs, also results in increased APTT (FIG.4I and FIG.11A). Finally, 306-N16B LNPs also lead to an increase in clotting time in vivo (FIG.8C). Thus, this clotting phenomenon is generalizable to different LNP and liposome formulations containing both ionizable and permanently cationic lipids. Notably, these side effects appear to be associated with a positive charge as anionic LNPs formulated with the negatively charged lipid DOPS do not induce clotting (FIG.11B and FIG.11C) LNPs with Cationic Lipids Aggregate and Activate Platelets Having proven that +DOTAP LNPs activate the coagulation cascade, we sought to assess if +DOTAP LNPs also cause activation and aggregation of platelets, since clots can also form due to platelet aggregates. We measured complete blood counts (CBCs) in blood drawn 30 min post-injection of +DOTAP LNPs. CBCs showed that +DOTAP LNPs greatly reduced the number of circulating platelets in mice with pre-existing inflammation (nebulized LPS), and also showed a trend towards reduced platelets in naive mice (FIG. 12A). This reduction in platelet count is dependent on both the number of +DOTAP LNPs injected and the mol fraction of DOTAP in the LNP formulation (FIG.9A and FIG.9C). Notably, LNPs targeted to the lung using antibodies against platelet endothelial cell adhesion molecule (PECAM) do not have the same effect on platelets showing that this is due to a positive charge (FIG.13A – FIG.13E). Furthermore, ionizable cationic 306- N16B LNPs also significantly decrease platelet count when injected into naïve mice (FIG. 12B). Reduced platelet count (thrombocytopenia) can indicate depletion of platelets from circulation due to incorporation in clots. We wanted to investigate if this thrombocytopenia was due to platelet activation by +DOTAP LNPs. To measure platelet activation, we employed flow cytometry. As shown in the schematic of FIG.12C, we used forward scatter (FSC) and side scatter (SSC) measurements to detect larger and more complex platelet aggregates versus small, isolated platelets. We stained for two specific markers of platelets: Glycoprotein V (GPV; CD42d), and P-selectin (CD62p) (FIG.14A). CD42d is constitutively found on the surface of all platelets. In resting platelets, P-selectin is generally stored in platelet granules, which are externalized when the platelet becomes activated. Platelet-rich plasma (PRP; FIG.12D) was either left untreated or incubated with fluorescently labeled −DOTAP or +DOTAP LNPs and stained for CD42d and P-selectin. FSC versus SSC plots for platelets (CD42d-positive events) in untreated, −DOTAP, and +DOTAP LNP-treated PRP show no difference between the untreated and −DOTAP LNP-treated conditions, but the shape of the +DOTAP LNP graph differs markedly from the other two (FIG.12E). The mean FSC and SSC values are elevated for platelets in the presence of +DOTAP LNPs, indicating the formation of aggregates (FIG. 12F and FIG.12G). Further evidence of platelet aggregation induced by +DOTAP LNPs is shown by increased CD42d signal for each CD42d-positive event (FIG.12H). In the presence of +DOTAP LNPs, each CD42d-positive event is a cluster of platelets, rather than an individual cell, so the mean CD42d signal is increased versus untreated and −DOTAP LNP-treated samples. Most interesting, however, is the 20-fold increase in P- selectin presentation induced by +DOTAP LNPs (FIG.12I). Since increased surface presentation of P-selectin is a marker of platelet activation, this demonstrates profound platelet activation induced by +DOTAP LNPs. Tracing LNP fluorescence in our flow cytometry measurements, we found that 100% of platelets were physically associated with fluorescently labeled +DOTAP LNPs, while only 50% of platelets were physically associated with −DOTAP LNPs, showing that the cationic lipid profoundly increases LNP adhesion to platelets (FIG.12J). Specifically, among platelets that had an LNP signal, we assessed the P-selectin signal. For −DOTAP- LNPs, only 30% of the LNP-associated platelets were P-selectin positive, indicating that −DOTAP LNPs had little effect on platelet activation. However, for +DOTAP LNPs, nearly 100% of LNP-positive platelets were also positive for P-selectin, indicating that platelet association with +DOTAP LNPs predicts platelet activation (FIG.12K). DOTAP LNPs bind to Fibrinogen, and Fibrinogen is Required for LNP-Induced Platelet Activation Having proven that +DOTAP LNPs cause clotting, initiate the coagulation cascade, and activate platelets, we sought to isolate components of the +DOTAP LNP protein corona that might lead to these effects. Upon IV injection, LNPs can form unique protein coronas based on their physicochemical properties and this could alter their functionality. The protein corona can therefore drive the biodistribution and cell-type localization of LNPs. However, the proteins adsorbed on LNPs could also induce side effects. Proteins can undergo conformational changes when adhering to surfaces and aggregates of proteins on surfaces do not often behave similarly to proteins in solution, including causing immunogenic or thrombotic side effects. We therefore hypothesized that +DOTAP LNPs could bind to coagulation proteins in plasma, the most abundant being fibrinogen. Fibrinogen is a soluble protein found in plasma. When it is cleaved by thrombin, fibrinogen undergoes conformational changes and aggregates to form fibrin strands, which in turn form the clot-stabilizing mesh. Fibrinogen / fibrin aggregates on surfaces could cause adhesion and activation of platelets. Fibrinogen has a net negative charge at physiological pH and contains domains with a high concentration of negatively charged residues, such as the E domain. Studies have linked the exposure of certain fibrinogen residues to specific side effects. For example, nanoparticle-induced exposure of a peptide sequence at the C-terminus of the fibrinogen γ- chain interacts with the integrin receptor Mac-1 and induces inflammation. We therefore hypothesized that the positively charged DOTAP in LNPs could interact with, and aggregate fibrinogen in a way that could lead to coagulation and platelet activation. This hypothesis is supported by our finding that +DOTAP LNPs decrease the expression of CD41 on platelets (FIG.14B). Since CD41 is the platelet receptor of fibrinogen / fibrin, this indicates that there is a blockage of this marker by fibrinogen induced by +DOTAP LNPs. We first tested if +DOTAP LNPs bind to fibrinogen in vitro. We added fluorescently labeled fibrinogen and LNPs to the plasma and incubated for 10 min. We diluted this sample and performed fluorescence-mode nanoparticle tracking analysis (NTA) to detect nanoparticle-sized aggregates of fluorescent fibrinogen, indicating fibrinogen aggregation on LNPs (FIG.15A). Approximately 38-fold more fibrinogen- positive particles were observed in the presence of +DOTAP LNPs versus −DOTAP LNPs (FIG.14B – FIG.14E). This agrees with our findings that +DOTAP LNPs aggregate significantly with fibrinogen in plasma and even after incubation with fibrinogen alone (FIG.16 and FIG.17A – FIG.17B). We further sought to determine if +DOTAP LNPs bind to fibrinogen in vivo and alter its biodistribution. Fibrinogen was radioactively labeled and injected into mice, followed by + or DOTAP LNPs 2 min later. Organs were harvested 30 min after LNP injection and the radioactivity in each organ was measured (FIG.15E). While −DOTAP LNPs do not alter fibrinogen biodistribution, there is a ≈2-fold increase in fibrinogen lung uptake in the presence of +DOTAP LNPs (FIG.15F and FIG.15G; FIG.18A). A similar +DOTAP LNP-induced increase in lung fibrinogen uptake was observed in mice with pre- existing inflammation (FIG.18B). Notably, the localization of fibrinogen to the lungs induced by +DOTAP LNPs persists even 4 h after injection with evidence of fibrinolysis only 24 h after (FIG.18C – FIG.18F). Confocal microscopy images of lung sections show visible aggregates of fibrinogen in +DOTAP LNP-injected mice and no visible fibrinogen in −DOTAP LNP-injected mice. This was quantified across multiple images, with the LNPs and fibrinogen having much more signal in the lungs of +DOTAP versus −DOTAP LNP mice (FIG.15H). Furthermore, fibrinogen is strongly associated with +DOTAP LNPs as shown by the percentage of fibrinogen signal overlap with that of LNPs or the percentage of LNP signal overlap with fibrinogen signal (FIG.15I). The data in FIG.15A – FIG.15I show that fibrinogen binds to +DOTAP LNPs (but not −DOTAP LNPs), and this binding correlates with clotting in the lungs. We then performed circular dichroism (CD) to determine if the binding of +DOTAP LNPs to fibrinogen induces changes in the protein's secondary structure. The CD spectra for fibrinogen alone show two negative peaks at 209 and 220 nm which is characteristic of a protein with an α-helix (FIG.15J). However, the addition of +DOTAP LNPs significantly reduces the ellipticity of fibrinogen at both 209 and 220 nm while −DOTAP LNPs do not. This indicates that the secondary structure of fibrinogen is significantly altered in the presence of +DOTAP LNPs which supports the hypothesis that this altered fibrinogen structure could induce platelet activation and coagulation. To further examine the effects of fibrinogen binding to +DOTAP LNPs, we depleted fibrinogen from plasma doped with +DOTAP LNPs. Since fibrinogen depletion would prevent coagulation, we checked if fibrinogen depletion also abrogated +DOTAP LNP-induced platelet activation. PRP was treated with tissue plasminogen activator (tPA) which converts plasminogen to plasmin, causing degradation of fibrinogen. We found that in fibrinogen-depleted plasma, +DOTAP LNPs do not induce significant platelet activation as measured by P-Selectin MFI (FIG.15K). Thus, fibrinogen is necessary for +DOTAP LNP-induced platelet activation. This fits with a mechanism in which positively charged LNPs first bind to fibrinogen, inducing a conformational change in the protein that activates platelets, which in turn activates the rest of the coagulation cascade. This fits with prior studies that show that immobilization of fibrinogen on surfaces can expose normally concealed domains that activate platelets. Anticoagulation Ameliorates Cationic LNP-Induced Clotting Below, we propose and test ways to prevent LNP-induced clotting. To determine whether anticoagulation can reduce the toxicity of +DOTAP LNPs, we compared clot formation between mice treated with +DOTAP LNPs alone and those pre-treated with the clinically used anticoagulants heparin and bivalirudin. Heparin functions by enhancing the activity of endogenous antithrombin while bivalirudin is a direct thrombin inhibitor. Anticoagulants were IV injected 5 min prior to +DOTAP LNP injection (62.5U per mouse for heparin and 400 µg per mouse for bivalirudin) and LNPs were allowed to circulate for 30 min. We found that both heparin and bivalirudin pre-treatment significantly reduce the formation of clots in the lung compared to +DOTAP LNP injection alone, as shown by lung histological samples (FIG.19A). We then investigated the effect of anticoagulation on +DOTAP LNP biodistribution and mRNA expression. We radiolabeled LNPs with indium-111 for biodistributions and injected mice with LNPs loaded with luciferase mRNA to trace mRNA expression (FIG.19B). Surprisingly, heparin pre-treatment shunted LNP localization from the lung to the liver and spleen and completely attenuated luciferase expression in the lungs (FIG.18C and FIG.19D; FIG.20A). We hypothesize that this unexpected effect is because heparin is a negatively charged polysaccharide, so it could have a charge interaction with +DOTAP LNPs and this could inhibit LNP functionality, though other mechanisms may exist. By contrast, bivalirudin pre-treatment preserves the localization of +DOTAP LNPs to the lung, as well as the LNP-induced mRNA luciferase expression (FIG.19B and FIG.19C; and FIG.20B). These studies illustrate that while the side effects of +DOTAP LNPs can be ameliorated with anticoagulation, the properties of each anticoagulant must be thoroughly examined to ensure that LNP activity is preserved. Based on this success with bivalirudin (a direct thrombin inhibitor), we tested the effects of a similar anticoagulant, dabigatran (FIG.21). Furthermore, we fabricated +DOTAP LNPs with a surface-conjugated direct thrombin inhibitor, PPACK (FIG.22A – FIG.22E). This might be a more convenient clinical solution than needing to transiently anticoagulate patients with bivalirudin. Clot formation in the lungs was also limited in mice treated with PPACK-conjugated +DOTAP LNPs (FIG.22D). Decreasing the Size of DOTAP LNPs Prevents Fibrinogen Binding and Clotting We serendipitously observed that the size of +DOTAP LNPs has a positive correlation with APTT. When we increased LNP size from 100 nm to 180 nm, APTT increased by ∼1.6-fold. However, when we decreased the LNP size to 80 nm, APTT was restored to naive levels indicating no significant activation of the intrinsic coagulation pathway (FIG.23A). Similarly, 80 nm +DOTAP LNPs did not significantly activate platelets, as measured by P-selectin MFI (FIG.23B). This led us to hypothesize that the size of +DOTAP LNPs could affect the amount of fibrinogen molecules that can be adsorbed on the LNP surface. We tested this hypothesis first in vitro using fluorescence NTA to probe for fibrinogen aggregation and found that 80 nm +DOTAP LNPs generate ≈79× fewer nanoparticle-associated fibrinogen aggregates than 100 nm +DOTAP LNPs (FIG.23C and FIG.23D). Additionally, the number of fibrinogen-nanoparticle aggregates detected with 80 nm +DOTAP LNPs is not significantly different from that under vehicle conditions, indicating a complete absence of detectable fibrinogen-nanoparticle aggregation. We subsequently tested how 80 nm +DOTAP LNPs affect fibrinogen biodistributions in vivo. As previously, we injected mice with radiolabeled fibrinogen followed by LNPs 2 min after for a circulation time of 30 min. While 100 nm +DOTAP LNPs almost double fibrinogen uptake in the lung, 80 nm +DOTAP LNPs do not alter fibrinogen biodistribution or localization ratio compared to control (FIG.23E and FIG. 23F). These data validate our hypothesis that 80 nm +DOTAP LNPs do not detectably bind to or cause aggregation of fibrinogen. Since this interaction is associated with thrombosis, these LNPs do not activate platelets or the intrinsic coagulation pathway (FIG. 23G). Furthermore, histological samples from the lungs of LNP-injected mice show that 80 nm +DOTAP LNPs do not generate visible clots like 100 nm +DOTAP LNPs (FIG. 23H). Finally, 80 nm +DOTAP LNPs still preserve their lung tropism, as shown by their biodistribution and mRNA expression profile (30-min and 4-h LNP circulation time respectively) (FIG.23I and FIG.23J). These data indicate that by altering the physical properties of +DOTAP LNPs, we can modulate the binding of fibrinogen and the subsequent initiation of clotting while maintaining lung tropism. References N. Chaudhary, D. Weissman, K. A. Whitehead, Nat. Rev. Drug Discovery 2021, 20, 817. H. Parhiz, V. V. Shuvaev, N. Pardi, M. Khoshnejad, R. Y. Kiseleva, J. S. Brenner, T. Uhler, S. Tuyishime, B. L. Mui, Y. K. Tam, T. D. Madden, M. J. Hope, D. Weissman, V. R. Muzykantov, J. Controlled Release 2018, 291, 106. M. Kim, M. Jeong, S. Hur, Y. Cho, J. Park, H. Jung, Y. Seo, H. A. Woo, K. T. Nam, K. Lee, H. Lee, Sci. Adv.2021, 7, abf4398. A. Akinc, W. Querbes, S. De, J. Qin, M. Frank-Kamenetsky, K. N. Jayaprakash, M. Jayaraman, K. G. Rajeev, W. L. Cantley, J. R. Dorkin, J. S. Butler, L. Qin, T. Racie, A. Sprague, E. Fava, A. Zeigerer, M. J. Hope, M. Zerial, D. W. Y. Sah, K. Fitzgerald, M. A. Tracy, M. Manoharan, V. Koteliansky, A. de Fougerolles, M. A. Maier, Mol. Ther.2010, 18, 1357. Q. Cheng, T. Wei, L. Farbiak, L. T. Johnson, S. A. Dilliard, D. J. Siegwart, Nat. Nanotechnol.2020, 15, 313. L. M. Kranz, M. Diken, H. Haas, S. Kreiter, C. Loquai, K. C. Reuter, M. Meng, D. Fritz, F. Vascotto, H. Hefesha, C. Grunwitz, M. Vormehr, Y. Hüsemann, A. Selmi, A. N. Kuhn, J. Buck, E. Derhovanessian, R. Rae, S. Attig, J. Diekmann, R. A. Jabulowsky, S. Heesch, J. Hassel, P. Langguth, S. Grabbe, C. Huber, Ö. Türeci, U. Sahin, Nature 2016, 534, 396. D. Zhang, E. N. Atochina-Vasserman, D. S. Maurya, M. Liu, Q. Xiao, J. Lu, G. Lauri, N. Ona, E. K. Reagan, H. Ni, D. Weissman, V. Percec, J. Am. Chem. Soc.2021, 143, 17975. S. A. Dilliard, Q. Cheng, D. J. Siegwart, Proc. Natl. Acad. Sci. U. S. A.2021, 118, 2109256118. S. T. LoPresti, M. L. Arral, N. Chaudhary, K. A. Whitehead, J. Controlled Release 2022, 345, 819. T. R. Blake, O. A. W. Haabeth, A. Sallets, R. L. McClellan, T. J. Del Castillo, J. G. Vilches-Moure, W. C. Ho, P. A. Wender, R. Levy, R. M. Waymouth, Bioconjug. Chem.2023, 34, 673. J. C. Kaczmarek, K. J. Kauffman, O. S. Fenton, K. Sadtler, A. K. Patel, M. W. Heartlein, F. DeRosa, D. G. Anderson, Nano Lett.2018, 18, 6449. Y. Yan, H. Xiong, X. Zhang, Q. Cheng, D. J. Siegwart, Biomacromolecules 2017, 18, 4307. J. W. Myerson, P. N. Patel, K. M. Rubey, M. E. Zamora, M. H. Zaleski, N. Habibi, L. R. Walsh, Y.-W. Lee, D. C. Luther, L. T. Ferguson, O. A. Marcos-Contreras, P. M. Glassman, L. L. Mazaleuskaya, I. Johnston, E. D. Hood, T. Shuvaeva, J. Wu, H.-Y. Zhang, J. V. Gregory, R. Y. Kiseleva, J. Nong, T. Grosser, C. F. Greineder, S. Mitragotri, G. S. Worthen, V. M. Rotello, J. Lahann, V. R. Muzykantov, J. S. Brenner, Nat. Nanotechnol.2022, 17, 86. Z. Wang, E. D. Hood, J. Nong, J. Ding, O. A. Marcos-Contreras, P. M. Glassman, K. M. Rubey, M. Zaleski, C. L. Espy, D. Gullipali, T. Miwa, V. R. Muzykantov, W.-C. Song, J. W. Myerson, J. S. Brenner, Adv. Mater.2022, 34, 2107070. S. Y. Fam, C. F. Chee, C. Y. Yong, K. L. Ho, A. R. Mariatulqabtiah, W. S. Tan, Nanomaterials 2020, 10, 787. X. Wu, H. Chen, C. Wu, J. Wang, S. Zhang, J. Gao, H. Wang, T. Sun, Y.-G. Yang, Biomaterials 2018, 156, 77. A. N. Ilinskaya, M. A. Dobrovolskaia, Nanomedicine 2013, 8, 969. C. F. Jones, R. A. Campbell, Z. Franks, C. C. Gibson, G. Thiagarajan, A. Vieira-de- Abreu, S. Sukavaneshvar, S. F. Mohammad, D. Y. Li, H. Ghandehari, A. S. Weyrich, B. D. Brooks, D. W. Grainger, Mol. Pharm.2012, 9, 1599. C. Oslakovic, T. Cedervall, S. Linse, B. Dahlbäck, Nanomedicine 2012, 8, 981. K. M. de la Harpe, P. P. D. Kondiah, Y. E. Choonara, T. Marimuthu, L. C. du Toit, V. Pillay, Cells 2019, 8, 1209. E. Fröhlich, Curr. Med. Chem.2016, 23, 408. L. S. Chase, M. H. Zaleski, L. J. Morrell, J. S. Brenner, Int. J. Pharm.2023, 645, 123369. C. T. Esmon, Thromb. Haemost.2013, 109, 416. M. Qiu, Y. Tang, J. Chen, R. Muriph, Z. Ye, C. Huang, J. Evans, E. P. Henske, Q. Xu, Proc. Natl. Acad. Sci. U. S. A.2022, 119, 2116271119. C. Maas, T. Renné, Blood 2018, 131, 1903. Y. Sang, M. Roest, B. de Laat, P. G. de Groot, D. Huskens, Blood Rev.2021, 46, 100733. B. E. J. Spurgeon, M. D. Linden, A. D. Michelson, A. L. Frelinger III, Curr. Protoc.2021, 1, 178. A. D. Blann, G. Y. Lip, Atherosclerosis 1997, 128, 135. G. Bashiri, M. S. Padilla, K. L. Swingle, S. J. Shepherd, M. J. Mitchell, K. Wang, Lab Chip 2023, 23, 1432. S. Wang, J. Zhang, H. Zhou, Y. C. Lu, X. Jin, L. Luo, J. You, J. Controlled Release 2023, 360, 15. A. D. Protopopova, N. A. Barinov, E. G. Zavyalova, A. M. Kopylov, V. I. Sergienko, D. V. Klinov, J. Thromb. Haemost.2015, 13, 570. S.-Y. Jung, S.-M. Lim, F. Albertorio, G. Kim, M. C. Gurau, R. D. Yang, M. A. Holden, P. S. Cremer, J. Am. Chem. Soc.2003, 125, 12782. H. Derakhshankhah, A. Hosseini, F. Taghavi, S. Jafari, A. Lotfabadi, M. R. Ejtehadi, S. Shahbazi, A. Fattahi, A. Ghasemi, E. Barzegari, M. Evini, A. A. Saboury, S. M. K. Shahri, B. Ghaemi, E.-P. Ng, H. Awala, F. Omrani, I. Nabipour, M. Raoufi, R. Dinarvand, K. Shahpasand, S. Mintova, M. J. Hajipour, M. Mahmoudi, Sci. Rep.2019, 9, 1558. Z. J. Deng, M. Liang, I. Toth, M. J. Monteiro, R. F. Minchin, ACS Nano 2012, 6, 8962. Z. J. Deng, M. Liang, M. Monteiro, I. Toth, R. F. Minchin, Nat. Nanotechnol.2011, 6, 39. B. Savage, E. Saldívar, Z. M. Ruggeri, Cell 1996, 84, 289. Y. Qiu, A. C. Brown, W. J. Jung, Y. Sakurai, R. Mannino, D. R. Myers, R. Tran, G. Bao, T. H. Barker, W. Lam, Blood 2012, 120, 384. R. Loncar, R. B. Zotz, C. Sucker, A. Vodovnik, M. Mihalj, R. E. Scharf, Thromb. J.2007, 5, 5. P. H. Mangin, M.-B. Onselaer, N. Receveur, N. L. Lay, A. T. Hardy, C. Wilson, X. Sanchez, S. Loyau, A. Dupuis, A. K. Babar, J. L. Miller, H. Philippou, C. E. Hughes, A. B. Herr, R. A. Ariëns, D. Mezzano, M. Jandrot-Perrus, C. Gachet, S. P. Watson, Haematologica 2018, 103, 898. R. J. Weiss, J. D. Esko, Y. Tor, Org. Biomol. Chem.2017, 15, 5656. J. Myerson, L. He, G. Lanza, D. Tollefsen, S. Wickline, J. Thromb. Haemost.2011, 9, 1292. P. Zeliszewska, A. Bratek-Skicki, Z. Adamczyk, M. Cieśla, Langmuir 2014, 30, 11165. H. Parhiz, J. S. Brenner, P. N. Patel, T. E. Papp, H. Shahnawaz, Q. Li, R. Shi, M. E. Zamora, A. Yadegari, O. A. Marcos-Contreras, A. Natesan, N. Pardi, V. V. Shuvaev, R. Kiseleva, J. W. Myerson, T. Uhler, R. S. Riley, X. Han, M. J. Mitchell, K. Lam, J. Heyes, D. Weissman, V. R. Muzykantov, J. Controlled Release 2022, 344, 50. O. A. Marcos-Contreras, C. F. Greineder, R. Y. Kiseleva, H. Parhiz, L. R. Walsh, V. Zuluaga-Ramirez, J. W. Myerson, E. D. Hood, C. H. Villa, I. Tombacz, N. Pardi, A. Seliga, B. L. Mui, Y. K. Tam, P. M. Glassman, V. V. Shuvaev, J. Nong, J. S. Brenner, M. Khoshnejad, T. Madden, D. Weissmann, Y. Persidsky, V. R. Muzykantov, Proc. Natl. Acad. Sci. U. S. A.2020, 117, 3405. Each and every patent, patent application, and publication, including websites and other publications cited throughout the specification, is incorporated herein by reference. US Provisional Patent Application No.63 / 514,723, filed July 20, 2023, is incorporated herein by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

CLAIMS:

1. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject an anticoagulant and / or a platelet inhibitor and a nanoparticle comprising the agent, wherein coagulation or thrombosis resulting from administration of the nanoparticle is reduced or prevented.

2. The method of claim 1, wherein the nanoparticle is a lipid nanoparticle (LNP) or liposome.

3. The method of claim 1 or 2, wherein the nanoparticle comprises an outer surface layer comprising a cationic lipid.

4. The method of claim 3, wherein the cationic lipid is 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 1,2-Dioleoyl-3-trimethylammonium propane (DOTMA), DLin-KC2-DMA, or D-Lin-MC3-DMA.

5. The method of any one of claims 1 to 4, wherein the nanoparticle comprises an outer surface layer comprising an ionizable lipid.

6. The method of claim 5, wherein the ionizable lipid is 306-N16B, lipid 29d, or 514O6,10.

7. The method of any one of claims 1 to 6, wherein the nanoparticle has a zeta potential greater than 0 mV, at least about 5 mV, at least about 10 mV, or at least about 15 mV, optionally wherein the zeta potential is measured in a solution at a pH of about 7.0 or about 7.0 to 7.

5.

8. The method of any one of claims 1 to 7, wherein the nanoparticle comprises a direct thrombin inhibitor or a coagulation cascade protease inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.

9. The method of claim 8, wherein the direct thrombin inhibitor is D-phenylalyl-L- prolyl-L-arginyl-chloromethyl ketone (PPACK).

10. The method of claim 8, wherein the direct thrombin inhibitor is hirudin, bivalirudin, desirudin, lepirudin, dabigatran, argatroban, inogatran, or melagatran.

11. The method of any one of claims 1 to 10, wherein the anticoagulant is a direct thrombin inhibitor or a coagulation cascade protease inhibitor.

12. The method of claim 11, wherein the direct thrombin inhibitor is hirudin, bivalirudin, desirudin, lepirudin, dabigatran, argatroban, inogatran, or melagatran.

13. The method of any one of claims 1 to 12, wherein the platelet inhibitor is acetylsalicylic acid, clopidogrel, prasugrel, ticagrelor, dipyridamole, eptifibatide, tirofiban, abciximab, cilostazol,or vorapaxar.

14. The method of any one of claims 1 to 13, wherein the nanoparticle is capable of binding fibrinogen present in the subject’s plasma following administration to the subject.

15. The method of any one of claims 1 to 14, where the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension.

16. The method of claim 15, wherein the nanoparticle is not capable of binding fibrinogen or has reduced binding to fibrinogen following administration to the subject.

17. The method of any one of claims 1 to 16, wherein the nanoparticle is administered intravenously or intra-arterially.

18. The method of any one of claims 1 to 17, wherein the nanoparticle and anticoagulant and / or the platelet inhibitor are administered to the subject via the same route.

19. The method of any one of claims 1 to 18, wherein the nanoparticle and anticoagulant and / or the platelet inhibitor are administered sequentially.

20. The method of any one of claims 1 to 19, wherein the agent is a therapeutic, diagnostic, or imaging agent.

21. The method of any one of claims 1 to 20, wherein the nanoparticle is administered intravenously and clotting in the lung is reduced or prevented.

22. The method of any one of claims 1 to 21, wherein administration of the anticoagulant and / or the platelet inhibitor does not alter localization of the nanocarrierwhen compared to delivery of the nanoparticle without administering the anticoagulant and / or the platelet inhibitor.

23. The method of any one of claims 1 to 22, wherein the coagulation resulting from administration of the therapeutic nanoparticles is nanoparticle-induced clotting (thrombosis) in a target organ of the subject.

24. The method of claim 23, wherein the target organ is the lung.

25. The method of any one of claims 1 to 24, wherein the nanoparticle comprises RNA and administration of the anticoagulant and / or the platelet inhibitor does not alter expression of the RNA when compared to delivery of the nanoparticle without administering the anticoagulant.

26. A nanoparticle comprising an agent and a direct thrombin inhibitor or a coagulation cascade protease inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.

27. The nanoparticle of claim 26, wherein the nanoparticle is a lipid nanoparticle (LNP) or liposome.

28. The nanoparticle of claim 26 or 27, wherein the direct thrombin inhibitor is D- phenylalyl-L-prolyl-L-arginyl-chloromethyl ketone (PPACK).

29. The nanoparticle of claim 26 or 27, wherein the direct thrombin inhibitor is hirudin, bivalirudin, desirudin, lepirudin, dabigatran, argatroban, inogatran, or melagatran.

30. The nanoparticle of any one of claims 26 to 29, wherein the nanoparticle comprises an outer surface layer comprising a cationic lipid.

31. The nanoparticle of claim 30, wherein the cationic lipid is 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 1,2-Dioleoyl-3-trimethylammonium propane (DOTMA), DLin-KC2-DMA, or D-Lin-MC3-DMA.

32. The nanoparticle of claim any one of claims 26 to 31, wherein the nanoparticle comprises an outer surface layer comprising a ionizable lipid.

33. The nanoparticle of claim 32, wherein the ionizable lipid is 306-N16B, lipid 29d, or 514O6,10.

34. The nanoparticle of any one of claims 26 to 33, wherein the nanoparticle has a zeta potential greater than 0 mV, at least about 5 mV, at least about 10 mV, or at least about 15 mV, optionally wherein the zeta potential is measured in a solution at a pH of about 7.0 or about 7.0 to 7.

5.

35. The nanoparticle of any one of claims 26 to 34, wherein the nanoparticle is capable of binding plasma fibrinogen.

36. The nanoparticle of any one of claims 26 to 34, where the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension.

37. The nanoparticle of claim 36, wherein the nanoparticle is not capable of binding fibrinogen or has reduced binding to fibrinogen following administration to the subject.

38. The nanoparticle of any one of claims 26 to 37, wherein the agent is a therapeutic, diagnostic, or imaging agent.

39. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject an anticoagulant and / or a platelet inhibitor and the nanoparticle of any one of claims 26 to 38, wherein coagulation or thrombosis resulting from administration of the nanoparticle is reduced or prevented.

40. The method of claim 39, wherein the anticoagulant is a direct thrombin inhibitor or a coagulation cascade protease inhibitor.

41. The method of claim 39, wherein the direct thrombin inhibitor is hirudin, bivalirudin, desirudin, lepirudin, dabigatran, argatroban, inogatran, or melagatran.

42. The method of any one of claims 39 to 47, wherein the platelet inhibitor is acetylsalicylic acid, clopidogrel, prasugrel, ticagrelor, dipyridamole, eptifibatide, tirofiban, abciximab, cilostazol,or vorapaxar.

43. The method of any one of claims 39 to 42, wherein the nanoparticle is administered intravenously or intra-arterially.

44. The method of any one of claims 39 to 43, wherein the nanoparticle and the anticoagulant and / or the platelet inhibitor are administered to the subject via the same route.

45. The method of any one of claims 39 to 44, wherein the nanoparticle and the anticoagulant and / or the platelet inhibitor are administered sequentially.

46. The method of any one of claims 39 to 45, wherein the nanoparticle is administered intravenously and clotting in the lung is reduced or prevented.

47. The method of any one of claims 39 to 46, wherein administration of the anticoagulant and / or the platelet inhibitor does not alter localization of the nanocarrier when compared to delivery of the nanoparticle without administering the anticoagulant.

48. The method of any one of claims 39 to 47, wherein the coagulation resulting from administration of the therapeutic nanoparticle includes clotting (thrombosis) in a target organ in the subject.

49. The method of claim 48, wherein the target organ is the lung.

50. The method of any one of claims 39 to 49, wherein the nanoparticle comprises RNA and administration of the anticoagulant and / or the platelet inhibitor does not alter expression of the RNA when compared to delivery of the nanoparticle without administering the anticoagulant.

51. The method of any one of claims 1 to 25 or 39 to 50, wherein the reduction or prevention of coagulation is determined by measuring platelet activation, platelet aggregation, partial thromboplastin time (PTT), activated partial thromboplastin time (aPTT), and / or thrombin–antithrombin (TAT) complex in a biological sample obtained from the subject, optionally wherein the biological sample is blood, serum, or plasma.

52. A pharmaceutical composition comprising the nanoparticle of any one of claims 26 to 38 and a pharmaceutically acceptable carrier, excipient, and / or preservative.

Images