Complement opsonization is among the biggest challenges facing nanomedicine. Nearly instantly after injection into blood, nanoparticles are opsonized by the complement protein C3, leading to ...clearance by phagocytes, fouling of targeting moieties, and release of anaphylatoxins. While surface polymers such as poly(ethylene glycol) (PEG) partially decrease complement opsonization, most nanoparticles still suffer from extensive complement opsonization, especially when linked to targeting moieties. To ameliorate the deleterious effects of complement, two of mammals’ natural regulators of complement activation (RCAs), Factors H and I, are here conjugated to the surface of nanoparticles. In vitro, Factor H or I conjugation to PEG‐coated nanoparticles decrease their C3 opsonization, and markedly reduce nanoparticle uptake by phagocytes. In an in vivo mouse model of sepsis‐induced lung injury, Factor I conjugation abrogates nanoparticle uptake by intravascular phagocytes in the lungs, allowing the blood concentration of the nanoparticle to remain elevated much longer. For nanoparticles targeted to the lung's endothelium by conjugation to anti‐ICAM antibodies, Factor I conjugation shifts the cell‐type distribution away from phagocytes and toward endothelial cells. Finally, Factor I conjugation abrogates the severe anaphylactoid responses common to many nanoparticles, preventing systemic capillary leak and preserving blood flow to visceral organs and the brain. Thus, conjugation of RCAs, like Factor I, to nanoparticles is likely to help in nanomedicine's long battle against complement, improving several key parameters critical for clinical success.
When nanoscale drug carriers enter the blood, they are rapidly bound by complement proteins, such as C3, which mark the nanocarriers for clearance by phagocytes and produce an anaphylaxis‐like reaction. In this work, nanocarriers are conjugated to the complement‐cleaving enzyme Factor I, and it abrogates several major problems facing nanomedicine.
Lipid nanoparticles (LNPs) have become the dominant drug delivery technology in industry, holding the promise to deliver RNA to up or down‐regulate any protein of interest. LNPs have mostly been ...targeted to specific cell types or organs by physicochemical targeting in which LNP’s lipid compositions are adjusted to find mixtures with the desired tropism. Here lung‐tropic LNPs are examined, whose organ tropism derives from containing either a cationic or ionizable lipid conferring a positive zeta potential. Surprisingly, these LNPs are found to induce massive thrombosis. Such thrombosis is shown in the lungs and other organs, and it is shown that it is greatly exacerbated by pre‐existing inflammation. This clotting is induced by a variety of formulations with cationic lipids, including LNPs and non‐LNP nanoparticles, and even by lung‐tropic ionizable lipids that do not have a permanent cationic charge. The mechanism depends on the LNPs binding to and then changing the conformation of fibrinogen, which then activates platelets and thrombin. Based on these mechanisms, multiple solutions are engineered that enable positively charged LNPs to target the lungs while ameliorating thrombosis. The findings illustrate how physicochemical targeting approaches must be investigated early for risks and re‐engineered with a careful understanding of biological mechanisms.
This study shows that lung‐tropic lipid nanoparticles (LNPs) containing either a cationic or a positive zeta potential conferring ionizable lipid, induce massive thrombosis. The mechanism depends on the LNPs binding to and then changing the conformation of fibrinogen and activating platelets. Specific anticoagulation techniques and tuning of LNP physical properties are shown to ameliorate thrombosis while preserving lung tropism.
Drug delivery by nanocarriers (NCs) has long been stymied by dominant liver uptake and limited target organ deposition, even when NCs are targeted using affinity moieties. Here we report a universal ...solution: red blood cell (RBC)-hitchhiking (RH), in which NCs adsorbed onto the RBCs transfer from RBCs to the first organ downstream of the intravascular injection. RH improves delivery for a wide range of NCs and even viral vectors. For example, RH injected intravenously increases liposome uptake in the first downstream organ, lungs, by ~40-fold compared with free NCs. Intra-carotid artery injection of RH NCs delivers >10% of the injected NC dose to the brain, ~10× higher than that achieved with affinity moieties. Further, RH works in mice, pigs, and ex vivo human lungs without causing RBC or end-organ toxicities. Thus, RH is a clinically translatable platform technology poised to augment drug delivery in acute lung disease, stroke, and several other diseases.
Hyperfibrinolysis is a systemic condition occurring in various clinical disorders such as trauma, liver cirrhosis, and leukemia. Apart from increased bleeding tendency, the pathophysiological ...consequences of hyperfibrinolysis remain largely unknown. Our aim was to develop an experimental model of hyperfibrinolysis and to study its effects on the homeostasis of the blood–brain barrier (BBB). We induced a sustained hyperfibrinolytic state in mice by hydrodynamic transfection of a plasmid encoding for tissue-type plasminogen activator (tPA). As revealed by near-infrared fluorescence imaging, hyperfibrinolytic mice presented a significant increase in BBB permeability. Using a set of deletion variants of tPA and pharmacological approaches, we demonstrated that this effect was independent of N-methyl-D-aspartate receptor, low-density lipoprotein–related protein, protease-activated receptor-1, or matrix metalloproteinases. In contrast, we provide evidence that hyperfibrinolysis-induced BBB leakage is dependent on plasmin-mediated generation of bradykinin and subsequent activation of bradykinin B2 receptors. Accordingly, this effect was prevented by icatibant, a clinically available B2 receptor antagonist. In agreement with these preclinical data, bradykinin generation was also observed in humans in a context of acute pharmacological hyperfibrinolysis. Altogether, these results suggest that B2 receptor blockade may be a promising strategy to prevent the deleterious effects of hyperfibrinolysis on the homeostasis of the BBB.
•Hydrodynamic transfection of plasmids encoding for plasminogen activators leads to a hyperfibrinolytic state in mice.•Hyperfibrinolysis increases BBB permeability via a plasmin- and bradykinin-dependent mechanism.
Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis ...factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood–brain barrier, thus benefiting numerous brain pathologies.
Current nucleoside-modified RNA lipid nanoparticle (modmRNA-LNP) technology has successfully paved the way for the highest clinical efficacy data from next-generation vaccinations against SARS-CoV-2 ...during the COVID-19 pandemic. However, such modmRNA-LNP technology has not been characterized in common pre-existing inflammatory or immune-challenged conditions, raising the risk of adverse clinical effects when administering modmRNA-LNPs in such cases. Herein, we induce an acute-inflammation model in mice with lipopolysaccharide (LPS) intratracheally (IT), 1 mg kg−1, or intravenously (IV), 2 mg kg−1, and then IV administer modmRNA-LNP, 0.32 mg kg−1, after 4 h, and screen for inflammatory markers, such as pro-inflammatory cytokines. ModmRNA-LNP at this dose caused no significant elevation of cytokine levels in naive mice. In contrast, shortly after LPS immune stimulation, modmRNA-LNP enhanced inflammatory cytokine responses, Interleukin-6 (IL-6) in serum and Macrophage Inflammatory Protein 2 (MIP-2) in liver significantly. Our report identifies this phenomenon as inflammation exacerbation (IE), which was proven to be specific to the LNP, acting independent of mRNA cargo, and was demonstrated to be time- and dose-dependent. Macrophage depletion as well as TLR3 −/− and TLR4−/− knockout mouse studies revealed macrophages were the immune cells involved or responsible for IE. Finally, we show that pretreatment with anti-inflammatory drugs, such as corticosteroids, can partially alleviate IE response in mice. Our findings characterize the importance of LNP-mediated IE phenomena in gram negative bacterial inflammation, however, the generalizability of modmRNA-LNP in other forms of chronic or acute inflammatory and immune contexts needs to be addressed.
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Ex vivo-loaded white blood cells (WBC) can transfer cargo to pathological foci in the central nervous system (CNS). Here we tested affinity ligand driven in vivo loading of WBC in order to bypass the ...need for ex vivo WBC manipulation. We used a mouse model of acute brain inflammation caused by local injection of tumor necrosis factor alpha (TNF-α). We intravenously injected nanoparticles targeted to intercellular adhesion molecule 1 (anti-ICAM/NP). We found that (A) at 2 h, >20% of anti-ICAM/NP were localized to the lungs; (B) of the anti-ICAM/NP in the lungs >90% were associated with leukocytes; (C) at 6 and 22 h, anti-ICAM/NP pulmonary uptake decreased; (D) anti-ICAM/NP uptake in brain increased up to 5-fold in this time interval, concomitantly with migration of WBCs into the injured brain. Intravital microscopy confirmed transport of anti-ICAM/NP beyond the blood–brain barrier and flow cytometry demonstrated complete association of NP with WBC in the brain (98%). Dexamethasone-loaded anti-ICAM/liposomes abrogated brain edema in this model and promoted anti-inflammatory M2 polarization of macrophages in the brain. In vivo targeted loading of WBC in the intravascular pool may provide advantages of coopting WBC predisposed to natural rapid mobilization from the lungs to the brain, connected directly via conduit vessels.
Our aim was to assess the spatiotemporal evolution of the cerebrovascular inflammation occurring after ischemic and hemorrhagic strokes using a recently developed, fast, and ultra-sensitive molecular ...MRI method.
We first assessed longitudinally the cerebrovascular inflammation triggered by collagenase-induced hemorrhage and by permanent/transient middle cerebral artery occlusion in mice, using MRI after injection of microparticles of iron oxide targeted to vascular cell adhesion molecule-1 (MPIOs-αVCAM-1). Thereafter, we used this method to study the anti-inflammatory effects of celecoxib, atorvastatin, and dipyridamole after stroke.
Using multiparametric MRI, we demonstrated that the level and the kinetics of cerebrovascular VCAM-1 expression depend on several parameters, including stroke pathogenesis, the natural history of the disease, and the administration of inflammation-modulating drugs. Interestingly, in transient middle cerebral artery occlusion and intracranial hemorrhage models, VCAM-1 expression was maximal at 24 hours and almost returned to baseline 5 days after stroke onset. In contrast, after permanent middle cerebral artery occlusion, VCAM-1 overexpression was sustained between 24 hours and 5 days, and was particularly significant in the peri-infarct areas. Our results suggest that these perilesional areas expressing VCAM-1 constitute an inflammatory penumbra that is recruited by the ischemic core during the subacute phase. Using MPIOs-αVCAM-1-enhanced imaging, we also provided evidence that celecoxib and atorvastatin (but not dipyridamole) alleviate VCAM-1 overexpression after stroke and prevent formation of the inflammatory penumbra.
MPIOs-αVCAM-1-enhanced imaging seems to be promising in the detection of individuals presenting with severe cerebrovascular responses after stroke, which could therefore benefit from anti-inflammatory treatments.
Recombinant activated human factor VII (rhFVIIa) is an established hemostatic agent in hemophilia, but its mechanism of action remains unclear. Although tissue factor (TF) is its natural receptor, ...rhFVIIa also interacts with the endothelial protein C receptor (EPCR) through its γ-carboxyglutamic acid (Gla) domain, with unknown hemostatic consequences in vivo. Here, we study whether EPCR facilitates rhFVIIa hemostasis in hemophilia using a mouse model system. Mouse activated FVII (mFVIIa) is functionally homologous to rhFVIIa, but binds poorly to mouse EPCR (mEPCR). We modified mFVIIa to gain mEPCR binding using 3 amino acid changes in its Gla domain (L4F/L8M/W9R). The resulting molecule mFVIIa-FMR specifically bound mEPCR in vitro and in vivo and was identical to mFVIIa with respect to TF affinity and procoagulant functions. In macrovascular injury models, hemophilic mice administered mFVIIa-FMR exhibited superior hemostatic activity compared with mFVIIa. This was abolished by blocking mEPCR and was absent in ex vivo whole blood coagulation assays, implicating a specific mFVIIa-FMR and endothelial mEPCR interaction. Because mFVIIa-FMR models the TF-dependent and EPCR binding properties of rhFVIIa, our data unmask a novel contribution of EPCR on the action of rhFVIIa administration in hemophilia, prompting the rational design of improved and safer rhFVIIa therapeutics.
•The binding of administered FVIIa to endogenous EPCR enhances its ability to bypass FVIII or FIX deficiency in vivo.•EPCR modulation of function of pharmacologic FVIIa administration may be exploited in protein or gene-based FVIIa therapeutics.
Intracerebral hemorrhage (ICH) is one of the most common causes of fatal stroke, yet has no specific drug therapies. Many attempts at passive intravenous (IV) delivery in ICH have failed to deliver ...drugs to the salvageable area around the hemorrhage. The passive delivery method assumes vascular leak through the ruptured blood-brain barrier will allow drug accumulation in the brain. Here we tested this assumption using intrastriatal injection of collagenase, a well-established experimental model of ICH. Fitting with hematoma expansion in clinical ICH, we showed that collagenase-induced blood leak drops significantly by 4 h after ICH onset and is gone by 24 h. We observed passive-leak brain accumulation also declines rapidly over ∼4 h for 3 model IV therapeutics (non-targeted IgG; a protein therapeutic; PEGylated nanoparticles). We compared these passive leak results with targeted brain delivery by IV monoclonal antibodies (mAbs) that actively bind vascular endothelium (anti-VCAM, anti-PECAM, anti-ICAM). Even at early time points after ICH induction, where there is high vascular leak, brain accumulation via passive leak is dwarfed by brain accumulation of endothelial-targeted agents: At 4 h after injury, anti-PECAM mAbs accumulate at 8-fold higher levels in the brain vs. non-immune IgG; anti-VCAM nanoparticles (NPs) deliver a protein therapeutic (superoxide dismutase, SOD) at 4.5-fold higher levels than the carrier-free therapeutic at 24 h after injury. These data suggest that relying on passive vascular leak provides inefficient delivery of therapeutics even at early time points after ICH, and that a better strategy might be targeted delivery to the brain endothelium, which serves as the gateway for the immune attack on the peri-hemorrhage inflamed brain region.
Description: In experimental intracerebral hemorrhage (ICH), endothelial targeting is an advantageous brain delivery strategy even during high vascular leakage (early ICH). This advantage increases with time, as vascular leakage resolves. Display omitted
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