Targeting lipid nanoparticles to the blood-brain barrier to ameliorate acute ischemic stroke.

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.

Effective Prevention of Arterial Thrombosis with Albumin-Thrombin Inhibitor Conjugates.

Thromboprophylaxis is indicated in patients at an elevated risk of developing thrombotic disorders, typically using direct oral anticoagulants or low-molecular-weight heparins. We postulated that transient thromboprophylaxis (days-weeks) could be provided by a single dose of an anticoagulant engineered for prolonged pharmacokinetics. In the present work, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone (PPACK) was used as a model anticoagulant to test the hypothesis that conjugation of thrombin inhibitors to the surface of albumin would provide durable protection against thrombotic insults. Covalent conjugates were formed between albumin and PPACK using click chemistry, and they were tested in vitro using a thrombin activity assay and a clot formation assay. Thromboprophylactic efficacy was tested in mouse models of arterial thrombosis, both chemically induced (FeCl3) and following ischemia-reperfusion (transient middle cerebral artery occlusion; tMCAO). Albumin-PPACK conjugates were shown to have nanomolar potency in both in vitro assays, and following intravenous injection had prolonged circulation. Conjugates did not impact hemostasis (tail clipping) or systemic coagulation parameters in normal mice. Intravenous injection of conjugates prior to FeCl3-induced thrombosis provided significant protection against occlusion of the middle cerebral and common carotid arteries, and injection immediately following ischemia-reperfusion reduced stroke volume measured 3 days after injury by ∼40% in the tMCAO model. The data presented here provide support for the use of albumin-linked anticoagulants as an injectable, long-circulating, safe thromboprophylactic agent. In particular, albumin-PPACK provides significant protection against thrombosis induced by multiple mechanisms, without adversely affecting hemostasis.

Selective targeting of nanomedicine to inflamed cerebral vasculature to enhance the blood-brain barrier.

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.

Local BDNF Delivery to the Injured Cervical Spinal Cord using an Engineered Hydrogel Enhances Diaphragmatic Respiratory Function.

We developed an innovative biomaterial-based approach to repair the critical neural circuitry that controls diaphragm activation by locally delivering brain-derived neurotrophic factor (BDNF) to injured cervical spinal cord. BDNF can be used to restore respiratory function via a number of potential repair mechanisms; however, widespread BDNF biodistribution resulting from delivery methods such as systemic injection or lumbar puncture can lead to inefficient drug delivery and adverse side effects. As a viable alternative, we developed a novel hydrogel-based system loaded with polysaccharide-BDNF particles self-assembled by electrostatic interactions that can be safely implanted in the intrathecal space for achieving local BDNF delivery with controlled dosing and duration. Implantation of BDNF hydrogel after C4/C5 contusion-type spinal cord injury (SCI) in female rats robustly preserved diaphragm function, as assessed by in vivo recordings of compound muscle action potential and electromyography amplitudes. However, BDNF hydrogel did not decrease lesion size or degeneration of cervical motor neuron soma, suggesting that its therapeutic mechanism of action was not neuroprotection within spinal cord. Interestingly, BDNF hydrogel significantly preserved diaphragm innervation by phrenic motor neurons (PhMNs), as assessed by detailed neuromuscular junction morphological analysis and retrograde PhMN labeling from diaphragm using cholera toxin B. Furthermore, BDNF hydrogel enhanced the serotonergic axon innervation of PhMNs that plays an important role in modulating PhMN excitability. Our findings demonstrate that local BDNF hydrogel delivery is a robustly effective and safe strategy to restore diaphragm function after SCI. In addition, we demonstrate novel therapeutic mechanisms by which BDNF can repair respiratory neural circuitry.SIGNIFICANCE STATEMENT Respiratory compromise is a leading cause of morbidity and mortality following traumatic spinal cord injury (SCI). We used an innovative biomaterial-based drug delivery system in the form of a hydrogel that can be safely injected into the intrathecal space for achieving local delivery of brain-derived neurotrophic factor (BDNF) with controlled dosing and duration, while avoiding side effects associated with other delivery methods. In a clinically relevant rat model of cervical contusion-type SCI, BDNF hydrogel robustly and persistently improved diaphragmatic respiratory function by enhancing phrenic motor neuron (PhMN) innervation of the diaphragm neuromuscular junction and by increasing serotonergic innervation of PhMNs in ventral horn of the cervical spinal cord. These exciting findings demonstrate that local BDNF hydrogel delivery is a safe and robustly effective strategy to maintain respiratory function after cervical SCI.

A hydrogel engineered to deliver minocycline locally to the injured cervical spinal cord protects respiratory neural circuitry and preserves diaphragm function.

We tested a biomaterial-based approach to preserve the critical phrenic motor circuitry that controls diaphragm function by locally delivering minocycline hydrochloride (MH) following cervical spinal cord injury (SCI). MH is a clinically-available antibiotic and anti-inflammatory drug that targets a broad range of secondary injury mechanisms via its anti-inflammatory, anti-oxidant and anti-apoptotic properties. However, MH is only neuroprotective at high concentrations that cannot be achieved by systemic administration, which limits its clinical efficacy. We have developed a hydrogel-based MH delivery system that can be injected into the intrathecal space for local delivery of high concentrations of MH, without damaging spinal cord tissue. Implantation of MH hydrogel after unilateral level-C4/5 contusion SCI robustly preserved diaphragm function, as assessed by in vivo recordings of compound muscle action potential (CMAP) and electromyography (EMG) amplitudes. MH hydrogel also decreased lesion size and degeneration of cervical motor neuron somata, demonstrating its central neuroprotective effects within the injured cervical spinal cord. Furthermore, MH hydrogel significantly preserved diaphragm innervation by the axons of phrenic motor neurons (PhMNs), as assessed by both detailed neuromuscular junction (NMJ) morphological analysis and retrograde PhMN labeling from the diaphragm using cholera toxin B (CTB). In conclusion, our findings demonstrate that local MH hydrogel delivery to the injured cervical spinal cord is effective in preserving respiratory function after SCI by protecting the important neural circuitry that controls diaphragm activation.

Hepatocyte Growth Factor Delivery to Injured Cervical Spinal Cord Using an Engineered Biomaterial Protects Respiratory Neural Circuitry and Preserves Functional Diaphragm Innervation.

A major portion of spinal cord injury (SCI) cases occur in the cervical region, where essential components of the respiratory neural circuitry are located. Phrenic motor neurons (PhMNs) housed at cervical spinal cord level C3-C5 directly innervate the diaphragm, and SCI-induced damage to these cells severely impairs respiratory function. In this study, we tested a biomaterial-based approach aimed at preserving this critical phrenic motor circuitry after cervical SCI by locally delivering hepatocyte growth factor (HGF). HGF is a potent mitogen that promotes survival, proliferation, migration, repair, and regeneration of a number of different cell and tissue types in response to injury. We developed a hydrogel-based HGF delivery system that can be injected into the intrathecal space for local delivery of high levels of HGF without damaging the spinal cord. Implantation of HGF hydrogel after unilateral C5 contusion-type SCI in rats preserved diaphragm function, as assessed by in vivo recordings of both compound muscle action potentials and inspiratory electromyography amplitudes. HGF hydrogel also preserved PhMN innervation of the diaphragm, as assessed by both retrograde PhMN tracing and detailed neuromuscular junction morphological analysis. Furthermore, HGF hydrogel significantly decreased lesion size and degeneration of cervical motor neuron cell bodies, as well as reduced levels surrounding the injury site of scar-associated chondroitin sulfate proteoglycan molecules that limit axon growth capacity. Our findings demonstrate that local biomaterial-based delivery of HGF hydrogel to injured cervical spinal cord is an effective strategy for preserving respiratory circuitry and diaphragm function.

Targeting of nanoparticles to the cerebral vasculature after traumatic brain injury.

Traumatic brain injury has faced numerous challenges in drug development, primarily due to the difficulty of effectively delivering drugs to the brain. However, there is a potential solution in targeted drug delivery methods involving antibody-drug conjugates or nanocarriers conjugated with targeting antibodies. Following a TBI, the blood-brain barrier (BBB) becomes permeable, which can last for years and allow the leakage of harmful plasma proteins. Consequently, an appealing approach for TBI treatment involves using drug delivery systems that utilize targeting antibodies and nanocarriers to help restore BBB integrity. In our investigation of this strategy, we examined the efficacy of free antibodies and nanocarriers targeting a specific endothelial surface marker called vascular cell adhesion molecule-1 (VCAM-1), which is known to be upregulated during inflammation. In a mouse model of TBI utilizing central fluid percussion injury, free VCAM-1 antibody did not demonstrate superior targeting when comparing sham vs. TBI brain. However, the administration of VCAM-1-targeted nanocarriers (liposomes) exhibited a 10-fold higher targeting specificity in TBI brain than in sham control. Flow cytometry and confocal microscopy analysis confirmed that VCAM-1 liposomes were primarily taken up by brain endothelial cells post-TBI. Consequently, VCAM-1 liposomes represent a promising platform for the targeted delivery of therapeutics to the brain following traumatic brain injury.

Dynamic natural components and morphological changes in nonculprit subclinical atherosclerosis in patients with acute coronary syndrome and mild chronic kidney disease at the 1-year follow-up and clinical significance at the 5-year follow-up.

INTRODUCTION: The natural outcome of coronary plaque in acute coronary syndrome (ACS) patients with chronic kidney disease (CKD) is unique, which can be analyzed quantitatively by optical flow ratio (OFR) software. METHODS: A total of 184 ACS patients with at least one nonculprit subclinical atherosclerosis (NSA) detected by optical coherence tomography (OCT) at baseline and 1-year follow-up were divided into non-CKD group (n = 106, estimated glomerular filtration rate (eGFR)> 90 mL/(min×1.73 m2)) and mild CKD group (n = 78, 60≤eGFR<90 mL/(min×1.73 m2)). Changes of normalized total atheroma volume (TAVn) of NSA was the primary endpoint at the 1-year follow-up. RESULTS: Patients with mild CKD showed more TAVn progression of NSA than non-CKD (p = 0.019) from baseline to the 1-year follow-up, which was mainly due to an increase in calcium TAVn (p<0.001). The morphological change in the maximal calcification thickness (p = 0.026) was higher and the change in the distance from the calcified surface to the contralateral coronary media membrane (ΔC-to-M) at the maximal cross-sectional calcium area was lower (p<0.001) in mild CKD group than in non-CKD group. Mild CKD had more NSA related MACEs at the 5-year follow-up than non-CKD (30.8% vs. 5.8%, p = 0.045). CONCLUSIONS: Mild CKD patients had more plaque progression of NSA which showed the increase of calcium component with more protrusion into the lumen morphologically at the 1-year follow-up and a higher corresponding incidence of NSA-related MACEs at the 5-year follow-up. TRIAL REGISTRATION: Clinical Trial registration ClinicalTrials.gov. NCT02140801. https://classic.clinicaltrials.gov/ct2/show/NCT02140801.

Nanocarriers' repartitioning of drugs between blood subcompartments as a mechanism of improving pharmacokinetics, safety, and efficacy.

For medical emergencies, such as acute ischemic stroke, rapid drug delivery to the target site is essential. For many small molecule drugs, this goal is unachievable due to poor solubility that prevents intravenous administration, and less obviously, by extensive partitioning to plasma proteins and red blood cells (RBCs), which greatly slows delivery to the target. Here we study these effects and how they can be solved by loading into nanoscale drug carriers. We focus on fingolimod, a small molecule drug that is FDA-approved for treatment of multiple sclerosis, which has also shown promise in the treatment of stroke. Unfortunately, fingolimod has poor solubility and very extensive partitioning to plasma proteins and RBCs (in whole blood, 86% partitions to RBCs, 13.96% to plasma proteins, and 0.04% is free). We develop a liposomal formulation that slows the partitioning of fingolimod to RBCs and plasma proteins, enables intravenous delivery, and additionally prevents fingolimod toxicity to RBCs. The liposomal formulation nearly completely prevented fingolimod adsorption to plasma proteins (association with plasma proteins was 98.4 ± 0.4% for the free drug vs. 5.6 ± 0.4% for liposome-loaded drug). When incubated with whole blood in vitro, the liposomal formulation greatly slowed partitioning of fingolimod to RBCs and also eliminated deleterious effects of fingolimod on RBC rigidity, morphology, and hemolysis. In vivo, the liposomal formulation delayed fingolimod partitioning to RBCs for over 30 min, a critical time window for stroke. Fingolimod-loaded liposomes showed improved efficacy in a mouse model of post-stroke neuroinflammation, completely sealing the leaky blood-brain barrier (114 ± 11.5% reduction in albumin leak into the brain for targeted liposomes vs. 38 ± 16.5% reduction for free drug). This effect was only seen for liposomes modified with antibodies to enable targeted delivery to the site of action, and not in unmodified, long-circulating liposomes. Thus, loading fingolimod into liposomes prevented partitioning to RBCs and associated toxicities and enabled targeted delivery. This paradigm can be used for tuning the blood distribution of small molecule drugs for the treatment of acute illnesses requiring rapid pharmacologic intervention.

A percolation-type criticality threshold controls immune protein coating of surfaces.

When a material enters the body, it is immediately attacked by hundreds of proteins, organized into complex networks of binding interactions and reactions. How do such complex systems interact with a material, "deciding" whether to attack? We focus on the "complement" system of ∼40 blood proteins that bind microbes, nanoparticles, and medical devices, initiating inflammation. We show a sharp threshold for complement activation upon varying a fundamental material parameter, the surface density of potential complement attachment points. This sharp threshold manifests at scales spanning single nanoparticles to macroscale pathologies, shown here for diverse engineered and living materials. Computational models show these behaviors arise from a minimal subnetwork of complement, manifesting percolation-type critical transitions in the complement response. This criticality switch explains the "decision" of a complex signaling network to interact with a material, and elucidates the evolution and engineering of materials interacting with the body.

Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions.

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.

Effect of negative remodeling of the side branch ostium on the efficacy of a two-stent strategy for distal left main bifurcation lesions: an intravascular ultrasound study.

OBJECTIVES: To investigate whether negative remodeling (NR) detected by intravascular ultrasound (IVUS) of the side branch ostium (SBO) would affect in-stent neointimal hyperplasia (NIH) at the one-year follow-up and the clinical outcome of target lesion failure (TLF) at the long-term follow-up for patients with left main bifurcation (LMb) lesions treated with a two-stent strategy. METHODS: A total of 328 patients with de novo true complex LMb lesions who underwent a 2-stent strategy of percutaneous coronary intervention (PCI) treatment guided by IVUS were enrolled in this study. We divided the study into two phases. Of all the patients, 48 patients who had complete IVUS detection pre- and post-PCI and at the 1-year follow-up were enrolled in phase I analysis, which aimed to analyze the correlation between NR and in-stent NIH at SBO at the 1-year follow-up. If the correlation was confirmed, the cutoff value of the remodeling index (RI) for predicting NIH ≥ 50% was analyzed next. The phase II analysis focused on the incidence of TLF as the primary endpoint at the 1- to 5-year follow-up for all 328 patients by grouping based on the cutoff value of RI. RESULTS: In phase I: according to the results of a binary logistic regression analysis and receiver operating characteristic (ROC) analysis, the RI cutoff value predicting percent NIH ≥ 50% was 0.85 based on the ROC curve analysis, with a sensitivity of 85.7%, a specificity of 88.3%, and an AUC of 0.893 (0.778, 1.000), P = 0.002. In phase II: the TLR rate (35.8% vs. 5.3%, P < 0.0001) was significantly higher in the several NR (sNR, defined as RI ≤ 0.85) group than in the non-sNR group. CONCLUSION: The NR of LCxO is associated with more in-stent NIH post-PCI for distal LMb lesions with a 2-stent strategy, and NR with RI ≤ 0.85 is linked to percent NIH area ≥ 50% at the 1-year follow-up and more TLF at the 5-year follow-up.

Marginated Neutrophils in the Lungs Effectively Compete for Nanoparticles Targeted to the Endothelium, Serving as a Part of the Reticuloendothelial System.

Nanomedicine has long pursued the goal of targeted delivery to specific organs and cell types but has yet to achieve this goal with the vast majority of targets. One rare example of success in this pursuit has been the 25+ years of studies targeting the lung endothelium using nanoparticles conjugated to antibodies against endothelial surface molecules. However, here we show that such "endothelial-targeted" nanocarriers also effectively target the lungs' numerous marginated neutrophils, which reside in the pulmonary capillaries and patrol for pathogens. We show that marginated neutrophils' uptake of many of these "endothelial-targeted" nanocarriers is on par with endothelial uptake. This generalizes across diverse nanomaterials and targeting moieties and was even found with physicochemical lung tropism (i.e., without targeting moieties). Further, we observed this in ex vivo human lungs and in vivo healthy mice, with an increase in marginated neutrophil uptake of nanoparticles caused by local or distant inflammation. These findings have implications for nanomedicine development for lung diseases. These data also suggest that marginated neutrophils, especially in the lungs, should be considered a major part of the reticuloendothelial system (RES), with a special role in clearing nanoparticles that adhere to the lumenal surfaces of blood vessels.

Preliminary study of the significance of reverberation by IVUS detection for patients with severe calcified lesions.

To explore the potential significance of the reverberation of calcification by comparing both intravascular ultrasound (IVUS) and optical coherence tomography (OCT) measurement post manual coregistration. The reverberation phenomenon is often detected by IVUS for severe calcified lesions post rotational atherectomy (RA), which is thought to be due to the glassy and smooth inner surfaces of calcifications. Because of the poor penetration of IVUS, it is impossible to measure the thickness of calcifications, and the relationship between multiple reverberations and the thickness of calcification lesions has not been reported before. A total of forty-nine patients with severe calcified coronary lesions that were detected by IVUS and OCT simultaneously were enrolled in our retrospective study. If reverberation phenomena were detected by IVUS, intravascular imaging (IVI) data (including distance between the IVUS catheter center and the inner surface of the reverberation signal, the intervals between all adjacent reverberation signals, the number of layers of reverberation in IVUS, and the thickness of the calcification in OCT) were measured at the same position and same direction (each cross-section had 4 mutually perpendicular directions) at 1-mm intervals. The correlation between each reverberation observational value and OCT data was the primary target in this retrospective study, and the correlation between reverberation and calcium crack post predilatation was analyzed in other 15 patients. Four hundred twenty-eight valid observational points were analyzed simultaneously by IVUS and OCT; among them, 300 points had a single layer of reverberation, 83 had double layers of reverberation and 42 had multiple layers (≥ 3 layers) of reverberation by IVUS detection post-RA. Multivariate logistic regression analysis showed that the number of layers of reverberation by IVUS was significantly related to the thickness of calcifications by OCT at the same point and in the same direction (p < 0.001). Single, double, and multiple layers of reverberation in IVUS correspond to median calcification thicknesses (interquartile ranges (IQRs)) of 0.620 mm (0.520-0.720), 0.950 mm (0.840-1.040) and 1.185 mm (1.068-1.373), respectively, by OCT detection. Another 100 points in other 15 patients with integrated IVUS data pre- and post-predilatation showed that only single layer of reverberation was related to calcium crack (p < 0.001). The number of layers of reverberation signal detected by IVUS is positively correlated with the thickness of calcifications measured by OCT post-RA and single layer of reverberation is correlated to calcium crack post-predilatation.

Morphologies and composition changes in nonculprit subclinical atherosclerosis in diabetic versus nondiabetic patients with acute coronary syndrome who underwent long-term statin therapy.

Although patients are undergoing similar lipid-lowering therapy (LLT) with statins, the outcomes of coronary plaque in diabetic mellitus (DM) and non-DM patients are different. Clinical data of 239 patients in this observational study with acute coronary syndrome was from our previous randomized trial were analyzed at 3 years, and 114 of them underwent OCT detection at baseline and the 1-year follow-up were re-anlayzed by a novel artificial intelligence imaging software for nonculprit subclinical atherosclerosis (nCSA). Normalized total atheroma volume changes (ΔTAVn) of nCSA were the primary endpoint. Plaque progression (PP) was defined as any increase in ΔTAVn. DM patients showed more PP in nCSA (ΔTAVn; 7.41 (- 2.82, 11.85) mm3 vs. - 1.12 (- 10.67, 9.15) mm3, p = 0.009) with similar reduction of low-density lipoprotein cholesterol (LDL-C) from baseline to 1-year. The main reason is that the lipid component in nCSA increases in DM patients and non-significantly decreases in non-DM patients, which leads to a significantly higher lipid TAVn (24.26 (15.05, 40.12) mm3 vs. 16.03 (6.98, 26.54) mm3, p = 0.004) in the DM group than in the non-DM group at the 1-year follow-up. DM was an independent predictor of PP in multivariate logistic regression analysis (OR = 2.731, 95% CI 1.160-6.428, p = 0.021). Major adverse cardiac events (MACEs) related to nCSA at 3 years were higher in the DM group than in the non-DM group (9.5% vs. 1.7%, p = 0.027). Despite a comparable reduction in LDL-C levels after LLT, more PP with an increase in the lipid component of nCSA and a higher incidence of MACEs at the 3-year follow-up was observed in DM patients.Trial registration: ClinicalTrials.gov. identifier: NCT02140801.

Targeted drug delivery to the brain endothelium dominates over passive delivery via vascular leak in experimental intracerebral hemorrhage.

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.

Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions.

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 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. In a popular approach, physicochemical targeting is accomplished by formulating with charged lipids. Negatively charged lipids localize LNPs to the spleen, and positively charged lipids to the lungs. Here we found that lung-tropic LNPs employing cationic lipids induce massive thrombosis. We demonstrate that thrombosis is induced in the lungs and other organs, and greatly exacerbated by pre-existing inflammation. This clotting is induced by a variety of formulations with cationic lipids, including LNPs and non-LNP nanoparticles. The mechanism depends on the LNPs binding to fibrinogen and inducing platelet and thrombin activation. Based on these mechanisms, we engineered multiple solutions which enable positively charged LNPs to target the lungs while not inducing thrombosis. Our findings implicate thrombosis as a major barrier that blood erects against LNPs with cationic components and illustrate how physicochemical targeting approaches must be investigated early for risks and re-engineered with a careful understanding of biological mechanisms.

Targeted Nanocarriers Co-Opting Pulmonary Intravascular Leukocytes for Drug Delivery to the Injured Brain.

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.

Targeting lipid nanoparticles to the blood brain barrier to ameliorate acute ischemic stroke.

After more than 100 failed drug trials for acute ischemic stroke (AIS), one of the most commonly cited reasons for the failure has been that drugs achieve very low concentrations in the at-risk penumbra. To address this problem, here we employ nanotechnology to significantly enhance drug concentration in the penumbra's blood-brain barrier (BBB), whose increased permeability in AIS has long been hypothesized to kill neurons by exposing them to toxic plasma proteins. To devise drug-loaded nanocarriers targeted to the BBB, we conjugated them with antibodies that bind to various cell adhesion molecules on the BBB endothelium. In the transient middle cerebral artery occlusion (tMCAO) mouse model, nanocarriers targeted with VCAM antibodies achieved the highest level of brain delivery, nearly 2 orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles loaded with either a small molecule drug (dexamethasone) or mRNA (encoding IL-10) reduced cerebral infarct volume by 35% or 73%, respectively, and both significantly lowered mortality rates. In contrast, the drugs delivered without the nanocarriers had no effect on AIS outcomes. Thus, VCAM-targeted lipid nanoparticles represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS. Graphical abstract: Acute ischemic stroke induces upregulation of VCAM. We specifically targeted upregulated VCAM in the injured region of the brain with drug- or mRNA-loaded targeted nanocarriers. Nanocarriers targeted with VCAM antibodies achieved the highest brain delivery, nearly orders of magnitude higher than untargeted ones. VCAM-targeted nanocarriers loaded with dexamethasone and mRNA encoding IL-10 reduced infarct volume by 35% and 73%, respectively, and improved survival rates.

Targeting vascular inflammation through emerging methods and drug carriers.

Acute inflammation is a common dangerous component of pathogenesis of many prevalent conditions with high morbidity and mortality including sepsis, thrombosis, acute respiratory distress syndrome (ARDS), COVID-19, myocardial and cerebral ischemia-reperfusion, infection, and trauma. Inflammatory changes of the vasculature and blood mediate the course and outcome of the pathology in the tissue site of insult, remote organs and systemically. Endothelial cells lining the luminal surface of the vasculature play the key regulatory functions in the body, distinct under normal vs. pathological conditions. In theory, pharmacological interventions in the endothelial cells might enable therapeutic correction of the overzealous damaging pro-inflammatory and pro-thrombotic changes in the vasculature. However, current agents and drug delivery systems (DDS) have inadequate pharmacokinetics and lack the spatiotemporal precision of vascular delivery in the context of acute inflammation. To attain this level of precision, many groups design DDS targeted to specific endothelial surface determinants. These DDS are able to provide specificity for desired tissues, organs, cells, and sub-cellular compartments needed for a particular intervention. We provide a brief overview of endothelial determinants, design of DDS targeted to these molecules, their performance in experimental models with focus on animal studies and appraisal of emerging new approaches. Particular attention is paid to challenges and perspectives of targeted therapeutics and nanomedicine for advanced management of acute inflammation.