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.

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.

Copper Oxide Nanoparticle-Induced Acute Inflammatory Response and Injury in Murine Lung Is Ameliorated by Synthetic Secoisolariciresinol Diglucoside (LGM2605).

Metal-oxide nanoparticles (MO-NPs), such as the highly bioreactive copper-based nanoparticles (CuO-NPs), are widely used in manufacturing of hundreds of commercial products. Epidemiological studies correlated levels of nanoparticles in ambient air with a significant increase in lung disease. CuO-NPs, specifically, were among the most potent in a set of metal-oxides and carbons studied in parallel regarding DNA damage and cytotoxicity. Despite advances in nanotoxicology research and the characterization of their toxicity, the exact mechanism(s) of toxicity are yet to be defined. We identified chlorination toxicity as a damaging consequence of inflammation and myeloperoxidase (MPO) activation, resulting in macromolecular damage and cell damage/death. We hypothesized that the inhalation of CuO-NPs elicits an inflammatory response resulting in chlorination damage in cells and lung tissues. We further tested the protective action of LGM2605, a synthetic small molecule with known scavenging properties for reactive oxygen species (ROS), but most importantly, for active chlorine species (ACS) and an inhibitor of MPO. CuO-NPs (15 µg/bolus) were instilled intranasally in mice and the kinetics of the inflammatory response in lungs was evaluated 1, 3, and 7 days later. Evaluation of the protective action of LGM2605 was performed at 24 h post-challenge, which was selected as the peak acute inflammatory response to CuO-NP. LGM2605 was given daily via gavage to mice starting 2 days prior to the time of the insult (100 mg/kg). CuO-NPs induced a significant inflammatory influx, inflammasome-relevant cytokine release, and chlorination damage in mouse lungs, which was mitigated by the action of LGM2605. Preventive action of LGM2605 ameliorated the adverse effects of CuO-NP in lung.

Nanocarriers for vascular delivery of anti-inflammatory agents.

There is a need for improved treatment of acute vascular inflammation in conditions such as ischemia-reperfusion injury, acute lung injury, sepsis, and stroke. The vascular endothelium represents an important therapeutic target in these conditions. Furthermore, some anti-inflammatory agents (AIAs) (e.g., biotherapeutics) require precise delivery into subcellular compartments. In theory, optimized delivery to the desired site of action may improve the effects and enable new mechanisms of action of these AIAs. Diverse nanocarriers (NCs) and strategies for targeting them to endothelial cells have been designed and explored for this purpose. Studies in animal models suggest that delivery of AIAs using NCs may provide potent and specific molecular interventions in inflammatory pathways. However, the industrial development and clinical translation of complex NC-AIA formulations are challenging. Rigorous analysis of therapeutic/side effect and benefit/cost ratios is necessary to identify and optimize the approaches that may find clinical utility in the management of acute inflammation.

Mechanisms that determine nanocarrier targeting to healthy versus inflamed lung regions.

Inflamed organs display marked spatial heterogeneity of inflammation, with patches of inflamed tissue adjacent to healthy tissue. To investigate how nanocarriers (NCs) distribute between such patches, we created a mouse model that recapitulates the spatial heterogeneity of the inflammatory lung disease ARDS. NCs targeting the epitope PECAM strongly accumulated in the lungs, but were shunted away from inflamed lung regions due to hypoxic vasoconstriction (HVC). In contrast, ICAM-targeted NCs, which had lower whole-lung uptake than PECAM/NCs in inflamed lungs, displayed markedly higher NC levels in inflamed regions than PECAM/NCs, due to increased regional ICAM. Regional HVC, epitope expression, and capillary leak were sufficient to predict intra-organ of distribution of NCs, antibodies, and drugs. Importantly, these effects were not observable with traditional spatially-uniform models of ARDS, nor when examining only whole-organ uptake. This study underscores how examining NCs' intra-organ distribution in spatially heterogeneous animal models can guide rational NC design.

Vascular Accessibility of Endothelial Targeted Ferritin Nanoparticles.

Targeting nanocarriers to the endothelium, using affinity ligands to cell adhesion molecules such as ICAM-1 and PECAM-1, holds promise to improve the pharmacotherapy of many disease conditions. This approach capitalizes on the observation that antibody-targeted carriers of 100 nm and above accumulate in the pulmonary vasculature more effectively than free antibodies. Targeting of prospective nanocarriers in the 10-50 nm range, however, has not been studied. To address this intriguing issue, we conjugated monoclonal antibodies (Ab) to ICAM-1 and PECAM-1 or their single chain antigen-binding fragments (scFv) to ferritin nanoparticles (FNPs, size 12 nm), thereby producing Ab/FNPs and scFv/FNPs. Targeted FNPs retained their typical symmetric core-shell structure with sizes of 20-25 nm and ∼4-5 Ab (or ∼7-9 scFv) per particle. Ab/FNPs and scFv/FNPs, but not control IgG/FNPs, bound specifically to cells expressing target molecules and accumulated in the lungs after intravenous injection, with pulmonary targeting an order of magnitude higher than free Ab. Most intriguing, the targeting of Ab/FNPs to ICAM-1, but not PECAM-1, surpassed that of larger Ab/carriers targeted by the same ligand. These results indicate that (i) FNPs may provide a platform for targeting endothelial adhesion molecules with carriers in the 20 nm size range, which has not been previously reported; and (ii) ICAM-1 and PECAM-1 (known to localize in different domains of endothelial plasmalemma) differ in their accessibility to circulating objects of this size, common for blood components and nanocarriers.

Glycation vs. glycosylation: a tale of two different chemistries and biology in Alzheimer's disease.

In our previous studies, we reported that the activity of an anti-oxidant enzyme, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) became decreased as the result of glycation in vitro and in vivo. Glycated Cu,Zn-SOD produces hydroxyl radicals in the presence of transition metals due to the formation of a Schiff base adduct and a subsequent Amadori product. This results in the site-specific cleavage of the molecule, followed by random fragmentation. The glycation of other anti-oxidant enzymes such as glutathione peroxidase and thioredoxin reductase results in a loss or decrease in enzyme activity under pathological conditions, resulting in oxidative stress. The inactivation of anti-oxidant enzymes induces oxidative stress in aging, diabetes and neurodegenerative disorders. It is well known that the levels of Amadori products and N(e)-(carboxylmethyl)lysine (CML) and other carbonyl compounds are increased in diabetes, a situation that will be discussed by the other authors in this special issue. We and others, reported that the glycation products accumulate in the brains of patients with Alzheimer's disease (AD) patients as well as in cerebrospinal fluid (CSF), suggesting that glycation plays a pivotal role in the development of AD. We also showed that enzymatic glycosylation is implicated in the pathogenesis of AD and that oxidative stress is also important in this process. Specific types of glycosylation reactions were found to be up- or downregulated in AD patients, and key AD-related molecules including the amyloid-precursor protein (APP), tau, and APP-cleaving enzymes were shown to be functionally modified as the result of glycosylation. These results suggest that glycation as well as glycosylation are involved in oxidative stress that is associated with aging, diabetes and neurodegenerative diseases such as AD.

Engineered Dual Antioxidant Enzyme Complexes Targeting ICAM-1 on Brain Endothelium Reduce Brain Injury-Associated Neuroinflammation.

The neuroinflammatory cascade triggered by traumatic brain injury (TBI) represents a clinically important point for therapeutic intervention. Neuroinflammation generates oxidative stress in the form of high-energy reactive oxygen and nitrogen species, which are key mediators of TBI pathology. The role of the blood-brain barrier (BBB) is essential for proper neuronal function and is vulnerable to oxidative stress. Results herein explore the notion that attenuating oxidative stress at the vasculature after TBI may result in improved BBB integrity and neuroprotection. Utilizing amino-chemistry, a biological construct (designated "dual conjugate" for short) was generated by covalently binding two antioxidant enzymes (superoxide dismutase 1 (SOD-1) and catalase (CAT)) to antibodies specific for ICAM-1. Bioengineering of the conjugate preserved its targeting and enzymatic functions, as evaluated by real-time bioenergetic measurements (via the Seahorse-XF platform), in brain endothelial cells exposed to increasing concentrations of hydrogen peroxide or a superoxide anion donor. Results showed that the dual conjugate effectively mitigated the mitochondrial stress due to oxidative damage. Furthermore, dual conjugate administration also improved BBB and endothelial protection under oxidative insult in an in vitro model of TBI utilizing a software-controlled stretching device that induces a 20% in mechanical strain on the endothelial cells. Additionally, the dual conjugate was also effective in reducing indices of neuroinflammation in a controlled cortical impact (CCI)-TBI animal model. Thus, these studies provide proof of concept that targeted dual antioxidant biologicals may offer a means to regulate oxidative stress-associated cellular damage during neurotrauma.

Physiologically based modeling of LNP-mediated delivery of mRNA in the vascular system.

RNA therapeutics are an emerging, powerful class of drugs with potential applications in a wide range of disorders. A central challenge in their development is the lack of clear pharmacokinetic (PK)-pharmacodynamic relationship, in part due to the significant delay between the kinetics of RNA delivery and the onset of pharmacologic response. To bridge this gap, we have developed a physiologically based PK/pharmacodynamic model for systemically administered mRNA-containing lipid nanoparticles (LNPs) in mice. This model accounts for the physiologic determinants of mRNA delivery, active targeting in the vasculature, and differential transgene expression based on nanoparticle coating. The model was able to well-characterize the blood and tissue PKs of LNPs, as well as the kinetics of tissue luciferase expression measured by ex vivo activity in organ homogenates and bioluminescence imaging in intact organs. The predictive capabilities of the model were validated using a formulation targeted to intercellular adhesion molecule-1 and the model predicted nanoparticle delivery and luciferase expression within a 2-fold error for all organs. This modeling platform represents an initial strategy that can be expanded upon and utilized to predict the in vivo behavior of RNA-containing LNPs developed for an array of conditions and across species.

Synthesis and antioxidant evaluation of (S,S)- and (R,R)-secoisolariciresinol diglucosides (SDGs).

Secoisolariciresinol diglucosides (SDGs) (S,S)-SDG-1 (major isomer in flaxseed) and (R,R)-SDG-2 (minor isomer in flaxseed) were synthesized from vanillin via secoisolariciresinol (6) and glucosyl donor 7 through a concise route that involved chromatographic separation of diastereomeric diglucoside derivatives (S,S)-8 and (R,R)-9. Synthetic (S,S)-SDG-1 and (R,R)-SDG-2 exhibited potent antioxidant properties (EC50=292.17±27.71 µM and 331.94±21.21 µM, respectively), which compared well with that of natural (S,S)-SDG-1 (EC50=275.24±13.15 µM). These values are significantly lower than those of ascorbic acid (EC50=1129.32±88.79 µM) and α-tocopherol (EC50=944.62±148.00 µM). Compounds (S,S)-SDG-1 and (R,R)-SDG-2 also demonstrated powerful scavenging activities against hydroxyl [natural (S,S)-SDG-1: 3.68±0.27; synthetic (S,S)-SDG-1: 2.09±0.16; synthetic (R,R)-SDG-2: 1.96±0.27], peroxyl [natural (S,S)-SDG-1: 2.55±0.11; synthetic (S,S)-SDG-1: 2.20±0.10; synthetic (R,R)-SDG-2: 3.03±0.04] and DPPH [natural (S,S)-SDG-1: EC50=83.94±2.80 µM; synthetic (S,S)-SDG-1: EC50=157.54±21.30 µM; synthetic (R,R)-SDG-2: EC50=123.63±8.67 µM] radicals. These results confirm previous studies with naturally occurring (S,S)-SDG-1 and establish both (S,S)-SDG-1 and (R,R)-SDG-2 as potent antioxidants and free radical scavengers for potential in vivo use.

Mechanisms by Which Liposomes Improve Inhaled Drug Delivery for Alveolar Diseases.

Diseases of the pulmonary alveolus, such as pulmonary fibrosis, are leading causes of morbidity and mortality, but exceedingly few drugs are developed for them. A major reason for this gap is that after inhalation, drugs are quickly whisked away from alveoli due to their high perfusion. To solve this problem, the mechanisms by which nano-scale drug carriers dramatically improve lung pharmacokinetics using an inhalable liposome formulation containing nintedanib, an antifibrotic for pulmonary fibrosis, are studied. Direct instillation of liposomes in murine lung increases nintedanib's total lung delivery over time by 8000-fold and lung half life by tenfold, compared to oral nintedanib. Counterintuitively, it is shown that pulmonary surfactant neither lyses nor aggregates the liposomes. Instead, each lung compartment (alveolar fluid, alveolar leukocytes, and parenchyma) elutes liposomes over 24 h, likely serving as "drug depots." After deposition in the surfactant layer, liposomes are transferred over 3-6 h to alveolar leukocytes (which take up a surprisingly minor 1-5% of total lung dose instilled) in a nonsaturable fashion. Further, all cell layers of the lung parenchyma take up liposomes. These and other mechanisms elucidated here should guide engineering of future inhaled nanomedicine for alveolar diseases.

Breast tumors interfere with endothelial TRAIL at the premetastatic niche to promote cancer cell seeding.

Endothelial cells (ECs) grant access of disseminated cancer cells to distant organs. However, the molecular players regulating the activation of quiescent ECs at the premetastatic niche (PMN) remain elusive. Here, we find that ECs at the PMN coexpress tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its cognate death receptor 5 (DR5). Unexpectedly, endothelial TRAIL interacts intracellularly with DR5 to prevent its signaling and preserve a quiescent vascular phenotype. In absence of endothelial TRAIL, DR5 activation induces EC death and nuclear factor κB/p38-dependent EC stickiness, compromising vascular integrity and promoting myeloid cell infiltration, breast cancer cell adhesion, and metastasis. Consistently, both down-regulation of endothelial TRAIL at the PMN by proangiogenic tumor-secreted factors and the presence of the endogenous TRAIL inhibitors decoy receptor 1 (DcR1) and DcR2 favor metastasis. This study discloses an intracrine mechanism whereby TRAIL blocks DR5 signaling in quiescent endothelia, acting as gatekeeper of the vascular barrier that is corrupted by the tumor during cancer cell dissemination.

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.

PECAM-targeted delivery of SOD inhibits endothelial inflammatory response.

Elevated generation of reactive oxygen species (ROS) by endothelial enzymes, including NADPH-oxidase, is implicated in vascular oxidative stress and endothelial proinflammatory activation involving exposure of vascular cell adhesion molecule-1 (VCAM-1). Catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet/endothelial cell adhesion molecule 1 (PECAM-1) bind specifically to endothelium and inhibit effects of corresponding ROS, H(2)O(2), and superoxide anion. In this study, anti-PECAM/SOD, but not anti-PECAM/catalase or nontargeted enzymes, including polyethylene glycol (PEG)-SOD, inhibited 2- to 3-fold VCAM expression caused by tumor necrosis factor (TNF), interleukin-1ß, and lipopolysaccharide. Anti- PECAM/SOD, but not nontargeted counterparts, accumulated in vascular endothelium after intravenous injection, localized in endothelial endosomes, and inhibited by 70% lipopolysaccharide-caused VCAM-1 expression in mice. Anti-PECAM/SOD colocalized with EEA-1-positive endothelial vesicles and quenched ROS produced in response to TNF. Inhibitors of NADPH oxidase and anion channel ClC3 blocked TNF-induced VCAM expression, affirming that superoxide produced and transported by these proteins, respectively, mediates inflammatory signaling. Anti-PECAM/SOD abolished VCAM expression caused by poly(I:C)-induced activation of toll-like receptor 3 localized in intracellular vesicles. These results directly implicate endosomal influx of superoxide in endothelial inflammatory response and suggest that site-specific interception of this signal attained by targeted delivery of anti-PECAM/SOD into endothelial endosomes may have anti-inflammatory effects.

Added to pre-existing inflammation, mRNA-lipid nanoparticles induce inflammation exacerbation (IE).

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.

Dual Affinity to RBCs and Target Cells (DART) Enhances Both Organ- and Cell Type-Targeting of Intravascular Nanocarriers.

A long-standing goal of nanomedicine is to improve a drug's benefit by loading it into a nanocarrier that homes solely to a specific target cell and organ. Unfortunately, nanocarriers usually end up with only a small percentage of the injected dose (% ID) in the target organ, due largely to clearance by the liver and spleen. Further, cell-type-specific targeting is rarely achieved without reducing target organ accumulation. To solve these problems, we introduce DART (dual affinity to RBCs and target cells), in which nanocarriers are conjugated to two affinity ligands, one binding red blood cells and one binding a target cell (here, pulmonary endothelial cells). DART nanocarriers first bind red blood cells and then transfer to the target organ's endothelial cells as the bound red blood cells squeeze through capillaries. We show that within minutes after intravascular injection in mice nearly 70% ID of DART nanocarriers accumulate in the target organ (lungs), more than doubling the % ID ceiling achieved by a multitude of prior technologies, finally achieving a majority % ID in a target organ. Humanized DART nanocarriers in ex vivo perfused human lungs recapitulate this phenomenon. Furthermore, DART enhances the selectivity of delivery to target endothelial cells over local phagocytes within the target organ by 6-fold. DART's marked improvement in both organ- and cell-type targeting may thus be helpful in localizing drugs for a multitude of medical applications.

Catalase and superoxide dismutase conjugated with platelet-endothelial cell adhesion molecule antibody distinctly alleviate abnormal endothelial permeability caused by exogenous reactive oxygen species and vascular endothelial growth factor.

Reactive oxygen species (ROS) superoxide anion (O(2)()) and hydrogen peroxide (H(2)O(2)) produced by activated leukocytes and endothelial cells in sites of inflammation or ischemia cause endothelial barrier dysfunction that may lead to tissue edema. Antioxidant enzymes (AOEs) catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet-endothelial cell adhesion molecule-1 (PECAM-1) specifically bind to endothelium, quench the corresponding ROS, and alleviate vascular oxidative stress and inflammation. In the present work, we studied the effects of anti-PECAM/catalase and anti-PECAM/SOD conjugates on the abnormal permeability manifested by transendothelial electrical resistance decline, increased fluorescein isothiocyanate-dextran influx, and redistribution of vascular endothelial-cadherin in human umbilical vein endothelial cell (HUVEC) monolayers. Anti-PECAM/catalase protected HUVEC monolayers against H(2)O(2)-induced endothelial barrier dysfunction. Polyethylene glycol-conjugated catalase exerted orders of magnitude lower endothelial uptake and no protective effect, similarly to IgG/catalase. Anti-PECAM/catalase, but not anti-PECAM/SOD, alleviated endothelial hyperpermeability caused by exposure to hypoxanthine/xanthine oxidase, implicating primarily H(2)O(2) in the disruption of the endothelial barrier in this model. Thrombin-induced endothelial permeability was not affected by treatment with anti-PECAM/AOEs or the NADPH oxidase inhibitor apocynin or overexpression of AOEs, indicating that the endogenous ROS play no key role in thrombin-mediated endothelial barrier dysfunction. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase, inhibited a vascular endothelial growth factor (VEGF)-induced increase in endothelial permeability, identifying a key role of endogenous O(2)() in the VEGF-mediated regulation of endothelial barrier function. Therefore, AOEs targeted to endothelial cells provide versatile molecular tools for testing the roles of specific ROS in vascular pathology and may be translated into remedies for these ROS-induced abnormalities.

Targeting antioxidant and antithrombotic biotherapeutics to endothelium.

The endothelium is one of the key targets for pharmacological interventions in oxidative stress and thrombosis, two conditions that are notoriously difficult to treat due to limited efficacy and precision of action of current drugs. Design of molecular and nano-devices that deliver potent antioxidant and antithrombotic therapeutic enzymes to the endothelium holds promise to improve the potency, localization, timing, specificity, safety, and mechanistic precision of these interventions. In particular, cell adhesion molecules expressed on the surface of resting and pathologically altered endothelial cells can be used for drug delivery to the endothelial surface (preferable for thrombolytics) and into intracellular compartments (preferable for antioxidants). Drug delivery platforms including protein conjugates, recombinant fusion constructs, and stealth polymer carriers designed to target these drugs to endothelium are reviewed in this article.

Targeting drug delivery in the vascular system: Focus on endothelium.

The bloodstream is the main transporting pathway for drug delivery systems (DDS) from the site of administration to the intended site of action. In many cases, components of the vascular system represent therapeutic targets. Endothelial cells, which line the luminal surface of the vasculature, play a tripartite role of the key target, barrier, or victim of nanomedicines in the bloodstream. Circulating DDS may accumulate in the vascular areas of interest and in off-target areas via mechanisms bypassing specific molecular recognition, but using ligands of specific vascular determinant molecules enables a degree of precision, efficacy, and specificity of delivery unattainable by non-affinity DDS. Three decades of research efforts have focused on specific vascular targeting, which have yielded a multitude of DDS, many of which are currently undergoing a translational phase of development for biomedical applications, including interventions in the cardiovascular, pulmonary, and central nervous systems, regulation of endothelial functions, host defense, and permeation of vascular barriers. We discuss the design of endothelial-targeted nanocarriers, factors underlying their interactions with cells and tissues, and describe examples of their investigational use in models of acute vascular inflammation with an eye on translational challenges.

Targeted detoxification of selected reactive oxygen species in the vascular endothelium.

Oxidative stress underlies diverse vascular diseases, but its management remains elusive, in part because of our inability to selectively detoxify reactive oxygen species (ROS) in pathological sites and our limited understanding which species need to be eliminated. The antioxidant enzymes (AOEs) superoxide dismutase (SOD) and catalase (which decompose and H(2)O(2), respectively), conjugated with an antibody to platelet-endothelial cell adhesion molecule-1 (PECAM-1), bind to endothelial cells and alleviate oxidative stress in cell culture models. Here, we studied the effects of these antioxidant conjugates in mouse models of vascular oxidative stress. Anti-PECAM/catalase and anti-PECAM/SOD conjugates, in contrast to control IgG/AOE conjugates, accumulated in the lungs and vascularized organs after intravenous injection in wild-type, but not PECAM KO mice. Anti-PECAM/catalase, but not anti-PECAM/SOD, protected mice from lung injury induced by H(2)O(2) produced by glucose oxidase deposited in the pulmonary vasculature. Anti-PECAM/catalase also reduced alveolar edema and attenuated decline in arterial oxygen in mice that underwent unilateral lung ischemia/reperfusion, whereas anti-PECAM/SOD was not effective, implying the key role of H(2)O(2) in tissue damage in this pathology. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase prevented oxidation of tetrahydrobiopterin and normalized vasoreactivity in the vessels of mice rendered hypertensive by pretreatment with angiotensin-II. This outcome agrees with reports implicating superoxide and peroxynitrite in altered endothelium-dependent vasodilatation in hypertension. Therefore, the use of endothelial cell-targeted antioxidants identifies the key specific species of ROS involved in various forms of vascular disease and holds promise for the mechanistically tailored treatment of these pathologies.