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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., Competing Interests: Declaration of interests J.N., P.M.G., V.V.S., H.P., D.W., J.S.B., V.R.M., and O.A.M-C. have pending patent applications fully disclosed by the University of Pennsylvania., (Copyright © 2024. Published by Elsevier Inc.)

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., Competing Interests: H.P. and D.W. are scientific founders and hold equity in Capstan Therapeutics. S.C.S. and B.L.M. are employees and hold equity in Acuitas Therapeutics. H.P. and D.W. receive research support from BioNTech. H.P., V.V.S., D.W., and V.R.M. are named on patents that describe the use of nucleoside-modified mRNA and targeted LNP., (© 2024 The Authors.)

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.

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.

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.

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., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Advanced NanoBiomed Research published by Wiley‐VCH GmbH.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 ( d ual a ffinity to R BCs and t arget 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.

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.

Targeted drug delivery to the endothelium has the potential to generate localized therapeutic effects at the blood-tissue interface. For some therapeutic cargoes, it is essential to maintain contact with the bloodstream to exert protective effects. The pharmacokinetics (PK) of endothelial surface-targeted affinity ligands and biotherapeutic cargo remain a largely unexplored area, despite obvious translational implications for this strategy. To bridge this gap, we site-specifically radiolabeled mono- (scFv) and bivalent (mAb) affinity ligands specific for the endothelial cell adhesion molecules, PECAM-1 (CD31) and ICAM-1 (CD54). Radiotracing revealed similar lung biodistribution at 30 minutes post-injection (79.3% ± 4.2% vs 80.4% ± 10.6% ID/g for αICAM and 58.9% ± 3.6% ID/g vs. 47.7% ± 5.8% ID/g for αPECAM mAb vs. scFv), but marked differences in organ residence time, with antibodies demonstrating an order of magnitude greater area under the lung concentration vs. time curve (AUC inf 1698 ± 352 vs. 53.3 ± 7.9 ID/g*hrs for αICAM and 1023 ± 507 vs. 114 ± 37 ID/g*hrs for αPECAM mAb vs scFv). A physiologically based pharmacokinetic model, fit to and validated using these data, indicated contributions from both superior binding characteristics and prolonged circulation time supporting multiple binding-detachment cycles. We tested the ability of each affinity ligand to deliver a prototypical surface cargo, thrombomodulin (TM), using one-to-one protein conjugates. Bivalent mAb-TM was superior to monovalent scFv-TM in both pulmonary targeting and lung residence time (AUC inf 141 ± 3.2 vs 12.4 ± 4.2 ID/g*hrs for ICAM and 188 ± 90 vs 34.7 ± 19.9 ID/g*hrs for PECAM), despite having similar blood PK, indicating that binding strength is more important parameter than the kinetics of binding. To maximize bivalent target engagement, we synthesized an oriented, end-to-end anti-ICAM mAb-TM conjugate and found that this therapeutic had the best lung residence time (AUC inf 253 ± 18 ID/g*hrs) of all TM modalities. These observations have implications not only for the delivery of TM, but also potentially all therapeutics targeted to the endothelial surface., (© 2020 Federation of American Societies for Experimental Biology.)

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., Competing Interests: Competing interest statement: O.A.M.-C., H.P., V.V.S., D.W., and V.R.M. are inventors on a patent filed on some aspects of this work. Those interests have been fully disclosed to the University of Pennsylvania.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Deformability of injectable nanocarriers impacts rheological behavior, drug loading, and affinity target adhesion. Here, we present atomic force microscopy (AFM) and spectroscopy measurements of nanocarrier Young's moduli, tune the moduli of deformable nanocarriers with cross-linkers, and demonstrate vascular targeting behavior that correlates with Young's modulus. Homobifunctional cross-linkers were introduced into lysozyme-dextran nanogels (NGs). Single particle-scale AFM measurements determined NG moduli varying from ∼50-150 kPa for unmodified NGs or NGs with a short hydrophilic cross-linker (2,2'-(ethylenedioxy)bis(ethylamine), EOD) to ∼350 kPa for NGs modified with a longer hydrophilic cross-linker (4,9-dioxa-1,12-dodecanediamine, DODD) to ∼10 MPa for NGs modified with a longer hydrophobic cross-linker (1,12-diaminododecane, DAD). Cross-linked NGs were conjugated to antibodies for plasmalemma vesicle associated protein (PLVAP), a caveolar endothelial marker that cannot be accessed by rigid particles larger than ∼100 nm. In previous work, 150 nm NGs effectively targeted PLVAP, where rigid particles of similar diameter did not. EOD-modified NGs targeted PLVAP less effectively than unmodified NGs, but more effectively than DODD or DAD modified NGs, which both yielded low levels of targeting, resembling results previously obtained with polystyrene particles. Cross-linked NGs were also conjugated to antibodies against intracellular adhesion molecule-1 (ICAM-1), an endothelial marker accessible to large rigid particles. Cross-linked NGs and unmodified NGs targeted uniformly to ICAM-1. We thus demonstrate cross-linker modification of NGs, AFM determination of NG mechanical properties varying with cross-linker, and tuning of specific sterically constrained vascular targeting behavior in correlation with cross-linker-modified NG mechanical properties.

The endothelium is a thin monolayer of specialized cells that lines the luminal wall of blood vessels and constitutes the critical innermost portion of the physical barrier between the blood and the brain termed the blood-brain barrier (BBB). Aberrant changes in the endothelium occur in many neuropathological states, including those with high morbidity and mortality that lack targeted therapeutic interventions, such as traumatic brain injury (TBI). Utilizing ligands of surface determinants expressed on brain endothelium to target and combat injury mechanisms at damaged endothelium offers a new approach to the study of TBI and new avenues for clinical advancement. Many factors influence the targets that are expressed on endothelium. Therefore, the optimization of binding sites and ideal design features of nanocarriers are controllable factors that permit the engineering of nanotherapeutic agents with applicability that is specific to a known disease state. Following TBI, damaged endothelial cells upregulate cell adhesion molecules, including ICAM-1, and are key sites of reactive oxygen species (ROS) generation, including hydrogen peroxide. Reactive oxygen species along with pro-inflammatory mediators are known to contribute to endothelial damage and loss of BBB integrity. The use of targeted endothelial nanomedicine, with conjugates of the antioxidant enzyme catalase linked to anti-ICAM-1 antibodies, has recently been demonstrated to minimize oxidative stress at the BBB and reduce neuropathological outcomes following TBI. Here, we discuss targeted endothelial nanomedicine and its potential to provide benefits in TBI outcomes and future directions of this approach.

Systemic administration of lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) leads predominantly to hepatic uptake and expression. Here, we conjugated nucleoside-modified mRNA-LNPs with antibodies (Abs) specific to vascular cell adhesion molecule, PECAM-1. Systemic (intravenous) administration of Ab/LNP-mRNAs resulted in profound inhibition of hepatic uptake concomitantly with ~200-fold and 25-fold elevation of mRNA delivery and protein expression in the lungs compared to non-targeted counterparts. Unlike hepatic delivery of LNP-mRNA, Ab/LNP-mRNA is independent of apolipoprotein E. Vascular re-targeting of mRNA represents a promising, powerful, and unique approach for novel experimental and clinical interventions in organs of interest other than liver., (Copyright © 2018. Published by Elsevier B.V.)

One of the goals of nanomedicine is targeted delivery of therapeutic enzymes to the sub-cellular compartments where their action is needed. Endothelial caveolae-derived endosomes represent an important yet challenging destination for targeting, in part due to smaller size of the entry aperture of caveolae (ca. 30-50 nm). Here, we designed modular, multi-molecular, ferritin-based nanocarriers with uniform size (20 nm diameter) for easy drug-loading and targeted delivery of enzymatic cargo to these specific vesicles. These nanocarriers targeted to caveolar Plasmalemmal Vesicle-Associated Protein (Plvap) deliver superoxide dismutase (SOD) into endosomes in endothelial cells, the specific site of influx of superoxide mediating by such pro-inflammatory signaling as some cytokines and lipopolysaccharide (LPS). Cell studies showed efficient internalization of Plvap-targeted SOD-loaded nanocarriers followed by dissociation from caveolin-containing vesicles and intracellular transport to endosomes. The nanocarriers had a profound protective anti-inflammatory effect in an animal model of LPS-induced inflammation, in agreement with the characteristics of their endothelial uptake and intracellular transport, indicating that these novel, targeted nanocarriers provide an advantageous platform for caveolae-dependent delivery of biotherapeutics., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Vascular endothelial cells represent an important therapeutic target in many pathologies, including inflammation, oxidative stress, and thrombosis; however, delivery of drugs to this site is often limited by the lack of specific affinity of therapeutics for these cells. Selective delivery of both small molecule drugs and therapeutic proteins to the endothelium has been achieved through the use of targeting ligands, such as monoclonal antibodies, directed against endothelial cell surface markers, particularly cell adhesion molecules (CAMs). Careful selection of target molecules and targeting agents allows for precise delivery to sites of inflammation, thereby maximizing therapeutic drug concentrations at the site of injury. A good understanding of the physiological and pathological determinants of drug and drug carrier pharmacokinetics and biodistribution may allow for a priori identification of optimal properties of drug carrier and targeting agent. Targeted delivery of therapeutics such as antioxidants and antithrombotic agents to the injured endothelium has shown efficacy in preclinical models, suggesting the potential for translation into clinical practice. As with all therapeutics, demonstration of both efficacy and safety are required for successful clinical implementation, which must be considered not only for the individual components (drug, targeting agent, etc.) but also for the sum of the parts (e.g., the drug delivery system), as unexpected toxicities may arise with complex delivery systems. While the use of endothelial targeting has not been translated into the clinic to date, the preclinical results summarized here suggest that there is hope for successful implementation of these agents in the years to come.

Molecular targeting of nanoparticle drug carriers promises maximized therapeutic impact to sites of disease or injury with minimized systemic effects. Precise targeting demands addressing to subcellular features. Caveolae, invaginations in cell membranes implicated in transcytosis and inflammatory signaling, are appealing subcellular targets. Caveolar geometry has been reported to impose a ≈50 nm size cutoff on nanocarrier access to plasmalemma vesicle associated protein (PLVAP), a marker found in caveolae in the lungs. The use of deformable nanocarriers to overcome that size cutoff is explored in this study. Lysozyme-dextran nanogels (NGs) are synthesized with ≈150 or ≈300 nm mean diameter. Atomic force microscopy indicates the NGs deform on complementary surfaces. Quartz crystal microbalance data indicate that NGs form softer monolayers (≈60 kPa) than polystyrene particles (≈8 MPa). NGs deform during flow through microfluidic channels, and modeling of NG extrusion through porous filters yields sieving diameters less than 25 nm for NGs with 150 and 300 nm hydrodynamic diameters. NGs of 150 and 300 nm diameter target PLVAP in mouse lungs while counterpart rigid polystyrene particles do not. The data in this study indicate a role for mechanical deformability in targeting large high-payload drug-delivery vehicles to sterically obscured targets like PLVAP., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ferritin subunits of heavy and light polypeptide chains self-assemble into a spherical nanocage that serves as a natural transport vehicle for metals but can include diverse cargoes. Ferritin nanoparticles are characterized by remarkable stability, small and uniform size. Chemical modifications and molecular re-engineering of ferritin yield a versatile platform of nanocarriers capable of delivering a broad range of therapeutic and imaging agents. Targeting moieties conjugated to the ferritin external surface provide multivalent anchoring of biological targets. Here, we highlight some of the current work on ferritin as well as examine potential strategies that could be used to functionalize ferritin via chemical and genetic means to enable its utility in vascular drug delivery., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Inflammatory mediators binding to Toll-Like receptors (TLR) induce an influx of superoxide anion in the ensuing endosomes. In endothelial cells, endosomal surplus of superoxide causes pro-inflammatory activation and TLR4 agonists act preferentially via caveolae-derived endosomes. To test the hypothesis that SOD delivery to caveolae may specifically inhibit this pathological pathway, we conjugated SOD with antibodies (Ab/SOD, size ~10nm) to plasmalemmal vesicle-associated protein (Plvap) that is specifically localized to endothelial caveolae in vivo and compared its effects to non-caveolar target CD31/PECAM-1. Plvap Ab/SOD bound to endothelial cells in culture with much lower efficacy than CD31 Ab/SOD, yet blocked the effects of LPS signaling with higher efficiency than CD31 Ab/SOD. Disruption of cholesterol-rich membrane domains by filipin inhibits Plvap Ab/SOD endocytosis and LPS signaling, implicating the caveolae-dependent pathway(s) in both processes. Both Ab/SOD conjugates targeted to Plvap and CD31 accumulated in the lungs after IV injection in mice, but the former more profoundly inhibited LPS-induced pulmonary inflammation and elevation of plasma level of interferon-beta and -gamma and interleukin-27. Taken together, these results indicate that targeted delivery of SOD to specific cellular compartments may offer effective, mechanistically precise interception of pro-inflammatory signaling mediated by reactive oxygen species., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Targeting drugs to endothelial cells has shown the ability to improve outcomes in animal models of inflammatory, ischemic and thrombotic diseases. Previous studies have revealed that certain pairs of ligands (antibodies and antibody fragments) specific for adjacent, but distinct, epitopes on PECAM-1 enhance each other's binding, a phenomenon dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL. This discovery has been leveraged to enable simultaneous delivery of multiple therapeutics to the vascular endothelium. Given the known role of PECAM-1 in promoting endothelial quiescence and cell junction integrity, we sought here to determine if CEPAL might induce unintended vascular effects. Using a combination of in vitro and in vivo techniques and employing human and mouse endothelial cells under physiologic and pathologic conditions, we found only modest or non-significant effects in response to antibodies to PECAM-1, whether given solo or in pairs. In contrast, these methods detected significant elevation of endothelial permeability, pro-inflammatory vascular activation, and systemic cytokine release following antibody binding to the related endothelial junction protein, VE-Cadherin. These studies support the notion that PECAM-1-targeted CEPAL provides relatively well-tolerated endothelial drug delivery. Additionally, the analysis herein creates a template to evaluate potential toxicities of vascular-targeted nanoparticles and protein therapeutics.

The pulmonary vasculature plays an important role in many lung pathologies, such as pulmonary arterial hypertension, primary graft dysfunction of lung transplant, and acute respiratory distress syndrome. Therapy for these diseases is quite limited, largely due to dose-limiting side effects of numerous drugs that have been trialed or approved. High doses of drugs targeting the pulmonary vasculature are needed due to the lack of specific affinity of therapeutic compounds to the vasculature. To overcome this problem, the field of targeted drug delivery aims to target drugs to the pulmonary endothelial cells, especially those in pathological regions. The field uses a variety of drug delivery systems (DDSs), ranging from nano-scale drug carriers, such as liposomes, to methods of conjugating drugs to affinity moieites, such as antibodies. These DDSs can deliver small molecule drugs, protein therapeutics, and imaging agents. Here we review targeted drug delivery to the pulmonary endothelium for the treatment of pulmonary diseases. Cautionary notes are made of the risk-benefit ratio and safety-parameters one should keep in mind when developing a translational therapeutic.

A highly efficient antioxidant is developed by encapsulating superoxide dismutase (SOD) within the aqueous interior of porous polymersomes. The porous polymersomes provide a permeable membrane that allows free superoxide radicals to pass into the aqueous interior and interact with the encapsulated antioxidant enzyme SOD. In vivo studies in the rat demonstrate that administration of SOD-encapsulated porous polymersomes can prevent neuropathic pain after nerve root compression more effectively than treatment with free antioxidant enzyme alone., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The intracellular scaffold KRIT1/CCM1 is an established regulator of vascular barrier function. Loss of KRIT1 leads to decreased microvessel barrier function and to the development of the vascular disorder Cerebral Cavernous Malformation (CCM). However, how loss of KRIT1 causes the subsequent deficit in barrier function remains undefined. Previous studies have shown that loss of KRIT1 increases the production of reactive oxygen species (ROS) and exacerbates vascular permeability triggered by several inflammatory stimuli, but not TNF-α. We now show that endothelial ROS production directly contributes to the loss of barrier function in KRIT1 deficient animals and cells, as targeted antioxidant enzymes reversed the increase in permeability in KRIT1 heterozygous mice as shown by intravital microscopy. Rescue of the redox state restored responsiveness to TNF-α in KRIT1 deficient arterioles, but not venules. In vitro, KRIT1 depletion increased endothelial ROS production via NADPH oxidase signaling, up-regulated Nox4 expression, and promoted NF-κB dependent promoter activity. Recombinant yeast avenanthramide I, an antioxidant and inhibitor of NF-κB signaling, rescued barrier function in KRIT1 deficient cells. However, KRIT1 depletion blunted ROS production in response to TNF-α. Together, our data indicate that ROS signaling is critical for the loss of barrier function following genetic deletion of KRIT1.

Traumatic brain injury (TBI) contributes to one third of injury related deaths in the US. Treatment strategies for TBI are supportive, and the pathophysiology is not fully understood. Secondary mechanisms of injury in TBI, such as oxidative stress and inflammation, are points at which intervention may reduce neuropathology. Evidence suggests that reactive oxygen species (ROS) propagate blood-brain barrier (BBB) hyperpermeability and inflammation following TBI. We hypothesized that targeted detoxification of ROS may improve the pathological outcomes of TBI. Following TBI, endothelial activation results in a time dependent increase in vascular expression of ICAM-1. We conjugated catalase to anti-ICAM-1 antibodies and administered the conjugate to 8 wk old C57BL/6J mice 30 min after moderate controlled cortical impact injury. Results indicate that catalase targeted to ICAM-1 reduces markers of oxidative stress, preserves BBB permeability, and attenuates neuropathological indices more effectively than non-targeted catalase and anti-ICAM-1 antibody alone. Furthermore, the study of microglia by two-photon microscopy revealed that anti-ICAM-1/catalase prevents the transition of microglia to an activated phenotype. These findings demonstrate the use of a targeted antioxidant enzyme to interfere with oxidative stress mechanisms in TBI and provide a proof-of-concept approach to improve acute TBI management that may also be applicable to other neuroinflammatory conditions.

Cardiovascular diseases are major contributors to global deaths and disability-adjusted life years, with hypertension a significant risk factor for all causes of death. The endothelium that lines the inner wall of the vasculature regulates essential haemostatic functions, such as vascular tone, circulation of blood cells, inflammation and platelet activity. Endothelial dysfunction is an early predictor of atherosclerosis and future cardiovascular events. We review the prognostic value of obtaining measurements of endothelial function, the clinical techniques for its determination, the mechanisms leading to endothelial dysfunction and the therapeutic treatment of endothelial dysfunction. Since vascular oxidative stress and inflammation are major determinants of endothelial function, we have also addressed current antioxidant and anti-inflammatory therapies. In the light of recent data that dispute the prognostic value of endothelial function in healthy human cohorts, we also discuss alternative diagnostic parameters such as vascular stiffness index and intima/media thickness ratio. We also suggest that assessing vascular function, including that of smooth muscle and even perivascular adipose tissue, may be an appropriate parameter for clinical investigations., Linked Articles: This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc., (© 2016 The British Pharmacological Society.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Monoclonal antibodies (mAbs) directed to extracellular epitopes of human and mouse Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) stimulate binding of other mAbs to distinct adjacent PECAM-1 epitopes. This effect, dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL, has been shown to enhance delivery of mAb-targeted drugs and nanoparticles to the vascular endothelium. Here we report new insights into the mechanism underlying this effect, which demonstrates equivalent amplitude in the following models: i) cells expressing a full length PECAM-1 and mutant form of PECAM-1 unable to form homodimers; ii) isolated fractions of cellular membranes; and, iii) immobilized recombinant PECAM-1. These results indicate that CEPAL is mediated not by interference in cellular functions or homophilic PECAM-1 interactions, but rather by conformational changes within the cell adhesion molecule induced by ligand binding. This mechanism, mediated by exposure of partially occult epitopes, is likely to occur in molecules other than PECAM-1 and may represent a generalizable phenomenon with valuable practical applications., Competing Interests: The authors have declared that no competing interests exist.

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.

Controlled endothelial delivery of SOD may alleviate abnormal local surplus of superoxide involved in ischemia-reperfusion, inflammation and other disease conditions. Targeting SOD to endothelial surface vs. intracellular compartments is desirable to prevent pathological effects of external vs. endogenous superoxide, respectively. Thus, SOD conjugated with antibodies to cell adhesion molecule PECAM (Ab/SOD) inhibits pro-inflammatory signaling mediated by endogenous superoxide produced in the endothelial endosomes in response to cytokines. Here we defined control of surface vs. endosomal delivery and effect of Ab/SOD, focusing on conjugate size and targeting to PECAM vs. ICAM. Ab/SOD enlargement from about 100 to 300nm enhanced amount of cell-bound SOD and protection against extracellular superoxide. In contrast, enlargement inhibited endocytosis of Ab/SOD and diminished mitigation of inflammatory signaling of endothelial superoxide. In addition to size, shape is important: endocytosis of antibody-coated spheres was more effective than that of polymorphous antibody conjugates. Further, targeting to ICAM provides higher endocytic efficacy than targeting to PECAM. ICAM-targeted Ab/SOD more effectively mitigated inflammatory signaling by intracellular superoxide in vitro and in animal models, although total uptake was inferior to that of PECAM-targeted Ab/SOD. Therefore, both geometry and targeting features of Ab/SOD conjugates control delivery to cell surface vs. endosomes for optimal protection against extracellular vs. endosomal oxidative stress, respectively., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

For a variety of diseases, effective therapy is severely limited or rendered impossible due to an inability to deliver medications to the intended sites of action. Multiple barriers exist through the body, which have evolved over time to limit the migration of foreign compounds from entering the tissues. Turning toward biology as inspiration, it has been the general goal of drug delivery to create carrier strategies that mimic, in part, features of bacteria/ viruses that allow them overcome these barriers. By packaging drugs into nano and micron scale vehicles, it should be possible to completely change the biodistribution and residence times of pharmaceutically active compounds. Recently, due to advances in formulation technologies, it has become possible to control not just the material selection, surface chemistry, and/or size, but also the overall geometry and plasticity of the drug carriers. These approaches aid in the formulation of nonspherical particles such as, discs, rods, and even unique structures such as cubes and nanodiamonds. The adjustment of size and shape can be used for the aid or prevention in cellular uptake and also to overcome the vascular and mucosal barrier. In this review, we present a summary of some approaches used to control carrier shape and the impact these geometries have upon drug transport across biological barriers.

Endothelium, a thin monolayer of specialized cells lining the lumen of blood vessels is the key regulatory interface between blood and tissues. Endothelial abnormalities are implicated in many diseases, including common acute conditions with high morbidity and mortality lacking therapy, in part because drugs and drug carriers have no natural endothelial affinity. Precise endothelial drug delivery may improve management of these conditions. Using ligands of molecules exposed to the bloodstream on the endothelial surface enables design of diverse targeted endothelial nanomedicine agents. Target molecules and binding epitopes must be accessible to drug carriers, carriers must be free of harmful effects, and targeting should provide desirable sub-cellular addressing of the drug cargo. The roster of current candidate target molecules for endothelial nanomedicine includes peptidases and other enzymes, cell adhesion molecules and integrins, localized in different domains of the endothelial plasmalemma and differentially distributed throughout the vasculature. Endowing carriers with an affinity to specific endothelial epitopes enables an unprecedented level of precision of control of drug delivery: binding to selected endothelial cell phenotypes, cellular addressing and duration of therapeutic effects. Features of nanocarrier design such as choice of epitope and ligand control delivery and effect of targeted endothelial nanomedicine agents. Pathological factors modulate endothelial targeting and uptake of nanocarriers. Selection of optimal binding sites and design features of nanocarriers are key controllable factors that can be iteratively engineered based on their performance from in vitro to pre-clinical in vivo experimental models. Targeted endothelial nanomedicine agents provide antioxidant, anti-inflammatory and other therapeutic effects unattainable by non-targeted counterparts in animal models of common acute severe human disease conditions. The results of animal studies provide the basis for the challenging translation endothelial nanomedicine into the clinical domain., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Targeting nanocarriers (NC) to endothelial cell adhesion molecules including Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1 or CD31) improves drug delivery and pharmacotherapy of inflammation, oxidative stress, thrombosis and ischemia in animal models. Recent studies unveiled that hydrodynamic conditions modulate endothelial endocytosis of NC targeted to PECAM-1, but the specificity and mechanism of effects of flow remain unknown. Here we studied the effect of flow on endocytosis by human endothelial cells of NC targeted by monoclonal antibodies Ab62 and Ab37 to distinct epitopes on the distal extracellular domain of PECAM. Flow in the range of 1-8dyn/cm(2), typical for venous vasculature, stimulated the uptake of spherical Ab/NC (~180nm diameter) carrying ~50 vs 200 Ab62 and Ab37 per NC, respectively. Effect of flow was inhibited by disruption of cholesterol-rich plasmalemma domains and deletion of PECAM-1 cytosolic tail. Flow stimulated endocytosis of Ab62/NC and Ab37/NC via eliciting distinct signaling pathways mediated by RhoA/ROCK and Src Family Kinases, respectively. Therefore, flow stimulates endothelial endocytosis of Ab/NC in a PECAM-1 epitope specific manner. Using ligands of binding to distinct epitopes on the same target molecule may enable fine-tuning of intracellular delivery based on the hemodynamic conditions in the vascular area of interest., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Lysozyme dextran nanogels (NG) have great potential in vitro as a drug delivery platform, combining simple chemistry with rapid uptake and cargo release in target cells with "stealth" properties and low toxicity. In this work, we study for the first time the potential of targeted NG as a drug delivery platform in vivo to alleviate acute pulmonary inflammation in animal model of LPS-induced lung injury. NG are targeted to the endothelium via conjugation with an antibody (Ab) directed to Intercellular Adhesion Molecule-1(ICAM-NG), whereas IgG conjugated NG (IgG-NG) are used for control formulations. The amount of Ab conjugated to the NG and distribution in the body after intravenous (IV) injection have been quantitatively analyzed using a tracer isotope-labeled [125I]IgG. As a proof of concept, Ab-NG are loaded with dexamethasone, an anti-inflammatory therapeutic, and the drug uptake and release kinetics are measured by HPLC. In vivo studies in mice showed that: i) ICAM-NG accumulates in mouse lungs (∼120% ID/g vs ∼15% ID/g of IgG-NG); and, ii) DEX encapsulated in ICAM-NG, but not in IgG-NG practically blocks LPS-induced overexpression of pro-inflammatory cell adhesion molecules including ICAM-1 in the pulmonary inflammation.

Targeted nanomedicine holds promise to find clinical use in many medical areas. Endothelial cells that line the luminal surface of blood vessels represent a key target for treatment of inflammation, ischemia, thrombosis, stroke, and other neurological, cardiovascular, pulmonary, and oncological conditions. In other cases, the endothelium is a barrier for tissue penetration or a victim of adverse effects. Several endothelial surface markers including peptidases (e.g., ACE, APP, and APN) and adhesion molecules (e.g., ICAM-1 and PECAM) have been identified as key targets. Binding of nanocarriers to these molecules enables drug targeting and subsequent penetration into or across the endothelium, offering therapeutic effects that are unattainable by their nontargeted counterparts. We analyze diverse aspects of endothelial nanomedicine including (i) circulation and targeting of carriers with diverse geometries, (ii) multivalent interactions of carrier with endothelium, (iii) anchoring to multiple determinants, (iv) accessibility of binding sites and cellular response to their engagement, (v) role of cell phenotype and microenvironment in targeting, (vi) optimization of targeting by lowering carrier avidity, (vii) endocytosis of multivalent carriers via molecules not implicated in internalization of their ligands, and (viii) modulation of cellular uptake and trafficking by selection of specific epitopes on the target determinant, carrier geometry, and hydrodynamic factors. Refinement of these aspects and improving our understanding of vascular biology and pathology is likely to enable the clinical translation of vascular endothelial targeting of nanocarriers.

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.

Pro-inflammatory activation of vascular endothelium is implicated in pathogenesis of severe conditions including stroke, infarction and sepsis. We have recently reported that superoxide dismutase (SOD) conjugated with antibodies (Ab/SOD) that provide targeted delivery into endothelial endosomes mitigates inflammatory endothelial activation by cytokines and agonists of Toll-like receptors (TLR). The goal of this study was to appraise potential utility and define the mechanism of this effect. Ab/SOD, but not non-targeted SOD injected in mice alleviated endotoxin-induced leukocyte adhesion in the cerebral vasculature and protected brain from ischemia-reperfusion injury. Transfection of endothelial cells with SOD, but not catalase inhibited NFκB signaling and expression of Vascular Cell Adhesion Molecule-1 induced by both cytokines and TLR agonists. These results affirmed that Ab/SOD-quenched superoxide anion produced by endothelial cells in response to proinflammatory agents mediates NFκB activation. Furthermore, Ab/SOD potentiates anti-inflammatory effect of NO donors in endothelial cells in vitro, as well as in the endotoxin-challenged mice. These results demonstrate the central role of intracellular superoxide as a mediator of pro-inflammatory activation of endothelium and support the notion of utility of targeted interception of this signaling pathway for management of acute vascular inflammation.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Oxidant stress caused by pathological elevation of reactive oxygen species (ROS) production in the endothelial cells lining the vascular lumen is an important component of many vascular and pulmonary disease conditions. NADPH oxidase (NOX) activated by pathological mediators including angiotensin and cytokines is a major source of endothelial ROS. In order to intercept this pathological pathway, we have encapsulated an indirect NOX inhibitor, MJ33, into immunoliposomes (Ab-MJ33/IL) targeted to endothelial marker platelet endothelial cell adhesion molecule (PECAM-1). Ab-MJ33/IL, but not control IgG-MJ33/IL are specifically bound to endothelium and attenuated angiotensin-induced ROS production in vitro and in vivo. Additionally, Ab-MJ33/IL inhibited endothelial expression of the inflammatory marker vascular cell adhesion molecule (VCAM) in cells and animals challenged with the cytokine TNF. Furthermore, Ab-MJ33/IL alleviated pathological disruption of endothelial permeability barrier function in cells exposed to vascular endothelial growth factor (VEGF) and in the lungs of mice challenged with lipopolysaccharide (LPS). Of note, the latter beneficial effect has been achieved both by prophylactic and therapeutic injection of Ab-MJ33/IL in animals. Therefore, specific suppression of ROS production by NOX in endothelium, attainable by Ab-MJ33/IL targeting, may help deciphering mechanisms of vascular oxidative stress and inflammation, and potentially improve treatment of these conditions., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Intracellular delivery of nanocarriers (NC) is controlled by their design and target cell phenotype, microenvironment, and functional status. Endothelial cells (EC) lining the vascular lumen represent an important target for drug delivery. Endothelium in vivo is constantly or intermittently (as, for example, during ischemia-reperfusion) exposed to blood flow, which influences NC-EC interactions by changing NC transport properties, and by direct mechanical effects upon EC mechanisms involved in NC binding and uptake. EC do not internalize antibodies to marker glycoprotein PECAM(CD31), yet internalize multivalent NC coated with PECAM antibodies (anti-PECAM/NC) via a noncanonical endocytic pathway distantly related to macropinocytosis. Here we studied the effects of flow on EC uptake of anti-PECAM/NC spheres (~180 nm diameter). EC adaptation to chronic flow, manifested by cellular alignment with flow direction and formation of actin stress fibers, inhibited anti-PECAM/NC endocytosis consistent with lower rates of anti-PECAM/NC endocytosis in vivo in arterial compared to capillary vessels. Acute induction of actin stress fibers by thrombin also inhibited anti-PECAM/NC endocytosis, demonstrating that formation of actin stress fibers impedes EC endocytic machinery. In contrast, acute flow without stress fiber formation, stimulated anti-PECAM/NC endocytosis. Anti-PECAM/NC endocytosis did not correlate with the number of cell-bound particles under flow or static conditions. PECAM cytosolic tail deletion and disruption of cholesterol-rich plasmalemma domains abrogated anti-PECAM/NC endocytosis stimulation by acute flow, suggesting complex regulation of a flow-sensitive endocytic pathway in EC. The studies demonstrate the importance of the local flow microenvironment for NC uptake by the endothelium and suggest that cell culture models of nanoparticle uptake should reflect the microenvironment and phenotype of the target cells.

Reactive oxygen species (ROS) are implicated as injurious and as signaling agents in human maladies including inflammation, hyperoxia, ischemia-reperfusion and acute lung injury. ROS produced by the endothelium play an important role in vascular pathology. They quench, for example, nitric oxide, and mediate pro-inflammatory signaling. Antioxidant interventions targeted for the vascular endothelium may help to control these mechanisms. Animal studies have demonstrated superiority of targeting ROS-quenching enzymes catalase and superoxide dismutase to endothelial cells over nontargeted formulations. A diverse arsenal of targeted antioxidant formulations devised in the last decade shows promising results for specific quenching of endothelial ROS. In addition to alleviation of toxic effects of excessive ROS, these targeted interventions suppress pro-inflammatory mechanisms, including endothelial cytokine activation and barrier disruption. These interventions may prove useful in experimental biomedicine and, perhaps, in translational medicine.

The endothelial lining of the lumen of blood vessels is a key therapeutic target for many human diseases. Polymeric filomicelles that self-assemble from polyethylene oxide (PEO)-based diblock copolymers are long and flexible rather than small or rigid, can be loaded with drugs, and--most importantly--they circulate for a prolonged period of time in the bloodstream due in part to flow alignment. Filomicelles seem promising for targeted drug delivery to endothelial cells because they can in principle adhere strongly, length-wise to specific cell surface determinants. In order to achieve such a goal of vascular drug delivery, two fundamental questions needed to be addressed: (i) whether these supramolecular filomicelles retain structural integrity and dynamic flexibility after attachment of targeting molecules such as antibodies, and (ii) whether the avidity of antibody-carrying filomicelles is sufficient to anchor the carrier to the endothelial surface despite the effects of flow that oppose adhesive interactions. Here we make targeted filomicelles that bear antibodies which recognize distinct endothelial surface molecules. We characterize these antibody targeted filomicelles and prove that (i) they retain structural integrity and dynamic flexibility and (ii) they adhere to endothelium with high specificity both in vitro and in vivo. These results provide the basis for a new drug delivery approach employing antibody-targeted filomicelles that circulate for a prolonged time yet bind to endothelial cells in vascular beds expressing select markers., (© 2011 American Chemical Society)

Background: Donor organ shortage represents a major problem in lung transplantation. Donation after cardiac death could help to expand the pool of organs, but the additional period of warm ischemia after cardiac arrest aggravates primary graft dysfunction. The pulmonary endothelium of the graft constitutes an important source and target of reactive oxygen species generated during ischemia and reperfusion. Targeted protection of graft pulmonary endothelial cells by the antioxidant enzyme catalase, conjugated with a platelet/endothelial cell adhesion molecule-1 (PECAM-1) antibody to nanosized particles (anti-PECAM/catalase conjugates), might improve outcome in lung transplantation using donors after cardiac death and prolonged hypothermic preservation., Methods: Left lung transplantation was performed in 18 pigs. Before cardiac arrest, donors received anti-PECAM/catalase, unconjugated component mixture or vehicle solution. After 90-min warm and 18-hr hypothermic ischemia, lungs were transplanted, and function was assessed during 6 hr after reperfusion. Samples of bronchoalveolar lavage fluid and lung tissue were taken thereafter. Six sham-operated animals served as controls., Results: During 6-hr reperfusion, anti-PECAM/catalase significantly ameliorated graft function, evidenced by major improvements of gas exchange and reduced intrapulmonary shunt fraction. Furthermore, lipid peroxidation, alveolar leakage, and edema formation were reduced in protected grafts. Similarly moderate lung pathology was seen after transplantation., Conclusions: Augmentation of the antioxidant capacity of graft pulmonary endothelial cells with anti-PECAM/catalase nanoparticles represents a straightforward approach to enable a safe transplantation of prolonged preserved donation after cardiac death lungs. Anti-PECAM/catalase protection alleviated oxidative stress and allowed immediate reconstitution of normal gas exchange and pulmonary microcirculation, a prerequisite for improved graft and patient outcome.

'Endothelial cells lining vascular luminal surface represent an important site of signaling and injurious effects of reactive oxygen species (ROS) produced by other cells and endothelium itself in ischemia, inflammation and other pathological conditions. Targeted delivery of ROS modulating enzymes conjugated with antibodies to endothelial surface molecules (vascular immunotargeting) provides site-specific interventions in the endothelial ROS, unattainable by other formulations including PEG-modified enzymes. Targeting of ROS generating enzymes (e.g., glucose oxidase) provides ROS- and site-specific models of endothelial oxidative stress, whereas targeting of antioxidant enzymes SOD and catalase offers site-specific quenching of superoxide anion and H(2)O(2). These targeted antioxidant interventions help to clarify specific role of endothelial ROS in vascular and pulmonary pathologies and provide basis for design of targeted therapeutics for treatment of these pathologies. In particular, antibody/catalase conjugates alleviate acute lung ischemia/reperfusion injury, whereas antibody/SOD conjugates inhibit ROS-mediated vasoconstriction and inflammatory endothelial signaling. Encapsulation in protease-resistant, ROS-permeable carriers targeted to endothelium prolongs protective effects of antioxidant enzymes, further diversifying the means for targeted modulation of endothelial ROS., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

Endothelial targeting of antioxidant enzymes attenuates acute vascular oxidative stress in animal studies. Superoxide dismutase (SOD) and catalase conjugated with antibodies to Platelet-Endothelial Cell Adhesion Molecule-1 (anti-PECAM/SOD and anti-PECAM/catalase) bind to endothelium, accumulate in the pulmonary vasculature, and detoxify reactive oxygen species. In order to define the role of conjugate size in the efficacy and specificity of endothelial targeting, we synthesized anti-PECAM/enzyme conjugates of controlled size (40nm-10,000nm). Binding of anti-PECAM/enzymes to endothelial cells increased with conjugate size from 300nm to 2μm (from 2.5 to 8.5% of bound fraction), and was specific, as conjugates did not bind to PECAM-negative cells. Pulmonary uptake of anti-PECAM/enzyme conjugates injected intravenously in mice also increased from 4.5 to 16% of injected dose for particles from 200 to 800nm. However, control conjugates larger than 300nm showed elevated non-specific pulmonary uptake, indicating that the targeting specificity of anti-PECAM/enzyme conjugates in vivo has a bell-shaped curve with a maximum close to 300-nm diameter. These results show that: i) the size of an antibody/enzyme conjugate modulates efficacy and specificity of targeting, and ii) a size optimum should be defined in vivo to account for parameters that are difficult to model in cell culture., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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-1β, 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.

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., (Thieme Medical Publishers.)

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.