key: cord-0859112-drxx49gm
authors: Glassman, Patrick M.; Myerson, Jacob W.; Ferguson, Laura T.; Kiseleva, Raisa Y.; Shuvaev, Vladimir V.; Brenner, Jacob S.; Muzykantov, Vladimir R.
title: Targeting drug delivery in the vascular system: Focus on endothelium()
date: 2020-06-21
journal: Adv Drug Deliv Rev
DOI: 10.1016/j.addr.2020.06.013
sha: 002c6a7e4b04193230529c0313c0368b2de6936a
doc_id: 859112
cord_uid: drxx49gm

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.

epitopes, for many reasons including rather relative character of the results provided by many methods (FACS, immunostaining, ELISA) and mostly enigmatic clustering of the epitopes. It seems reasonable to categorize epitope density based on the B max (maximal number of binding sites per cell determined using directly radiolabeled ligands), as high (10 5 and above), intermediate (10 3 -10 5 sites per cell) and low (less than few thousands per cell).

Under pathological conditions, endothelial cells undergo many changes that could impact targeting. Some constitutive surface molecules, such as TM, are shed from the endothelium in pathologies. For example, following acute lung injury, delivery of anti-TM mAbs to the lung was reduced by 50% [53] . This observation is in agreement with the notion that TM is shed by pulmonary endothelial cells following an inflammatory insult [54] . Another classical endothelial target, ACE, is also shed by the endothelium in pathologies [55] . This process has been shown to be mediated by several agents, including pro-inflammatory cytokines and oxidants, resulting in reduced uptake of ACEtargeted agents [56, 57] . In thoracic imaging studies, it was revealed that sarcoidosis led to reduced pulmonary uptake of anti-ACE in patients [44] . Animal studies have revealed a similar reduction in lung targeting of anti-ACE in several models, including pulmonary edema, ischemiareperfusion, and endotoxemia [57] [58] [59] .

Disappearance of endothelial determinants in pathologies occurs via multiple mechanisms. They include activation on the cell surface specific proteases and peptidases that cleave off transmembrane glycoproteins such as ACE and TM [48, 60, 61] . In addition, enzymes cleaving chondroitin sulfate, heparan sulfate, and other sugar moieties from ACE, TM, and many other endothelial surface glycoproteins shed these components of glycocalyx, which unmasks new binding sites on normally hidden epitopes. Reactive oxygen species, proteases, and other highly aggressive entities, including hypochlorous acid (bleach), released by activated leukocytes modify endothelial membrane components directly and via damage to endothelial cells themselves [62] [63] [64] .

Stable constitutive molecules, such as Platelet-Endothelial Cell Adhesion Molecule (PECAM), can be used for prophylactic and/or therapeutic delivery [39, 49] . Inflammation enhances Intercellular Adhesion Molecule 1 (ICAM-1) level in many cell types, including endothelia [65, 66] . Pathological endothelia commonly express inducible markers including Aminopeptidase N (APN), Tumor Endothelial Marker 1 (TEM-1), Vascular Cell Adhesion Molecule 1 (VCAM-1), and E-and P-selectins [25, 67] . These determinants are preferable for diagnostic imaging and therapeutic interventions due to their specificity for pathologicallyaltered endothelium [6, 23, [68] [69] [70] ]. Selectins exposed by pathological endothelium facilitate adhesion of WBC and platelets [71] . The kinetics of upregulation of surface epitopes are dependent on the mechanism of increased surface exposure. For example, P-selectin is mobilized from intracellular stores within 10-30 min [72] . However, E-selectin [73] and VCAM-1 [74] surface expression requires hours to increase due to the requirement for de novo protein synthesis ( Figure 2 ).

In massive hemorrhage, sepsis, severe sterile tissue injury, such as reperfusion or cytokine release syndrome, and other pathological conditions, endothelial cells cease their quiescent phenotype. Instead of maintaining blood fluidity and confinement to the vascular space, pathological endothelium aggravates thrombosis and inflammation. It promotes blood clotting via exposure of phosphatidylserine and exteriorization of von Willebrand Factor and Pselectin [75] . Activated endothelial cells release chemoattractants and cytokines luring migrating host defense cells and the luminal surface becomes pro-adhesive to leukocytes and blood components and the monolayer loses the barrier function as endothelial cells contract and the VE-cadherin "zipper" opens up [76, 77] . These changes, concomitant with loss of antithrombotic and anti-inflammatory mechanisms, including TM/APC, CD39, and nitric oxide (converted into dangerous peroxinitrite ONOOby elevated influx of reactive oxygen species) ignite and propagate the vicious cycle of acute vascular damage, multi-organ failure, and demise [78] [79] [80] . Rapidly emerging pathological and clinical data implicate this transformation in the pathogenesis of COVID-19, in particular contributing to severe morbidity and mortality ensuing from the pulmonary microvascular injury [81] [82] [83] . ICAM-1 and VCAM-1 expression in the tetraspanin domains is increased, largely via de novo synthesis. Accessibility to endothelial junction proteins PECAM-1 and VE-Cadherin may change due to reduced cell-cell interactions. P-selectin is upregulated on the membrane through mobilization of intracellular stores found in Weibel-Palade Bodies. Other proteins such as ACE and thrombomodulin are lost from the cell surface through a shedding mechanism.

Beyond the endothelium, activated platelets also serve as a target for P-selectin binding agents [84] . In general, both selectins (e.g., P-selectin, E-selectin) and molecules such as VCAM-1 have a low and transient surface expression on endothelium. Because of this, targeting to these epitopes could provide excellent specificity in diagnostic imaging using radioisotopes [68] or ultrasound contrasts [84, 85] to visualize pathologically activated endothelium. In general, these molecules are more closely associated with activated endothelia, in tissues such as the skin [86] . VCAM-1 is expressed relatively selectively in the inflamed cerebrovascular endothelium and VCAM-targeted DDS selectively and effectively target drugs, including mRNA, to the blood-brain barrier and normalize its pathological alterations in mouse models [87] (Figure 3 ). Figure 3 : Targeting to VCAM-1 enables selective delivery to the inflamed cerebral vasculature. Left panel: Absolute uptake of anti-VCAM-1 mAb in the brain of mice injured via an intrastriatal injection of TNF-α exceeds that of the 'gold standard' for brain delivery, Transferrin Receptor (TfR), by an order of magnitude. Middle panel: Flow cytometry on brain homogenates reveals that over 50% of endothelial cells are positive for VCAM-targeted agents (either mAb or liposome) following IV injection. Following IV injection of fluorescently labeled mAbs and liposomes under the same conditions as in the left panel, brains were disaggregated and stained to determine mAb/liposome association with leukocytes and endothelial cells. Inset: Typical dot plot showing cell types identified via this approach. Right panel: SPECT imaging of VCAM-targeted liposomes labeled with 111 In demonstrates selective uptake in the injured hemisphere of the brain. Units in the scale bar are presented as arbitrary intensity units. Figures adapted from [87] . Colors are consistent across panels.

Hydrodynamic conditions may also modulate endothelial processing of targeted carriers [88, 89] . It was observed that nanoparticles targeted to either ICAM or PECAM had reduced uptake by endothelial cells following flow adaptation [90, 91] , and their uptake in endothelium in vivo appears to be inferior in arterioles vs. capillaries [91] . In contrast, acute increses in shear stress stimulates endocytosis of PECAM-targeted nanocarriers [90] . Activation of endothelial cells using cytokines (e.g. TNF-α) leads to enhanced internalization of ICAM-1-targeted nanoparticles, compared to naïve, quiescent cells [91] . leukocyte adhesion and migration, are largely found on the apical surface of the membrane within specific micro-domains [94] [95] [96] , or 'rafts' [97] . Within the pulmonary endothelium, GP85 is found on the luminal surface within an organelle-free portion of the cell [98, 99] .

Various forms of endocytosis (e.g. clathrin, etc.) are used by endothelial cells to internalize ligands bound to selectins [100] [101] [102] . This process allows entry of a variety of Eselectin targeted agents, such as liposomes [103] , drugs [103, 104] , and nucleic acids [105] . However, not all endothelial surface epitopes are internalized as efficiently. Studies revealed that anti-PECAM mAbs are not internalized and that anti-ICAM-1 mAbs are internalized, but are subsequently recycled back to the cell surface [106] . This feature of CAMs is desirable for therapeutics that require prolonged exposure in the vessel lumen, such as anti-thrombotics [107] . It should be noted that this process can be modulated by the avidity of the drug carrier, as multivalent protein conjugates and nanoparticles targeted to PECAM and ICAM-1 are able to enter endothelial cells via an inducible CAM-mediated endocytosis mechanism [108] . By tuning the valency of a DDS, sub-cellular delivery of CAM-targeted agents can be controlled, to a certain extent.

Accessibility of particles to specific endothelial epitopes is dependent on the localization of the epitope within the membrane and vascular conditions [109] . Epitopes that may not be suitable for affinity targeting include those that are masked by the glycocalyx, located deep within cell-cell junctions, or are inside membrane invaginations [110] . Pathological changes in tissues can alter the accessibility of target epitopes. A feature of certain pathologies is the shedding of the endothelial glycocalyx, which may increase accessibility of certain target epitopes, such as ICAM-1 [111] . However, the endothelial surface may become coated with leukocytes and thrombi, which would reduce accessibility to target determinants [58] . Recent advances in techniques such as proteomics [112] in vivo phage display [113] [114] [115] have provided additional insights into accessibility of membrane targets. As carrier size increases, accessibility to epitopes becomes increasingly more important (e.g., accessibility is less of a concern for peptides than for immunoliposomes).

Proteins located within caveolae, such as Aminopeptidase P (APP) and Plasmalemma Vesicle Associated Protein-1 (PLVAP) [116] , are accessible only to relatively small drug carriers. This is due to the nature of the flask-shaped membrane invaginations that are characteristic of caveolae. It is generally thought that the caveolar mouth has a diameter of about 50 nm [117, 118] . Functions of caveolae include endothelial trafficking and cellular signaling [119] [120] [121] [122] [123] [124] . A key feature of caveolae that distinguishes them from other cholesterol-rich domains (e.g., lipid rafts) is the presence of the protein caveolin-1. Caveolar pathway(s) transport ligands including albumin [125] and chemically-modified albumin [126] across the endothelium [127] . In rats, mAbs directed against several caveolar antigens are able to efficiently target and transmigrate across the pulmonary vasculature, consistent with the large number of caveolae present in the pulmonary endothelium [6] . systems, which may not be reflective of true in vivo endothelial physiology [116, 129, 130] . The spatial accessibility of caveolar targets to circulating nanocarriers is a hot topic. For example, flexible anti-PLVAP nanogels with a diameter of 150-300 nm enter caveolae and accumulate in the lungs, whereas their rigid nanoparticle counterparts do not [131] .

Ferritin-based nanocarriers have been investigated as a DDS that can be used in a modular and stimuli-responsive manner [132] . Ferritin particles conjugated with anti-PLVAP accumulate in the lungs after intravenous injection and enter caveolae, since their size does not exceed 20 nm. Since the ferritin carriers are so small, conjugation of the ligand and enzymatic cargo to external and internal facets of ferritin nanocages, respectively, improves targeting [133] . Interestingly, ferritin nanocarriers targeted to ICAM also appear to have greater access to the endothelial surface in the pulmonary vasculature compared to larger carriers [134, 135] .

A conventional approach to achieve intracellular delivery utilize affinity ligands capable of anchoring DDS to epitopes that are permissive of endocytosis. Diverse endothelial determinants have been implicated in internalization [50, [136] [137] [138] . In general, identification and selection of affinity ligands is empirical in nature [139, 140] . Approaches such as phage display facilitate selection of internalizable ligands [141] [142] [143] . In one example, phage display was used to select several VCAM-1-binding peptides [144] . Peptides that were rapidly endocytosed were used for molecular imaging of vascular inflammation [86, 144] . Antibodies to APP, PLVAP or GP90 enter caveolae [6] and those to E-selectin or VCAM-1 [103, 104] enter clathrin-mediated endocytosis [101, 102] .

In some cases, coating carriers with affinity ligands permits internalization to occur at least as efficiently as monovalent ligands, if not more efficiently. This may occur due to multivalent carriers eliciting strong cell signaling and actin rearrangement [145] . Both VCAM-1 antibodies [104, 146] and VCAM-1 targeted DDS have been reported to enter endothelial clathrin-mediated endocytosis [86, 144] . Anti-transferrin receptor (TfR) mAbs bound to TfR and free transferrin also enter endothelial cells via clathrin-mediated endocytosis [147, 148] . However, coupling ligands to carriers may actually reduce cellular uptake due to their exceeding size limits of endocytosis. Caveolar and clathrin-mediated endocytosis have size limits of 50-70 [149] and 200-300 nm [70] , respectively. Findings made using in vitro, static systems should be taken with a grain of salt, as they may use non-physiologically-relevant endocytic pathways that have not been confirmed in vivo thus far [127] .

The uptake and trafficking of ICAM-targeted DDS vary. Endothelial cells are not effective in internalizing monovalent fusion proteins that do not initiate clustering of epitopes, whereas bivalent anti-ICAM and anti-ICAM conjugates recycle rapidly to the luminal surface, unlike multivalent nanoparticles targeted to ICAM, which traffic to the lysosomes [91, 106, 108, [150] [151] [152] [153] [154] . Binding to different epitopes may also change the efficiency of entrance into cells [110] . Nanocarrier geometry may also affect uptake and subcellular trafficking [108, 155] . For example, disk shaped particles targeted to ICAM-1 have a lower rate of endocytosis compared to spheres of a similar size. Once inside the cell, the rate of lysosomal trafficking is inversely related to size (e.g., small particles traffick more quickly) [155] . Silvia Muro, whose studies greatly advanced the field of endothelial intracellular delivery, devised a novel DDS platform for ICAM-1 targeted delivery of nanocarriers loaded with enzyme replacement therapies for lysosomal storage diseases (LSD) [153, 156] . LSDs are morbid conditions caused by mutation of lysosomal enzymes [157] . In LSD, enzyme replacement therapy (ERT) is the standard of care. Clinically, this approach is applied by chronic injections of recombinant forms of the deficient lysosomal enzyme [158] . The systemically injected proteins are then internalized by cells in a manner dependent on mannosylation of proteins, via either mannose receptor and/or mannose-6-phosphate receptor [159] . Currently approved therapies for treatment of LSD include ERT, substrate reduction inhibitors, and small molecule chaperones [160] [161] [162] . Type B Niemann-Pick disease (NPD) is caused by deficiency/malfunction of the protein acid sphingomyelinase (ASM). Deficiency of ASM results in increased deposition of sphingomyelin and cholesterol enzymes [157] .

In LSD, endothelial cells are often a key pharmacologic target [156] , but endothelial delivery of ERT is not very effective [161, 163] . It has been reported that endothelial cells have increased ICAM-1 expression in many LSD, which may provide a route for improved delivery of recombinant enzymes [164, 165] . Delivery of ERT using ICAM-1-targeted nanoparticles has been shown to improve endothelial targeting and pharmacologic effects in several animal models of LSD [153, 166, 167] . In a mouse model of Pömpe disease, an ~600-fold improvement in lung delivery was observed for ICAM-1-targeted acid α-glucosidase (the enzyme deficient in this disease), relative to untargeted enzyme [168] . By adjusting geometric features of the carrier, delivery via ICAM-1 targeting can be optimized. Use of relatively small (100-200 nm), spherical carriers provided maximal delivery to the lysosomes as compared to particles with other features (e.g., discoidal, micron-sized/spherical) [169] .

The Muro group has also identified that interactions between nanocarriers and endothelial ICAM-1 results in activation of enzymes responsible for sphingomyelin metabolism in the membrane and endocytosis of a wide range of sizes of targeted particles [170] . However, this pathway is dependent on sphingomyelinase expression and coating of particles with both anti-ICAM-1 and ASM permits internalization in NPD [166] . Additionally, it was shown that larger carriers (>200 nm) coated with ASM and anti-TfR were also internalized [171] .

CAM-mediated endocytosis achieved via targeting to ICAM-1 has been shown to be more efficient than clathrin-mediated endocytosis via TfR targeting in delivery of enzymes to the lysosome [154] . However, dual targeting to ICAM-1 and TfR provided a different tissue distribution than either mono-or non-targeted carriers [171] . The utility and significance of these findings represent an area of active investigations [156] .

selectin/ICAM-1 targeted particles carrying equimolar ratios of both ligands were reported to bind to the inflamed endothelium better than mono-targeted particles [172, 174, 175] . This dual targeting approach improved imaging in mouse models of inflammation using contrast enhancing particles targeted to either P-selectin and VCAM [176] or P-selectin and ICAM-1 [177] . Dual targeting of anti-ICAM/anti-TfR nanoparticles has shown that individual ligands could promote targeting to a specific tissue vasculature (ICAM-1: inflamed lungs, TfR: brain) [171] .

Dual therapeutic targeting to vascular lumen can be achieved by utilizing a recently discovered phenomenon where distinct monoclonal antibodies directed to adjacent epitopes in the extracellular region of PECAM stimulate binding of each other [178] . The effect was dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL, and is dissimilar to generally observed binding patterns of ligands to same target molecule inhibiting binding of each other [48, 179, 180] . This unusual finding can be explained by increased accessibility of an epitope to its ligand due to conformational changes in the PECAM molecule induced by binding of a paired "stimulatory" ligand [181] . In-depth investigation of mechanisms underlying this effect revealed several findings: CEPAL is independent of PECAM-PECAM homophilic interactions in the plasma membrane and can be observed with recombinant PECAM protein [181] . Given the known roles of PECAM-1 in promoting endothelial quiescence and in maintaining cell junction integrity [182] , investigations of possible unintended effects of CEPAL on vascular function have shown only modest or insignificant effects. Interestingly, these effects were observed in response to antibodies to PECAM-1, whether given solo or in pairs [183] .

CEPAL was applied to achieve enhanced effects of protein therapeutics using anti-PECAM scFv fusion proteins [178] . A key advantage of this dual targeting strategy was demonstrated by codelivery of thrombomodulin (TM) and its natural endothelial binding partner, endothelial protein C receptor (EPCR) using paired anti-PECAM scFv fusions. Co-administered scFv/TM and scFv/EPCR partnered in activation of protein C (PC) and reduced markers of lung inflammation in a model of acute lung injury [184] . Interestingly, the CEPAL effect also increased delivery of PECAM-targeted nanocarriers, a finding that opens up a new avenue for the field of nanomedicine in optimizing and enhancing delivery of diverse therapeutic cargoes encapsulated in endothelial-targeted nanocarriers [185] . The CEPAL mechanism, occurring by exposure of partially occult epitopes on PECAM protein, may be a generalizable phenomenon and is likely to occur for other molecules and antibody pairs. was demonstrated to be optimal at an intermediate ligand density, which balanced efficient uptake and detachment from target following transport [150] .

In addition to a carrier's affinity, its geometry and flexibility regulate pharmacokinetics, circulation, tissue permeation, and interactions with cells [190] [191] [192] [193] [194] . Carrier size is a factor in route of administration, target access, and pharmacokinetics. Carriers smaller than 20-30 nm can be administered via diverse routes, while larger carriers generally require vascular injection for systemic administration [193, [195] [196] [197] [198] . Once in circulation, carriers of small (<10 nm) diameter are prone to rapid renal clearance [199] . A size range of ~100-200 nm generally avoids filtration to the space of Disse in the liver and entrapment in the splenic sinusoids, giving longer circulation and more opportunities for target engagement [200] . For instance, it has been noted that larger ICAM-and PECAM-targeted particles have enhanced uptake in the RES, and a reduction of specificity of vascular delivery relative to untargeted carriers [155, 201] . Indeed, at larger extremes of injectable particle size (>1-3 µm), both greater splenic retention and non-specific entrapment in lung capillaries is observed [202] [203] [204] .

Larger carriers generally have lesser access to targets and adhered carriers may experience greater dragging force from blood, leading to target detachment [198, 205, 206] . For instance, larger carriers were shown to be incapable of targeting PLVAP, expressed in caveolae in lung endothelium [128] . However, in studies of targeting to PECAM, increasing carrier size from 40 nm to 300 nm increased specific adhesion in the lungs, implying that greater engagement with accessible endothelial targets may be achieved for larger carriers [201] .

Nanocarrier shape, and interplay between shape and size, is also an important factor in targeting behavior [192, 207] . Carriers that are non-spherical in nature may circulate longer than similarly sized spheres due to reduced recognition by host defense cells [109, 208] . Discoid, ellipsoid, and filamentous carriers have prolonged circulation relative to spheres [155, 209, 210] , in part due to reduced uptake by macrophages [211] . Rods and discoid particles avoid side effects associated with engagement with host defense cells in the lungs, unlike spherical carriers [210] .

Eluding clearance from circulation and non-specific uptake may lead to non-spherical nanocarriers with more specific targeting to vascular endothelium. ICAM-targeted disks adhered to the pulmonary vascular endothelium more specifically than spherical counterparts [155] . Nonspherical and spherical particles have distinct propensity for margination in flow [212, 213] , modulating their affinity interactions under flow [214] [215] [216] and facilitating efficient non-spherical particle targeting to endothelial cells in the lungs and brain [217] . However, at an extreme of carrier elongation, worm-like filomicelles align with flow and avoid binding to vascular cells [218] . Filomicelles targeted to ICAM-1 had similar specificity of targeting as spheres, but a lower magnitude of uptake in the lung [209] .

Generally, greater flexibility correlates to prolonged circulation of nanocarriers [195, 218, 219] . In part, prolonged circulation of soft nanoparticles may be attributable to reduced non-specific uptake in phagocytic cells [223, 224] . In macrophages and other immune cells, harder particles are internalized more readily than soft particles [195, 219, 224, 225] . A relationship between particle flexibility and ease of particle uptake may also be emerging in results with endothelial cells, where in vitro and in silico studies show passive and affinity-mediated nanoparticle uptake being impeded in softer particles [195, 219, 226] . Further, recent results show that harder (GPa bulk modulus) nontargeted 200 nm spheres associated with and transported across brain endothelium in a microfluidic model to a greater degree than softer (MPa bulk modulus) spheres [227] .

Changes in the mechanical properties of nanoparticles have yielded changes in affinitydirected targeting behavior. For PEG hydrogel nanoparticles, softer nanoparticles outperformed similar rigid particles in targeting to ICAM in the lungs [195] . However, in vivo observations with soft nanoparticles must consider the impact of prolonged circulation times on the extent of engagement with target molecules. The softer PEG nanoparticles also maintained higher circulating concentrations, meaning the lung to blood localization ratio was largely unaffected by flexibility of the ICAM-targeted particles in this case. Controlling for this factor, a microfluidic study showed that soft disc nanoparticles outperform rigid counterpart discs in adhesion to a target surface under flow [216] . It is hypothesized that this result may reflect the capacity of soft particles for increased target engagement and for resistance to target disengagement via fluid forces on the nanoparticles.

A specific application for soft particles in endothelial targeting was noted in studies of nanoparticles targeted to PLVAP. As noted above, PLVAP localizes to caveolae in the lungs and targeting to caveolae is limited for particles larger than the caveolar neck (>50-100nm) [128] . However, soft (~50 kPa) dextran nanogels of 150-300 nm diameter successfully targeted to PLVAP in the lungs ( Figure  4) , where analogous rigid polystyrene particles, functionalized with the same antibody, did not [131] . Further, tuning the modulus of the dextran nanogels via crosslinking resulted in abrogation of PLVAP targeting in inverse proportion to Young's modulus of the crosslinked particles [228] ( Figure 4D ).

Therefore, engineering of nanoparticle softness may affect particle access to caveolae, representing an important route for internalization in endothelial cells and possibly for transcytosis. Carrier mechanical properties can affect how well carriers access targets (e.g. by soft particles "squeezing" into small spaces, as depicted in (a)). PLVAP is located in a sterically concealed position, the caveolar neck, in lung endothelial cells. Soft dextran particles (a) were able to target PLVAP more effectively than crosslinked dextran particles (b) or rigid polystyrene particles (c) of similar size. As evaluated in radioisotope tracing (d) and fluorescence (e) studies, PLVAP targeting efficacy in the lungs decreased as nanoparticle Young's modulus (as determined by AFM) increased (d). Adapted from [131, 228] .

Compared to other routes of administration, the intravenous route has a distinct advantage in pulmonary delivery, as the lungs receive the entire first pass of venous blood [229] . In addition to first pass benefits, delivery to the pulmonary endothelium is particularly attractive, as it comprises ¼ of the entire surface area of endothelium. Because of the high perfusion of lungs and large endothelial surface area, delivery to endothelial antigens (e.g. ICAM-1, PECAM) is pharmacokinetically favored in the pulmonary vasculature.

An infusion via vascular catheters in conduit vessels favors uptake in the microvasculature immediately downstream of the injection site. This principle was successfully employed for PECAM and ICAM-targeted DDS: local infusion enhanced uptake in the vasculature in the brain, mesentery, and heart in several species, including mice, rats, dogs, and pigs [229] [230] [231] (Figure 5a ). Further, infusion in the isolated organs bypasses numerous factors impeding targeting and boosts local delivery, providing mechanistic insights and potential utility of vascular targeting for improving grafts for organ transplantation.

The intravascular route can be further manipulated by using red blood cells (RBC) as a transport vehicle for DDS that are both actively and passively targeted [232, 233] . Nanocarrier DDS can massively increase the mass of drug delivered to the target organ, but are not without their inherent problems, such as short half-life due to rapid clearance by the reticuloendothelial system [234] . The liver, spleen, and intravascular leukocytes, aided by the complement cascade, all contribute to this rapid clearance.

Red blood cell hitchhiking (RBC-H) was devised to evade this rapid clearance by the RES. Early studies used passive adsorption to non-covalently attach polystyrene nanoparticles onto RBCs and found that the half-life of RBC-adsorbed nanocarriers was longer than that of free nanoparticles. Additionally, the lung-to-clearing organ (liver & spleen) ratios were significantly improved using RBC-H [232] . This landmark work; however, was limited to rigid nanoparticles having no drug delivery utility, nonspecific attachment to RBC, and a circulation time of less than 24 hours. Notably, increased delivery to the target tissue was achieved by injecting RBC-coupled nanoparticles immediately upstream of target organ, thus increasing firstpass delivery (Figure 5b ).

Upon careful loading that does not compromise RBC biocompatibility, RBC carrying surface-bound cargoes do not accumulate in any organ, including the reticuloendothelial system. However, the surface-bound cargoes that are loaded on RBC in a reversible fashion detach from RBC and transfer to the microvascular bed encountered by RBC. In the case of intravascular injections of RBC carrying such cargoes, the first major microvascular bed downstream injection site, i.e., first pass. In IV injection this is the pulmonary vasculature.

Additional studies incorporated biocompatible nanocarriers into RBC-H using liposomes, polymeric nanocarriers, and adeno-associated virus (AAV). The first pass effect was again amplified using RBC-H with 40-fold increase in lung targeting compared to free nanocarrier after intravenous injection. Importantly, this effect was also observed after intra-arterial injection via the carotid artery and measuring uptake in the immediate downstream organ, the brain, achieving brain uptake 143-fold than that of free nanocarriers [232] . Studies have shown therapeutic benefit using this technique in vivo in mouse models of acute pulmonary embolism and lung metastasis [232, 233] .

RBC-H has shown many advantages over intravenous delivery of free nanocarriers, but is not without room for improvement. The initial work focused largely on first-pass delivery to the immediate downstream organ, relying on the interaction of RBCs with the endothelium to encourage mechanical dissociation of nanoparticle from RBC vehicle. The promise of RBC-H as a drug delivery system that successfully evades the RES will likely need to incorporate better control over the RBC-nanoparticle association (and thus dissociation) and effect on the carrier RBC. One method to increase control of RBC-nanocarrier interaction is the use of targeted binding rather than passive adsorption. The interaction of RBC with RBC membrane targeting moieties has been characterized using drug-coupled fusion proteins. Specifically, anti-RBCmembrane scFv fused to thrombomodulin were evaluated in vitro in a microfluidic system.

While some targeting to certain RBC surface epitopes caused pathologic increases in membrane rigidity (Band 3/ Glycophorin A), targeting to others (RhCE) did not. Importantly, therapeutic efficacy of fusion-TM was retained when compared to soluble TM [235] . Future generations of RBC-H may utilize nanocarriers targeted to the RBC membrane to improve control of RBCnanocarrier association, dissociation, and ultimately prolong time in circulation beyond the first pass effect. 

Many groups utilize antibody fragments (e.g. Fab, scFv, nanobody, etc.) as affinity ligands to achieve targeted drug delivery. Despite many advantages, such as ease of production in microbial systems and lack of Fc fragment-induced toxicities, these fragments are often inferior to full mAbs as isolated targeting ligands due to rapid blood clearance and poor stability of binding due to their monovalent nature. Additionally, commonly used amine-based conjugation strategies often inhibit the function of these small affinity ligands [236] . As such, there have been a myriad of protein engineering strategies proposed to improve the drug delivery properties of antibody fragments.

Improving circulation time of affinity ligands can be critical in conferring them with favorable biodistribution properties for therapeutic applications. This has been achieved in many ways, including fusion to long-circulating proteins (IgG and albumin) [237] [238] [239] [240] or attachment of hydrophilic polymers (PEG) [241, 242] to reduce renal elimination. Fusion to proteins such as albumin or albumin-binding molecules confers a long half-life, in part, due to interactions with the neonatal Fc receptor (FcRn), which serves as a salvage receptor preventing the intracellular catabolism of albumin [243] and IgG [244] [245] [246] . We have recently reported that fusion of a VCAM-1-targeting nanobody to an albumin-binding nanobody leads to 25-40-fold improvements in uptake in target tissues over a 24 hour period, relative to the anti-VCAM-1 nanobody alone [247] .

To overcome challenges in coupling of therapeutic cargoes to small affinity ligands, sitespecific conjugation strategies are becoming more prevalent. These typically involve building in motifs for conjugation near the C-terminus of the antibody fragment. Some of the more common strategies for site-specific conjugation of antibodies and fragments include Sortase A transpeptidation (recognizing the sequence LPXTG) [248] and conjugation to unpaired cysteines introduced into the molecule. These strategies have been successfully used to attach therapeutic cargoes and radiolabels to targeting ligands without adverse effects on their function [236, [249] [250] [251] [252] [253] . In addition to engineering of antibody fragments, our group has recently reported a strategy for engineering full-length mAbs for site-specific modification by Sortase A via CRISPR/Cas9-mediated engineering of hybridoma cells [254, 255] . The use of site-specific bioconjugation strategies permits oriented coupling of targeting ligands on the surface of J o u r n a l P r e -p r o o f nanoparticles, which has been demonstrated to improve their specificity for endothelial targets [252, 256] .

Nucleic acid aptamers are a recently developed class of affinity ligands that selectively bind to targets with high affinity via unique three-dimensional structures [257] . Aptamers are short, single-stranded DNA oligonucleotides that can be developed for a variety of target molecules using techniques analogous to phage display for antibody selection [258] . Aptamers directed against TfR have been used to deliver therapeutics both to [259] and across [260] the blood-brain barrier in animal models. This presents another potential approach for selective delivery to the endothelium. [6, 19, 41, 44, 45, 69, 138, [261] [262] [263] have demonstrated affinity targeting to both normal and pathologically altered endothelium [40, [264] [265] [266] . The delivery of enzyme replacement therapies to improve lysosome storage disease treatment was discussed above in the section on intracellular endothelial delivery. Here we provide a brief overview of a few areas of potential medical utility of this approach, with a focus on thrombosis, acute vascular inflammation, and ischemia ( Figure 6 ). Applications of endothelial targeting in select pathologies are summarized in Table 3 .

Endothelial targeted DDS can deliver a great variety of anti-thrombotic agents, with different mechanisms for release and or activation at the intended site. They include plasminogen activators [230, [267] [268] [269] and thrombomodulin [64, [270] [271] [272] fused with scFv binding to surface endothelial epitopes and exert their activities in the lumen directly. Alternatively, DDS targeted to the same epitopes carrying encapsulated small drug molecules such as anti-inflammatory drugs (which have both direct and indirect anti-thrombotic effects) employ release of drugs from the cell-bound DDS either in blood or in endothelial cells [205, 273] . Further, delivery of mRNA for TM (and likely other anti-thrombotic proteins) deliver their cargo into the cytosol of endothelial cells enabling synthesis and luminal exposure of the therapeutic transgene [87] . 6.1. Acute thrombosis. In many pathologies, affected blood vessels have an increased propensity for thrombosis, in no small part due to suppression of the endothelium's natural antithrombotic mechanisms [274] . Delivery of ATA, such as TM or plasminogen activators (tissue type, tPA, or urokinase, uPA) to the luminal surface of the endothelium may help to subside some prothrombotic states. These results have been supplemented by nucleic acid-based delivery of these proteins [275] .

In cell-based models, targeting anticoagulants to activated endothelium using anti-Eselectin mAbs was shown to inhibit thrombin expression, providing proof-of-principle of this approach [67] . As investigations moved into rodent studies, both tPA and uPA were shown to be concentrated within the lungs by targeting to ACE; however, there were limited pharmacologic benefits due to rapid endocytosis [99, 276] . However, delivery of ATA to non-internalizing endothelial epitopes (e.g., ICAM-1, PECAM) permitted not only efficient targeting of the lung, but also clot dissolution [269] . Fusion proteins between anti-PECAM scFv and ATA were able to provide thromboprophylaxis due to prolonged residence in the pulmonary lumen [267, 277, 278] . Local infusion of scFv/uPA via the carotid artery prevented cerebrovascular thrombosis [230] .

Fusions containing uPA mutant activated by thrombin [279] [280] [281] had similar high degree of lung uptake and thromboprophylaxis, with additional improvements in reductions of fibrin deposition and improvement of arterial oxygen tension relative to a non-thrombin-dependent scFv/uPA [268] . Factors that promote intracellular uptake (with retention of drug conjugate and recycling of receptor) include multivalent targeting moieties and low-flow state. Factors that modify uptake include ligand density, carrier geometry, and size. Ischemia/reperfusion injury (A) and acute vascular inflammation (B) both result in increased leukocyte recruitment and leaky tight junctions between endothelial cells. Treatment requires drug to be delivered intracellularly to the endothelium and to the interstitial space. Drug targeting to the specific receptors shown has resulted in treatment effect in animal models of these pathologies. (C) Shown is a blood clot within the lumen. Thrombosis and embolism both require the drug to remain in the vessel lumen to achieve direct therapeutic effect. Drug targeting to the receptors shown has resulted in treatment effect in animal models of thrombosis.

In rat models, pharmacologic benefits have been observed using both recombinant soluble TM [34] and tissue factor-targeted TM [282] , but this fusion rapidly disappeared from the vascular lumen. PECAM-targeted scFv/TM fusion accumulated in the pulmonary vasculature, and reduced both thrombosis and other tissue damage in mice, without causing bleeding [270] . By fusing TM with scFvs directed against PECAM and ICAM, the antithrombotic and anti-inflammatory effects of J o u r n a l P r e -p r o o f untargeted soluble TM are dwarfed, due to prolonged endothelial anchoring of TM [268] . Targeting scFv/TM fusions to ICAM afforded more potent protection than to PECAM, consistent with the notion of ICAM localization close to the TM cofactor EPCR in the plasmalemma [271] . As discussed above, by taking advantage of the CEPAL effect observed for PECAM-targeted ligands, TM and EPCR were able to be co-delivered, providing a substantially increased pharmacologic effect. This benefit was likely due to enhanced targeting (CEPAL effect) and optimal special arrangement of TM and EPCR, allowing enhanced interactions between the partner molecules [184] . Thrombin-activated, targeted ATA have the potential to provide a safe thrombophrophlyactic option in patients at high risk of acute thrombosis.

Thrombosis and inflammation are closely intertwined. Endothelium controls vascular permeability of leukocytes in response to tissue damage and infection, but also become a victim of injurious inflammatory mediators. Targeting anti-inflammatory drugs to protect the endothelium from friendly fire without impeding host defense is an attractive strategy. Inducible and constitutive endothelial determinants involved in inflammation appear to be good targets for this approach.

The use of liposomes targeted to E-selectin has shown utility in delivering dexamethasone (DEX) to a variety of tissues in animal models of chronic inflammation. For example, these particles showed accumulation in skin and kidneys in models of dermal [283] and renal [284] inflammation. In the latter model, this treatment conferred anti-inflammatory effects in mice [284] and in rats [285] . Within minutes of injection, similar formulations accumulated in the inflamed eye and reduced the level of inflammatory markers [286] . Similar DDS were used for nucleic acid delivery to the kidney and tumors [287, 288] . Both E-selectin and ICAM-1 have been used as target molecules for delivery of siRNA in vitro [289, 290] . Coupling of an E-selectin targeting ligand to adenovirus allowed targeting to the kidneys and displayed functional activity in a glomerulonephritis model [289] .

Delivery of DEX liposomes to integrins was achieved in a rat arthritis model, using RGD peptides. These particles provided improved protection when compared to untargeted particles [291] . A similar strategy was also used to delivery anti-inflammatory siRNA in animals [292] . Delivery of the anti-inflammatory prostaglandin, PGE 2 , was achieved by targeting to VCAM-1. Chronic administration of VCAM-1-targeted particles provided improved PGE 2 delivery and therapeutic benefits in a mouse model of 'atherosclerosis' [293] . Targeting dexamethasone-loaded liposomes and nanogels to ICAM, PECAM and PLVAP supersedes protective effects of untargeted counterparts in a mouse model of ARDS [273, 294] .

A plethora of recent findings have revealed close links between endocytosis and signaling pathways that have changed our understanding of the role that endocytosis plays in a cell's make-up [295, 296] . Traditionally, endocytosis and signaling were considered as distinct processes. However, it has now become clear that receptor-regulated endocytosis is critical for modulation of numerous signaling pathways, including attenuation of signal, its prolongation, activation, signal distinction from several possible paths, etc. [297] . This is also the case for many cytokine receptors, including TNFR1 and IL-1R [298] . TNFR1 activated by interaction with TNF co-localizes with caveolin in lipid raft and receptor complex internalization may modulate TNF signaling outcome [299] [300] [301] . Similarly, IL-1R J o u r n a l P r e -p r o o f translocates to caveolae upon activation and receptor internalization is required for complete NF-κB signaling processes [302, 303] .

Redox-active endosomes (i.e. containing reactive oxygen species (ROS)) may play an important role in pro-inflammatory signaling [304, 305] indicating potential therapeutic applications of antioxidant drug delivery [306] . PECAM-and ICAM-targeted delivery of SOD is able to considerably block the inflammatory response caused by cytokines and by TLR activation [307] [308] [309] . In addition, caveolar targeting of nanocarriers may have significant potential, particularly for endothelial and trans-endothelial delivery, due to the abundance of caveolae in endothelial cells [310] . Effective caveolar targeting has been demonstrated using albumin-coated nanocarriers [130] . The profound involvement of caveolae in cellular signaling [311] should be of particular interest for the drug delivery field (Figure 7 ).

Cytokine or PAMP binds to counterpart receptor, causing NOX activation and the complex internalization forming redox-active endosome. Generated ROS transfers into cytosol via CLC3 channel and stimulates proinflammatory NFκB cascade. Delivery of antioxidant to the endosome or overexpression of cytosolic SOD can reduce the inflammatory signaling and attenuate cellular injury [307, 308] . Inset shows SOD-loaded nanoparticles (green) enter endosomes (red), SOD-containing endosomes seen as yellow. Cell nuclei are shown blue.

The role of localization and receptor trafficking of TLRs (and particularly TLR4 as the best characterized TLR) was clarified from recent works [312, 313] . However, the mechanisms of TLRs endocytosis and its exact role in cellular response to pro-inflammatory signals still require more studies for our understanding. Moreover, it may be cell-specific. Indeed, TLR4 endocytosis in macrophages is not required for their response. In contrast, astrocytes internalized activated TLR4 by both caveolae and clathrin endocytosis, and caveolar endocytosis is required for both MyD88-and J o u r n a l P r e -p r o o f TRIF-dependent signaling pathways [314] , while epithelial cells are characterized by intracellular localization of TLR4 at Golgi apparatus and CD14-dependent trafficking of LPS via clathrindependent endocytosis [315, 316] . Interestingly, the proinflammatory effects of LPS are significantly attenuated in caveolin-1 knockout mice. NF-κB activation by LPS is decreased; ICAM expression is lower as well as PMN accumulation in lungs, while NO level is higher in Cav-1 -/mice compared to wild type [317] suggesting an important role of caveolae in LPS-induced response in vivo. Indeed, caveolar targeting and delivery of SOD using PLVAP has a significant advantage compared to less specific delivery via PECAM and ICAM [133, 318] . Caveolar targeting potentially can deliver SOD right to the signaling endosomes responsible for activation of NFκB signal transduction pathway.

Endothelial involvement in oxidative mechanisms of inflammation and other conditions is not limited to mediation of proinflammatory signaling via endosomal pathways discussed above. Endothelial cells are common victims of ROS in ischemia-reperfusion injury, acute inflammation, and infections. ROS-quenching enzymes, catalase and SOD, conjugated with mAbs directed against several endothelial targets (PECAM, ICAM-1, ACE) improved protection in several models of acute oxidative stress compared to untargeted formulations [263, 277, 278, [319] [320] [321] [322] [323] [324] [325] [326] [327] [328] [329] .

PECAM-and ICAM-targeted catalase protected against H 2 O 2 -induced vascular leak [330] , alleviated vascular [331] and oxidative stresses [332] , and pulmonary ischemia-reperfusion injuries [278, 321] . These results include lung transplantation in multiple animal models [324, 325] . Targeting to PECAM and ICAM of SOD, liposomal SOD, and liposomal SOD/catalase mimetic, as well as inhibitors of ROS-producing enzymes, reduced ROS toxicities in endothelial cells [205, 306, 326] , normalized vasoconstriction in mice [321] , attenuated VEGF-induced endothelial leakage [330] , and endotoxin-induced acute pulmonary inflammation in animals [333] .

Pharmacological agents decelerating vesicular trafficking prolong endothelial antioxidant protection conferred by ICAM-and PECAM-targeted catalase and SOD via delaying their lysosomal degradation [106, 152] . Targeting to PECAM of catalase loaded into nanocarriers selectively permeable for ROS provided prolonged protection [334] [335] [336] [337] .

Overall, delivery of both antioxidants and anti-inflammatories to the endothelium provides a variety of mechanistically precise, spatiotemporally controlled protective effects in the variety of animal models emulating pathological pathways typical of acute human diseases and conditions associated with or driven by vascular oxidative stress, ischemia-reperfusion and inflammation. 7. Current challenges and perspectives. 7.1. Modeling of vascular targeting. Many reasons drive attempts to minimize in vivo investigations by pursuing in vitro models, despite the fact that no in vitro system fully recapitulates the situation in vivo and most, if not all, lack effects of an organism on behavior of a drug or DDS systemically (PK, clearance, effect of pathology, as well as humoral, nervous and defense factors) and locally (cellular microenvironment, histological, and pathological factors). In case of vascular targeting, these challenges are aggravated by rapid phenotypic bastardization of cultivated endothelial cells, which quickly lose specific markers like ACE [338] .

The introduction of HUVEC (human umbilical vein endothelial cells) as an in vitro endothelial model by Eric Jaffe, Michael Gimbrone, Jordan Pober and colleagues nearly half a century ago was a truly major breakthrough [339] . Further advances, such as adaptation of the Boyden's chamber to study endothelial permeability, upgrades of static cell cultures to flowadapted and flow-exposed cell cultures, microfluidic systems and flow chambers with cells grown on semi-permeable supports, and recent advent of the "organ on the chip", including exquisite 3D multichannel models of vascular segments have yielded significant results [91, 272, [340] [341] [342] [343] .

In vitro models are useful to address specific questions, such as analysis of hydrodynamic parameters and effects of flow adaptation on binding, uptake, vesicular transport, and effects of DDS targeted to endothelium. Static 2D cultures are useful to determine specificity and other parameters of binding DDS to target vs. non-target cells (K d , B max , kinetics of binding and dissociation).

Sometimes, there is no choice but to retreat to in vitro models, since our methods do not work in vivo. Thus, parameters of resolution, dynamics, objectivity, and attribution to histological and cellular structures of current methods afford rather limited information on DDS sub-cellular addressing and intracellular transport. Here, endothelial cell culture seems to be a reasonable resolution. More technically challenging models of perfusion of isolated vessels and organs provide a valuable resource, permitting real-time direct measurements of the uptake, tissue localization, and activity of a DDS in conditions maximally close to in vivo [39, 319] .

Computational modeling and systems biology represent further interesting in silico approaches. Major challenges in this area include variable compatibility and quality of the feed data, oversimplification of conditions, parameters, and margins, and relatively arbitrary selection of the focus and readouts. Yet, mutually enriching collaboration of computational researchers with experimentalists and physicians may enable modeling with high predictive value that allows for a reduction of in vivo experimentation [344] [345] [346] [347] .

A second approach that is often reported in the literature is optical tracing of fluorescent components of a DDS. While fluorescent labels are often useful for visualizing distribution on a subtissue level, the presence of high (and variable) background signals in biological matrices and nonlinear signal vs. concentration relationships reduces their utility in measuring PK/BD. Finally, mass spectrometry (MS) is being used frequently for PK measurements in both preclinical and clinical investigations [350] [351] [352] . Due to the complex nature of many DDS, it would be challenging to validate an assay for direct measurement of unlabeled DDS. However, MS should be considered as the gold standard for assessment of PK/BD of small molecule cargo drugs, as it is able to make highly precise measurements in both blood and tissues.

Collection of high quality PK/BD data permits development of mathematical models that can be used to guide further development and optimization of DDS. The types of models that can be developed range in terms of complexity and degree of mechanistic information, from empirical models that simply describe the concentration vs. time curve, to physiologically-based models that are able to predict drug behavior. At the preclinical academic level, more mechanistic models are often useful as tools to predict the impact of DDS properties and pathology on behavior, permitting optimization of a delivery strategy early in development.

Additionally, physiologically-based models have the potential for relatively straightforward scaling to higher species, permitting prediction of clinical PK/PD profiles [353] [354] [355] . Once a DDS moves into the clinical domain, modeling often shifts to simpler models, with the goal of identifying patient-specific factors, termed covariates that may impact drug behavior and patient outcomes. This application of population PK is then used to identify special patient populations (e.g. renal failure, pediatrics, obesity, etc.) that may require dose adjustments from the 'standard' dose [356, 357] .

Ultimately, preclinical studies culminate in analysis of the beneficial vs. unintended and adverse effects of the devised DDS in animal models. Admittedly, no animal model fully recapitulates human diseases, but, as physicians know, no individual patient's condition fully recapitulates another patient's condition. Of course, the closer to human the species is and the closer the pathogenesis is to the relevant human condition, the greater the confidence in the translatability of results.

Parameters of the amplitude, timing of the onset, and duration of the desirable vs. unintended effects are the key readouts deciding the fate of a DDS. Statistics are necessary, but not always sufficient to prove the therapeutic significance. The rigor and scrutiny of characterization of DDS effects are painstaking. These studies, performed in healthy and sick animals in an obligatory double-blinded manner, must include direct comparison of experimental groups with controls including healthy and sick animals treated with: A) Untargeted agents -free and loaded in untargeted DDS; B) A mixture of targeted DDS and free drug; C) Drugless targeted and untargeted DDS. Not all, but many studies appraising benefit/risk ratio of vascular targeting in fact meet these draconian criteria, giving a certain level of comfort and confidence that this drug delivery approach will inevitably reach the clinical utility.

The wealth of knowledge garnered in three decades of studies of vascular targeting is enormous. These studies yielded diverse endothelial target molecules for drug delivery, J o u r n a l P r e -p r o o f multitudes of ligands, nanocarriers, and methodologies for their conjugation and assembly. These means provide specific and effective targeting of drugs to, into and across of the endothelia in several important organs and vascular areas of significant biomedical interest. It seems safe to state that the general progress in this area of the drug delivery field is impressive, encouraging and brings us to the brink of industrial development and clinical translation.

These studies have also advanced our understanding of physiological and pathological processes localized in or involving endothelial interface, especially in the lungs and brain. Targeted pharmacological interventions designed to yield functional responses in the specific components and compartments of vascular system and blood represent valuable investigational tools. Indeed, both positive and negative outcomes of such interventions may give mechanistic insight about the role of implicated molecules and cells, on the sine qua none condition that delivery, localization and activity of the targeted pharmacological agent are confirmed "beyond any reasonable doubt", and therefore, the lack of the effect is attributable to the intricacies of the pathophysiological mechanism, not failure of delivery.

Notwithstanding, the main biomedical purpose of drug delivery is, of course, advancement of the treatment of patients -diagnostic, prophylaxis and therapy. The pressure to come up with the winning formulations is palpable, but it would be a mistake to put on the back burner, suspend or abandon devising and research of new DDS, especially in academia, for the sake of concentrating efforts on the industrial development and clinical testing of DDS showing favorable profile in experimental settings. It is unpredictable which specific DDS iterations will emerge as new therapeutic options. Yet, focus on promising candidates seems timely and necessary. Selection of these lead candidates is a complex and multifaceted interdisciplinary affair that must involve basic scientists, clinical investigators, industrial, and regulatory counterparts.

J o u r n a l P r e -p r o o f Increased J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f Journal Pre-proof

A proposal linking clearance of circulating lipoproteins to tissue metabolic activity as a basis for understanding atherogenesis

Endothelium and haemostasis

Vascular heterogeneity in the kidney

Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms

Caveolae in CNS arterioles mediate neurovascular coupling

Targeting endothelium and its dynamic caveolae for tissuespecific transcytosis in vivo: a pathway to overcome cell barriers to drug and gene delivery

Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis

Targeting therapeutics to endothelium: are we there yet?

Targeting Tumor Endothelial Cells with Nanoparticles

Significant role of cationic polymers in drug delivery systems

Targeting of apoptotic macrophages and experimental atheroma with radiolabeled annexin V: a technique with potential for noninvasive imaging of vulnerable plaque

Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand -based mechanisms

Liposome clearance from blood: different animal species have different mechanisms

Delivering nanoparticles to lungs while avoiding liver and spleen through adsorption on red blood cells

Neutrophil-mediated transport is crucial for delivery of short-circulating magnetic nanoparticles to tumors

Neutrophils, as "Trojan horses", participate in the delivery of therapeutical PLGA nanoparticles into a tumor based on the chemotactic effect

Lipopeptide nanoparticles for potent and s elective siRNA delivery in rodents and nonhuman primates

Ionizable lipid nanoparticles encapsulating barcoded mRNA for accelerated in vivo delivery screening

Molecular heterogeneity of the vascular endothelium revealed by in vivo phage display

To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery

Immunotargeting of drugs to the pulmonary vascular endothelium as a therapeutic strategy

Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue -specific therapy

Vascular targeting as a strategy for cancer therapy

Rat monoclonal antibody distribution in mice: an epitope inside the lung vascular space mediates very efficient localization

Immunotargeting of liposomes to activated vascular endothelial cells: a strategy for site-selective delivery in the cardiovascular system

Dynamic and cellular interactions of nanoparticles in vascular-targeted drug delivery

Targeted drug delivery and penetration into solid tumors

Molecular imaging and therapy of atherosclerosis with targeted nanoparticles

Development of ligand-targeted liposomes for cancer therapy

Folate-targeted therapies for cancer

Monoclonal antibody targeting of liposomes to mouse lung in vivo

Lipid composition is important for highly efficient target binding and retention of immunoliposomes

Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects

Inflammation and the activated protein C anticoagulant pathway, Seminars in thrombosis and hemostasis

Antibodies to thrombomodulin induce receptor-mediated endocytosis in human saphenous vein endothelial cells

Anti-thrombomodulin antibodies and venous thrombosis

Selective rat lung endothelial targeting with a new set of monoclonal antibodies to angiotensin I-converting enzyme

Monoclonal antibodies to native mouse angiotensin -converting enzyme (CD143): ACE expression quantification, lung endothelial cell targeting and gene delivery

Lung uptake of antibodies to endothelial antigens: key determinants of vascular immunotargeting

Radioimmunoimaging of lung vessels: an approach using indium-111-labeled monoclonal antibody to angiotensin-converting enzyme

Lung is the target organ for a monoclonal antibody to angiotensin -converting enzyme

Angiotensin I converting enzyme and the changes in our concepts through the years. Lewis K. Dahl memorial lecture

Angiotensin -(1-7)-Stimulated Nitric Oxide and Superoxide Release From Endothelial Cells

Targeting of radiolabeled monoclonal antibody against ACE to the pulmonary endothelium

Combined transductional and transcriptional targeting improves the specificity of transgene expression in vivo

A targetable, injectable adenoviral vector for selective gene delivery to pulmonary endothelium in vivo

Interaction of mAb to angiotensin-converting enzyme (ACE) with antigen in vitro and in viv o: antibody targeting to the lung induces ACE antigenic modulation

Epitope-specific antibody-induced cleavage of angiotensin-converting enzyme from the cell surface

Vascular immunotargeting of glucos e oxidase to the endothelial antigens induces distinct forms of oxidant acute lung injury: targeting to thrombomodulin, but not to PECAM-1, causes pulmonary thrombosis and neutrophil transmigration

Targeting of superoxide dismutase and catalase to vascular endothelium

Targeting endothelial cells with adenovirus expressing nitric oxide synthase prevents elevation of blood pressure in stroke-prone spontaneously hypertensive rats

Bone morphogenetic protein type 2 receptor gene therapy attenuates hypoxic pulmonary hypertension

Factors modulating the delivery and effect of enzymatic cargo conjugated with antibodies targeted to the pulmonary endothelium

Gene expression profiling of the early pulmonary response to hyperoxia in mice

Angiotensin-converting enzyme: clinical applications and laboratory investigations on serum and other biological fluids

Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells

Systemic administration of platelet -activating factor in rat reduces specific pulmonary uptake of circulating monoclonal antibody to angiotensin -converting enzyme

Endotoxin reduces specific pulmonary uptake of radiolabeled monoclonal antibody to angiotensin -converting enzyme

Normoxic lung ischemia/reperfusion accelerates shedding of angiotensin converting enzyme from the pulmonary endothelium

A novel angiotensin I-converting enzyme mutation (S333W) impairs N-domain enzymatic cleavage of the anti-fibrotic peptide

Protein C and acu te inflammation: a clinical and biological perspective

Inhibition of inflammation induced shedding of the endothelial glycocalyx with low molecular weight heparin

Relative shedding of glycosaminoglycans from the endothelial glycocalyx during inflammation and their contribution to stiffness of the glycocalyx

Platelet endothelial cell adhesion molecule targeted oxidant -resistant mutant thrombomodulin fusion protein with enhanced potency in vitro and in vivo

Targeting of antioxidant and anti-thrombotic drugs to endothelial cell adhesion molecules

Endothelial cells at inflammatory sites as target for therapeutic intervention

Immunoselective targeting of an antithrombin agent to the surface of cytokine-activated vascular endothelial cells

Imaging vascular endothelial activation: an approach using radiolabeled monoclonal antibodies against the endothelial cell adhesion molecule E-selectin

Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature

Endothelial endocytic pathways: gates for vascular drug delivery

Adhesion receptors of the immune system

The role of P-selectin and ICAM-1 in acute lung injury as determined using blocking antibodies and mutant mice

Integrins, ICAMs, and selectins: role and regulation of adhesion molecules in neutrophil recruitment to inflammatory sites

Endothelial and epithelial cell adhesion molecules

Blood coagulation-dependent inflammation. Coagulation-dependent inflammation and inflammation-dependent thrombosis

Leukocyte-endothelial interactions in inflammation

Endothelial Cell Junctional Adhesion Molecules: Role and Regulation of Expression in Inflammation

Targeting vascular (endothelial) dysfunction

The Endothelium in Sepsis

The vascular endothelium and human diseases

COVID-19: the vasculature unleashed

Overcoming Barriers: The Endothelium As a Linchpin of Coronavirus Disease 2019 Pathogenesis?

SARS-CoV-2 and viral sepsis: observations and hypotheses

Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin

Biomedical aspects of drug targeting

In vivo imaging of activated endothelium using an anti-VCAM-1 magnetooptical probe

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

Multifactorial determinants that govern nanoparticle uptake by human endothelial cells under flow

Internalization of microparticles by endothelial cells promotes platelet/endothelial cell interaction under flow

Acute and Chronic Shear Stress Differently Regulate Endothelial Internalization of Nanocarriers Targeted to Platelet -Endothelial Cell Adhesion Molecule-1

Effect of flow on endothelial endocytosis of nanocarriers targeted to ICAM-1

Cellular and molecular aspects of PECAM -1

VE-cadherin and endothelial adherens junctions: active guardians of vascular integrity

Endothelial tetraspanin microdomains regulate leukocyte firm adhesion during extravasation

Vascular cell adhesion molecule-1 expression and signaling during disease: regulation by reactive oxygen species and antioxidants

Apical topography and modulation of ICAM-1 expression on activated endothelium

Tetraspanin microdomains in immune cell signalling and malignant disease

A novel, 85 kDa endothelial antigen differentiates plasma membrane macrodomains in lung alveolar capillaries

Vascular immuno targeting to endothelial surface in a specific macrodomain in alveolar capillaries

Cytokine -activated endothelial cells internalize E-selectin into a lysosomal compartment of vesiculotubular shape. A tubulin -driven process

Rapid transport of internalized P-selectin to late endosomes and the TGN: roles in regulating cell surface expression and recycling to secretory granules

Evidence for endocytosis of E-selectin in human endothelial cells

Investigation of the cellular uptake of E-Selectin-targeted immunoliposomes by activated human endothelial cells

Selective intracellular delivery of dexamethasone into activated endothelial cells using an Eselectin-directed immunoconjugate

Targeting an adenoviral gene vector to cytokine-activated vascular endothelium via E-selectin

ICAM-1 recycling in endothelial cells: a novel pathway for sustained intracellular delivery and prolonged effects of drugs

Advanced drug delivery systems that target the vascular endothelium

A novel endocytic pathway induced by clustering endothelial ICAM-1 or PECAM-1

Vascular targeting of nanocarriers: perplexing aspects of the seemingly straightforward paradigm

Differential intra-endothelial delivery of polymer nanocarriers targeted to distinct PECAM-1 epitopes

Role of glycocalyx in leukocyte-endothelial cell adhesion

Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture

A search for reliable molecular markers of prognosis in prostate cancer: a study of 240 cases

Tumor-targeted gene delivery using molecularly engineered hybrid polymers functionalized with a tumor-homing peptide

Screening phage display libraries for organ-specific vascular immunotargeting in vivo

Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung

Endothelial cell-surface gp60 activates vesicle formation and trafficking via G(i)-coupled Src kinase signaling pathway

Evidence of transcellular permeability pathway in microvessels

Molecular mechanisms of endocytosis

Plasmalemmal vesicles represent the large pore system of continuous microvascular endothelium

Structure and function of endothelial caveolae

Vesicle formation and trafficking in endothelial cells and regulation of endothelial barrier function

Caveolae: from basic trafficking mechanisms to targeting transcytosis for tissue -specific drug and gene delivery in vivo

Separation of caveolae from associated microdomains of GPI-anchored proteins

Quantitative ana lysis of albumin uptake and transport in the rat microvessel endothelial monolayer

Transport of nitrated albumin across continuous vascular endothelium

The multiple faces of caveolae

Endothelial targeting of polymeric nanoparticles stably labeled with the PET imaging radioisotope iodine-124

Uptake of ricinB-quantum dot nanoparticles by a macropinocytosis-like mechanism

Size and dynamics of caveolae studied using nanoparticles in living endothelial cells

Flexible Nanoparticles Reach Sterically Obscured Endothelial Targets Inaccessible to Rigid Nanoparticles

Ferritin -based drug delivery systems: Hybrid nanocarriers for vascular immunotargeting

Spatially controlled assembly of affinity ligand and enzyme cargo enables targeting ferritin nanocarriers to caveolae

Ferritin Nanocages with Bio logically Orthogonal Conjugation for Vascular Targeting and Imaging

Antioxidant strategies in respiratory medicine

Probing the structural and molecular diversity of tumor vasculature

Vascular targeting and antigen presentation

Selection of monoclonal antibodies which induce internalization and phosphorylation of p185HER2 and growth inhibition of cells with HER2/NEU gene amplification

Selection and use of ligands for receptor-mediated gene delivery to myogenic cells

Selection and characterization of cell binding and internalizing phage antibodies

Internalizing cancer antibodies from phage libraries selected on tumor cells and yeast-displayed tumor antigens

Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library

Detection of vascular adhesion molecule-1 expression using a novel multimodal nanoparticle

Challenges in design and characterization of ligand -targeted drug delivery systems

VCAM-1 is internalized by a clathrin-related pathway in human endothelial cells but its alpha 4 beta 1 integrin counter-receptor remains associated with the plasma membrane in human T lymphocytes

Kinetics of transit of transferrin and epidermal growth factor through clathrin-coated membranes

Trafficking of Gold Nanoparticles Coated with the 8D3 Anti-Transferrin Receptor Antibody at the Mouse Blood -Brain Barrier

Caveolae: mining little caves for new cancer targets

Intertwined mechanisms define transport of anti-ICAM nanocarriers across the endothelium and brain delivery of a therapeutic enzyme

Slow intracellular trafficking of catalase nanoparticles targeted to ICAM-1 protects endothelial cells from oxidative stress

Control of intracellular trafficking of ICAM-1-targeted nanocarriers by endothelial Na+/H+ exchanger proteins

Lysosomal enzyme delivery by ICAM -1-targeted nanocarriers bypassing glycosylation-and clathrin-dependent endocytosis

Comparative binding, endocytosis, and biodistribution of antibodies and antibody -coated carriers for targeted delivery of lysosomal enzymes to ICAM-1 versus transferrin receptor

Control of Endothelial Targeting and Intracellular Delivery of Therapeu tic Enzymes by Modulating the Size and Shape of ICAM-1-targeted Carriers

New biotechnological and nanomedicine strategies for treatment of lysosomal storage disorders, Wiley interdisciplinary reviews

Lysosomal storage diseases: natural history and ethical and economic aspects

Delivery of lysosomal enzymes for therapeutic use: gluco cerebrosidase as an example

The role of mannosylated enzyme and the mannose receptor in enzyme replacement therapy

Enzyme replacement and enhancement therapies: lessons from lysosomal disorders

In fusion of recombinant human acid sphingomyelinase into niemann -pick disease mice leads to visceral, but not neurological, correction of the pathophysiology

FDA orphan drug designations for lysosomal storage disorders -a cross-sectional analysis

Comparative effects of recombinant acid sphingo myelinase administration by different routes in niemann-pick disease mice

VCAM-1 and ICAM-1

Profile of endothelial and leukocyte activation in Fabry patients

Delivery of acid sphingomyelinase in normal and niemann -pick disease mice using intercellular adhesion molecule-1-targeted polymer nanocarriers

Enhanced endothelial delivery and biochemical effects of α-galactosidase by ICAM-1-targeted nanocarriers for Fabry disease

Enhanced delivery of α-glucosidase for Pompe disease by ICAM-1-targeted nanocarriers: comparative performance of a strategy for three distinct lysosomal storage disorders

Lysosomal enzyme replacement therapies: Historical development, clinical outcomes, and future perspectives

Intercellular adhesion molecule 1 engagement modulates sphingomyelinase and ceramide, supporting uptake of drug carriers by the vascular endothelium

Biological Functionalization of Drug Delivery Carriers To Bypass Size Restrictions of Receptor-Mediated Endocytosis Independently from Receptor Targeting

In vitro characterization of leukocyte mimetic for targeting therapeutics to the endothelium using two receptors

In vivo performance of polymer nanocarriers dually-targeted to epitopes of the same or different receptors

Superior sensitivity of novel molecular imaging probe: simultaneously targeting two types of endothelial injury markers

Interplay between rolling and firm adhesion elucidated with a cellfree system engineered with two distinct receptor-ligand pairs

Magnetic resonance imaging of endothelial adhesion molecules in mouse atherosclerosis using dual-targeted microparticles of iron oxide

Targeted ultrasound contrast agents: in vitro assessment of endothelial dysfunction and multi-targeting to ICAM-1 and sialyl Lewisx

Collaborative enhancement of antibody binding to distinct PECAM-1 epitopes modulates endothelial targeting

Structure-function analysis of angiotensin I-converting enzyme using monoclonal antibodies. Selective inhibition of the amino -terminal active site

Fine epitope mapping of monoclonal antibodies 9B9 and 3G8 to the N domain of angiotensin -converting enzyme (CD143) region involved in regulating angiotensin -converting enzyme dimerization and shedding

Mechanism of Collaborative Enhancement of Binding of Paired Antibodies to Distinct Epitopes of

The role of PECAM-1 in vascular cell biology

Vascular endothelial effects of collaborative binding to platelet/endothelial cell adhesio n molecule-1 (PECAM-1)

Dual targeting of therapeutics to endothelial cells: collaborative enhancement of delivery and effect

Collaborative Enhancement o f Endothelial Targeting of Nanocarriers by Modulating Platelet-Endothelial Cell Adhesion Molecule-1/CD31 Epitope Engagement

Effect of ligand density, receptor density, and nanoparticle size on cell targeting

Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers

Controlling ligand surface density optimizes nanoparticle binding to ICAM-1

Reduction of Nanoparticle Avidity Enhances the Selectivity of Vascular Targeting and PET Detection of Pulmonary Inflammation

Intravascular delivery of particulate systems: does geometry really matter?

Reductively Responsive Hydrogel Nanoparticles with Uniform Size, Shape, and Tunable Composition for Systemic siRNA Delivery in Vivo

Non -affinity factors modulating vascular targeting of nano-and microcarriers

Calibration -quality cancer nanotherapeutics

Towards programming immune tolerance through geometric manipulation of phosphatidylserine

Elasticity of nanoparticles influences their blood circulation, phagocytosis, endocytosis, and targeting

Lung vascular targeting through inhalation delivery: insight from filamentous viruses and other shapes

From stealthy polymersomes and filomicelles to "self" Peptide -nanoparticles for cancer therapy

Novel platforms for vascular carriers with controlled geometry

Renal clearance of quantum dots

Strategies in the design of nanoparticles for therapeutic applications

Endothelial Targeting of Antibody -Decorated Polymeric Filomicelles

Microcirculation of the spleen: and open or closed circulation?

Acute hemodynamic effects and blood pool kinetics of polystyrene microspheres following intravenous administration

The targeting of drugs parenterally by use of microspheres

Endothelial targeting of liposomes encapsulating SOD/catalase mimetic EUK-134 alleviates acute pulmonary inflammation

Cell-mediated delivery of nanoparticles: taking advantage of circulatory cells to target nanoparticles

The shape of things to come: importance of design in nanotechnology for drug delivery

Materials for drug delivery: innovative solutions to address complex biological hurdles

Endothelial targeting of antibody -decorated polymeric filomicelles

Bypassing adverse injection reactions to nanoparticles through shape modification and attachment to erythrocytes

Polymer particle shape independently influences binding and internalization by macrophages

Sedimentation of an ellipsoid inside an infinitely long tube at low and intermediate Reynolds numbers

Modeling particle shape-dependent dynamics in nanomedicine

Flow and adhesion of drug carriers in blood vessels depend on their shape: a study using model synthetic microvascular networks

Platelet mimetic particles for targeting thrombi in flowing blood

Platelet-like nanoparticles: mimicking shape, flexibility, and surface biology of platelets to target vascular injuries

Using shape effects to target antibody-coated nanoparticles to lung and brain endothelium

Shape effects of filaments versus spherical particles in flow and drug delivery

Impact of particle elasticity on particle-based drug delivery systems

The role of antibody synergy and membrane fluidity in the vascular targeting of immunoliposomes

Nanoparticle elasticity directs tumor uptake

Nanogel Carrier Design for Targeted Drug Delivery

Soft Discoidal Polymeric Nanoconstructs Resist Macrophage Uptake and Enhance Vascular Targeting in Tumors

Effect of mechanical properties of hydrogel nanoparticles on macrophage cell uptake

Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target

Tunable rigidity of (polymeric core)-(lipid shell) nanoparticles for regulated cellular uptake

Size, shape, and flexibility influence nanoparticle transport across brain endothelium under flow

Cross -linker-Modulated Nanogel Flexibility Correlates with Tunable Targeting to a Sterically Impeded Endothelial Marker

Platelet-endothelial cell adhesion molecule-1-directed immunotargeting to cardiopulmonary vasculature

Delivery of anti-platelet-endothelial cell adhesion molecule single-chain variable fragment-urokinase fusion protein to the cerebral vasculature lyses arterial clots and attenuates postischemic brain edema

Combining vascular targeting and the local first pass provides 100-fold higher uptake of ICAM-1-targeted vs untargeted nanocarriers in the inflamed brain

Red blood cell-hitchhiking boosts delivery of nanocarriers to chosen organs by orders of magnitude

Erythrocyte leveraged chemotherapy (ELeCt): Nanoparticle assembly on erythrocyte surface to combat lung metastasis

Pharmacokinetic and Pharmacodynamic Properties of Drug Delivery Systems

Biocompatible coupling of therapeutic fusion proteins to human erythrocytes

Site-Specific Modification of Single-Chain Antibody Fragments for Bioconjugation and Vascular Immunotargeting

Half-life extension through albumin fusion technologies

Albumin as a versatile platform for drug half-life extension

An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates

Enhanced circulating halflife and hematopoietic properties of a human granulocyte colony -stimulating factor/immunoglobulin fusion protein

Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce immunogenicity

Pegylation: a novel process for modifying pharmacokinetics

The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan

Abnormally short serum half-lives of IgG in beta 2-microglobulin-deficient mice

The protection receptor for IgG catabolism is the beta2-microglobulincontaining neonatal intestinal transport receptor

Increased clearance of IgG in mice that lack beta 2-microglobulin: possible protective role of FcRn

Greineder, Molecularly Engineered Nanobodies for Tunable Pharmacokinetics and Drug Delivery

Sortase-mediated protein ligation: a new method for protein engineering

Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency

Sortase A-mediated site-specific labeling of camelid single-domain antibody-fragments: a versatile strategy for multiple molecular imaging modalities

Legomedicine-A Versatile Chemo-Enzymatic Approach for the Preparation of Targeted Dual-Labeled Llama Antibody-Nanoparticle Conjugates

Vascular Targeting of Radiolabeled Liposomes with Bio-Orthogonally Conjugated Ligands: Single Chain Fragments Provide Higher Specificity than Antibodies

Site -specific labeling of cysteine-tagged camelid single-domain antibody-fragments for use in molecular imaging

Molecular engineering of antibodies for site -specific covalent conjugation using CRISPR/Cas9

CRISPR/Cas9-Mediated Genetic Engineering of Hybridomas for Creation of Antibodies that Allow for Site-Specific Conjugation

Unprecedently high targeting specificity toward lung ICAM-1 using 3DNA nanocarriers

SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands

Nucleic Acid Aptamers: An Emerging Tool for Biotechnology and Biomedical Sensing

Inhibition of cerebral vascular inflammation by brain endothelium-targeted oligodeoxynucleotide complex

Bifunctional Aptamer-Doxorubicin Conjugate Crosses the Blood-Brain Barrier and Selectively Delivers Its Payload to EpCAM-Positive Tumor Cells

Lu-ECAM-1-mediated adhesion of melanoma cells to endothelium under conditions of flow

Isolation, cloning, and localization of rat PV-1, a novel endothelial caveolar protein

Immunotargeting of antioxidant enzyme to the pulmonary endothelium

Drug delivery and targeting

Cationic liposomes enhance targeted d elivery and expression of exogenous DNA mediated by N-terminal modified poly(L-lysine)-antibody conjugate in mouse lung endothelial cells

Targetability of novel immunoliposomes modified with amphipathic poly(ethylene glycol)s conjugated at their distal terminals to monoclonal antibodies

Endothelial targeting of a recombinant construct fusing a PECAM -1 single-chain variable antibody fragment (scFv) with prourokinase facilitates prophylactic thrombolysis in the pulmonary vasculature

Prophylactic thrombolysis by thrombin -activated latent prourokinase targeted to PECAM-1 in the pulmonary vasculature

ICAM-directed vascular immunotargeting of antithrombotic agents to the endothelial luminal surface

Anchoring fusion thrombomodulin to the endothelial lumen prote cts against injury-induced lung thrombosis and inflammation

Vascular immunotargeting to endothelial determinant ICAM-1 enables optimal partnering of recombinant scFv-thrombomodulin fusion with endogenous cofactor

ICAM-1-targeted thrombomodulin mitigates tissue factor-driven inflammatory thrombosis in a human endothelialized microfluidic model

Icam-1 targeted nanogels loaded with dexamethasone alleviate pulmonary inflammation

Inflammation and thrombosis

Enhanced in vivo antithrombotic effects of endothelial cells expressing recombinant plasminogen activators transduced with retroviral vectors

Targeting of antibodyconjugated plasminogen activators to the pulmonary vasculature

Streptavidin facilitates internalization and pulmonary targeting of an anti-endothelial cell antibody (platelet-endothelial cell adhesion molecule 1): a strategy for vascular immunotargeting of drugs

Immunotargeting of catalase to the pulmonary endothelium alleviates oxidative stress and reduces acute lung transplantation injury

Single-chain urokinase-type plasminogen activator does not possess measurable intrinsic amidolytic or plasminogen activator activities

The activation of pro -urokinase by plasma kallikrein and its inactivation by thrombin

Design and evaluation of a thrombinactivable plasminogen activator

Amplified anticoagulant activity of tissue factor-targeted thrombomodulin: in-vivo validation of a tissue factorneutralizing antibody fused to soluble thrombomodulin

In Vitro Cellular Handling and in Vivo Targeting of E-Selectin-Directed Immunoconjugates and Immunoliposomes Used for Drug Delivery to Inflamed Endothelium

Site -Specific Inhibition of Glomerulonephritis Progression by Targeted Delivery of Dexamethasone to Glomerular Endothelium

Inhibition of proinflammatory genes in anti-GBM glomerulonephritis by targeted dexamethasone-loaded AbEsel liposomes

Highefficacy site-directed drug delivery system using sialyl-Lewis X conjugated liposome

Polycation-siRNA nanoparticles can disassemble at the kidney glomerular basement membrane

Lipid nanoparticle siRNA systems for silencing the androgen receptor in human prostate cancer in vivo

Targeted adenovirus mediated inhibitio n of NF-kappaB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo

Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro --a quantitative study

Targeting of angiogenic endothelial cells at sites of inflammation by dexamethasone phosphatecontaining RGD peptide liposomes inhibits experimental arthritis

Targeted delivery of small interfering RNA to angiogenic endothelial cells with liposome -polycation-DNA particles

LipoCardium: endothelium-directed cyclopentenone prostaglandin-based liposome formulation that completely reverses atherosclerotic lesions

Liposome-encapsulated dexamethasone attenuates ventilator-induced lung inflammation

The endocytic matrix

Endocytosis and signaling: cell logistics shape the eukaryotic cell plan

Endocytosis and signalling: intertwining mo lecular networks

Endocytic regulation of cytokine receptor signaling

Caveolae participate in tumor necrosis factor receptor 1 signaling and internalization in a human endothelial cell line

Targeting of tumor necrosis factor receptor 1 to low density plasma membrane domains in human endothelial cells

Recruitment of TNF receptor 1 to lipid rafts is essential for TNFalpha-mediated NF-kappaB activation

Ethanol mimics ligand-mediated activation and endocytosis of IL-1RI/TLR4 receptors via lipid rafts caveolae in astroglial cells

Regulation of NF-kappaB-dependent gene expression by ligand-induced endocytosis of the interleukin-1 receptor

The basic biology of redoxosomes in cytokine -mediated signal transduction and implications for disease-specific therapies

Compartmentalization of Redox Signaling through NADPH Oxidase-derived ROS

Targeted modulation of reactive oxygen species in the vascular endothelium

Anti-inflammatory effect of targeted delivery of SOD to endothelium: mechanism, synergism with NO donors and protective effects in vitro and in vivo

PECAMtargeted delivery of SOD inhibits endothelial inflammatory response

Size and targeting to PECAM vs ICAM control endothelial delivery, internalization and protective effect of multimolecular SOD conjugates

Delivery of nanoparticle: complexed drugs across the vascular endothelial barrier via caveolae

Caveolae as plasma membrane sensors, protectors and organizers

Localisation and trafficking of Toll-like receptors: an important mode of regulation

The function and biological role of toll-like receptors in infectious diseases: an update

LPS or ethanol triggers clathrin-and rafts/caveolae-dependent endocytosis of TLR4 in cortical astrocytes

Response of human pulmonary epithelial cells to lipopolysaccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways: evidence for an intracellular compartmentalization of TLR4

Intracellular recognition of lipopolysaccharide by toll-like receptor 4 in intestinal epithelial cells

Caveolin -1 regulates NF-kappaB activation and lung inflammatory response to sepsis induced by lipopolysaccharide

Targeting superoxide dismutase to endothelial caveolae profoundly alleviates inflammation caused by endotoxin

Immunotargeting of catalase to ACE or ICAM-1 protects perfused rat lungs against oxidative stress

Cell-selective intracellular delivery of a foreign enzyme to endothelium in vivo using vascular immunotargeting

Targeted detoxification of selected reactive oxygen species in the vascular endothelium

Immunotargeting of the pulmonary endothelium via angiotensin -converting-enzyme in isolated ventilated and perfused human lung

Gene therapy by targeted adenovirus -mediated knockdown of pulmonary endothelial Tph1 attenuates hypoxia-induced pulmonary hypertension

Pre-ischaemic conditioning of the pulmonary endothelium by immunotargeting of catalase via angiotensinconverting-enzyme antibodies

Immunotargeting of catalase to lung endothelium via anti-angiotensin-converting enzyme antibodies attenuates ischemia-reperfusion injury of the lung in vivo

Platelet -endothelial cell adhesion molecule-1-directed endothelial targeting of superoxide dismutase alleviates oxida tive stress caused by either extracellular or intracellular superoxide

Directed targeting of immunoerythrocytes provides local protection of endothelial cells from damage by hydrogen peroxide

Protection of cultured endothelial cells from hydrogen peroxide-induced injury by antibody-conjugated catalase

PECAM -directed immunotargeting of catalase: specific, rapid and transient protection against hydrogen p eroxide

Catalase and superoxide dismutase conjugated with plateletendothelial cell adhesion molecule antibody distinctly alleviate abnormal endothelial permeability ca used by exogenous reactive oxygen species and vascular endothelial growth factor

Endothelial cells internalize monoclonal antibody to angiotensin-converting enzyme

PECAM-directed delivery of catalase to endothelium protects against pulmonary vascular oxidative stress

Antioxidant protection by PECAM-targeted delivery of a novel NADPH-oxidase inhibitor to the endothelium in vitro and in vivo

Polymer nanocarriers protecting ac tive enzyme cargo against proteolysis

Loading PEG-catalase into filamentous and spherical polymer nano carriers

Filamentous Polymer Nanocarriers of Tunable Stiffness that Encapsulate the Therapeutic Enzyme Catalase

Endothelial targeting of semi-permeable polymer nanocarriers for enzyme therapies

Modulation of angiotensin -converting enzyme in cultured human vascular endothelial cells

Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria

Acute and chronic shear stre ss differently regulate endothelial internalization of nanocarriers targeted to platelet -endothelial cell adhesion molecule-1

Biomimetic channel modeling local vascular dynamics of pro-inflammatory endothelial changes

Biomimetic microfluidic platform for the quantification of transient endothelial monolayer permeability and therapeutic transport under mimicked cancerous conditions

Microphysiological Engineering of Self-Assembled and Perfusable Microvascular Beds for the Production of Vascularized Three-Dimensional Human Microtissues

Computational model for nanocarrier binding to endothelium validated using in vivo, in vitro, and atomic force microscopy experiments

Nanocarrier Hydrodynamics and Binding in Targeted Drug Delivery: Challenges in Numerical Modeling and Experimental Validation

Biophysically inspired model for functionalized nanocarrier adhesion to cell surface: roles of protein expression and mechanical factors

Fluorescence Microscopy Imaging Calibration for Quantifying Nanocarrier Binding to Cells During Shear Flow Exposure

Programmable nanoparticle functionalization for in vivo targe ting

Multimodal Nanocarrier Probes Reveal Superior Biodistribution Quantification by Isotopic Analysis over Fluorescence

Mass spectrometry in drug metabolism and pharmacokinetics: Current trends and future perspectives

ADME Considerations and Bioanalytica l Strategies for Pharmacokinetic Assessments of Antibody-Drug Conjugates, Antibodies (Basel)

Spatial heterogeneity of nanomedicine investigated by multiscale imaging of the drug, the nanoparticle and the tumour environment

Scale-up of a physiologically-based pharmacokinetic model to predict the disposition of monoclonal antibodies in monkeys

Physiologically-based pharmacokinetic modeling to predict the clinical pharmacokinetics of monoclonal antibodies

Dual physiologically based pharmacokinetic model of liposomal and nonliposomal amphotericin B disposition

Population pharmacokinetics. A regulatory perspective

Covariate pharmacokinetic model building in oncology and its potential clinical relevance

The reversion -inducing cysteinerich protein with Kazal motifs (RECK) interacts with membrane type 1 matrix metalloprot einase and CD13/aminopeptidase N and modulates their endocytic pathways

Integrin signalling at a glance

Integrins: bidirectional, allosteric signaling machines

Integrin trafficking in cells and tissues

Endothelial permeability and VE-cadherin: a wacky comradeship

TEM1 expression in cancer-associated fibroblasts is correlated with a poor prognosis in patients with gastric cancer

CD44: A Multifunctional Cell Surface Adhesion Receptor Is a Regulator of Progression and Metastasis of Cancer Cells

A requirement for the CD44 cytoplasmic domain for hyaluronan binding, pericellular matrix assembly, and receptor-mediated endocytosis in COS-7 cells

Targeted endothelial delivery of nanosized catalase immunoconjugates protects lung grafts donated after cardiac death

Endothelial targeting of nanocarriers loaded with antioxidant enzymes for protection against vascular oxidative stress and inflammation

Endothelium-targeted delivery of dexamethasone by anti-VCAM-1 SAINT-O-Somes in mouse endotoxemia

Targeted drug delivery via caveolae-associated protein PV1 improves lung fibrosis