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CARDIOVASCULAR
Institute for Environmental Medicine (S.M., T.D., W.Q., J.L., X.C., E.B., V.R.M.), Targeted Therapeutics Program of the Institute for Translational Medicine and Therapeutics (S.M., T.D., V.M.), Department of Pharmacology (S.M., V.M.), School of Medicine, and Department of Bioengineering (W.Q.), School of Engineering and Applied Sciences, University of Pennsylvania, Philadelphia, Pennsylvania
Received November 23, 2005; accepted February 23, 2006.
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Abstract |
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200 nm diameter spheres carrying 200 anti-ICAM molecules). Anti-ICAM/PNCs, but not control IgG/PNCs 1) rapidly (t1/2 5 min) and specifically bound to tumor necrosis factor-activated ECs in a dose-dependent manner (Bmax 350 PNC/cell) at both static and physiological shear stress conditions and 2) bound to ECs and accumulated in the pulmonary vasculature after i.v. injection in mice. Anti-ICAM/PNCs displayed markedly higher EC affinity versus naked anti-ICAM (Kd 80 pM versus 8 nM) in cell culture and, probably because of this factor, higher value (185.3 ± 24.2 versus 50.5 ± 1.5% injected dose/g) and selectivity (lung/blood ratio 81.0 ± 10.9 versus 2.1 ± 0.02, in part due to faster blood clearance) of pulmonary targeting. These results 1) show that reformatting monomolecular anti-ICAM into high-affinity multivalent PNCs boosts their vascular immuno-targeting, which withstands physiological hydrodynamics and 2) support potential anti-ICAM/PNCs utility for medical applications.
; Song et al., 2002; Oh et al., 2004; Muzykantov, 2005). Among other EC surface determinants, intercellular adhesion molecule-1 (CD54 or ICAM-1, a 110-kDa Ig-like transmembrane constitutive endothelial adhesion molecule) is a good candidate target for this goal. ICAM-1 is 1) exposed to blood primarily by ECs and readily accessible to the circulation, 2) up-regulated by pathological factors, and 3) implicated in pathogenesis of many disease conditions including inflammation, ischemia-reperfusion, atherosclerosis, thrombosis, and oxidative stress (Marlin and Springer, 1987; Jevnikar et al., 1990; Muro and Muzykantov, 2005). Because of the latter factor, blocking of leukocyte interaction with ICAM-1 provides beneficial anti-inflammatory effects (Hallahan and Virudachalam, 1997; Hopkins et al., 2004).
ICAM-1 targeting can be achieved by coupling ICAM-1 ligands to drugs or drug carriers [e.g., polymer nanocarriers (PNCs), ranging from <100 nm to <1 µm in diameter]. Such ligands include high-affinity monoclonal ICAM antibodies (anti-ICAM) or their F(ab')2, Fab, and single chain Fv fragments, some of which undergo clinical testing (Takei et al., 1996; Luo et al., 2003; Muro and Muzykantov, 2005). Numerous studies have reported targeting to ECs in cell cultures, perfused organs, and in vivo using anti-ICAM-conjugated isotopes, ultrasound and magnetic resonance imaging contrast probes (Villanueva et al., 1998; Weiner et al., 2001; Weller et al., 2002), therapeutic agents (Murciano et al., 2003; Muro et al., 2005, 2006), and drug carriers including liposomes, PNCs, and micron-size polymer particles (Bloemen et al., 1995; Sakhalkar et al., 2003). Hence, ICAM targeting is a promising area of molecular diagnostics and drug delivery (Muro and Muzykantov, 2005).
These empirical successes justify analysis of factors that control ICAM-1 targeting including anti-ICAM/PNC affinity, valency, size, shape, plasticity, and biological factors such as ICAM-1 expression and organization in the EC plasmalemma, and hydrodynamic conditions. For example, effects of flow and levels of endothelial ICAM-1 expression on binding of micron-size particles coated with ICAM-1 ligands have been studied by several groups to test their targeting potential and obtain numerical data for simplified models of leukocyte adhesion (Sakhalkar et al., 2003; Eniola et al., 2005).
Conceivably, internalizable nanoscale carriers (e.g., PNCs or liposomes) may be more suitable for vascular targeting versus micron-scale carriers (Bronich et al., 2002; Davda and Labhasetwar, 2002), yet parameters of anti-ICAM/PNC binding to ECs have not been characterized. Changing carrier size and anti-ICAM affinity and density on the carrier surface may profoundly alter targeting. For example, modulation of valency and size of anti-ICAM-based targeting systems alters their internalization by ECs: monomeric anti-ICAM and micron-size anti-ICAM conjugates bind to ECs without internalization, whereas 100- to 500-nm diameter anti-ICAM/PNCs are internalized by ECs via a unique pathway, cell adhesion molecule-mediated endocytosis (Muro et al., 2003). Multivalent delivery systems targeting ICAM-1 provide intracellular delivery of antioxidant and lysosomal enzymes, optimal for their therapeutic effects (Muro et al., 2005, 2006). These experimental results imply potential medical utility of anti-ICAM/PNCs.
In this study, we used isotope tracing and fluorescence microscopy to determine and visualize EC targeting in vitro, ex vivo, and in vivo of multivalent spherical anti-ICAM/PNCs carrying 200 anti-ICAM molecules per carrier (final diameter 200 nm) based on prototype polystyrene or biodegradable polymers. For the latter iteration, we produced anti-ICAM/PNCs using biocompatible poly(lactic-coglycolic) acid (PLGA), a Food and Drug Administration-approved candidate material (Davda and Labhasetwar, 2002; Dziubla and Muzykantov, 2006).
We determined amplitude and kinetics of anti-ICAM/PNC binding to ECs under static and flow conditions in cell cultures and their biodistribution and pulmonary uptake, reflecting endothelial targeting after intravenous injection in animals. Of particular interest, comparison of anti-ICAM/PNCs versus naked maternal anti-ICAM showed that reformatting monomolecular anti-ICAM into nanoscale multivalent PNCs iteration greatly enhances affinity and targeting to ECs in vitro and in vivo.
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Materials and Methods |
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), YN1 (Jevnikar et al., 1990), and 1A29 (Christensen et al., 1993), respectively. Secondary antibodies were from Molecular Probes (Eugene, OR). Green fluorescent polystyrene latex beads, 100 nm in diameter, were from Polysciences (Warrington, PA). Poly(lactic-coglycolic) acid (50:50 monomer ratio, 11.2 kDa mol. wt.) was obtained from Alkermes (Cambridge, MA). Na125I was obtained from Perkin Elmer (Wellesley, MA), and Iodogen was purchased from Pierce Biotechnology (Rockford, IL). Unless otherwise stated, all other reagents were from Sigma Chemical (St. Louis, MO).
Synthesis of PLGA Nanocarriers. A solvent extraction emulsification procedure was used to form PLGA nanoparticles. In brief, 25 mg of polymer was dissolved in dry acetone for a final concentration of 10 mg/ml (1 wt% of 125I-labeled polytyrosine (10–40 kDa mol. wt.) was added for in vivo tracing studies). This solution was then slowly pipetted into 100 ml of PBS solution containing 3 wt% Pluronic F68 [polyethylene glycol (PEG)-polypropylene glycol-PEG triblock copolymer]. The acetone was evaporated off at room temperature overnight. After this time, particles were collected by centrifuging at 20,000g for 25 min and were then washed twice more in 40 ml of pure PBS solution. Purified particles were stored in PBS. Final particle concentration was determined using an enzymatic assay for lactic acid as previously published, and residual surfactant content was evaluated by a spectrophotometer PEG assay (Dziubla et al., 2005). Size and 
potential of uncoated PLGA nanoparticles were determined on a Brookhaven 90Plus potential and dynamic light scattering apparatus.
Preparation of Anti-ICAM Nanocarriers. Anti-ICAM nanocarriers (anti-ICAM/PNCs) and control IgG/PNCs were prepared by coating green fluorescent polystyrene beads with either anti-human, anti-murine, or anti-rat ICAM-1, or nonspecific IgG, respectively (Muro et al., 2003). Radiolabeled PNCs contained a mix of anti-ICAM and 125I-IgG at 95:5 M ratio (Muro et al., 2005). After separation of the free anti-ICAM or IgG by centrifugation, the amount of 125I-tracer coated onto the nanocarriers was determined in a gamma counter. A saturating antibody density on the PNC surface (220 ± 10 molecules/PNC, 7000 antibody molecules/µm2) was used in the study. The same procedure was used to prepare coated PLGA nanocarriers. The diameter of the final anti-ICAM/PNCs and IgG/PNCs ranged from 150 to 300 nm, as determined by dynamic light scattering.
Cell Culture. Pooled human umbilical vein endothelial cells (HUVECs) (Clonetics, San Diego, CA) were cultured at 37°C, 5% CO2, and 95% relative humidity in supplemented M199 medium (Gibco BRL, Grand Island, NY) and used at passage 4 to 5. For experiments under static conditions, cells were seeded onto 12 mm2 gelatin-coated coverslips in 24-well plates. For experiments under flow conditions, cells seeded in gelatin-coated 22 x 40 mm coverslips were grown to confluence, then placed in a parallel plate flow chamber (RC-30HV; Warner Instruments Inc., Hamden, CT) and perfused overnight at 9 dynes/cm2 using a peristaltic pump (Pharmacia Biotech AB, Appsala, Sweden). Cells were always treated overnight with TNF-
before experiments.
Binding of Anti-ICAM and Anti-ICAM Nanocarriers to Endothelial Cells. Varying concentrations of 125I-labeled free anti-ICAM or green fluorescent multivalent anti-ICAM/PNCs were incubated with activated HUVECs for 1 h at 4°C. In the case of free antibody, the cells were washed and lysed with 1% Triton X-100 in 1 M NaOH, and the amount of 125I-anti-ICAM was determined in a gamma counter. In the case of multivalent anti-ICAM/PNCs, the cells were washed, fixed in cold with 2% paraformaldehyde, and analyzed by phase-contrast and fluorescence microscopy (Eclipse TE2000-U; Nikon, Melville, NY) using a 40x/NA1.4 PlanApo objective (Nikon). PLGA anti-ICAM/PNCs were visualized by staining fixed cells with fluorescein isothiocyanate-labeled goat anti-mouse IgG. For experiments under flow conditions, green fluorescent anti-ICAM/PNCs were perfused at 9 dynes/cm2 shear stress at 37°C in a flow chamber with TNF-activated HUVEC adapted to these flow conditions for 24 h. The amount of nanocarriers bound per cell was determined from micrographs obtained with an Orca-1 charge-coupled device camera (Hamamatsu, Bridgewater, NJ) and analyzed using ImagePro 3.0 software (Media Cybernetics, Silver Spring, MD).
Targeting to Endothelium in Isolated Vasculature. The mesentery was isolated from anesthetized 200-g male Sprague-Dawley rats and perfused at 1.5 ml/min using a peristaltic pump with Krebs-Ringer buffer containing green fluorescent anti-ICAM/PNCs at 37°C. Fluorescent micrographs were collected at varying periods of time during 30 min to detect accumulation of anti-ICAM/PNCs in the mesenteric vessels.
Targeting to Vascular Endothelium in Mice. Anesthetized C57BL/6 male mice were injected intravenously with 125I-labeled anti-ICAM or control IgG (0.12 mg/kg b.wt.), polystyrene anti-ICAM/PNCs or IgG/PNCs (2.8 mg of IgG and 5.2 x 1013 PNCs/kg b.wt.), PLGA anti-ICAM/PNCs, or untargeted PLGA nanocarriers (10 mg of polymer/kg b.wt.). Blood samples were collected from the retro-orbital plexus at 1, 15, and 30 min postinjection, and organs (heart, kidneys, liver, spleen, and lungs) were collected at 30 min postinjection. The radioactivity and weight of the samples were determined to calculate nanocarrier targeting parameters, including percentage of injected dose (ID) per gram, localization ratio (LR) (organ/blood ratio of percentage of ID per gram), and immunospecificity index (ratio of LR targeted versus nontargeted formulation in an organ) (Murciano et al., 2003). In parallel experiments, accumulation of green fluorescent anti-ICAM/PNCs or IgG/PNCs in mice organs was visualized by fluorescence microscopy from 10-µm-thick frozen sections. These studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health.
Statistics. Unless otherwise stated, the data were calculated as means ± S.D., where statistical significance was determined by Student's t test.
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Results |
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First, we determined PNC coating by radiolabeled anti-ICAM or control IgG and found that a saturating concentration (800 antibody molecules added/PNC) provides noncovalent coupling of 250 IgG molecules/200 nm of PNCs (Fig. 1A). Hence, surface density of anti-ICAM on PNCs prepared at a saturating anti-ICAM concentration was 7000 molecules/µm2.
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-activated HUVECs (Fig. 1B). Anti-ICAM/PNCs bound to the apical surface of cells with relatively uniform distribution. Computer analysis of images showed binding of 174 ± 6 anti-ICAM/PNCs per TNF-activated EC, which exceeded IgG/PNC binding by 90 times (2 ± 2 IgG/PNCs per cell). Targeting of anti-ICAM/PNCs to Endothelial Cells under Flow. The EC phenotype degenerates in static culture. On the other hand, PNC binding to and detachment from ECs in vivo may be also affected by hydrodynamic conditions. To test the effects of these factors, we perfused anti-ICAM/PNCs in a flow chamber containing a monolayer of HUVECs adapted for 24 h to a physiological level of shear stress, 9 dynes/cm2. Anti-ICAM/PNC binding to flow-adapted cells (note cell alignment to flow direction in Fig. 2A) under flow was indistinguishable from that to static cells (138.7 ± 6.5 versus 121.6 ± 10.1 after a 60-min incubation). The kinetics of anti-ICAM/PNC binding to HUVECs was very rapid (t1/2 = 5 min) under both static and flow conditions (Fig. 2B).
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To test binding to ECs in intact vasculature under physiological flow conditions, we perfused anti-ICAM/PNCs ex vivo through the blood vessels in isolated rat mesentery (Fig. 3). Bright punctate fluorescence associated with the luminal surface of either large (Fig. 3, A–C) and small (Fig. 3D) vessels revealed anti-ICAM/PNC binding to ECs in this model. In agreement with cell culture data, endothelial targeting was rapid and clearly detectable as early as 5 min after the start of perfusion (Fig. 3B).
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30% of total endothelial surface, represent the first major vascular network after i.v. injection, and receive 100% of venous cardiac output of blood flowing via a high-capacity, low-resistance pulmonary vasculature at a relatively slow rate, which favors binding to endothelium (Muzykantov, 2005). ICAM antibodies and anti-ICAM conjugates bind to ECs and accumulate in lungs after i.v. injection in naive animals, due to a high constitutive level of ICAM-1 expression by ECs in vivo (Murciano et al., 2003; Muro et al., 2005). Thus, pulmonary uptake serves as an indicator of endothelial targeting of anti-ICAM/PNCs after systemic intravascular administration.
Both radiolabeled anti-ICAM/PNCs and control IgG/PNCs were rapidly eliminated from the circulation (inset in Fig. 4A). The total amount of PNC-coupled antibody in circulation was 4.7 ± 0.8 versus 22.3 ± 1.2 µg at 1 min after injection and 3.4 ± 0.7 versus 2.6 ± 0.6 µg at 15 min after injection, for anti-ICAM/PNCs versus control IgG/PNCs, respectively. Blood clearance reflects uptake of anti-ICAM/PNC and IgG/PNCs in the reticuloendothelial system in liver and spleen (Fig. 4, A and B), at least in part due to Fc fragment-mediated phagocytosis.
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Visualization of PNCs in organs by fluorescence microscopy confirmed isotope tracing results. Both anti-ICAM/PNCs and IgG/PNCs showed high levels of uptake and similar tissue distribution in the liver (Fig. 4B) and spleen (not shown). In contrast, microscopy showed highly specific pulmonary targeting anti-ICAM/PNCs and revealed their relatively uniform distribution in the alveolar capillaries (Fig. 4C) and their presence in the luminal surface of larger vessels (not shown). Specific targeting of anti-ICAM/PNCs was also seen in the kidneys, albeit to a significant lesser extent (Fig. 4D).
High Affinity and Targeting Effectiveness of Multimeric Anti-ICAM/PNCs. We reported that reformatting a monomolecular anti-ICAM into multimeric conjugates or anti-ICAM/PNCs alters their subcellular targeting: ECs did not internalize anti-ICAM, yet internalized anti-ICAM conjugates and anti-ICAM/PNCs, due to clustering of ICAM-1 in the EC plasmalemma (Murciano et al., 2003; Muro et al., 2003). In this study we tested the role of the multivalent nature of anti-ICAM/PNCs in binding to ECs, the first step of targeting.
Both anti-ICAM and anti-ICAM/PNCs displayed dose-dependent binding to TNF-activated HUVECs (Fig. 5). Scatchard analysis revealed 1.6 x 106 binding sites for anti-ICAMs [which agrees well with previous reports by other groups (Remy et al., 1999)] and 300 binding sites for anti-ICAM/PNCs per cell (insets in Fig. 5). However, the affinity of anti-ICAM/PNC binding to cells calculated as an effective Kd was practically two orders of magnitude higher than that of anti-ICAM (77 pM versus 8.5 nM), reflecting the multivalent character of anti-ICAM/PNC binding involving many copies of ICAM-1.
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40 fold higher versus that of anti-ICAMs (Fig. 6B). This was probably due to higher effectiveness both of targeting and blood clearance of anti-ICAM/PNCs. The pulmonary immunospecificity index (ISI), the ratio of levels of immune and nonimmune counterparts in an organ corrected for their blood levels, of anti-ICAM/PNC doubled this parameter of maternal anti-ICAM.
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; Dziubla et al., 2005).
The final stock of PLGA PNCs contained 9.0 ± 0.8 mg/ml PLGA with an average PNC diameter of 120 nm and surface charge of –19.5 ± 3.3 mV, similar to prototype polystyrene PNCs (100 nm and 20 mV) used elsewhere in this study. This PLGA PNC stock preparation containing little residual surfactant (36.6 ± 8.6 µg/ml, which translates into <0.4% of the total particle mass) was used to produce biocompatible PLGA-based anti-ICAM/PNCs and IgG/PNCs.
PLGA-based anti-ICAM/PNCs, but not IgG/PNCs, bound to TNF-activated HUVECs (Fig. 7A), reproducing and validating data obtained with prototype polystyrene carriers (Fig. 1B). Furthermore, PLGA-based anti-ICAM/PNCs, but not the nontargeted counterpart, accumulated in the lungs after intravenous injection in mice, with amplitude, selectivity, and specificity parameters similar to those of polystyrene anti-ICAM/PNCs (Fig. 7, B–D).
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Discussion |
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; Song et al., 2002; Oh et al., 2004; Kelly et al., 2005; Muzykantov, 2005; Muro et al., 2006). Caveolar antigens, ecto-peptidases, receptors for hormones, transferrin, and albumin, and markers of pathological endothelia provide targets for drug delivery to, into, and across ECs (Danilov et al., 2001; McIntosh et al., 2002; Song et al., 2002; Muzykantov, 2005). Adhesion molecules represent an attractive class of targets including inducible selectins and vascular cellular adhesion molecule-1, useful for specific recognition of pathological endothelia (Lindner et al., 2001; Everts et al., 2002; Kelly, 2005), and constitutively expressed adhesion molecules ICAM-1 and platelet cellular adhesion molecule-1, useful for both prophylactic and therapeutic endothelial drug delivery (Muro and Muzykantov, 2005).
In particular, vascular immunotargeting of ICAM-1 provides delivery of imaging and therapeutic agents to ECs (Danilov et al., 2001; Murciano et al., 2003; Sakhalkar et al., 2003; Muzykantov, 2005; Muro et al., 2006). Anti-ICAM-targeted ultrasound contrasts bind to activated ECs, facilitating imaging of acute cardiac transplant rejection (Villanueva et al., 1998). ICAM-targeted isotopes detect acute inflammation sites (Weiner et al., 2001). Anti-ICAM immunoliposomes bind to ECs in vitro and in animal studies (Bloemen et al., 1995). Anti-ICAM delivers conjugated therapeutic and reporter enzymes to ECs in vitro and in vivo (Murciano et al., 2003).
ECs bind and internalize multimeric anti-ICAM conjugates and polymer nanocarriers (anti-ICAM/PNCs, 100–500 nm) providing intracellular delivery of antioxidant enzymes for containment of vascular oxidative stress (Muro et al., 2005). Lysosomal trafficking of anti-ICAM/PNCs offers a new effective pathway for delivery of enzyme replacement therapies for lysosomal storage disorders, obviating defunct native delivery pathways (Muro et al., 2006).
Anti-ICAM-blocking leukocyte adhesion showed anti-inflammatory and protective effects (Kavanaugh et al., 1997) in animal models of stroke, cardiac ischemia-reperfusion, lung injury, and organ transplantation and in clinical trails (for a review see Muro and Muzykantov, 2005). Thus, ICAM-1 targeting may provide secondary anti-inflammatory benefits.
Yet, despite these empirical successes, our understanding of factors regulating targeting carriers to ICAM-1 is deficient. Anti-ICAM affinity and surface density on carriers have not been determined in many studies (Table 1), which in most cases utilized micron-scale, not nano-scale, carriers (Weller et al., 2002; Sakhalkar et al., 2003; Eniola et al., 2005). Studies with anti-ICAM/liposomes, a nano-scale drug delivery system, confirm endothelial targeting, but provide limited quantitative analyses (Bloemen et al., 1995). However, targeting of liposomes versus PNCs may differ. Lateral diffusion of antibody molecules in liposome membrane can facilitate multivalent binding. Liposomes can also flatten on the cell surface, minimizing the detaching force and anchoring additional binding sites. These advantages are unlikely for PNCs.
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However, there are no data on ICAM-targeted PNC binding characteristics. Furthermore, no studies quantitatively analyzed PNC targeting in vitro and in vivo to any endothelial antigen. The goal of this study was to define and test factors that control binding of ICAM-targeted PNCs to ECs using tracing of anti-ICAM/PNCs in static and flow-perfused EC cultures (Figs. 1 and 2) and vascular perfusion ex vivo (Fig. 3) and in vivo (Figs. 4, 5, 6, 7).
Because of the size difference with free anti-ICAMs, anti-ICAM/PNCs diffuse slower, which lowers the potential number of cell collisions necessary for binding to ECs. Yet, this feature may be offset by the greater momentum of PNCs, which may allow for more frequent wall collisions in vasculature. It is likely that the detaching force applied toward anti-ICAM/PNCs anchored on the EC is higher versus that of anti-ICAMs. This parameter is likely to depend on the shear stress in the binding system, which varies from a relatively low 1 dyne in capillaries to 100 dynes in large arteries.
However, the multivalent character of the anti-ICAM/PNC interaction with ECs facilitates anchoring with a very high affinity (70 pM) and binding capacity (Bmax 300 PNCs/cell) for 200 nm diameter PNCs possessing 250 antibodies/PNC (7000 antibodies/µm2). As expected, the maximum binding sites available to PNC-coupled anti-ICAMs was lower versus that for naked anti-ICAMs (7.5 x 104 versus 1.6 x 106 per cell). Free antibody can potentially interact with every ICAM-1 molecule in the plasma membrane. Yet, only those antibodies properly oriented onto the surface of PNCs facing the EC plasma membrane can engage ICAM-1. PNCs bulk bound to the EC surface may impair (due to its size) the access of additional PNCs to nonengaged ICAM-1 molecules. Access of PNC to ICAM-1 located in certain plasma membrane furrows may also be impaired due to steric hindrance. However, the effective affinity of anti-ICAM/PNCs exceeded that of naked anti-ICAM by 100 times, probably due to the strong multivalent binding to ECs.
The time to reach equilibrium and equilibrium binding efficiency (Fig. 2) was identical for stagnant versus flow-adapted ECs under shear stress (9 dynes/cm2, 130 l/s shear rate). Because this shear rate would be expected to provide a significant detachment force, the lack of appreciable negative effect of flow further emphasizes the importance of the high affinity of the multivalent PNCs in targeting. In fact, anti-ICAM/PNCs greatly outperformed pulmonary targeting of monomolecular anti-ICAM in mice. Therefore, reformatting of anti-ICAMs into nanoscale multivalent PNCs enhances targeting. This outcome correlates with a recent report that multivalent particles directed to cell adhesion molecules block leukocyte adhesion more effectively then their monomolecular counterparts (Ali et al., 2004).
Table 1 shows that the value of anti-ICAM/PNC binding to ECs is more than 100 time higher than that of micron-scale anti-ICAM/carriers (Kiani et al., 2002; Weller et al., 2002; Sakhalkar et al., 2003; Eniola et al., 2005). It is likely that size governs the effectiveness of EC targeting. Yet, there are too many variables between these studies (antibody affinity and surface density, carrier concentration, and shear rate). To fully elucidate the effects of size upon binding, one must compare carriers of variable sizes coated by antibodies with known affinity at known density. This aim is beyond the scope of this article and represents the subject of ongoing work.
Most of our experiments used prototype polystyrene PNCs, which can be replaced by PNCs made of biodegradable, biocompatible materials (Fig. 7). To exclude targeting variability due to different mode of anti-ICAM coating (which can, for example, alter steric freedom or orientation of carrier-coupled anti-ICAM), we used the same antibody-coupling technique as for polystyrene carriers, hydrophobic absorption of anti-ICAM. PNC materials (PLGA and Pluronic F68 surfactant) and synthesis were chosen to obtain PNCs with surface hydrophobicity similar to that of polystyrene PNCs. Other groups have demonstrated that surfactants [in these cases, poly(vinyl alcohol] can be irreversibly adsorbed to PNC during formulation (Scholes et al., 1999). In our study, F68 was shown to account for a very low residual mass (<0.4 wt%) after three washings. Furthermore, particle stability was maintained by a slight negative surface charge, which, along with the hydrophobic surface, permitted antibody coating via surface adsorption. As a result, targeting of PLGA PNCs in vitro and in vivo was comparable with that for polystyrene PNCs, supporting the utility of the targeting parameters of prototype PNCs to PLGA PNCs, more compatible with medical use.
Translation of multivalent ICAM-targeted PNCs into the medical domain will require rigorous testing of the safety of this approach. ICAM-1 blocking by antibodies and their fragments is well tolerated by laboratory animals and has been utilized to attenuate inflammation and infections (Hallahan and Virudachalam, 1997; Hopkins et al., 2004). However, EC response(s) to ICAM-1 clustering induced by multivalent anti-ICAM/PNCs may differ from those to the maternal antibody.
ICAM-1 engagement may cause cytosolic Ca2+ elevation and cytoskeletal remodeling (Etienne-Manneville et al., 2000). Thus, in theory, cytoskeletal reorganization in response to ICAM-1 engagement may modulate vascular permeability (Stevens et al., 2000). ICAM-1 clustering in ECs may also induce reactive oxygen species production, cytokine generation, and expression of cell adhesion molecules (vascular cellular adhesion molecule-1 and ICAM-1 itself) (Hubbard and Rothlein, 2000). Although these effects may vary among ECs from different vascular beads (Hubbard and Rothlein, 2000; Stevens et al., 2000; Wang et al., 2002), it is believed that these responses to ICAM-1 engagement serve as a physiological, transient signal for EC activation (Hubbard and Rothlein, 2000). Whether ECs respond to ICAM-1 clustering by nanocarriers in a similar manner remains to be determined.
However, anti-ICAM/PNCs did not induce systemic release of pro-inflammatory factors after i.v. injection in mice (Muro et al., 2005) and neither did they affect endothelial endocytosis and intracellular trafficking of fluid phase markers or HUVEC monolayer morphology or viability (Muro et al., 2005). Furthermore, cytoskeletal reorganizations in cultured ECs induced by ICAM-1 clustering by anti-ICAM/PNCs are transient and do not cause changes in vascular permeability in vitro and in vivo (S. Muro and V. Muzykantov, unpublished observations). These data corroborate with a recent report that uptake of PLGA nanocarriers did not induce endothelial damage (Davda and Labhasetwar, 2002) and imply that the side effects of multivalent anti-ICAM/PNCs may be benign, transient, and well tolerated.
In summary, reformatting monomolecular anti-ICAM into multivalent anti-ICAM/PNCs, markedly increasing endothelial affinity, highlights the potential utility of this platform for diagnostic and therapeutic applications, for example, for the delivery of enzyme replacement or antioxidant therapies into endothelial cells (Dziubla et al., 2005; Muro et al., 2006; Dziubla and Muzykantov, 2006).
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: EC, endothelial cell; ICAM, intercellular adhesion molecule(s); PNC, polymer nanocarrier; PLGA, poly(lactic-coglycolic) acid; PBS, phosphate-buffered saline; PEG, polyethylene glycol; HUVEC, human umbilical vein endothelial cell; TNF, tumor necrosis factor; ID, injected dose; LR, localization ratio; ISI, immunospecificity index.
Address correspondence to: Dr. Vladimir R. Muzykantov, Institute for Environmental Medicine, 1 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-6068. E-mail: muzykant{at}mail.med.upenn.edu
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References |
|---|
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Ali M, Hicks AE, Hellewell PG, Thoma G, and Norman KE (2004) Polymers bearing sLex-mimetics are superior inhibitors of E-selectin-dependent leukocyte rolling in vivo. FASEB J 18: 152–154.
Bloemen PG, Henricks PA, van Bloois L, van den Tweel MC, Bloem AC, Nijkamp FP, Crommelin DJ, and Storm G (1995) Adhesion molecules: a new target for immunoliposome-mediated drug delivery. FEBS Lett 357: 140–144.[CrossRef][Medline]
Bronich TK, Ouyang M, Kabanov VA, Eisenberg A, Szoka FC Jr, and Kabanov AV (2002) Synthesis of vesicles on polymer template. J Am Chem Soc 124: 11872–11873.[CrossRef][Medline]
Christensen PJ, Kim S, Simon RH, Toews GB, and Paine R 3rd (1993) Differentiation-related expression of ICAM-1 by rat alveolar epithelial cells. Am J Respir Cell Mol Biol 8: 9–15.
Danilov SM, Gavrilyuk VD, Franke FE, Pauls K, Harshaw DW, McDonald TD, Miletich DJ, and Muzykantov VR (2001) Lung uptake of antibodies to endothelial antigens: key determinants of vascular immunotargeting. Am J Physiol 280: L1335–L1347.
Davda J and Labhasetwar V (2002) Characterization of nanoparticle uptake by endothelial cells. Int J Pharm (Amst) 233: 51–59.
Dziubla TD, Karim A, and Muzykantov VR (2005) Polymer nanocarriers protecting active enzyme cargo against proteolysis. J Controlled Release 102: 427–439.[CrossRef][Medline]
Dziubla TD and Muzykantov V (2006) Synthetic carriers for the delivery of protein therapeutics. Biotechnol Genet Eng Rev 22: 267–299.[Medline]
Eniola AO, Krasik EF, Smith LA, Song G, and Hammer DA (2005) I-domain of lymphocyte function-associated antigen-1 mediates rolling of polystyrene particles on ICAM-1 under flow. Biophys J 89: 3577–3588.[CrossRef][Medline]
Etienne-Manneville S, Manneville JB, Adamson P, Wilbourn B, Greenwood J, and Couraud PO (2000) ICAM-1-coupled cytoskeletal rearrangements and transendothelial lymphocyte migration involve intracellular calcium signaling in brain endothelial cell lines. J Immunol 165: 3375–3383.
Everts M, Kok RJ, Asgeirsdottir SA, Melgert BN, Moolenaar TJ, Koning GA, van Luyn MJ, Meijer DK, and Molema G (2002) Selective intracellular delivery of dexamethasone into activated endothelial cells using an E-selectin-directed immunoconjugate. J Immunol 168: 883–889.
Hallahan DE and Virudachalam S (1997) Intercellular adhesion molecule 1 knockout abrogates radiation induced pulmonary inflammation. Proc Natl Acad Sci USA 94: 6432–6437.
Hopkins AM, Baird AW, and Nusrat A (2004) ICAM-1: targeted docking for exogenous as well as endogenous ligands. Adv Drug Deliv Rev 56: 763–778.[CrossRef][Medline]
Hubbard AK and Rothlein R (2000) Intercellular adhesion molecule-1 (ICAM-1) expression and cell signaling cascades. Free Radic Biol Med 28: 1379–1386.[CrossRef][Medline]
Jevnikar AM, Wuthrich RP, Takei F, Xu HW, Brennan DC, Glimcher LH, and Rubin-Kelley VE (1990) Differing regulation and function of ICAM-1 and class II antigens on renal tubular cells. Kidney Int 38: 417–425.[Medline]
Kavanaugh AF, Schulze-Koops H, Davis LS, and Lipsky PE (1997) Repeat treatment of rheumatoid arthritis patients with a murine anti-intercellular adhesion molecule 1 monoclonal antibody. Arthritis Rheum 40: 849–853.[Medline]
Kelly K, Allport JR, Tsourkas A, Shinde-Patil VR, Josephson L, and Weissleder R (2005) Detection of vascular adhesion molecule-1 expression using a novel multimodal nanoparticle. Circ Res 96: 327–336.
Kiani MF, Yuan H, Chen X, Smith L, Gaber MW, and Goetz DJ (2002) Targeting microparticles to select tissue via radiation-induced upregulation of endothelial cell adhesion molecules. Pharm Res (NY) 19: 1317–1322.
Lindner JR, Song J, Christiansen J, Klibanov AL, Xu F, and Ley K (2001) Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin. Circulation 104: 2107–2112.
Luo GX, Kohlstaedt LA, Charles CH, Gorfain E, Morantte I, Williams JH, and Fang F (2003) Humanization of an anti-ICAM-1 antibody with over 50-fold affinity and functional improvement. J Immunol Methods 275: 31–40.[CrossRef][Medline]
Marlin SD and Springer TA (1987) Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1). Cell 51: 813–819.[CrossRef][Medline]
McIntosh DP, Tan XY, Oh P, and Schnitzer JE (2002) Targeting endothelium and its dynamic caveolae for tissue-specific transcytosis in vivo: a pathway to overcome cell barriers to drug and gene delivery. Proc Natl Acad Sci USA 99: 1996–2001.
Molema G (2002) Tumor vasculature directed drug targeting: applying new technologies and knowledge to the development of clinically relevant therapies. Pharm Res (NY) 19: 1251–1258.
Murciano JC, Muro S, Koniaris L, Christofidou-Solomidou M, Harshaw DW, Albelda SM, Granger DN, Cines DB, and Muzykantov VR (2003) ICAM-directed vascular immunotargeting of antithrombotic agents to the endothelial luminal surface. Blood 101: 3977–3984.
Muro S, Gajewski C, Koval M, and Muzykantov VR (2005) ICAM-1 recycling in endothelial cells: a novel pathway for sustained intracellular delivery and prolonged effects of drugs. Blood 105: 650–658.
Muro S and Muzykantov VR (2005) Targeting of antioxidant and anti-thrombotic drugs to endothelial cell adhesion molecules. Curr Pharm Des 11: 2383–2401.[CrossRef][Medline]
Muro S, Schuchman EH, and Muzykantov VR (2006) Lysosomal enzyme delivery by ICAM-1-targeted nanocarriers bypassing glycosylation- and clathrin-dependent endocytosis. Mol Ther 13: 135–141.[CrossRef][Medline]
Muro S, Wiewrodt R, Thomas A, Koniaris L, Albelda SM, Muzykantov VR, and Koval M (2003) A novel endocytic pathway induced by clustering endothelial ICAM-1 or PECAM-1. J Cell Sci 116: 1599–1609.
Muzykantov V (2005) Targeting drugs to pulmonary endothelium. Expert Opin Drug Deliv 2: 909–926.[CrossRef][Medline]
Oh P, Li Y, Yu J, Durr E, Krasinska KM, Carver LA, Testa JE, and Schnitzer JE (2004) Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy. Nature (Lond) 429: 629–635.[CrossRef][Medline]
Remy M, Valli N, Brethes D, Labrugere C, Porte-Durrieu MC, Dobrova NB, Novikova SP, Gorodkov AJ, and Bordenave L (1999) In vitro and in situ intercellular adhesion molecule-1 (ICAM-1) expression by endothelial cells lining a polyester fabric. Biomaterials 20: 241–251.[CrossRef][Medline]
Sakhalkar HS, Dalal MK, Salem AK, Ansari R, Fu J, Kiani MF, Kurjiaka DT, Hanes J, Shakesheff KM, and Goetz DJ (2003) Leukocyte-inspired biodegradable particles that selectively and avidly adhere to inflamed endothelium in vitro and in vivo. Proc Natl Acad Sci USA 100: 15895–15900.
Scholes PD, Coombes AG, Illum L, Davis SS, Watts JF, Ustariz C, Vert M, and Davies MC (1999) Detection and determination of surface levels of poloxamer and PVA surfactant on biodegradable nanospheres using SSIMS and XPS. J. Controlled Release 59: 261–278.[CrossRef][Medline]
Song BW, Vinters HV, Wu D, and Pardridge WM (2002) Enhanced neuroprotective effects of basic fibroblast growth factor in regional brain ischemia after conjugation to a blood-brain barrier delivery vector. J Pharmacol Exp Ther 301: 605–610.
Stevens T, Garcia JG, Shasby DM, Bhattacharya J, and Malik AB (2000) Mechanisms regulating endothelial cell barrier function. Am J Physiol 279: L419–L422.
Takei Y, Nishimura Y, Kawano S, Nagai H, Ohmae A, Fusamoto H, and Kamada T (1996) Expression of ICAM-1 is involved in the mechanism of liver injury during liver transplantation: therapeutic usefulness of the F(ab')2 fragment of an anti-ICAM-1 monoclonal antibody. Transplant Proc 28: 1103–1105.[Medline]
Villanueva FS, Jankowski RJ, Klibanov S, Pina ML, Alber SM, Watkins SC, Brandenburger GH, and Wagner WR (1998) Microbubbles targeted to intercellular adhesion molecule-1 bind to activated coronary artery endothelial cells. Circulation 98: 1–5.
Wang Q, Pfeiffer GR 2nd, Stevens T, and Doerschuk CM (2002) Lung microvascular and arterial endothelial cells differ in their responses to intercellular adhesion molecule-1 ligation. Am J Respir Crit Care Med 166: 872–877.
Weiner RE, Sasso DE, Gionfriddo MA, Thrall RS, Syrbu S, Smilowitz HM, and Vento J (2001) Early detection of oleic acid-induced lung injury in rats using (111)In-labeled anti-rat intercellular adhesion molecule-1. J Nucl Med 42: 1109–1115.
Weller GE, Villanueva FS, Klibanov AL, and Wagner WR (2002) Modulating targeted adhesion of an ultrasound contrast agent to dysfunctional endothelium. Ann Biomed Eng 30: 1012–1019.[CrossRef][Medline]
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, compares monomeric versus PNC-coupled anti-ICAM. *, compares PNC-coupled IgG versus anti-ICAM. #, 