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

Silvia Muro,1 Xiumin Cui,1 Christine Gajewski,1 Juan-Carlos Murciano,1,2 Vladimir R. Muzykantov,1,2 and Michael Koval1,3

1Institute for Environmental Medicine and Departments of 2Pharmacology and 3Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Submitted 14 March 2003 ; accepted in final form 14 July 2003


    ABSTRACT
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Nanotechnologies promise new means for drug delivery. ICAM-1 is a good target for vascular immunotargeting of nanoparticles to the perturbed endothelium, although endothelial cells do not internalize monomeric anti-ICAM-1 antibodies. However, coupling ICAM-1 antibodies to nanoparticles creates multivalent ligands that enter cells via an amiloride-sensitive endocytic pathway that does not require clathrin or caveolin. Fluorescence microscopy revealed that internalized anti-ICAM nanoparticles are retained in a stable form in early endosomes for an unusually long time (1-2 h) and subsequently were degraded following slow transport to lysosomes. Inhibition of lysosome acidification by chloroquine delayed degradation without affecting anti-ICAM trafficking. Also, the microtubule disrupting agent nocodazole delayed degradation by inhibiting anti-ICAM nanoparticle trafficking to lysosomes. Addition of catalase to create anti-ICAM nanoparticles with antioxidant activity did not affect the mechanisms of nanoparticle uptake or trafficking. Intracellular anti-ICAM/catalase nanoparticles were active, because endothelial cells were resistant to H2O2-induced oxidative injury for 1-2 h after nanoparticle uptake. Chloroquine and nocodazole increased the duration of antioxidant protection by decreasing the extent of anti-ICAM/catalase degradation. Therefore, the unique trafficking pathway followed by internalized anti-ICAM nanoparticles seems well suited for targeted delivery of therapeutic enzymes to endothelial cells and may provide a basis for treatment of acute vascular oxidative stress.

drug delivery; endocytosis; microtubules; lysosomes


NANOTECHNOLOGIES offer the opportunity for the design of novel carriers for more effective, specific, and safe drug delivery (4, 24). For example, impressive advances have been achieved in synthesis of nanoparticles with controlled rates of drug release (8, 30, 41, 44). Specific affinity for targets and favorable subcellular addressing are some parameters that are critically important for optimizing the therapeutic potential of nanoparticles as drug delivery vehicles (2, 4, 20, 39).

The vascular endothelium is a prime target for drug delivery. Endothelial cells represent a barrier for drug delivery from the bloodstream to target tissues (42). Conversely, the endothelium is involved in diverse pathological processes including inflammation, oxidative stress, and thrombosis and, therefore, itself represents an important drug delivery target (17, 48, 50). One particularly good target for drug delivery to perturbed endothelial cells is intercellular adhesion molecule-1 (ICAM-1) (3, 51, 52), a plasma membrane protein that is upregulated and functionally involved in inflammation and thrombosis (11, 14, 23, 38). However, ICAM-1 and another Ig superfamily cell adhesion molecule, platelet endothelial cell adhesion molecule-1 (PECAM-1), are not readily internalized by endothelial cells (33, 37, 53).

Nevertheless, despite the inability of these cell adhesion molecules (CAM) to act as receptors to mediate endocytosis of monomeric antibodies, endothelial cells internalize multimeric anti-PECAM nanoparticles and anti-ICAM nanoparticles <300 nm in diameter (34, 37, 53). Importantly, internalization of these anti-CAM nanoparticles is distinct from clathrin- and caveolin-mediated endocytosis. Instead, anti-CAM nanoparticle uptake depends on signaling induced by CAM clustering and represents a unique actin-dependent process requiring activation of protein kinase C, Src kinase, and Rho kinase (CAM-mediated endocytosis) (34). Furthermore, anti-CAM nanoparticles enable vascular delivery of diverse active and reporter cargoes in vivo to pulmonary and cardiac endothelium (1, 9, 33, 37, 47). However, little is known about the intracellular trafficking and fate of anti-CAM nanoparticles. This is a critical component in the design of drug carriers, given that delivery to the appropriate cellular compartment can increase therapeutic efficacy.

Here we examine the intracellular trafficking, activity, and fate of anti-ICAM nanoparticles in endothelial cells. The antioxidant activity of anti-ICAM nanoparticles loaded with catalase, an H2O2-degrading enzyme potentially useful for antioxidant protection in the cardiovascular system (35, 36), was also tested. Our data show that 1) the kinetics of anti-ICAM nanoparticle trafficking to lysosomes are remarkably slow; 2) nanoparticle degradation by endothelial cells can be further delayed by auxiliary pharmacological agents; and 3) anti-ICAM/catalase nanoparticles permit effective protection against oxidative stress for several hours after internalization. These results indicate that internalization via ICAM-1 permits nanoparticles to be retained in intracellular compartments where they can avoid degradation for a relatively prolonged time. This feature of ICAM targeting and CAM-mediated endocytosis suggests that drug carriers may be designed to be retained in a protected early endocytic compartment with the potential to enhance their therapeutic effects.


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Antibodies and reagents. Mouse anti-human ICAM-1 (antibody R6.5) was affinity purified from the hybridoma HB-9580 (ATCC, Manassas, VA) (33, 34). Polyclonal antibodies to catalase, human early endosome antigen-1 (EEA-1), or lysosome-associated membrane protein-1 (LAMP-1) were from Calbiochem (La Jolla, CA), Affinity BioReagents (Golden, CO), and BD Biosciences/PharminGen (Franklin Lakes, NJ), respectively. Secondary fluorescent antibodies were from Jackson ImmunoResearch (West Grove, PA) and Molecular Probes (Eugene, OR). Fluoresbrite YG microspheres, which are polystyrene latex microspheres 100 nm in diameter containing a fluorochrome compatible with FITC fluorescence, were from Polysciences (Warrington, PA). Unless otherwise stated, all other reagents were from Sigma (St. Louis, MO).

Preparation of anti-ICAM nanoparticles. Nanoparticles were prepared as described previously (34) by coating on fluorescently labeled latex microspheres with either anti-ICAM-1 alone (anti-ICAM nanoparticles) or anti-ICAM-1 and biotinylated catalase (biotin-catalase) at a 1:0.5 molar ratio (anti-ICAM/catalase nanoparticles). The final effective diameter of resulting nanoparticles was determined by dynamic light scattering (53). In each case, these protocols yielded preparations with diameter ranging from 100 to 300 nm.

Cell culture. Human umbilical vein endothelial cells (HUVEC), pooled from several donors, were from Clonetics (San Diego, CA). The cells were cultured in M199 medium (GIBCO BRL, Grand Island, NY) supplemented with 15% heat-inactivated fetal bovine serum, 2 mM glutamine, 15 µg/ml endothelial cell growth supplement, 100 µg/ml heparin, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were maintained at 37°C, 5% CO2, and 95% relative humidity. When seeded for experiments, HUVEC between passages 4 and 5 were cultured onto 12-mm2 gelatin-coated coverslips in 24-well plates and then activated by overnight incubation with TNF-{alpha}.

Internalization, trafficking, and stability of anti-ICAM nanoparticles. TNF-{alpha}-activated confluent HUVEC were incubated at 4°C for 30 min with FITC-labeled anti-ICAM or anti-ICAM/catalase nanoparticles to enable binding to the cell surface. The cells were then washed, warmed to 37°C for varying amounts of time, cooled to 4°C, and washed, and the cells were fixed with 2% paraformaldehyde at room temperature (RT) for 15 min. Intracellular delivery of catalase was confirmed by labeling permeabilized cells with rabbit anti-catalase followed by Alexa Fluor 350-conjugated goat anti-rabbit IgG. Also, to preferentially label nanoparticles bound to the cell surface, nonpermeabilized cells were treated with Texas red-conjugated goat anti-mouse IgG (to label anti-ICAM-1). The samples were analyzed with an Olympus IX70 fluorescence microscope using x10 or x40 PlanApo objectives and filters optimized for FITC, Texas red, and Alexa Fluor 350 fluorescence. Images were obtained with a Hamamatsu Orca-1 charge-coupled device camera and analyzed using ImagePro 3.0 software. With this approach, internalized nanoparticles were imaged as single-labeled green particles and surface-bound nanoparticles were double-labeled in yellow. Merged micrographs were scored automatically by image analysis to obtain the percentage of cell-associated particles that were internalized. Images of cells from two to five independent determinations were combined to form the average percentage of internalized particles, where n reflects the total number of cells averaged for a given value. To examine the effect of inhibitors on nanoparticle uptake, TNF-{alpha}-activated HUVEC were pretreated at 37°C for 30 min in the presence of 3 mM amiloride, 50 µM monodansyl cadaverine (MDC), or 1 µg/ml filipin before incubation with nanoparticles.

To identify compartments containing internalized particles, TNF-{alpha}-activated HUVEC were incubated with nanoparticles as described above. After surface labeling of nonpermeabilized cells, the cells were permeabilized with a 15-min incubation with 0.2% Triton X-100 at RT, washed, and further labeled with polyclonal rabbit anti-human EEA-1 followed by Texas red-goat anti-rabbit IgG or with phycoerythrin-labeled rabbit anti-human LAMP-1. To determine the intracellular stability of anti-ICAM or anti-ICAM/catalase nanoparticles, internalized particles were counterstained with either Texas red-goat anti-mouse IgG (to recognize nondegraded anti-ICAM-1) or rabbit anti-catalase followed by Texas red-goat anti-rabbit IgG. Alternatively, cells were incubated with nanoparticles prepared with either 125I-labeled anti-ICAM or biotin-catalase for varying amounts of time. The cells were then harvested, and proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride. Degradation of 125I-anti-ICAM-1 was determined by densitometric analysis of autoradiograms, and intact biotincatalase was analyzed using an avidin-horseradish peroxidase (HRP) blot. Lanes were normalized to total cell protein, which was loaded in the range of 10-20 µg/lane. When indicated, cells were pretreated with either 20 µM nocodazole or 300 µM chloroquine before incubation with nanoparticles. To measure kinetics of fluid-phase endocytic delivery to lysosomes, cells were incubated for varying amounts of time at 37°C with 2 mg/ml amine-fixable 10-kDa Texas red-dextran (Molecular Probes) and then fixed with 2% paraformaldehyde and counterstained with FITC-conjugated rabbit anti-human LAMP-1.

Antioxidant effect of anti-ICAM/catalase nanoparticles. The antioxidant effect of anti-ICAM/catalase nanoparticles was tested at different periods of time after their internalization within control HUVEC or cells treated with either 20 µM nocodazole or 300 µM chloroquine. The cells containing either internalized control anti-ICAM nanoparticles or anti-ICAM/catalase nanoparticles were incubated for 15 min at RT with 5 mM H2O2 in Phenol red-free RPMI. The cells were washed after H2O2 treatment, incubated with 0.1 µM calcein-AM and 1 µM ethidium (Live/Dead kit; Molecular Probes) for 15 min at 37°C, and finally scored to determine the percentage of surviving (calcein positive/ethidium negative) cells. Unless stated otherwise, the data were calculated as means ± SE, where statistical significance was determined by Student's t-test.


    RESULTS
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Anti-ICAM nanoparticles, with a size range between 100 and 300 nm in diameter, are internalized by endothelial cells through a unique endocytic pathway, CAM-mediated endocytosis (34). Therefore, these nanoparticles have the potential to provide intracellular delivery of therapeutics for the treatment of pathologically altered endothelium. Given this, we analyzed the intracellular trafficking and fate of anti-ICAM nanoparticles. Internalization of anti-ICAM nanoparticles by HUVEC was temperature dependent. Cells were incubated with anti-ICAM nanoparticles for 1 h at either 4 or 37°C and then fixed and incubated with Alexa Fluor 350-goat anti-mouse IgG to double label nanoparticles remaining on the cell surface. As shown in Fig. 1, HUVEC incubated at 4°C showed green/blue double-labeled nanoparticles retained on the cell surface. In contrast, cells incubated at 37°C showed little, if any, blue labeling, indicating near complete anti-ICAM nanoparticle internalization. For trafficking studies described below, we routinely counterstained cells using this approach to identify noninternalized nanoparticles. Typically, there was little, if any, blue nanoparticle labeling in these experiments, suggesting that there was near complete internalization of anti-ICAM nanoparticles in the colocalization experiments described.



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Fig. 1. Temperature dependence of anti-intercellular adhesion molecule (ICAM) nanoparticle uptake. Human umbilical vein endothelial cells (HUVEC) were incubated for 30 min at 4°C with green fluorescent anti-ICAM nanoparticles to permit binding. Cells were washed, incubated at 4°C (a and b) or 37°C (c and d) for 1 h, and fixed, and then surface-bound anti-ICAM nanoparticles were counterstained with goat anti-mouse IgG conjugated to blue Alexa Fluor 350. Phase-contrast images are shown in a and c, where n indicates the position of the nucleus and dashed lines denote regions of the plasma membrane in contact with adjacent cells. Immunofluorescence images show extensive blue labeling of anti-ICAM nanoparticles when cells were incubated at 4°C (b), consistent with a lack of internalization at this temperature. In contrast, after 37°C incubation (d), there was little, if any, blue colocalization, confirming that virtually all cell-associated anti-ICAM nanoparticles were internalized after incubation for 1 h at 37°C. Bar, 10 µm.

 

As shown in Fig. 2, A and B, internalized anti-ICAM nanoparticles partially colocalized with EEA-1-positive endosomes in the perinuclear region 1 h after internalization by HUVEC. With time at 37°C the nanoparticles redistributed from EEA-1-positive endosomes to LAMP-1-positive lysosomal compartments. The lysosomal trafficking of anti-ICAM nanoparticles was remarkably slow compared with that of the fluid phase marker Texas red-dextran, which HUVEC trafficked to LAMP-1-positive vesicles after a 15-min incubation (Fig. 2Bd).



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Fig. 2. Slow lysosomal trafficking anti-ICAM nanoparticles. A: HUVEC were incubated for 30 min at 4°C with green fluorescent anti-ICAM nanoparticles to permit binding. Cells were washed and then incubated at 37°C for 1 (a), 2 (b), or 3 h (c) to allow nanoparticle internalization and trafficking. Cells were fixed, surface-bound anti-ICAM nanoparticles were counterstained with goat anti-mouse IgG conjugated to blue Alexa Fluor 350, the cells were permeabilized, and early endosomes were stained with rabbit anti-early endosome antigen-1 (EEA-1) and Texas red-goat anti-rabbit IgG. Cells showed little, if any, blue fluorescence, confirming that virtually all cell-associated anti-ICAM nanoparticles were internalized under these conditions (compare with Fig. 1b). Yellow color in a and b reflects anti-ICAM nanoparticles localized to early endosomes. B: HUVEC were incubated with fluorescent anti-ICAM nanoparticles as in A, except that lysosomes were labeled with Texas red-conjugated rabbit anti-lysosome-associated membrane protein-1 (LAMP-1) (a-c). To measure fluid-phase trafficking to lysosomes, HUVEC were incubated with FITC-conjugated dextran for 15 min (d), 1 h (e), or 3 h (f) before fixation and immunolabeling with Texas red anti-LAMP-1. Yellow color reflects anti-ICAM nanoparticles or FITC-dextran localized to lysosomes. Bars, 10 µm. C: the extent of colocalization of anti-ICAM nanoparticles and EEA-1 or LAMP-1 was quantified by image analysis and plotted as a function of incubation time. Data are means ± SD and represent %colocalization for n = 20-25 cells.

 

Because trafficking of anti-ICAM nanoparticles to lysosomes was slow, we examined degradation of anti-ICAM nanoparticles using a qualitative immunofluorescence assay (Fig. 3A). Anti-ICAM nanoparticles internalized by HUVEC lost immunoreactivity after a 2- to 3-h incubation at 37°C, a rate of degradation consistent with the slow rate of delivery to lysosomes. This was confirmed directly by examining the degradation of 125I-anti-ICAM by HUVEC, where ~30% of the HUVEC associated anti-ICAM remained intact after a 3-h incubation (Fig. 3E). These data suggest that degradation of anti-ICAM nanoparticles, which occurred at an unusually slow rate, takes place after delivery to lysosomes.



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Fig. 3. Stability of internalized anti-ICAM nanoparticles. HUVEC were either untreated (A) or preincubated for 30 min at 37°C in the presence of either 300 µM chloroquine (B) or 20 µM nocodazole (C) before incubation with anti-ICAM nanoparticles. Stability of intracellular anti-ICAM-1 was tested by incubating cells with anti-ICAM nanoparticles as described in Fig. 2 and then immunolabeling permeabilized cells with Texas red-goat anti-mouse IgG, which binds nondegraded anti-ICAM-1 to produce yellow double-labeled particles (arrows). Arrowheads denote green nanoparticles, which did not bind Texas red-goat anti-mouse IgG, suggesting that anti-ICAM was degraded. Bars, 10 µm. D: cells were either untreated (lanes 2-4) or treated with chloroquine (lanes 5-7) or nocodazole (lanes 8-10) as described above and then incubated with anti-ICAM nanoparticles containing 125I-labeled IgG for varying amounts of time. Cells were harvested, proteins were resolved by SDS-PAGE, and the amount of cell-associated 125I-IgG was determined by densitometric analysis of autoradiograms (E, controls; F, nocodazole and chloroquine). Lane 1 contained 125I-anti-ICAM nanoparticles loaded directly onto the gel; other lanes were loaded with equal amounts of total cell protein. Densitometric values are means ± SD (n = 3) normalized to values obtained for the corresponding 1 h time point.

 

Therefore, we determined whether pharmacological agents that interfere with lysosome activity could inhibit nanoparticle degradation. Pretreatment of HUVEC with either chloroquine or nocodazole before incubation with anti-ICAM nanoparticles inhibited anti-ICAM degradation (Fig. 3). The mechanism of action for these agents was distinct. Pretreatment of HUVEC with the weak base chloroquine did not affect trafficking of anti-ICAM nanoparticles from early endosomes to lysosomes (Fig. 4, A and B) but markedly prolonged their stability (Fig. 3F), consistent with decreased lysosome acidification in the presence of chloroquine and suggesting that that anti-ICAM nanoparticles were degraded by acidic proteolytic enzymes. Comparable results were obtained by using bafilomycin to inhibit lysosome acidification (Muro S, Muzykantov VR, and Koval M, unpublished observations).



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Fig. 4. Chloroquine did not inhibit anti-ICAM nanoparticle transport to lysosomes. HUVEC were treated as described in Fig. 2, except that cells were preincubated for 30 min at 37°C in the presence of 300 µM chloroquine before incubation with anti-ICAM nanoparticles and immunolabeling for surface nanoparticles (blue), EEA-1 (A), or LAMP-1 (B). Cells showed little, if any, blue fluorescence, confirming that virtually all cell-associated anti-ICAM nanoparticles were internalized under these conditions (compare with Fig. 1b). Yellow color reflects localization of anti-ICAM nanoparticles to early endosomes (A) or lysosomes (B). Bars, 10 µm. C: the extent of colocalization of anti-ICAM nanoparticles and EEA-1 or LAMP-1 was quantified by image analysis and plotted as a function of incubation time. Data are means ± SD and represent %colocalization for n = 10-12 cells.

 

In contrast, nocodazole pretreatment blocked anti-ICAM nanoparticle trafficking by HUVEC to lysosomes by disrupting the microtubule network of the cell (46, 49). Previously, we found that nocodazole did not inhibit nanoparticle uptake (34). Instead, for nocodazole-treated cells, anti-ICAM nanoparticles were internalized and scattered throughout the cell periphery instead of distributing to the perinuclear area. Consistent with blocking anti-ICAM nanoparticle trafficking to lysosomes (Fig. 5B), nocodazole markedly decelerated degradation of anti-ICAM nanoparticles (Fig. 3F). Also, nocodazole-treated cells showed little, if any, colocalization of internalized anti-ICAM nanoparticles with EEA-1 (Fig. 5A), suggesting that microtubules may be required for nanoparticle trafficking to EEA-1-positive early endosomes as well as to late endocytic compartments.



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Fig. 5. Nocodazole inhibited trafficking of anti-ICAM nanoparticles to lysosomes. HUVEC were labeled as described in Fig. 2, except that cells were preincubated for 30 min at 37°C in the presence of 20 µM nocodazole before to incubation with anti-ICAM nanoparticles and immunolabeling surface nanoparticles (blue), EEA-1 (A), or LAMP-1 (B). Cells showed little, if any, blue fluorescence, confirming that virtually all cell-associated anti-ICAM nanoparticles were internalized under these conditions (compare with Fig. 1b). Yellow color reflects localization of anti-ICAM nanoparticles in early endosomes (A) or lysosomes (B). Bars, 10 µm. C: the extent of colocalization of anti-ICAM nanoparticles and EEA-1 or LAMP-1 was quantified by image analysis and plotted as a function of incubation time. Data are means ± SD and represent %colocalization for n = 15-18 cells.

 

These findings imply that anti-ICAM nanoparticles could deliver a drug into endothelial cells that might be transiently stable for several hours and that could be further stabilized by using auxiliary agents that inhibit lysosomal traffic and/or degradation, features that make anti-ICAM nanoparticles good candidates for antioxidant delivery. To test this, we prepared anti-ICAM nanoparticles carrying catalase, a classic antioxidant enzyme that protects cells from H2O2 generated during oxidative stress (35). Anti-ICAM/catalase nanoparticles were internalized by HUVEC (Fig. 6A). Neither MDC nor filipin significantly inhibited internalization of anti-ICAM/catalase nanoparticles (14 ± 2 and 23 ± 3% inhibition of control levels of internalization, respectively; n = 10-15 cells from 2 independent experiments), showing that neither clathrin- nor caveolin-mediated endocytic pathways are responsible for internalization. However, the uptake was significantly inhibited by amiloride (Fig. 6, B and C; 40 ± 3% inhibition of control level of internalization; n = 10-15 cells from 2 independent experiments), consistent with internalization of anti-ICAM/catalase nanoparticles by CAM-mediated endocytosis (34).



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Fig. 6. Intracellular delivery of catalase nanoparticles by ICAM-mediated internalization. A: HUVEC were incubated for 1 h at 37°C with green fluorescent anti-ICAM/catalase nanoparticles. Cells were washed, fixed, and counterstained with Texas red-goat anti-mouse IgG. Cells were then permeabilized and stained using rabbit anti-catalase and blue Alexa Fluor 350-goat anti-rabbit IgG. Internalized nanoparticles containing catalase show a blue-green color (arrow), and surface-bound triple-labeled nanoparticles appear white (arrowhead). The internalization of anti-ICAM/catalase nanoparticles was tested in control HUVEC (B) or cells treated with 3 mM amiloride (C), where surface-bound particles were counterstained with Texas red-goat anti-mouse IgG without cell permeabilization. Internalized anti-ICAM/catalase nanoparticles were single labeled green (arrow), and nanoparticles at the cell surface were double-labeled yellow (arrowhead). Bar, 10 µm.

 

Internalized anti-ICAM/catalase nanoparticles were transported to lysosomes (Fig. 7A), where degradation occurred (Fig. 7B). Both trafficking and degradation kinetics were slow (3 h) and similar to that observed for control anti-ICAM nanoparticles. Also, the kinetics for biotin-catalase degradation, as assessed by HRP-avidin blot (Fig. 7D), were comparable to the kinetics for anti-ICAM degradation (Fig. 3E). These data are consistent with the notion that the catalase cargo did not affect the mechanism of uptake, traffic, and degradation of anti-ICAM nanoparticles.



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Fig. 7. Slow lysosomal trafficking and degradation of internalized anti-ICAM/catalase nanoparticles. A: HUVEC were incubated with Texas red-dextran to label lysosomes and then incubated with anti-ICAM/catalase nanoparticles for 30 min at 4°C, washed, and incubated at 37°C for different periods of time to permit internalization. Surface-bound particles were counterstained with blue Alexa Fluor 350-goat anti-mouse IgG. Cells showed little, if any, blue fluorescence, confirming that virtually all cell-associated anti-ICAM/catalase nanoparticles were internalized under these conditions (compare with Fig. 1b). Yellow color reflects localization of green fluorescent nanoparticles to Texas red-labeled lysosomes. B: surface-bound particles were counterstained with blue Alexa Fluor 350-goat anti-mouse IgG, and then the stability of intracellular anti-ICAM/catalase nanoparticles was tested by immunolabeling permeabilized cells with rabbit anti-catalase and Texas red-goat anti-rabbit IgG, as described in Fig. 3. Bars, 10 µm. C: cells were either untreated (lanes 2-4) or treated with chloroquine (lanes 5-7) or nocodazole (lanes 8-10) as described above and incubated with anti-ICAM nanoparticles containing biotinylated catalase for varying amounts of time. Cells were harvested, proteins were resolved by SDS-PAGE and transferred to membranes, and the amount of cell-associated biotinylated catalase was determined by densitometric analysis of horseradish peroxidase (HRP)-avidin blots (D, controls; E, nocodazole and chloroquine). Lane 1 contains biotinylated catalase nanoparticles loaded directly onto the gel; other lanes were loaded with equal amounts of cell protein. Densitometric values are means ± SD (n = 3) normalized to values obtained for the corresponding 1 h time point.

 

To test the antioxidant capacity of catalase delivered within the cells by anti-ICAM nanoparticles, HUVEC were incubated after internalization of anti-ICAM/catalase nanoparticles at 37°C for varying amounts of time and then subsequently exposed to 5 mM H2O2 for 15 min. Cells pretreated with unloaded anti-ICAM nanoparticles showed significant H2O2-mediated toxicity; only ~60% of the cells survived 15 min after H2O2 shock (Fig. 8, Ad-Af and B). In contrast, anti-ICAM/catalase nanoparticle-treated cells were resistant to H2O2 (>90% cell survival; Fig. 8, Ag, Ah, and B), indicating that catalase nanoparticles delivered inside the cells had a protective effect. The protective effect of anti-ICAM/catalase nanoparticles diminished 3 h after internalization, consistent with the kinetics of lysosomal trafficking and degradation of these nanoparticles (Fig. 8Ai).



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Fig. 8. Prolonged antioxidant protection by intracellular delivery of anti-ICAM/catalase nanoparticles. A: HUVEC were incubated with either control anti-ICAM nanoparticles (a-f) or anti-ICAM/catalase nanoparticles (g-i) for 30 min at 4°C and then incubated at 37°C for 1 (a, d, g), 2 (b, e, h), or 3 h (c, f, i). Cells were washed and then mock-treated (a-c) or treated with 5 mM H2O2 for 15 min at room temperature (d-i). Cell viability was then assessed using the Live/Dead assay, which reveals live, intact cells as calcein positive/ethidium negative (green) and dead cells as calcein negative/ethidium positive (red). Bar, 50 µm. B: cell viability was quantified by fluorescence microscopy from at least 500 cells per condition. Data are means ± SE and represent the percentage of survival. Cells treated with anti-ICAM/catalase nanoparticles (catalase + H2O2) were resistant to H2O2 damage, compared with untreated cells (control + H2O2). C: HUVEC were pretreated with either 300 µM chloroquine or 20 µM nocodazole and then incubated with anti-ICAM/catalase nanoparticles, and protection to H2O2 injury was analyzed as described above. Cells pretreated with nocodazole or chloroquine had prolonged resistance to H2O2 damage, compared with control H2O2-treated cells that received catalase nanoparticles alone (catalase + H2O2). Dashed line shows the mean value for %survival of cells that were not exposed to H2O2; dotted line shows the mean value for %survival of cells treated with unloaded anti-ICAM nanoparticles and H2O2. *P < 0.05 compared with H2O2-exposed cells that were not treated with anti-ICAM/catalase nanoparticles.

 

To determine whether auxiliary pharmacological agents prolong the therapeutic effect of anti-ICAM/catalase nanoparticles, HUVEC were pretreated with either chloroquine or nocodazole. Both agents markedly prolonged the duration of antioxidant protective effect of anti-ICAM/catalase nanoparticles (Fig. 8C) and inhibited catalase degradation (Fig. 7 E). In particular, nocodazole enabled internalized catalase to remain active for at least 5 h after internalization, which doubled the duration of the antioxidant activity of anti-ICAM/catalase nanoparticles.


    DISCUSSION
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 METHODS
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 DISCUSSION
 REFERENCES
 
The present data demonstrate that functionally active, catalase-carrying anti-ICAM nanoparticles are internalized by endothelial cells using CAM-mediated endocytosis and were retained in an early endosomal compartment for an unusually long period of time (1-2 h). Anti-ICAM nanoparticles were ultimately delivered to lysosomes after 2-3 h, where they were degraded and inactivated. By comparison, ligands internalized via other pathways including clathrin-mediated or fluid-phase endocytosis and phagocytosis are delivered to lysosomes within minutes (6, 21, 31). The slow kinetics of anti-ICAM nanoparticle trafficking to lysosomes proved advantageous for the intracellular targeting of catalase, which had prolonged antioxidant protective activity due to the prolonged (1-2 h) residence time in a nondegrading early endosomal compartment. Furthermore, catalase activity was extended when cells were treated with pharmacological agents that delayed proteolysis by decreasing lysosome acidification or nanoparticle delivery to lysosomes.

The multivalent nature of anti-ICAM nanoparticles is likely to contribute to the delay in trafficking to lysosomes. Consistent with this, Marsh et al. (27) found that oligomerized transferrin was retained in an early endocytic compartment, where it was resistant to degradation. Furthermore, oligomerized transferrin ultimately was recycled to the plasma membrane, consistent with the trafficking of natural transferrin. This is in contrast to our results, where anti-ICAM-1 nanoparticles were ultimately transported to lysosomes. Thus, although the multivalent nature of the ligands affects the kinetics of transport through endocytic compartments, the destination of internalized multivalent ligands appears to depend more on the receptor, rather than on ligand valency.

The intracellular trafficking of internalized ligands is influenced by receptor determinants (6, 31). However, Ig superfamily CAM, such as ICAM-1 and PECAM-1, are not typically thought of as endocytic receptors. In fact, monomeric anti-ICAM-1 and anti-PECAM-1 are poorly internalized by endothelial cells (33, 37, 53). Nevertheless, endothelial cells internalize multivalent anti-ICAM and anti-PECAM nanoparticles in the size range of 100-300 nm in diameter through CAM-mediated endocytosis, an endocytic mechanism that is distinct from clathrin- and caveolin-mediated endocytosis, phagocytosis, and macropinocytosis (34, 53). Given this, the trafficking of internalized anti-CAM nanoparticles could not be predicted on the basis of known properties of ICAM-1 or PECAM-1 trafficking. Nonetheless, it seems plausible that internalization by CAM-mediated endocytosis may be critical for targeting and retention of anti-CAM nanoparticles to an early endosomal compartment.

Both in vitro and in vivo results suggest that CAM-mediated internalization may have the potential to be useful for the specific intracellular immunotargeting of nanoparticles (33, 34, 53). Although agents such as catalase can be active when bound to the endothelial cell surface, internalization offers several advantages. For instance, receptor shedding has the potential to decrease the efficacy of anti-ICAM/catalase complexes by releasing them from the cell surface. Consistent with this possibility, dimeric ICAM-1 was found to be shed into the pleural space in response to inflammation (29). Also, because internalized catalase will have access to intracellular oxidants diffusing from all directions, internalized catalase has the potential to be more effective than catalase localized to the cell surface. The ability to target catalase to specific intracellular compartments also has the additional potential for intercepting oxidants near sites of generation or sites sensitive to damage (such as nuclear DNA).

The trafficking of anti-ICAM/catalase nanoparticles underscores the notion that the fate of therapeutic cargoes should and can be modulated to optimize drug delivery strategies. For example, fast endosomal traffic and escape are critical for activity of immunotoxins (45). Conversely, targeting drugs to parasitophorous vacuoles or lysosomes is preferable for antiparasitic and certain types of enzyme replacement therapies (15, 25, 55), and nuclear delivery is necessary for gene therapies (7, 19, 40). Furthermore, ligands targeted to antigens involved in caveolin-mediated endocytosis have the potential to be transcytosed by endothelial cells via a pathway that eludes lysosomes and has the potential to circumvent the endothelial barrier (28, 43).

ICAM-1 represents a particularly attractive target for targeting antioxidant enzymes because it is upregulated by stressed endothelial cells and is functionally involved in vascular oxidative stress, ischemiareperfusion injury, and inflammation (12, 13, 18, 32). Intracellular delivery of antioxidants may permit effective interception and detoxification of reactive oxygen species produced inside endothelial cells and diffusing through the plasma membrane from released activated leukocytes (16, 26, 54). Also, anti-ICAM nanoparticles may also have the capacity to occupy ICAM-1 at the cell surface and thus block leukocyte adhesion to endothelial cells, which could provide a secondary anti-inflammatory benefit (5, 10, 11, 23).

Catalase delivered by anti-ICAM nanoparticles afforded antioxidant protection for over 2 h after internalization (Fig. 7). In fact, this time frame for antioxidant protection is consistent with animal studies showing that anti-CAM/catalase nanoparticles protect the lung from acute oxidative injury during transplantation (22). Here, we found that this therapeutic time window can be further extended by the use of auxiliary drugs, which either inhibit pH-dependent lysosomal proteolysis or prevent microtubule-dependent lysosomal trafficking. This raises the possibility that pharmacological agents already approved for clinical use (e.g., chloroquine) can be used in combination with anti-CAM nanoparticles to improve the in vivo efficacy of nanoparticle-based drug delivery vehicles, an approach with the potential to enhance therapeutic interventions in acute pathological conditions associated with vascular oxidative stress, including acute lung injury, hyperoxia, and ischemia-reperfusion injury.


    DISCLOSURES
 
This work was supported by National Institutes of Health (NIH) Specialized Center of Research in Acute Lung Injury Grant HL-60290, Project 4 (to V. R. Muzykantov); NIH Grants HL/GM-71175-01 (to V. R. Muzykantov), GM-61012 (to M. Koval), and P01-HL-19737-26, Project 3 (M. Koval); and Department of Defense Grant PR 012262 (to V. R. Muzykantov). S. Muro was supported by a fellowship from Fundación Ramón Areces (Spain).


    ACKNOWLEDGMENTS
 
We thank Samira Tliba and Dr. Vladimir Shuvaev for technical assistance and advice.


    FOOTNOTES
 

Address for reprint requests and other correspondence: V. R. Muzykantov (drug delivery and vascular immunotargeting), Univ. of Pennsylvania School of Medicine, Institute for Environmental Medicine, 1 John Morgan/6068, 3620 Hamilton Walk, Philadelphia, PA 19104 (E-mail: muzykant{at}mail.med.upenn.edu); or M. Koval (cell biology and endocytosis), Univ. of Pennsylvania School of Medicine, Dept. of Physiology, B-400 Richards Bldg./6085, 3700 Hamilton Walk, Philadelphia, PA 19104 (E-mail: mkoval{at}mail.med.upenn.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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