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

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doi:10.1016/j.ymthe.2005.07.687

Lysosomal Enzyme Delivery by ICAM-1-Targeted Nanocarriers Bypassing Glycosylation- and Clathrin-Dependent Endocytosis Silvia Muro,1,2,* Edward H. Schuchman,3 and Vladimir R. Muzykantov1,2 1

Institute for Environmental Medicine and 2Department of Pharmacology, University of Pennsylvania Medical School, 1 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-6068, USA 3 Department of Human Genetics, Mount Sinai School of Medicine, Room 14-20A, 1425 Madison Avenue, New York, NY 10029, USA *To whom correspondence and reprint requests should be addressed. Fax: +1 215 898 0868. E-mail: [email protected].

Available online 8 September 2005

Enzyme replacement therapy, a state-of-the-art treatment for many lysosomal storage disorders, relies on carbohydrate-mediated binding of recombinant enzymes to receptors that mediate lysosomal delivery via clathrin-dependent endocytosis. Suboptimal glycosylation of recombinant enzymes and deficiency of clathrin-mediated endocytosis in some lysosomal enzyme-deficient cells limit delivery and efficacy of enzyme replacement therapy for lysosomal disorders. We explored a novel delivery strategy utilizing nanocarriers targeted to a glycosylation- and clathrin-independent receptor, intercellular adhesion molecule (ICAM)-1, a glycoprotein expressed on diverse cell types, up-regulated and functionally involved in inflammation, a hallmark of many lysosomal disorders. We targeted recombinant human acid sphingomyelinase (ASM), deficient in types A and B Niemann– Pick disease, to ICAM-1 by loading this enzyme to nanocarriers coated with anti-ICAM. Anti-ICAM/ ASM nanocarriers, but not control ASM or ASM nanocarriers, bound to ICAM-1-positive cells (activated endothelial cells and Niemann–Pick disease patient fibroblasts) via ICAM-1, in a glycosylation-independent manner. Anti-ICAM/ASM nanocarriers entered cells via CAM-mediated endocytosis, bypassing the clathrin-dependent pathway, and trafficked to lysosomes, where delivered ASM displayed stable activity and alleviated lysosomal lipid accumulation. Therefore, lysosomal enzyme targeting using nanocarriers targeted to ICAM-1 bypasses defunct pathways and may improve the efficacy of enzyme replacement therapy for lysosomal disorders, such as Niemann– Pick disease. Key Words: enzyme replacement therapy, lysosomal storage disorder, Niemann–Pick, acid sphingomyelinase, ICAM-1, immunotargeting, nanocarrier delivery

INTRODUCTION Enzyme replacement therapy (ERT) is available for three lysosomal storage diseases (LSDs) and is under development for several others [1–3]. This approach capitalizes on the fact that lysosomal enzymes contain oligosaccharide residues (e.g., mannose and/or mannose 6-phosphate (M6P)) that bind to cellular receptors (e.g., mannose or M6P receptors (MR or M6PR)), leading to internalization of the enzymes by clathrin-mediated endocytosis and subsequent trafficking to lysosomes [4–6]. However, inadequate glycosylation of recombinant enzymes and/or lack of appropriate receptors in LSD target cells are common obstacles affecting enzyme delivery and the efficacy of ERT. High doses of enzymes, required to compensate for ineffective delivery, may result in harmful side effects and rapid saturation of receptor-mediated uptake [7].

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Novel strategies have been designed to circumvent these problems, such as enzyme modification to expose mannose residues on h-glucosidase for improved targeting to MR in Gaucher disease macrophages [8,9]. In addition, pharmacological agents can be used to increase receptor expression and reduce saturation, such as dexamethasone, which increases MR surface density [10]. However, due to preferential expression of MR in the reticuloendothelial system (RES), other cells and tissues are precluded from being efficiently targeted by these methods [11,12]. Other receptors such as M6PRs can be utilized for enzyme delivery to cell types other than RES [13,14], but the lack of M6P residues in some recombinant enzymes may lead to inefficient targeting [15]. Recently, innovative approaches focused on chimeric enzymes containing alternative targeting moieties

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(e.g., a peptide derived from insulin-like growth factor II [16] or the receptor-associated protein [17]). However, chimeric enzymes often exhibit diminished activity and also the targeted receptors (the M6PR and LDLR family) are internalized via clathrin-mediated pathways, which are defective in some lysosomal enzyme-deficient cells [18]. Together, these factors currently hinder the efficacy of LSD ERT and justify the design of alternative strategies bypassing endogenous pathways for lysosomal enzyme delivery. In this context, a recently defined endocytic pathway employing intercellular adhesion molecule (ICAM)-1 is of particular interest. ICAM-1-positive cells internalize nanocarriers coated with anti-ICAM (anti-ICAM/NCs) via a peculiar pathway, CAM-mediated endocytosis, induced upon ICAM-1 clustering by multivalent antiICAM/NCs [19]. CAM-mediated endocytosis is distinct from clathrin- and caveoli-mediated pathways, yet efficiently delivers materials to lysosomes [20,21], an ideal feature of a delivery system for LSD ERT. ICAM-1, a transmembrane glycoprotein from the Iglike superfamily of adhesion molecules, may indeed provide a unique target for LSD ERT: (i) it is expressed by cell types affected in diverse forms of LSDs, including endothelial (EC), epithelial, glial, and Schwann cells; leukocytes; myocytes; and other cells [22]; (ii) it is accessible to ligands administered via intravascular, intratracheal, or intracerebral routes; (iii) ICAM-1 levels are enhanced by pathological factors pertinent to LSDs, such as inflammation [20,23]; and (iv) ICAM-1 targeting blocks its function as an anchor for leukocytes, thus providing anti-inflammatory benefits [20]. Results of cell culture and in vivo studies showed that CAM-targeted systems, including anti-ICAM nanocarriers, deliver reporter and model enzymes into target cells (immunotargeting) [20,24–28]. Here we tested this strategy in the context of lysosomal ERT and found that anti-ICAM/NCs greatly enhance binding, internalization, and lysosomal trafficking of recombinant human acid sphingomyelinase (ASM; the hydrolase deficient in types A and B Niemann–Pick disease (NPD)). Most importantly, in situ functional activity of ICAM-targeted recombinant ASM alleviated cellular lipid storage, either induced by imipramine treatment of normal cells or inherent to NPD cells.

RESULTS

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DISCUSSION

Cellular Targeting of Recombinant Human ASM (rhASM) Loaded onto Anti-ICAM Nanocarriers The use of specific cell surface receptors as target molecules to deliver therapeutics is an emerging field with a wide spectrum of potential clinical applications, including drug, enzyme, and gene delivery [24,29,30]. In comparison to other targets previously used in the context of ERT for LSDs (e.g., MPR, or megalin and

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LRP1 for the LDLR family [16,17]) and cell-specific target molecules (e.g., the surface adhesion molecule PECAM-1 or selectins [24,27,28,31,32]), widespread expression of ICAM-1 by numerous cell types [22] may broaden its applicability for delivery of lysosomal enzymes. However, since cells do not internalize monomeric anti-ICAM [26,33], synthesis of conjugates or carriers in the nano(submicrometer) scale is a key component of the proposed delivery system. We produced anti-ICAM nanocarriers carrying ASM (anti-ICAM/ASM/NCs) by coupling recombinant human ASM and anti-ICAM to 100-nm nanocarriers. Anti-ICAM/ASM/NCs showed substantial ASM activity and mean size of 150 to 250 nm in diameter, consistent with the presence of a layer of enzyme and antibody molecules on the carrier surface (Supplementary Fig. 1). This configuration of anti-ICAM/NCs has been demonstrated to provide effective targeting and intracellular delivery in animal studies [34] and various cell systems, including endothelial, mesothelioma, the hybrid endothelial–epithelial EAhy926 cells [19,21, 33,34], and macrophages (Muro et al., unpublished data). We studied interactions of anti-ICAM/ASM/NCs with ECs, given that these constitute the first and most proximal, large cell interface encountered by nanocarriers injected into the bloodstream. In addition, pathologically altered ECs represent direct therapeutic targets for several LSDs, as well as a route for delivery to other tissues. Our results indicate that anti-ICAM/ASM/NCs provided effi-

FIG. 1. Anti-ICAM/NCs mediate efficient targeting of rhASM to ICAM-1expressing cells. TNF-a-activated HUVEC were incubated with FITC-labeled ASM/Ns, either (A) nonimmunotargeted or (B) targeted to ICAM-1, for 30 min at 48C. Cell borders are marked by a dashed line. Magnification bar, 10 Am. (C) The number of NCs bound per cell was determined by fluorescence microscopy. (D) The targeting specificity of anti-ICAM/ASM/NCs and control anti-ICAM/NCs was determined in the presence of competitors of the two potential targeting moieties, anti-ICAM or mannose 6-phosphate (M6P). Data are means F SEM (n z 10 cells from two independent experiments).

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doi:10.1016/j.ymthe.2005.07.687

FIG. 2. Anti-ICAM/ASM/NCs are efficiently endocytosed, bypassing clathrinmediated pathways. (A) Internalization of FITC-labeled anti-ICAM/ASM/NCs by TNF-a-activated HUVEC (2 h at 378C). Nanocarriers were counterstained with Texas red goat anti-mouse; therefore, double-labeled yellow particles are nanocarriers bound to the cell surface (arrowhead) and single-labeled green particles are nanocarriers internalized within the cells (arrow). (B) Kinetics of internalization of FITC-labeled anti-ICAM/ASM/NCs determined at both 4 and 378C and compared to that of control anti-ICAM/NCs. (C and D) Micrographs of cells treated with inhibitors of anti-ICAM/NC-induced CAM-mediated endocytosis (C; amiloride) or (D) clathrin-coated pits (MDC), and (E) quantification of nanocarrier internalization under these conditions. Data are means F SD (n z 10 cells from two independent experiments). Magnification bar, 10 Am.

cient binding to TNF-a-activated human umbilical vein endothelial cells (HUVEC) in culture, while control ASM/ NCs showed very low, if any, binding to the cells (Fig. 1). Binding of anti-ICAM/ASM/NCs to ECs was inhibited by blocking ICAM-1 with excess free antibody, but not by blocking the M6PR by M6P (Fig. 1D and Supplementary Fig. 2). Therefore, although rhASM employed in this study possesses M6P residues and can bind to M6PR [15], binding of anti-ICAM/ASM/NCs to the cell surface is predominantly mediated by anti-ICAM. This is likely due to high ICAM-1 surface density (1–2  105 molecules/ cell), which can be further stably up-regulated by numerous pathological factors (Supplementary Fig. 3), including those pertinent to NPD and other LSDs [20,23]. Thus ICAM-1 targeting overcomes typical delivery obstacles for LSD ERT, such as lack or inaccessibility of sugar-binding residues in recombinant lysosomal enzymes or saturation of the endogenous receptors (MR or M6PR). Endocytosis and Lysosomal Delivery of rhASM by Anti-ICAM Nanocarriers Intracellular delivery mediated by anti-ICAM/NCs has been documented in a variety of ICAM-1-expressing cell types [19,21,33]. The molecular mechanisms involved in interactions between ICAM-1-expressing cells and CAM-

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targeted delivery systems (including PKC, Src, and ROCK signaling upon ICAM-1 clustering, regulatory activity of dynamin and amiloride-sensitive sodium exchangers, and actin cytoskeleton remodeling) have also been recently characterized [19,21,34]. This information provides the basis for rational design of ICAM-delivery systems, which may be adjusted to specific therapeutic needs, including LSD ERT [20,35]. Consistent with this hypothesis, anti-ICAM/ASM/NCs were rapidly and efficiently internalized by HUVEC at 378C, but not at 48C, a temperature precluding endocytosis (Figs. 2A and 2B). Targeting ICAM-1 increased internalization of the nanocarriers (N100 anti-ICAM/ ASM/NCs per cell vs. b10 ASM/NCs per cell by 1 h at 378C) and also markedly increased intracellular delivery of 125I-rhASM (7.1 F 0.2 ng/well anti-ICAM/125I-ASM/ NCs vs. 0.7 F 0.1 ng/well 125I-rhASM or 0.6 F 0.7 ng/ well 125I-ASM/NCs). In addition, anti-ICAM/ASM/NC internalization kinetics was similar to that of antiICAM/NCs lacking the enzyme cargo: half-time for internalization was 20 min and the maximum internali-

FIG. 3. Internalized anti-ICAM/ASM/NCs traffic to lysosomes. (A) HUVEC lysosomes were prelabeled with Texas red dextran, then the cells were incubated with FITC-labeled anti-ICAM/ASM/NCs for 30 min at 48C, warmed to 378C after washing of nonbound materials, incubated for the indicated times at 378C, and counterstained with secondary labeled antibodies, to stain surfacebound nanocarriers in blue (the absence of blue staining indicates that nanocarriers are internalized). Green particles (arrowhead) are nanocarriers in prelysosomal compartments, while yellow color (arrow) shows colocalization of anti-ICAM/ASM/NC with red-labeled lysosomes. Magnification bar, 10 Am. (B) Colocalization of anti-ICAM/NCs, either containing rhASM or not, with lysosomes is plotted as a function of time after internalization. Data are means F SEM (n N 10 cells from two independent experiments).

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standpoint of other therapies, the lysosomal destination of anti-ICAM delivery vehicles represents a limitation, since proteolytic degradation of the cargo shortens duration of the therapeutic effects. However, in the context of ERT for LSDs, lysosomal destination of antiICAM/NCs is ideal. As shown in Fig. 3, internalized antiICAM/ASM/NCs trafficked to lysosomal compartments within 3 h, with kinetics similar to that observed in the case of anti-ICAM/NCs lacking rhASM, consistent with a CAM-mediated pathway [21]. Thus, as verified by direct, visual identification methods [39], ICAM-1 targeting deviates rhASM from glycosylation- and the clathrindependent delivery route (defective in ASM-deficient cells [18]) and provides efficient lysosomal delivery.

FIG. 4. Anti-ICAM/ASM/NCs are internalized and delivered to lysosomes in ASM-deficient cells. Internalization of FITC-labeled anti-ICAM/ASM/NCs at 378C was tested in two ASM-deficient cell models: (A) HUVEC pretreated for 48 h with imipramine (IMP), which degrades endogenous ASM, and (B) skin fibroblasts from NPD patients. Also, trafficking of FITC–anti-ICAM/ASM/NCs to Texas red dextran-labeled lysosomes was studied in (C) imipramine-treated HUVEC and (D) NPD fibroblasts. Data are means F SD (n N 10 cells from two independent experiments).

zation level attained 85% of the nanocarriers bound to the plasma membrane (Fig. 2B). These results suggest that the mechanism of internalization of anti-ICAM/ ASM/NCs is consistent with endocytosis induced by ICAM-1 clustering [19]. To verify this we utilized drugs known to inhibit specifically CAM-mediated endocytosis (amiloride) or endocytosis via clathrin-coated pits (monodansyl cadaverine, or MDC). Amiloride, but not MDC, inhibited endocytosis of anti-ICAM/ASM/NCs (Figs. 2C, 2D, and 2E), indicating that rhASM targeted to ICAM-1 enters cells via CAM-mediated endocytosis. Bypassing clathrinmediated pathways, such as those utilized to deliver enzymes targeted to MR, M6PR, or the LDLR family, may be advantageous in the context of ERT for LSDs, given that this pathway has been observed to be defective in cells from animal models of lipid storage [18]. In addition, it has been observed that ICAM-1, internalized by CAM-mediated endocytosis, recycles to the plasma membrane, thus a single ICAM-1 molecule can provide multiple rounds of nanocarrier binding and internalization by this pathway [34]. In theory, lysosomal enzyme delivery by nanocarriers can also take advantage of this feature of CAM endocytosis, since nanocarriers can be tailored for recurrent and sustained delivery, as is the case for stealth PEG-coated nanocarriers with decelerated blood clearance [19,34,36–38]. Importantly, contrary to the recycling route followed by ICAM-1 upon internalization, anti-ICAM/NCs have been shown to traffic to lysosomes [21,34]. From the

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Anti-ICAM Nanocarriers Deliver rhASM into ASM-Deficient Cells In the experiments above we treated cells with TNF-a to model inflammatory activation, an important pathology pertinent to NPD and other LSDs [20,23]. However, these cells express endogenous ASM and do not accumulate lipids, which can secondarily impair endocytosis and lysosomal trafficking. We therefore tested lysosomal delivery of anti-ICAM/ASM/NCs in a model of ASMdeficient ECs, by treating HUVEC with imipramine, an agent that irreversibly inhibits ASM by degradation [40,41]. Imipramine-treated HUVEC abnormally accumulated sphingomyelin in intracellular compartments (Supplementary Fig. 4), yet efficiently endocytosed antiICAM/ASM/NCs (Fig. 4A), which accumulated in lysosomal vesicles (Fig. 4C and Supplementary Fig. 5). We also utilized cells genetically deficient in ASM, e.g., skin fibroblasts from NPD patients, which manifest pathological alterations characteristic of ASM-deficient NPD. FACS analysis showed that these cells express and up-regulate ICAM-1 in response to TNF-a (Supplementary Fig. 3), providing a more adequate cell model to study anti-ICAM/ASM/NC delivery. NPD fibroblasts rapidly and efficiently internalized anti-ICAM/ASM/NCs (Fig. 4B), which trafficked to lysosomal compartments (Fig. 4D). Therefore, both normal cells and cells with excessive lipid stores induced by either pharmacological or hereditary ASM deficiency showed similar uptake and lysosomal delivery of anti-ICAM/ASM/NCs (Figs. 2, 3, and 4). This is likely due to specific features of the endocytic pathway mediating uptake of anti-ICAM/ASM/NCs, CAM-mediated endocytosis, which functions independent of clathrin-coated pits, despite the presence of rhASM containing sugar-targeting moieties. Recombinant ASM, Delivered by Anti-ICAM Nanocarriers, Is Stable and Functionally Active within Lysosomal Compartments We examined the stability of anti-ICAM/ASM/NCs internalized by target cells (Fig. 5A and Supplementary Fig. 6). An immunofluorescence assay showed the loss of immu-

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noreactivity against the anti-ICAM targeting moiety after 2 h postinternalization, consistent with lysosomal degradation of this protein moiety, as observed in previous studies using radiolabeled anti-ICAM [21]. In contrast, recombinant 125I-rhASM delivered intracellularly to HUVEC by anti-ICAM/NCs was stable in this environment and showed very little, if any, degradation even 5 h after internalization within the cells. This likely reflects high natural resistance of ASM to proteolysis, characteristic of lysosomal enzymes. In addition, the delivered enzyme, a 75-kDa secreted ASM isoform [15], was partially processed to a smaller molecular weight form migrating as an ~65-kDa band, which may represent a

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mature lysosomal isoform similar to the endogenous enzyme [42]. Finally, we determined the activity of anti-ICAM/ASM/ NCs delivered intracellularly to ASM-deficient cells, e.g., NPD fibroblasts (Fig. 5B) or imipramine-treated HUVEC (not shown) by testing their ability to revert intracellular accumulation of BODIPY-FLC12-sphingomyelin (Fl-SM) in situ. Three hours after internalization, cells treated with rhASM showed only a partial reduction of Fl-SM stores, due to rather limited binding, uptake, and lysosomal delivery via endogenous pathways. In sharp contrast, FlSM levels were reduced to background in cells treated with anti-ICAM/ASM/NCs, indicating that rhASM delivery by anti-ICAM/NCs may provide significant biochemical effects and that the delivered enzyme is active within intracellular compartments (Fig. 5B). Together, these results highlight the utility of ICAMtargeted nanocarriers as an effective delivery means for lysosomal enzymes, which may permit reduction of enzyme doses necessary to attain therapeutic benefits. In addition, other features of ICAM-1 targeted systems make these vehicles attractive for LSD ERT. Particularly important in the case of types A and B NPD, targeting to ICAM-1 causes ICAM-1 ligation, which increases endogenous ASM activity in nondiseased cells [43]. Given that CAM endocytosis shares numerous common features with the pathways by which some pathogens enter ICAM-1-expressing cells [44], ICAM-1 blockage by nanocarriers may be secondarily beneficial by hindering opportunistic infections using this receptor. Previous studies have shown that targeting-induced ICAM-1 blockage is well tolerated by laboratory animals and has been utilized to attenuate inflammation [45,46]. For example, anti-ICAM/NCs does not seem to induce FcRmediated, systemic release of inflammatory factors within several hours after iv injection [34]. Importantly, FIG. 5. Anti-ICAM/NC-delivered ASM is stable and functionally active within intracellular compartments. (A) TNF-a-activated HUVEC were incubated with either FITC-labeled anti-ICAM/ASM/NCs or anti-ICAM/125I-ASM/NCs at 48C to permit binding, then washed, and warmed to 378C to permit internalization for 1, 2, or 5 h. Immunoreactive anti-ICAM on internalized nanocarriers was quantified from fluorescence micrographs in permeabilized cells. 125I-ASM was estimated by densitometry of radioblots of cell lysates. Data are means F SEM (n = 2 independent experiments). (B) Skin fibroblasts from NPD patients were incubated with BODIPY-FLC12-sphingomyelin (Fl-SM) for 48 h at 378C, then the cells were washed, treated for 3 h at 378C with control medium or medium containing either ASM or anti-ICAM/ASM/NCs, and analyzed by fluorescence microscopy. Red fluorescence in addition to green fluorescence indicates an abnormally high concentration of Fl-SM in intracellular compartments. Attenuation of lipid stores is indicated by absence of red fluorescence. Magnification bar, 10 Am. (C) Nanocarriers containing rhASM and targeting anti-ICAM bind to ICAM-1-expressing cells and are subsequently internalized via CAM-mediated endocytosis. Thus, anti-ICAM/ASM/NCs bypass glycosylation-dependent targeting and clathrin-dependent internalization and provide efficient trafficking through the endolysosomal route. Recombinant hASM is finally delivered to lysosomes in an active form and catalyzes conversion of accumulated sphingomyelin (SM) to ceramide (Cer) and phosphocholine (Pc).

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ICAM-1 is employed by leukocytes for extravasation [22] and by some pathogens as a means for tissue invasion and to overcome the blood–brain barrier [44]. Thus it is tempting to speculate that targeting lysosomal enzymes to ECs via ICAM-1 may also favor paracellular transport or transcytosis to adjacent tissues in particular vascular beds by similar mechanisms. In conclusion, this work attempted for the first time to translate emerging technologies for immunotargeting and nanocarrier delivery of therapeutics into ERT for LSDs. We have shown that delivery of lysosomal enzymes, in this case rhASM, by nanocarriers targeted to the surface molecule, ICAM-1, bypasses glycosylationand clathrin-mediated pathways and markedly enhances: (i) binding, (ii) internalization, (iii) lysosomal delivery, and (iv) in situ catalytic activity of the delivered enzyme (Fig. 5C). These features of the delivery method described, together with the additional advantages derived from utilizing ICAM-1 as a target and nanocarriers as delivery vehicles, may improve ERT for LSDs.

MATERIALS

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METHODS

Antibodies and reagents. The monoclonal antibody against human ICAM-1 was mAb R6.5 [19]. Secondary antibodies were from Molecular Probes (Eugene, OR, USA). Green fluorescent polystyrene latex beads, 100 nm in diameter, were from Polysciences (Warrington, PA, USA). rhASM was produced in CHO cells and purified as described [15]. Unless otherwise stated, all other reagents were from Sigma (St. Louis, MO, USA). Preparation of anti-ICAM/ASM/NCs. FITC latex beads were coated with rhASM, anti-ICAM, or a mixture (50:50) of rhASM and anti-ICAM [21]. Radiolabeled nanocarriers were prepared by mixing 125I-rhASM and antiICAM at varying ratios (1:99, 5:95, 50:50, 100:0). After separation of the free counterparts by centrifugation, the amount of 125I-rhASM coated onto the nanocarriers was determined in a gamma counter. The final diameter of the coated nanocarriers ranged from 200 to 300 nm, as determined by dynamic light scattering [33]. Cell culture. HUVEC (Clonetics, San Diego, CA, USA) and skin fibroblasts from type B Niemann–Pick disease patients (NPD fibroblasts) were cultured at 378C, 5% CO2, and 95% relative humidity in supplemented M199 or DMEM (Gibco BRL, Grand Island, NY, USA), respectively [19]. For experiments, cells were seeded onto 12-mm2 gelatin-coated coverslips in 24-well plates. To generate an endothelial cell model for ASM deficiency, HUVEC were treated with 10 AM imipramine to degrade endogenous ASM [40,41]. Cells were always treated overnight with TNF-a prior to experiments. Binding and internalization of ASM nanocarriers by cultured cells. HUVEC, imipramine-treated HUVEC, or NPD fibroblasts were utilized to compare the delivery capacity of FITC ASM/NCs, anti-ICAM/NCs, or antiICAM/ASM/NCs. Nanocarrier binding to the cell surface was determined by fluorescence microscopy after 30 min incubation at 48C. For competition experiments, cells were incubated with nanocarriers in the presence of control medium or medium containing 33.5 ng/Al anti-ICAM or 25 mM mannose 6-phosphate. Internalization of nanocarriers within the cells was determined after incubation for varying periods of time at 378C. Cells were washed, fixed, and treated with Texas red goat anti-mouse IgG to label nanocarriers bound to the cell surface. The mechanism of nanocarrier uptake was determined in the presence of an inhibitor of CAM-mediated endocytosis, 3 mM amiloride, or of clathrin-mediated pathways, 50 AM MDC [19]. Samples were analyzed by fluorescence microscopy (Nikon Eclipse TE2000-U) using 60 PlanApo objectives. Images were obtained

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with a Hamamatsu Orca-1 CCD camera and analyzed using ImagePro 3.0 software to determine the amount of nanocarriers internalized (green labeled) with respect to the total number of nanocarriers (green + red labeled) associated with the cells [39]. In parallel, the amount of rhASM intracellularly delivered was determined in a gamma counter after incubation of cells with free 125I-rhASM, 125 I-ASM/NCs, or anti-ICAM/125I-ASM/NCs for 1 h at 378C [39]. Lysosomal trafficking of anti-ICAM/ASM nanocarriers. Lysosomes of TNF-a-activated HUVEC, imipramine-treated HUVEC, or NPD fibroblasts were labeled with Texas red dextran (10,000 MW) [34]. The cells were then incubated with nanocarriers for 30 min at 48C, then washed, and warmed to 378C for varying periods of time [39]. Cells were fixed, incubated with blue Alexa Fluor 350 goat anti-mouse IgG to stain nanocarriers bound to the cell surface, and then analyzed by fluorescence microscopy to determine colocalization of green internalized nanocarriers within Texas red dextran lysosomes. The results were confirmed by immunolabeling lysosomes with anti-LAMP-1 (Calbiochem, La Jolla, CA, USA). Stability of anti-ICAM/ASM nanocarriers delivered intracellularly. AntiICAM on the surface of internalized FITC nanocarriers was detected in permeabilized HUVEC by staining with Texas red goat anti-mouse IgG and analysis by fluorescence microscopy [21]. In parallel, the stability of 125 I-rhASM internalized via anti-ICAM/NCs was estimated from cell lysates containing similar amounts of total cell protein, as described ibid. These were separated by SDS–PAGE and blotted onto a nitrocellulose membrane. 125I-rhASM protein bands were visualized by exposing an Xray film to the radioblot and quantified by densitometry. Functional activity of anti-ICAM/ASM nanocarriers in situ. NPD fibroblasts and HUVEC were incubated overnight with fluorescent FlSM and Fl-SM plus 10 AM imipramine, which degrades endogenous ASM [40,41], respectively. Fl-SM fluoresces at 530 nm (green) when sphingomyelin or its product, ceramide, is present at physiological concentrations in cells. However, Fl-SM fluoresces at 620 nm (red) at high concentrations, permitting qualitative estimation of intracellular accumulation of Fl-SM by fluorescence microscopy. Cells with high levels of Fl-SM were treated with rhASM or anti-ICAM/ASM/NCs for 30 min at 378C, then washed, and analyzed 3 h after to determine whether delivered rhASM could revert lysosomal accumulation of sphingomyelin in situ. Statistics. Unless otherwise stated, the data were calculated as the means F standard deviation, where statistical significance was determined by Student’s t test.

ACKNOWLEDGMENTS We thank John Leferovich and Christine Gajewski for their outstanding technical assistance. This work was supported by American Heart Association 0435181N (S.M.), National Institutes of Health HD 28607 (E.S.) and HL/GM 71175-01 (V.M.), and Department of Defense PR 012262 (V.M.). RECEIVED FOR PUBLICATION APRIL 12, 2005; REVISED JULY 19, 2005; ACCEPTED JULY 19, 2005.

APPENDIX A. SUPPLEMENTARY DATA Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ymthe. 2005.07.687. REFERENCES 1. Brady, R. O. (2003). Enzyme replacement therapy: conception, chaos and culmination. Philos. Trans. R. Soc. London B Biol. Sci. 358: 915 – 919. 2. Desnick, R. J., and Schuchman, E. H. (2002). Enzyme replacement and enhancement therapies: lessons from lysosomal disorders. Nat. Rev. Genet. 3: 954 – 966. 3. Futerman, A. H., and van Meer, G. (2004). The cell biology of lysosomal storage disorders. Nat. Rev. Mol. Cell. Biol. 5: 554 – 565.

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