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

Effect of flow on endothelial endocytosis of nanocarrierstargeted to ICAM-1

Tridib Bhowmicka, Erik Berkb, Xiumin Cuib, Vladimir R. Muzykantovb,c,*, and Silvia Muroa,d,*

aInstitute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD,20742, USAbInstitute for Environmental Medicine, University of Pennsylvania School of Medicine,Philadelphia, PA 19104, USAcDepartment of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA19104, USAdFischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA

AbstractDelivery of drugs into the endothelium by nanocarriers targeted to endothelial determinants mayimprove treatment of vascular maladies. This is the case for intercellular adhesion molecule 1(ICAM-1), a glycoprotein overexpressed on endothelial cells (ECs) in many pathologies. ICAM-1-targeted nanocarriers bind to and are internalized by ECs via a non-classical pathway, CAM-mediated endocytosis. In this work we studied the effects of endothelial adaptation tophysiological flow on the endocytosis of model polymer nanocarriers targeted to ICAM-1 (anti-ICAM/NCs, ~180-nm diameter). Culturing established endothelial-like cells (EAhy926 cells) andprimary human umbilical vein ECs (HUVECs) under 4 dyn/cm2 laminar shear stress for 24 hresulted in flow adaptation: cell elongation and formation of actin stress fibers aligned to the flowdirection. Fluorescence microscopy showed that flow-adapted cells internalized anti-ICAM/NCsunder flow, although at slower rate versus non flow-adapted cells under static incubation (~35%reduction). Uptake was inhibited by amiloride, whereas marginally affected by filipin andcadaverine, implicating that CAM-endocytosis accounts for anti-ICAM/NC uptake under flow.Internalization under flow was more modestly affected by inhibiting protein kinase C, whichregulates actin remodeling during CAM-endocytosis. Actin recruitment to stress fibers thatmaintain the cell shape under flow may delay uptake of anti-ICAM/NCs under this condition byinterfering with actin reorganization needed for CAM-endocytosis. Electron microscopy revealedsomewhat slow, yet effective endocytosis of anti-ICAM/NCs by pulmonary endothelium after i.v.injection in mice, similar to that of flow-adapted cell cultures: ~40% (30 min) and 80% (3 h)internalization. Similar to cell culture data, uptake was slightly faster in capillaries with lowershear stress. Further, LPS treatment accelerated internalization of anti-ICAM/NCs in mice.Therefore, regulation of endocytosis of ICAM-1-targeted nanocarriers by flow and endothelial

© 2011 Elsevier B.V. All rights reserved.*Correspondence should be addressed to: Silvia Muro (endocytosis and intracellular transport of drug delivery systems): Institutefor Bioscience and Biotechnology Research, 5115 Plant Sciences Building, College Park, MD 20742-4450, Tel. 1+301-405-4777,Fax. 1+301-314-9075, [email protected]. *Vladimir Muzykantov (endothelial targeting of drug delivery systems): University ofPennsylvania, 1 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-4215, USA, Tel. 1+215-898-9823, Fax.1+215-898-0868, [email protected].

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NIH Public AccessAuthor ManuscriptJ Control Release. Author manuscript; available in PMC 2013 February 10.

Published in final edited form as:J Control Release. 2012 February 10; 157(3): 485–492. doi:10.1016/j.jconrel.2011.09.067.

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status may modulate drug delivery into ECs exposed to different physiological (capillaries vs.arterioles/venules) or pathological (ischemia, inflammation) levels and patterns of blood flow.

KeywordsVascular endothelium; ICAM-1; nanocarrier uptake; endocytosis; flow adaptation

INTRODUCTIONTargeting of nanocarriers into endothelial cells (ECs) in the vasculature holds promise tooptimize drug delivery for cardiovascular, metabolic and oncologic diseases. In particular,intercellular adhesion molecule 1 (ICAM-1) [1] is a good target for endothelial drugdelivery, as shown in numerous works describing effective endothelial accumulation oftherapeutic cargoes, imaging agents, and their carriers in cell cultures and animal models [2–9]. In addition, ECs poorly internalize ICAM-1 antibodies (anti-ICAM) [10]. However,multivalent binding of nanocarriers (e.g., spherical polymer particles with diameter <1,000nm) coated with multiple copies of anti-ICAM leads to endocytic uptake and intracellulardelivery of carriers and their cargoes by ECs [7, 8, 11–14].

Understanding the factors regulating endothelial internalization of nanocarriers is necessaryfor effective intra-endothelial drug delivery. Studies in EC cultures revealed that uptake ofanti-ICAM nanocarriers (anti-ICAM/NCs) occurs via cell adhesion molecule (CAM)-mediated endocytosis, a unique pathway sensitive to inhibition by amiloride and distinctfrom previously known endothelial endocytic mechanisms [13, 15]. It has been also foundthat CAM-endocytosis involves signaling through protein kinase C (PKC) and Rho-dependent kinase (among others), which mediate transient rearrangement of the actincytoskeleton into stress fibers [13, 15]. This cytoskeletal rearrangement is necessary forinternalization of ICAM-1-targeted nanocarriers by ECs [13, 15].

These mechanistic studies were performed in static cell cultures, and electron microscopyhas showed that anti-ICAM/NCs injected in mice are internalized by vascular ECs,confirming cell culture data [8, 16]. Several works have focused on the effects of flow onendothelial binding of CAM-targeted carriers, where typically shear stress negativelyimpacts binding of said carriers to the endothelial surface [17–22]. However, the effects offlow and flow adaptation on the endocytosis of these nanocarriers within endothelial cellshave not been previously explored and, therefore, we decided to focus on this aspect in thiswork. The pace, efficacy and mechanism of endocytosis of ICAM/NCs may be different atphysiological conditions involving ECs exposed to flow. Flow affects adhesive interactionof carriers with endothelium (rolling, binding, and detachment) [17–19, 21, 22]. Mostimportantly for endocytosis, flow-adapted ECs display phenotypic features that are differentfrom those displayed by cells grown in static cultures, which may affect internalization. Forexample, flow-induced activation of PKC and Rho [23, 24] might stimulate nanocarrierendocytosis. Flow causes cells to align and rearrange their cytoskeleton (including actinfilaments) differently from that in static endothelial cultures [23, 25, 26].

The net effect of flow adaptation on endothelial endocytosis of anti-ICAM/NCs is difficultto predict, yet crucial in order to evaluate the potential of this drug delivery strategy inphysiological conditions. To address this question, in the present study we have investigatedendocytosis of model anti-ICAM/NCs (~180 nm diameter) under flow conditions in cellculture and mice using multi-label fluorescence microscopy, radioisotope labeling, andtransmission electron microscopy.

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MATERIALS AND METHODSAntibodies and reagents

Monoclonal antibodies to human or mouse ICAM-1 (anti-ICAM) were R6.5 [1] and YN1[27]. Secondary antibodies were from Jackson Immunoresearch (West Grove, PA). FITC-labeled 100-nm diameter polystyrene particles were from Polysciences (Warrington, PA).Na125I and Pierce iodination beads were from Perkin Elmer - Analytical Sciences(Wellesley, MA) and Thermo Scientific (Rockford, IL). Cell media and supplements werefrom Cellgro (Manassas, VA) or Gibco BRL (Grand Island, NY). All other reagents werefrom Sigma Aldrich (St. Louis, MO).

Anti-ICAM nanocarriersPrototype anti-ICAM nanocarriers (anti-ICAM/NCs) were prepared by adsorbing anti-ICAM or a mix of anti-ICAM and 125I-IgG (95:5 ratio) on the surface of 100-nm diameterpolystyrene particles, as described [12]. Non-bound counterparts were separated bycentrifugation [12]. This protocol rendered coated particles of ~180 nm in diameter, as perdynamic light scattering [12].

Cell cultureHuman umbilical vein ECs, HUVECs (Clonetics, San Diego, CA), were cultured insupplemented M199 medium (GibcoBRL, Grand Island, NY). EAhy926 cells [28], werecultured in supplemented DMEM medium [13]. For control experiments herein referred toas static conditions, cells were seeded onto gelatin-coated coverslips, grown to confluenceand incubated with anti-ICAM/NCs, both under static conditions. For experiments hereinreferred to as flow conditions, cells seeded on coverslips were first grown to confluenceunder static conditions, then placed in a parallel plate flow chamber (RC-30HV, WarnerInstruments Inc, Hamden, CT) and subjected for 24 h to laminar flow by perfusion using aperistaltic pump (Pharmacia Biotech AB, Appsala, Sweden) [19]. These cells, hereinreferred to as flow-adapted cells, were incubated with anti-ICAM/NCs under shear stress.All cells were treated overnight with TNF- to mimic ICAM-1 overexpression in manydiseases, as described [12].

Flow alignment of cellsCells were perfused at 37°C under a shear stress of 4 dyn/cm2 for 24 h, fixed with 2%paraformaldehyde, permeabilized by 0.2% Triton X-100, and incubated with Texas Redphalloidin to label filamentous actin [13]. As a control for endothelial phenotype, cells werestained with anti-PECAM-1 (mAb 62 [29]) and a Texas Red-secondary antibody. Sampleswere analyzed by phase-contrast and fluorescence microscopy (Eclipse TE2000-U, Nikon,Melville, NY) using a 40x/NA1.4 PlanApo objective (Nikon, Melville, NY). Cellmorphology (elongation in the direction of flow) and cytoskeletal reorganization(appearance of actin stress fibers aligned to the flow) were visualized from micrographsobtained with an Orca-1 CCD camera (Hamamatsu, Bridgewater, NJ) using ImagePro 3.0(Media Cybernetics, Silver Spring, MD), and compared to that of cells cultured in staticconditions [19].

Targeting and internalization of anti-ICAM nanocarriersTNF-α activated, 24 h-flow adapted cells were perfused at 37°C with FITC anti-ICAM/NCs(4.5 × 1010 particles/mL, 4 dyn/cm2 shear stress) in a continuous manner, allowing bindingand endocytosis as in a physiological situation. When indicated, cells were perfused withanti-ICAM/NCs for 15 min to allow binding, after which non-bound carriers were washed tobetter synchronize endocytosis of pre-bound nanocarriers [13]. Similar experiments were

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conducted in the presence of 3 mM amiloride or 0.5 µM phorbol 12-myristate 13-acetate -PMA- (which inhibit CAM endocytosis), 50 µM monodansylcadaverine (MDC, an inhibitorof clathrin-coated pits), or 1 µg/ml filipin (which inhibits caveolar endocytosis) [13]. Cellsgrown and incubated with anti-ICAM/NCs under standard static conditions were used forcomparison. Surface-bound nanocarriers were stained using Texas Red goat anti-mouse IgGthat recognizes anti-ICAM on particles located on the cell surface (yellow color) but is notaccessible to those endocytosed within the cell (green color) [13]. Samples were imaged byfluorescence microscopy and phase-contrast was used to delimit the cell borders [13].

In vivo targeting and visualization of uptake of anti-ICAM nanocarriersControl C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine, USA) or mice pre-treatedfor 16 h with 1 mg/kg body-weight bacterial lipopolysaccharide (LPS) administeredintraperitonealy, were anesthetized, injected intravenously (i.v.) with anti-ICAM/NCs (1.8 ×1013 particles/kg), and euthanized 30 min or 3 h after injection. Targeting of nanocarrierswas assessed by measuring the radioactivity in lungs collected without blood removal byperfusion or, alternatively, collected after blood perfusion to remove blood, and calculatedas the percentage of the injected dose (%ID). Alternatively, samples were processed fortransmission electron microscopy (TEM). Lungs were fixed in 2.5% glutaraldehyde in 0.1 Msodium cacodylate buffer, treated with 2% osmium tetroxide in 0.1 M cacodylate,dehydrated, and embedded in LR White resin (EM Sciences, Malvern, PA). Ultrathinsections (~90 nm) were cut using an Ultracut microtome (Reichert, Wien, Austria) andstained with 1% uranyl acetate and lead citrate. TEM micrographs were taken using 2100FField Emission TEM (Jeol, Waterford, VA). Endocytosis of anti-ICAM/NCs by pulmonaryECs in capillaries (≤5-µm diameter) versus 10–50-µm diameter vessels (arterioles andvenules) was quantified as the percentage of all carriers visualized in association with thecells (surface-bound and internalized). These studies were performed according to IACUCregulations and University of Maryland standards.

StatisticsData were calculated as mean ± standard error of the mean (SEM).

RESULTSEfficient binding of anti-ICAM nanocarriers to flow-adapted endothelial cells

Primary ECs (HUVECs) and established endothelial-like cells (EAhy926) were firstincubated for 24 h under a laminar shear stress of 4 dyn/cm2 (Fig. 1 and Supp. Fig. 1,respectively), a flow in the range of that in arterioles and venules [30]. Under this condition,both cell types showed evident signs of flow adaptation absent in static counterparts. Thiswas clearly detectable by visualization of the cell morphology by phase-contrast andfluorescence microscopy of filamentous actin, showing cell elongation in the direction of theflow and massive rearrangement of actin into flow-aligned stress fibers (Fig. 1A and Supp.Fig. 1). Staining of the endothelial marker CD31, primarily localized at intracellular borders(Supp. Fig. 2), confirmed the integrity and confluence of the cell monolayer despite such arearrangement of the cytoskeleton.

To trace binding of nanocarriers under flow conditions in flow-adapted cell cultures, weused FITC-labeled anti-ICAM/NCs (diameter ~180 nm), as in our previous studies understatic conditions [12, 13] or in mice [7]. Anti-ICAM/NCs bound similarly well under flowconditions to flow-adapted ECs as to non flow-adapted cells under static conditions, even atshort periods of time (e.g., 5 min and 15 min, Fig. 2B). Semi-quantitative imaging analysisrevealed that maximal amplitude of binding under flow approached ~125±10 nanocarriers/cell at 2 h, in the case of primary HUVECs, in agreement with previous results on this cell

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type under static conditions [13]. This pattern was also observed in the case the endothelial-like cell line EAhy926, where nanocarrier binding to flow-adapted cells under flowconditions reached 133.3 28.4% of binding to non-flow adapted cells in static cultures (notshown).

Rate of uptake of anti-ICAM nanocarriers by flow-adapted endothelial cellsWe next used a double-label fluorescence microscopy technique that allows us to distinguishbetween carriers bound to the cells surface vs those internalized within the cell body [13](Materials and Methods). Despite the fact that no major differences were observed inbinding of anti-ICAM/NCs to flow-adapted cells under flow conditions vs non flow-adaptedcells under static conditions, internalization was slower and less effective in the former:flow-adapted HUVECs internalized ~30% less anti-ICAM/NCs at 30 min of perfusion vsHUVECs grown and incubated with nanocarriers under static conditions (Fig. 2). Also,nanocarrier uptake under flow reached a saturation level ~15% lower than in static cells(Fig.2). This outcome was also observed for EAhy926 cells, where the level of nanocarrieruptake was reduced by ~30% vs that in non-flow adapted cells under static conditions(73.1±8.6% of static cultures, Supp. Fig. 3).

To better define differences in the early phases of endocytosis, we used a pulse-chaseincubation mode. Non flow-adapted cells or flow-adapted cells were incubated under staticconditions or perfused under flow, respectively, with anti-ICAM/NCs for 15 min to allowanchoring to the cell surface. Then, non-bound nanocarriers were eliminated, followed bystatic incubation or perfusion for 2 h. This protocol allows to discern the effect of flowadaptation on carrier endocytosis, eliminating potential effect of flow on binding that mayconfound the results. Under these conditions, where the short binding time imitates in vivodrug delivery more appropriately than continuous incubation, flow-adapted HUVECsincubated with carriers under flow displayed lower efficacy of uptake vs static controls(~40% reduction, Fig. 3).

Nevertheless, even under these restrictive conditions allowing only 15 min binding-timeinterval, primary flow-adapted ECs internalized anti-ICAM/NCs under flow with an efficacylevel at the same order of magnitude as non flow-adapted cells incubated with nanocarriersunder static condition. Indeed, under this pulse-chase exposure, the maximal level ofendocytosis achieved at 2 h perfusion was ~82.3±2.7% of the uptake of nanocarriersperfused in a continued manner (Fig. 3B).

Flow-adapted endothelial cells internalize anti-ICAM nanocarriers via CAM-mediatedendocytosis

To test whether delayed rate of the uptake of anti-ICAM/NCs by flow-adapted endotheliumis due to utilization of an alternative endocytic mechanism, cells were pretreated withinhibitors that affect endocytosis mediated by CAM (amiloride and PMA), clathrin pits(MDC), or caveoli (filipin) [13]. However, studies using these pharmacological agentsshowed that maximal inhibition of endocytosis of anti-ICAM/NCs was achieved byamiloride (Fig.4), similarly to previous results in non flow-adapted cells in static conditions[13]. PMA, which affects the role of PKC in actin remodeling during CAM endocytosis,inhibited uptake less markedly than in non flow-adapted cells under static conditions (Fig. 4and [13]). Filipin and MDC less effectively affected uptake (Fig. 4).

Endothelial targeting and endocytosis of anti-ICAM nanocarriers in vivoThese data indicate that ECs under physiological conditions of blood flow in vessels mayinternalize anti-ICAM/NCs, although with slower kinetics than in static cell cultures. To testthis hypothesis, we evaluated the endocytosis of anti-ICAM/NCs in the pulmonary

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vasculature, which represents a major fraction of the total body vasculature receiving thewhole venous cardiac output and, therefore, is a preferential site for specific endothelialtargeting by anti-ICAM/NCs after i.v. administration [7, 8, 12].

We first injected anti-ICAM/NCs i.v. in C57Bl/6 mice and determined their accumulation inthe lungs. As previously reported [7, 8, 12], anti-ICAM/NCs specifically and efficientlyaccumulated in this organ: 37.2±3.5% of the injected dose (% ID) by 30 min, whichrepresents 16-fold higher accumulation over the pulmonary uptake of control IgG/NCs (Fig.5A). Effective pulmonary targeting led to fast blood clearance of anti-ICAM/NCs (Fig.5B).Further reflecting specificity, LPS treatment to mimic inflammation increased pulmonaryaccumulation of anti-ICAM/NCs by 1.5-fold (data not shown).

This level of accumulation of anti-ICAM/NCs in the lungs corresponded to nanocarriersfirmly bound to and/or internalized within the pulmonary endothelium rather than carrierscirculating through this organ. This was indicated by the fact that anti-ICAM/NCs rapidlydisappeared from blood and were only retained at low level in the circulation for severalhours: only 8.9±2.9% ID and 4.5±0.2% ID anti-ICAM/NCs remained in the circulation at 30min and 3 h after injection, respectively (Fig. 5B). Further confirming this, perfusion of theblood off the lungs did not decrease the level of anti-ICAM/NCs (94.9±8.4% of non-perfused condition) and this level of accumulation did not vary between 30 min and 3 h (Fig.5C).

We then injected anti-ICAM/NCs in mice and imaged by TEM the pulmonary vasculaturepostmortem to evaluate endocytosis of anti-ICAM/NCs in capillaries (≤ 5 µm diametervessels) and vessels ranging between 10–50 µm in diameter (arterioles and venules). Asshown in Fig. 6A, anti-ICAM/NCs were found bound to the cell surface and internalizedwithin vesicular compartments (indicative of endocytosis) in EC of the pulmonaryvasculature. Endocytosis of anti-ICAM/NCs occurred within minutes and markedlyincreased after several hours: ranging from ~30–40% to ~65–70% of all cell-associatednanocarriers at 30 min and 3 h, respectively (Fig. 6B). This suggests that endocytosis ofanti-ICAM/NCs in vivo correlates better with that of flow-adapted ECs under flow (~45% at30 min) rather than non-flow adapted cells under static conditions (~70%, 30 min).Nanocarrier internalization was slightly faster in capillaries, although a similar level ofendocytosis was reached in both types of vessels by 3 h (Fig. 6B). LPS treatment acceleratedendocytosis in capillaries (Fig. 6C), indicating that intracellular drug delivery by anti-ICAM/NCs may gain efficiency in areas pathologically altered.

DISCUSSIONTargeting drugs into the endothelium by ICAM-1-addressed nanocarriers may improvetreatment of vascular maladies. This strategy enhances delivery of a variety of agents intothe vascular endothelium in cell cultures and animal models, including probes forinflammation and oxygen sensing, imaging, and therapeutic enzymes for anti-oxidantinterventions or genetic lysosomal conditions [2, 3, 7–9, 12, 14, 15]. Several works hadreported how the shear stress of the blood flow affects binding of nanocarriers targeted tocell adhesion molecules [17–22], yet the intracellular delivery of anti-ICAM/NCs inendothelial cells under physiological and inflammatory conditions had not been examined.The work presented here reveals that CAM-mediated endocytosis of anti-ICAM/NCs ismodulated by effects of flow and the EC status: endothelial adaptation to flow delayedendocytosis of anti-ICAM/NCs in control conditions (Fig. 2, Supp. Fig. 3), yet inflammationseemed to favor uptake (Fig. 6). This phenomenon was observed in primary ECs (HUVEC),an EC-like cell line (EAhy926 cells), and pulmonary ECs in vivo, indicating that these cells

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use a similar mechanism (CAM-mediated endocytosis) for internalization of anti-ICAM/NCs [13].

Delayed endocytosis of anti-ICAM/NCs by flow-adapted ECs may be, at least in part, due torecruitment of the actin cytoskeleton to maintenance of the cell shape under flow, celljunctions that regulate the vascular permeability, and focal adhesions to the substrate [23,25, 26, 31, 32]. Both HUVECs and EAhy926 cells displayed formation of actin stress fibersaligned to the direction of flow (Fig. 1 and Supp. Fig. 1). Commitment of actin in flow-induced stress fibers may impair the rapid reorganization of the actin cytoskeleton neededfor CAM-mediated endocytosis [13, 15]. Cell-bound anti-ICAM/NCs align with endocyticactin stress fibers induced by the anti-ICAM/NCs binding during CAM-mediatedendocytosis in static cells [13, 15]. This may be mediated by interaction of actin cross-linkers such as α-actinin, cortactin, or ezrin-radixin-moesin family proteins with thecytosolic domain of ICAM-1 [13, 33, 34]. It is possible that this supports the formation ofmembrane invaginations and endocytic vesicles that transport anti-ICAM/NCs into the ECbody [13, 15]. Therefore, conflicting commitments of actin recruitment to the stress fibersinduced by flow vs anti-ICAM/NCs can contribute to explain why the latter process occursmore slowly in flow-adapted vs static ECs (Figs. 2, 3 and Supp. Fig. 3).

Although involvement of the actin cytoskeleton is a feature common to most endocyticpathways, this feature typically relates to actin polymerization into thin filaments or actincups around endocytic vesicles, whereas formation of thick bundles of actin are rather aunique feature of CAM-mediated endocytosis [35]. Formation followed by shortening ofsaid fibers have been observed along with progression of endocytosis of anti-ICAM/NCsand impairing formation of actin stress fibers inhibits internalization of these carriers [13,15]. Generation of such fibers is linked to activation of protein kinase C (PKC), RhoA andthe Rho-dependent kinase ROCK, triggered upon binding of anti-ICAM/NCs to ICAM-1 onthe endothelial surface [13, 15, 36]. Treatment of flow-adapted ECs with PMA (to impairPKC) inhibits uptake of anti-ICAM/NCs, indicating that PKC-mediated signaling is stillinvolved in CAM-endocytosis under flow. However, this inhibition was less markedcompared to non flow-adapted ECs under static conditions (Fig. 4 and [13]), supporting thenotion that commitment of actin stress fibers to maintain cell structures under flow may playa role in delayed actin remodeling needed for CAM-mediated endocytosis of anti-ICAM/NCs.

In contrast to PKC inhibition, the effect of amiloride (a drug that inhibits sodium/protonexchanger proteins -NHEs- involved in CAM endocytosis) on the endocytosis of anti-ICAM/NCs by flow-adapted ECs was similar to that in static cell cultures (Fig. 3 and [13]).In particular, we previously showed that NHE1 is recruited to areas of the endothelialplasmalemma where ICAM-1 is engaged by anti-ICAM/NCs, and subsequently interactswith ICAM-1 in these regions [15]. Given that NHE1 serves as an actin cross-linker [37], wepostulated that interaction of NHE1 with ICAM-1 may provide support to ICAM-1 linkageto the cytoskeleton during CAM-endocytosis [15]. Yet, in view of the results shown hereunder flow conditions it is likely than other function(s) of NHE1 may mediate CAM-endocytosis.

From a general perspective, similar molecules mediate signaling and actin remodelingduring other endocytic events, e.g., PKC is involved actin remodeling in certain clathrin-and caveolar-mediated pathways, and NHE1 ion exchange is involved in macropinocytosis[38–40]. However, signaling associated to ICAM-1 engagement by anti-ICAM/NCs differsin that molecules such as phospholipase C (PLC) or phosphatidylinositol 3 kinase (PI3K) donot signal upstream of these molecular players in the case of CAM-mediated endocytosis[13, 15]. It is unknown whether flow affects endothelial endocytosis of nanocarriers targeted

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to markers of alternative endocytic pathways, e.g., associated to clathrin-coated pits orcaveolar compartments. Several works suggest that this may be the case, given that elementscommonly involved in endocytosis, such as the actin cytoskeleton, signaling, protein coats,lipid raft platforms, and other elements are differently regulated under static vs flowconditions [17, 23–26].

It is noteworthy from a translational standpoint that our results in cell cultures under flowconditions agreed well with the rate of endocytosis of anti-ICAM/NCs in the lungvasculature in mice (Figs. 2 and 6). As an example, pulmonary ECs in capillaries andarterioles/venules effectively internalized anti-ICAM/NCs under physiological flow of thecirculation (~80% uptake 3 h after injection), supporting the potential of this strategy forvascular drug delivery. Internalization occurred gradually at a rate similar to that of flow-adapted cells in culture (~30–40% uptake 30 min after injection, vs ~70–80% in staticconditions), indicating that CAM-endocytosis may be delayed in vivo. This could decreasethe bioavailability of therapeutics intended for intracellular delivery (e.g., lysosomalenzymes [7, 8]), while favoring surface retention of nanocarriers, e.g., useful for delivery ofthrombolytics [10]. Delayed internalization could also thus hinder therapeutic effects, e.g., ifthere is relatively fast release of a cargo from the carrier while located in the cell surface.

Also, endocytosis occurred somewhat faster in smaller vessels, in agreement with theirlower shear stress. Pathological stimuli, such as LPS-induced systemic inflammation(known to enhance ICAM-1 expression) accelerated internalization of anti-ICAM/NCs inpulmonary capillaries, which may favor intra-endothelial drug delivery in vascular areasaffected by pathology, especially conducive of ICAM-1 targeted nanocarriers. We havepreviously found that concomitant endocytosis of ICAM-1 during uptake of the anti-ICAM/NC removes ICAM-1 from the endothelial plasma membrane only temporarily followed byICAM-1 reappearance on the cell surface within 15–30 min, where it supports new roundsof binding and endocytosis of anti-ICAM/NCs [5]. Taken together with present data, theseresults indicate that anti-ICAM/NCs provide a robust approach for sustained intraendothelialdrug delivery under physiological and pathological conditions in the vasculature.

CONCLUSIONSWe have identified a new factor, i.e., flow and physiological adaptation to flow, controllingintra-endothelial delivery via CAM in the vasculature. Regulation of endothelial CAM-endocytosis by flow may modulate drug delivery mediated by ICAM-1-targetednanocarriers into ECs exposed to different levels and patterns of blood flow, e.g., incapillaries vs larger vessels and, perhaps, in venous vs arterial vascular beds, or in areas ofpathological flow reduction such as those affected by ischemia. Studies similar to this one inthe context of other endocytic pathways may benefit our understanding of the behavior andoptimization of drug delivery vehicles targeted for intra-endothelial drug delivery

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank Dr. Marian Nakada (Centocor Ortho Biotech Inc., Horsham, PA) for providing anti-PECAM, Dr. VirajMane (Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD) for helpon in vivo experiments, and the Nanocenter and Laboratory for Biological Ultrastructure (University of Maryland,College Park, MD) for assistance on TEM. We acknowledge grants R01 HL087036 and R01 EB006818 (VM),AHA 09BGIA2450014 and R01 HL098416 (SM).

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Abbreviations

CAM cell adhesion molecule

FITC fluorescein isothiocyanate

HUVEC human umbilical vein endothelial cells

ICAM-1 intercellular adhesion molecule 1

IgG immunoglobulin G

i.v. intravenous

LPS lipopolysaccharide

MDC monodansylcadaverine

NCs nanocarriers

PKC protein kinase C

PMA phorbol 12-myristate 13-acetate

ROCK Rho-dependent kinase

TEM transmission electron microscopy

TNF-α tumor necrosis factor α

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Figure 1. Binding of anti-ICAM/NCs to flow-adapted ECs under flow conditionsTNF-α activated HUVECs were cultured under static conditions (left panels) or subjected toa laminar flow of 4 dyn/cm2 shear stress for 24 h (right panels). (A) Cells were then fixed,permeabilized, incubated with Texas red-labeled phalloidin, and analyzed by phase-contrastand fluorescence microscopy to visualize the cell morphology and actin filaments,respectively. The arrows show the direction of flow. (B) Fluorescence micrographs showbinding of FITC anti-ICAM/NCs to TNF-α-activated HUVECs at either 5 min or 15 minunder static conditions (37°C), or carriers perfused for the same time and temperature at 4dyn/cm2 over TNF-α activated, flow-adapted HUVECs. Scale bar = 10 µm.

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Figure 2. Effect of shear stress on the uptake of anti-ICAM/NCs by flow-adapted ECs(A) Fluorescence micrographs showing internalization of FITC-labeled (green, arrows) anti-ICAM/NCs incubated with TNF-α activated, non flow-adapted HUVECs for 2 h at 37°Cunder static conditions, or perfused for 2 h at 37°C and 4 dyn/cm2 over TNF-α activated,flow-adapted HUVECs. Counterstaining with Texas red-labeled secondary antibody revealssurface-accessible, non-internalized anti-ICAM/NCs (yellow, arrowheads). The arrow on theupper-left corner of the panel marked as “Flow” shows the direction of the flow. Scale bar =10 µm. (B) Automatic quantification from fluorescence micrographs compares theinternalization kinetics of anti-ICAM/NCs by non flow-adapted cells under static vs flow-adapted cells under flow conditions, as a function of the maximum uptake obtained in static

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cells for 120 min. Data are Mean ± SEM (n≥ 15 cells, 2 experiments). * P ≤ 0.05, ** P ≤0.01, *** P ≤ 0.001, by Student’s t test.

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Figure 3. Reduced, yet significant, uptake under flow of anti-ICAM/NCs by flow-adapted ECs(A) Fluorescence micrographs showing internalization of FITC (green, arrows) anti-ICAM/NCs incubated at 37°C with TNF-α activated, non flow-adapted HUVECs under staticconditions, or perfused at 4 dyn/cm2 over flow-adapted cells. In both cases, cells wereincubated with anti-ICAM/NCs for 15 min to allow binding, then non-bound carriers werewashed, and internalization was assessed by incubation for 2 h. Counterstaining with Texasred-secondary antibody revealed surface-accessible, non-internalized anti-ICAM particles(yellow, arrowheads). The arrow on the upper-left corner of the “Flow” panel shows thedirection of flow. Scale bar = 10 µm. (B) Quantification from fluorescence micrographscompares the level of internalization of anti-ICAM/NCs under flow conditions by flow-adapted cells to that observed under static conditions in non flow-adapted cultures,depending on whether cells were subjected to 2 h continuous incubation (Cnt) with anti-ICAM/NCs or 15 min-binding pulse followed by 2 h-internalization chase (Pls-Chs). Dataare Mean ± SEM (n 10 cells, 2 experiments). * P ≤ 0.05 by Student’s t test.

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Figure 4. Mechanism of uptake under flow of anti-ICAM/NCs by flow-adapted ECs(A) Fluorescence micrographs showing internalization of FITC (green, arrows) anti-ICAM/NCs perfused for 2 h at 37°C and 4 dyn/cm2 over TNF-α activated, flow-adapted HUVECs,either in control cell media (Ctrl) or media containing an inhibitor of CAM-mediatedendocytosis, 3 mM amiloride (Amil). Counterstaining with Texas red-secondary antibodyreveals surface-accessible, non-internalized anti-ICAM particles (yellow, arrowheads). Thearrow on the bottom-left corner of the panel marked as “Ctrl” shows the direction of flow.Scale bar = 10 µm. (B) Quantification of fluorescence micrographs compares the level ofinternalization of anti-ICAM/NCs under flow, in flow-adapted cells treated with 3 mMamiloride, 50 µM monodansyl cadaverine (MDC, an inhibitor of clathrin endocytosis), 1 µg/

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mL filipin, (Fil, a cholesterol binding agent), or 0.5 µM phorbol 12-myristate 13-acetate(PMA, a PKC inhibitor). Data are Mean ± SEM (n≥ 10 cells, 2 experiments). **P ≤ 0.01,***P ≤ 0.001, by Student’s t test.

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Figure 5. Targeting of anti-ICAM/NCs to pulmonary ECs in miceAnti-ICAM/NCs were injected iv in control or LPS challenged C57BL/6 mice, and bloodand lungs (subjected or not to perfusion to remove the blood in the organ) were collected atthe indicated times after injection. The 125Iodine content of the samples was measured tocalculate the percentage of injected dose (%ID) of anti-ICAM/NCs: (A) accumulated in thelungs, as compared to control IgG/NCs, and (B) in circulation. (C) The comparative fractionof anti-ICAM/NCs targeted to the lungs in LPS-challenged mice 30 min after injection, lungretention of the carriers after perfusion of this organ at this time, and lung retention after aprolonged time (3 h), were also assessed. Data are mean±SEM (n≥3 mice). *P≤0.05,***P≤0.001, by Student’s t-test.

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Figure 6. Endocytic uptake of anti-ICAM/NCs by pulmonary ECs in mice(A) Transmission electron micrographs showing anti-ICAM/NCs either bound (arrowheads)or internalized within vesicular compartments (arrows) by pulmonary endothelial cells (EC)3 h after intravenous injection in C57Bl/6 mice. As an example, a capillary (≤ 5-µmdiameter) and a larger vessel (5–10-µm diameter) are shown. Right top and bottom panelsare 4X and 8X magnifications of the insets marked in the corresponding left panels. Top andbottom scale bars = 1 µm and 2 µm, respectively. RBC = red blood cell. (B) Quantificationof the internalization (see Methods) of anti-ICAM/NCs from micrographs. Data are Mean ±SEM (n 10 micrographs from ≥2 mice). # P≤0.05, ## P≤0.01, *** P≤0.001, where *compares different times within the same condition and # compares different conditionswithin the same time point, by Student’s t-test.

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

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