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. Author manuscript; available in PMC: 2012 Jun 5.
Published in final edited form as: Int J Transp Phenom. 2011 Jan 1;12(1-2):63–75.

Effect of Glycocalyx on Drug Delivery Carriers Targeted to Endothelial Cells

Andres J Calderon 1, Madiha Baig 2, Ben Pichette 3, Vladimir Muzykantov 4, Silvia Muro 5, David M Eckmann 6
PMCID: PMC3367256  NIHMSID: NIHMS247398  PMID: 22679359

Abstract

Animal models have shown that coupling ligands, targeted to endothelium surface receptors, with drug delivery carriers (DDC) can optimize the treatment of diseases by specific vascular delivery. The endothelium is exposed to hydrodynamic forces that modulate the expression of these cellular adhesion molecules (CAMs) and affect the structural and biological activity of endothelial cells (ECs). In order to investigate how delivery of targeted DDC can be optimized, we investigated carriers binding to flow adapted ECs under flow conditions. Comparison of live ECs to fixed cells from our previous experiments give insight into the effect of receptor motility on the cell surface as well as the effect of other factors such as glycocalyx (a protective layer of carbohydrates on the surface of cells) and actin remodeling. A flow chamber model is used to investigate how DDC size variation alters binding under flow conditions. Binding experiments were done with and without glycocalyx in order to elucidate its protective effect. Using fluorescence microscopy we determined the real time binding and rolling speeds of DDC under flow conditions. We also demonstrate the presence of glycocalyx and image actin filament remodeling. The binding of 1 µm carriers to ECs decreased after flow adaptation, in both non-activated and TNF-α activated ECs compared to non-flow adapted live cells. After removal of the glycocalyx by degrading enzymes binding increased in quiescent ECs, but only increased in activated cells after 2 hr of perfusion with particles. The binding with 100 nm carriers also decreased after flow adaptation but to a lesser extent and partially increased after enzyme degradation. These experiments give insight as to how tunable affinity parameters can be optimized to enhance therapeutic capabilities.

Keywords: glycocalyx, shear stress, flow, targeted delivery, carriers, endothelium, ICAM-1

INTRODUCTION

Delivery of therapeutics to vascular diseases can be improved by the use of targeted carriers against surface present cell adhesion molecules (CAMs) (Muzykantov, 2005; Torchilin, 2000) on endothelial cells (ECs). ECs line the lumen of the vasculature and are involved in most of the pathologies of vascular diseases including arteriosclerosis, acute lung injury, hypertension, aneurysms and thrombosis, among others (Cines et al., 1998; Cunningham & Gotlieb, 2005; Cybulsky & Gimbrone, 1991; Henninger et al., 1997; Matharu, Rainger, Vohra, & Nash, 2006). The use of functionalized drug delivering vehicles which bind to receptors on the EC surface is an attractive method for delivering therapeutics to specific diseased regions of the vasculature (Ding, Dziubla, Shuvaev, Muro, & Muzykantov, 2006). Pathological factors, including inflammatory cytokines such as interleukin-1-β and tumor necrosis factor-α (TNF-α) increase the expression of CAMs, for example, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) (Mulligan, Vaporciyan, Miyasaka, Tamatani, & Ward, 1993; S. Muro, 2007). Previous experiments have also shown that the shear stress experienced by the cells also regulate the expression of these two CAMs (Chiu et al., 2004; Cunningham & Gotlieb, 2005; S. Muro, 2007) as well as the thickness of the glycocalyx (Gouverneur, Berg, Nieuwdorp, Stroes, & Vink, 2006; Gouverneur, Spaan, Pannekoek, Fontijn, & Vink, 2006).

The glycocalyx is a negatively charged surface on top of ECs composed of polysaccharides that contributes to the antiadhesive properties of ECs under normal disease free conditions (Gouverneur, Berg et al., 2006). It functions as a protective barrier to prevent white and red blood cells to bind to ECs in normal pathophysiological conditions (Gouverneur, Berg et al., 2006). This sugar like protective layer may also hinder the binding of targeted carriers to ECs. In this study, we investigate the effect of the glycocalyx in the binding of ICAM-1 targeted carriers to ECs under flow conditions.

The glycocalyx is a hair like structure composed of glycoconjugates, with a reported thickness of 0.5–3 µm (van den Berg, Vink, & Spaan, 2003; van Haaren, VanBavel, Vink, & Spaan, 2003). This thickness can potentially interfere with the ligand-receptor interaction of therapeutic carriers and the endothelium, in the same way that it prevents other cells from binding to the endothelium (Gouverneur, Berg et al., 2006). Studies of glycocalyx removal in the context of leukocyte interaction with the endothelium have shown that removal of this layer decreases the rolling speeds and increases the binding of leukocytes to ECs (Constantinescu, Vink, & Spaan, 2003; Mulivor & Lipowsky, 2002). Enzyme degradation of the glycocalyx increases the permeability of the endothelium (Henry & Duling, 1999). The cytokine TNF-α can modify the structure of the glycocalyx, leading to an increase in permeability by macromolecules (Henry & Duling, 2000). TNF-α up-regulates the expression of ICAM-1 on the surface of ECs by up to 60 to 100 fold (Murciano, Muro et al., 2003). This increase of ICAM-1 receptors leads to an increase biding of anti-ICAM targeted carriers to ECs compared to resting ECs in-vitro and in-vivo (Murciano, Muro et al., 2003; S. Muro, Muzykantov, V.R., Murciano, J., 2004). Computational analysis indicates that the glycocalyx offers a resistance to binding of targeted carriers and that its removal increases the multivalency and probability of binding of functionalized carriers (Agrawal & Radhakrishnan, 2007). Understanding how the glycocalyx layer influences the binding of targeted therapeutic carriers to the endothelium will improve the design of such carriers for specific vascular diseases.

The glycocalyx layer thickness increases with higher levels of shear stress (Gouverneur, Berg et al., 2006), but large gradients of shears stress can diminish the thickness of the glycocalyx (van den Berg, Spaan, Rolf, & Vink, 2006). Gradients of shear occur in regions of the vasculature that have flow separation and high flow velocities. These regions of high shear gradients also increase the expression of ICAM-1 (Chiu et al., 2004; Cunningham & Gotlieb, 2005) and are associated with arthrosclerosis (Constantinescu et al., 2003; Cunningham & Gotlieb, 2005). Disturbance of the glycocalyx may play a role in the high risk atherogenic regions (Gouverneur, Berg et al., 2006). These regions of low vasculoprotective capacity associated with increased expression of ICAM-1 and other inflammatory receptors are excellent targets for delivering therapeutic carriers to protect and treat atherosclerosis. Carriers targeted to ICAM-1 can bind to these regions to deliver therapeutic material or imaging agents with a more specific efficacy than intravenous drugs. The binding of these carriers may also decrease the binding of inflammatory and plaque-developing agents that are commonly associated with atherosclerosis and scar tissue (Anderson & Siahaan, 2003; Jois & Teruna, 2003).

To elucidate how the glycocalyx layer affects the delivery of carriers targeted to ICAM-1 we have used a flow assay to determine if the disturbance of this layer increases the binding of anti-ICAM carriers to flow adapted ECs. We previously studied how shear stress and antibody density on carriers affected binding to statically cultured fixed ECs (Calderon, Muzykantov, Muro, & D.M., 2009). These cells were non-flow adapted and likely lacked the presence of a continuous or thick glycocalyx. The cells were fixed, preventing diffusion of receptors on the surface and carrier internalization due to CAM mediated endocytosis (Agrawal & Radhakrishnan, 2007; S. Muro, Wiewrodt et al., 2003). In this study we flow adapted ECs to uniform shear for 24 hr, and observed alignment of the ECs and actin remodeling. The presence of the glycocalyx layer was determined by fluorescence microscopy using ruthenium red (RR); which attaches to the glycosaminoglycans (GAGs) that are a major component of the glycocalyx (Jones, Roth, & Sanders, 1969). We show that there is a glycocalyx layer present in flow adapted cells but not in non-flow adapted cells. We used model polystyrene particles of 1 µm functionalized with R6.5 anti-ICAM antibody (Marlin & Springer, 1987) and examined how these carriers bound to ECs under flow conditions.

This study is a first step in understanding the importance of the glycocalyx as it pertains to targeted drug delivering carriers to the endothelium. These experiments will offer important information that can be used in the design of therapeutic strategies using targeted drug delivering carriers.

MATERIALS AND METHODS

Antibodies and Reagents

Green-Yellow fluorescent 1 µm diameter spherical polystyrene latex particles (Polysciences, Warrington, PA) were coated with monoclonal antibody R6.5 (Marlin & Springer, 1987) against human ICAM-1. Phalloidin was purchased from Invitrogen (Carlsbad, CA). Ruthenium Red (RR), Hyaluronidase, Heparinase I, and Neuraminidase were from Sigma-Aldrich (St. Louis, MO). The rest of the chemicals were from Sigma-Aldrich unless stated otherwise.

Particle Preparation

Anti-ICAM particles having a diameter of 1 µm were prepared by coupling anti-ICAM antibody R6.5 with yellow-green latex particles (Calderon et al., 2009). The suspension was incubated for one hour followed by centrifugation to remove unbound antibody in the suspension. The precipitate was re-suspended in PBS-1% BSA, and sonicated 30–40 times at low speed in order to break up aggregated particles. Particle diameters were measured using Dynamic Light Scattering (DLS) (S. Muro, Dziubla et al., 2006).The effective diameters of the particles after R6.5 coating were 170–210 nm and 0.93–1.35 µm for 100 nm and 1 µm particles, respectively. For all the experiments the surface concentration of R6.5 was 2.34×10−9 µg/µm2.

Cell Culture Preparation

Human umbilical vein endothelial cells (HUVEC) (LONZA, San Diego, CA) were cultivated as in [3]. HUVEC were cultured on 22 × 40 mm gelatin coated glass cover slips in a prepared mix of M-199 treated with 15% fetal bovine serum, 2 mM glutamine, 15-µg/ml endothelial cell growth supplement, 100 U/ml penicillin and 100 µg/ml heparin. The prepared cells were incubated at 37°C with 5% CO2 until they reached confluency. To simulate inflammatory activation of endothelial cells and cause maximum expression of ICAM-1, cells were treated overnight with 10 ng/ml of TNF-α.

Flow Adaptation of Cell Culture

Alignment of Cells

For flow adaptation assays we mounted cover slips with confluent ECs in a parallel-plate flow chamber (RC-30HV; Warner Instruments Inc., Hamden, CT), Figure 1. Continuous uniform shear stress of 15 dynes/cm2 was applied by perfusion with complete M199 media by means of a peristaltic pump (Rainin, Oakland, CA). Complete media was perfused for at least 24 hours in order to align cells in the direction of the applied flow. Cells were imaged before and after flow adaptation to categorize the alignment of the ECs to flow. In order to check for stress fiber or actin remodeling of the aligned ECs, the cells were fixed with 2% paraformaldehyde. Following fixation the cells were permeabilized with 0.2% Triton X-100, and stained with phalloidin conjugated with Alexa Flour 594. The coverslips were washed with phosphate buffered saline, and mounted on microscope slides for observation and analysis. Images were taken with a fluorescence microscope (Eclipse TE2000-U; Nikon, Melville, NY) using 40 X/NA1.4 PlanApo or 10X objective (Nikon) and Image-Pro software 3.0 (Media Cybernetics, Silver Spring).

Figure 1. Schematic of flow chamber setup.

Figure 1

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In order to flow adapt cells the flow chamber was mounted and the cells were exposed to a shear stress of 15 dynes/cm2 for at least 24 hr with M199 complete media without carriers. The experiments with carriers used the same setup but the media had a suspension of carriers.

Glycocalyx and Cells

We also tested for the presence of a glycocalyx layer in non-flow and flow adapted cells. We stained the GAGs of the glycocalyx with RR (Waller, Fox, Fox, Fox, & Price, 2004) to observe if the layer was present on the ECs. A fixative solution was prepared with 0.1% of RR mixed in 200 mM cacodylate buffer and 5% of glutaraldehyde in order to fix and stain the glycocalyx (Mayberry-Carson, Tober-Meyer, Smith, Lambe, & Costerton, 1984). The fixative was diluted to 1/100 in cacodylate buffer and the ECs were immersed in the fixative for 30 minutes. Then the cells were washed with cacodylate buffer 3 times. Following fixation fluorescence microscopy images of cells were obtained using a Texas Red filter. To examine the effect of the glycocalyx layer on carrier binding, we used glycocalyx degrading enzymes to diminish the glycocalyx. We used a mixture of Hyaluronidase (10 U/mL), Heparinase I (2 U/mL), and Neuraminidase (10 mU/mL) (Murciano, Medinilla et al., 2003) in complete media and perfused it for 2 hr through the already flow adapted ECs. The cells were washed with plain M199 and then perfused with the suspension of particles in complete media.

Particle Binding and Rolling Experiments

To observe the binding of particles to cells the media was seeded with 1 µm particles and then perfused through the flow chamber at 1 dyne/cm2. Time-lapse fluorescence and phase-contrast series microscopy images were obtained using an Orca-1 charge-coupled device camera (Hamamatsu, Bridgewater, NJ) and analyzed off line using Image Pro 3.0 software. Twenty real time consecutive images were captured, of 3–5 separate fields of views, at 5, 15, 30, 45, 60, 120 and 180 min. These images were then averaged in order to distinguish between bound and transient particles (Calderon et al., 2009). The particles were then counted and averaged per cell. We also observed qualitative delivery results by combining the fluorescent and phase-contrast images.

Rolling speeds were calculated using Image Pro 3.0 software. The complete method is described in detail in (Calderon et al., 2009). Briefly, we combine two consecutive images focused on the cell surface and determine the distance between particles. All particles that are in focus while displacing more than 100 µm are considered to be rolling on the cell surface.

Statistical Analysis

The data were calculated as mean ± standard error. For all experiments that involved carriers per cell the n was at least 40 cells. Statistical significance for rolling speeds differences was determined by two-tailed Student’s t test and taken as p<0.05.

RESULTS AND DISCUSSION

Glycocalyx in Non-Flow Adapted ECs

The glycocalyx layer may prevent cell surface receptors of ECs from being accessible to functionalized carriers. We have previously performed experiments of carrier binding under flow conditions to fixed non-flow adapted ECs with and without treatment of enzymes that degrade the glycocalyx. We perfused 1 µm anti-ICAM carriers through activated (TNF-α) non-flow adapted ECs and compared them with enzyme treated non-flow adapted activated cells at 1 dyne/cm2. Figure 2 shows that enzyme treatment did alter binding of anti-ICAM carriers. This preliminary result suggested that the glycocalyx, if present, did not have any effect on carrier binding. Previous results have shown that the thickness of the glycocalyx in statically cultured cells is very thin if at all present (Ueda, Shimomura, Ikeda, Yamaguchi, & Tanishita, 2004). Research has also suggested that TNF-α can change the nature of the glycocalyx, making it more permeable to macromolecules (Henry & Duling, 2000). These results may explain why the degrading enzymes did not have a significant effect on carrier binding to non-flow adapted fixed endothelial cells. Our preliminary results led us to evaluate a more complete flow based assay to study binding to live flow adapted cells.

Figure 2. Preliminary results of carriers binding to fixed non-flow adapted ECs.

Figure 2

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1 µm carriers prepared with 10 µg of R6.5 were perfused through 2% paraformaldehyde fixed activated ECs at 5 dynes/cm2. Treatment with enzymes had no significant effect on the binding of carriers to ECs.

Glycocalyx and Stress Fibers in Flow Adapted Cells

We cultured confluent ECs and adapted them to flow at a shear stress of 15 dynes/cm2 for at least 24 hrs. Adaptation to flow changes the morphology and biological activity of ECs. Under shear stress ECs align to flow and their cytoskeleton changes (Noria et al., 2004). Figure 3 shows phase contrast images of our flow adapted cells (A and C) and non-flow adapted cells (B). The cells in Fig. 3 A and C are stretched and are aligned axially in the same direction as the flow. The resultant cell shape is a result of cytoskeleton remodeling caused by the shear stress exposure. Stress fibers mainly consisting off actin filaments align with the flow and form strands from one end of the cell to the other. This is not the case in cells that are not flow adapted. Figure 4 shows this difference between cells that align to flow and cells that are not flow adapted.

Figure 3. Phase images of flow and non flow adapted endothelial cells.

Figure 3

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Cells were mounted in a parallel plate flow chamber and adapted to flow for at least 24 hrs at 15 dynes/cm2. (A) Flow adapted cells, 10× magnification. (B) Non-flow adapted cells, 10× magnification. (C) Cells adapted to flow, 40× magnification. Cell stretching is observed. The arrow represents the direction of flow.

Figure 4. Phase and fluorescent actin filament staining images of flow and non-flow adapted cells.

Figure 4

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A and B (Top) show phase and phalloidin stating of stress fibers for cells adapted to flow, respectively. C and D (Bottom) show phase and phalloidin staining of stress fibers for cells that were not adapted to flow, respectively. Images are taken at 40×.

The rearrangement of the cytoskeleton is important to the delivery of therapeutics that utilize CAMs as targets. The internalization of carriers that are functionalized to CAMs occurs via CAM mediated endocytosis, which has been shown to use actin filaments in their internalization process (S. Muro, Cui et al., 2003; S. Muro, Gajewski, Koval, & Muzykantov, 2005; S. Muro, Mateescu et al., 2006). Anti-ICAM carriers are internalized more slowly by ECs that are flow adapted vs. non-flow adapted cells [Unpublished observation, S. Muro]. More evidence of this process will be shown in this study as well.

Our interest lies in elucidating the role of the glycocalyx in facilitating or inhibiting binding of anti-ICAM carriers to ECs. The glycocalyx is a dynamic structure, reactive to, and remodeled by, shear stress (Nieuwdorp et al., 2005; Ueda et al., 2004). To evaluate the presence of the glycocalyx in our experiments, we have used RR as a staining agent to label the GAGs that are constituents of its structure. We expect cells lacking a glycocalyx to have a lower number of GAGs and therefore less red intensity, whereas cells having a more prominent glycocalyx should have a greater number of GAGs and higher red intensity. Figure 5 shows the presence of a glycocalyx in flow adapted cells but no significant fluorescence in the non-flow adapted cells and in the cells treated with the enzymes. This method does not permit determination of the thickness of the layer, which itself may play an important role in governing carrier binding by altering the accessibility of ICAM-1 receptors for cell-carrier interactions. This is a parameter that merits additional study in future experiments.

Figure 5. Glycocalyx staining with Ruthenium Red, for flow and non-flow adapted cells.

Figure 5

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A and B. Cells adapted to flow. C and D. Cells not adapted to flow. Staining of the glycocalyx was observed in the cells that were flow adapted while very little staining was observed in the non-flow adapted cells. Images were taken at 40× magnification.

The glycocalyx establishes a protective layer against leukocyte binding to the endothelium (Gouverneur, Berg et al., 2006; Mulivor & Lipowsky, 2002). Its removal decreases leukocyte rolling speeds and increases firm arrest probably by increasing encounters between surface molecules mediating adhesion to the endothelium (Constantinescu et al., 2003; Mulivor & Lipowsky, 2004). This suggests that the glycocalyx functions as a physical barrier to ligand-receptor bond formation. By this logic, removing the glycocalyx should make EC surface receptors more accessible to the ligands on anti-CAM coated carriers. However drug delivering carriers are typically 1–2 orders of magnitude smaller than leukocytes (S. Muro et al., 2008). Carrier size may play an important role in the ability to bypass the glycocalyx layer. Our experiments performed with 1 µm carriers were designed to investigate how the glycocalyx modulates carrier binding.

Glycocalyx and Carrier Binding

Figure 6 shows the binding of 1 µm carriers to quiescent and activated ECs. Binding is higher using activated non-flow adapted cells, reaching maximum binding of ~ 40 carriers/cell at 2 hr. These results are very similar to previous published findings of the same carriers binding to fixed ECs (Calderon et al., 2009). The binding of the quiescent non-flow adapted cells is higher than the previous published results for fixed quiescent cells. This difference may result from increased expression of ICAM-1 resulting from the shear stress exposure (McKinney, Rinker, & Truskey, 2006) used for flow adaptation, which was absent in the previous study. At 2 hr the binding of carriers is the same in both non-flow adapted cells and flow adapted cells treated with enzymes. These results imply that expression of ICAM-1 in flow adapted cells is increased, and that removal of the glycocalyx makes the receptors more accessible for interaction with carriers, while in non-flow adapted cells the ICAM-1 expression is initially lower, but increases with shear stress exposure during carrier perfusion. This accounts for the observed similarities in binding occurring at long times. TNF-α activated cells already express ICAM-1 maximally; the presence of shear stress does not further alter receptor expression. Thus, the binding of carriers for non flow adapted cells should be high. The flow adapted cells treated with enzymes should have higher binding than flow adapted cells with an intact glycocalyx. This only becomes apparent at long times.

Figure 6. Binding of anti-ICAM carriers to ECs under flow conditions.

Figure 6

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A and B, show the binding of anti-ICAM carriers to activated and quiescent ECs for the three different binding conditions, respectively.

Figure 6 also shows that adapting cells to flow decreases the binding of 1 µm carries compared to the non-flow adapted cells. We hypothesize that this observed decrease in binding is due to the presence of the glycocalyx preventing the carriers to bind efficiently to the ICAM-1 receptors. With flow adaptation cells have still higher binding than the quiescent cells, as becomes apparent after 45 minutes. These results along with the results from Figures 2 and 5 demonstrate that the presence of the glycocalyx decreases binding of 1 µm carriers to ECs. This is most likely due to the protective nature of the glycocalyx preventing an adequate interaction between ICAM-1 molecules and the anti-ICAM antibody present on the carriers. The adhesion-protective properties of the glycocalyx may have a larger effect on the carriers than on leukocytes, which, once activated, release chemicals that can cleave, and decrease the barrier effect of, the glycocalyx (Henry & Duling, 2000).

Adding glycocalyx degrading enzymes to the cells for 2 hours increases the binding of anti-ICAM carriers significantly for quiescent cells at all time points. The enzymes did not have the same effect on activated ECs since the increase in binding only becomes apparent after 120 min. It is not clear why there was no increase from 5–60 min in activated cells. Although the binding of carriers increased compared to that occurring with flow adapted cells without enzymes, the binding is still less than that occurring with non-flow adapted cells. Since the thickness of the glycocalyx could not be determined in this study, there remains the possibility that the glycocalyx was not removed completely after the enzyme treatment. Any residual layer may still potentially confer some protection against binding.

Carrier Internalization

Another possibility is that the internalization process of the anti-ICAM carriers occurs. Carriers begin to internalize after 15 min of incubation time (S. Muro, Cui et al., 2003). The process of CAM endocytosis depends on actin filaments remodeling. Most experiments demonstrating this have been performed under static conditions. As seen in Figure 3, actin filaments are normally not stretched and can reorganized very easily to accommodate carrier internalization. For flow adapted cells the filaments are already aligned, stretching the entire cell length. Because of this alignment the process of internalization takes longer in flow adapted cells (S. Muro, Unpublished). Thus carriers remain attached to the surface of the cells for longer periods of time before being internalized. Non flow adapted cells internalize carriers more rapidly, leaving more space available on the cell surface for additional carriers to bind. Since we do not distinguish between carriers that are intracellular and carriers located on the cell surface, and since it is difficult to distinguish between them in real time, some of the carriers imaged may actually reside within, and not on, the cell.

It is also known that ICAM-1 receptors recycle back at approximately 60 minutes after internalization (S. Muro et al., 2005), but that if internalization takes longer the receptors will recycle slower to the cell surface. Thus, in the non-flow adapted cells new carriers are more likely to bind at 2 and 3 hr than in flow adapted cells.

CAM endocytosis has several unique characteristics, one being that once the carriers are internalized, they travel to endosomes, lysosomes and finally they cluster around the nucleus of the cell(S. Muro, Cui et al., 2003). This process takes approximately 3 hr (S. Muro, Cui et al., 2003). Figure 7 shows the clustering of carriers around the nucleus of some non-flow adapted cells while there is no clustering of carriers in flow adapted cells at 3 hours. This is evidence that the internalization and trafficking process take longer with flow adapted cells. This is reasonable since the internalization process requires actin remodeling to occur, and hindrances of actin remodeling in cells restrict internalization from occurring (S. Muro, Cui et al., 2003; S. Muro, Mateescu et al., 2006). Flow adapted cells have aligned actin filaments and stress fibers that are required to maintain the cell shape in presence of shear stress. When the CAM meditated endocytosis is triggered due to the clustering of the multivalent anti-ICAM carriers (S. Muro, Wiewrodt et al., 2003), actin filaments are needed for the internalization and specially to traffic carriers to internal cell compartments (S. Muro, Cui et al., 2003). The actin filaments in flow adapted cells require greater energy to reorganize the cytoskeleton and to remodel the cell interior due to the CAM endocytosis signal cascade.

Figure 7. Peri-nuclear clustering of carriers in non-flow adapted cells.

Figure 7

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Phase and fluorescent images were captured and merged. A. Peri-nuclear clustering of carriers is observed in non–flow adapted cells. B. Peri-nuclear clustering did not occur at 180 minutes in flow adapted cells.

Binding experiments with 100 nm carriers were also performed. These experiments show that there is a decrease in carrier binding in flow adapted cells. The effect is less than that observed with 1 µm carriers. The glycocalyx hair-like structure of carbohydrates may prevent large particles or cells from interacting with the cell surface, but smaller carriers or molecules may permeate it more readily. Removing the glycocalyx increased the binding of the carriers compared to flow adapted cells but not as much as the non flow adapted cells. Additional experiments with 100 nm carriers are needed in order to better quantify this effect. The extremely small size of these particles requires that more highly refined real-time fluorescence microscopy techniques and image analysis methods be devised to quantify accurately reliable results regarding carrier interactions with cells and to derive the kinetics of carrier binding that will guide optimization experiments. Such experiments are on going in our lab and will serve as a critical guide for future efficient design of targeted drug delivering carriers having nanometer dimensions.

Carrier Rolling Speeds

We also calculated the rolling speeds of 1 µm carriers over the ECs using the method previously described (Calderon et al., 2009). We observed carrier rolling over endothelial cells, with the higher antibody density carriers having the lower rolling speeds (Calderon et al., 2009). Other investigators have observed that carriers also attach to cells without rolling occurring prior to firm arrest (Chen, Alon, Fuhlbrigge, & Springer, 1997; Marshall et al., 2003; Sakhalkar et al., 2003). We did not observe this behavior with 1 µm carriers, but we did observe similar behavior with 100 nm carriers. It was unclear if 100 nm carriers rolled over the cell surface since we could not discriminate between their motion along the cell surface or displacement in the free stream close to the cell surface. However, some 100 nm carriers moving in the free stream abruptly attached to the cell surface, without any subsequent detachment. The number of anti-ICAM-ICAM bonds needed for firm attachment of 100 nm carriers is predicted to be ~ 1–3 (Agrawal & Radhakrishnan, 2007; Calderon et al., 2009) at a shear stress of 1 dyne/cm2. Therefore, once a 100 nm carrier interacts with the cell surface receptors, there is a high probability that firm binding to the cell surface will follow.

We did not observe any 1 µm carriers firmly binding to the cell surface from the free stream. We believe the interaction of the surface anti-ICAM of the 1 µm carriers with ICAM-1 on the cell surface decreases the rolling speed until there are enough anti-ICAM-ICAM bonds to attach the carrier to the cell. It is possible that 1 µm carriers are less able to form stable bonds if the glycocalyx is present accessible ICAM-1 receptors are relatively inaccessible. Our results in Table 1 show that 1 µm carriers rolling over flow adapted ECs have the highest rolling velocity compared to non-flow adapted cells and flow adapted cells treated with enzymes. This supports the notion that the glycocalyx serves a barrier function preventing receptor-ligand interactions from occurring on the cell surface. This prevents carriers from decelerating as they roll and may reduce carriers binding.

Table 1.

Rolling speeds (µm/sec) of 1 µm carriers over endothelial cells.

TNF-α Non-Flow
Adapted		 Flow Adapted Flow Adapted
with Enzymes
− 26.1 ± 1.73 42.0 ± 4.63** 34.4 ± 2.53*
+ 24.2 ± 1.60 44.0 ± 3.62** 22.6 ± 1.65
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*

Statistically significant to non-flow adapted quiescent cells.

**

Statistically significant to non-flow adapted cells and to flow adapted cells with enzymes.

After the use of enzymes the rolling speeds decreased significantly compared to flow adapted cells without enzymes. These results suggest that removal of the glycocalyx eliminates its anti-adhesive properties by making surface receptors on the cell more readily accessible to form bonds with the anti-ICAM carriers. As expected, the speeds were higher for all the carriers rolling over quiescent cells than for activated cells. These results is attributed to the greater number of receptors present on activated cells than on quiescent cells.

The flow profile over the cells was uniform and no recirculation regions were noted. There was some transient behavior from the pulsatility from the peristaltic pump, but did not affect the behavior of the particles to attach to the cells. The pulsatility that is present in the flow chamber is similar to the pulsatility experienced by cells in-vivo. It was noted that the flow should be similar to flow between two parallel plates with a small transient component.

Limitations and Future Work

There were some limitations with the procedure using RR. It was difficult to wash the RR at some concentrations. This led to several experiments to optimize the adequate concentration of RR. Future experiments are ongoing and planned in order to quantify the presence of the glycocalyx before and after enzyme treatment, as well as the thickness of the glycocalyx at different shear stresses. Future work is also needed to understand the internalization process of different carrier sizes under flow conditions. There are still limitations with the imaging techniques and how to accurately follow carriers and distinguish if the carriers are in the surface or inside the cell. Future work is planned in order to identify the location of carriers at different time points under flow conditions in real time. We have also performed preliminary experiments with 100 nm carriers and plan to investigate the binding and internalization of 100 nm carriers in real time.

CONCLUSION

In summary we have adapted endothelial cells to flow and have confirmed axial alignment and reorganization of the cell cytoskeleton. We have demonstrated the glycocalyx layer to be present following flow adaptation and that this cell surface layer is diminished or absent following enzymatic degradation. The binding of 1 µm anti-ICAM carriers increased with non-flow adapted cells and decreased with cells that were flow adapted. Following glycocalyx removal binding increased and rolling speeds decreased compared to flow adapted cells bearing an intact glycocalyx. We further demonstrated that carriers bound to non-adapted cells were internalized and trafficked to cluster around the nucleus at 180 min. Such clustering did not occur in flow adapted cells.

This study is helpful in identifying the specific roles of the glycocalyx and of cell flow adaptation in targeted carriers binding and internalization. Important applications to efficient carrier delivery for therapeutic or diagnostic purposes will be dependent on such work.

ACKNOWLEDGMENTS

Funding for this work was provided by NIH R01 EB006818 (DME) and NIH R01 HL60230-S1 (DME).

Contributor Information

Andres J. Calderon, Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, PA 19104

Madiha Baig, Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, PA 19104.

Ben Pichette, Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, PA 19104.

Vladimir Muzykantov, Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104.

Silvia Muro, Center for Biosystem Research, University of Maryland Biotechnology Institute, College Park, MD, 20742.

David M. Eckmann, Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, PA 19104

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