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Biomimetic Glycoliposomes as Nanocarriers for Targeting P-Selectin on Activated Platelets Junmin Zhu,† Jie Xue,‡ Zhongwu Guo,*,‡ Linda Zhang,† and Roger E. Marchant*,† Department of Biomedical Engineering, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, and Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202. Received June 14, 2007; Revised Manuscript Received July 2, 2007
The cell glycocalyx is an attractive model for surface modification of liposomes with the objectives of tissue targeting and prolonged circulation time. Here, we reported on glycocalyx-mimicking liposomes, prepared by incorporating a glycolipid of 3′-sulfo-Lewis a (SuLea)-PEG-DSPE with a headgroup of SuLea and a spacer of poly(ethylene glycol) (PEG) linked to two hydrophobic tails. This PEG spaced structure is used to mimic the extended structure of P-selectin glycoprotein ligand 1 (PSGL-1) on activated leukocytes, in order to facilitate the specific binding of liposomes to the receptor of P-selectin expressed on activated platelets. Our results indicate that SuLea-PEG-DSPE can form stable, narrowly distributed liposomes with 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC) and cholesterol, with a vesicle size of 113.3 nm. The resultant SuLea-PEG-liposomes can facilitate their binding to the receptor of P-selectin 22 times higher than SuLea-liposomes without a PEG spacer. Further studies by fluorescence microscopy show that SuLea-PEG-liposomes can bind to activated platelets in Vitro effectively. It suggests that biomimetic SuLea-PEG-liposomes may be used as nanocarriers to target activated platelets for drug delivery to the injury sites of cardiovascular diseases.
The glycocalyx of cells like endothelial cells and erythrocytes is a membrane-bound carbohydrate-rich layer consisting of various glycoproteins, proteoglycans, and glycosaminoglycans. This carbohydrate-rich layer plays an important role in mediating cell-specific interactions and preventing nonspecific protein adsorption (1, 2). Therefore, the cell glycocalyx is an attractive model for surface modification of liposomes with the objectives of tissue targeting and prolonged circulation time. Here, we report on glycocalyx-mimicking glycoliposomes, as shown in Figure 1, prepared by 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol with incorporation of a glycolipid with a headgroup of 3′-sulfo-Lewis a (SuLea) and a spacer of poly(ethylene glycol) (PEG) linked to two hydrophobic tails. This PEG spaced structure is supposed to mimic the extended structure of P-selectin glycoprotein ligand 1 (PSGL-1) on activated leukocytes, in order to facilitate the specific binding of liposomes to the receptor of P-selectin expressed on activated platelets in the processes of thrombosis, inflammation, and atherosclerosis. Platelets play a central role in thrombosis and atherosclerotic lesions (3-5), including angina, coronary artery disease, myocardial infarction, and ischemic stroke, which are main causes of death and disability in the Western countries (6, 7). Platelets can be activated upon adhesion to extracellular matrix proteins in the vascular injury region, foreign material surface, or by thrombin generated in the coagulation process (8). A number of receptors have been recognized on activated platelets (9), including GPIIb/IIIa, GPIb, P-selectin, CD9, CD36, and CD63. P-selectin is one of the important receptors expressed on activated platelets with about 10 000 copies/cell (10). It is a member of the selectin family of adhesion molecules, which * Corresponding authors. R. E. Marchant, Tel: 216-368-3005, Fax: 216-368-4969, E-mail:
[email protected]. Z. Guo, Tel: 313577-2557, Fax: 313-577-8822, E-mail:
[email protected]. † Case Western Reserve University. ‡ Wayne State University.
Figure 1. Model of glycocalyx-mimetic liposome with a PEG spacer between SuLea and the liposome surface. Cholesterol is not included in order to simply the liposome model.
Figure 2. Model for interactions between P-selectin and PSGL-1. EGF, endothelial growth factor; SCR, short consensus repeat.
includes E-, L-, and P-selectin (11). P-selectin is stored in platelet R-granules, as well as endothelial Weibel-Palade bodies. After the activation of platelets, P-selectin is expressed on the platelet surface, which can bind to its receptor of PSGL1, a dimeric membrane mucin on circulating leukocytes (12), as shown in Figure 2. P-selectin on activated platelets has a crucial function in the early phases of inflammation regulating
10.1021/bc700212b CCC: $37.00 © 2007 American Chemical Society Published on Web 08/11/2007
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Figure 3. Structures of SuLea-DSGA (1) and PEG-DSPE (2, with PEG Mw of 2000).
leukocyte-platelet interactions and mediating the recruitment of leukocytes and platelets to the sites of vascular injury (1315). Therefore, P-selectin on activated platelets is an attractive candidate for targeted drug delivery to the sites of vascular injury. P-selectin binds with high affinity to the extreme N-terminal extracellular region of mature PSGL-1, which begins at residue 42 (12). Specifically, tyrosine sulfate (Sulfo-Tyr) residues and sialylated O-glycans within this region have been considered essential for P-selectin binding (Figure 2). Much effort has been directed at identifying their carbohydrate mimetics as P-selectin ligands (16). One of the most important is Lewis x/a (Lex/a) antigens, including sialyl Lex/a (SLex/a) (17), and sulfo-Lex/a (SuLeaxa) as found on epithelial mucins (18). To explore new therapeutic strategies for inflammatory diseases based on using biomimetic glycoliposomes, we have synthesized a SuLea glycolipid 1 (designated as SuLea-DSGA, Figure 3) by conjugating SuLea with 1,2-distearoyl-rac-glyceroglutaric acid (DSGA). SuLea-DSGA has been incorporated into liposomes for studying vesicle size and stability (19, 20). However, PSGL-1 is a thin, highly extended molecule with an average length of 54 nm. From the natural interaction between PSGL-1 and P-selectin, the binding site for P-selectin is at the N-terminus of PSGL-1, which is about 50 nm away from the leukocyte surface (Figure 2) (21, 22). Therefore, we propose that a spacer between SuLea
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Figure 4. Vesicle size and distribution of liposomes with 5 mol % incorporation of SuLea-PEG-DSPE.
and the liposome surfaces may better mimic the native PSGL-1 structure with enhanced binding affinity to P-selectin, compared with SuLea-liposomes without a spacer. PEG is a good candidate for using as this kind of spacer due to its flexibility and biocompatibility (23). Also, it has another advantage to control the spacer length through adjusting its molecular weight. Here, we choose PEG with Mw of 2000 because it has been widely used for surface modification of liposomes (24, 25). For example, PEG-1,2-distearoyl-sn-glycero3-phosphoethanolamine (DSPE) (2, with PEG Mw of 2000) (Figure 3) has been incorporated into liposomes to enhance the circulation time of vesicles in ViVo. Thus, a glycolipid, SuLeaPEG-DSPE 9 was designed and synthesized with a PEG spacer (Mw 2000) inserted between the headgoup of SuLea and two hydrophobic tails, as shown in Scheme 1. The synthesis of 9 was based on the conjugation of our previously developed intermediate, a protected amino derivative of SuLea 3 with DSPE-PEG-NHS 4 (with PEG Mw of 2000 and an activated carboxylic group by N-hydroxylsuccinimide), followed by deprotection of p-methoxybenzyl [Bzl(OMe)] groups, 3′-sulfatation, and deprotection of allyl (All) and benzyl (Bzl) groups (19, 20). In the natural form, Lewis antigens are attached through their reducing ends to lipids and/or proteins. If Lewis antigen conjugates have the lipids attached to the
Scheme 1. Synthesis of SuLea-PEG-DSPEa
a Reagents and conditions: (a) dichloromethane, RT, 36 h; (b) (NH4)2Ce(NO3)6, CH3CN, CHCl3/H2O, RT, 2 h; (c) pyridine, (CH3)3N.SO3, RT, 48 h; (d) PdCl2, NaOAc, HOAc/H2O, RT, 48 h; (e) H2, 10% Pd/C, CHCl3/MeOH/H2O (1:5:1), RT, 48 h.
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Table 1. Properties of Liposomes with 5% Glycolipid Incorporation lipid
Dv (nm)
PDI
ζ (mV)
IC50 (mM)
PEG-DSPE SuLea-PEG-DSPE SuLea-DSGA SuLea
108.5 113.3 105.8 N/A
0.12 0.03 0.19 N/A
0.41 -5.66 -8.46 N/A
N/Aa 0.13 2.92 5.61b
a Not applicable. b Using free SuLea as a positive control for IC 50 measurements.
Figure 5. Inhibition of sPSGL-1 binding to recombinant rat P-selectinIgG coated on plates by liposomes or carbohydrates.
2-amino group of the glucosamine residue with the reducing end capped by an unreactive group, it may affect the conformation of the antigen and their binding to selectins (26). SuLeaPEG-DSPE was synthesized with the lipid attachment through the reducing end of SuLea. Therefore, it has the advantage of stability under physiological conditions due to the lower susceptibility of the glycosyl ether linkage to glycosidases, compared with natural O-glycosides with reducing ends. SuLea-PEG-DSPE (5 mol %) was used with DSPC (55 mol %) and cholesterol (40 mol %) to prepare glycoliposomes using a freeze-thraw and extrusion method through 100 nm membranes (27). The vesicle size and distribution was determined by photon correlation spectroscopy (PCS), as shown in Figure 4. The mean vesicle size for SuLea-PEG-liposomes is 113.3 nm after extrusion, which is close to the vesicle size of SuLealiposomes (105.8 nm) and PEG-liposomes (108.5 nm) with 5 mol % incorporation of SuLea-DSGA 1 and PEG-DSPE 2 (with PEG Mw of 2000, Figure 3), respectively, as listed in Table 1. In addition, the vesicles of SuLea-PEG-liposomes were narrowly distributed with a polydispersity index (PDI) of 0.03, maybe due to the steric stabilization from PEG and the electrostatic effect from the charged head group of SuLea. Zeta (ζ)-potential was measured to verify whether the negatively charged SuLea groups are on the liposome surface. The results (Table 1) show that the ζ-potential of SuLea-PEGliposomes was -5.66 mV, which is comparable to that of SuLea-
liposomes (-8.46 mV). It indicates that SuLea-PEG-DSPE was successfully incorporated into liposomes and the negatively charged headgroup of SuLea was present on the liposome surface. The stability of liposomes was determined by measuring the vesicle size change over time at room temperature. SuLeaPEG-liposomes with 5% of glycolipid incorporation maintained their vesicle size in the same range in storage over a 1 month period, comparable with results for PEG-liposomes. It demonstrates that SuLea-PEG-liposomes is stable enough for practical uses, and may maintain vesicle integrity while in the circulation. Enzyme-linked immunosorbent assay (ELISA) was carried out to determine the binding affinity of the surface-modified liposomes to P-selectin. Briefly, recombinant rat P-selectin-IgG was coated on plates, followed by addition of human-soluble PSGL-1 (sPSGL-1) and glycoliposomes or carbohydrate ligands. After incubation, the plates were rinsed and the adherent sPSGL-1 was linked with horseradish peroxidase (HRP) through biotin-steptavidin interactions, followed by incubation with a substrate solution of tetramethylbenzidine (TMD). The absorbance of developed color was measured at 450 nm. The inhibition of sPSGL-1 binding to P-selectin by liposomes or carbohydrate ligands was expressed as the percentage of the sPSGL-1 control in the absence of any inhibitors, as shown in Figure 5. SuLea-PEG-liposomes show significant inhibition of sPSGL binding to P-selectin, while PEG-liposome shows no inhibition. IC50 was used to compare the binding ability of liposomes, which was the concentration of carbohydrate ligands that inhibited sPSGL-1 binding to P-selectin by 50%. The IC50 values were obtained from Figure 5, as listed in Table 1. The results show that the free SuLea saccharide has an IC50 of 5.61 mM (Figure 4), but IC50 of SuLea-liposomes was 2.92 mM. The IC50 of SuLea-PEG-liposomes was 0.13 mM. That means the binding ability of the ligands on SuLea-PEG-liposomes was increased 43 and 22 times, compared to the free SuLea ligand and SuLealiposomes, respectively. Therefore, the PEG spacer can increase the mobility of the ligands, and thereafter increase the binding ability. To further confirm and visualize the specific binding of SuLea-PEG liposomes with P-selectin, liposomes were labeled by incorporating 1 mol % of nitrobenzodiazole-modified phosphatidylcholine (NBD-PC), and human platelets were activated and adsorbed on collagen-coated glass coverslips. The binding was studied by the fluorescence microscopy method (28). It was observed that the SuLea-PEG-liposomes had much higher binding to activated platelets (Figure 6b) than the control of PEG-liposomes (Figure 6a). The significantly increased binding of the former to activated platelets is attributed to the specific interactions between SuLea on the glycoliposome and the P-selectin on activated platelets. In summary, we reported on novel glycocalyx-mimicking liposomes, SuLea-PEG-liposomes from a glycolipid, SuLeaPEG-DSPE with a headgroup of SuLea and a PEG spacer linked to two hydrophobic tails. Our results indicate that SuLea-PEG-
Figure 6. Fluorescence micrographs of platelet binding with NBD-labeled liposomes from PEG-DSPE (a) and SuLea-PEG-DSPE (b).
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DSPE can form stable, narrowly distributed liposomes with DSPC and cholesterol, with a vesicle size of 113.3 nm. The resultant SuLea-PEG-liposomes can mimic the extended structure of PSGL-1 on activated leukocytes and facilitate their binding to the receptor of P-selectin 22 times higher than SuLealiposomes without a PEG spacer. Further studies by fluorescence microscopy show that SuLea-PEG-liposomes can bind to activated platelets in Vitro effectively. It suggests that biomimetic SuLea-PEG-liposomes may be used as nanocarriers to target activated platelets for drug delivery to the injury sites of cardiovascular diseases.
ACKNOWLEDGMENT This project was supported by the National Institutes of Health (grant HL-70263-2). We gratefully thank the facilities provided by Center for Cardiovascular Biomaterials. Supporting Information Available: Details of experimental section. This material is available free of charge via the Internet at http://pubs.acs.org.
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