Bioconjugate Chem. 2006, 17, 849−854
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TECHNICAL NOTES Targeted Liposomes: Convenient Coupling of Ligands to Preformed Vesicles Using “Click Chemistry” Fatouma Said Hassane, Benoıˆt Frisch, and Francis Schuber* Institut Gilbert Laustriat, UMR 7175 CNRS/Universite´ Louis Pasteur (Strasbourg I), De´partement de Chimie Bioorganique, Faculte´ de Pharmacie, 74 route du Rhin, 67400 Illkirch, France. Received October 24, 2005; Revised Manuscript Received February 28, 2006
An efficient and convenient chemoselective conjugation method based on “click chemistry” was developed for coupling ligands to the surface of preformed liposomes. It can be performed under mild conditions in aqueous buffers; the use of a water soluble Cu(I) chelator, such as bathophenanthrolinedisulfonate, was essential to obtain good yields in reasonable reaction times. A model reaction was achieved in which, in a single step, an unprotected R-D-mannosyl derivative carrying a spacer arm functionalized with an azide group was conjugated to the surface of vesicles presenting a synthetic lipid carrying a terminal alkyne function. When liposomes composed of saturated phospholipids were used, the reaction conditions developed in the present work did not damage the membranes as measured by the absence of leakage of entrapped 5,6-carboxyfluorescein. Moreover, as assessed by agglutination experiments using concanavalin A, the mannose residues were perfectly accessible on the surface of the targeted vesicles.
INTRODUCTION Liposomes, which are self-closed vesicular structures composed of (phospho)lipid bilayers, have attracted considerable interest because of their potential applications in various fields such as membrane models, diagnostics, and drug/gene delivery (1). Several liposomal formulations have reached clinical application during this past decade especially for the treatment of cancer and opportunistic diseases (2). The discovery of “stealth liposomes”sliposomes coated with, e.g., poly(ethylene glycol) (PEG) chains characterized by much increased circulation times and modified biodistributionssprovoked a new impetus for the design of targeted bioactive molecule delivery systems. Such liposomes carry ligands that provide specific interactions with receptors/antigens expressed at the surface of target cells. The design of targeted liposomes is heavily dependent on the development of well-controlled bioconjugation reactions (for reviews see refs 3-6) which in most cases involve the coupling of ligands to the surface of preformed vesicles that carry functionalized (phospho)lipid anchors. Among the many conjugation methods available, the most popular ones involve the reaction of thiol-containing ligands with anchors carrying thiol-reactive functions such as maleimide, bromoacetyl, or 2-pyridyldithio linkages, generating thioether or disulfide bonds (7-9). More recently peptide ligands were also coupled to the surface of liposomes via hydrazone and R-oxo hydrazone linkages (10). Chemically controlled conjugation between preformed liposomes and ligands should ideally combine several features such as mild reaction conditions in aqueous media, high yields, and chemoselectivity. In search of new coupling strategies we have * To whom correspondence should be addressed. Phone: + 33 390 24 41 72. Fax: +33 390 24 43 06. E-mail: francis.schuber@ pharma.u-strasbg.fr.
explored the application of “click chemistry” (11, 12) to this field; it involves a copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes yielding 1,4disubstituted 1,2,3-triazole linked conjugates (13, 14). This reaction is particularly appealing because it is highly regiospecific, chemoselective, and tolerant to a wide variety of other functional groups. Moreover the click reaction, which can also be performed almost quantitatively under mild conditions in aqueous buffers, was recently extended to bioconjugation applications (see, e.g., refs 15-23). Herein we report the successful application of click chemistry to the conjugation, in a single step, of an unprotected R-D-mannosyl derivative carrying a spacer arm functionalized with an azide group, to the surface of liposomes that incorporate a synthetic lipid carrying a terminal triple bond. Reaction conditions were optimized for this model reaction, and mannosylated vesicles were obtained in excellent yield. As assessed by agglutination experiments with the lectin concanavalin A (Con A), the mannose residues were perfectly accessible on the surface of the vesicles and could engage into multivalent interactions.
EXPERIMENTAL PROCEDURES General. Reagent-grade solvents were used without further purification. The phospholipids 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) and 1,2-dipalmitoyl-rac-glycero-3phospho-rac-(1-glycerol) sodium salt (DPPG) were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France), and their purity (over 99%) was assessed by TLC. Cholesterol was recrystallized in methanol. The amine 5 (Scheme 2), that contains 12 ( 2 ethyloxide units, was a kind gift from Dr. L. Lebeau. The lectin concanavalin A, type VI (Jack Bean), was obtained from Sigma. L-Ascorbic acid sodium salt and bathophenanthrolinedisulfonic acid disodium salt are from AcrosOrganics (Noisy-le-Grand, France) and Lancaster-Synthesis (Strasbourg-Bischheim, France), respectively. Silica gel 60 F
10.1021/bc050308l CCC: $33.50 © 2006 American Chemical Society Published on Web 04/18/2006
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Scheme 1. Model Reaction
Scheme 2. Synthesis Azidomannose, 7
of
the
Functionalized
Ligand
254 plates for thin-layer chromatography (TLC) were purchased from Merck (Darmstadt, Germany). Column chromatographic separations were carried out as flash chromatography using silica gel (Merck) with a particle size of 0.040-0.063 mm. 1H and 13C NMR were recorded on Bruker Avance 200 (200 MHz) and DPX-300 (300 MHz) spectrometers. Chemical shifts are expressed as parts per million using CHCl3 (δ ) 7.27 ppm for 1H and 77.7 ppm for 13C) or CH OH (δ ) 3.35 ppm for 1H and 3 49.0 ppm for 13C) as the internal standard (δ ) 0.0 ppm). Mass spectra (ES-MS) were recorded on a ESI/TOF Mariner mass spectrometer by electrospray. 4-[1-(2-(2-(2-(2-Hydroxyethoxy)ethoxy)ethoxy)ethyl)-1H1,2,3-triazol-4-yl]butanoic Acid (3). To 5-hexynoic acid (Lancaster-Synthesis) (21 mg, 0.187 mmol) in 200 µL of t-BuOH were added 200 µL of freshly prepared solutions of CuSO4‚ 5H2O (1 mg, 4 µmol) and sodium ascorbate (3.6 mg, 18.2 µmol) in water. Compound 1 (24) (40 mg, 0.182 mmol) was then added dissolved in 200 µL of water. The reaction mixture was stirred under argon at room temperature for 25 h. The solvent was then evaporated to dryness, and the product 3 was obtained without any purification. The yield was estimated at 91% according to NMR analysis. Rf: 0.51 (CH2Cl2:MeOH 19:1). 1H NMR (200 MHz, CDCl3:CD3OD, 1:2): δ 7.56 (s, 1H, CH triazole); 4.524.47 (t, J ) 4.9 Hz, 2H, CH2N); 3.85-3.80 (t, J ) 4.9 Hz, 2H, CH2CH2N); 3.73-3.57 (m, 10H, CH2OCH2CH2OCH2CH2OH); 3.40-3.06 (t, J ) 5.0 Hz, 2H, CH2OCH2CH2N); 2.81-2.74 (t, J ) 7.2 Hz, 2H, CH2CCH); 2.39-2.32 (t, J ) 7.1 Hz, 2H, CH2COOH); 2.06-1.91 (m, 2H, CH2CH2COOH). ES-MS m/z: 332.2656 ([M + H]+), calcd 331.1743 for C14H25N3O6. N-[(2-(2-Azidoethoxy)ethoxy10)ethyl-(1-deoxy-2,3,4,6-tetraO-acetyl-thio-r-D-mannopyranosyl)]propionamide (6). To a
solution of 4 (25) (149 mg, 0.342 mmol) in 3 mL of CH2Cl2 were added DCC (85 mg, 0.41 mmol) and NHS (47 mg, 0.41 mmol). After 45 min of stirring at room temperature under argon, the amine 5 (26) (210 mg, 0.342 mole) in 1 mL of CH2Cl2 containing DIEA (47.5 µL, 0.273 mol) was added to the reaction mixture. After 18 h, 20 µL of DIEA and 0.2 equiv of 5 were again added. The stirring was continued during 48 h. The precipitate was then removed by filtration, and the organic phase was washed with 2 × 10 mL of a citric acid solution (5%, w/v) followed by water. After passage over MgSO4, the solvent was evaporated to dryness. The reaction product 6 (129 mg; yield 30%, yellow oil) was obtained after purification by chromatography on silica gel eluted with CH2Cl2:MeOH 30:1. Rf: 0.42 (CH2Cl2:MeOH 9:1). 1H NMR (200 MHz, CDCl3): δ 5.39-5.10 (m, 4H, 3 CH Man + 1 CHR); δ 5.39-5.10 (m, 4H, CH Man); 4.28-4.23 (m, 2H, CH2 Man); 4.04-3.99 (m, 1H, CH-CH2OH Man); 3.56-3.30 (m, 52H, CH2NH, CH2OCH2, CH2CH2NH, CH2N3); 2.88-2.81 (t, J ) 6.9 Hz, 2H, CH2S); 2.47-2.40 (t, J ) 7.04 Hz, CH2CH2S); 2.08, 2.02, 1.97, 1.90 (s, 12H, CH3CO). 13C NMR (50 MHz, CDCl3): δ 170.99 (CONH); 170.13 (COOCH3); 83.03 (C1); 71.37, 71.00, 70.68, 70.44 (CH2O-CH2, CH2CH2NH); 70.15 (C2); 69.81 (C5); 69.43 (C3); 66.75 (C4); 62.87 (C6); 51.08 (CH2N3); 39.79 (CH2NH); 36.56 (CH2CO); 27.33 (CH2S); 21.30 (CH3OCO). ES-MS m/z: 1055.6430 ([M + Na+]), calcd 1032,4672 for C43H76N4O22S. N-[2-(2-Azidoethoxy)ethoxy10)-ethyl(1-deoxy-1-thio-r-Dmannopyranosyl)]propionamide (7). Compound 6 (100 mg, 0.097 mmol) was dissolved in 3 mL of MeOH, and K2CO3 (20 mg, 0.145 mmol) was then added with a few drops of water. The deprotection reaction was performed under argon at room temperature for 4 h. The reaction product 7 (84 mg, yield 100%) was obtained as a yellow oil after filtration to remove the salts and evaporation of the solvents. Rf: 0.30 (CH2Cl2:MeOH 8:2). 1H NMR (200 MHz, CD OD): δ 5.26 (s, 1H, CHR); 3.923 3.87 (m, 2H, CH2 Man); 3.67-3.32 (m, 55H, CH Man, CH2NH, CH2OCH2, CH2CH2NH, CH2N3); 3.02-2.86 (m, 2H, CH2S); 2.64-2.48 (m, 2H, CH2CH2S). 13C NMR (50 MHz, CD3OD): δ 174.22 (CONH); 87.48 (C1); 75.04 (C2); 73.63 (C5); 73.21 (C3); 70.79 (CH2O-CH2, CH2CH2NH,); 68.90 (C4); 62.85 (C6); 51.70 (CH2N3); 40.22 (CH2NH); 37.34 (CH2CH2S); 28.82 (CH2S). ES-MS m/z: 903.4658 ([M + K+]), calcd 864.4249 for C35H68N4O18S. N-[2-(2-(2-(2-(2,3-Bis(hexadecyloxy)propoxy)ethoxy)ethoxy)ethoxy)ethyl]hex-5-ynamide (8). DCC (52 mg, 0.25 mmol) and NHS (12 mg, 0.105 mmol) were added to a solution of 5-hexynoic acid (2) (23 mg, 0.21 mmol) in CH2Cl2 (4 mL). 2-(2-(2-(2,3-Bis(hexadecyloxy)propoxy)ethoxy)ethoxy)ethoxyethanolamine (150 mg, 0.21 mmol), synthesized as outlined in ref 27, and DIEA (43 µL, 0.25 mmol) were then added to the mixture. After 22 h of reaction with stirring at room temperature, and under argon, the formed precipitate was removed by filtration and the organic phase was washed with 2 × 10 mL of a solution of citric acid (5%, w/v) followed by 2 × 10 mL of brine. After passage over MgSO4, the solvent was evaporated to dryness. The reaction product 8 (98 mg; yield 58%) was obtained after purification by chromatography on silica gel eluted with CH2Cl2:AcOEt 9:1 to 7:3. A white powder was obtained by recrystallization from CH2Cl2:MeOH (1:9, v/v). Rf: 0.54 (CH2Cl2:MeOH 19:1). 1H NMR (300 MHz, CDCl3): δ 3.65-3.39 (m, 25H, CH2NH, CH2O, HC(CH2)2O); 2.332.22 (m, 4H, NHCOCH2, CH2CCH); 1.98-1.96 (t, J ) 2.3 Hz, 1H, CCH); 1.90-1.80 (m, 2H, CH2CH2CH2); 1.56-1.51 (m, 4H, CH2CH2CH2O); 1.24 (s, 52H, CH2 alkyl chains); 0.890.84 (t, J ) 6.39, 6H, CH3). 13C NMR (75 MHz, CDCl3): δ 172.90 (CONH); 84.26 (CCH); 78.54 (CH2CHCH2); 72.33, 72.10 (OCH2CHCH2O); 71.51 (CH2CH2CH2O); 71.37, 71.25, 70.91, 70.54 (CH2O); 69.77 (CCH); 39.84 (CH2N); 35.65
Technical Notes
(NHCOCH2); 32.57 (CH2CH2CH3); 30.76(CH2CH2CH2O); 30.35, 30.17, 30.01 (alkyl chains); 26.76 (CH2CH2CH2O); 24.86 (CH2CH2CCH); 23.34 (CH2CH3); 18.54 (CH2CCH); 14.76 (CH3). ES-MS m/z: 832.8311 ([M + Na+]), calcd 809.7109 for C49H95NO7. Liposome Preparation and Characterization. Small unilamellar vesicles (SUV) were prepared by sonication. Briefly, phospholipids (DPPC, DPPG) and cholesterol (70/20/50 molar ratio) dissolved in chloroform/methanol (9:1, v/v) were mixed in a round-bottom flask. For functionalized vesicles, the lipid anchor 8 dissolved in chloroform/methanol (9:1, v/v) was added at given concentrations (between 5 and 10 mol %). After solvent evaporation under high vacuum, the dried lipid film was hydrated by addition of 1 mL of HBS (10 mM HEPES, 145 mM NaCl, pH 6.5) to obtain a final concentration of 10 µmol of lipid/mL. The mixture was vortexed, and the resulting suspension was sonicated for 1 h at 60 °C, i.e., above the Tm of the lipids, using a 3 mm diameter probe sonicator (Vibra Cell, Sonics and Materials Inc., Danbury, CT). The liposome preparations were then centrifuged at 10000g during 10 min to remove the titanium particles originating from the probe. The size of the liposomes was determined by dynamic light scattering using a submicron particle analyzer (Coulter, Hialeah, FL) or a Zetamaster 3000 (Malvern Instrument, Orsay, France). The different vesicle preparations were homogeneous in size and exhibited an average diameter between 90 and 130 nm. The phospholipid content in liposomes was determined according to Rouser (28) with sodium phosphate as standard. Azidomannose Coupling to Liposomes by Click Chemistry. Solutions containing 8 mM CuSO4‚5H2O (solution A), 145 mM sodium ascorbate (solution B), and 28 mM bathophenanthrolinedisulfonic acid disodium salt (ligand L, Scheme 3) (solution C) were freshly prepared in 10 mM HEPES (pH 6.5) completed with NaCl to obtain an osmolarity of 300 mOsm. To a suspension of functionalized liposomes in 200 µL of HBS adjusted to a 1 mM concentration of anchor 8 (i.e., ∼0.5 mM surface available alkyne group) were added solutions A and B (respectively 286 and 355 µL) followed by 164 µL of solution C. Following addition of 15 µL of a 13.9 mM solution of azidomannose 7 in water, the reaction mixture was gently stirred under argon at room temperature for 1-24 h. Alternatively, the ligand L was first added to the liposome suspensions, followed by the extratemporaneously made mixture of solution A and solution B and finally by azidomannose 7. Control experiments were performed under the same conditions in the absence of the ligand 7. After the conjugation step, the liposomes were purified by exclusion chromatography on a 1 × 18 cm Sephadex G-75 (Amersham Biosciences) column equilibrated in HBS. Liposome Stability under Coupling Conditions. Liposomes were prepared as above in 10 mM Hepes buffer (pH 6.5) containing 100 mM NaCl and 40 mM 5,6-carboxyfluorescein. Nonencapsulated dye was eliminated by gel filtration (see above). The dye-loaded liposomal suspensions were treated as above for the click-chemistry coupling step. The leakage of 5,6carboxyfluorescein was assessed by determining the fluorescence increase (λex 490 nm; λem 520 nm) observed in the presence of excess detergent. To measure the total fluorescence intensity corresponding to 100% dye release, Triton X-100 (0. 1% w/v final) was added to the vesicles. The percentage of dye release caused by the coupling conditions was calculated using the equation (F - F0) × 100/(Ft - F0), where F is the fluorescence intensity measured after exposing the vesicles to the coupling conditions and F0 and Ft respectively are the intensities obtained before the coupling conditions and after Triton X-100 treatment (29). Ft values were corrected for dilutions caused by the Triton X-100 addition.
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Lectin Binding Assay and Analytical Techniques. The lectin Con A (125 µg) was added to suspensions (1 mL final volume) of liposomes carrying mannose residues (60 nmol of lipids and ∼3 nmol of surface coupled mannose residue) in HBS. The increase in turbidity was recorded at 360 nm at room temperature. Mannose coupled to the liposomes was quantified by the resorcinol-sulfuric acid method (30). The standard curve was established as follows: To a 12.2 × 100 mm glass tube containing 2-12 µg of reference mannose in 200 µL water were added successively 20 µL of 0.1% (w/v) Triton X-100, 200 µL of a 6 mg/mL aqueous solution of resorcinol, and 1 mL of 75% (v/v) H2SO4. The tubes were vortexed and covered with aluminum foil before heating in a boiling water bath for 12 min and then cooled to room temperature. The optical density was then recorded at 430 nm. Aliquots of conjugated liposomes (about 0.4 µmol of lipid) were first dried under vacuum using a Speed Vac (Savant Instrument Inc., Holbrook, NY). HBS (200 µL) was then added to the tubes and the mixtures, after vortex mixing, were treated as indicated above for the standards. Before optical density reading the samples were filtered through a 0.45 µm PTFE filter. The spectrophotometric determination of copper present in the final liposomal preparations was carried out using an atomic absorption spectrophotometer Varian SpectrAA 50B calibrated with a standard solution of CuSO4‚5H2O (0.1953.9 µg/mL). Samples (1 mL, about 0.65 µmol of phospholipids) were added to a solution of 5% nitric acid (5 mL) completed to 7 mL with water. The mixtures were vortexed and filtered on a 0.22-µm membrane (Millex, Millipore), and the copper content was determined.
RESULTS AND DISCUSSION Chemistry. The synthesis of the azidomannose ligand 7 (Scheme 2), destined to be conjugated at the surface of preformed liposomes using a click reaction (Scheme 3), consisted in a coupling between the known mannosyl derivative 4 (25) and a PEG chain (n ∼12) functionalized with amine and azide functions (compound 5) synthesized as described previously (26). The carboxylic acid function of 7 was classically activated with dicyclohexylcarbodiimide and N-hydroxysuccinimide, at room temperature, in the presence of diisopropylethylamine. Compound 5 was then added, and the reaction was carried out for 48 h to give 6, which, after quantitative deprotection of the acetyl groups with potassium carbonate in methanol, gave the ligand 7. For the synthesis of the functionalized lipid anchor 8 (Scheme 3), 2-(2-(2-(2,3-bis(hexadecyloxy)propoxy)ethoxy)ethoxy)ethoxyethanolamine was first synthesized in 7 steps starting from glycerol, triethylene glycol, and palmityl alcohol by an adaptation of a procedure we have described previously (27). The free amine of this compound was then reacted with the hydroxysuccinimide ester of 5-hexynoic acid, previously obtained by reaction of its acid function with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide. The coupling reaction was carried out for 12 h, and the final product 8 was obtained with a satisfactory yield (58%). Model Reaction. As a proof-of-concept we have explored the possibility to prepare mannosylated liposomes (Scheme 3) using click chemistry. Such liposomes, which have been prepared before (see, e.g., refs 27, 31-34), are easily characterized and serve as vehicles to target cells such as antigen presenting cells (35-37). For the conjugation step both alkyne and azide-terminated spacer arms between the mannose residue and the hydrophobic anchor associated with the liposomes can be envisaged. For that reason, in a first approach we have studied a model click reaction between a short PEG spacer functionalized with an azide (1, Scheme 1) and 5-hexynoic acid (2) catalyzed by copper(I) in solution. The reaction was performed in an aqueous solvent system with reagents that could be
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Scheme 3. Coupling of the Ligand 7 to Preformed Liposomes That Incorporate the Alkyne Functionalized Anchor 8 by Click Chemistry in the Presence of the Cu(I) Ligand Bathophenanthrolinedisulfonate
extended, in principle, to the coupling of ligands to the surface of preformed vesicles. The procedure involving in situ generation of active Cu(I) catalyst via reduction of CuSO4 by sodium ascorbate (13) proved highly satisfactory and reliable. When starting with an equimolar mixture of 1 and 2 the expected product 3 was obtained over 90% yield after 1 day of reaction, at room temperature, using a copper(II) (2 mol % with respect to reactants)/Na ascorbate (10 mol %) system. In the absence of copper catalyst no reaction was observed. The reaction mechanism of this “ligand-free” Cu(I) catalyzed process (see below), which involves the formation Cu-acetylide, has been outlined recently (38, 39). Conjugation of Mannose Residues to the Surface of Preformed Liposomes. To perform the coupling of unprotected R-D-mannosyl residues to the surface of preformed liposomes with the click reaction we have first synthesized the ligand 7 that carries an azide function (Scheme 2). We have given a preference to an R-thiomannosyl derivative because this glycosidic bond is very resistant, in vivo, especially toward the action of glycosidases. The mannosyl derivative 4 (25) was reacted with a relatively long PEG spacer arm of defined length (26), the purpose being to provide an optimal accessibility of the liposomal mannose ligands to their receptors (40). Next we have synthesized the anchor 8 (Scheme 3) that carries the alkyne moiety. This function was selected for the anchor because of its chemical “inertness” in vivo andsconsidering that about 50% of the anchor is oriented toward the interior of the vesiclessits absence of reactivity with (bio)molecules that can be entrapped within the liposomes. The molecule 8 was quantitatively incorporated in the liposomes during their formation. Small unilamellar vesicles composed of saturated phospholipids (DPPC, DPPG) and cholesterol (70/20/50 molar ratio) and containing 10 mol % of the anchor 8 were prepared (average size: 90-130 nm). For the conjugation of the mannose ligand 7 (Scheme 3) to the preformed liposomes several conditions were tested in an aqueous buffer (pH 6.5). We have first tried to adapt the conditions that gave excellent yields in the model reaction (Scheme 1): i.e., generation of the catalytic Cu(I) by reduction of Cu(II) by sodium ascorbate. Keeping constant the concentrations of liposome (0.1 mM surface accessible anchor 8) and azidomannose 7 (0.2 mM), the reaction time (24 h), the temperature (25 °C), and the ascorbate/CuSO4 ratio ()5), we have varied the concentration of CuSO4 (1-10 µM). Under these conditions, compared to Scheme 1, covalent coupling of
mannose to the liposomes in appreciable yields failed. In fact, much higher concentrations of the ascorbate/CuSO4 couple were needed to observe a conjugation which nevertheless remained relatively modest, i.e., about 25% coupling was observed in the presence of 2.3 mM CuSO4 and 51.5 mM sodium ascorbate. This observation is in agreement with other works which showed that, for bioconjugations involving for example large molecular complexes, catalytic quantities of the ascorbate/CuSO4 system are not enough to drive click reactions to completion. To circumvent this limitation we have added a ligand of copper ions. Indeed, stabilization of the Cu(I) oxidation state by specific heterocyclic ligands was shown to largely accelerate the 1,3dipolar cycloaddition reaction between azides and alkynes (41, 42). In our case, bathophenanthrolinedisulfonate (L; Scheme 3) provided a water soluble (41) and potent chelate catalyst for the click reaction. It allowedswhen used in a 2-fold excess over copper to reduce the agglutination of the vesiclessa large increase in the yield of mannosylation and a decrease in the reaction time. For example, under standard conditions (24 h) the coupling yield increased from 23%, in the absence of L, to nearly completion in its presence. Moreover reaction times could also be decreased because, within 1 h in the presence of L, the observed conjugation of 7 was already about 80%. Altogether, we have routinely conjugated, in high yield, ligands such as 7 (2-fold molar excess) to the surface of preformed liposomes carrying alkyne functions in the presence of CuSO4/Naascorbate/L (2.3, 50, and 4.6 mM) in an aqueous buffer (pH 6.5) at room temperature for 6 h (standard conditions). In the liposome field it is of importance to verify the integrity of the vesicles after the coupling steps. To that end, using a dynamic light scattering technique, we have first verified whether the reaction conditions described above altered the size of the vesicles. Some changes were noticeable using our standard conditions, i.e., about 50% diameter increases were noted even under control conditions in the absence of ligand 7. However, we found that this effect could be efficiently limited just by changing the order of reactants added to the liposome suspensions. Thus, when ligand L was added before the CuSO4/ ascorbate mixture followed by the mannosyl ligand 7, no significant increase in size of the vesicles could be observed and, importantly, an identical yield of conjugation was measured. Next, to test whether the experimental conditions used for the click reaction could provoke some leakage of the vesicles, we have exposed to our standard conditions the same type of
Technical Notes
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it should be underlined that the mandatory use of copper catalysts in click chemistry could represent a limitation; indeed, vesicles made of unsaturated phospholipids are known to be readily (per)oxidized by copper ions in the presence of oxygen and become leaky (43, 44). Alternative means of promoting catalyst-free ligation reactions between azides and alkynes have been described recently. They involve an activation of the alkyne partner either in strain-promoted activation of cycloalkynes in [3 + 2] cycloaddition reactions with azides (45) or 1,3-dipolar cycloadditions of azides with electron-deficient alkynes (46). These methods could constitute attractive extensions of the click reactions to biological systems that are sensitive to the action of copper ions.
ACKNOWLEDGMENT Figure 1. Turbidity changes of suspensions, in HBS (pH 6.5), of targeted liposomes composed of DPPC, DPPG, and Chol (70/20/50 molar ratio) after addition of Con A (125 µg/mL). The coupling time by click reaction (Scheme 3) for the targeted vesicles (-9-) that contained 10 mol % anchor 8 was 3 h; control liposomes (-b-). Liposome concentration ) 60 µM lipids.
liposomes having encapsulated self-quenching concentrations of 5,6-carboxyfluorescein (29). Using a well-established method (29) based on fluorescence quenching determinations (see Experimental Procedures) we could demonstrate that, compared to vesicles incubated in the absence of the coupling reagents, essentially no leakage was triggered by the conjugation reaction. Importantly, these controls indicate that the standard conditions established here are harmless for liposomes made of saturated phospholipids. Finally, residual copper in our targeted liposome preparations could be a potential concern. Determination by atomic absorption spectroscopy showed, that for our standard conditions, final copper concentrations in our constructs were in the low µM range, whereas using the modified protocol the presence of this ion was no more measurable (
