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ARTICLES Uptake by Macrophages of a Biotinylated Oligo-a-deoxythymidylateby Using Mannosylated Streptavidin? Edwige Bonfils, Christina Mendes, Annie-Claude Roche, Michel Monsigny, and Patrick Midoux’ Laboratoire de Biochimie des Glycoconjugubs et Lectines endoghes, Centre de Biophysique Molhculaire, CNRS et Universith d‘orlbans, 1,rue Haute 45071, Orlbans Cedex 02, France. Received December 23, 1991 Streptavidin substituted with mannose residues increased by 20-fold the intracellular concentration of a biotinylated dodecakis(a-deoxythymidylate) in macrophages by comparison with the uptake of free oligodeoxynucleotide. Streptavidin, the bacterial homologue of the very basic avidin, which does not contain any carbohydrate moieties and is a neutral protein, was substituted with 12 mannose residues in order to be recognized and internalized by mannose-specific lectins on the surface of macrophages. A 3’-biotinylated and 5’-fluoresceinylated dodecakis(a-deoxythymidylate)was synthesized and bound onto mannosylated streptavidin. The conjugate was isolated, and by using flow cytometry, it was shown that the uptake of fluoresceinylated oligodeoxynucleotides bound to mannosylated streptavidin by macrophages is 20-fold higher than that of free oligodeoxynucleotides and that the uptake was competively inhibited by mannosylated serum albumin. Glycosylated streptavidin conjugates recognizing specific membrane lectins on different cells provide the possibility to target biotinylated antisense oligodeoxynucleotides and to increase the biological effect of these chemotherapeutic agents.
INTRODUCTION
Synthetic oligodeoxynucleotides and particularly antisense oligodeoxynucleotideshave been found to control the gene expression in various systems and could be used as antiviral drugs (for reviews see refs 1 and 2). However, their use in therapy is impaired by their absence of cell specificity and their limited cell uptake. In order to increase oligodeoxynucleotideuptake by cells, derivatives with cholesterol (3), phospholipid ( 4 ) and poly(L-lysine) (5, 6) have been described. Although these derivatives increase the oligodeoxynucleotide cellular uptake, they do not allow specific targeting. The attachment of an antisenseoligodeoxynucleotideto a specificmacromolecule recognized and internalized by cells via a receptormediating endocytosis will offer both a specific targeting and an increase of the intracellular uptake. Since (i) macrophages are potential targets for viruses, (ii) macrophages possess several membrane lectins (sugar binding receptors), one of them induces endocytosis of mannose(iii) mannosylated neoglyterminated glycoproteins (7), coproteins and mannosylated neutral polymers have been used to target an immunomodulator (8-11) and an antiviral drug (121,(iv) the linking of biotin to oligodeoxynucleotides has been reported to be either onto the 5‘- or 3‘ends or onto the purine or pyrimidine bases (13-15),and (v) the interaction between biotin and streptavidin is very M) with a very low dissociation rate strong (& = ~
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* To whom reprint request should be addressed. + Abbreviationsused biotin-HPDP, N- (6-biotinamidohexy1)3-(2’-ppidy1dithio)propionamide; BSA, bovine serum albumin; DABCO, 1,4-diazobicyclo[2.2.2]octane;EDTA, ethylenediaminetetraacetic acid; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; F1, fluoresceinyl; Fl-(a-dT)lrRSSHPbiotin, 5’-fluoresceinylated 3’-biotinylated dodecakii(a-deoxythymidylate); Fl-(a-dT)12-RSCH2CONH2,5’-fluoresceinyl 3‘thioacetamide dodecakis(a-deoxythymidylate);Man-BSA,mannosylated BSA; PBS, phosphate-buffered saline; a-dT, a-deoxythymidine; SDS, sodium dodecyl sulfate.
constant (9 X 8-9 (16,17), we planed to use mannosylated streptavidin to target biotinylated oligodeoxynucleotidesto macrophagesand to increase their intracellular concentration. In this report, we describe the synthesis of a 3‘-biotinylated 5‘-fluoresceinylated dodecakis(a-deoxythymidylate) and the preparation of mannosylated streptavidin and demonstrate by flow cytometry that mannosylated streptavidin increases 20-fold the internalization of this oligodeoxynucleotide into a macrophage cell line. EXPERIMENTAL PROCEDURES
Streptavidin, bovine serum albumin fraction V (BSA), a-deoxythymidine,and 1,4diazobicyclo[2.2.21octane (DABCO) were purchased from Sigma (St. Louis, MO); 1,l’carbonyldiimidazole, hexamethylenediamine, iodoacetamide, and L-glutamine were from Merck (Darmstadt, Germany); dithiothreitol (DTT) was from Serva (Heidelberg, Germany);fluorescein isothiocyanate (FITC isomer I) was from Molecular Probes (La Jolla, CAI; N(6-biotinamidohexyl)-3-(2’-pyridyldithio)propionamide (biotinHPDP) was from Pierce (Rockford, IL); monensin was from Calbiochem (La Jolla, CAI; Trisacryl GF05 and U1trogel ACA54 were from Sepracor (Villeneuvela Garenne, France); sodium dodecyl sulfate (SDS) and Biogel P2 were from Bio-Rad (Richmond, CA); fetal bovine serum was from Gibco (Reufrewshire);penicillin (100units/mL) and streptomycin (0.1 mg/mL) were from Eurobio (Paris, isothiocyanateand France). 4-a-~-mannopyran~ylphenysylphenyl mannosylated BSA (Man-BSA) containing 20 mannose residues were prepared as described (18, 19). Preparation of Mannosylated Streptavidin. Streptavidin (6.5 mg, 0.1 pmol) in 1 mL of 0.3 M NaC1, 0.1 M sodium carbonate buffer (pH 9) was reacted for 20 h at isothiocyanate (2 4 OC with 4-a-~-mannopyranosylphenyl mg, 6 pmol in 70 pL of DMSO). The conjugate was purified by gel filtration on Trisacryl GF05 (column size 20 X 2 cm) in PBS. The number of mannose residues bound per 0 1992 American Chemical Society
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streptavidin molecule was determined by the resorcinol sulfuric micromethod (20). The streptavidin concentration was determined spectrophotometrically by using = 34 (17). The concentration of mannosylated streptavidin was determined spectrophotometrically at 300 nm (where mannose residues did not absorb) by using E;&,,/E;$~ = 5.2. OligonucleotideSynthesis. Preparation of 5'-Fluoresceinylated Dodecakis(a-deoxythymidylate)[F1(a-dT)12-RSSROH]Where R = -(CH2)20(CH2)20(CH2)2-. A dodecakis(a-deoxythymidylate)was obtained by solid-phase synthesis on a Milligen DNA synthesizer 7500 (Milligen, Saint Quentin en Yvelines, France) using 5'-0-(4,4'-dimethoxytrityl)-2'-deoxy-lJ-a-thymidine 3'42cyanoethyl NJV-diisopropylphosphoramidite) prepared from a-thymidine and the phosphoramidite method (21, 22). The chain elongation was performed on a modified solid support (10-pmol scale) allowing the synthesis of an oligomer bearing a 3'4hiol function m previously described (23). After detritylation, the free 5'-hydroxyl group of the oligodeoxynucleotide5'-end was activated on the solid support at 20 "C for 5 h in 5 mL of 0.3 M 1,l'-carbonyldiimidazole in dioxane. After washing with dioxane and then with dry pyridine, the activated 5'-hydroxyl was reacted for 12 h at 20 "C with 5 mL of 0.45 M hexamethylenediamine in dry pyridine (24). The cleavage and the deprotection of the oligodeoxynucleotide from the support was conducted for 4 h at 20 "C in concentrated ammonia. The oligodeoxynucleotide was purified by HPLC ion exchange chromatography on a Mono Q 5/10 column (Pharmacia, Uppsala, Sweden) with a FPLC GP 250/500 apparatus (Pharmacia) using a linear NaCl gradient from 0 to 0.9 M in 20 min in 0.01 M phosphate buffer (pH 6.8) containing 20% acetonitrile. The oligodeoxynucleotide was eluted with a retention time of 10.1 min (flow rate, 4 mL/min). Acetonitrile was removed under reduced pressure. The oligodeoxynucleotide solution was desalted by gel filtration on Biogel P2 equilibrated with water and then freeze-dried. Fluorescein isothiocyanate (20 mg, 52 pmol in 1.5 mL of DMF) was reacted a t 20 "C for 5 h with the 5'-end amine function of the above oligodeoxynucleotide (5 mg, 0.13 pmol) in 3 mL of 0.1 M carbonate/bicarbonate buffer (pH 9.6). Excess of unbound fluorescein was removed by gel filtration on Biogel P2 equilibrated with water. Then, the fluoresceinylated oligodeoxynucleotide was further purified by reverse-phase chromatography on C18 column (Lichrocart 250 mm X 10 mm packed with 7-pm Lichrospher 100 RP-18 from Merck) using a Waters G 25 LC system apparatus and a Waters 600 E (system controller) equipped with a photodiode-array detector Waters 990. The fluoresceinylated oligodeoxynucleotidewas purified using a linear gradient of acetonitrile from 5% to 27.5% in 30 min in 0.1 M triethylammonium acetate (pH 7) and eluted with a retention time of 22.3 min (flow rate, 4 mL/ min). Acetonitrile was removed under reduced pressure and finally the oligodeoxynucleotidesolution was freezedried. Preparationof 5~-Fluowsceinyl3'-Thiol Dodecakis(a-deoxythymidylate) [F1-(a-dT)12-RSHI. The fluoresceinylated oligodeoxynucleotide (1mg, 0.026 pmol) was reduced by treatment for 6 h a t 20 "C with 0.1 M DTT in 0.4 mL of 0.1 M NaC1,O.l M sodium acetate buffer (pH 4.5) in the presence of 1.7 M EDTA. Then, DTT was removed by gel filtration on Biogel P2 equilibrated with the acetate buffer. Preparation of 5'-Fluoresceinyl3'-Thioacetamide Dodecakis(a-deoxythymidylate)[Fl-(a-dT)lrRSCH2-
Bonflls et al.
CONHz]. The Fl-(a-dT)lz-RSH oligodeoxynucleotidein acetate buffer was reacted with iodoacetamide in order to block the 3'-thiol function. The pH of the solution was adjusted to 7 with 0.2 M sodium carbonate, and solid iodoacetamide (1%final concentration) was added. Fl-(a~ T ) I ~ - R S C H ~ C O Nwas H Zpurified by gel filtration on Biogel P2 equilibrated with water and used as "free oligodeoxynucleotide" in uptake experiments. Biotinylation of the Oligodeoxynucleotide[Fl-(adT)~z-RSS-HPbiotin].Biotin-HPDP (1.4 mg, 2.6 pmol in 300 pL of DMF) was added toFl-(a-dT)Iz-RSH (0.5 mg, 0.11 pmol) in 0.6 mL of 0.1 M acetate buffer (pH 4.5) with stirring. The solution pH was adjusted to 7.4 with a 0.2 M sodium carbonate and the solution stirred for 20 h at 20 "C. Fl-(a-dT)12-RSSHPbiotin was purified by gel filtration on Biogel P2 equilibrated with PBS. Fluoresceinylated Oligodeoxynucleotideand Mannosylated Streptavidin Conjugate. Mannosylated streptavidin (1.2 mg, 0.02 pmol) in 1mL of PBS was mixed with Fl-(a-dT)lz-RSSHPbiotin (0.4 mg, 0.09 pmol) in 1.3 mL of PBS andstirred for 3 h a t 20 "C. Then, the conjugate was purified by gel filtration on Ultrogel ACA54 (column size 1 X 40 cm) equilibrated with complete PBS (PBS containing 1mM CaCl2 and 0.5 mM MgC12). The number of molecules bound per mannosylated streptavidin molecule was spectrophotometrically determined by using = 12.6 for the fluoresceinylated oligodeoxynucleotide. Gel Electrophoresis. Polyacrylamide gel electrophoresiswas performed in the presence of sodium dodecyl sulfate (SDS/PAGE). Electrophoresis was carried out for 6 h at 70 mA with 5% stacking gel and either 15% or 20% separating gels. The running buffer was 0.1 % SDS (w/v) in 25 mM Tris-glycine buffer (pH 8.9). For electrophoresis performed under reducing conditions, samples contained 1% SDS, 0.1 M DTT,and 0.8% bromophenol blue in PBS. Samples were reduced to their monomer form(@ upon boiling for 3 min. Then 50% (w/v) saccharose was added. For electrophoresis performed under nonreducing conditions, samples contained 0.1% SDS, 0.8% bromophenol blue, and 50% (w/v) saccharose in PBS and were not boiled. After electrophoresis, gels were stained with 0.1 % Coomassie Brillant Blue R 250 in a mixture of methanol, acetic acid, and water ( 5 2 5 , v/v/v), destained in the same solvent, and dried under vacuum. Cells. The 5774 clone E cell line (5774E),kindly given by Dr. P. Stahl (Wmhington University Medical School, St. Louis, MO) is a variant of murine macrophage-like 5774 cell line, selected, upon treatment with 5-azacytidine, because it expressed mannose membrane lectin (25). Cells were grown at 37 "C in RPMI medium (RPMI supplemented with 109% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and antibiotics). Cells were passaged by harvesting in PBS for 3 min at 37 "C in the presence of 2.5 pg/mL trypsin and 0.02% EDTA. Cells were mycoplasma free as evidenced by the BVC-Kanamycin A staining method (26). The cells were plated and incubated with fluoresceinylated compounds in either complete PBS containing 1%BSA, RPMI containing 1% BSA, or RPMI medium for a long incubation period. Flow Cytometry Analysis. Cells were washed and suspended in sheath fluid (134 mM NaC1,3.75 mM KC1, 1.9 mM KH2PO4, 16.53 mM Na2HP04, 15.24 mM NaF, 0.2 5% 2-phenoxyethanol) (27). The cell fluorescence intensity was measured using a FACS analyzer (Becton Dickinson, Sunnyvale, CA) equipped with the FACSlite unit (Becton Dickinson). A 488nm excitation wavelength was produced by a 25-mW cold-air argon laser. A 520 f
Macrophage Uptake of Ollgo-adeoxythymldylates
Figure 1. Structure of the 5'-fluoresceinylated 3'-biotinylated dodecakis(a-deoxythymidylate);R = -(CH2)20(CHz)zO(CH2)2-.
10 nm interference filter was set to collect the emitted green fluorescence. Three parameters (90" light scatter, electric volume, and green fluorescence) were simultaneously recorded in the list mode. The data from 5000 cells collected at 300 events/s were analyzed using the Consort 30 device (Hewlett-Packard). The cell fluorescence intensity was measured before and after a postincubation a t 4 "C with 50 p M monensin in order to estimate the uptake efficacy as previously described (19, 27, 28). Cell fluorescence intensity values were expressed as the mean value statistically calculated from the data of cells contained in the gated cell population. Fluoresceinylated beads (19) were used both to compare experiments done at different times and to quantitate the amount of fluoresceinylated oligodeoxynucleotide associated to the cells. Microscopy Analysis. Cell-associated fluorescence was localized with a confocal fluorescence microscope (Lasereharp MRC-600,Bio-Rad, Oxfordshire,U.K.) operating with a 488-nm argon laser excitation wavelength. RESULTS
A dodecakis(a-deoxythymidylate) [Fl-(a-dT)Iz-RSSROHI bearing a fluoresceinyl group a t the 5'-end and a disulfide group at the 3'-end was synthesized using the solid support previously described (23).Then, the 3'-thiol function, liberated upon reduction with DTT, was reacted with the dithiopyridinyl group of biotin-HPDP, leading to a 5'-fluoresceinylated oligodeoxynucleotide bearing a biotinyl group at the 3'-end (Figure 1). Streptavidin was mannosylated in order to prepare a specific macromolecular ligand for macrophages. Streptavidin was substituted with an activated mannose derivative @-isothiocyanatophenyl a-D-mannopyranoside)which reacted with the c-amino groups of the lysine residues of streptavidin. The absorbance spectrum of purified streptavidin substituted with mannose residues was modified between 240 and 290 nm compared to native streptavidin, because N[@-a-~mannopyranosylpheny1)thioamidyllresidues bound to streptavidin also absorb in this wavelength region (Figure 2). The number of mannose residues bound per streptavidin molecule was determined by resorcinol sulfuric micromethod (20)and according to the method of ref 28 by using 14 000 Da as the streptavidin subunit molecular mass (16, 17). Mannosylated streptavidin was found to contain three mannose residues per subunit molecule (12 mannose residues per tetramer and 35 pg of mannose per mg of protein). The purity and the molecular size of mannosylated streptavidin was checked and estimated by SDS/PAGE analysis (Figure 3). Under nonreducing conditions, the apparent molecular masses of mannosylated and native streptavidin were 62 OOO and 68 100 Da, respectively. Mannosylated streptavidin migrated faster, corresponding to an apparent smaller molecular mass than native streptavidin. Under reducing conditions, the apparent molecular mass of the subunit of both mannosylated and native streptavidin was 14 000 Da. Substitution of a large number of lysine residues with sugar residues may have altered the streptavidin structure and led to an increase in ita migration in polyacrylamide gel in the presence of SDS.
Bloconjugate Chem., Vol. 3, No. 4, 1992 279
A fluorescent oligonucleotide-mannosylated streptavidin conjugate was prepared by mixing in PBS the 3'biotinylated 5'-fluoresceinylated dodecakis(a-deoxythymidylate) and the mannosylated streptavidin (oligonucleotide to protein molar ratio was 41), and the conjugate was purified by gel filtration on a Ultrogel ACA 54 column. On the basis of the absorbance a t 495 nm, the main peak was found to contain the mannosylated streptavidin with three biotinylated and fluoresceinylated dodecakis(a-deoxythymidylate) molecules per mannosylated streptavidin molecule. Oligonucleotide Endocytosis. 57743 cells were incubated a t 37 "C in the presence of various concentrations of fluoresceinylated oligodeoxynucleotideseither free [Flor bound to mannosylated (~-~T)~z-RSCHZCONHZI streptavidin. After 2 h, the cell fluorescence intensity was analyzed by flow cytometry before and after a monensin posttreatment (Figure4). Monensin, a proton/sodium ionophore, was used to neutralize (29) acidic compartments (endosomes and/or lysosomes) of the cells, in order to recover the total fluorescence intensity of fluoresceinylated oligodeoxynucleotides inside the cells. The fluorescence intensity of cells incubated with the fluoresceinylated oligodeoxynucleotide was very low, even after a monensin posttreatment, indicating that the oligodeoxynucleotide was poorly internalized by 57743 cells. The fluorescence intensity of cells incubated in the presence of the fluoresceinylated oligodeoxynucleotide bound to mannosylated streptavidin was much higher than that of cells incubated with free oligodeoxynucleotide. The enhancement of the cell fluorescence intensity upon monensin treatment demonstrates that the oligodeoxynucleotide linked to mannosylated streptavidin was mainly internalized into acidic compartments. The binding and the uptake of mannosylated streptavidin carrying fluoresceinylated oligodeoxynucleotideswere saturable at 2pM oligodeoxynucleotide, in agreement with an endocytosis mediated by receptors (Figure 4). The uptake of fluoresceinylated oligodeoxynucleotides-mannosylated streptavidin conjugate was inhibited (up to 75% in the presence of an excess of mannosylated BSA, showing that mannosylated streptavidin was recognized by receptors specific for mannose (Figure 5 ) . Upon incubation of cells with nonbiotinylated oligonucleotide, either in the absence or in the presence of mannosylated streptavidin, the cell fluorescence intensities were identically low (Figure 6). This result indicates that nonbiotinylated oligonucleotide does not interact with mannosylated streptavidin to form a nonspecific conjugate. Therefore, biotinylated oligonucleotides were conjugated with mannosylated streptavidin by a specific binding to biotin sites of streptavidin, and the endocytosis of the conjugate was mediated by the mannose membrane lectins of 57743 cells. The cells were also analyzed by fluorescencemicroscopy using a confocal microscope (Figure 7). The fluorescence microscopy patterns of cells incubated with the fluoresceinylated oligodeoxynucleotides-mannosylated streptavidin conjugate showed that the conjugate entered the cells more efficiently than the unconjugated oligonucleotide. Furthermore, the fluorescence was mainly localized inside intracellular vesicles, a very mild fluorescence in the cytosol and almost nothing in nucleus. These results are in agreement with data obtained by using flow cytometry and specially with the enhancement of the cell fluorescence intensity measured after a postincubation with monensin, indicating that the fluorescent material was mainly in an acidic environment. Time Course of the OligonucleotideUptake. Flu-
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Figure 2. Absorbance spectra of (A) mannosylated streptavidin (2.0 p M ) and (B) streptavidin (3.5 pM)in PBS.
oresceinylated oligodeoxynucleotide (2 pM) either free or bound to mannosylated streptavidin was incubated at 37 "Cfor 24 h with 5774 clone E cells and the cell fluorescence intensity was measured at different times before and after a postincubation with monensin (Figure 8). The uptake of oligodeoxynucleotide bound to mannosylated streptavidin increased significantly during 6 h whereas the uptake of the free oligodeoxynucleotide remained very low even after 24 h of incubation. After 24-h incubation, the fluorescence intensity of cells incubated with the oligodeoxynucleotides-streptavidin conjugate was close to that obtained after 6-h incubation. The amount of fluoresceinylated oligodeoxynucleotide associated with a single cell was calculated from the enhancement of the cell fluorescence intensity measured after monensin treatment and using calibrated fluoresceinylated beads as previously described (28). The intracellular concentrations of oligodeoxynucleotide bound to mannosylated streptavidin were found to be 3 and 12.2pM after 1and 6 h, respectively (assuming a cell volume of 1.76 X lo4 p L ) . Under similar conditions, cells incubated with free oligodeoxynucleotide contained 0.14 and 0.60 pM oligodeoxynucleotide,respectively. DISCUSSION
In this report, we describe the use of streptavidin bearing mannose residues to allow an efficient and specific uptake of a synthetic oligodeoxynucleotide by a macrophage-like cell line. Streptavidin, a protein which does not contain any carbohydrate moieties, binds four biotin molecules with a dissociation constant of about 10-15M (16). SDS/ PAGE analysis of streptavidin from Sigma used in our experiments showed an apparent molecular mass of 68 100 and 14 OOO for streptavidin and its subunits, respectively. These values are close to those published by Green (17) and are in agreement with data reported by Bayer et al. (30)showing that commercial streptavidin is a truncated form of streptavidin related to a proteolytic digestion occurring during the isolation process. Streptavidin containing 16 lysine residues (17)which are not directly involved in the binding of biotin (31)was substituted with 12 mannose residues by allowing 4-a-~-mannopyranosylphenyl isothiocyanate to react with the lysine amino group of the protein. A mannosylated streptavidin
substituted with 12 mannose residues is supposed to be recognized by mannose-specific lectins of the macrophage surface as glycoproteins exposing terminal mannose residues do. 57743 cells, a macrophage cell line bind and internalize mannosylated protein via their mannosespecific membrane lectin (25). A dodecakis(a-deoxythymidylate) [ ( a - d T ) ~resistant ~l to nuclease degradation was synthesized and modified at the 5'-end by coupling a fluoresceinyl group and a t the 3'-end by coupling a biotinyl group via a disulfide bridge. The fluoresceinylated and biotinylated oligodeoxynucleotide strongly interacted with mannosylated streptavidin, which was easily substituted by three 3'-biotinylated 5'-fluoresceinylated dodecakis(a-deoxythymidylate)molecules. This indicated that the glycosylation of streptavidin by 12 mannose residues did not impair the accessibility of the biotin binding sites. The dissociation rate constant (k,ff = 9 X 10-8 8-11 (17) between biotin and streptavidin is so low that the biotinylated oligodeoxynucleotide-mannosylated streptavidin conjugate is as stable as a conjugate in which oligodeoxynucleotides would have been bound by a covalent linkage. This type of conjugate can be isolated by chromatographic methods. The 3'-thiol and 5'-fluoresceinyl dodecakis(a-deoxythymidylate)is linked to biotin by a disulfide bond. It was shown previously that this type of linkage is stable in culture medium: a 5'radioionidated oligodeoxyribonucleotideconjugated to 6phosphomannosylated bovine serum albumin (neoglycoprotein) via a disulfide bridge was not released from the carrier after 4 h at 37 "C in the culture medium supplemented with heat-inactivated fetal calf serum. Conversely, when 57743 cells were incubated for 2 h in the presence of the oligonucleotide-neoglycoprotein conjugate, it was found by cell fractionation that the cell-associated radiolabeled oligonucleotide was no longer linked with the carrier (32, and unpublished results). The release of the oligonucleotide may come from the reduction of the disulfide bond, reduction that may occur somewhere from the plasma membrane (33, 34) to any of the internal compartments where the conjugate may reach. The endocytosis of both fluoresceinylated oligodeoxynucleotide free or bound to mannosylated streptavidin was measured by a flow cytometry method as previously described (19,27,28).The uptake of macromolecules by
Macrophage Uptake of Oligo-adeoxythymidylates
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Bioconjugate Chem., Vol. 3, No. 4, 1992 281
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Figure 4. Flow cytometry analysis of the uptake by 57743 cells of 5'-fluoresceinylateddodecakis(a-deoxythymidylate) free and bound to mannosylated streptavidin. Cells were incubated at 37 "C for 2 h in RPMI containing 1%BSA in the presence of F1(a-dT)12-RSCH2CONH2(0,o)or 5'-fluoresceinylated dodecakis(a-deoxythymidylate) bound to mannosylated streptavidin (A, A). Cell fluorescence intensities were measured by flow cytometry before (0, A) and after (0,A) a postincubation for 30 min a t 4 "C with 50 p M monensin.
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Figure 3. SDS/PAGE pattern of mannosylated and native streptavidin. Mannosylated and native streptavidin were subjected to SDS/PAGE: (A) 15% gel and unboiled samples and (B) 20% under reducing conditions; proteins were stained by Coomassie Brillant Blue; lanes a, 25 pg of streptavidin; lanes b, 40 pg of mannosylated streptavidin; lanes c, the standard protein markers from Bio-Rad [myosin (MW 200 000), &galactosidase (MW 116 250), phosphorylase B (MW 97 400),bovine serum albumin (MW 66 200), ovalbumin (MW 42 700),bovine carbonic anhydrase (MW 31 000),soybean trypsin inhibitor (MW 21 500), and hen egg white lysozyme (MW 14 400)].
receptor-mediated endocytosis generally occurs via acidic vesicles-such as endosomes and ultimately lysosomes-in which the fluorescein fluorescence is partially quenched (35-37). The internalization of fluoresceinylated macromolecules,can be evidenced by cell fluorescence intensity measurements before and after a postincubation in the presence of monensin, which allows neutralization of the acidic compartments. The enhancement of the cell fluorescence intensity after monensin treatment proves that the fluoresceinylatedmacromoleculeswere contained in acidic vesicles upon endocytosis. We showed by using flow cytometry that the fluoresceinylated dodecakis(adeoxythymidylate) conjugated with mannosylated streptavidin was intensely internalized by 57743 cells by a receptor-mediated endocytosis process using the mannose
Figure 5. Specificity of the binding and uptake of 5'-fluoresceinylated dodecakis(a-deoxythymidylate) bound to mannosylated streptavidin. 57743 cells were first incubated a t 37 "C for 30 min in complete PBS containing 1% BSA in the absence (A) or presence (m) of mannosylated bovine serum albumin (1mg/ mL; 13 pM),and then 1 h a t 37 "C with 5'-fluoresceinylated dodecakis(a-deoxythymidylate)bound to mannosylated streptavidin. Cell fluorescence intensities were measured by flow cytometry after a postincubation for 30 min at 4 "C with 50 pM monensin.
membrane lectin of these cells. Conversely, the carrierfree fluoresceinylated oligonucleotide entered the cells poorly. The endocytosed oligonucleotide targeted by mannosylated streptavidin as well as the carrier-free fluoresceinylated oligonucleotide are mainly located in vesicles, as suggested by flow cytometry analysis and confirmed by fluorescence confocal microscopy analysis. Intracellular oligodeoxynucleotideconcentration was only 30 % of the extracellular concentration when cells were incubated with free oligodeoxynucleotide. Zamecnik et al. reported that 1.5 pM of an eicosakis(6-deoxythymidylate)was found in Hela cells after 15-min incubation in the presence of 20 pM oligodeoxynucleotide(38). Incubating cellswith 2 pM oligodeoxynucleotide bound to mannosylated streptavidin gave an intracellular oligodeoxynucleotide concentration 1.5 and 6 times higher than the extracellular concentration after 1 and 6 h, respectively. Therefore, mannosylated streptavidin concentrated the dodecakis(a-deoxythymidylate) inside 57743 cells. Moreover, the cellular uptake of oligodeoxynucleotide bound to mannosylated streptavidin was 20-fold higher than that of free
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Biocon/ugate Chem., Vol. 3, No. 4, 1992 h
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A B C Figure 6. Flow cytometry analysis of the uptake by 57743 cells of 5'-fluoresceinylated dodecakis(a-deoxythymidylate) conjugated with mannosylated streptavidin. Cells were incubated at 37 "C for 2 h in RPMI containing 1% BSA in the presence of (A) 5 p M 5'-fluoresceinylated dodecakis(a-deoxythymidylate)bound to mannosylated streptavidin (40 pg/mL), (B) 5 pM Fl-(a-dT)12RSCH2CONH2, and (C) 5 pM F ~ - ( ( Y - ~ T ) ~ ~ - R S C H ~ mixed CONH~ with 40 pg/mL mannosylated streptavidin. Cell fluorescence intensities were measured by flow cytometry before (open) and after (hatched) a postincubation for 30 min at 4 "C with 50 p M monensin.
Figure 7. Intracellular localization of 5'-fluoresceinylated dodecakis(a-deoxythymidylate)either free or bound to mannosylated streptavidin. 57743 cells were incubated at 37 "C for 2 h in RPMI containing 1%BSA in the presence of either (A) 5 pM 5'-fluoresceinylated dodecakis( a-deoxythymidylate) conjugated with 40 pg/mL mannosylated streptavidin or (B) 5 pM Fl-(adT)12-RSCH&ONH2. Cells were washed, incubated for 30 min at 4 "C with 50 pM monensin, and fixed at 4 "C in 1% paraformaldehyde in PBS. Cells were mounted in PBS/glycerol (v/v) containing 1%DABCO as antifade agent (scale bar, 10 pm).
oligodeoxynucleotide. These values are close to those obtained using cholesterol,phospholipid, or poly(L-lysine) oligodeoxynucleotidederivatives. Attachment of a cholesterol residue at the 3'-end of a decakis(P-deoxythymidylate) increased 6- and %fold its intracellular concentration
Time (hours)
Figure 8. Time course of the 57743 cell uptake of 5'-fluoresceinylated dodecakis(a-deoxythymidylate) either free or bound to mannosylated streptavidin. Cells were incubated a t 37 "C in RPMI medium in the presence of 2 p M Fl-(a-dT)12-RSCH20 ) or 2 p M 5'-fluoresceinylated dodecakis(a-deoxCOQH2 (0; ythymidylate) bound to mannosylated streptavidin (15 pg/mL) (A;A). Cell fluorescence intensities were measured by flow cytometry before (0;A) and after (0;A) a postincubation for 30 min a t 4 "C with 50 pM monensin.
and 15-and 20-fold its internalization by comparison with those of underivatized oligodeoxynucleotidewhen 2.5 and 0.5 p M cholesterol-derivatized oligodeoxynucleotideconjugates were incubated for 2 h with L929 and Krebs-2 cells, respectively (3). Attachment of a phospholipid tail to the 5'-end of a pentadecakis(0-oligodeoxynucleotide) increased 10-fold its cellular uptake after 4-h incubation by comparison with the uptake of phosphorothioate and phosphodiester derivatives and concentrated 4-fold the oligodeoxynucleotide inside L929 cells (4). Attachment of a fluoresceinylated pentadecakis(P-oligodeoxynucleotide) to the polycationic poly(L-lysine) increased its internalization 12-fold by comparison with that of free oligodeoxynucleotidewhen L929 cells were incubated for 4 h with 1 p M fluoresceinylated oligodeoxynucleotidepolylysine conjugate (6). Recently, a 4-fold increase of the uptake by brain capillaries of a biotinylated antisense uneicosakis(P-oligodeoxynucleotide) was obtained after conjugation with avidin (39). In this case, it is the cationic properties of avidin that mediated the uptake of the conjugate. Indeed, when streptavidin, which is a neutral protein, was used, there was no increase in the uptake of the uneicosakis(P-oligodeoxynucleotide) (39). The use of poly(L-lysine) and avidin cannot be considered as true targeting since polycationic molecules will interact with any cells. Biotinylated oligodeoxyribonucleotidescan be easily synthesized and conjugated to streptavidin substituted with mannose to allow targeting to macrophages. A conjugatecontainingthree oligodeoxynucleotide molecules was easily obtained and isolated from free oligodeoxynucleotides with a greater yield than conjugates prepared using covalentlinkagesbetween oligodeoxynucleotides and protein. Membrane lectins have been evidenced at the surface of many normal cells (including hepatocytes, monocytes, macrophages, endothelial cells, and lymphocytes) and tumor cells and specific glycoconjugates have been used to target drugs (for reviews see refs 40-42). Streptavidin could be substituted by various monosaccharides or complex oligosaccharides,provided that the biotin binding site accessibility be preserved and so could be used to target membrane lectins on different cell types. Various glycosylated streptavidins may be obtained and therefore open the possibility (i) to target antisense oligodeoxynucleotides to different cells; (ii) to increase their cellular uptake, and (iii) to study their intracellular traffic, in a chemotherapeutic strategy. Streptavidin could also be
Macrophage Uptake of Oligo-adeo~ymldydylates
used as an intermediate carrier to bind biotinylated antisense oligonucleotides to specific biotinylated vectors such as monoclonal antibodies, glycoproteins, cytokines, or growth factors. Antisense oligonucleotides, which have been found to control the gene expression in cultured cells, enter the cells mainly by pinocytotic vesicles (43); few molecules may reach, by a still unknown process, the cytosol and/or the nucleus, and, there, interact with their targets (mRNA translation initiation sites, pre-mRNA splicing sites, or genes). The use of macromolecular carriers such as mannosylated streptavidin allows an increased uptake of fluoresceinylated dodecakis(a-deoxythymidylate)into macrophages by a receptor-mediated endocytotic process. The increased intracellular concentration of the oligonucleotide is clearly observed inside endocytotic vesicles. According to the fact that the internalized oligonucleotide is carrier free, it may be expected that the cytosolic and/or the nuclear oligonucleotideconcentration will also increase. The mechanism of the uptake of free oligonucleotides is not yet understood; it has been proposed that specific receptors are involved (43),but even in this case, the free oligonucleotides are taken up by an endocytotic process. Therefore, oligonucleotides targeted by mannosylated streptavidin have the opportunity to enter the cells and to reach a higher intracellular concentration, which may in turn significantly increase the cytosolic and nuclear concentrations. ACKNOWLEDGMENT
We thank Dr. C. Helene for helpful discussions and his interest in this work, and Dr. N. T. Thuong for his valuable help in oligonucleotidesynthesis. This work was supported by grantsfrom ‘Agence National de Recherche sur le Sida” (ANRS). E.B. and C.M. received afellowship from ANRS and MRT, respectively. A.C.R. is “Directeur de recherche INSERM” and P.M. is ‘Charge de recherche INSERM”. LITERATURE CITED (1) HBlBne, C., and ToulmB, J. J. (1990) Specific regulation of gene expression by antisense, sense and antigene nucleic acid. Biochim. Biophys. Acta 1049, 99-125. (2) Uhlmann, E., and Peyman, A. (1990) Antisense oligonucleotides: A new therapeutic principle. Chem.Rev. 90,543584. (3) Boutorin, A. S., Gus’kova, L. V., Ivanova, E. M., Kobetz, N. D., Zarytova, V. F., Ryte, A. S., Yurchenko, L. V., and Vlassov, V. V. (1989)Synthesis of alkylating oligonucleotidederivatives containing cholesterol or phenazinium residues at their 3’terminus and their interaction with DNA within mammalian cells. FEBS Lett. 254, 129-132. (4) Shea, R. G., Marsters, J. C., and Bischofberger, N. (1990) Synthesis, hybridization properties and antiviral activity of lipid-oligodeoxynucleotideconjugates. Nucleic Acids Res. 18, 3771-3783. (5) Lemaitre, M., Bayard, B., and Lebleu, B. (1987) Specific antiviral activity of a poly(L-lysine)-conjugate oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site. h o c . Natl. Acad. Sei. U.S.A. 84, 64-52. (6) Leonetti, J. P., Degols, G., and Lebleu, B. (1990) Biological activity of oligonucleotide-Poly(L-lysine) conjugates: Mechanism of cell uptake. Bioconjugate Chem. 1, 149-153. (7) Wileman, T., Harding, C., and Stahl, P. (1985) Receptormediated endocytosis. Biochem. J. 232, 1-14. (8) Monsigny, M., Roche, A. C., and Bailly, P. (1984) Tumoricidal activation of murine alveolar macrophages by muramyl dipeptide substituted mannosylatedserum albumin. Biochem. Biophys. Res. Commun. 121,579-584.
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