Bioconjugate Chem. 1995, 6,695-701
695
Cell-Type Specific and Ligand Specific Enhancement of Cellular Uptake of Oligodeoxynucleoside-MethylphosphonatesCovalently Linked with a Neoglycopeptide, YEE(ah-Ga1NAc)s J o n J. Hangeland, Joel T. Levis, Yuan C. Lee,",' a n d Paul 0. P. Ts'o" Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 21205, and Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218. Received May 17, 1 9 9 P
A novel, structurally defined, and homogeneous oligodeoxynucleoside methylphosphonate (oligo-MP) neoglycopeptide conjugate, CYEE(~~-G~~NAC)~]-SMCC-AET-~U~~'IJ, has been synthesized. The linkage between the carbohydrate ligand and the oligo-MP is a metabolically stable thioether. Experiments establish that uptake of this conjugate by human hepatocellular carcinoma (Hep G2) is cell-type specific when compared with its uptake by human fibrosarcoma (HT 1080) and human promyleocytic leukemia (HL-60). Uptake of the conjugate with Hep G2 cells can be totally inhibited by the addition of a 100-fold excess of free YEE(ah-GalNAc13in the culture medium indicating the observed cell uptake is ligand specific. The conjugate is rapidly taken in by Hep G2 cells in a linear fashion reaching a saturation plateau of 26 pmol per lo6 cells afker 24 h. Conjugation of oligo-MPs to ligands for hepatic carbohydrate receptors, such as YEE(ah-GalNA&, represents a n efficient and ligand-specific method for the intracellular delivery of oligo-MPs.
INTRODUCTION
The antisense (anticode) or antigene strategy for drug design is based on the sequence-specific inhibition of protein synthesis due to the binding and masking of the target mRNA or genomic DNA, respectively, by the synthetic oligodeoxynucleotide (oligo-dN)' and their analogs (1). Implicit in this strategy is the ability of oligodNs to cross the cellular membrane(s), thereby gaining access to the cellular compartments containing their intended target, and to do so in sufficient amounts for binding to its target to take place. Among the many oligo-dN analogs for application as antisense agents, nonionic oligonucleoside methylphosphonates (oligo-MPs) have been extensively studied (2). Oligo-MPs are totally resistant to nuclease degradation (3) and are effective antisense agents with demonstrative in vitro activity against herpes simplex virus type 1 (41, vesicular stomatitis virus (51,and human immunodeficiency virus (6) and are able to inhibit the expression of ras p21(7). For oligo-MPs to exhibit antisense activity, however, they must be present in the extracellular medium in concentrations up to 100 pM (4-7).
* P.O.P.T.: Phone: (410) 955-3172. Fax (410) 955-4392. Y.C.L.: Phone: (410) 516-7041. Fax (410) 516-8716. Department of Biology. Abstract published in Advance ACS Abstracts, September 15, 1995. Abbreviations: AET, aminoethanethiol; ATCC, American Type Culture Collection, Rockville, MD; ATP, adenosine triphosphate; BAP, bacterial alkaline phosphatase; CPG, controlled pore glass support; DIPEA, diispropylethylamine; D-MEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; dN, 2'-deoxynucleotide; D-PBS, Dulbecco's phosphate buffered saline; D l T , dithiothreitol; EDAC, l-ethyl-3-[3-(dimethylamino)propyllcarbodiimide; EDTA, ethylenediaminetetraacetate; FCS, fetal calf serum; HBSS, Hank's balanced salt solution; HSA, human serum albumin; HSV-1, herpes simplex virus type 1; MEM, minimal essential medium with Earle's salts; MP, methylphosphonate; PAGE, polyacrylamide gel electrophoresis; PNK, bacteriophage T4 polynucleotide kinase; SMCC, N-hydroxysuccinimidyl4-(N-methylmaleimido)cyclohexane-l-carboxylate; Tris, tris(hydroxylmethy1)amine. +
@
Delivery of exogenous DNA into the intracellular medium is greatly enhanced by coupling its uptake to receptor-mediated endocytosis. Pioneering work by Wu and Wu (8)showed that foreign genes (8a-c)or oligo-dNs (8d),electrostatically complexed to poly-L-lysine linked to asialoorosomucoid, are efficiently and specifically taken into human hepatocellular carcinoma (Hep G2) cells through direct interaction with the asialoglycoprotein receptor. Since this initial study, other examples of receptor-mediated delivery of DNA have appeared including a tetra-antennary galactose neoglycopepideolyL-lysine conjugate (91,folate conjugates (IO),an antibody conjugate (11),transferrin conjugate (12),and 6-phosphomannosylated human serum albumin (HSA)covalently linked to a n antisense oligo-dNs via a disulfide bond (13). Recently, the triantennary N-acetylgalactosamine neoglycopeptide, YEE(ah-GalNAc)s (141, conjugated to human serum albumin which was in turn linked to polyL-lysine, was shown to effectively deliver DNA into Hep G2 cells (15). In each instance, structurally heterogeneous conjugates were utilized to deliver DNA or oligodNs into cells. This strategy for the targeting and delivery of DNA can also be exploited for the targeting and delivery of oligo-MPs to specific cell types. In this paper, the synthesis, characterization, and cellular uptake profile of a structurally defined and homogeneous neoglycopeptide-oligo-MP conjugate, TYEE(ah-GalNAc),lSMCC-AET-pU"pT7 (6;Figure 2), is described.2 EXPERIMENTAL PROCEDURES
Synthesis of [5-32Pl-[YEE(ah-GalNAc)31 -SMCCAET-pUmpT, (6). General. Methyl phosphonamidite synthons were a generous gift from JBL Scientific, Inc. All other reagents for the automated synthesis of 2 were purchased from Glen Research. HiTrap Q anion exchange columns were purchased from Pharmacia LKB 2 UmpT7: U m is 2'-0-methyluridine. The oligo-MP was constructed with a 5' terminal phosphodiester. T 7 denotes seven thymidine nucleosides linked by methylphosphonate diesters.
1043-1802/95/2906-0695$09.00/0 0 1995 American Chemical Society
696 Bioconjugate Chem., Vol. 6,No. 6,1995
Biotechnology. Reversed phase high-performance liquid chromatography was carried out using a Microsorb C-18 column purchased from Rainin Instrument Co., Inc. Cystamine hydrochloride, l-ethyl-3-[3-(dimethylamino)propyllcarbodiimide (EDAC), 1-methylimidazole, anhyd dimethyl sulfoxide (DMSO), dithiothreitol (DTT), and Ellman's reagent were purchased from Aldrich and were used without further purification. Diisopropylethylamine (DIPEA)was purchased from Aldrich and was redistilled from calcium hydride prior to use. N-Hydroxysuccinim(SMidyl 4-(N-methylmaleimido)cyclohexanecarboxylate CC) was purchased from Pierce. Waters SepPak C-18 cartridges were purchased from Millipore Corp. YEE(ah-GalNAc)3 was synthesized according to Lee et al. (14a) and was stored a t 4 "C as an aqueous solution. Adenosine triphosphate (ATP) and [y-32Pl-ATPwere purchased from P-L Biochemicals, Inc., and Amersham, respectively. Polyacrylamide gel electrophoresis (PAGE) was carried out with 20 cm x 20 cm x 0.75 mm gels that contained 15% polyacrylamide, 0.089 M Tris, 0.089 M boric acid, 0.2 mM EDTA, pH 8.0 (1 x TBE), and 7 M urea. Samples were dissolved in loading buffer containing 90% formamide, 10% 1 x TBE, 0.2% bromophenol blue, and 0.2% xylene blue. Synthesis of Ump_T7 (2). The oligodeoxynucleoside methylphosphonate was synthesized on a controlled pore glass support (CPG) using 5'-O-(dimethoxytrity1)-3'-0(methyl-N,N-diisopropy1phosphonamido)thymidineand deprotected according to established methods (16).The final synthon incorporated into the oligomer a t its 5' end was 5'-0-(dimethoxytrityl)-2'-O-methyl-3'-(2-cyanoethylN,N-diisopropy1phosphoramido)uridine. The final coupling step positioned a phosphodiester linkage between the terminal 5'-nucleoside and the adjacent nucleoside, which permitted phosphorylation of the 5'-terminal hydroxyl group with bacteriophage T4 polynucleotide kinase (PNK) and ensured the stability of the phosphodiester linkage toward endonuclease cleavage due to the presence of the 2'-O-methyl group (17). The crude oligo-MP was purified by HiTrap Q anion exchange chromatography (load with water containing '25% acetonitrile; elute with 0.1 M sodium phosphate, pH 5.8) and preparative reversed-phase chromatography (Microsorb C-18) using a linear gradient (solvent A: 50 mM sodium phosphate, pH 5.8, 2% acetonitrile; solvent B: 50 mM sodium phosphate, pH 5.8, 50% acetonitrile; gradient: 0-60% B in 30 min). The oligomer thus purified was ca. 97% pure by analytical HPLC contaminated by a small amount of the n-1 species. Synthesis of [5'-32PI-5'-0-[(N-(2-Mercaptoethyl)phosphoramidate]-Ump_T7 (5). The purified oligomer (168 nmol), ATP (160 nmol), HzO (75 pL), l o x PNK buffer (5 mM DTT, 50 mM TrismHCl, 5 mM MgC12, pH 7.6; 10 pL), [Y-~~PI-ATP (1.1x l O I 4 Bqlmmol, 3.7 x lo6 Bq; 10 pL), and PNK (150 U in 5 pL) were combined and incubated a t 37 "C for 16 h and evaporated to dryness. The residue was redissolved in 0.2 M 1-methylimidazole, pH 7.0 (100 pL), and 1.0 M cystamine hydrochloride, pH 7.2, containing 0.3 M EDAC (100 pL), and heated a t 50 "C for 2 h (18). The excess reagents were removed by SepPak (loaded with 50 mM sodium phosphate, pH 5.8, 5% acetonitrile; washed with 5% acetonitrile in water; eluted with 50% acetonitrile in water). The solvent was evaporated in U ~ C U Oand crude cystamine adduct redissolved in 10 mM phosphate containing 50 mM DTT (200 pL) and heated to 37 "C for 1h. The buffer salts and excess reductant were removed from the reaction mixture as before, and the crude product was dried in uacuo. The title compound 5, produced in 57% yield from 2, was used in the next step without further purification.
Hangeland et al.
Synthesis of [~-32Pl-~E(ah-CalNAc)37-SMCC-AETpUmp_T7 (6). The neoglycopeptide 1 (336 nmol) was dissolved in anhyd DMSO (40 pL) and treated with DIPEA (336 nmol) and SMCC (336 nmol). The reaction was allowed to stand a t rt for 4 h and then added to the freshly prepared thiol 5. The reaction mixture was degassed and allowed to slowly concentrate under vacuum a t rt. The crude 6 was dissolved in formamide loading buffer (100 pL), purified by PAGE (4 V/cm, 1.5 h), and recovered by the crush and soak method (50% acetonitrile in water). The overall yield of pure 6 was 25%. Upon treatment with 0.1 M HC1 (37 "C, 1 h), 6 produced [5'32Plphosphorylated2 due to hydrolysis of the P-N bond; however, 6 was unreactive toward DTT (50 mM, pH 8, 37 "C, 1h), 3-maleimidopropionic acid (50 mM, pH 8, 37 "C, 1 h), Ellman's reagent (50 mM, pH 8, 37 "C, 1 h), and bacterial alkaline phosphatase (BAP; 70 U, 65 "C, 1 h). Sequential treatment of 6 with 0.1 N HC1 and BAP resulted in complete loss of [32P]-labelas anticipated. Stoichiometric analysis of a n unlabeled sample of 6 prepared identically showed it to contain 3 mol of N-acetylgalactosamine for each mole of c ~ n j u g a t econ,~ sistent with the proposed structure, while pneumatically assisted electrospray mass spectrometry (Scripps Research Institute Mass Spectrometry Facility, La Jolla, CA) produced a single parent ion (negative ion mode) a t m / z 4080 (calcd 4080.71, confirming the homogeneity of the sample. Cellular Uptake Experiments. General. Minimal essential medium with Earle's salts supplemented with L-glutamine (MEM),Dulbecco's modified Eagle's medium (D-MEM), RMPI medium 1640 supplemented with Lglutamine (RMPI), Dulbecco's phosphate-buffered saline (D-PBS), fetal calf serum (FCS), sodium pyruvate (100 mM), nonessential amino acids (10 mM),aqueous sodium bicarbonate (7.5%), and trypsin (0.25%; prepared in HBSS with 1.0 mM EDTA) were purchased from GIBCO BRL. Human hepatocellular carcinoma (Hep G2), human fibrosarcoma (HT 10801, and human promyleocytic leukemia (HL-60) cells were purchased from ATCC and were maintained in 1 x MEM supplemented with 10% FCS, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids (Hep G2), 1 x D-MEM supplemented with 10%FCS (HT-1080)or 1 x RMPI supplemented with 10% FCS (HL-60) in a Forma Scientific Model 3158 incubator maintained at 37 "C and 5% COZ. Silicon oil was a generous gift from General Electric (product no. SF 1250). Cells were counted using a Coulter Cell Counter Model ZBI. Uptake Experiments with Hep G2 Cells or HT 1080. Cells were passaged into 2 cm wells and grown in the appropriate medium to a density of ca. lo6 cells per well. The maintenance medium was aspirated, and the cells were incubated at 37 "C with 0.5 mL of medium that contained 2% FCS and was made 1pM in [5'-32P]-labeled 6. After the prescribed time had elapsed, a 5 pL aliquot of the medium was saved for scintillation counting and the remainder aspirated from the well. The cells were washed with D-PBS (2 x 0.5 mL), treated with 0.25% trypsin (37 "C, 2 m i d , and suspended in fresh growth medium containing 10% FCS. The suspended cells were layered over silicon oil (0.5 mL) in a 1.7 mL conical microcentrifuge tube and pelleted by centrifugation a t 14 000 rpm (12 OOOg) for 30 s. The supernatent was The molar absorbance of U m p 3was calculated to be 59 750 Umol-cm by taking the sum of the molar absorbance values for each of the nucleosides contained in the structure. This value was in excellent agreement with the number of moles of GalNAc residues found to be contained in the conjugate.
Enhanced Cell Uptake of Oligomethylphosphonates
carefully decanted, and the cell pellet was lysed with 100 pL of a solution containing 0.5%NP 40, 100 mM sodium chloride, 14 mM TrivCl, and 30% acetonitrile. The quantity of [32Pl-labeledmaterial and, by inference, the amount of 6 (or its breakdown product^)^ associated with the cell lysate, were determined by scintillation counting. Uptake Experiments with HL-60Cells. RMPI medium supplemented with 2% FCS and made 1pM in [5’-32Pl-6 was pre-treated with 7.5 x lo6 HL 60 cells for 5 min a t rt. The cells were removed by centrifugation (5 min). The medium (31 mL) was decanted and added to 7.5 x lo6 fresh HL-60 cells. The cells were evenly suspended and the cell suspension divided into six 0.4 mL portions. The remainder was discarded. The cells were incubated in medium containing the conjugate 6 for the prescribed time and then collected by centrifugation (5 min), resuspended in 0.5 mL D-PBS, and layered onto silicon oil in a 1.7 mL conical microfuge tube. The cells were pelleted by centrifugation (12000g, 30 s) and lysed, and the amount of [32Pl-labeledmaterial associated with the cells was determined by scintillation counting.
Bioconjugate Chem., Vol. 6, No. 6,1995 697
GalNAc
w
HO
1
RESULTS AND DISCUSSION
Synthesis of [YEE(ah-GalNAc),l-SMCC-AETpUmpz, (6). Synthesis and purification of YEE(ah) Umpx7(2) (16) were carried out GalNAc)3 (1) ( 1 4 ~and according to established procedures. In order to form a covalent link between 1 and 2, we chose to modify the 5‘-end of 2 using the method of Orgel (18). This introduced a disulfide into the oligo-MP, which in turn could be reduced with DTT to give a 5’-thiol. The neoglycopeptide 1was modified in a complementary fashion using the heterobifunctional cross-linking reagent, SMCC, capable of combining specifically with the N-terminal amino group of 1. Coupling of the maleimido group introduced by SMCC and the 5‘4hiol of the modified oligo-MP resulted in linkage of the oligo-MP and neoglycopeptide via a metabolically stable thioether (Figure 2). To begin the synthesis, 2 was phosphorylated using PNK and 0.95 equiv of [32Pl-ATP. Successful 5’-phosphorylation was confirmed by a n increase in the electrophoretic mobility of the product compared to the parent oligo-MP owing to the increased negative charge from -1 to -3 upon addition of a 5’-phosphate and incorporation of 32Pinto the structure (band A Figure 3). Formulation of the end-labeling reaction in this way ensured that ca. 90% of the ATP was consumed, allowing efficient use of the [32Pl-ATPto radioactively label the conjugate. Modification of the 5’-phosphate was accomplished in two steps. The 5’-end-labeled oligo-MP was incubated at 50 “C with 0.5 M cystamine hydrochloride in a buffer containing 0.1 M 1-methylimidazole a t pH 7.2 in the presence of 0.15 M EDAC to give the 5‘-cystamine phosphoramidate in 65% yield. PAGE analysis of the reaction mixture showed the product to migrate significantly slower than the 5’-end-labeled oligo-MP. This observation is consistent with the change in charge from -3 to -1 due to the loss of a single oxyanion on the 5’phosphate upon formation of the P-N bond and neutralThe exact structures of breakdown products of conjugate 6 have not been established. It is expected, however, that the neoglycopeptide will undergo significant biodegradation (glycolysis and proteolysis) upon endocytosis and partitioning to lysosomes. We report here the extent to which 5’-[32Pl-labeled 6 and the products of its biodegradation are associated with the cells. It is worth noting that the oligo-MP moiety of conjugate 6 is resistant toward endo- and exonuclease degradation (3, 17) and, therefore, is expected to remain unaltered inside the cell over the course of the experiment.
HO 2 R=H O H
7 R = - T - N d NH; 0Figure 1. Structures of neoglycopeptide YEE(ah-GalNAc)s (1) and oligo-MP UmpT7(2) and 5’-ethylenediamine capped UmpT7 (7).
ization of a second negative charge by the positively charged protonated primary amine present on the terminus of the cystamine group (compare bands A and B; Figure 3). Up to 35% of thymidine-modified oligo-MP was produced during this reaction (band C; Figure 3), and despite attempts to modify the reaction conditions (e.g., lowering the temperature and reducing the concentration of EDAC), its production could not be eliminated without concomitant reduction in yield of the desired cystamine adduct. This side product presumably arises due to reaction of EDAC with N-3 of thymidine to form a thymidine-EDAC adduct (18, 19). Reduction of the disulfide with 50 mM DT” a t pH 8 was quantitative5 and was accompanied by mobility shift to a faster migrating species due to the loss of the positively charged protonated primary amino group (compare bands B and C; Figure 3). In a separate reaction, 1 was combined with 1 equiv each of SMCC (3) and DIPEA in anhydrous DMSO and incubated a t room temperature. Combination of this reaction mixture with thiol 5 could be carried out without complete consumption of SMCC by 1 since 5 Although we chose to introduce a thiol onto the oligo-MP postsynthetically, in part to allow introduction of 32Penzymatically at the 5’-terminus, the construction of the conjugate could as easily be carried out by introduction of a thiol linker during the solid phase synthesis of the oligo-MP using, for example, 6-(tritylthio)hexyl phosphoramidite (19)commercially available from Glen Research.
698 Bioconjugate Chem., Vol. 6,No. 6, 1995
Hangeland et al.
I
DMSO DlPEA
1. PNK, ATP 2. imidazole, EDAC, cystamine 3. D l T
0
0 5
4
Hfl S-~-y-~-~mp~,
[YEE(ah-GalNAc)&NH
00
6 Figure 2. Reaction scheme for the synthesis of TYEE(ah-GalNAc)31-SMCC-AET-pUmpT, (6). 1
2
3
4
>
xc*
>
Figure 3. PAGE analysis (15%polyacrylamide, 4 V/cm, 2 h) of intermediates in the synthesis of conjugate 6. Lane 1: [5’32Pl-labeled2 (band A). Lane 2: [5’-32Pl-cystamineadduct (band B) and corresponding thymidine-EDAC adducts (bands C). Lane 3: [5’-32P]-thiol5 (band D) and corresponding thymidineEDAC adducts (bands E). Lane 4: [5’-32P]conjugate6 (band F) and corresponding thymidine-EDAC adducts (bands G). the reactive groups present on 1, 3, and 5 combined regiospecifically, thereby yielding a structurally defined and homogeneous conjugate. As anticipated, the addition of the modified neoglycopeptide to the 5’-end of the activated oligo-MP was accompanied by a substantial slowing of its mobility by PAGE since the mass of the
conjugate 6 is significantly larger than that of the parent oligo-MP (band F; Figure 3). Following this scheme, 5 was completely converted to 6 when 2 equiv (based on starting oligo-MP 2) of the neoglycopeptide 1 was used. The overall yield of the conjugate 6 was 24%(average of three syntheses) based on oligo-MP 2. The homogeneity of 6 was confirmed by the detection of a single parent ion (negative ion mode) by electrospray mass spectrometry. Cellular Uptake Experiments. We first investigated the cellular uptake of the conjugate 6, both alone and in the presence of 100 equiv of free neoglycopeptide 1, by Hep G2 cells to demonstrate that uptake by the cells was a result of binding of the neoglycopeptide moiety of 6 to the hepatic carbohydrate receptor. As a control, an oligo-MP modified a t the 5’-end with ethylenediamine (7; Figure 1)6 was also incubated with Hep G2 cells under identical conditions. In each instance, the modified oligoMP was present a t a concentration of 1pM in medium containing 2% fetal calf serum (FCS) and incubations were carried out a t 37 “C. The concentration of FCS was lowered from 10% to 2% in order to decrease the possibility of nonspecific binding of the oligo-MP conjugate with medium associated proteins. The uptake of conjugate 6 by the cells was rapid when incubated alone, loading the cells in a linear fashion to the extent of 7.8 pmol per lo6 cells aRer only 2 h (Figure 4A14 In contrast, when a 100-fold excess of free 1 was present with 1pM conjugate, association of 6 was only 0.42 pmol per lo6 cells, a value essentially identical to that obtained with the control oligo-MP 7 (0.49 pmol per lo6 cells). As an additional control, Hep G2 cells were incubated with 7 in the presence of a 10-fold excess of 1 to assess the Miller, P. S., and Levis, J. T. Unpublished results. Modification of the Ei’-phosphatewith ethylenediamine was accomplished by incubation of 5’-phosphorylated 2 with 0.1 M EDAC in a buffer containing 0.1 M imidazole a t pH 7 a t 37 “C for 2 h followed by overnight incubation with an aqueous solution 0.3 M ethylenediamine hydrochloride buffered to pH 7.0. This modification prevents removal of the 5’-phosphate by cellular phosphatase activity.
Enhanced Cell Uptake of Oligomethylphosphonates
Biocon/ugate Chem., Vol. 6,No. 6, 1995 699 30 25
HepG2 HL-60
0
.r
t
HT 1080 v)
20
0 W
W
2
2 & -P 0 E,
8Q 5 15 0
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10 5 0
0.0
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1.5
2.0
2.5
-
I
3
time (hours) Figure 5. Tissue specific uptake of conjugate 6 by Hep G2, HL-60, and HT 1080 cells. Cells were collected and the amount of [32Plwas determined at 3 and 24 h for each cell line.
time (hours)
Experiments were done in triplicate and the data expressed as the average & one standard deviation.
0
4
8
12
16
20
24
28
time (hours) Figure 4. (A) Time course for the uptake by Hep G2 cells of 1 ,uM conjugate 6, alone ( 0 )and in the presence of 100 equiv of free 1 (01,and oligo-MP 7,alone (v)and in the presence of 10 equiv of free 1 (VI. Cells were incubated at 37 "C for 0, 1, and 2 h, and samples were collected as described in the Experimental Procedures. Each data point represents the average of three trials f one standard deviation. (B)24 h time course for the uptake of conjugate 6 by Hep G2 cells. Cells were incubated at 37 "C and the cells collected as described in the Experimental Procedures. Each data point represents the average of three experiments zk one standard deviation.
possibility that, despite the absence of a covalent link between 1 and 7 , l could cause enhanced uptake of 7 by the Hep G2 cells. Under these conditions, the amount of cell associated 7 following a 2-h incubation was only 0.60 pmol per lo6 cells, significantly less than found with the conjugate 6. In addition, we have examined the uptake of 6 by Hep G2 cells for longer times (1 p M conjugate, 37 "C)and found uptake of 6 to be linear up to ca. 24 h reaching a value of 26 pmol per lo6 cells (Figure 4B). Over the course of the experiment, the cells continued to divide and increased in number by a factor of 1.5. The increase in cell number would, in part, contribute to the continued uptake of the conjugate by the cell mass. However, uptake of the conjugate 6 a t 24 h is 3.7-fold greater than a t 2 h, suggesting that all the cells, whether newly formed or not, are continuing to
actively take in the conjugate over the entire 24 h period and that the concentration of the conjugate within the cells had not yet reached a steady state. We conclude from the results of these experiments that (1) the observed enhanced uptake of conjugate 6 by Hep G2 cells occurs as a result of specific binding to the asialoglycoprotein receptor; (2) a covalent link between the oligoMP and neoglycopeptide is essential for the observed enhancement of uptake; and (3) uptake of 6 by Hep G2 cells does not appear to reach a steady state up to ca. 24 h under the conditions used in this study. The second issue to be addressed was that of cell-type specificity. It is well established that the asialoglycoprotein receptor is found on the surface of hepatocytes and represents an efficient means for selectively targeting this tissue for intracellular delivery of a variety of therapeutic agents (21).We examined tissue specificity by incubating three human cell lines, Hep G2, HL-60, and HT 1080, in medium containing 1 p M conjugate 6 and 2% FCS a t 37 "C for 3 and 24 h. As was anticipated, the only cell line to exhibit signficant uptake of 6 was Hep G2. After incubation for 3 and 24 h, 8.5 and 26 pmol per lo6 cells, respectively, was associated with the cells (Figure 5). In contrast, after 24 h only 0.10 and 0.53 pmol per lo6 cells were associated with the HL-60 cells and HT 1080 cells, respectively. This result is consistent with previous findings, which showed the conjugate YEE(ahGalNAc)3-HSA-poly-~-lysine to deliver DNA primarily to the liver of mice (15). CONCLUDING REMARKS
We have examined the cellular uptake and cell-type specificity of a novel, structurally defined and homogeneous oligo-MP-neoglycopeptide conjugate, [YEE(ahGalNAcIsl-SMCC-AET-pUmpT-, (61, using three human cell lines. The cellular uptake of 6 by Hep G2 cells is remarkably efficient and appears to be linear up to 24 h, reaching maximum level of 26 pmol per lo6 cells. Using a n approximation that lo6 cells represents a volume of 1pL, then the intracellular concentration of this conjugate and its breakdown products can be as high
700 Bioconjugate Chem., Vol. 6,No. 6, 1995
as 26 pM. In addition, little conjugate associates with HL-60 or HT 1080 cells, demonstrating that the neoglycopeptide 1 is capable of delivering the oligo-MP 2 in a highly selective manner to hepatocytes. Further work is being carried out to measure the rate of efflux from Hep G2 cells, to observe the interior distribution of the conjugate inside the cell, to identify the metabolites produced following uptake and efflux of the conjugate, and to assess fully the biological efficacy of antisense oligo-MP-neoglycopeptide bioconjugates in this cell uptake process. In collaboration with the immunology and virology laboratory of Dr. Laure Aurelian of the University of Maryland, we have recently obtained preliminary results from an in vitro biological assay that has shown another conjugate, [YEE(ah-GalNAc3)]-SMCCAET-pTpTCCTCCTGCGG, which contains an oligo-MP complementary to the splice acceptor site of immediateearly pre-mRNAs 4 and 5 of herpes simplex virus type 1 (HSV-11, was ca. 25-fold more effective a t inhibiting infection of Hep G2 cells by HSV-1 than was its parent oligo-MP, pTpTCCTCCTGCGG (4). A complete description of this study will be disclosed in due course. ACKNOWLEDGMENT
We gratefully acknowledge many productive discussions with Dr. Paul s.Miller regarding the synthesis and characterization of the conjugate 6 , Dr. Reiko T. Lee for providing YEE(ah-GalNAc)a,Dr. K. B. Lee for the determination of N-acetylgalactosamine content in the conjugate, and Sarah Kipp for the preparation of oligo-MPs. J.J.H. and J.T.L. were supported through Genta-JHU Postdoctoral Fellowships. LITERATURE CITED (1) Mirabelli, C. K., and Crooke, S. T. (1993) Antisense oligonucleosides in the context of modern molecular drug discovery and development. In Antisense research and applications (S. T. Crooke and B. LeBleu, Eds.) pp 7-35, CRC Press, Boca Raton. (2) Ts'o, P. 0. P., Aurelian, L., Chang, E., and Miller, P. S. (1992) Nonionic oligodeoxynucleotide analogs (Matagen as anticodic agents in duplex and triplex formation. Ann. N.Y. Acad. Sci. 600, 159-177. (3) Miller, P. S., McParland, K. B., Javaraman, K., Ts'o, P. 0. P. (1981) Biochemical and biological effects of nonionic nucleic acid methylphosphonates. Biochemistry 20, 1874-1880. (4) (a) Smith, C. C., Aurelian, L., Reddy, M. P., Miller, P. S., and Ts'o, P. 0. P. (1986)Antiviral effect of an oligo(nuc1eoside methylphosphonate) complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5. Proc. Natl. Acad. Sei. U.S.A. 83,2787-2791. (b) Kulka, M., Smith, C. C., Aurlian, L., Fishelevich, R., Meade, K., Miller, P., and Ts'o, P. 0. P. (1989) Site specificity of the inhibitory effects of oligo(nuc1eoside methylphosphonatels complementary to the acceptor splice junction of herpes simplex virus type 1 immediate early mRNA 4. Proc. Natl. Acad. Sei. U.S.A. 86, 6868-6872. (c) Kulka, M., Wachsman, M., Miura, S., Fishelevich, R., Miller, P. S., Ts'o, P. 0. P., and Aurelian, L. (1993) Antiviral effect of oligo(nuc1eoside methylphosphonates) complementary to the herpes simplex virus type 1 immediate early mRNAs 4 and 5. Antiviral Res. 20, 115-120. (d) Kulka, M., Smith, C. C., Levis, J., Fishelevich, R., Hunter, J. C. R., Cushman, C. D., Miller, P. S., Ts'o, P. 0. P., and Aurelian, L. (1994) Synergistic antiviral activities of oligonucleoside methylphosphonates complementary to herpes simplex virus type 1 immediate-early mRNAs 4, 5, and 1.Antimicrob. Agents Chemother. 38, 675-680. (5) Agris, C. H., Blake, K. R., Miller, P. S., Reddy, M. P., and Ts'o, P. 0. P. (1986) Inhibition of vesicular stomatitis virus protein synthesis and infection by sequence-specific oligodeoxyribonucleoside methyphosphonates. Biochemistry 25,62686275.
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