Enhanced Biorecognition and Internalization of HPMA Copolymers

The Hebrew University of Jerusalem, Faculty of Medicine, School of ... N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing ... of the glyco...
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Bioconjugate Chem. 2001, 12, 890−899

Enhanced Biorecognition and Internalization of HPMA Copolymers Containing Multiple or Multivalent Carbohydrate Side-Chains by Human Hepatocarcinoma Cells Ayelet David,† Pavla Kopecˇkova´,‡ Abraham Rubinstein,*,† and Jindrˇich Kopecˇek*,‡ The Hebrew University of Jerusalem, Faculty of Medicine, School of Pharmacy, P.O. Box 12065, Jerusalem, 91120, Israel, and Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112. Received February 9, 2001; Revised Manuscript Received June 14, 2001

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing pendant saccharide moieties (galactosamine, lactose, and triantennary galactose) were synthesized. The relationship between the content of saccharide moieties and three-dimensional arrangement of galactose residues and their biorecognition and internalization by human hepatocarcinoma HepG2 cells was investigated. The results obtained clearly indicated preferential binding of the trivalent galactose and the lactosecontaining copolymers to these cells. The higher the saccharide moieties content in HPMA copolymers, the higher the levels of binding. The biorecognition of the glycosylated HPMA copolymers by HepG2 cells was inhibited by free lactose. The data on the internalization and subcellular trafficking of HPMA copolymer conjugates obtained by confocal fluorescence microscopy correlated well with the flow cytometric analysis of their biorecognition by target cells. Structural features of the glycosides responsible for the specific recognition of the HPMA copolymers have been identified. The results underline the potential of glycosylated HPMA copolymers for delivery of pharmaceutical agents to hepatocarcinoma cells.

INTRODUCTION

The extensive participation of saccharides in recognition processes has led to the concept of targetable glycosylated drug delivery systems (1-3). Carbohydrate specific cell surface receptors play essential roles in various biological recognition processes, including intercellular recognition, adhesion, cell growth regulation, and cell differentiation (4, 5). Receptors expressed at the surface of cells, which mediate endocytosis of their ligands, are attractive targets for delivering bioactive macromolecules to specific cells (6, 7). Several cell surface lectins have been discovered that sort their ligands into the cell. For example, it was recognized that liver parenchymal cells have cell surface receptors specific for galactose (7), phagocytic cells have receptors specific to mannose (8), and L1210 leukemia cells have receptors specific to fucose (9). The galactose binding receptor of hepatocytes has been widely investigated, and its specific interactions with asialoglycoproteins (ASGP1) (10) and neoglycoproteins (11) have been documented. The asialoglycoprotein receptor (ASGP-R), also known as the hepatic lectin, is a recycling endocytotic receptor that recognizes terminal galactose (Gal) and N-acetylgalactosamine (GalNAc) (10). The ASGP-R is composed of three or more subunits that project their CRD (carbohydrate recognition domain) extracellularly (1). The affinity of a ligand for this receptor is highly dependent on the valency of sugar residues, as well as on their threedimensional arrangement (12). Schwartz et al. have previously described the presence of the galactose-recep* Corresponding authors: E-mails: Jindrich.Kopecek@ m.cc.utah.edu; [email protected]. † The Hebrew University of Jerusalem. ‡ University of Utah.

tor on human hepatoma cell line HepG2, suggesting approximately 150000 binding sites per cell (13, 14). Glycosylated polymers, in which saccharide residues are displayed from side-chains as pendant groups, appear to increase binding affinity (15). Saccharide-substituted polymer derivatives have diverse potential uses, such as multivalent inhibitors of cell or virus binding, specific binding to cell surface, artificial antigens, and drug delivery agents (16, 17). The chemically well-defined, water-soluble carbohydrate containing synthetic copolymers, the “glycopolymers”, can be readily synthesized for various biological assays (18). Most studies of carbohydrate-substituted polymers have focused on the polyacrylamide backbone (19-21), polylysine (22), or HPMA copolymers (23, 24). These glycopolymers may have lower molecular weight and higher sugar densities compared with synthetic glycoproteins. Water-soluble polylactoside copolymers have increased binding properties and have demonstrated enhanced binding affinity to plant lectins 1 Abbreviations: AIBN, 2,2′-azobis(isobutyronitrile); AH, aminohexyl; AHT, (6-aminohexanamido)tris(hydroxymethyl)methane; ASGP, asialoglycoprotein; ASGP-R, asialoglycoprotein receptor; Cbz, benzyloxycarbonyl; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DPBS, Dulbecco’s phosphate-buffered saline; EEDQ, N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline; FBS, fetal bovine serum; FITC, fluorescein-5-isothiocyanate; Gal, galactose; GalN, galactosamine; GalNAc, N-acetylgalactosamine; Hex, hexanoyl; HPMA, N-(2-hydroxypropyl)methacrylamide; Lac, lactose; MA, methacryloyl; MALDI-TOF MS, matrixassisted laser desorption/ionization time-of-flight mass spectrometry; MEM-R, minimum essential medium; Mw, weight average molecular weight; ONp, p-nitrophenoxy; P, HPMA copolymer backbone; PNp, p-nitrophenol; SEC, size-exclusion chromatography; TLC, thin-layer chromatography; TNBS, 2,4,6trinitrobenzenesulfonic acid; TriGal, tris-based galactoside; Tris, 2-amino-2-(hydroxymethyl)-1,3-propanediol.

10.1021/bc010026v CCC: $20.00 © 2001 American Chemical Society Published on Web 10/25/2001

Biorecognition and Internalization of HPMA Copolymers

(25). Poly-L-lysine substituted with mannose and gluconoyl residues displays a high specificity toward membrane lectins (26). It is expected that sugar residues exposed on macromolecular delivery systems will mediate targeting to corresponding receptors expressed on certain organs or cells. During the past decade, targetable N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates have been designed, developed, and evaluated for their potential use in cancer therapy (reviewed in ref 27). Phase 1 clinical trial data indicate their great potential for applications in oncology (28, 29). Various targeting moieties, including carbohydrates (30, 31) have been used to promote HPMA copolymer conjugate recognition at the plasma membrane. It has been found that HPMA copolymers containing side chains terminating in N-acylated galactosamine behaved similarly to the ASGP and were biorecognizable in vivo. Their biorecognition by the ASGP-R of hepatocytes was dependent on the number of saccharides displayed on the polymer (23). The polymeric backbone of HPMA copolymers could be designed for a desired property such as biodegradation and stability (27). The design, activity, and mechanism of action of HPMA copolymer drug conjugates have been reviewed in detail (32). Incorporation of certain glycoside moieties (targeting residues) into HPMA copolymer conjugates has been used to facilitate targeting of anthracyclines to the vicinity of tumor cells. One of the most successful polymer-drug-saccharide conjugate systems reported are the HPMA copolymer-adriamycin (ADR)-sugar conjugates, in which both ADR and sugar were attached to the polymer backbone via lysosomally cleavable oligopeptide spacers (33). The conjugates containing galactosamine moieties were reported to accumulate in the liver selectively (34). Our knowledge of the ligand binding preferences of the ASGP-R may be exploited for targeting molecules to hepatocytes (1). However, one of the main difficulties associated with using carbohydrates to target lectins is their low binding affinity. The interactions of individual saccharide groups are generally of low affinity (35), and dissociation constants (Kd) are generally in the mM to µM range. To obtain adequate binding affinities for some proteins, multiple carbohydrate moieties are often needed. Many naturally occurring glycoproteins contain multiantennary structures (36). When the density and relative spatial arrangements of the carbohydrate residues incorporated are appropriate, glycoconjugates can induce a marked enhancement of binding affinity toward proteins due to the multivalent recognition of ligand (37). The incorporation of numerous biorecognizable moieties into one macromolecule can enormously enhance its rate of uptake by cells, by enhancing adherance to the plasma membrane being internalized. Such polyvalent interactions can collectively be much stronger than the corresponding monovalent interactions (38). The most comprehensive description of an enhancement in binding affinity for a series of polymeric sialosides was reported by Sigal et al. (17). The inhibitory properties of polyacrylamides containing pendant sialoside groups illustrate the value of cooperative polyvalent interaction in the design of potent inhibitors of virus-mediated agglutination of erythrocytes. Similar polyvalency effect (cooperative binding) was observed for synthetic glycosylated systems as well (23, 24). Thus, it may be possible to generate synthetic glyconjugates, organized as multivalent (cluster) structures that compensate for weak-binding interactions (39, 40). Classical studies by Lee et al., (41) have established the “glycoside cluster effect”, which is defined

Bioconjugate Chem., Vol. 12, No. 6, 2001 891

as an “affinity enhancement over and beyond what would be expected from the concentration increase for the determinant sugar in a multivalent ligand” (42). Triantennary glycopeptide with three terminal galactose residues showed approximately a 105-affinity enhancement over a monovalent ligand for the human hepatic lectin expressed on the human hepatoma cell line HepG2. In the same study, data from partial structures of the triantennary glycopeptide suggested that the affinity increased about 1000-fold per galactoside residue. This paper describes the synthesis of novel HPMA copolymers bearing pendant saccharide moieties of different structure and content, which facilitates targeting. The saccharides were chosen based on their specific interactions with the asialoglycoprotein receptor of hepatocytes. The ultimate aim of our research is to investigate the influence of the structure and content of the targeting saccharide group on the specific recognition of saccharidecontaining HPMA copolymers by hepatocarcinoma cells. The purpose of this characterization is to identify saccharide moieties with improved attachment properties. The influence of multiple sugar residues attached to individual monomers composing HPMA copolymers (clustering) and the degree of polymer substitution (monitored as sugar concentration: 10-30 mol %) were assessed with regard to delivery efficacy to hepatocarcinoma cells. EXPERIMENTAL SECTION

Materials. All sugars were of D configuration and in the pyranose form. 2,2′-Azobis(isobutyronitrile) (Polysciences, Warrington, PA) was recrystallized from methanol. Solvents were freshly distilled. D-(+)-Galactosamine hydrochloride, D-(+)-galactose, and β-D-lactose, and Dulbecco’s phosphate-buffered saline (DPDS) were obtained from Sigma, St. Louis, MO. Palladium-on-charcoal (Aldrich, Milwaukee, WI) and 6-(benzyloxycarbonamido)hexanoic acid (Bachem, King of Prussia, PA) were used without further purification. 2,3,4,6-Tetra-O-acetyl-β-Dgalactopyranosyl bromide (43) and 4-bromo-2,3,6,2′,3′,4′,6′hepta-O-acetyl-β-lactoside (44) were prepared by the published methods indicated. MEM-R medium and FBS (fetal bovine serum) were obtained from HyClone (Logan, UT). Monensin (sodium salt) was purchased from Calbiochem, La Jolla, CA. Cell Line. The human hepatocarcinoma cell line HepG2 was purchased from the American Type Culture Collection (ATCC, Rockville, MD). HepG2 cells were cultured in MEM-R medium supplemented with 10% FBS. Cells were grown at 37 °C in a humidified atmosphere of 5% (v/v) CO2 in air. Methods. The intermediary and final products were characterized by classical methods (SEC, UV spectrophotometry, 1H NMR, elemental analysis) and by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Colorimetric quantification of natural sugars by the phenol-sulfuric-acid assay and of N-acetylated sugars by the Morgan-Elson assay were conducted by the published procedures (45). Amino groups were quantified by modified TNBS assay (46) using glycine as the standard. The TLC examination of reaction mixtures and products was performed on silica gel layers (F254) precoated on aluminum sheets. Carbohydrate-containing compounds were visualized by spraying the sheets with 15% sulfuric acid in 50% ethanol and heating on a hot plate at ∼150 °C. Amino-containing components were detected by spraying the sheets with 0.5% ninhydrin in ethanol and heating the plates briefly. Aromatic groups were detected by exposing the plate to iodine vapor in an enclosed chamber for a few minutes.

892 Bioconjugate Chem., Vol. 12, No. 6, 2001

Figure 1. Scheme of the synthesized monomers.

Monomer Synthesis. N-(2-Hydroxypropyl)methacrylamide [HPMA] [1] and 5-[3-(methacryloylaminopropyl)thioureidyl] fluorescein [MA-AP-FITC] [2] were prepared according to previously described procedures (47 and 48, respectively). N-Methacryloylglycylglycylgalactosamine (MAGG-GalN) [3]. N-Methacryloylglycylglycine p-nitrophenyl ester (MA-Gly-Gly-ONp) and N-methacryloylglycylglycylgalactosamine [MA-Gly-Gly-GalN] were prepared as previously described (49). Briefly, MA-Gly-Gly-ONp was synthesized by reacting methacryloyl chloride (MACl) with glycylglycine (Gly-Gly-OH) in a basic solution to yield methacryloylglycylglycine (MA-Gly-Gly-OH) (yield 82%), which was then coupled with p-nitrophenol (PNp) in DMF in the presence of DCC to give MA-Gly-Gly-ONp in 79% yield. MA-Gly-Gly-GalN was prepared by reacting MA-Gly-Gly-ONp (3.21 g, 0.01 mol) with galactosamine (2.80 g, 0.013 mol) at 0 °C, in dry DMSO (20 mL), in the presence of triethylamine (1.5 mL, 0.015 mol). The reaction mixture was continuously stirred at 20 °C for 6 h. The obtained methacryloylglycylglycyl-galactosamine was purified and crystallized to give 2.31 g (65%) of MAGG-GalN; mp 179-181 °C (dec). Its 1H NMR (200 MHz, D2O) spectrum and elemental analysis were in agreement with the structure given (32): δ 1.8 (s, 3H), 3.5-3.7 (m, 4H), 3.9 (m, 6H), 5.1 (d, 1H), 5.3 (s, 1H), 5.7 (s, 1H). Anal. Calcd for C14H23N3O8 (361): C, 46.53; H, 6.41; N, 11.64. Found: C, 46.37; H, 6.51; N, 11.69. The structure of MAGG-GalN is shown in Figure 1. 4-Aminophenyl 2,3,6,2′,3′,4′,6′-hepta-O-acetyl-βlactoside [4] was prepared as previously described by Roy et al. (25). Briefly, transformation of lactose into acetobromolactose through the intermediary of octa-Oacetyl-lactose was carried out using 45% w/v HBr in glacial acetic acid (44) to give acetobromo-lactose in 55% overall yield. The 2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-lactopyranosyl bromide was then glycosylated with PNp using

David et al.

phase transfer-catalyzed (PTC) condition, in the presence of tetrabutylammonium hydrogen sulfate (TBAHS), to result in 4-nitrophenyl 2,3,6,2′,3′,4′,6′-hepta-O-acetyl-βlactoside in 53% yield. Transformation of the nitro group into the amino one was performed by using 10% Pd/C in the presence of ammonium formate. A solution of 4-nitrophenyl β-lactoside (241.5 mg, 0.319 mmol) and ammonium formate (400 mg) in 40 mL of MeOH containing 10% Pd/C (50 mg) was gently warmed for 5-10 min in a stoppered round-bottom flask. After cooling, the reaction mixture was filtered and rinsed with methylene chloride. TLC in hexane/EtOAc (1:1) indicated a complete conversion of the nitro into amino. The reaction product gave a positive response in a ninhydrin test. The solvents were evaporated, and the residue was dissolved in methylene chloride. The organic phase was washed with water and dried with sodium sulfate. The residue obtained from the solvent gave pure 4-aminophenyl-2,3,6,2′,3′,4′,6′-heptaO-acetyl-β-lactoside (240 mg, 96%), which failed to crystallize from ethanol/methanol. 1H NMR spectrum and elemental analysis of intermediary and compounds are identical to the given structures (13,24). mp 104-124 °C. Anal. Calcd for C32H41NO18: C, 52.82; H, 5.68; N, 1.92. Found: C, 52.19; H, 5.74; N, 1.89. 1H NMR (200 MHz, CDCl3): δ 6.74 (d, 2 H), 6.55 (d, 2 H), 5.29-4.76 (6dd, 6H), 4.43 (d, 2 H), 4.07-4.02 (m, 3 H), 3.85 (m, 2 H), 3.63 (m, 1 H), 2.10-1.89 (7s, OAc). 4-Methacrylamidophenyl 2,3,6,2′,3′,4′,6′-hepta-Oacetyl-β-lactoside [5] was synthesized according to the procedure described by Roy et al. (25). Compound [4] (460 mg, 0.632 mmol) and triethylamine (385 µL, 3.82 mmol) in CH2Cl2 (44 mL) were cooled to 0 °C. Methacryloyl chloride (89 µL, 1.25 equiv) in CH2Cl2 (16.5 mL) was added dropwise, while the temperature was kept at 0 °C for a further 30 min, after which it was allowed to reach room temperature. MeOH (2 mL) was then added, and the reaction mixture was stirred for a further hour. Water (60 mL) was then added, and the organic phase was washed successively with 0.5 M HCl, saturated NaHCO3, and water. The dried (Na2SO4) organic phase was filtered and evaporated to dryness. The residue was purified by silica gel column chromatography with CHCl3/EtOAc to give pure 4-methacrylamidophenyl 2,3,6,2′,3′,4′,6′-heptaO-acetyl-β-lactoside, (480 mg, 95%), which failed to crystallize. Anal. Calcd For C36H45NO19 (795): C, 54.34; H, 5.66; N, 1.76. Found: C, 54.85; H, 6.32; N, 1.50. 1H NMR (200 MHz, CDCl3): δ 7.41 (d, 2 H), 6.89 (d, 2 H), 5.71 (s, 1 H), 5.40 (s, 1 H), 5.28-4.91 (m, 4 H), 4.43 (d, 2 H), 4.05-3.81 (m, 5 H), 2.09-1.90 (7s, OAc), 1.53 (t, 3 H), 1.19 (t, 3 H), 0.82 (t, 2 H). 4-Methacryloylamidophenyl-β-lactoside (MA-PhLac) [6] was prepared by de-O-acetylation of 5 in methanol containing a catalytic amount of sodium methoxide. Compound 5 (480 mg, 0.603 mmol) was dissolved in warm MeOH (15 mL) containing 0.5 M sodium methoxide (40 µL). After a few hours at room temperature, an additional 15 mL of MeOH containing sodium methoxide was added, and the solution was warmed again. The solution was stirred overnight at room temperature. Ether (25 mL) was added to the cold solution. Filtration yielded with pure 6 (336 mg, 67%). mp 208212 °C. Anal. Calcd for C22H31NO12 (501): C, 52.69; H, 6.19; N, 2.73. Found: C, 51.73; H, 6.32; N, 2.65. 1H NMR (200 MHz, CDCl3): δ 7.25 (d, 2 H), 7.01 (d, 2 H), 5.67 (s, 1 H), 5.42 (s, 1 H), 5.01 (d, 1 H), 4.34 (d, 1 H), 3.80-3.20 (m, 11 H), 1.67 (s, 3 H), 1.03 (t, 1 H). The structure of MA-Ph-Lac is shown in Figure 1. 6-(Aminohexanamido)tris(2,3,4,6-tetra-O-acetylβ-D-galactopyranosyloxymethyl)methane, (AHT-

Biorecognition and Internalization of HPMA Copolymers

[Gal(OAc)4]3) [7]. According to the method reported by Lee et al. (49), Tris was coupled with Cbz-aminohexanoic acid in absolute ethanol in the presence of 2-ethoxy-N(ethoxycarbonyl)-1,2-dihydroquinoline (EEDQ) to afford a 65% yield of [6-(benzyloxycarbonamido)hexanamido]tris(hydroxymethyl)methane (Cbz-AHT). Transformation of galactose into 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl bromide was carried out using 45% w/v HBr in glacial acetic acid (43) to effect a 97% yield of 1-Br-Gal(OAc)4. The acetylated sugar was glycosylated by coupling with Cbz-AHT, as described by Polidori et al. (51), in acetonitrile in the presence of mercuric cyanide (catalyst) followed by sonication under nitrogen atmosphere to give a 66.5% yield of the [6-(benzyloxycarbonamido)hexanamido]tris(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxymethyl)methane (Cbz-AHT-[Gal(OAc)4]3) conjugate. Hydrogenolysis in 60% acetic acid in the presence of 10% palladium on carbon was performed, to give 74% yield of AHT-[Gal(OAc)4]3. 1H NMR analysis (CDCl3) data indicated the removal of the benzyl group, showing the presence of aminohexanoyl group at 5.6 ppm and the presence of acetylated galactose in correct ratios. Mass spectrum analysis (+FAB): 1225.7 (M + H+). Furthermore, colorimetric analysis for the amino group (TNBS method) and galactose (phenol-sulfuric acid method) indicated the ratio of these groups to be 1:3. The 1H NMR spectrum and MS analysis of intermediary and final product are in agreement with the given structures (45, 52, 53). N-Methacryloyl-6-(aminohexanamido)-tris-(2,3,4,6tetra-O-acetyl-β- D-galactopyranosyloxymethyl)methane (MA-AHT-[Gal(OAc)4]3) [8]. The monomer in the acetylated form was synthesized by coupling AHT[Gal(OAc)4]3 with methacryloyl chloride (MA-Cl) in CH2Cl2 in the presence of triethylamine at 0 °C. A solution of AHT-[Gal(OAc)4]3 (20 mg, 0.0163 mmol) and triethylamine (20 µL, 11 equiv) in CH2Cl2 (1.0 mL) was cooled to 0 °C. Methacryloyl chloride (2 µL, 1.1 equiv) in CH2Cl2 (0.5 mL) was added dropwise while the temperature was allowed to reach room temperature. The reaction mixture was stirred for 2 h. Water (2 mL) was then added, and the organic phase was successively washed with 0.5 M HCl, saturated NaHCO3, and water. The dried (Na2SO4) organic phase was filtered and evaporated to dryness. MA-AHT-[Gal(OAc)4]3 was obtained in 65% yield. TLC in EtOAc:acetic acid:H2O (3:2:1) indicated complete consumption of the amino-compound. 1H NMR spectra (200 MHz, CDCl3) confirmed the appropriate functional groups (acetylated galactose, aminohexanoyl, oxymethylene, and double bond at 5.4 and 5.5 ppm). Mass spectrum analysis (+FAB): 1316 (M + Na+). N-Methacryloyl-6-(aminohexanamido)tris(β-D-galactopyranosyloxymethyl)methane (MA-AHT-(Gal)3) [9]. De-O-acetylation of MA-AHT-[Gal(OAc)4]3 was performed in methanol containing a catalytic amount of sodium methoxide. MA-AHT-[Gal(OAc)4]3 (20 mg, 0.0154 mmol) was dissolved in dry methanol (800 µL) containing 0.5 M sodium methoxide (20 µL). After 24 h stirring at room temperature, an additional amount of dry methanol (800 µL) containing 0.5 M sodium methoxide (20 µL) was added, and the reaction mixture was stirred at room temperature for a further 24 h. Sixty-percent acetic acid (200 µL) was then added (deionization), evaporated, and coevaporated with EtOH/water. The TLC in mixed solvents of EtOAc:acetic acid:H2O (3:2:1) and EtOAc:2propanol:H2O (5:3:2) indicated a conversion of the perO-acetylated derivative into a more polar product, MAAHT-(Gal)3 in a yield of 74%. 1H NMR spectra (200 MHz, D2O): δ 5.44 (s, 1H, dCH2), 5.68 (s, 1H, dCH2), 4.7-5.0

Bioconjugate Chem., Vol. 12, No. 6, 2001 893 Table 1. Characteristics of HPMA Copolymer Conjugates Containing Pendant Saccharide Moieties polymer P-Gal P-Lac P-TriGal P-F

polymer no.

targeting moiety

10a 10b 10c 11a 11b 11c 12a 12b 12c 13

GalN GalN GalN lactose lactose lactose tri-galactose tri-galactose tri-galactose -

mol % mol % approx Mw of sugara FITCb conjugate (Da)c 14.8 20.4 29.1 11.1 19.9 27.6 8.47 13.6 24.4 -

2.04 2.42 2.64 1.58 1.76 1.90 2.23 2.34 2.52 2.51

15000 16000 16000 18000 20000 19500 14500 14000 14000 20000

a The contents of galactose and lactose residues were determined spectrophotometrically using the phenol-sulfuric acid assay; the content of galactosamine was determined by the MorganElson assay. b The content of FITC was determined spectrophotometrically using  ) 82000 M-1 cm-1 (FITC; 495 nm, pH 9.1 (0.1 M sodium borate). c Weight average molecular weights (Mw) of copolymers were estimated by SEC using a Superose 12 column, FPLC system, PBS buffer pH 7.3, calibrated with poly(HPMA) fractions.

(m, 3H, R-H), 3.5-4.5 (m, 24H, sugar), 3.28 (m, 2H, CH2N), 2.30 (t, 2H, CH2O), 1.95 (s, 3H CH3), 1.5-1.7 (m, 2H, CH2), 1.2-1.4 (m, 2H, CH2), 0.8-1.1 (m, 2H, CH2). Mass spectrum analysis (+FAB): 788.3 (M + H+), 809.8 (M + Na+). The structure of MA-AHT-(Gal)3 is shown in Figure 1. Polymer Synthesis. Polymer glycoconjugates containing fluorescein were synthesized by radical precipitation copolymerization as described below. The polymers are of the general structure shown in Figure 2. The characterization of polymers and the method of preparation are shown in Table 1. All the polymerizations were conducted using free radical copolymerization of three monomers: HPMA, MA-AP-FITC, and the saccharidecontaining monomer (either MA-GG-GalN, or MA-PhLac, or MA-AHT-(Gal)3). Three copolymers of each saccharide moiety (i.e., galactosamine, lactose, or trivalentgalactose) were synthesized. The monomer feed concentrations were 3 mol % of MA-AP-FITC; 10 mol %, 20 mol %, and 30 mol % of the saccharide-containing monomer, and 87 mol %, 77 mol % and 67 mol % of HPMA, respectively. The concentration of monomers, with respect to initiator and solvent, was in the weight ratio of 12.5:1.0:86.5, respectively. The monomer mixture of the control copolymer, which did not contain carbohydrate moiety, was composed of 98 mol % of HPMA to make up the lack of carbohydrate-containing monomer. The polymerization was initiated using AIBN. Synthesis of P-(AP-FITC)-GalN [10a-c]. Galactosecontaining HPMA copolymers were synthesized by copolymerization of HPMA, MA-GG-GalN, and MA-AP-FITC in acetone/DMSO (7:3). All monomer mixtures contained 3 mol % (17.7 mg) of MA-AP-FITC; the contents of MAGG-GalN were 10 mol % (40 mg), 20 mol % (80 mg), and 30 mol % (120 mg), and of HPMA were 87 mol % (138 mg), 77 mol % (122.2 mg), and 67 mol % (106.4 mg), respectively. MA-GG-GalN and MA-AP-FITC were dissolved in DMSO, HPMA, and AIBN were dissolved in acetone. The solutions were mixed, transferred to an ampule, bubbled for 10 min with nitrogen and sealed, and the mixture was polymerized in a water bath at 50 °C for 40 h while stirring. After polymerization, the ampules were cooled to room temperature and opened, and the volume was reduced under vacuum. The viscous liquid thus obtained was precipitated into a large excess of acetone. The precipitated polymer was dissolved in distilled water, dialyzed against water for 24 h, and

894 Bioconjugate Chem., Vol. 12, No. 6, 2001

David et al.

Figure 2. Synthesis of the saccharide-containing HPMA copolymers, their structure, and the corresponding amino-sugar (R-H).

lyophilized. The copolymers were denoted 10% P-Gal [10a], 20% P-Gal [10b], and 30% P-Gal [10c] reflecting the amount of MA-GG-GalN in the monomer mixture. Yields: 10% P-Gal, 140 mg (71%); 20% P-Gal, 150 mg (68%); 30% P-Gal, 160 mg (66%). The structures of the polymers are shown in Figure 2. The results of their characterization and methods used are listed in Table 1. Synthesis of P-(AP-FITC)-Lac [11a-c]. The lactosecontaining HPMA copolymers were synthesized by the same procedure as described for the galactosaminecontaining copolymers. The feed weights were: 3 mol % of MA-AP-FITC (9.5 mg); 10 mol % (30 mg), 20 mol % (60 mg), and 30 mol % (90 mg) of MA-Ph-Lac, and 87 mol % (74.6 mg), 77 mol % (66.0 mg), and 67 mol % (57.5 mg) of HPMA, respectively. The solvent system was MeOH. After polymerization, volume was reduced under vacuum. The crude compound thus obtained was dissolved in water and the high Mw copolymer was purified on Sephadex G-25M (Amersham Pharmacia Biotech, Piscataway, NJ). The copolymers 10% P-Lac, 20% P-Lac, and 30% P-Lac are denoted as 11a, 11b, 11c, respectively. Yields: 10% P-Lac, 90 mg (79%); 20% P-Lac, 90 mg (67%); 30% P-Lac 120 mg (76%). The structure of polymers and the results of their characterization are shown in Figure 2 and Table 1.

Synthesis of P-(AP-FITC)-TriGal [12a-c]. The trivalent galactoside-containing HPMA copolymers were synthesized by the same procedure as described for the galactosamine-containing copolymers. The feed weights were: 3 mol % of MA-AP-FITC (4.1 mg); 10 mol % (20 mg), 20 mol % (40 mg), and 30 mol % (60 mg) of MAAHT-(Gal)3, and 87 mol % (32 mg), 77 mol % (28.3 mg) and 67 mol % (25.0 mg) of HPMA, respectively. The solvent system was MeOH. After polymerization, volume was reduced under vacuum. The crude compound thus obtained was dissolved in water, and the high Mw copolymer was purified on Sephadex G-25M. The copolymers 10% P-TriGal, 20% P-TriGal, and 30% P-TriGal are denoted as 12a, 12b, 12c, respectively. Yields: 10% P-TriGal, 30 mg (53%); 20% P-TriGal, 40 mg (55%); 30% P-TriGal, 40 mg (45%). The structure of polymers and the results of their characterization are shown in Figure 2 and Table 1. Control fluorescein labeled HPMA copolymer (P-F) [13]. A fluorescein labeled polymer conjugate without any carbohydrate moiety was synthesized as a control. The procedure used was the same as that used for the galactosamine-containing copolymer. The feed weights were: 98 mol % of HPMA (320 mg) and 2 mol % of MA-AP-FITC (24 mg). The solvent system was: ac-

Biorecognition and Internalization of HPMA Copolymers

etone. Yields: 97% (271 mg). The characterization of copolymer is shown in Table 1. Flow Cytometric Analysis. Cell suspensions were prepared by trypsinization of cell monolayers. Cells (1 × 106) were seeded in 24-well plates in 2 mL of MEM-R medium supplemented with 10% FBS. The cells were incubated separately with 500 ng/mL of the saccharidecontaining HPMA copolymers (P-Gal, P-Lac, and PTriGal) for 24 h at 37 °C under 5% CO2 atmosphere. Control cells were incubated with the same concentration of the FITC-labeled HPMA copolymer without sugar moiety (P-F). Inhibition experiments were performed with the same concentrations of the saccharide-containing copolymers, but in the presence of 150 mM lactose in the incubation medium. After incubation, the polymeric solutions were removed from the well, and the cells were washed twice with 1 mL of cold PBS containing 1mM CaCl2, 1mM MgCl2 and 0.5% BSA. Cells were harvested by trypsinization with 0.25% (w/v) trypsin and further incubated for 30 min at 4 °C in PBS containing 50mM monensin. Cell suspensions were immediately analyzed by flow cytometry using fluorescence activated cell sorter (FACS, Becton Dickinson). Cells were gated according to light-scattering parameters. Confocal Microscopy. The intrinsic fluorescence of FITC was also used to monitor the subcellular fate of the saccharide-containing HPMA copolymer conjugates (PGal, P-Lac and P-TriGal) with different contents of pendant saccharide moieties. After trypsinization, 2 × 104 cells were seeded on a N1 coverslip (Corning) and cultured for 24 h. The culture medium was replaced with medium containing polymer conjugates (20 µg/mL) and cultured for 24 h at 37 °C. Unbound conjugates were removed by washing the cell layer three times with cold PBS. Cells were fixed with 3% paraformaldehyde for 10 min at room temperature and washed again. The fate of glycoconjugates in cells was analyzed on a BioRad (Hercules, CA) MRC-600 laser scanning confocal imaging system with a Zeiss (Oberkochen, Germany) Axioplan microscope with a plan-apo objective (60x, NA)1.4, oil) and a krypton/argon laser. Images were acquired with a Fluor filter block (excitation at 488 nm, emmision collected with a 515 nm barrier filter). Settings were adjusted so that control (untreated) cells always yielded dark images. Fluorescent images were scaled to 256 Gy levels. Quantitative analysis was performed using ImagePro Plus 4.0 (Media Cybernetics). Fluorescence intensities were expressed in arbitrary units per square micron.

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Figure 3. Flow cytometry analysis of the binding of 10% P-Gal (10 µg/mL) to HepG2 cells, in the presence of 150-mM lactose (solid bars) and the absence of lactose (open bars), after 2 and 24 h incubation at 37 °C. HPMA copolymer (P-F), which did not contain sugar moiety served as control (gray bars).

RESULTS

The saccharide-containing monomers (MA-GG-GalN, MA-Ph-Lac, and MA-AHT-(Gal)3) copolymerized with HPMA and small amounts of MA-AP-FITC gave high molecular weight compounds soluble in water. Analysis of the sugar content in the copolymers (Table 1) confirmed that the saccharide-containing monomer units present in the polymerization mixture were polymerized to give a final product in which the ratio of monomer units was close to their initial ratio in the reaction mixture. SEC analysis of the final preparations confirmed that the weight average molecular weight (Mw) of all copolymers was similar and did not alter under the influence of saccharide (substitution) attachment. These data indicate that the monomers synthesized did not contain minute amounts of dimethacryloylated impurities. The latter would act as cross-linking (branching) agents increasing the molecular weight with increasing content of saccharide-containing monomers in the polymerization mixture.

Figure 4. Flow cytometry analysis of the binding of the saccharide-containing HPMA copolymers (500 ng/mL) to HepG2 cells after 24 h incubation at 37 °C. Upper panel (4A): Binding in the presence (solid bars) and absence of free lactose (150mM). In all studies P-F (gray bars, left) served as control. (P-Gal, open bars; P-Lac, hatched bars; P-TriGal, dotted bars. Lower panel (4B): Binding dependency on the type and the content of the sugar moiety in the copolymer.

The results of binding of the fluorescein-labeled glycoconjugates to HepG2 cells are shown in Figures 3 and 4. The total amount of cell-associated conjugate increased

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David et al.

Figure 5. Confocal fluorescence images ofHepG2 cells incubated with the saccharide-containing HPMA copolymer conjugates, after 24 h incubation at 37 °C. Internalized glycoconjugates were transported into the lysosomal compartment of the cells. Upper three images (left to right): 10% P-Gal, 20% P-Gal, and 30% P-Gal: Centered three images (left to right): 10% P-Lac, 20% P-Lac, and 30% P-Lac. Lower three images (left to right): 10% P-TriGal, 20% P-TriGal, and 30% P-TriGal.

when the incubation period was raised (Figure 3). At 37 °C the fluorescence intensity of all cells incubated with the glycoconjugates was greater than that of the cells incubated at 4 °C, suggestive of an endocytic process (data not shown). The cell-associated fluorescence of HepG2 cells after incubation with the HPMA copolymer conjugates for 24 h at 37 °C is summarized in Figure 4. The flow cytometry analysis revealed strong binding of the copolymers to HepG2 cells. The fluorescence intensity indicated that their biorecognition by hepatocarcinoma cells was dependent on the type and the content of the pendent saccharide moieties in the copolymer. The increased glycosylation level of copolymers with galactose and lactose, caused an increase in cell binding. In general, the binding of the trivalent galactoside and the lactosidecontaining copolymers was found to be greater than that of the galactoside-containing copolymers (Figure 4). In the lowest sugar concentration (10 mol %), a 5.6-fold difference in binding intensity was observed between the trivalent-galactoside- and the monovalent-galactosidecontaining copolymer. Twenty mol % concentration resulted in a difference of 4.5-fold, whereas in the highest concentration (30 mol %) the difference was only 2 times greater between the trivalent and monovalent galactoside-containing copolymers. The specificity of lectinglycocopolymer interactions was verified by an inhibition assay. The incubation of cells with 150 mM lactose (competitive inhibitor) completely abolished cell binding of copolymers and indicated sugar specificity of the process involved (Figures 3 and 4). In addition, copoly-

mers with no sugar bound very weakly to the cells, ruling out nonspecific reactivity (Figure 3). The uptake and subcellular trafficking of FITC-labeled HPMA copolymer conjugates by HepG2 cells were followed by confocal fluorescence microscopy. Cells were incubated with the HPMA copolymer conjugates for 24 h at 37 °C, before optical sections were taken. Confocal images indicated the lysosomotropism of the conjugates, i.e., internalized glycoconjugates were transported into the lysosomal compartment of the cells (Figure 5). The internalization rate of conjugates was dependent on the type and the content of the pendant carbohydrate moiety in the copolymers. The higher the contents of each sugar moiety in the HPMA copolymer, the brighter the image. Furthermore, the internalization rates correlated well with the biorecognition of the saccharide moieties by the cells, as detected by flow cytometry, i.e., the trivalent galactoside- and the lactoside-containing copolymers were internalized more efficiently than the galactoside-containing copolymers (Figure 6). The fluorescence images of cells stained with the untargeted copolymer P-F (which did not contain sugar) were dimmer than the targeted polymer at the same polymer concentration, indicating no presence of the nontargeted copolymer inside the cells. DISCUSSION

HPMA copolymers designed in this study can be used as drug carriers to achieve optimal targeting to hepatocarcinoma cells. The incorporation of sugar residues into HPMA copolymers results in effective biorecognition by

Biorecognition and Internalization of HPMA Copolymers

Figure 6. Fluorescence intensity of HepG2 cells incubated with the saccharide containing HPMA copolymer-FITC conjugates, after 24 h at 37 °C. The fluorescence intensity is expressed in arbitrary units per square micron.

liver cells (52). The copolymers were evaluated for their potential to induce a marked enhancement of binding affinity toward hepatocytes. The copolymers used in this study were synthesized by radical copolymerization of HPMA, MA-AP-FITC, and the saccharide-containing monomers. From the similarity in the structure of the polymerizable parts of the comonomers, it was expected that the copolymerization parameters would be close (49) and consequently the monomers units randomly distributed along the polymer backbone. In addition, the distribution of chemical composition of the resulting copolymers was expected to be narrow, despite the high polymerization yields. Of the mammalian lectins discovered to date, the ASGP-R has been studied in greatest detail (10, 13, 14, 53, 54). Although the ASGP-R binds and internalizes most galactose-terminated ligands, binding affinity is strongly influenced by the ligand structure. Lee et al. demonstrated that monovalent ligands such as galactose, lactose, and monoantennary galactosides bind the ASGP-R with a milimolar dissociation constant (12, 41). Ligands containing a cluster of three galactose residues bind to the receptor with a nanomolar dissociation constant which appear to be the affinity threshold needed to achieve appreciable targeting of glycoconjugates under physiological conditions (37). This relationship between sugar-clustering and enhanced binding affinity is related to the structure of the ASGP-R, which is composed of three subunits, that each of them contains a galactose binding site (1, 37). Although small polyvalent ligands often display enhanced binding affinity for target proteins, glycosylated polymers could achieve better biorecognition just by increasing the concentration of the biorecognizable sugar moiety along the polymeric chain (15). To learn more about the relationship between the structural features of glycosylated polymers containing multiple glycoside residues and their biorecognition, we synthesized HPMA copolymers containing side-chains terminated with galactosamine, lactose, or trivalentgalactose. The relationship between the content of saccharide moieties, the three-dimensional arrangement of galactose residues, and their biorecognition and internalization by human hepatocarcinoma cells was investigated. The observed strong binding of saccharide-containing HPMA copolymers to hepatocarcinoma cells in these

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studies supports the hypothesis that the saccharide groups serve as ligands for hepatic lectins. Furthermore, it was demonstrated that efficient internalization of the saccharide-containing polymers by hepatocarcinoma cells occurred. This internalization increased with increasing the content of the sugar residues bound to the polymer. The endocytosis of the glycoconjugates was not clearly defined in the flow cytometry experiments. However, when considered together with the confocal data, there is a strong evidence to suggest that receptor-mediated endocytosis was responsible for the increased internalization of the glycopolymers. The internalization rates were dependent on the type of sugar bound to the polymer. Inhibition studies in the presence of a competitive inhibitor (free lactose) revealed a complete blockage of copolymer binding, indicating the sugar specificity of the process involved. Moreover, the binding intensity of the monovalent galactoside containing copolymer to HepG2 cells is rather weak. However, the trivalent-galactosidebearing copolymer displayed stronger binding intensities, which were attributed to the clustering of galactose residues. To achieve biorecognition of monosaccharides at levels comparable to multiantennary structures, HPMA copolymers must contain a high content (above 20 mol %) of randomly distributed carbohydrate moieties at side chain termini. About 30 mol % of galactose in the copolymer was needed to achieve binding similar to the 10% trivalent-galactose. Nevertheless, the enhancement in binding affinity observed in these studies was somehow less dramatic than expected (as described by Lee (37) and Whitesides (38)). The increase in valency was only weakly reflected in the reactive potency of the glycoconjugates to enhance binding affinity. This factor did not exceed 5.6-fold binding enhancement of the triantennary over the monoantennary galactoside-containing copolymer. A possible explanation could be that in these binding studies the polymer concentration was relatively high. At high polymer concentrations, receptor saturation may occur and consequently decrease the efficiency of targeting to the cells (55). If that was the case, it could explain why high concentrations of the lactoside- and trivalentgalactoside-containing copolymers maintained, more or less, similar binding intensities. Our findings are consistent with the data reported by Vansteenkiste et al., indicating that dextran substituted with trigalactose units was captured by the liver only 1.6times faster than the mono-galactose-substituted dextran (56). Interestingly we found that the HPMA copolymer substituted with triantennary galactose exhibits a larger binding intensity difference between the triantennaryand monoantennary-substituted copolymers than the substituted-dextran, at the same concentrations (5.6-fold difference for HPMA compared with 1.6-fold difference for dextran). This difference may be attributed to the influence of the polymer backbone on the recognition, i.e., HPMA copolymer backbone is more flexible than dextran, thus the chain may have a conformation in which several sugar moieties from the same chain interact with one receptor. The fact that the ratio of tri- to mono-binding decreases with increasing contents of sugar in the copolymer also supports this point. Affinity studies of ligands containing glycoside residues were traditionally been undertaken using glycoconjugates labeled with radioactive probes. This approach allows binding affinities to be tested even at the nanomolar concentration range. Perhaps incubation studies at these low concentrations may be more indicative of the extent of copolymer uptake by receptor mediated endocytosis, and will exhibit more pronounced clustering effect.

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In conclusion, the binding of the saccharide-containing HPMA copolymers to HepG2 cells was demonstrated by flow cytometry. While the results of the binding experiments could not distinguish endocytosis from surface adherence, the use of confocal microscopy did show the presence of the glycoconjugates inside the cells. The receptor-mediated endocytosis is suggested since the nontargeted copolymer could hardly be detected inside the cells under similar conditions. The data are in agreement with the results of Omelyanenko et al. (50) who observed in human ovarian carcinoma cells that subcellular organelles containing HPMA copolymer conjugates were morphologically similar to organelles containing a lysosomal marker, Neutral Red. The relative rate or extent of endocytosis of the glycoconjugates was dependent on the type and the content of the sugar moiety. Our results suggest that the copolymers containing multivalent carbohydrate clusters may be considered as a targetable delivery system with enhanced binding affinity toward hepatocytes. It appears that high sugar concentrations facilitate multiple binding domains and therefore enhance the binding of the polymer to recognition sites on hepatocytes. Since the ASGP-R is expressed on HepG2 cells, it is reasonable to speculate that the increased binding of the saccharide-containing HPMA copolymers reported here is due to endocytosis mediated by the asialoglycoprotein receptor. ACKNOWLEDGMENT

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