Carbohydrate-Specific Uptake of Fucosylated Polymeric Micelles by

Jun 9, 2015 - In this work, we show that fucosylated micelles reveal an increased uptake by pancreatic, lung, and ovarian carcinoma cell lines, wherea...
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Carbohydrate-Specific Uptake of Fucosylated Polymeric Micelles by Different Cancer Cell Lines Krzysztof Babiuch,†,∥ Aydan Dag,†,‡,∥ Jiacheng Zhao,† Hongxu Lu,† and Martina H. Stenzel*,† †

Centre for Advanced Macromolecular Design, School of Chemistry, University of New South Wales, Kensington, Sydney, New South Wales 2052, Australia ‡ Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Bezmialem Vakif University, 34093 Fatih, Istanbul Turkey S Supporting Information *

ABSTRACT: Inspired by upregulated levels of fucosylated proteins on the surfaces of multiple types of cancer cells, micelles carrying β-L-fucose and β-D-glucose were prepared. A range of block copolymers were synthesized by reacting a mixture of 2-azidoethyl β-L-fucopyranoside (FucEtN3) and 2azideoethyl β- D -glucopyranoside (GlcEtN 3 ) with poly(propargyl methacrylate)-block-poly(n-butyl acrylate) (PPMAb-PBA) using copper-catalyzed azide−alkyne cycloaddition (CuAAC). Five block copolymers were obtained ranging from 100 mol % fucose to 100% glucose functionalization. The resulting micelles had hydrodynamic diameters of around 30 nm. In this work, we show that fucosylated micelles reveal an increased uptake by pancreatic, lung, and ovarian carcinoma cell lines, whereas the uptake by the healthy cell lines (CHO) is negligible. This finding suggests that these micelles can be used for targeted drug delivery toward cancer cells.

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INTRODUCTION Glycosylation is one of the most common forms of co- and post-translational modifications, with over 50% of all proteins carrying various carbohydrate structures.1 In mammals, glycans containing L-fucose (6-deoxy-L-galactose) are involved in a multitude of processes including blood transfusion reactions, selectin-mediated adhesion of leukocytes and microbes as well as signaling events by different receptors.2 Fucosylation was identified as one of the key oligosaccharide alterations in cancer and inflammation.3 Increased levels of fucose-modified glycolipids were first found in hepatoma cells, as compared with normal hepatocytes.4 Further research into this subject has led to the discovery of a highly specific fucosylated glycobiomarkers for early detection of hepatocellular carcinoma in human sera.5 Additionally, increases of fucose-modified haptoglobin in sera of patients with different cancers such as pancreatic, lung, liver, ovarian, and breast have been reported.6 The altered cell surface protein fucosylation patterns disrupt normal cellular functions and ultimately lead to their metastasis.7 Fucose is an essential component of the carbohydrate ligands (Lewis antigens) for selectin family of cell adhesion receptors expressed on endothelia as well as platelets.2 The levels of sialyl Lewisx and sialyl Lewisa are upregulated in numerous cancers as well as associated with poor prognosis and malignancy. Moreover, the upregulation of FUT8 enzyme (α1−6 fucosyltransferase), responsible for core fucosylation of proteins, has been reported in all the cancers except melanoma.7 Because most of the cancer cell lines reveal the © XXXX American Chemical Society

upregulation of fucosylation patterns, L-fucose should be an attractive ligand, which actively and specifically targets these cells. Fucosylation levels in normal liver and colon are relatively low but increase during carcinogenesis; consequently, the fucose-specific lectins on cancer cells need further investigation.8 In mammalian cells, all fucosyltransferases employ nucleotide-activated fucose (GDP-fucose) in the biosynthesis of fucosylated glycans. GDP-fucose can either be synthesized de novo from GDP-mannose or salvaged from extracellular or lysosomal sources.2 Relatively little is known about the exact method of fucose transport across the cellular and lysosomal membrane. Fucose, upon cleavage by fucosidase from glycolipids and glycoproteins inside the lysosomal compartments, can be transported into the cytosol by facilitated diffusion through a relatively uncharacterized transport system.9 Nevertheless, the salvage pathway may have great potential in actively targeting cancer cells through fucosylated delivery vehicles. Despite the importance of fucose in cancer, this carbohydrate has rarely been exploited as a mean to target carcinoma cells. Especially, the decoration of a nanoparticle with fucose could create an advanced drug delivery carrier. Nanomicellar drug carriers based on functional polymers have found a wide range of applications within the field of anticancer therapy,10 imaging Received: March 4, 2015 Revised: June 5, 2015

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DOI: 10.1021/acs.biomac.5b00299 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

Scheme 1. Schematic Representation of the Synthesis of Amphiphilic Block Glycopolymers with Various Glc/Fuc Ratios

a

Determined from SEC in DMAc (relative to PS standards).

of tumors11 as well as simultaneous imaging and therapy (theranostics).12 However, all of these therapeutics accumulate in the diseased tissues predominantly by “passive” targeting, taking advantage of the enhanced permeability and retention (EPR) effect.13 An increased delivery of therapeutic/imaging agents can be achieved by “active” targeting using ligands specific for the receptors on the cellular membranes of the diseased tissue.14 When the nanoparticles concentrate inside the tumor tissue via the EPR effect, the receptor-mediated internalization of particles by cells through the actively targeting ligands present on the surface of the particles will facilitate the uptake. As the ideal result, the residence at the site of action of the therapeutic agent will be prolonged, leading to an enhanced therapeutic efficiency and decreased side-effects. Sugars, peptides, vitamins, lectins, growth factors, or antibodies are the most commonly employed actively targeting ligands.15−17 Out of these ligands, carbohydrates (e.g., fucose) offer the highest stability toward environmental changes, such as variation in pH, temperature, salt concentration as well as oxidative stress. The combination of physical properties (size, shape, charge) with bioactive targeting motifs on the surface of nanoparticles should result in the development of systems, which deliver a drug precisely and safely to its site of action. Glycopolymers, synthetic polymers carrying pendant sugar units, have been shown to target carbohydrate receptors on cancer cells.18−20 Due to multimeric representation of targeting ligands, the glycopolymers exhibit much higher affinities than the monomeric ligands. Furthermore, particles consisting of self-assembled, amphiphilic, galactosylated block glycopolymers revealed an enhanced uptake by human hepatocellular carcinoma cell line (HepG2), as compared to the water-soluble homopolymers.21 Additionally, clinical trials on galactosaminemodified polymers revealed liver-specific doxorubicin delivery with visible accumulation in primary hepatocellular tumors.22 Therefore, carbohydrate-modified polymers and micelles thereof can be applied as cancer-targeted drug delivery systems. In this publication, we report the synthesis of fluorescent, amphiphilic glycopolymers (Scheme 1), their self-assembly into

nanosized micellar aggregates, and their carbohydrate-dependent internalization by pancreatic, lung, and ovarian carcinoma cell lines.

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MATERIALS AND METHODS All materials were reagent grade and used as received, unless otherwise specified: methacryloyl chloride, 3-(trimethylsilyl) 1propanol, fluorescein O-methacrylate, n-butyl acrylate (99%), Lfucose, D-glucose, acetic anhydride, sodium acetate, 2bromoethanol, boron trifluoride diethyl etherate, sodium azide, sodium methoxide, anhydrous dichloromethane (≥99.8%), N,N-dimethylformamide (≥99.8%), anhydrous methanol (99.8%) were all from Sigma-Aldrich. Triethylamine, tetrahydrofuran (THF), and acetone were from Chem-Supply Pty Ltd. Australia, and diethyl ether (99%) was obtained from Univar. n-Butyl acrylate (BA) was deinhibited by passing through a column of activated basic alumina. Deinhibited monomers were stored at below 4 °C. 2,2′-Azobis(isobutyronitrile) (AIBN) was recrystallized twice from methanol. Deionized (DI) water was produced by a Milli-Q reverse osmosis system and had a resistivity of 19.6 MΩ·cm. The RAFT agent, 4-cyanopentanoic acid dithiobenzoate (CPDAB) was synthesized according to the literature.23 All compounds described herein gave NMR spectral data in accordance with their structures. Synthesis of 1,2,3,4-Tetra-O-acetyl-L-fucose (AcFuc). Seventy milliliters of acetic anhydride (0.74 mol) and 5.5 g of sodium acetate (0.067 mol) were heated to 140 °C, with stirring, in a round-bottomed flask. Ten grams (0.061 mol) of L-fucose was slowly added to the boiling mixture and stirred for 45 min under reflux conditions. The reaction was quenched by pouring onto 200 mL of crushed ice. The mixture was stirred for 2 h and left overnight at 4 °C. The organic phase was extracted with ethyl acetate two times and washed with water, saturated sodium bicarbonate solution (three times), and water again. Ethyl acetate phase was collected and dried over sodium sulfate. The mixture was filtered, concentrated on rotary evaporator, and dried in vacuo to yield the peracetylated B

DOI: 10.1021/acs.biomac.5b00299 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

3.62 g, 62%; 1,2,3,4,6-penta-O-acetyl-β- D -glucopyranose (AcGlc)) 1H NMR (CDCl3, 300 MHz): δH 5.73 (1Hanomeric, d, J = 8.2 Hz), 5.33−5.10 (3H, m), 4.31 (1H, dd, J = 12.5 Hz, 4.5 Hz), 4.13 (1H, dd, J = 12.5 Hz, 2.2 Hz), 3.92−3.82 (1H, m), 2.13 (3H, s), 2.10 (3H, s), 2.05 (6H, s), 2.03 (3H, s). Synthesis of 2-Bromoethyl 2,3,4,6-tetra-O-acetyl-β-Dglucopyranoside (AcGlcEtBr). AcGlc (5.00 g, 12.80 mmol) and 2-bromoethanol (1.92 mL, 15.37 mmol) were added into a 250 mL two-neck round-bottomed flask together with 4 Å molecular sieves. Anhydrous CH2Cl2 (80 mL) was added to the stirred flask under nitrogen atmosphere. The reaction was initiated by introduction of boron trifluoride diethyl etherate (9.09 mL, 64.04 mmol), dropwise, over 30 min via a gastight syringe while maintaining the flow of nitrogen through the flask. After purging the solution for another 20 min, the flask was sealed and left stirring at room temperature for 40 h. The reaction mixture was filtered through a sintered glass funnel, poured into iced water (100 mL), and extracted with CH2Cl2. The solution was washed with saturated solution of sodium bicarbonate and water. The organic phase was dried over Na2SO4, filtered, and concentrated. Upon drying in vacuo, an oily, yellow mixture was obtained. The anomers were separated by column chromatography using hexane/ethyl acetate (3:2) as eluent. 2-Bromoethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (AcGlcEtBr) was obtained as a white solid (3.62 g, 62%). 1H NMR (CDCl3, 300 MHz): δH 5.24 (1H, t, J = 9.4), 5.15−4.99 (2H, m), 4.60 (1Hanomeric, d, J = 7.9 Hz), 4.33−4.12 (3H, m), 3.90−3.79 (1H, m), 3.77−3.69 (1H, m), 3.53−3.45 (2H, m), 2.12 (3H, s), 2.09 (3H, s), 2.05 (3H, s), 2.03 (3H, s). Synthesis of 2-Azidoethyl 2,3,4,6-tetra-O-acetyl-β-Dglucopyranoside (AcGlcEtN3). Both 3.2 g (7.0 mmol) of AcGlcEtBr and 0.65 g (10.0 mmol) of sodium azide were stirred overnight in 60 mL of acetone and 20 mL of water at reflux temperature. Upon full conversion of the bromide to azide, as shown by TLC analysis, the acetone was evaporated, and the aqueous reaction mixture was extracted with CH2Cl2 twice. Safety note: sodium azide is known to generate highly explosive organic azides with halogenated solvents. The solvents should not be heated above 35 °C. The organic phases were combined and dried over sodium sulfate. After filtration, the solvent was evaporated in vacuo to give 2.78 g (94.8%) of the title compound in the form of white solid (2azidoethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranose (AcGlcEtN3)). 1H NMR (CDCl3, 300 MHz): δH 5.21 (1H, t, J = 9.4), 5.15−4.96 (2H, m), 4.60 (1Hanomeric, d, J = 7.9), 4.33−4.10 (2H, m), 4.09−3.97 (1H, m), 3.77−3.62 (2H, m), 3.56−3.42 (1H, m), 3.36−3.22 (1H, m), 2.08 (3H, s), 2.05 (3H, s), 2.02 (3H, s), 2.00 (3H, s). Synthesis of 2-Azidoethyl O-β-D-glucopyranoside (GlcEtN3) and 2-Azidoethyl O-β-L-fucopyranoside (FucEtN3). Both carbohydrate derivatives were deacetylated following the standard Zemplén procedure. In brief, the peracetylated sugar was dissolved in dry methanol. Sodium methoxide (25% solution in methanol), 0.05 mol equiv, was added dropwise via syringe. Upon completion of the reaction, as monitored by TLC, DOWEX 50W × 2−200 (a cation exchange resin) was added to neutralize the mixture. After filtration, methanol was evaporated, and the product was dried under vacuum. 2-Azidoethyl O-β-D-glucopyranoside (GlcEtN3) was obtained as a colorless oil (0.54 g, 90.4% yield). 1H NMR (DMSO-d6, 300 MHz): δH 4.99 (1H, d, J = 5.0), 4.94 (1H, d, J = 4.8), 4.90 (1H, d, J = 5.1), 4.49 (1H, t, J = 5.8), 4.19 (1Hanomeric, d, J = 7.8), 3.95−3.83 (1H, m), 3.74−3.60 (2H, m),

carbohydrate in the form of yellow oil (19.6 g, 95% yield; 1,2,3,4-tetra-O-acetyl-L-fucose (AcFuc, α:β = 0.3:1, NMR data presented for β anomer)). 1H NMR (CDCl3, 300 MHz): δH 5.69 (1Hanomeric, d, J = 8.3 Hz), 5.32 (1H, m), 5.27 (1H, m), 5.08 (1H, dd, J = 10.8 Hz, 3.6 Hz), 3.96 (6.62 Hz, 0.9 Hz), 2.19 (3H, s), 2.11 (3H, s), 2.04 (3H, s), 1.99 (3H, s), 1.23 (3H, d, J = 6.3 Hz). 13C NMR (CDCl3, 125 MHz): δC 170.5, 170.0, 169.4, 169.1, 92.2, 71.2, 70.2, 69.9, 67.9, 20.8, 20.7, 20.6, 20.6, 20.5, 15.9). Synthesis of 2-Bromoethyl 2,3,4-tri-O-acetyl-β-L-fucopyranoside (AcFucEtBr). AcFuc (5.00 g, 15.0 mmol) and bromoethanol (2.2 mL, 3.0 mmol) were dissolved in 20 mL of dry dichloromethane, in a round-bottomed flask. The reaction vessel was sealed with a rubber septum and cooled to 0 °C in an ice water bath. Subsequently, 10 mL (6.0 mmol) of boron trifluoride diethyl etherate was added dropwise via a gastight syringe. The mixture was stirred at 0 °C in the dark for 4 h and left for 3 days at 4 °C. TLC analysis (silica on glass) in ethyl acetate/hexane (2:3) confirmed full conversion of AcFuc to AcFucEtBr (Rf ≈ 0.5). The reaction mixture was washed with water, saturated sodium bicarbonate solution (three times), and water again. The organic phase was collected and dried over sodium sulfate. The mixture was filtered, concentrated on rotary evaporator, and dried in vacuo to yield approximately 6 g of crude anomeric mixture of AcFucEtBr. The anomers were separated by column chromatography (silica gel, ethyl acetate/ hexane (1:4). 4.63 g (77.5% yield) of pure β-AcFucEtBr was obtained (2-bromoethyl 2,3,4-tri-O-acetyl-β-L-fucopyranose (AcFucEtBr)). 1H NMR (CDCl3, 300 MHz): δH 5.27−5.18 (2H, m), 5.04 (1H, dd, J = 10.4 Hz, 3.5 Hz), 4.52 (1Hanomeric, d, J = 7.9 Hz), 4.25−4.16 (1H, m), 3.90−3.76 (2H, m), 3.55−3.44 (2H, m), 2.19 (3H, s), 2.09 (3H, s), 2.00 (3H, s), 1.26 (3H, d, J = 7.2 Hz). Synthesis of 2-Azidoethyl 2,3,4-tri-O-acetyl-β-L-fucopyranoside (AcFucEtN3). Initially, 4.5 g (11.0 mmol) of AcFucEtBr and 1.10 g (17.0 mmol) of sodium azide were stirred overnight in 50 mL of DMF at 70 °C. Upon full conversion of the bromide to azide, as shown by TLC analysis, the reaction mixture was diluted with chloroform and washed with water (two times). Saftey note: sodium azide is known to generate highly explosive organic azides with halogenated solvents. The solvents should not be heated above 35 °C. The organic phase was collected and dried over sodium sulfate. After filtration, the solvent was evaporated in vacuo to give 2.0 g (49.1%) of the title compound in the form of yellowish oil (2′-azidoethyl-2,3,4-tri-O-acetyl-β- I -fucopyranose (AcFucEtN3)). 1H NMR (CDCl3, 300 MHz): δH 5.30−5.20 (2H, m), 5.11−5.00 (1H, m), 4.56 (1Hanomeric, d, J = 8.0), 4.13−4.04 (1H, m), 3.85 (1H, dd, J = 12.8 Hz, 0.9 Hz), 3.75−3.65 (1H, m), 3.59−3.48 (1H, m), 3.37−3.27 (1H, m), 2.20 (3H, s), 2.09 (3H, s), 2.01 (3H, s), 1.25 (3H. d, J = 6.42). Synthesis of 1,2,3,4,6-Penta-O-acetyl-β-D-glucopyranose (AcGlc). To a 250 mL, round-bottomed flask, sodium acetate (6.1 g, 0.074 mol) and acetic anhydride (65 mL, 0.69 mol) were added, and the mixture was heated at 140 °C. Glucose (11.9 g, 0.056 mol) was added slowly to the boiling mixture and stirred for 45 min. Then the reaction mixture was poured into ice-cold water (500 mL) with vigorous stirring, and precipitation of the acetylated compound was observed. The solid phase was collected by filtration and washed extensively with water. The crude product was then purified by crystallization from ethanol. Subsequently, the obtained white powder was lyophilized to complete dryness (AcGlc, yield = C

DOI: 10.1021/acs.biomac.5b00299 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules 3.53−3.38 (3H, m), 3.20−2.89 (4H, m). 2-Azidoethyl O-β-Lfucopyranoside (FucEtN3) was obtained as yellowish oil (1.1 g, 92.5% yield). 1H NMR (DMSO-d6, 300 MHz): δH 4.84−4.73 (1H, br), 4.71−4.59 (1H, br), 4.41 (1H, d, J = 4.7 Hz), 4.13 (1Hanomeric, d, J = 7.5 Hz), 3.89−3.77 (1H, m), 3.67−3.56 (1H, m), 3.57−3.48 (1H, m), 3.45 (2H, t, J = 5.1 Hz), 3.42−3.38 (1H, br), 3.30−3.23 (2H, br), 1.13 (3H, d, J = 6.4 Hz). Synthesis of 3-(Trimethylsilyl)prop-2-yn-1-yl Methacrylate. A mixture of 3-(trimethylsilyl) 1-propanol (10.0 g, 78.0 mmol) and Et3N (14.2 mL, 101.3 mmol) in CH2Cl2 (100 mL) was cooled to 0 °C, and a solution of methacryloyl chloride (8.8 mL, 93.0 mmol) in CH2Cl2 (50 mL) was added dropwise over 30 min. The mixture was stirred at this temperature for 30 min, then at ambient temperature overnight. The precipitated ammonium salts were removed by filtration, and the volatiles were removed under reduced pressure. The residue was redissolved in CH2Cl2 and was washed with two 100 mL portions of water to remove the remaining impurities of the triethylammonium chloride. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. This dark orange oil was purified by column chromatography over silica gel eluting with petroleum spirit/diethyl ether (50:1) giving the title compound as colorless liquid (11.2 g, 73% yield; 3-(trimethylsilyl)prop-2-yn-1-yl methacrylate). 1H NMR (CDCl3, 300 MHz): δH 6.21−6.20 (1H, m), 5.66−5.64 (1H, m), 4.79 (2H, s), 2.00−1.99 (3H, m), 0.21 (9H, s). Synthesis of Poly(Trimethylsilyl-Protected Propargyl Methacrylate) (PTMSPMA). PTMSPMA was synthesized using RAFT polymerization. 3-(Trimethylsilyl)prop-2-yn-1-yl methacrylate (2.50 g, 12.73 mmol), fluorescein O-methacrylate (0.051 g, 0.127 mmol), and CPDAB RAFT Agent (0.071 g, 0.254 mmol) were loaded into a 10 mL round-bottom flask along with 2.42 mL of dioxane and 2.13 mL of toluene as solvent. AIBN (0.025 mmol) was introduced from fresh stock solution in toluene (0.056 g, in 10 mL) by taking the appropriate quantity (0.741 mL). The polymerization solution was purged with nitrogen for 1 h. The flask was placed in a preheated oil bath at 70 °C to react for 5 h. The monomer conversions were determined using 1H NMR by taking aliquots of crude mixtures and dissolving the samples in CDCl3. The conversion was calculated from the relative integration of monomer’s vinylic peak (CHCH2, 6.30−5.58 ppm) and the polymer’s OCH2 peak at 4.76−4.56 ppm. The final polymer from the crude solution was purified by precipitation (twice) into 10-fold excess of cold methanol and dried in vacuum oven at 40 °C for 24 h (poly(trimethylsilyl-protected propargyl methacrylate) (PTMSPMA)). 1H NMR (CDCl3, 300 MHz): δH 4.76−4.56 (64H, br), 2.20−1.76 (96H, br), 0.40−0.09 (288H, br). Synthesis of PTMSPMA-block-poly(n-Butyl Acrylate) (PTMSPMA-b-PBA). PTMSPMA was used as macro RAFT agent and chain-extended with n-BA. PTMSPMA (0.555 g, 0.085 mol; based on Mn,NMR (Table 1)) and n-BA (2.251 mL, 15.359 mmol) were combined with 6.0 mL of toluene in a Schlenk tube. AIBN (0.017 mmol) was introduced through stock solution of toluene (0.056 g, in 10 mL) and by taking the appropriate aliquot (0.496 mL). The reaction mixture was degassed by three freeze−pump−thaw (FPT) cycles and left under nitrogen then stirred at 70 °C for 4 h. The diblock copolymer was purified by precipitation in cold methanol twice. The obtained polymer was dried in the vacuum oven at 40 °C for 24 h (PTMSPMA-block-poly(n-butyl acrylate) (PTMSPMA-b-PBA)). 1H NMR (CDCl3, 300 MHz): δH

4.76−4.56 (64H, br), 4.20−3.90 (291H, br), 2.47−2.21 (140 H, br), 2.09−1.79 (135H, br), 1.78−1.53 (576H, br), 1.50− 1.32 (357H, br), 1.20−1.07 (40H, br), 1.06−0.84 (498H, br), 0.32−0.12 (295H, br). Synthesis of Poly(Propargyl Methacrylate)-blockpoly(n-Butyl Acrylate) (PPMA-b-PBA). The trimethylsilylprotected block copolymer (500 mg) was dissolved in THF (20 mL). The colorless solution was bubbled with nitrogen (ca. 10 min) and cooled to 0 °C. A 1 M solution of TBAF in THF (1.5 equiv mol/mol with respect to the alkyne-trimethylsilyl groups) was added dropwise via syringe (ca. 2−3 min). The resulting turbid mixture was stirred at this temperature for 30 min and then warmed to ambient temperature. The deprotection was complete in less than 2 h. The reaction solution was passed through a cake of silica in order to remove the excess of TBAF, and the cake was subsequently washed with additional THF. The resulting solution was then concentrated under reduced pressure, and the polymer was precipitated in cold methanol. The obtained polymer (PPMA-b-PBA)) was dried in the vacuum oven at 40 °C for 24 h (poly(propargyl methacrylate)block-poly(n-butyl acrylate) (PPMA-b-PBA)). 1 H NMR (CDCl3, 300 MHz): δH 4.74−4.54 (64H, br), 0.4.19−3.90 (355H, br), 2.65−2.46 (36H, br), 2.45−2.22 (166 H, br), 2.12−1.8 (overlapped with THF, br), 1.79−1.52 (617H, br), 1.51−1.24 (441H, br), 1.21−1.08 (44H, br), 1.07- 0.82 (596H, br). General Procedure for Preparation of Glycopolymers via “Click” Reaction. PPMA-b-PBA (0.100 g, 0.431 μmol, based on Mn,NMR, (Table 1)), carbohydrate azide (0.207 mmol), PMDETA (0.008 mL, 0.042 mmol), CuBr (0.006 g, 0.42 mmol), and DMF (1 mL) were added to a 10 mL Schlenk tube. Reaction mixture was degassed by three FPT cycles, left under nitrogen, and stirred overnight at room temperature. Subsequently, the solution was filtered through a column filled with neutral alumina to remove copper complex and purified by dialysis against Milli-Q water (molecular weight cutoff, MWCO 1000 Da) for 2 days, and lyophilized. Representative 1H NMR (300 MHz, DMSO-d6) spectra and signal assignments for all the glycosylated polymers are presented in Figure 1. Self-Assembly of Amphiphilic Block Glycopolymers. Micelles of the diblock glycopolymers were prepared according to the following protocol. Eight milligrams of respective polymers were dissolved in 1 mL of dimethyl sulfoxide (DMSO). Subsequently, 4 mL of DI water was added to this solution at the rate of 0.2 mL/h. Finally, the DMSO was removed by dialysis, yielding solutions with a 2 mg/mL of micelle concentration. Size-Exclusion Chromatography (SEC). The molecular weight and polydispersity of synthesized polymers were analyzed via size exclusion chromatography (SEC). A Shimadzu modular system comprising a SIL-10AD autoinjector, DGU12A degasser, LC-10AT pump, CTO-10A column oven and a RID-10A refractive index detector was used. A 5.0-lm bead-size guard column (50 × 7.8 mm) followed by four 300 × 7.8 mm linear columns (500, 103, 104, and 105 Å pore size, 5 μm particle size) were employed for analysis. N,N-Dimethylacetamide (DMAc; HPLC grade, 0.05% w/v 2,6-dibutyl-4methylphenol (BHT) and 0.03% w/v LiBr) with a flow rate of 1 mL/min at 50 °C was used as mobile phase. Fifty microliters of polymer solution with a concentration of 2 mg/ mL in DMAc was used for every injection. The calibration was performed using commercially available narrow-polydispersity polystyrene standards (0.5−1000 kDa, Polymer Laboratories). D

DOI: 10.1021/acs.biomac.5b00299 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules Table 1. Reaction Conditions and Selected Characterization Data for the Obtained Polymers and Micelles polymer PTMSPMA PTMSPMA-b-PBA PPMA-b-PBA P1 (100 mol % Fuc; 0 P2 (80 mol % Fuc; 20 P3 (50 mol % Fuc; 50 P4 (20 mol % Fuc; 80 P5 (0 mol % Fuc; 100 a

mol mol mol mol mol

% % % % %

Glc)) Glc) Glc) Glc) Glc)

time (h)

conv. (%)

Mn,theor (g/mol)

Mn,SECa (g/mol)

Đ

micelle

sizeb (nm)

Đp b

5 9 2 24 24 24 24 24

63 80 100 100 100 100 100 100

6500 23 150 20 800 28 250 28 300 28 300 28 300 28 300

7200 20 850 20 050 31 350 32 450 33 250 34 950 35 050

1.13 1.23 1.22 1.31 1.29 1.32 1.34 1.32

M1 M2 M3 M4 M5

26 24 21 20 33

0.22 0.26 0.27 0.34 0.35

According to polystyrene standards in DMAc. bDetermined by DLS.

Cytotoxicity Tests. AsPC-1 cells were seeded in 96-well cell culture plates at 4000 cells per well and cultured at 37 °C for 1 or 3 days. Solutions were sterilized by filtration through a 0.22 μm membrane in a biosafety cabinet and then serially diluted (2 × dilution) with sterile water. The medium in the cell culture plate was discarded, and 100 μL of fresh 2× concentrated RPMI 1640 serum medium was added. The samples were added into the plate at 100 μL per well for 72 h. The cell viability was measured using a sulforhodamine B (SRB) assay. The incubation with micelles was finished by addition of cold trichloroacetic acid for 30 min. After a complete washing with distilled water, 100 μL of 0.4% sulforhodamine B (SRB) solution in 1% acetic acid (w/v) was added to each well. After staining, unbound dye was removed by washing with 1% acetic acid, and plates are airdried. Finally, the SRB was solubilized with 200 μL of 10 mM Tris buffer, and the optical density was read on a Bio-Rad BenchMark microplate reader at 490 nm. Confocal Laser Scanning Microscopy. Cells were seeded in 35 mm Fluorodish (World Precision Instruments) at a density of 2 × 105 per dish and cultured for 1 day with RPMI 1640 cell culture medium. Micelle solution was loaded to the cells at a working concentration of 40 μg mL−1 polymer and incubated at 37 °C for 24 h. After incubation, the cells were washed three times with PBS and observed under a laser scanning confocal microscope system (Zeiss LSM 780). The system was equipped with a Diode 405-30 laser, an argon laser, and a DPSS 561-10 laser (excitation and absorbance wavelengths: 405 nm, 488 and 561 nm, respectively) connected to a Zeiss Axio Observer. Z1 inverted microscope ( × 20/1.4 NA objective lens). The ZEN2011 imaging software (Zeiss) was used for image acquisition and processing. Flow Cytometry. Cells were seeded in six-well plates at a density of 5 × 105 cells per well and incubated at 37 °C with 5% CO2 for 1 day prior to micelle treatment. During treatment, the medium was replaced with RPMI1640 cell culture medium containing micelles (40 μg/mL working concentration) at 37 °C for 24 h. The cell monolayer was washed three times with cold PBS and treated with trypsin/EDTA to detach the cells. The cells were collected, centrifuged, and resuspended in cold serum-free culture medium. The cells suspensions were used for flow cytometry analysis on BD FACSCanto II Analyzer (BD Biosciences, San Jose, USA), collecting results from at least 50 000 events. Competition Assay with Free Fucose. AsPC-1 cells were seeded in six-well tissue culture plates at a density of 5 × 105 cell per well and incubated for 2 days at 37 °C with 5% CO2 before fucose competition assay. Micelles were prepared with the polymer of 50% Fuc and 50% Glc. Nile red was encapsulated in the micelles as a fluorescent model drug.

Nuclear Magnetic Resonance (NMR) Spectroscopy. NMR general characterization was conducted using a Bruker Avance DPX 300 spectrometer (1H, 300.2 MHz). Samples were analyzed in the solvents DMSO-d6. All chemical shifts are stated in ppm (δ) relative to tetramethylsilane (δ = 0 ppm), referenced to the chemical shifts of residual solvent resonances (1H and 13C). Fourier-Transform Infrared (FT-NIR) Spectroscopy. FT-IR spectroscopy was used to determine conversions of triple bond and confirm absence of the free sugar azides (Supporting Information). A Bruker IFS 66\S Fourier transform spectrometer equipped with a tungsten halogen lamp, a CaF2 beam splitter, and a liquid nitrogen cooled InSb detector was used. Dynamic Light Scattering Measurements. Particle sizes (the average diameters and size distributions) were determined using a Malvern Zataplus particle size analyzer (laser, 35 mW, λ = 632 nm, angle = 90°) at a polymer concentration of 1 mg/ mL. Samples were prepared in deionized water and purified from dust using a micro filter (0.45 μm) prior to the measurements. Transmission Electron Microscopy (TEM). The TEM micrographs were obtained using a FEI Tecnai G2 20 TEM transmission electron microscope. The instrument operates at an accelerating voltage of 200 kV. Samples were negative stained with uranyl acetate (2 w/w %). The particles were cast onto a Formvar-coated grid by placing a droplet a micelle aqueous solution for 15 min onto its surface. Subsequently, the excess of the solution was removed using filter paper. In the staining process, the cast grid was gently put onto the surface of a drop of uranyl acetate for 1 min. The stained grids were dried on air. Fluorescence Spectroscopy. Fluorescence spectra of 40 μg/mL micelle suspensions were obtained on Cary Eclipse Fluorescence Spectrophotometer (Aglient Technologies). The excitation and emission wavelengths were 490 and 512 nm, respectively. In Vitro Cell Culture. Human pancreatic carcinoma (AsPC1) cells, human ovarian carcinoma (OVCAR-3) cells, human lung carcinoma (A549) cells, and Chinese Hamster Ovary cells (CHO) cells were cultured in T25 cell culture flask with 5% CO2 at 37 °C. The cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mM sodium pyruvate. After the cells reached confluence, the cells were washed with phosphate-buffered saline (PBS) and detached by trypsin/EDTA treatment. The cells were collected, centrifuged, and resuspended in culture medium for the further experiments. E

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be carried out under extremely mild reaction conditions. It has been widely applied for the synthesis of glycopolymers.30−33 For glycopolymer P1 and P5, either FucEtN3 or GlcEtN3 was “clicked” onto the polymer chain to yield the homofucose or homoglucose glycopolymer, whereas for glycopolymer P2, P3, P4, fucose azide and glucose azide were both reacted simultaneously at different ratios. The “clicking” strategy is attractive as it allowed the preparation of a range of materials featuring identical macromolecular properties (polymer architecture, Mn, Đ) that only differ in their binding epitope (Fuc) density. Furthermore, the use of glucose as the nonbinding sugar ensured that the amphiphilic character of the blocks remained almost unchanged. Additionally, this approach ensures random distribution of both carbohydrates along the hydrophilic block of the polymeric chain. Therefore, by increasing Glc content, the distances between Fuc moieties are also increased. The organization of sugars on the chain can have strong influence on biological interactions; however, it is difficult to determine which carbohydrate is predominantly displayed on the surface of the spherical particles. 1H NMR (Figure 1) and FT-IR analysis (Supporting Information)

Before micelle loading, the cells were treated with 0.5 mM fucose for 1 h in serum-free RPMI1640 (with 0.1% bovine serum albumin (BSA)) after being washed with PBS twice. Micelles at different concentrations (0, 10, 50, and 100 μg/mL) were loaded onto the cells together with 0.5 mM fucose in serum free medium (with 0.1% BSA) for 1.5 h. The cells were then washed with cold PBS three times and collected with trypsin treatment. After trypsin removal by centrifugation, the cells were then resuspended in cold HBSS (Hank’s Balanced Salt Solution). Flow cytometry analysis was carried out using the method described as in the above sessions.

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RESULTS AND DISCUSSION Synthesis of Amphiphilic Block Glycopolymers. Reaction conditions and selected characterization data for all of the obtained polymers and micelles are summarized in Table 1. The monomer TMSPA was prepared and polymerized using RAFT polymerization similar to earlier procedures.24 Fluorescein O-methacrylate (1 mol %) was added to the polymerization mixture to introduce the fluorescent label. The size-exclusion chromatography (SEC) characterization of the obtained polymer (PTMSPMA) revealed that it was welldefined, with dispersity index (Đ) of 1.13 and molar mass of 7200 g/mol (in N,N-dimethylacetamide (DMAc), according to polystyrene standards). NMR analysis revealed the monomer conversion of 63%, corresponding to approximately 32 repeating units. Subsequently, PTMSPMA was used as macro-RAFT agent in the chain extension polymerization of n-butyl acrylate (nBA). Polymers were targeted with large hydrophilic, upon functionalization with carbohydrates, fractions (>70% by weight) such that they would yield spherical micelles under standard solution assembly conditions. The chain-extended block copolymer with conversion of 80% (according to NMR analysis), corresponding to approximately 130 repeating units of nBA, was quite well-defined with dispersity of 1.23 and molar mass of 20 850 g/mol, as revealed by SEC in DMAc using polystyrene standards. To obtain the alkyne-functionalized polymeric backbone, the TMS protecting group was removed from the diblock copolymer using tetrabutylammonium fluoride. The SEC analysis revealed that, as expected, the hydrodynamic volume of the deprotected polymer PPMA-b-PBA decreased, whereas the Đ (1.22) remained almost unchanged, signifying no occurrence of undesired side reactions during the deprotection. The RAFT endfunctionality is usually cleaved under these conditions, which results in a colorless polymer. Earlier mass spec analysis has shown that the thiocarbonylthio functionality is replaced by a hydrogen.25 The polymers in this work are however too long to obtain meaningful mass spectrometry data, and we can therefore only speculate about the actual end group, which could be altentaively a thiol functionality. The reactive carbohydrates, 2-azidoethyl O-β-L-fucopyranoside (FucEtN3) and 2-azidoethyl O-β-D-glucopyranoside (GlcEtN3), were prepared by protection of fucose and glucose, followed by reaction with ethanol bromide, subsequent nucleophilic substitution of the bromide by azide, and deacetylation under standard Zemplén conditions.26−29 After the four-step synthesis, both building blocks were obtained at an overall yield of 33% for both of the derivatives. To obtain the amphiphilic block glycopolymers, coppercatalyzed azide−alkyne cycloaddition (CuAAC) was employed. The advantage of using CuAAC is that it belongs to the group of “click chemistry” reactions, which are very efficient and can

Figure 1. 1H NMR spectra (300 MHz, DMSO-d6) of synthesized block glycopolymers with various Fuc/Glc ratios.

confirmed that the conversion of the alkyne groups into triazoles was achieved at close to 100% yield, whereas the dispersity Đ of the polymers increased only slightly. SEC revealed a slight increase in the Mn values with the increase of glucose content, which may be related to increased hydrophilic character and better solvation of the glucosylated polymers. All of the glycopolymers were purified from the copper complex by passing through a column filled with neutral alumina and dialysis against distilled water for 2 days. After lyophilization, the resulting colorless powder or resulting DMSO solutions thereof did not reveal any bluish tinge indicating the removal of cytotoxic copper ions. Copper was removed following a procedure described in literature,29 but it should be noted that careful optimization is often required to yield a copper-free product.34 Synthesis and Characterization of the Polymeric Micelles. The micelles were prepared using standard aqueous self-assembly procedure, namely, the solvent displacement method.35 Briefly, DI water was slowly (0.2 mL/h) added to stirred DMSO solutions of the respective block copolymers, F

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Fucose-Dependent Uptake of Polymeric Micelles by Cancer Cells. Before commencing the cell uptake studies, the cytotoxicity of the micelles toward Chinese hamster ovary (CHO) and human pancreatic carcinoma (AsPC-1) cell lines was investigated using sulforhodamine B assay. After 1 and 3 days, no decrease in cell viability was found (Figure 3) for the concentration (40 μg/mL) employed in the internalization studies. To identify the underlying reason for the toxicity, cells were grown with different amounts of fucose, revealing that high fucose concentration can inhibit cell proliferation. Interestingly, very small amounts for added fucose seems to accelerate the cell metabolism although the difference to the control is not statistically significant (Support Information, Figure S2). The uptake of fluorescent micelles carrying different amounts of fucose was investigated on AsPC-1 cells, human ovarian carcinoma (OVCAR-3) cells, human lung carcinoma (A549) cells, and CHO cells by flow cytometry and confocal laser scanning microscopy (CLSM) (Figure 4). Appropriate cell numbers were cultured for 24 h at 37 °C in medium containing 40 μg/mL of micelles, according to the procedures described in the Materials and Methods. Such a long time of incubation allowed the cells to reach the state of equilibrium between endo- and exocytosed material. The cells were washed thoroughly, three times, with phosphate-buffered saline (PBS) before trypsinization and analysis by flow cytometry. The obtained histograms were normalized according to the specific fluorescence intensities obtained from the fluorescence spectra of the micelles (Supporting Information, Table S1). The results (Figure 4) revealed the fucose-dependent increase of the uptake by cancer cell lines. The higher fucosylation and lower glucosylation of the micelles the more they were internalized by the cells, with the highest endocytosis for the particles (M1)

followed by extensive dialysis to remove any remaining organic solvents and traces of copper, yielding solutions with a 2 mg/ mL concentration of micelles. Dynamic light scattering (DLS) measurements revealed the sizes of the resulting micelles as well as the size distributions thereof (Table 1). In addition, the micelles exhibited similar, slightly negative zeta potential (−5 to −8 mV). All of the micelles showed hydrodynamic diameters in the range of 20 to 30 nm. Transmission electron microscopy (TEM) images (Figure 2) further confirmed the presence of

Figure 2. Transmission electron microscopy images of obtained micelles stained with uranyl acetate.

assemblies of this size, however, with some larger agglomerates resulting, most probably, from the dry sample preparation, because no larger structures were observed in DLS measurements. TEM also revealed the spherical shape of the micelles, albeit some of the micelles were squeezed together and deformed on the grid. Micelles of this size and shape have been reported to avoid renal clearance and penetrate as well as accumulate in tightly packed pancreatic cancer tissues in vivo.36 In addition, DLS measurements revealed stability of these micelles at dilutions used in the cell uptake studies. Lack of agglomeration and dissolution in the presence of blood components is one of the prerequisites for nanomedicines.37

Figure 3. Cytotoxicity of M1 (100% Fuc/0% Glc), M3 (50% Fuc/50% Glc), M5 (0% Fuc/100% Glc) against the pancreatic cell line AsPC-1 and CHO. G

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Figure 4. Confocal laser scanning microscopy images (M5 − top row, M1 − bottom row) and results of flow cytometric uptake assay (cell count vs log of normalized fluorescence).

formed from the block copolymer (P1) containing 100 mol % of fucose. The fully glucosylated micelles (M5) as well as the ones prepared from the block copolymer (P4) carrying 20 mol % of Fuc and 80 mol % of Glc (M4) did not reveal any clear uptake, with fluorescence histograms in the same range as the blank (PBS) samples. Additionally, the noncancerous CHO cells did not internalize the fucosylated particles in observable amounts. The fluorescence of the inoculated cells did not increase above their natural autofluorescence. This might be due to very low nonspecific uptake at the chosen low concentration (40 μg/mL) of the polymer, although higher micelle concentrations could theoretically enforce more cell uptake.38 The CLSM images (Figure 4) further confirmed the flow cytometry results, demonstrating that the fucosylated micelles are specifically internalized by cancer cells. The particles accumulated in cytosol, most probably within lysosomal compartments. The micrographs of CHO cells did not reveal any preferential uptake of M1 over M5 and their overall internalization was low. In order to study the receptors responsible for the increased uptake of M1, we have carried out competition assays using serum free RPMI1640 medium supplemented with additional free fucose at the concentrations of 0.5 mM (Figure 5). In previous study by Yoshida et al., fucosylated liposomes were efficiently internalized by pancreatic cancer cell lines, including AsPC-1, which expressed CA19−9 (sialylated Lewisa antigen), a tumor marker found in sera of pancreatic and colon cancer patients.39 Excess of L-fucose but not D-glucose, D-mannose, Dxylose, or D-galactose inhibited the degree of uptake. Additionally, by applying 14C-L-fucose receptor binding assay using AsPC-1 cells, the researchers have found a high affinity (3.25 × 106 receptors/cell, Kd = 28.74 nM) receptor, which is specific for this sugar. In this work, the addition of free L-fucose led to

Figure 5. Results of the flow cytometric analysis of the AsPC-1 uptake of the M3 (50% Fuc/50% Glc) micelles with or without the competition of free fucose. Micelles were loaded onto cells with the presence of free fucose for 1.5 h after a 1 h treatment with free fucose. The uptake with fucose competition was normalized to the uptake without fucose.

decreased internalization compared with that without free fucose treatment, as measured by flow cytometry. Figure 5 displays the difference in uptake between M3 (50% Fuc/50% Glc) micelles with and without added fucose. Although the added fucose did not block all of the internalization, the competition of fucose decreased 18.5%, 15.4%, and 26.6% in uptake for 10, 50, and 100 μg/mL micelles, respectively. The results revealed that there might be another internalization pathway for fucosylated polymeric micelles other than via fucose receptors. H

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(7) Christiansen, M. N.; Chik, J.; Lee, L.; Anugraham, M.; Abrahams, J. L.; Packer, N. H. PROTEOMICS 2014, 14 (4−5), 525−546. (8) Takeda, Y.; Shinzaki, S.; Okudo, K.; Moriwaki, K.; Murata, K.; Miyoshi, E. Cancer 2012, 118 (12), 3036−3043. (9) Michalski, J. C.; Klein, A. Biochim. Biophys. Acta 1999, 1455 (2− 3), 69−84. (10) Abeylath, S. C.; Ganta, S.; Iyer, A. K.; Amiji, M. Acc. Chem. Res. 2011, 44 (10), 1009−1017. (11) Barreto, J. A.; O’Malley, W.; Kubeil, M.; Graham, B.; Stephan, H.; Spiccia, L. Adv. Mater. 2011, 23 (12), H18−H40. (12) Koo, H.; Huh, M. S.; Sun, I.-C.; Yuk, S. H.; Choi, K.; Kim, K.; Kwon, I. C. Acc. Chem. Res. 2011, 44 (10), 1018−1028. (13) Acharya, S.; Sahoo, S. K. Adv. Drug Del Rev. 2011, 63 (3), 170− 183. (14) Bertrand, N.; Wu, J.; Xu, X. Y.; Kamaly, N.; Farokhzad, O. C. Adv. Drug Del Rev. 2014, 66, 2−25. (15) Pearce, T. R.; Shroff, K.; Kokkoli, E. Adv. Mater. 2012, 24 (28), 3803−3822. (16) Mahon, E.; Salvati, A.; Bombelli, F. B.; Lynch, I.; Dawson, K. A. J. Controlled Release 2012, 161 (2), 164−174. (17) Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J. J. Cell Biol. 2010, 188 (6), 759−768. (18) Babiuch, K.; Stenzel, M. H., Synthesis and Application of Glycopolymers. In Encyclopedia of Polymer Science and Technology; John Wiley & Sons, Inc.: New York, 2014; pp 1−54. (19) Ahmed, M.; Narain, R. Biomaterials 2012, 33 (15), 3990−4001. (20) Chen, W.; Meng, F.; Cheng, R.; Deng, C.; Feijen, J.; Zhong, Z. J. Mater. Chem. B 2015, 3, 2308−2317. (21) Babiuch, K.; Pretzel, D.; Tolstik, T.; Vollrath, A.; Stanca, S.; Foertsch, F.; Becer, C. R.; Gottschaldt, M.; Biskup, C.; Schubert, U. S. Macromol. Biosci. 2012, 12 (9), 1190−1199. (22) Seymour, L. W.; Ferry, D. R.; Anderson, D.; Hesslewood, S.; Julyan, P. J.; Poyner, R.; Doran, J.; Young, A. M.; Burtles, S.; Kerr, D. J. J. Clin. Oncol. 2002, 20 (6), 1668−1676. (23) Mitsukami, Y.; Donovan, M. S.; Lowe, A. B.; McCormick, C. L. Macromolecules 2001, 34 (7), 2248−2256. (24) Withey, A. B. J.; Chen, G. J.; Nguyen, T. L. U.; Stenzel, M. H. Biomacromolecules 2009, 10 (12), 3215−3226. (25) Quemener, D.; Le Hellaye, M.; Bissett, C.; Davis, T. P.; BarnerKowollik, C.; Stenzel, M. H. J. Polym. Sci., Polym. Chem. 2008, 46 (1), 155−173. (26) Ladmiral, V.; Mantovani, G.; Clarkson, G. J.; Cauet, S.; Irwin, J. L.; Haddleton, D. M. J. Am. Chem. Soc. 2006, 128 (14), 4823−4830. (27) Geng, J.; Lindqvist, J.; Mantovani, G.; Haddleton, D. M. Angew. Chem. Int. Ed. 2008, 47 (22), 4180−4183. (28) Hetzer, M.; Chen, G. J.; Barner-Kowollik, C.; Stenzel, M. H. Macromol. Biosci. 2010, 10 (2), 119−126. (29) Vinson, N.; Gou, Y.; Becer, C. R.; Haddleton, D. M.; Gibson, M. I. Polym. Chem. 2011, 2 (1), 107−113. (30) Slavin, S.; Burns, J.; Haddleton, D. M.; Becer, C. R. Eur. Polym. J. 2011, 47 (4), 435−446. (31) Lu, J.; Zhang, W.; Richards, S.-J.; Gibson, M. I.; Chen, G. Polym. Chem. 2014, 5 (7), 2326−2332. (32) Li, X.; Chen, G. Polym. Chem. 2015, 6 (9), 1417−1430. (33) Huang, J.; Bonduelle, C.; Thévenot, J.; Lecommandoux, S.; Heise, A. J. Am. Chem. Soc. 2012, 134 (1), 119−122. (34) Abd Karim, K. J.; Binauld, S.; Scarano, W.; Stenzel, M. H. Polym. Chem. 2013, 4 (22), 5542−5554. (35) Zhao, J.; Babiuch, K.; Lu, H.; Dag, A.; Gottschaldt, M.; Stenzel, M. H. Chem. Commun. 2014, 50 (100), 15928−15931. (36) Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Terada, Y.; Kano, M. R.; Miyazono, K.; Uesaka, M.; Nishiyama, N.; Kataoka, K. Nat. Nanotechnol. 2011, 6 (12), 815−823. (37) Li, Y.; Budamagunta, M. S.; Luo, J.; Xiao, W.; Voss, J. C.; Lam, K. S. ACS Nano 2012, 6 (11), 9485−9495. (38) Kim, Y.; Pourgholami, M. H.; Morris, D. L.; Stenzel, M. H. Macromol. Biosci 2011, 11 (2), 219−233.

It was observed to be rather crucial to evaluate the effect of additionally added fucose on the cell uptake after a short incubation time only. Incubation with the a mixture of micelles and added fucose led to an increased uptake of the fucosylated micelle M1 while M5, which only carried glucose, was not affected by the presence of fucose. It is not clear what effect fucose has on the events inside the cell in such a scenario, but it can safely be assumed that after 24 h exocytosis of nanoparticle can be prevalent (Supporting Information, Figure S3).

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CONCLUSIONS In summary, the previous studies have reported L-fucose dependent uptake of fucosylated liposomes by pancreatic cancer cells in vitro as well as in vivo.39 Here, we have examined other cancer cell lines using 20−30 nm micellar aggregates, which differed only in the degree of fucosylation. The results have confirmed the preferential and specific uptake of nanoparticulates having higher content of fucose. Furthermore, the presented synthetic methodology can be extended to formation of potential micellar drug carriers with low cytotoxicity, stability at low concentrations and in the presence of serum proteins, and the ability to passively and actively target tumor tissues of various types of cancer.

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ASSOCIATED CONTENT

S Supporting Information *

FT-IR of the functionalized polymer and fluorescence intensity of each micelle type with normalization factors used for flow cytometry. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.biomac.5b00299.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ∥

These authors contributed equally (K.B. and A.D.).

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Mark Wainwright Analytical Centre at UNSW for support. A.D. acknowledges the financial support from the The Scientific & Technological Research Council of Turkey (TUBITAK) (Project No: 1059B191200208). M.S. thanks the Australian Research Council (ARC) for funding (DP130101625).

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REFERENCES

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