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Carbohydrate Conjugated Amino Acid Based Fluorescent Block Copolymers: Their Self-Assembly, pH Responsiveness and/or Lectin Recognition Sonu Kumar, Binoy Maiti, and Priyadarsi De Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b02245 • Publication Date (Web): 10 Aug 2015 Downloaded from http://pubs.acs.org on August 15, 2015
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Carbohydrate
Conjugated
Amino
Acid
Based
Fluorescent
Block
Copolymers: Their Self-Assembly, pH Responsiveness and/or Lectin Recognition Sonu Kumar, Binoy Maiti, Priyadarsi De* Polymer Research Centre, Department of Chemical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur - 741246, Nadia, West Bengal, India *
Corresponding author. E-mail:
[email protected] (P. De).
ABSTRACT: An effective strategy has been documented to combine both carbohydrate and amino acid biomolecules in a single synthetic polymeric system via reversible additionfragmentation chain transfer (RAFT) polymerization technique. The resultant unique block copolymer was engineered to form uniform micelles with desired projection of either selective or both amino acid/sugar residues on the outer surface with “multivalency”, providing pH-based stimuli-responsiveness and/or lectin recognition. The self-assembly process was studied in detail by field emission scanning electron microscopy (FE-SEM), dynamic light scattering (DLS) and UV-visible spectroscopy. The enhanced lectin binding behaviour was observed for glyconanoparticle with both amino acid/sugar entities on shell as compared to the only glycopolymer nanoparticle, because of higher steric hindrance factor in the case of only glycopolymer nanoparticle. The fluorophore conjugation by postpolymerization functionalization was further exploited by fluorescence spectroscopy for evidencing the lectin recognition process.
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INTRODUCTION Synthetic polymers having carbohydrate moieties are known as ‘glycopolymers’ and this class of polymers are of considerable interests in the field of drug delivery and therapeutics.1 Carbohydrates are well-known to interact with specific proteins, known as lectins and play a key role in wide range of biological events such as cell-surface recognition, detection, inflammation, signalling and infection of pathogens.2-4 Such biospecific protein-carbohydrate interactions are usually very weak due to monovalent binding, but can be increased dramatically by displaying multiple copies of carbohydrate on their synthetic polymer conjugates, to exhibit polyvalent interactions (multivalency).5-7 Synthetic glycopolymers are conceptually similar to natural polysaccharides in terms of structural properties.8 Glycopolymers are found to be impressive as the complex nature of polysaccharides makes their precise synthesis with narrow dispersities difficult for chemists. On the other hand, conjugation of amino acids, peptides or proteins into the synthetic polymers have been widely used for applications in the areas of medicine, biotechnology and materials science.9-11 In particular, naturally occurring amino acids, which are the constitutional component of proteins/peptides, based biohybrid materials create an important class of polymer therapeutics.12-14 Thus, incorporation of both amino acids and carbohydrate biomolecules to the synthetic polymers as a single hybrid material can provides unique opportunities to combine properties of two individual components, with entirely new synergism.15-17 The resultant hybrid materials may create a new class of nonbiological macromolecules which may mimic the biological structures and properties of amino acids as well as bioactivities of natural polysaccharides.18,19 Though there are several reports available for such hybrid materials in the form of glycosylated synthetic polypeptides (glycopolypeptides),17,20 limited reports are available for their side-chain hybrid polymers carrying those biomolecules in the 2
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pendants.21,22 The strategy to prepare side-chain glycosylated polymers via postpolymerization modification is generally ineffective to provide quantitative functionalization, while the direct polymerization of corresponding monomers can facilitates inherently functionalized side-chains. In general, the lack of the functional sites (free –NH2 or –COOH terminus) on incorporated amino acid residues, the limitation for selection of amino acid and the tedious synthetic methods, limits the versatile application of glycopolypeptides, prepared from direct ring-opening polymerization of glycosylated α-amino acid based Ncarboxyanhydrides.23 Therefore, preparation of side-chain hybrid polymers by direct polymerization of amino acid and sugar based monomers can offer significant opportunities as it may provides unique features such as controlled synthesis with narrow dispersity, appropriate for a range of amino acid residues, presence of functionalization sites, preserved individual properties such as specific biological functions of amino acids and lectin binding properties of carbohydrates. On the other hand, creations of materials that responds to pH based biological stimuli encountered in cells and organisms, are highly desirable for wide range of biomedical applications.24 To have pH-responsive feature in the synthetic system, the utilization of biocompatible natural residues such as amino acids are much advantageous, compared to the other non-natural pH-responsive moieties.13 Importantly, there are several reports available for the thermo-based stimuli responsive glycopolymers,25,26 while the pH-based stimuli responsive glycopolymers are relatively unexplored.27-31 In this context, we have revealed the feasibility of side-chain hybrid polymeric system comprised of both sugar and amino acid constituents with pH responsive property. This ‘smart’ block copolymer can be selectively tuned to form higher-order micellar structures having selective or dual projection of these biomolecules in the outer surface with ‘multivalency’, thus provides desired properties of pH responsiveness by amino acids and/or lectin recognition by carbohydrates. To the best of our 3
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knowledge, this is the first report on the synthesis of such dual responsive sugar/amino acid based block copolymer conjugates. Additionally, conjugating fluorescent tag to these scaffolds as one multifunctional package is very attractive for study of protein-carbohydrate interactions due to their intrinsic fluorescent properties.32 Therefore, successive postpolymerization functionalization approach for fluorescent biohybrid polymer was further established to sense the lectin recognition process.
EXPERIMENTAL SECTION Materials. Boc-L-valine (Boc-V-OH, 99%), and trifluoroacetic acid (TFA, 99.5%) were obtained from Sisco Research Laboratories Pvt. Ltd., India and used as received. The 4dimethylaminopyridine (DMAP, 99%), 1-hydroxybenzotriazole hydrate (HOBt hydrate, 97%), dicyclohexylcarbodiimide (DCC, 99%), 2-hydroxyethyl methacrylate (HEMA, 97%), β-D-glucose pentaacetate (98%), boron trifluoride diethyl etherate (BF3.Et2O, 46.5%), fluorescein isothiocyanate (FITC, 90%), Concanavalin A from Canavalia ensiformis (ConA, Type IV), pyrene (98%), Dowex® 50WX8 and anhydrous N,N-dimethylformamide (DMF, 99.9%) were purchased from Sigma. The 2,2′-azobisisobutyronitrile (AIBN, Sigma, 98%) was recrystallized twice from methanol. NMR solvents such as CDCl3 (99.8% D), CD3OD (99.8% D) and DMSO-d6 (99.9% D) were obtained from Cambridge Isotope Laboratories, Inc., USA. The solvents such as hexanes (mixture of isomers), acetone, ethyl acetate, tetrahydrofuran (THF), and dichloromethane (CH2Cl2) were purified by standard procedures. 4-Cyano-4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid (CDP) was synthesized as reported elsewhere.33 Instrumentation. The (average) molar mass and molar mass distributions (dispersity, Ð) were determined by gel permeation chromatography (GPC) in DMF at 35 °C, using a flow rate of 0.9 mL min-1 (Viscotek pump; columns: one PolarGel-M guard column (50 × 7.5 mm)
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and two PolarGel-M analytical columns (300 × 7.5 mm)). Detection was performed using a Viscotek refractive index (RI) detector operating at λ = 660 nm, and a Viscotek model 270 series platform, consisting of a laser light scattering detector at 3 mW with λ = 670 nm, detection angles of 7° and 90°, and a four-capillary viscometer. The system was calibrated with poly(methyl methacrylate) (PMMA) standards of narrow molar mass distribution. The solution state 1H NMR spectroscopy was carried out on a Bruker AVANCEIII 500 MHz spectrometer, at 25 oC. Positive mode electrospray ionization mass spectroscopy (ESI-MS) was carried out on a Q-TOF Micro YA263 high resolution (Waters Corporation) mass spectrometer. Solid state FT-IR spectra were obtained on a FT-IR Perkin-Elmer spectrometer at a nominal resolution of 2 cm-1 with the KBr disk technique. Fluorescence emission spectrum was recorded on a fluorescence spectrometer (Horiba Jobin Yvon, Fluromax-3, Xe150 W, 250-900 nm) at an excitation wavelength 490 nm. The UV-visible (UV-vis) spectroscopic
measurements
were
carried
out
on
a
Perkin-Elmer
Lambda
35
spectrophotometer, with a scan rate of 240 nm min-1, equipped with a peltier system. Particle size and zeta potential (ξ) were determined by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively, using a Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, UK) equipped with a 4.0 mW He–Ne laser beam operating at λ = 658 nm. All samples were measured in aqueous environment at room temperature at a scattering angle of 173°. Each measurement was repeated three times to obtain the average ξ value for 1.0 mg mL-1 solution. The self-assembled morphology of various polymers was investigated on a high resolution field emission scanning electron microscopy (FE-SEM). The micrographs were taken from Zeiss microscope; SUPRA 55VP-Field Emission Scanning Electron Microscope. Synthesis of Amino Acid Based Monomer. To the stirring solution of Boc-V-OH (10.0 g, 46.0 mmol) and HEMA (6.6 g, 50.6 mmol) in dry CH2Cl2 (150 mL) under dry N2 atmosphere, a solution of DCC (10.5 g, 50.6 mmol) and DMAP (1.1 g, 9.2 mmol) in 10 mL of dry CH2Cl2 were added drop-wise in ice-water bath condition. The reaction mixture was allowed to react at room temperature for 24 h. After removing insoluble N,N′5
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dicyclohexylurea (DCU) by suction filtration, the organic layer was further washed with 1.0 N HCl, saturated NaHCO3 and brine solution, and dried over anhydrous Na2SO4 and evaporated by using vacuum. The crude product was purified by silica gel column chromatography using hexanes:ethyl acetate (3:1 v/v) as mobile phase, to get a white solid compound Boc-V-EMA (1, yield: 86%) and was characterized by 1H NMR (Figure 1A). Synthesis of Carbohydrate Based Monomer. To the stirring solution of β-D-glucose pentaacetate (16.5 g, 42.3 mmol) and HEMA (5.0 g, 38.4 mmol) in anhydrous CH2Cl2 (100 mL), 5.0 g of 4 Å molecular sieves was added. The solution was cooled down to 0 °C under N2 atmosphere and BF3.Et2O (16.4 g, 115.3 mmol) was added drop-wise to this solution. The solution was further left for stirring at room temperature for 18 h. The final suspension was filtered and washed with saturated NaCl solution (150 mL) and the filtrate was evaporated under vacuum. The crude product was purified by silica gel column chromatography using CH2Cl2:ethyl acetate (4:1 v/v) as mobile phase, to get the pure product Ac-G-EMA (2) as colorless oil, with a yield of 75%, and was further characterized by ESI-MS and 1H NMR (see Figure S1 and S2A in the Supporting Information). General Method for RAFT Polymerization. Typically, monomer 2 (1.0 g, 2.17 mmol), CDP (17.5 mg, 0.434 µmol), AIBN (0.713 mg, 4.34 µmol; 0.1 mL solution of 71.3 mg AIBN in 10 mL DMF), DMF (4.0 mL) and a magnetic stir bar were taken in a 20 mL septa sealed glass vial. The vial was purged with dry N2 for 20 min and placed in a preheated reaction block at 70 ºC. Samples were removed periodically by a N2 purged syringes and analyzed by NMR spectroscopy for the monomer conversion data. Polymerization was quenched by cooling the vial on an ice-water bath and exposing the solution to air, diluted with acetone and precipitated into cold hexanes. The sugar based polymer, P(Ac-G-EMA) (4), was reprecipitated four times form acetone/hexanes and dried under high vacuum at room temperature to a constant weight. Similar procedure was followed for the synthesis of amino acid based homopolymer P(Boc-V-EMA) (3).
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Synthesis of Glycosylated Amino Acid Based Block Copolymer. A typical block copolymerization procedure is described as follows: monomer 2 (104.6 mg, 0.227 mmol), macro-CTA 3 [number-average molar mass (Mn,GPC) = 11,000 g/mol, Ð = 1.19, 50.0 mg, 4.55 µmol], AIBN (0.075 mg, 0.045 µmol; 0.1 mL solution of 7.5 mg AIBN in 10 mL DMF), DMF (0.3 mL) and a magnetic stir bar were sealed in a 20 mL glass vial. After N2 purging (20 min), the vial was placed in a preheated reaction block at 70 ºC. Samples were removed periodically by a N2 purged syringes and analyzed by NMR spectroscopy for the monomer conversion data. After a predetermined time, the vial was cooled in an ice-water bath, and diluted with acetone as necessary. The polymer was precipitated into cold hexanes and allowed to settle down, and then the solvent was decanted off. Finally, the block copolymer, P(Boc-V-EMA)-b-P(Ac-G-EMA) (5), was reprecipitated four times from acetone/hexanes and dried under vacuum at room temperature. Selective Acetyl Deprotection of Sugar Constituents of Block Copolymer. Typically, to a stirring solution of glycosylated amino acid based block copolymer 5 (70.0 mg) in CHCl3/MeOH (1:1 v/v, 2 mL) under N2 atmosphere at room temperature, 1 mL of a freshly prepared 1M solution of NaOMe in MeOH was added and left for stirring for 1 h. The solution was neutralized by adding Dowex cation-exchange resin (30 mg) and was stirred for further 20 min. The filtered solution was transferred to a 6-8 kDa molecular weight cut off (MWCO) dialysis bag (Spectra/Pro Membrane) and was dialyzed against water for 24 h. Finally, the solution was lyophilized to provide block copolymer P(Boc-V-EMA)-b-P(HO-GEMA) (9). Similar procedure was followed for the deprotection of acetyl group for the synthesis of glycosylated homopolymer P(HO-G-EMA) (7). Selective Boc- Deprotection of Amino Acid Segments of Block Copolymer. Typically, glycosylated amino acid based block copolymer 5 (30.0 mg) was taken in 1.0 mL CH2Cl2. To this solution 0.3 mL TFA was added drop-wise at ice-water bath condition. Then, the reaction mixture was stirred at room temperature for 1 h. The resulting selective Bocdeprotected polymer salt form, P(CF3COO-.H3N+-V-EMA)-b-P(Ac-G-EMA), was isolated by
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precipitation from diethyl ether (× 3), and dried under high vacuum at room temperature. Further to remove the TFA salt, these polymers were dissolved in 1.0 mL of water and 1N NaOH solution was added drop-wise to its stirring solution until the appearance of precipitates. The precipitate was isolated by centrifugation, washed with DI water thrice, weakly acidified by adding very diluted HCl solution and finally lyophilized to obtain free primary amino group carrying block copolymer P(HCl•H2N-V-EMA)-b-P(Ac-G-EMA) (8) as a white powder. Similar procedure was followed for the deprotection of Boc- group of block copolymer 9 and homopolymer 3 for the synthesis of polymer P(HCl•H2N-V-EMA)-b-P(HOG-EMA) (10) and P(HCl•H2N-V-EMA) (6), respectively. Fluorescent Copolymer Synthesis by Post-Polymerization Modification. A typical post-polymerization modification procedure for dye functionalization of –NH2 containing polymers was achieved as follows: copolymer 10 (Mn,theo = 17,750 g/mol, 10.0 mg, 0.56 mmol) was dissolved in 20 mL methanol and 10.0 µL of triethylamine was added to the solution and the mixture was stirred for 30 min under dry N2 atmosphere. FITC (7.2 mg, 18.3 mmol) in 1.0 mL methanol solution was then added to the reaction mixture and was further stirred for 6 h at room temperature. Then, the solution was transferred to a 6-8 kDa MWCO dialysis bag and was dialyzed against methanol for 48 h. The dialysed solution was finally precipitated into cold hexanes and allowed to stand, and then the solvent was decanted off. Finally, the FITC functionalized block copolymer 11 was reprecipitated four times from acetone/hexanes and dried under high vacuum at room temperature. ConA Turbidimetry Binding Assay. A solution of 1.0 mg mL-1 ConA lectin was prepared in the phosphate buffer solution (PBS, pH 7.4). Turbidity measurements were performed by adding 0.5 mL of this lectin solution to a dry quartz micro-cuvette and placed into the holding block of the UV-vis spectrophotometer for temperature equilibration at 25 °C. 1 mg of glycopolymers was dissolved in 0.1 mL of PBS buffer at pH 7.4 and the solution was sonicated for 15 min. A small volume of this solution was added to the cuvette containing the lectin solution. The solution in the cuvette was quickly mixed for ~5 s using a
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micropipette and immediately returned into the spectrometer. Absorbance data were recorded at 420 nm as a function of time. ConA Recognition by Fluorescent Copolymer. A solution of 2.0 mg mL-1 FITCfunctionalized copolymer 11 was prepared in PBS, pH 7.4. An aliquot of 12 µL of this polymer solution was added to 2.5 mL of PBS solution in a quartz cuvette having final concentration of 9.6 µg mL-1 and was recorded its absorbance and fluorescence (λexcit. = 490 nm) at 25 °C by using UV-vis and fluorescence spectrophotometer, respectively. Then sequential addition of small volume of PBS solution of ConA (2.0 mg mL-1) was carried out to the copolymer solution in the cuvette and the mixture was quickly mixed for ~5 s using a micropipette and immediately returned to the spectrometer for monitoring absorbance and fluorescence, for each added volume of ConA. Micelle Preparation from Polymeric Amphiphiles. Typically, 10 mg of amphiphilic copolymer 9 was dissolved in 5 mL acetone, and the solution was transferred to a dialysis bag (MWCO: 6-8 kDa) and dialyzed against DI water for 48 h (water was replaced after every 2 to 8 h). The prepared micellar solutions were further diluted and sonicated for 3 h. Then, an aliquot of the solution were drop casted on a silicon wafer and dried by slow evaporation. It was allowed to further dry under vacuum for two days and were coated with gold:palladium (20:80) before analyzing by FE-SEM. Determination of Critical Aggregation Concentration (CAC) by Fluorescence Spectroscopy. A predetermined amount of pyrene in acetone was prepared in 50 mL volumetric flasks and then acetone was evaporated completely. Different concentrations of polymer solutions were added to the flask to make the pyrene concentration in the final solution 6.0 × 10−7 mol/L, and left to equilibrate with pyrene overnight. The fluorescence intensities of solutions were measured with the excitation wavelength set at 339 nm and the ratio of the pyrene probe emission fluorescence intensities of the first and third vibrational
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peaks at 372 and 382 nm (I1/I3 = I372/I382) were plotted as a function of the logarithm of polymer concentrations. The CAC value was obtained from the intersection of two tangent plots of intensity ratio I372/I382 versus the logarithm of polymer concentrations (log C).34
Scheme 1. Chemical structure of block copolymer 5, and representation of synthetic routes to obtain various structures, their self-assembly in aqueous medium and lectin binding
RESULTS AND DISCUSSION In order to prepare multifunctional block copolymer, we first synthesized L-valine derived representative amino acid based methacrylate monomer 1 (Scheme S1), and glucose conjugated representative carbohydrate based methacrylate monomer 2 (Scheme S2, Supporting Information), by coupling reaction with HEMA. Structures of the monomers were confirmed by 1H NMR and mass spectroscopy measurements (Figure 1, and Figure S1 and S2 in the Supporting Information). The N-terminus of as synthesized monomer 1 was intentionally masked by tert-butyloxycarbonyl (Boc-) group whereas hydroxyl groups of 10
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glucose was protected with acetyl (Ac-) groups for the feasibility of successive RAFT polymerization and having opportunity for selective alteration of their physical characteristics. Homopolymerization of monomer 1 and 2 was carried out via RAFT method to prepare corresponding well defined side-chain polymers 3 and 4, respectively. To the best of our knowledge, this is the first example of valine based such polymer via RAFT technique. The homopolymers were characterized by 1H NMR spectroscopy, where the typical resonance signals of the different protons for the repeating units of the respective polymers are clearly assigned (Figure 1B and Figure S2B in the Supporting Information). The homopolymers 3 and 4 exhibited narrow dispersity (Ð
