Glycoconjugated Amphiphilic Polymers via Click-Chemistry for the

Article pubs.acs.org/Langmuir

Glycoconjugated Amphiphilic Polymers via Click-Chemistry for the Encapsulation of Quantum Dots Christian Schmidtke,*,†,‡ Anna-Marlena Kreuziger,†,‡ Dirk Alpers,§ Anna Jacobsen,§ Yevgeniy Leshch,§ Robin Eggers,†,‡ Hauke Kloust,†,‡ Huong Tran,†,‡ Johannes Ostermann,†,‡ Theo Schotten,∥ Joachim Thiem,§ Julian Thimm,§ and Horst Weller*,†,‡,∥ †

Institute of Physical Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany The Hamburg Center for Ultrafast Imaging, University of Hamburg, Luruper Chaussee 149, 22761 Hamburg, Germany § Institute of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany ∥ Center for Applied Nanotechnology, Grindelallee 117, 20146 Hamburg, Germany ‡

S Supporting Information *

ABSTRACT: Herein, we present a strategy for the glycoconjugation of nanoparticles (NPs), with a special focus on fluorescent quantum dots (QDs), recently described by us as “preassembly” approach. Therein, prior to the encapsulation of diverse nanoparticles by an amphiphilic poly(isoprene)-bpoly(ethylene glycol) diblock copolymer (PI-b-PEG), the terminal PEG appendage was modified by covalently attaching a carbohydrate moiety using Huisgen-type click-chemistry. Successful functionalization was proven by NMR spectroscopy. The terminally glycoconjugated polymers were subsequently used for the encapsulation of QDs in a phase transfer process, which fully preserved fluorescence properties. Binding of these nanoconstructs to the lectin Concanavalin A (Con A) was studied via surface plasmon resonance (SPR). Depending on the carbohydrate moiety, namely, D-manno-heptulose, D-glucose, D-galactose, 2deoxy-2-{[methylamino)carbonyl]amino}-D-glucopyranose (“des(nitroso)-streptozotocin”), or D-maltose, the glycoconjugated QDs showed enhanced affinity constants due to multivalent binding effects. None of the constructs showed toxicity from 0.001 to 1 μM (particle concentration) using standard WST and LDH assays on A549 cells.

■

processes,7 leveraging on their high photostability and adjustable size-dependent emission wavelength.8 Various glycoconjugated nanoparticles have been described previously, for example, metal,9,10 metal oxide,11,12 semiconductor nanoparticles (NPs),13,14 polymeric micelles,15,16 and glycopolymers.16−19 Pellegrino et al. developed a polymeric encapsulation strategy for various NPs with an amphiphilic copolymer.20 Using this system, glycol-conjugation of polymer coated Au NPs with carbohydrates was demonstrated.21 Recently, we published a versatile micellar encapsulation technique, suitable for a broad range of NPs, including quantum dots (QDs), iron oxide, and gold NPs, using an amphiphilic, cross-linkable poly(isoprene)-b-poly(ethylene glycol) diblock copolymer (PI-b-PEG).22 Subsequent cross-linking led to exceptional stable polymer shells, fully preserving the physical properties of the diverse core particles. By employing living anionic polymerization (LAP) for the synthesis of the PIb-PEG copolymer, in situ chain termination with different electrophiles allowed convenient functionalization for successive glycoconjugation.23

INTRODUCTION Carbohydrates play a crucial role in various utmost important biological processes. Commonly, carbohydrates are perceived as pivotal substrates in energy metabolism such as, for example, glycolysis, Krebs cycle, or gluconeogenesis, as constitutive elements of DNA, or of structural support matrices of plants, for example, in celluloses, lignans, pectins, glycans, and so forth. Curiously, carbohydrates are much less recognized as key elements in signaling cascades, cellular recognition, cell−cell communication, and molecular and cellular targeting, occurring in processes, like for example, bacterial and viral infections, cancer metastasis, and inflammatory reactions. In fact, detailed studies of bacterial and viral interactions or cancer metastasis to date frequently reveal not protein−protein, but lectin−sugar interactions.1−4 Lectins are proteins that specifically bind to definite glyco structures. Whereas a single monosaccharide moiety displays only negligible affinity to a lectin, linear or branched oligosaccharide clusters exhibit binding constants in the nanomolar range or even below. This strong binding of glycoclusters is a result of polyvalent interactions.3 Therefore, we were intrigued to use nanoparticles as structural scaffolds for spatially arranging carbohydrates to clusters and presenting these nanoscale superstructures to lectins and lectin receptors.4−6Advantageously, a fluorescent quantum dot (QD) as a carrier of glycoclusters would allow imaging of biological © XXXX American Chemical Society

Received: July 24, 2013 Revised: September 11, 2013

A

dx.doi.org/10.1021/la402826f | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

Table 1. Synthesis of Glycoconjugated PI-b-PEG Diblock Copolymers 4a−g compd

abbreviation

sugar [mg]

m (sugar) [mg]

polymer 3 [mg]

m (CuI) [mg]

m (DABCO) [mg]

yield %

coupling yield %

4a 4b 4c 4d 4e 4f 4g

PI-b-PEG-Glc(1)Ac4 PI-b-PEG-Glc(6)Ac4 PI-b-PEG-Gal(1)Ac4 PI-b-PEG-Gal(6)Ac4 PI-b-PEG-Glc(1)-(4→1)-GlcAc7 PI-b-PEG-STZAc4 PI-b-PEG-Man-HeptAc5

7 8 9 10 11 12 13

26.0 24.6 30.1 26.6 27.8 24.7 26.0

106.7 103.5 98.3 101.0 106.0 99.2 50.0

3.7 1.9 3.0 4.0 4.4 5.6 5.0

5.8 7.4 8.1 6.2 4.5 7.1 5.0

79 78 78 86 76 74 89

55 61 50 60 40 19 20

backscatter system using He−Ne laser illumination at 633 nm. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on an AV I 400, AV II 400, or DRX 500 spectrometer (Bruker). Chemical shifts in 1H NMR spectra are reported in δ units (ppm) relative to deuterated solvent signal as internal standard (chloroform D1 signal at 7.26 ppm). Transmission electron microscopy (TEM) images were recorded with a Jeol JEM-1011 microscope. Methods. Cytotoxicity Tests. For each assay, A549 cells were plated at a density of 104 cells/mL in a 96-well plate and grown for one day before use. Different concentrations of nanoparticles in DMEM + 10% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer were tested. LDH and WST8 assays were run after 24 h incubation at 37 °C in a humidified atmosphere containing 5% CO2, according to BioVision Research Products (Mountain View, CA). Absorbance was measured in a 96-well plate with an Infinite 2000 microplate reader (Tecan, Switzerland) at 460 nm and a reference wavelength of 620 nm. SPR Experiments: Lectin Immobilization. CM5 sensorchip (carboxymethylated dextran matrix covalently attached to a gold surface, Series S Sensor Chip CM5, GE Healthcare) was used for the experiments. The HEPES buffer was filtered through a 0.22 μm PTFE filter (Millipore) and degassed. The lectin Con A (Concanavalin A from Canavalia ensiformis (Jack Bean, Type VI, lyophilized powder) was immobilized by the following procedure at a flow rate of 10 μL/ min: the sample channel of the chip was activated with 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) (0.5/0.1 M, 198 μL) solution. Con A in 0.01 M HEPES buffer (138 μL, pH 6.5) was injected. The reactive groups on the surface were blocked with ethanolamine solution (1 M, 139 μL). The reference channel was treated the same, except for the lectin injection. SPR Measurements. After equilibration of the system by HEPES buffer until a stable baseline was observed, the sample solutions were injected in the channels at a flow rate of 10 μL/min. Volumes of 100− 250 μL with increasing concentrations were used for the experiments. Contact time was 180 s, dissociation time was 300 s and the temperature was 25 °C. For evaluation the Biacore T100 Evalution Software was used. Nanoparticle Synthesis. CdSe/CdS/ZnS core/shell/shell nanoparticles were synthesized via a bottom-up procedure in hexadecylamine (HDA) using a modified protocol according to Talapin et al.24 Bis(trimethylsilyl) sulfide and diethylzinc were replaced with hydrogen sulfide and zinc(II) acetate. Sodium oleate capped Fe3O4 nanoparticles (5 nm) were produced in high-boiling ether solvents according to Yu et al.25 Citrate stabilized Au nanoparticles were synthesized according to Bastús et al.26 These NPs were rendered hydrophobic by a biphasic (water/CHCl3 = 1:1) ligand exchange with hexadecylamine (1000 equiv per particle). Synthesis of Polymers. Polymers PI and PI-b-PEG (Mn = 10.5 kDa) (1) were synthesized by living anionic polymerization (LAP) as previously described.22 Poly(isoprene)-diethylentriamine (PI-DETA, Mn = 1.2 kDa) was synthesized by CDI coupling of PI to diethylenetriamine (DETA). Synthesis of PI-b-PEG−O−CH2−CCH (3). Alternatively to the in situ chain termination of the living polymer with propargyl bromide,23 a two-step procedure was used. Potassium salt 2 of PI-b-PEG−OH diblock copolymer (1000 mg, 9.524 × 10−5 mol) in THF (10 mL) was generated by titration with diphenylmethyl potassium (DPMK) at

Accordingly, we quenched the living anion with propargyl bromide, thus introducing terminal prop-2-yn-1-yloxy moieties for subsequent copper catalyzed Huisgen 1,3-dipolar cycloaddition (click-chemistry) with various azido pyranose derivatives, that is, 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide (7), 6-azido-6-deoxy-1,2,3,4-tetra-O-acetyl-α/β-D-glucopyranose (8), 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl azide (9), 6-azido-6-deoxy-1,2,3,4-tetra-O-acetyl-α/β-D-galactopyranose (10), 2,3,6,2′,3′,4′6′-hepta-O-acetyl-a-D-glucopyranosyl1→4-β-D-glucopyranosyl azide (11), 1,3,4,6-tetra-O-acetyl-2deoxy-2-(4′-azidobutyl-ureido)-β-D-glucopyranose (a derivative of streptozotocin [STZ]) (12), and 4′-azidobutyl-1,3,4,5,7penta-O-acetyl-α-D-manno-hept-2-ulopyranoside (13). The glycoconjugation did not affect the propensity of the polymers for encapsulation, as evidenced by QDs, which fully retained fluorescence. Furthermore, gold and iron oxide NPs were successfully encapsulated, demonstrating the versatility of the method. The carbohydrate surface functionalization of NPs was proofed by lectin binding tests via surface plasmon resonance (SPR). Interestingly, the unprecedented glycoconjugation of Dmanno-heptulose provided NPs with very high affinity to the lectin Concanavalin A (Con A) in the nM range. Toxicity studies were performed in vitro using a standard cell line (A549). All glycoconjugated constructs were devoid of toxic effects over the biologically relevant concentration range, making them promising candidates testing on lectin receptor expressing cells. These results will be reported shortly elsewhere.

■

EXPERIMENTAL SECTION

Materials. Air or water sensitive chemical compounds were handled under inert conditions with N2/Ar using the Schlenk technique or glove boxes. Cadmium acetate anhydrous (99.99%) and zinc acetate hydrate (99.9%) were purchased from ChemPur, selenium shots (1−5 mm, 99.9%) from Alfa Aesar, and Con A (from Canavalia ensiformis, type VI) from Sigma. Chloroform (anhydrous, amylene stabilized), copper(I) iodide (≥99.5%), 1,4diazabicyclo[2.2.2]octane (DABCO, ≥99%), dichlormethane (anhydrous, amylene stabilized), and pyridine (anhydrous, 99.8%) were obtained from Sigma-Aldrich. Amberlite IR120 (hydrogen form), 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl azide (97%), 6-azido-6deoxy-α/β-D-galactose (≥98%), 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide, 6-azido-6-deoxy-α/β-D-glucose (≥98%), propargyl bromide (80% in toluene), and trioctyl phosphine (TOP, 90%) were purchased from Aldrich, acetone from Grüssing, and chloroform D1 (CDCl3), methanol (MeOH, ≥99.9%), tetrahydrofuran (THF, 99.9%), tetrahydrofuran D8 (99.5% D) from Carl Roth. Acetic anhydride, N,N-dimethylformamide (DMF, ≥99.8%), (≥99.5%) and sodium methoxide (NaOMe, ≥97.0%) were obtained from Fluka. Instrumentation. Ultraviolet−visible (UV−vis) absorption spectra were collected on Cary 50 (Varian). Fluorescence spectra were recorded on Eclipse (Varian) and on FluoroMax-4 (Horiba). Hydrodynamic light scattering (DLS) was performed on Zetasizer Nano ZS system (Malvern) equipped with a single angle 173° B


Langmuir

Article

(86% yield according to Zissis et al.30) (500 mg, 1.08 × 10−3 mol) was dissolved in CHCl2 (8 mL) and 4-azidobutan-1-ol (250 mg, 2.16 × 10−3 mol) was added. Boron trifluoride diethyl etherate (221 μL, 1.08 × 10−3 mol) was added dropwise at 0 °C, and the reaction mixture was warmed up to room temperature. After stirring for 48 h, the reaction mixture was neutralized with triethylamine (1 mL), diluted with CH2Cl2, and washed with water. The organic phase was dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography (PE/EE 1:1) to obtain the product as colorless oil (170 mg, 30% yield). Synthesis of 1-Azido-4-(2, 5-dioxopyrrolidin-1-yl-oxycarbonylamino)-butane. 1-Amino-4-azido-butane was obtained from 1,4butanediol by triflation, subsequent substitution to the 1,4-diazide and Staudinger reaction of one azido group according to Lee et al.31 A solution of 1-amino-4-azido-butane (3.09 g, 27.1 mmol) in anhydrous acetonitrile (100 mL) was added dropwise within 2 h to a solution of N,N′-disuccinimidyl carbonate (8.33 g, 30.9 mmol) in anhydrous acetonitrile (250 mL) at room temperature and stirred for 48 h. After completion, the solvent was removed under vacuum. The crude product was purified by column chromatography (EE) to obtain the product (3.99 g) as colorless oil in 58% yield. Synthesis of D-manno-Heptulose (14). Starting from D-mannose, D-manno-heptulose was synthesized in seven steps according to Waschke et al.32 Ligand Exchange with PI-DETA. NPs in n-hexane were incubated with a 500 molar excess of PI18-DETA (average Mw 1.3 kDa). After 2 h, the NPs were precipitated with ethanol and collected by centrifugation. The PI-DETA coated NPs were stored in n-hexane under light exclusion. Ligand Addition. An aliquot of PI-DETA coated QDs (25 nmol) was dried under nitrogen flow, and resuspended in THF (2 mL) with a 70−300 times excess of glycoconjugated PI-b-PEG. AIBN was added in the half mass ratio as the used diblock copolymer. The solution was injected into water (18 mL) and incubated for 15 − 30 min at room temperature, followed by heating to 80 °C for 4 h for cross-linking. The particle solution was filtered through a syringe filter (CE; hydrophilic; 0.45 μm) and washed twice with water (8 mL) in centrifugal filter units (Amicon Ultra-15; 100 kDa membrane).

ambient temperature. To this solution propargyl bromide (119 mg, 1.00 × 10−3 mol) in toluene (0.035 mL) was added. After stirring for 24 h at room temperature, an equimolar amount of acetic acid was added. All volatiles were evaporated, and the yellowish syrup dissolved in CHCl3 (2 mL) and acetone (45 mL). After precipitation at −20 °C, the colorless precipitate thus formed was collected by centrifugation (6 min, 8000g). The pellet was further purified by two additional precipitation/centrifugation steps in cold acetone (2 × 45 mL). The colorless sediment was redissolved in deionized water (20 mL) and lyophilized (723 mg, 73% yield). Glycoconjugation via Click-Chemistry (4a−g). The peracetylated azido sugar (6.0 × 10−5 mol), PI-b-PEG−O−CH2-CCH (6.0 × 10−6 mol), copper(I) iodide (1.0 × 10−5 mol), and DABCO (5.0 × 10−5 mol) were dissolved in CHCl3 (5 mL). After stirring for 24 h under reflux, the yellowish-orange solution was cooled to room temperature and filtered through a syringe filter (0.2 μm, PTFE). The reaction mixture was evaporated and the remaining residue was dissolved in CHCl3 (0.5 mL) and precipitated by addition of acetone (10 mL) at −20 °C. After centrifugation (4 min, 12 000g), the supernatant was discarded and the precipitation/centrifugation step in acetone at −20 °C was repeated once. The product was dissolved in deionized water (25 mL) and lyophilized, providing the protected glycoconjugated polymer (Table 1). Deprotection of the Glycoconjugated Polymers (5a−g). Deprotection was accomplished under Zemplén conditions by dissolving the foregoing compounds (3−4 × 10−6 mol) in CHCl3 (6 mL) and adding a freshly prepared solution of NaOMe in MeOH (12 mL, 0.1 M). The solution was stirred at room temperature for 3 h. After neutralization with Amberlite IR120 (hydrogen form) for 0.5 h and filtration, the filtrate was concentrated under reduced pressure to dryness. The crude product was precipitated from acetone (10 mL) at −20 °C. The precipitate was isolated by centrifugation (4 min, 12 000g), and the precipitation process was repeated once. The product was dissolved in deionized water (25 mL) and lyophilized yielding the desired compound: PI-b-PEG-Glc(1) (5a), 90%; PI-b-PEG-Glc(6) (5b), 91%; PI-b-PEG-Gal(1) (5c), 86%; PI-b-PEG-Gal(6) (5d), 91%; PI-b-PEG-Glc(1)-(4→1)-Glc (5e), 83%; PI-b-PEG-STZ (5f), 68%; PI-b-PEG-Man-Hept (5g), 86%. Synthesis of 6-Azido-6-deoxy-1,2,3,4 -tetra-O-acetyl-α/β-D-glucopyranose (8). A solution of 6-azido-6-deoxy-α/β-D-glucose (33.5 mg, 1.63 × 10−4 mol) in pyridine (4000 μL) was cooled at 10 °C and acetic anhydride (400 μL) was slowly added. After stirring for 18 h, acetic anhydride and pyridine were removed in vacuo and the remaining residue was taken up in CH2Cl2 (2000 μL). Evaporation gave the crystalline product (60.1 mg, 99% yield) which was directly used in the next steps without further purification. Synthesis of 6-Azido-6-deoxy-1,2,3,4 -tetra-O-acetyl-α/β-D-galactopyranose (10). A solution of 6-azido-6-deoxy-α/β-D-galactose (16.5 mg, 8.05 × 10−5 mol) in pyridine (2 mL) was cooled at 10 °C and acetic anhydride (200 μL) was slowly added. After stirring for 18 h, acetic anhydride and pyridine were removed in vacuo and the remaining residue was taken up in CH2Cl2 (2 mL). Evaporation gave the crystalline product (26.6 mg, 89% yield) which was directly used in the next steps without further purification. Synthesis of 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-(4′-azidobutyl-ureido)-β-D-glucopyranose (12). Starting from D-glucosamine hydrochloride, 1,3,4,6-tetra-O-acetyl-β-D-glucosamine hydrochloride was obtained in three steps according to Silva et al.27 and Ran et al.28 1,3,4,6-Tetra-O-acetyl-β-D-glucosamine hydrochloride (286 mg, 7.44 mmol) and 1-azido-4-(2,5-dioxopyrrolidin-1-yl-oxycarbonylamino)butane (950 mg, 3.72 mmol) were suspended in anhydrous acetonitrile (30 mL). The reaction mixture was treated with triethylamine (2.00 mL, 27.1 mmol) and stirred for 24 h. After removing the solvent under vacuum, the crude residue was subjected to column chromatography (PE/EE 1:1) to obtain pure product 12 (1.07 g) as a colorless solid in 59% yield. Synthesis of 4-Azidobutan-1-ol. The azido alcohol was prepared in a two-step procedure according to Bates and Dewey.29 Synthesis of 4′-Azidobutyl-1,3,4,5,7-penta-O-acetyl-α-D-mannohept-2-ulopyranoside (13). Peracetylated D-manno-heptulose (15)

■

RESULTS AND DISCUSSION Phase Transfer. High-quality NPs with a high crystallinity and monodispersity were mostly produced in organic solvents at high temperatures via bottom-up procedure. Rendering these hydrophobic particles water-soluble demands for a suitable phase transfer process, which preserves their original features. Hence, CdSe/CdS/ZnS core/shell/shell QDs were synthesized

Scheme 1. Schematic Representation of the PI-b-PEG Diblock Copolymer Encapsulation of QDs with an Unfunctionalized (A) and a Glycoconjugated Polymer (B)

C


Langmuir

Article

Scheme 2. Schematic Representation of the Glycoconjugation Strategy and Chemical Structures of the Compoundsa

a

(I) DPMK, THF; (II) propargyl bromide, THF; (III) peracetylated azido-pyranose, CuI, DABCO, CHCl3; (IV) NaOMe, MeOH, Amberlite IR120 (hydrogen form); (V) acetic anhydride, pyridine; (VI) 4-azidobutan-1-ol, BF3·Et2O, CH2Cl2.

Functionalization. We modified the protocol described above by replacing the hydroxy terminated PI-b-PEG diblock copolymer with terminally glycoconjugated PI-b-PEG diblock copolymer. Since the functionalization of the polymer was performed prior to the micellar self-assembly, we named our approach “preassembly”.23 This process gave excellent control over the surface coverage of the nanocapsule by using defined mixtures of diversely end group modified diblock copolymers.34 However, two conditions have to be complied to: The end group must not affect the propensity of micellar self-assembly and furthermore has to survive radical induced cross-linking. To this end, herein we applied well-established copper-catalyzed Huisgen 1,3-dipolar cycloaddition (“Sharpless click chemistry”)35,36 between azido pyranoses and PI-b-PEG−O−CH2− CCH) (see Scheme 2). In general, the reaction provides high yields under mild conditions and tolerates many functional

in hexadecylamine and transferred into water using an amphiphilic poly(isoprene)-b-poly(ethylene glycol) diblock copolymer (PI-b-PEG) applying the three-step procedure previously reported (Scheme 1).15,16 First, the native ligands of the NP were replaced with a multidentate poly(isoprene)diethylentriamine (PI-DETA) prepolymer. Second, after ligand exchange, the PI-DETA coated NPs in THF were mixed with PI-b-PEG diblock copolymer and injected into water. In a spontaneous self-assembly process micelles are formed which stably enclose the QDs. Finally, the inner hydrophobic PI-part of the micelle was cross-linked with a radical initiator, providing exceptionally stable water-soluble QDs with high fluorescence quantum yields up to 55%.33 This protocol gave also excellent results for rendering a broad range of nanoparticles, for example, FeOx, GdPO4, NaYF4, or Au NPs water-soluble. D


Langmuir

Article

Figure 1. Superimposed NMR spectra of glycoconjugated PI-b-PEG in different solvents: PI-b-PEG-Glc(1)Ac4 in CDCl3 (blue), PI-b-PEG-Glc (5a) after deacetylation in CDCl3 (red), and in THF-d8 (green).

refluxing chloroform) smoothly provided glycoconjugated PI-bPEG diblock copolymers 4a−g in very good yields (Table 1, Scheme 2). All structures were in accordance with NMR spectra in CDCl3, however some of the corresponding 1,5triazole isomers were also detected. Assumedly, complexation by the polyethylene glycol chain38 led to depletion of copper species, thus compromising the stereospecificity of the reaction. Curiously, after Zemplén deacetylation all proton signals from the unprotected pyranose rings of 5a−g disappeared (signals in red in Figure 1 for compound 5a), presumably due to the formation of inverse micelles in CDCl3, evidenced by corresponding DLS measurements. Nevertheless, structural integrity of 5a−g was confirmed in deuterated THF, and also after retransformation to 4a−g. The here described preassembly functionalization consists of several reaction steps, but leads to well-defined, pure products and the coupling yield can be determined by NMR. The postassembly approach, however, where the functionalization takes place on the ready assembled nanocontainer,23 seems to be faster and easier. Yan et al. reported facile postassembly photocoupling chemistry of carbohydrates to NPs (silica, Au, and FeOX NPs).4 Disadvantageously, this strategy provides no control over the coupling position of the carbohydrate and to determine the coupling yield on the NP’s surface is difficult. The glycoconjugated polymers were successfully used for the micellar encapsulation of QDs (see Figure 2A) following the phase transfer method described above. The glycoconjugated PI-b-PEG encapsulated QDs showed excellent fluorescence quantum yields and UV−vis spectra, identical to the corresponding unfunctionalized nanocapsules (for superimposed spectra and TEM images, see the Supporting Information). This also strongly indicated that no carryover of copper species from the “click” reaction occurred, because even minute amounts copper ions would induce instantaneous fluorescence quenching.39 In DLS, average hydrodynamic diameters of glycoconjugated nanocapsules were considerably increased from 30 nm (volume) to 34−50 nm compared to the nonfunctionalized PI-b-PEG−OH micelles (see the Supporting Information). Assumedly, the branched multiple hydroxyl

Figure 2. (A) Aqueous solutions of glycoconjugated QDs under visible (bottom) and UV light (top). (B) Aqueous solutions of FeOx, Au NPs, and QDs functionalized with D-mannose under visible (left) and UV light (right).

groups. Remarkably, in our hands, no concomitant cross reaction of the polyisoprene double bonds was observed. Starting PI-b-PEG block copolymer was synthesized via living anionic polymerization (LAP) providing narrow molecular weight distributions.23,37 O-Alkylation with propargyl bromide was either achieved by quenching the living anion with propargyl bromide, or by alkylation of the PI-b-PEG−OH diblock copolymer (see Scheme 2 compound 1) after deprotonation with DMPK leading to the desired alkynylated polymer 3. Copper-catalyzed “click” reaction with 10 equivalents of peracetyl azido pyranoses 7−13 under optimized conditions (1:1 ratio of copper(I) iodide/carbohydrate in E


Langmuir

Article

manno-heptulose functionalized construct 5g displayed the lowest dissociation constant (KD = 8.5 nM), whereas the single D-manno-heptulose derivative 13 has a higher KD of 0.166 mM. Structurally D-manno-heptulose resembles D-manno configuration, but due to the additional carbon atom it possesses different biological properties. To the best of our knowledge, Dmanno-heptulose and D-manno-heptulose NP conjugates have not been characterized via SPR yet. All other glycoconjugates 5a−f showed low response units and low (unspecific) affinities as expected. As a result, polyvalent binding causes a 4 orders of magnitude higher binding force. The corresponding SPRsensograms, the association (kon) and dissociation (koff) rate constants of compound 13, and functionalized QDs with 5g (binding) and 5d (no binding) are given in the Supporting Information. Cellular Toxicity. All glycoconjugated QD-containing nanocapsules were tested on adenocarcinoma human alveolar basal epithelial cells (A549) using standard water-soluble tetrazolium salt (WST8) and lactate dehydrogenase (LDH) toxicity assays. Particles were incubated at a concentration range from 0.001 to 1 μM for 20 h on the cell line, which corresponds to a cell to QD-micelle ratio between 1 to 7.5 × 1010 and 7.5 × 1013. Within this range, none of glycoconjugated-QDs showed toxicity (see Figure 3).

■

CONCLUSION In conclusion, we used well-established Huisgen-type “click” chemistry for the PEG-terminal glycoconjugation of narrow dispersed amphiphilic PI-b-PEG diblock copolymers with various pyranoses (D-manno-heptulose, D-glucose, D-galactose, de(methyl(nitroso))-streptozocin, or D-maltose). Notably, the glycoconjugates formed inverse micelles in chloroform, as proven by NMR and DLS measurements. Interestingly, the substitution did not compromise the micelle-forming propensity of the modified polymers in the aqueous phase transfer process, making them highly suitable for the encapsulation of diverse nanoparticles. In particular, sensitive organic QDs were rendered watersoluble under perfect preservation of their optical properties, providing multiple glycofunctionalized fluorescent nanocapsules. Accordingly, polyvalent binding effects were detected for a hitherto unprecedented mannoheptosylated nanoconstruct, which showed high affinity to Concanavalin A with a binding constant in the nanomolar range. Suitability of the glycosylated nanocapsules as potential theranostics in general was proven by toxicological testing on standard WST8 and LDH assays on the A549 cell line showing no toxicity up to 1 μM concentrations. Further testing on cellular lectin receptors will be reported elsewhere. Utmost important pathogeneses, like, for example, bacterial and viral infections, cancer metastasis, and inflammatory reactions, are frequently mediated by lectin−sugar interactions, elicited by the spatial arrangement of specific glycoclusters on surfaces of viruses, bacteria and eukaryotic cells.3 Nanoparticles, attributed with fluorescent, superparamagnetic, plasmonic, or radioactive properties, all highly interesting for biomedical research, diagnosis, and therapy, herein may act as perfect functional scaffolds for the presentation of glycoclusters to lectins and lectin receptors, in order to gain insight in these intriguing processes, finally aiming for remedies. Consequently, we feel that the methods described herein, will contribute to this goal.

Figure 3. LDH (top) and WST8 assay (bottom) of PI-b-PEG encapsulated CdSe/CdS/ZnS QDs (5a−g) tested on A549 cells.

groups of the glycoconjugated micelles generate an increased hydration shell. The hydrodynamic diameter of the unfunctionalized and glycoconjugated QDs does not change significantly in the presence of NaCl concentrations up to 1 M (see ref 22 and the Supporting Information). Analogously, glycoconjugated micelles containing superparamagnetic FeOx and Au NPs were prepared (see Figure 2B). Biological Testing. Affinity to Concanavalin A. The accessibility of the carbohydrate moieties on the QDs was tested via SPR using Con A immobilized on a CM5-chip in combination with a Biacore instrument was monitored. Con A is a tetramer at pH > 7 with four binding sites.4 It is known to bind α-D-mannosyl and α-D-glucosyl residues via their hydroxyl groups on C-3, C-4, and C-6.4,40 Owed to the high molecular weights of glycoconjugated nanocapsules (∼800−1000 kDa) and in order to avoid spurious viscosity effects we adjusted the Biacore technology to much lower concentrations (0.83 nM to 0.425 μM) than originally recommended. Clearly these NPs do not behave as ideal monomeric species,41,42 but rather as substrates, for the formation of carbohydrate clusters, hence prompting polyvalent binding events. As expected, the DF


Langmuir

■

Article

(13) Babu, P.; Sinha, S.; Surolia, A. Sugar−Quantum Dot Conjugates for a Selective and Sensitive Detection of Lectins. Bioconjugate Chem. 2007, 18, 146−151. (14) Kikkeri, R.; Lepenies, B.; Adibekian, A.; Laurino, P.; Seeberger, P. In Vitro Imaging and in Vivo Liver Targeting with Carbohydrate Capped Quantum Dots. J. Am. Chem. Soc. 2009, 131, 2110−2112. (15) Freichels, H.; Alaimo, D.; Auzély-Velty, R.; Jérôme, C. α-Acetal, ω-Alkyne Poly(ethylene oxide) as a Versatile Building Block for the Synthesis of Glycoconjugated Graft-Copolymers Suited for Targeted Drug Delivery. Bioconjugate Chem. 2012, 23, 1740−1752. (16) Sunasee, R.; Narain, R. Glycopolymers and Glyco-nanoparticles in Biomolecular Recognition Processes and Vaccine Development. Macromol. Biosci. 2013, 13, 9−27. (17) Becer, C. R.; Gibson, M. I.; Geng, J.; Ilyas, R.; Wallis, R.; Mitchell, D. A.; Haddleton, D. M. High-Affinity Glycopolymer Binding to Human DC-SIGN and Disruption of DC-SIGN Interactions with HIV Envelope Glycoprotein. J. Am. Chem. Soc. 2010, 132, 15130− 15132. (18) Chen, Q.; Cui, Y.; Zhang, T. L.; Cao, J.; Han, B.-H. Fluorescent Conjugated Polyfluorene with Pendant Lactopyranosyl Ligands for Studies of Ca2+-Mediated Carbohydrate-Carbohydrate Interaction. Biomacromolecules 2010, 11, 13−19. (19) Gou, Y.; Geng, J.; Richards, S.-J.; Burns, J.; Becer, C. R.; Haddleton, D. M. A Detailed Study on Understanding Glycopolymer Library and Con A Interactions. J. Polym. Sci., Part A-1: Polym. Chem. 2013, 51, 2588−2597. (20) Pellegrino, T.; Manna, L.; Kudera, S.; Liedl, T.; Koktysh, D.; Rogach, A. L.; Keller, S.; Rädler, J.; Natile, G.; Parak, W. J. Hydrophobic Nanocrystals Coated with an Amphiphilic Polymer Shell: A General Route to Water Soluble Nanocrystals. Nano Lett. 2004, 4, 703−707. (21) Lin, C.-A. J.; Sperling, R. A.; Li, J. K.; Yang, T.-Y.; Li, P.-Y.; Zanella, M.; Chang, W. H.; Parak, W. J. Design of an Amphiphilic Polymer for Nanoparticle Coating and Functionalization. Small 2008, 4, 334−341. (22) Pöselt, E.; Schmidtke, C.; Fischer, S.; Peldschus, K.; Salamon, J.; Kloust, H.; Tran, H.; Pietsch, A.; Heine, M.; Adam, G.; Schumacher, U.; Wagener, C.; Förster, S.; Weller, H. Tailor-Made Quantum Dot and Iron Oxide Based Contrast Agents for in Vitro and in Vivo Tumor Imaging. ACS Nano 2012, 6, 3346−3355. (23) Schmidtke, C.; Pöselt, E.; Ostermann, J.; Pietsch, A.; Kloust, H.; Tran, H.; Schotten, T.; Bastús, N. G.; Eggers, R.; Weller, H. Amphiphilic, Cross-Linkable Diblock Copolymers for Multifunctionalized Nanoparticles as Biological Probes. Nanoscale 2013, 5, 7433− 7444. (24) Talapin, D. V.; Mekis, I.; Gotzinger, S.; Kornowski, A.; Benson, O.; Weller, H. CdSe/CdS/ZnS and CdSe/ZnSe/ZnS Core−Shell− Shell Nanocrystals. J. Phys. Chem. B 2004, 108, 18826−18831. (25) Yu, W. W.; Falkner, J. C.; Yavuz, C. T.; Colvin, V. L. Synthesis of Monodisperse Iron Oxide Nanocrystals by Thermal Decomposition of Iron Carboxylate Salts. Chem. Commun. 2004, 20, 2306−2307. (26) Bastús, N. G.; Comenge, J.; Puntes, V. Kinetically Controlled Seeded Growth Synthesis of Citrate-Stabilized Gold Nanoparticles of up to 200 nm: Size Focusing versus Ostwald Ripening. Langmuir 2011, 27, 11098−11105. (27) Silva, D. J.; Wang, H.; Allanson, N. M.; Jain, R. K.; Sofia, M. J. Stereospecific Solution- and Solid-Phase Glycosylations. Synthesis of β-Linked Saccharides and Construction of Disaccharide Libraries Using Phenylsulfenyl 2-Deoxy-2-Trifluoroacetamido Glycopyranosides as Glycosyl Donors. J. Org. Chem. 1999, 64, 5926−5929. (28) Ran, C.; Pantazopoulos, P.; Medarova, Z.; Moore, A. Synthesis and Testing of Beta-Cell-Specific Streptozotocin-Derived Near-Infrared Imaging Probes. Angew. Chem., Int. Ed. 2007, 46, 8998−9001. (29) Bates, R. W.; Dewey, M. R. A Formal Synthesis of Swainsonine by Gold-Catalyzed Allene Cyclization. Org. Lett. 2009, 11, 3706−3708. (30) Zissis, E.; Stewart, L. C.; Richtmyer, N. K. The Formation of 2,7-Anhydro-β-D-manno-heptulopyranose by the Action of Acid on Dmanno-Heptulose and of Alkali on Phenyl α-D-manno-Heptulopyrano-

ASSOCIATED CONTENT

* Supporting Information S

(1) NMR and analytical data of the polymeric/organic compounds; (2) the physicochemical characterization of the glycoconjugated nanoparticles (absorption and emission spectra, TEM, and DLS); (3) and the SPR-sensograms. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the EU within the FP 7 program (Vibrant, EU 228933). We would like to thank Matthias Wulf for providing us the maltose derivative and Alexander Stark for valuable discussions (both from the Institute of Organic Chemistry, University of Hamburg). Dr. Sebastian Meinke is acknowledged for the help with the SPR measurements. Furthermore, we thank 2layers and in particular Frederik Schmidtke for the graphic design.

■

REFERENCES

(1) Varki, A. Biological Roles of Oligosaccharides: All of the Theories Are Correct. Glycobiology 1993, 3, 97−130. (2) Lis, H.; Sharon, N. Protein Glycosylation. Structural and Functional Aspects. Eur. J. Biochem. 1993, 218, 1−27. (3) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Polyvalent Interactions in Biological Systems: Implications for Design and Use of Multivalent Ligands and Inhibitors. Angew. Chem., Int. Ed. 1998, 37, 2754−2794. (4) Wang, X.; Ramström, O.; Yan, M. Glyconanomaterials: Synthesis, Characterization, and Ligand Presentation. Adv. Mater. 2010, 22, 1946−1953. (5) Lee, Y.; Lee, R. Carbohydrate-Protein Interactions: Basis of Glycobiology. Acc. Chem. Res. 1995, 28, 321−327. (6) Schumacher, U. A Critical Evaluation of Neoglycoprotein Binding Sites in Vivo and in Sections of Mouse Tissues. Histochemistry 1992, 97, 95−99. (7) Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Immunofluorescent Labeling of Cancer Marker Her2 and Other Cellular Targets with Semiconductor Quantum Dots. Nat. Biotechnol. 2003, 21, 4−46. (8) Burda, C.; Chen, X.; Narayanan, R.; El-Sayed, M. A. Chemistry and Properties of Nanocrystals of Different Shapes. Chem. Rev. 2005, 105, 1025−1102. (9) Hernáiz, M. J.; de la Fuente, J. M.; Barrientos, Á . G.; Penadés, S. A Model System Mimicking Glycosphingolipid Clusters to Quantify Carbohydrate Self-Interactions by Surface Plasmon Resonance. Angew. Chem., Int. Ed. 2002, 41, 1554−1557. (10) Huang, H.; Yuan, Q.; Yang, X. Preparation and Characterization of Metal−Chitosan Nanocomposites. Colloids Surf., B 2004, 39, 31− 37. (11) Moros, M.; Hernáez, B.; Garet, E.; Dias, J. T.; Sáez, B.; Grazú, V.; González-Fernández, A.; Alonso, C.; de la Fuente, J. M. Monosaccharides versus PEG-Functionalized NPs: Influence in the Cellular Uptake. ACS Nano 2012, 6, 1565−1577. (12) El-Boubbou, K.; Zhu, D. C.; Vasileiou, C.; Borhan, B.; Prosperi, D.; Li, W.; Huang, X. Magnetic Glyco-Nanoparticles: A Tool To Detect, Differentiate, and Unlock the Glyco-Codes of Cancer via Magnetic Resonance Imaging. J. Am. Chem. Soc. 2010, 132, 4490− 4499. G


Langmuir

Article

side; also 1,6-Anhydro-β-D-mannopyranose from D-Mannose in Acid Solution. J. Am. Chem. Soc. 1957, 79, 2593−2597. (31) Lee, J. W.; Jun, S. I.; Kim, K. An Efficient and Practical Method for the Synthesis of Mono-N-Protected α,ω-Diaminoalkanes. Tetrahedron Lett. 2001, 42, 2709−2711. (32) Waschke, D.; Thimm, J.; Thiem, J. Highly Efficient Synthesis of Ketoheptoses. Org. Lett. 2011, 13, 3628−3631. (33) Schmidtke, C.; Lange, H.; Tran, H.; Ostermann, J.; Kloust, H.; Bastús, N. G.; Merkl, J.-P.; Thomsen, C.; Weller, H. Radical Initiated Reactions on Biocompatible CdSe-Based Quantum Dots: Ligand Crosslinking, Crystal Annealing and Fluorescence Enhancement. J. Phys. Chem. C 2013, 117, 8570−8578. (34) Gu, F.; Zhang, L.; Teply, B. A.; Mann, N.; Wang, A.; RadovicMoreno, A. F.; Langer, R.; Farokhzad, O. C. Precise Engineering of Targeted Nanoparticles by Using Self-Assembled Biointegrated Block Copolymers. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2586−2591. (35) Huisgen, R.; Szeimies, G.; Möbius, L. 1.3-Dipolare Cycloadditionen, XXXII. Kinetik der Additionen organischer Azide an CCMehrfachbindungen. Chem. Ber. 1967, 100, 2494−2507. (36) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem., Int. Ed. 2001, 40, 2004−2021. (37) Webster, O. W. Living Polymerization Methods. Science 1991, 251, 887−893. (38) Karakoti, A. S.; Das, S.; Thevuthasan, S.; Seal, S. PEGylated Inorganic Nanoparticles. Angew. Chem., Int. Ed. 2011, 50, 1980−1994. (39) Bernardin, A.; Cazet, A.; Guyon, L.; Delannoy, P.; Vinet, F.; Bonnaffé, D.; Texier, I. Copper-Free Click Chemistry for Highly Luminescent Quantum Dot Conjugates: Application to in Vivo Metabolic Imaging. Bioconjugate Chem. 2010, 21, 583−588. (40) Weis, W. I.; Drickamer, K. Structural Basis of LectinCarbohydrate Recognition. Annu. Rev. Biochem. 1996, 65, 441−473. (41) Sigmundsson, K.; Másson, G.; Rice, R.; Beauchemin, N.; Ö brink, B. Determination of Active Concentrations and Association and Dissociation Rate Constants of Interacting Biomolecules: An Analytical Solution to the Theory for Kinetic and Mass Transport Limitations in Biosensor Technology and Its Experimental Verification. Biochemistry 2002, 41, 8263−8276. (42) Tassa, C.; Duffner, J. L.; Lewis, T. A.; Weissleder, R.; Schreiber, S. L.; Koehler, A. N.; Shaw, S. Y. Binding Affinity and Kinetic Analysis of Targeted Small Molecule-Modified Nanoparticles. Bioconjugate Chem. 2010, 21, 14−19.

H


Glycoconjugated Amphiphilic Polymers via Click-Chemistry for the

Recommend Documents