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Aug 6, 2012 - produce well-defined copolymers, CP1 and CP2. 1H NMR along with FT-IR spectroscopy characterization confirms the copolymer formation ...
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Norbornene-Derived Poly‑D‑lysine Copolymers as Quantum Dot Carriers for Neuron Growth Vijayakameswara Rao N,† Abhinoy Kishore,‡ Santu Sarkar,† Jayasri Das Sarma,*,‡ and Raja Shunmugam*,† †

Polymer Research Centre, Department of Chemical Sciences and ‡Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata (IISER K), India S Supporting Information *

ABSTRACT: Synthesis of norbornene derived phosphonate (1), poly-D-lysine (2), and phopholipid (3) monomers and their complete characterizations are studied. Ring-opening metathesis polymerizations (ROMP) of monomers (1−3) produce well-defined copolymers, CP1 and CP2. 1H NMR along with FT-IR spectroscopy characterization confirms the copolymer formation, while gel permeation chromatography (GPC) analysis suggests the formation of polymers with fairly narrow molecular weight distributions. Upon following the well-known ligand exchange methods these copolymers produce CdSe-bound copolymers, CP3 and CP4. Dynamic light scattering and transmission electron microscopy measures the size of these CdSe bound copolymers, while 31P NMR suggests the formation of CP3 and CP4. The results from the experiments of these copolymers on Neuro2A cells suggest that the novel PDL-anchored nanomaterial show their ability to polarize neuronal growth and differentiation.



INTRODUCTION The application of quantum dots (QDs)1 in biology needs to address the issue of toxicity induced by the presence of cadmium in them.2 The recent study shows that the toxicity of QDs can be reduced drastically if they are quoted with biocompatible molecules like polyethylene glycol (PEG) or poly-D-lysine (PDL).3 We are very specific here about PDL because of its cationic nature and unique adhesive property with biomolecules. Moreover, its biocompatibility with cell behavior shows its major effect in the epidermal growth factor of serum proteins. PDL simply enhances its adhesiveness with plastic and glass surfaces for many anchorage-dependent cells and specifically primary cell adhesion. As a linker PDL has several advantages4 in cell adhesion, biocompatibility, flexible molecular backbone, and good water solubility. So it will be ideal for cell imaging and isolation studies, if the PDL anchored nanomaterial is designed. Ring-opening metathesis polymerization (ROMP),5 assisted by various Grubbs’ catalysts, has attracted great attention. This is mainly due to the efficiency, control, and tolerability of the ruthenium catalysts to a variety of pendant functional groups. Consequently, the synthesis of well-defined oxanorbornene functionalized polymers and copolymers will find significant use in the generation of highly functional materials. ROMP of monomers substituted with a nonprotected amino group6 is observed to be unsuccessful with ruthenium catalyst due to the incompatibility of amines with ruthenium. So it has also been in practice that protection of amine group is necessary in the acyclic diene metathesis polycondensation of amines with ruthenium © 2012 American Chemical Society

catalyst. Very recently, it has been reported that a 7oxanorbornene substituted with dimethylamino groups (protected amino group) undergoes ROMP with the third generation Grubbs’ catalyst, but until now there are very few reports on ROMP of monomers with primary and secondary amino groups.6 Motivated by the cell binding prospects of PDL along with fascinating physical properties of nano materials; we investigate the possibility of the potential application of newly designed PDL functionalized matrix in the neuronal phenotype and neurites formation. N2A is a mouse neural crest-derived cell line that has been extensively used to study neuronal differentiation, axonal growth and signaling pathways. A characteristic advantage of these cells is their ability to differentiate into neurons within few days.7 However, most differentiation methods for N2A cells in the literature do not provide information about the neuronal types obtained after each treatment. Several studies are going on to evaluate the effect of number of growth factors and hormones to induce neuronal differentiation. This manuscript explores the possibility of using PDL-coated CdSe copolymers as scaffolds for neuronal growth and differentiation.8 In this article, we report a completely different approach to design and synthesis of QDs attached copolymers that have the potential application in the cell imaging and isolation studies. Received: June 24, 2012 Revised: August 4, 2012 Published: August 6, 2012 2933

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Figure 1. 1H NMR spectrum of (a) monomer 2 in MeOD; (b) phosphonate derived norbornene 1 in CDCl3; (c) exo-5-norbornene-2-carboxylic acid in DMSO-d6. dride, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-hydroxy succinimide (NHS), triphenylphosphine, dicyclohexylcarbodiimide (DCC), diisopropyl azodicarboxylate (DIAD), ethyl vinyl ether, trioctyl phosphine oxide (TOPO), poly(ethylene glycol) methyl ether, second generation Grubbs’ catalyst (G2), CDCl3, MeOD, and DMSO-D6 were purchased from Sigma Aldrich. 4-Dimethylamino pyridine (DMAP), triethylamine, and selenium powder were received from Oligo chemicals. Tetradecylphosphonic acid (TDPA), diethyl (hydroxyl methyl) phosphonate, and trioctyl phosphine were purchased from Alfa Aesar. Cadmium oxide (CdO) was purchased from Rankem Chemicals. Reagent-grade dichloromethane (DCM), methanol (MeOH), ethyl acetate, toluene, diethyl ether, N,N-dimethylformamide (DMF), and tetrahydrofuran (THF) were obtained from Oligo or Merck and were purified by vacuum distillation. Characterization. Gel Permeation Chromatography (GPC). Molecular weights and PDIs were measured by Waters gel permeation chromatography in THF relative to PMMA and PS standards on systems equipped with Waters Model 515 HPLC pump and Waters Model 2414 Refractive Index Detector at 35 °C with a flow rate of 1 mL/ min. HRMS analyses were performed with Q-TOF YA263 high resolution (Waters Corporation) instruments by +ve mode electrospray ionization.

Motivated by the cell binding prospects of PDL along with fascinating physical properties of QDs, this work investigates (i) synthesis of phosphonate (1), PDL (2), and phopholipid (3) derived norbornene monomers, (ii) polymerization of monomers, 1−3 by ring-opening metathesis polymerization (ROMP) to produce copolymers CP1 and CP2, and (iii) binding of copolymers with CdSe nanoparticles to produce copolymers CP3 and CP4. There is a strong growing interest in the field of research to functionalize nanomaterials with suitable cell substratum for making an appropriate cell matrix for imaging and isolation studies. In this article an effective approach is made to conceive a novel polymer that can have potential application in neuronal growth and differentiation. To best of our knowledge this is the first report on ROMP of PDL-derived norbornene monomers that containing more than one nonprotected amino group in each monomer.



EXPERIMENTAL SECTION

Materials. 5-Norborene-2-carboxylic acid (mixture of endo and exo isomers), poly-D-lysine hydrobromide [PDL; Mw = 512; Ln = 4 by MALDI, (Figure S1)], cis-5-norbornene-endo-2,3-dicarboxylic anhy2934

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Figure 2. 1H NMR spectrum of homopolymer from monomers 1 and 2, respectively; 1H NMR spectrum of (a) homopolymer 1 in CDCl3; (b) homopolymer 2 in CDCl3. Synthesis of (Diethoxyphosphoryl) methyl bicyclo[2.2.1]hept-5ene-2-carboxylate 1. 100 mg (0.72 mmol, 1equiv) of norbornene exo carboxylic acid, 201 mg (0.769 mmol, 1.0625 equiv) of triphenylphosphine and 129.3 mg (0.769 mmol, 1.0625 equiv) of diethyl(hydroxylmethyl)phosphonate were added to a properly dried 4 neck flask. Then, 5 mL of dry THF was added as solvent and the reaction mixture was cooled to 0−5 °C. 155.5 mg (0.769 mmol, 1.0625 equiv) of diisopropyl azodicarboxylate was added via syringe at 0−5 °C and the reaction mixture was stirred at room temperature for 15 h. This reaction mixture was concentrated to 3 mL and was dissolved in toluene (10 mL) to remove excess triphenylphosphine oxide. Then the mixture was kept for almost 8 h at −20 °C. The solid was filtered off and the filtrate was concentrated. Crude product was purified by column chromatography on silica. (70 mg, 70% yield) 1 H NMR (DMSO−D6, 400 MHz) (Figure 1b): δ 1.28−1.30 (t, 6H, J = 6.88 Hz, CH3), 1.81−1.84 (m, 2H), 2.24−2.25 (m, 1H), 2.934 (s, 1H), 3.067 (s, 1H), 4.155−4.177 (q, 4H), 4.351−4.428 (dd, 2H), 6.106− 6.146 (m, 2H). 13C NMR (CDCl3, 400 MHz) (Figure S4): 175.15, 138.35, 135.42, 62.73, 61.9, 48, 42.6, 46.3, 43, 29, 16.34. IR (KBr, cm−1): 3272, 2922, 2850, 1745, 1237, 1178, 1021, 965, 750. MS (ESI) calculated for C13H21O5P [M + NH4]+; 288.11, found 288.21. Synthesis of PDL derived norbornene 2. 50 mg (0.362 mmol, 1equiv) of exo norbornene carboxylic acid, 300 mg (0.583 mmol, 1.61 equiv) of PDL, and 100 mg (0.485 mmol, 1.34 equiv) of DCC were dissolved in 20 mL of DMF. The reaction mixture was stirred at 50 °C for 24 h under nitrogen flow. After filtering of the DCU solid, 5 mL of water, 25 mL of ethyl acetate were added and the mixture was stirred for 15 min at room temperature. The organic layer was separated and dried over MgSO4. Then the dried layer was concentrated under vacuum to yield compound 2 as free white color powder. (140 mg, 46% yield). [Mw = 682; by MALDI, (Figure S2)] 1 H NMR (MeOD, 400 MHz) (Figure 1a): δ 1.30−1.86 (m, 8H), 2.334−2.346 (m, 1H), 2.886−2.924 (m, 2H), 3.53−3.54 (m, 2H), 4.007−4.037 (m, 1H), 6.097−6.146 (m, 2H). 13C NMR (MeOD, 400 MHz) (Figure S5): 175.9, 155.9, 139.3, 137.1, 56.0, 50.0, 44.0, 42.0, 35.0, 33.2, 32.4, 32.0, 27.0, 26.6, 26.07. IR (KBr, cm−1): 3270, 3052, 2928, 2852, 2342, 1654, 1536, 1448, 1384, 1219, 1078, 892, 709.

Fluorometry. Fluorescence emission spectra were recorded on a Fluorescence spectrometer (Horiba Jobin Yvon, Fluoromax-3, Xe-150 W, 250−900 nm). Nuclear Magnetic Resonance (NMR). The 1H NMR spectroscopy was carried out on a Bruker 500 MHz spectrometer using CDCl3 as a solvent. 1H NMR spectra of solutions in CDCl3 were calibrated to tetramethylsilane as internal standard (δH 0.00). Fourier Transform Infra Red (FT-IR). FT-IR spectra were obtained on FT-IR Perkin-Elmer spectrometer at a nominal resolution of 2 cm−1. Ultra Violet (UV) Spectroscopy. UV−visible absorption measurements were carried out on U-4100 spectrophotometer HITACHI UV− vis spectrometer, with a scan rate of 500 nm/min. Dynamic Light Scattering (DLS). Particle size of QDs were measured by dynamic light scattering (DLS), using a Malvern Zetasizer Nano equipped with a 4.0 mW He−Ne laser operating at λ = 633 nm. All samples were measured in aqueous as well as methanol at room temperature and a scattering angle of 173°. Transmission Electron Microscopy (TEM). Low resolution transmission electron microscopy (TEM) was performed on a JEOL 200 CX microscope. TEM grids were purchased from Ted Pella, Inc. and consisted of 3−4 nm amorphous carbon film supported on a 400-mesh copper grid. MALDI analysis. The sample was mixed with alpha cyano-4-hydroxy cinnamic acid (CHCA, 5 mg/mL in 50/50 acetonitrile, 0.1% TFA) and 1 μL spotted onto a standard 96 well MALDI target. The sample was analyzed using a MALDI SYNAPT G2 HDMS mass spectrometer. Red phosphorus was used to calibrate. Isolation of 5-Norbornene-2-exocarboxylic acid. Twenty-five g of Exo-5-norbornene-2-carboxylic acid was separated from the commercially available mixture of endo and exo 5-norbornene-2-carboxylic acid by the iodolactonization method of Ver Nooy and Rondestvedt.9 (5 g, 20% yield) 1H NMR (DMSO−D6, 400 MHz) (Figure 1c): δ 1.13−1.17 (m, 2H), 1.28−1.29 (d, J = 8.5 Hz, 1H), 1.66−1.71 (m, 1H), 1.97−2.05 (dt, J = 12.7 Hz, 1H), 2.76 (s, 1H), 2.9 (s, 1H), 6.03−6.05 (m, 2H), 12.00 (br, 1H). 13C NMR (CDCl3, 500 MHz) (Figure S3): 182.7, 138.1, 135.7, 46.7, 46.4, 43.2, 41.7, 30.3. IR (KBr, cm−1): 2919, 2852, 1700, 1421, 1218, 909, 766. MS (ESI) calculated for C8H10O2Na [M + H]+, 138.07; observed 138.09. 2935

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Synthesis of Phospholipid derived norbornene 3.10 1.75 g (0.81 mmol, 1 equiv) of PEG-Norbornene carboxylic acid and 0.14 g (1.21 mmol, 1.5 equiv) of N-hydroxysuccinimide were dissolved in dry DCM in a round-bottom flask. 0.33 g (1.62 mmol, 2 equiv) of N,N′dicyclohexylcarbodiimide (DCC) was added into the reaction mixture and stirred for 5 h at room temperature. Solid portion of the reaction mixture was filtered. The filtrate was treated thrice with diethyl ether and white solid was obtained after evaporating under reduced pressure. Without further purification, the activated carboxylic acid (180 mg, 1 equiv) was used for the next step. 55 mg solution of (0.08 mmol, 1 equiv) of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine in 20 mL of dry DCM was prepared in a flask. To this, triethylamine (0.1 mL, 10 equiv) was added to it, followed by the previously synthesized activated carboxylic ester (180 mg, 1 equiv), and 4-di(methylamino) pyridine (DMAP) (2 mg, cat). The reaction mass was concentrated under vacuum and the product was isolated by precipitation using excess diethyl ether. Crude product was purified by column chromatography on Silica (CH2Cl2/MeOH: 92/8) obtained as a white solid (90 mg, 50% yield). 1 H NMR (CDCl3, 400 MHz): δ 0.86 (t, J = 6.8 Hz, 6H), 1.24 (m, 24H), 1.57−1.9 (m, 4H), 2.28 (m, 4H), 3.07 (s, 3H), 3.30 (s, 3H, OCH3), 3.60−3.65 (H-PEG), 3.9−4.2 (m, 2H), 4.1−4.2 (m, 2H), 4.2− 4.4 (m, 2H), 5.22 (m, 1H), 6.13−6.69 (m, 2H). 13C NMR (CDCl3, 400 MHz): 173.40, 172.97, 172.32, 135.72, 133.76, 70.20, 70.07, 63.48, 62.34, 58.99, 48.61, 47.17, 44.73, 31.89, 29.33, 14.10. IR (KBr, cm−1): 3431, 2901, 1715, 1464, 1342, 1212, 1101, 944, 840. Homopolymerization of phosphonate derived norbornene 1. 10 mg (0.0347 mmol) of monomer 1 was added into a separate Schlenk flask under an atmosphere of nitrogen, and dissolved in 1 mL of dry DCM. Into another Schlenk flask, a desired amount of second generation Grubbs’ catalyst 1.4 mg (G2, 20 mol %) was added, flushed with nitrogen, dissolved in minimum (0.5 mL) anhydrous dichloromethane. All two flasks were degassed three times by freeze−pump− thaw cycles. Monomer 1 was transferred to the flask containing the catalyst via a cannula. The reaction was allowed to stir at room temperature until the polymerization was complete (50 min) before it was quenched with vinyl ethyl ether (0.5 mL). An aliquot was taken for GPC analysis, and the remaining product was precipitated with diethyl ether, dissolved it again THF, passed it through neutral alumina to remove the catalyst and precipitated again with diethyl ether to get pure polymer (8 mg, 80% yield). Gel permeation chromatography (GPC) was done in tetrahydrofuran (flow rate =1 mL/1 min). The molecular weight of homopolymer 1, was measured using polystyrene standards. Mn = 18,000 and PDI = 1.2 suggested the homopolymerization of 1 (Figure S11a). 1H NMR (CDCl3, 400 MHz,) (Figure 2a): δ 1.2−1.306 (m, protons of y), 1.67−1.96, (m, c, e), 2.44−2.68 (m, protons of d), 2.93−3.11 (m, protons of bb′), 3.89−4.29 (m, protons of x), 4.28−4.39 (m, protons of z), 5.0−5.4 (m, protons of a, new olefinic protons). Homopolymerization of PDL derived norbornene 2. 20 mg (0.0070 mmol) of macromonomer 2 was weighed into a separate Schlenk flask, placed under an atmosphere of nitrogen, and dissolved in 3 mL of dry DCM. Into another Schlenk flask, a desired amount of second generation Grubbs’ catalyst 1.13 mg (G2, 5 mol %) was added, flushed with nitrogen, dissolved in minimum (1 mL) anhydrous dichloromethane. All two flasks were degassed three times by freeze−pump− thaw cycles. The Monomer 2 was transferred to the flask containing the catalyst via a cannula. The reaction was allowed to stir at room temperature until the polymerization was complete (3 h) before it was quenched with vinyl ethyl ether (1 mL). An aliquot was taken for GPC analysis, and the remaining product was precipitated with diethyl ether, dissolved it again THF, passed it through neutral alumina to remove the catalyst, and precipitated again with diethyl ether to get pure polymer (12 mg, 60% yield). Gel permeation chromatography (GPC) was done in tetrahydrofuran (flow rate = 1 mL/1 min). The molecular weight of homopolymer 2, was measured using polystyrene standards. The observed Mn = 17000 and PDI = 1.4 suggested the polymerization of 2 (Figure S11b). 1H NMR (CDCl3, 400 MHz; Figure 2b): δ 1.29−1.90, (m, protons of PDL, i, h, j, e, s), 2.50−2.87 (m, protons of c and d′), 2.93−3.45 (m, protons of k, b, d, d′), 3.2−3.3 (m, protons of b), 3.4−3.5

Figure 3. 1H NMR spectrum of (a) monomer 2 in MeOD; (b) phosphonate derived norbornene 1 in CDCl3; (c) 1H NMR spectrum of CP1 in CDCl3. Synthesis of PEG-Norbornene Carboxylic acid. 1 g (Mn = 2000, 0.5 mmol, 1 equiv) of poly(ethylene glycol) methyl ether was dissolved in 5 mL of dry DCM. 0.27 mL (4 equiv) of dry triethylamine was added via syringe followed by 0.165 g (1 mmol, 2 equiv) of 5-norbornene-endo2,3 dicarboxylic anhydride. The reaction mixture was stirred at room temperature for 15 h. Then 0.5 N HCl (10 mL) was added into the reaction mixture and pH was adjusted to 7. Organic layer was treated with 20 mL of water, then dried over MgSO4 and the layer was concentrated under vacuum. The product was isolated by precipitation using excess diethyl ether as white color solid. (0.9 g, 90% yield). 1 H NMR (CDCl3, 500 MHz) (Figure S6): δ 1.2−1.5 (m, 2H), 3.14 (m, 2H), 3.33 (s, 2H), 3.37 (s, 3H), 3.45−3.82 (H-PEG), 3.98 (m, 1H), 6.0−6.4 (m, 2H). 13C NMR (CDCl3, 400 MHz) (Figure S7): 174.0, 173.70, 172.23, 135.20, 134.73, 134.27, 70.40, 70.60, 69.91, 63.48, 58.99, 48.61, 47.99, 46.17. IR (KBr, cm−1): 3545, 2860, 1954, 1739, 1464, 1343, 1255, 1094, 943, 841. 2936

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Figure 4. (a) 1H NMR spectrum of CP2 in CDCl3; (b) list of molecular weights and PDIs of polymers. (m, protons of g), 3.8 (m, protons of k), 4.17 (m, protons of g), 5.11− 5.29 (m, protons of a, new olefinic protons). Copolymerization of Monomers 1 and 2. Known amounts of monomers 1 (10 mg, 0.03472 mmol, 1 equiv) and 2 (25 mg, 0.03472 mmol, 1 equiv) were weighed into two separate Schlenk flasks, placed under an atmosphere of nitrogen, and dissolved in 1 mL of dry DCM. Into another Schlenk flask, a desired amount of second generation Grubbs’ catalyst 1.4 mg (G2, 20 mol %) was added, flushed with nitrogen, and dissolved in a minimum amount of (1 mL) anhydrous dichloromethane. All three flasks were degassed three times by freeze− pump−thaw cycles. Monomers 1 and 2 were transferred to the flask containing the catalyst via a cannula. The reaction was allowed to stirr at room temperature until the polymerization was complete (50 min) before it was quenched with vinyl ethyl ether (1 mL). An aliquot was taken for GPC analysis, and the remaining product was precipitated with diethyl ether, dissolved again in THF, passed through neutral alumina to remove the catalyst, and precipitated again with diethyl ether to get pure polymer (18 mg, 72% yield). Gel permeation chromatography (GPC) was done in tetrahydrofuran (flow rate =1 mL/1 min). The molecular weight of CP1 was measured using polystyrene standards. Mn = 14000 and PDI = 1.38 suggested the polymerization of CP1 (Figure S13). 1H NMR (CDCl3, 400 MHz; Figure 3c): δ 1.2−1.306 (m, protons of p), 1.58−1.90, (m, protons of PDL, i, h, j and c, e), 2.3−2.7 (m, protons of b′), 2.93−3.09 (m, protons of dd′), 3.2−3.3 (m, protons of b), 3.4−3.5 (m, protons of g), 3.8 (m, protons of k), 4.0−4.13 (q, protons of I), 4.28−4.39 (m, protons of r), 5.0−5.4 (m, protons of m, new olefinic protons). Copolymerization of Monomers 2 and 3. Known amounts of monomers 2 (10 mg, 0.0146 mmol, 1 equiv) and 3 (25 mg, 0.0879 mmol, 6 equiv) were weighed into two separate Schlenk flasks, placed under an atmosphere of nitrogen, and dissolved in 1 mL of dry DCM. Into another Schlenk flask, a desired amount of second generation Grubbs’ catalyst 1.5 mg (G2, 20 mol %) was added, flushed with

nitrogen, and dissolved in a minimum amount of (1 mL) anhydrous dichloromethane. All three flasks were degassed three times by freeze− pump−thaw cycles. The monomers 2 and 3 were transferred to the flask containing the catalyst via a cannula. The reaction was allowed to reflux until the polymerization was complete (24 h) before it was quenched with vinyl ethyl ether (1 mL). An aliquot was taken for GPC analysis, and the remaining product was precipitated with diethyl ether, dissolved again in THF, passed through a neutral alumina to remove the catalyst, and precipitated again with diethyl ether to get pure polymer CP2 (16 mg, 64% yield). Gel permeation chromatography (GPC) was done in tetrahydrofuran (flow rate = 1 mL/1 min). The molecular weight of CP2 was measured using polystyrene standards. The observed Mn = 9279 and PDI = 1.70 of the polymer suggested the polymerization of CP2. 1 H NMR (CDCl3, 400 MHz; Figure 4a): δ 0.849−0.855 (t, J = 6.8 Hz, z protons), 1.24 (m, H-Alk), 1.59−1.95 (m, protons of q, r, s, b, j, w), 2.221−2.325 (m, x protons), 2.765−3.240 (protons of g, e, e′), 3.37 (s, OCH3), 3.4−3.810 (H-PEG), 4.133−4.1624 (m, protons of t, n, h, v), 5.121 (m, protons of m), 5.295−5.339 (m, new olefinic protons). CdSe Nanoparticles Attachment to Copolymer CP1. CdSe nanoparticles were prepared using trioctylphosphine oxide (TOPO) as stabilizing agent.12 Quantum dots in hexane were precipitated in methanol and dissolved in 2 mL of dry DCM. Copolymer CP1 was added to it and stirred overnight at room temperature.12 DCM from the reaction mixture was evaporated and hexane was added to precipitate CP3. Then the supernatant was discarded to remove unreacted TOPO capped CdSe nanoparticles. This process was repeated twice to produce CdSe nanoparticles attached to copolymer CP3. A similar procedure was followed to attach CdSe nanoparticles to copolymer CP2 to get the CdSe attached copolymer CP4. Procedure for the Neuron Growth. Neuro 2A (N2A), a mouse neuroblastoma cell line, was obtained from ATCC and maintained in MEM with 10% FBS, 1% penicillin (10000 units/mL), and streptomycin (10000 μg/mL) at 37 °C in CO2 incubator for 2−5 passages. Cells were 2937

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placed on poly-D-lysine coated coverslips and maintained for confluency. Nonneuronal cells were eliminated from the confluent monolayer cells by adding a mixture of fluorodeoxyuridine and uridine containing medium (10 μM). After 24 h, fluorodeoxyuridine and uridine containing medium were replaced with fresh N2A specified medium with 5 μg of CP1 to CP4 and cells were incubated for 24, 48, and 72 h. The cells were then washed with fresh medium and visualized by fluorescence microscopy using an Olympus IX-81 microscope system with a 40× UPlanApo objective (1.0 numerical aperture). Images were acquired with a Hamamatsu Orca-1 CCD camera and analyzed by Image ProPLus image analysis software (Media Cybernetics, Silver Spring, MD). Cytotoxicity assay (MTT assay): For MTT assay N2A cells were placed on 96-well plates coated with poly-D-lysine and maintained until confluency. At confluency cells were treated with fluorodeoxyuridine and uridine containing medium (10 μM) for 24 h to eliminate the nonneuronal cells, as described earlier, followed by media change with fresh N2A medium containing four different doses (5, 10, 50, and 100 μg) of CP1 to CP4 and incubated for either 24 or 48 h and processed for MTT assay, as described previously.13

was observed at 12 ppm (br, 1H), as shown in Figure 1c. The signals at 6.02−6.06 ppm (m, 2H) were corresponding to norbornene olefinic protons while the signals at 1.97−2.045 ppm (dt, J = 12.7 Hz, 1H) and 1.66−1.71 (m, 1H) were due to norbornene bridged hydrogens. From the FT-IR analysis, the stretching mode at 1700 cm−1 was due to carboxylic functionality of exonorbornene carboxylic acid, as shown in Figure S9b. Monomer 1 was prepared by Mitsunobu coupling reaction as shown in the Scheme 1.11 The alcohol derivative, diethyl (hydroxymethyl)phosphonate, was treated with exonorbornene carboxylic acid (Figure 1c) in the presence of diisopropylazodicarboxylate (DIAD) and triphenylphosphine in THF to produce the monomer 1. Then it was characterized by 1H NMR (Figure 1b) and FT-IR spectroscopy (Figure S8c). From the 1H NMR analysis, the signal due to carboxylic functionality at 12 ppm was absent; because it was evident that exo-norbornene carboxylic acid completely reacted with diethyl hydroxymethyl phosphonate. It was further confirmed by FT-IR spectroscopy. The stretching mode of carboxylic functionality of exonorbonene carboxylic acid at 1700 cm−1 (Figure S8a), which was shifted to 1745 cm−1 in Figure S8c, confirmed the formation of new ester bond. Monomer 2 was prepared by acid-amine coupling of norbornene exo carboxylic acid and poly-D-lysine by using DCC as coupling reagent in DMF. The attachment was confirmed by FT-IR (Figure S9c) and 1H NMR spectroscopy (Figure 1a). The FT-IR spectrum confirmed (Figure 3c) the shift in the norbornene carboxylic acid stretching mode from 1700 to 1654 cm−1 due to the amide bond. Molecular weight of this macromonomer (Mw = 682) was obtained from MALDI analysis (Figure S2). Synthesis of monomer 3 was done by following the procedure reported in the literature. Poly(ethylene glycol) methyl ether (Mn = 2000) was treated with cis-5-norbornene-endo-2,3dicarboxylic anhydride by using triethyl amine as a base to get 3. 1H and 13C NMR spectroscopy confirmed the formation of the product. The signals at 3.65−4.3 ppm were due to the PEG (HPEG), while the signals at 3.37 (3H) were for the methyl group of poly(ethylene glycol) methyl ether. The signals at 1.2 ppm (d, 1H) and 1.5 ppm (d, 1H) were responsible for the norbornene bridged protons. The signals at 6.0−6.4 ppm (m, 2H) were corresponding to norbornene olefinic protons. The resulting acid was activated by N-hydroxy succinimide and DCC. The activated ester was further made to react with 1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine. The covalent attachment of norbornene carboxylic acid and phospholipid was further confirmed by FTIR analysis where the shift in the carboxylic acid stretch mode was observed from 1700 to 1654 cm−1 (amide bond; Figure S10c). Homopolymerization of Monomers. Homopolymerization of monomer 1 was carried out by using second generation Grubbs’ catalyst (50 mol %) at room temperature in dry DCM solvent and was monitored by 1H NMR spectroscopy. New signals were observed at 5.0−5.4 ppm (Figure 2a) and norbornene olefinic protons were disappeared at 6.10−6.14 ppm, indicating the formation of the product. The molecular weight of homopolymer 1, was measured using polystyrene standards. The observed Mn = 18000 and PDI = 1.2 from the GPC analysis suggested the polymerization of 1 (Figure S11a). Homopolymerization of monomer 2 was carried out by using second generation Grubbs’ catalyst (15 mol %) at room temperature in dry DCM solvent and was monitored by 1H NMR. New signals were observed at 5.11−5.29 ppm (Figure 2b). The observed Mn = 17000 and PDI = 1.4 from the GPC analysis suggested the polymerization of 2 (Figure S11b).



RESULTS AND DISCUSSIONS Monomer Synthesis. Exo-5-norbornene-2-carboxylic acid was separated from the commercially available mixture of endoand exo-5-norbornene-2-carboxylic acid by the iodolactonization methods of Ver Nooy and Rondestvedt.9 Carboxylic acid peak Scheme 1. Schematic Representation of the Synthesis of Monomers (1 and 2) and Copolymers CP1 and CP3

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Figure 5. 31P NMR spectrum (decoupled) of (a) TOPO capped CdSe; (b) CdSe quantum dot attached copolymer (CP3) in CDCl3. All the 31P NMR measurements were performed in chloroform-d for the same period of time. Inset: Picture of red emission of CP3 in THF under the hand-held UV light.

Copolymerization. Copolymerization of monomers 1 and 2 was carried out by second generation Grubbs’ catalyst (20 mol %) at room temperature in dry DCM solvent and was monitored by 1H NMR spectroscopy. New signals were observed at 5.0−5.3 ppm and norbornene olefinic protons were disappeared at 6.0− 6.3 ppm, as shown in (Figure 3c). Gel permeation chromatography (GPC) was done in tetrahydrofuran (flow rate = 1 mL/1 min). The molecular weight of CP1, was measured using polystyrene standards. The observed Mn = 14000 and PDI = 1.38 of the polymer suggested the polymerization of CP1. Different molecular weights with fairly narrow PDIs were obtained (Figures S12 and S13) by varying the ratio of the monomer to catalyst (100 and 125 mol %). Copolymerization of monomers 2 and 3 was carried out by second generation Grubbs’ catalyst (20 mol %) in dry DCM solvent. The whole reaction was monitored by 1H NMR spectroscopy. New signals were observed at 5.0−5.3 ppm and norbornene olefinic protons were disappeared at 6.0−6.3 ppm, as shown in Figure 4a, which indicated the formation of the product. Gel permeation chromatography (GPC) was done in tetrahydrofuran (flow rate = 1 mL/1 min). The molecular weight of CP2 was measured using polystyrene standards. The observed Mn = 9500 and PDI = 1.70 of the polymer suggested the polymerization of CP2 (Figure 4b). It was expected to observe relatively broader PDIs for these monomers due to highly polar nature. It is a well-known fact among the researchers working on

bioactive polymers that, with certain types of polymers, the targeted molecular weight has never been achieved.5i,j QD Incorporation. Monomers 1 and 3 were designed to incorporate the QDs. Copolymers CP1 and CP2, upon treatment with TOPO-capped QDs, produced CP3 and CP4, respectively. The CP1 was sparingly soluble in water. Hence, the possibility of using PEG along with PDL was explored so that the presence of little amount of PEG would increase the solubility of the system. The PDL not only would reduce the toxicity of the CdSe by surface coating but also help the cell binding. Because of this, especially the monomer 3 was produced in addition to monomer 1. TOPO-capped CdSe nanoparticles were prepared12 and purified by precipitating in methanol to remove unreacted parts. TOPO covered CdSe nanoparticles were characterized by UV and fluorescence spectroscopy (see Supporting Information, Figures S14 and S15). The absorbance peak at 540 nm and emission at 560 nm were observed as mentioned in the reported procedure. Average diameter of CdSe nanoparticles was around 8 nm as measured by DLS (Figure S16). Replacement of TOPO from the surface of CdSe nanoparticles was done by copolymer CP1 and CP2 separately by established procedure.14 Copolymer CP1 and freshly prepared CdSe nanoparticles were dissolved in dry DCM and stirred for overnight at room temperature under inert atmosphere. DCM from the reaction mixture was made to be evaporated and the product was treated with hexane to 2939

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Scheme 2. Schematic Representation of Synthesis of Copolymers CP2 and CP4

Figure 7. Determination of CMC of CP3 in water.

attached copolymer CP3. 1H and 31P NMR spectroscopy suggested the formation of CP3. From the 1H NMR spectroscopy (Figure S17) of CP3, it was learned that all the peaks of CP1 were retained during the process of attaching the QDs. 31P NMR spectroscopy (decoupled spectrum) confirmed the formation of CP3 as it showed a broad signal at 59 ppm that was from phosphonate ester (Figure 5b). The broadening of the 31P NMR signal was probably due to the inhomogeneous distribution of magnetic environments.15 Because the experiment was monitored in the NMR tube, the observed signals at 63 and 90.3 ppm were responsible for the unreacted CP3 and CdSe-capped TOPO, respectively.15 The P-31 NMR peak for free TOPO appeared around 49−50.15d The ligand exchanged copolymers were purified to remove excess TOPO using the usual procedure of precipitation by addition of methanol then it was again dispersed in hexane. It was necessary to repeat this procedure three times to ensure that free phosphine ligands were completely removed. Similar procedure was followed to attach CdSe nanoparticles to the copolymer CP2 to get the CdSeattached copolymer CP4 (Scheme 2). Polymer-coated QDs

remove unreacted TOPO capped CdSe nanoparticles. This process was repeated twice to produce CdSe nanoparticles

Figure 6. TEM images of CdSe attached copolymer, CP3, in methanol as well as THF drop-casted on a 400 mesh carbon-coated copper grid. Scale bar = 50 nm. Below: Cartoon representation of behavior of CP3 in methanol and THF, as observed in TEM images. 2940

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(CMC) to confirm that the observed aggregates were micelles. The CMC was measured by using pyrene as an extrinsic probe.17 Pyrene (4 μg) was dissolved in water. Several samples were prepared with different concentration of CP3. The CP3 was dissolved in 5 mL of water. The fluorescence intensity of prepared solutions was measured by spectrofluorometer. The concentration of pyrene was maintained at 0.2 μM and final concentration of CP3 was changed from 10 to 1600 μg/mL, with the excitation wavelength set at 339 nm; the emission intensity was determined at 371, 382, and 396 nm. The relative emission fluorescence intensity of 396/371 nm was varied as a function of CP3 concentration (Figure 7). The CMC was determined by taking copolymer concentration value at which the relative fluorescence intensity ratio began to change. The observed CMC was 0.79 μg/mL. Dynamic light scattering (DLS) analysis was performed on the solution that was used to measure the CMC. The size of the micelles was measured as 38 nm with 0.29 PDI (Figure S18). Neuronal Phenotype and Neurites Formation. Cytotoxicity assay by MTT revealed that all the compounds (CdSe, CP1, CP2, CP3, and CP4) were toxic to the N2A cells at higher dose (more than 5 μg; Figure 8). Based on this observation, all our microscopic studies were done in 5 μg concentration. Interestingly CP1−CP4 showed their ability to polarize neuronal growth and differentiation within this concentration. N2A cells were monitored for its neurite growth formation and differentiation for every 24 h up to 72 h. In the experimental setup, THF and CdSe nanoparticles were used as negative control, while the copolymers (CP1−CP4) were used as experimental samples. Phase images were obtained in Olympus IX81 with Hamamatsu cool CCD camera and the neurite formation was measured by using Image ProPlus software. Our microscopic observations demonstrated that, in the case of both THF and CdSe, the neurite growth was very minimal even after 72 h (Figure 9). It was observed that the neurite differentiation and process formation (length wise) was varied from CP1 to CP4. In cases of CP1 and CP3 treated culture, larger neuronal processes were observed after 24 h of treatment in comparison to CdSe-treated culture. Because CP2 and CP4 were functionalized with biocompatible PEG molecule along with PDL, a larger neurite formation was observed in comparison to the neurite formed from the CP1 and CP3 treated cultures. It was interesting to note that the free CdSe-treated culture was unable to produce the neurite formation and differentiation. But when CdSe was attached with the copolymers CP3 and CP4, it demonstrated the ability to polarize the neuronal growth and differentiation. In summary, among the molecules tested for the neuronal growth (including the control molecules), it was found that CP4 significantly enhanced the formation of neuritis (Figure 10). Taken together, this study provided a simple and reliable method to generate differentiated and polarized neurons, which could be readily used for rapid and efficient physiological and pharmacological assays.

Figure 8. Plot of % survival vs concentration (a) for 24 h and (b) for 48 h.

(CP3 and CP4) showed almost the same fluorescence intensity compared to TOPO-capped CdSe nanoparticles as shown in the Figure 5 (inset). The water solubility of the CP4 would open the door to many experiments in cell imaging and isolation studies. The CP3 was also characterized by two independent sizemeasurement methods. Transmission electron microscopy was used to measure the diameter of CP3 in THF, which, as expected, increased from 8 to 12 nm with the copolymer attachment (Figure 6). Results from dynamic light scattering were highly correlated with respect to hydrodynamic diameter after addition of the polymer coating. Interestingly, when the solvent was changed from THF to methanol, the Rh increased dramatically (to 35 nm). Here, the formation of aggregates could be the cause of this dramatic increase in Rh. The TEM images (Figure 6) also showed clusters of micelles, but it was difficult to tell if the clusters were present in solution or were formed while evaporation of the solvent. From size analysis of these TEM images, the mean diameter of the individual aggregates was 30 ± 2 nm. It was very interesting to observe the aggregation due to self-assembly16 of these copolymers by just changing the solvent polarity. Despite the random nature of CP3, the proposed cartoon structure had shown the PDL functionality in the peripheral due to its water-soluble nature. The exact origin and detailed investigation on these assemblies by changing the polarity would be the future report. Determination of Critical Micelle Concentration of CP3. Formation of the individual aggregates in the polar solvent prompted us to measure the critical micelle concentration



CONCLUSION Synthesis and complete characterization of novel monomers that contained phosphonate, PDL and phospholipid (1−3) were discussed in detail. ROMP of monomers 1−3 produced welldefined homopolymers and copolymers (CP1−CP4). 1H NMR, IR, MALDI, and GPC studies confirmed the polymer architecture and its molecular weights. CdSe quantum dot synthesis and its successful attachment to the newly designed copolymers were also clearly exhibited. It was very 2941

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Figure 9. CLFM phase images of CdSe (a-i−iii), control (b-i−iii), CP1 (c-i−iii), CP2 (d-i−iii), CP3 (e-i−iii), and CP4 (f-i−iii) are shown for the neurites growth after 24, 48, and 72 h; (g) plot of total neurite length per neuron after 24, 48, and 72 h. 2942

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Figure 10. A cartoon representation of neuronal growth on a self-assembled CP3. W. C. W. Small 2010, 6, 138−144. (h) Sudeep, P. K.; Early, K. T.; McCarthy, K. D.; Odoi, M. Y.; Barnes, M. D.; Emrick, T. J. Am. Chem. Soc. 2008, 130 (8), 2384−2385. (2) (a) Daerfus, A. M.; Chaan, W. C. W.; Bhatia, S. N. Nano Lett. 2004, 4 (1), 11−18. (b) Kirchner, C.; Liedl, T.; Kudera, S.; Pellegrino, T.; Javier, A. M.; Gaub, H. E.; Stölzle, S.; Fertig, N.; Parak, W. J. Nano Lett. 2005, 5 (2), 331−338. (3) Liu, W.; Howarth, M.; Greytak, A. B.; Zheng, Y.; Nocera, D. G.; Ting, A. Y.; Bawendi, M. G. J. Am. Chem. Soc. 2008, 130 (4), 1274. (4) Calvert, P. Nature 1999, 399, 210−211. (b) Murthy, V. S.; Cha, J. N.; Stucky, G. D.; Wrong, M. S. J. Am. Chem. Soc. 2004, 126 (16), 5292− 5299. (5) Grubbs, R. H. Handbook of Metathesis; Wiley-VCH: New York, 2003; 3. (a) Watson, K. J.; Park, S. J.; Lm, J. H.; Nguyen, S. T. Macromolecules 2001, 34 (11), 3507−3509. (b) Mortell, K. H.; Gingras, M.; Kiessling, L. L. J. Am. Chem. Soc. 1994, 116 (26), 12053−12054. (c) Maynard, H. D.; Sheldon, Y. O.; Grubbs, R. H. Macromolecules 2000, 33 (17), 6239−6248. (d) Pollino, J. M.; Stubbs, L. P.; Weck, M. Macromolecules 2003, 36 (7), 2230−2234. (e) Schubert, U. S.; Eschbaumer, C. Angew. Chem., Int. Ed. 2002, 41 (16), 2892−2926. (f) Meyer, E.; Castellano, R. K.; Diederich, F. Angew. Chem., Int. Ed. 2003, 42 (11), 1210−1250. (g) Bergbreiter, D. E. Angew. Chem., Int. Ed. 1999, 38 (19), 2870−2872. (h) Mane, S. R.; Rao, V. N.; Shunmugam, R. ACS Macro Lett. 2012, 1, 482−488. (i) Lienkamp, K.; Madkour, A. E.; Musante, A.; Nelson, C. F.; Nusslein, K.; Tew, G. N. J. Am. Chem. Soc. 2008, 130 (30), 9836−9843. (j) Liu, Y.; Victor, P, III.; Marcus, W. Polym. Chem. 2011, 2, 1964−1975. (6) Sutthira, S.; Masashi, S.; Hideki, M.; Toshio, M.; Fumio, S. Macromolecules 2010, 43 (4), 1815−1822. (b) Sterling, F. A.; Lienkamp, K.; Madkour, A. E.; Tew, G. N. J. Polym. Sci., Part A: Polym. Chem. 2008, 46 (19), 6672−6676. (7) Hui, H.; Yingchun, N.; Swadhin, K. M.; Vedrana, M.; Bin, Z.; Haddon, R. C.; Parpura, V. J. Phys. Chem. B 2005, 109, 4285−4289. (8) Edward, J.; Nicholas, A. K. Nano Lett. 2007, 7 (5), 1123−1128. (9) Ver Nooy, C. C.; Rondestvedt, C. S. J. Am. Chem. Soc. 1955, 77 (13), 3583−3586. (10) Travert-Branger, N.; Dubois, F.; Carion, O.; Carrot, G.; Mahler, B.; Dubertret, B.; Doris, E.; Mioskowski, C. Langmuir 2008, 24 (7), 3016−3019. (11) Eren, T.; Tew, G. N. J. Polym. Sci., Part A: Polym. Chem. 2009, 47 (15), 3949−3956. (12) Peng, Z. A.; Peng, X. J. Am. Chem. Soc. 2001, 123 (1), 183−184. (13) Rao, V. N.; Mane, S. R.; Kishore, A.; Das Sarma, J.; Shunmugam, R. Biomacromolecules 2012, 13, 221−230.

interesting to observe the aggregated structures of the QD attached copolymers influenced by the solvent polarity. The detailed experiments on N2A cells with the newly designed copolymers suggested the ability to polarize neuronal growth and differentiation. Going forward, the ability to synthesize and organize functionalized QDs with biologically interacting molecules presents new opportunities in self-directed selfassembly that might be exploited in cell isolation, imaging, biosensors, and other applications.



ASSOCIATED CONTENT

S Supporting Information *

Additional analytical data, synthetic scheme, procedures, and references. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Phone: 9748897367. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS V.N. and A.K. thank IISER-Kolkata for the research fellowship. S.S. thanks CSIR, New Delhi, for research fellowship. R.S. thanks Department of Science and Technology, New Delhi, for Ramanujan Fellowship. R.S. and J.D.S. thank IISER-Kolkata for providing the infrastructure and start up funding. J.D.S. thanks CSIR, India, and DBT, India, for the funding. All the authors thank Dr. M. Jayakannan for the MALDI characterization.



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