imidazole based biocompatible polymer coating in deriving

9
Imidazole Based Biocompatible Polymer Coating in Deriving <25 nm Functional Nanoparticle Probe for Cellular Imaging and Detection Nikhil R. Jana,* ,† Pranab K. Patra, § Arindam Saha, SK Basiruddin, and Narayan Pradhan †,‡ Centre for AdVanced Materials and Department of Materials Science, Indian Association for the CultiVation of Science, Kolkata 700032, India, and Jubilant Chemsys Ltd., B-34, Sector 58, Noida 201301, India ReceiVed: June 17, 2009; ReVised Manuscript ReceiVed: NoVember 10, 2009 Although synthetic methods for high quality near-monodispersed nanoparticles of metal and semiconductor are well established, their conversion into various functional nanoparticles is challenging. We report here an imidazole based polymer coating that can be used in deriving <25 nm diameter water-soluble functional nanoparticles. The advantage of this coating method is that it is applicable to various hydrophobic metal and semiconductor nanoparticles, provides different chemical functionality and surface charge to the coated particle, induces minimum cytotoxicity, and can be used in deriving a bifunctional cellular nanoprobe for simultaneous imaging and surface enhanced Raman spectroscopy based detection. Introduction Metal and semiconductor nanoparticles have size and shape dependent tunable optical properties 1 useful for various exciting applications that includes quantum dot based fluorescence imaging for biomedical diagnostic; 2 noble metal nanoparticle based dark field imaging, 3 ultrasensitive detection, 4 and photo- thermal therapy; 5 and composite nanoparticle based multimodal imaging and detection. 6 Although synthetic methods are well advanced to prepare high quality nanoparticles of different sizes and shapes, 7 prior to application, they need to be transformed into functional nanoparticles via appropriate coating and conjugation chemistry. 8 Unfortunately, current coating methods such as thiol based small molecule coating, lipid coating, silica coating, and coating by various polymers have many limitations in deriving a wide range of functional nanoparticles. 2,8,9 One major limitation is the poor colloidal stability of weakly adsorbed coating (shell) that often desorbs by another competi- tive molecule or due to the reaction of surface bound functional group (e.g., chemisorbed thiol reacts with maleimide based reagent) during conjugation chemistry. 9 Among those coatings, silica coating, ligand bridging, and some polymer coatings are proved to be most powerful as they introduce the cross-linked shell structure that protects the nanoparticle from adverse chemical and physical environments during conjugation chem- istry or physiological conditions. 8a,c,d,f,i However, these cross- linked coatings often produce coated particles of larger overall diameters (>10 nm) where coating contributes to a 5-50 times increase in particle diameter. 8d-f Such particles which are larger in size than proteins have limitations in subcellular imaging, protein imaging/dynamics studies, and other applications. 2 Thus researchers are focusing on new coating methods that can produce colloidally stable and water-soluble functional nano- particles of smaller size. 10 In another direction, various com- posite nanoparticle based multimodal imaging and detection are under intense investigation, but their larger diameter (in the range of 50 nm to a few micrometers) again limits many interesting applications. 6 For example, the quantum dot (QD)- iron oxide nanocomposite is useful for fluorescence and magnetic resonance imaging (MRI) as well as for magnetic separation, and the iron oxide-gold nanocomposite is useful for magnetic separation and surface enhanced Raman (SERS) based detection; however, their larger diameter limits cellular delivery and subcellular targeting. 6c,d Herein we report a <25 nm diameter stable bifunctional cellular nanoprobe with a cross-linked coating. The nanoprobe consists of a 2-10 nm diameter Au/ Ag/Au-Ag core, coated with an imidazole based cross-linked polymeric shell and functionalized with a bioaffinity peptide. In a nanoprobe labeled cell, the Au/Ag/Au-Ag core is detected via scattering based dark field imaging and the imidazole coating is detected via SERS. The coating method is also extended to CdSe-ZnS based quantum dots. The bifunctional nanoprobe reported here consists of an imidazole based cross-linked polymer coating. This coating method has been developed to convert 2-10 nm diameter hydrophobic nanoparticles into 10-20 nm diameter stable and water-soluble coated nanoparticles. Imidazolium salts and imidazole derivatives have been widely used as room temper- ature ionic liquids, green solvents for organic synthesis, a source of carbene precursors, and a stabilizer for nanoparticle 11,12 Recently, imidazole based polymeric microparticles have been synthesized and used as metal ion adsorbents and heterogeneous organometallic catalysts. 13 We have used imidazole based polymer coatings on different inorganic nanoparticles and found that this coating is useful in deriving various water-soluble functional nanoparticles. Experimental Section General. Gold(III) chloride, silver acetate, tetrabutylammo- nium borohydride, Igepal CO-520, imidazole, imidazole-4 acetic acid sodium salt, L-histidine, L-histidinol dihydrochloride, 1,4- dibromo-2,3-butanediol, 2,4,6-tris(bromomethyl)mesitylene, and N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC) were purchased from Sigma-Aldrich and used as- received without further purification. TAT peptide with the sequence CGRKKRRQRRR (MW-1499.8) was purchased from GL Biochem (Shanghai) Ltd. with 97% purity. * To whom correspondence should be addressed. E-mail: camnrj@ iacs.res.in. Centre for Advanced Materials. Department of Materials Science. § Jubilant Chemsys Ltd. J. Phys. Chem. C 2009, 113, 21484–21492 21484 10.1021/jp905685a 2009 American Chemical Society Published on Web 12/08/2009

Upload: narayan

Post on 03-Feb-2017

213 views

Category:

Documents


1 download

TRANSCRIPT

Page 1: Imidazole Based Biocompatible Polymer Coating in Deriving

Imidazole Based Biocompatible Polymer Coating in Deriving <25 nm FunctionalNanoparticle Probe for Cellular Imaging and Detection

Nikhil R. Jana,*,† Pranab K. Patra,§ Arindam Saha,† SK Basiruddin,† and Narayan Pradhan†,‡

Centre for AdVanced Materials and Department of Materials Science, Indian Association for the CultiVation ofScience, Kolkata 700032, India, and Jubilant Chemsys Ltd., B-34, Sector 58, Noida 201301, India

ReceiVed: June 17, 2009; ReVised Manuscript ReceiVed: NoVember 10, 2009

Although synthetic methods for high quality near-monodispersed nanoparticles of metal and semiconductorare well established, their conversion into various functional nanoparticles is challenging. We report here animidazole based polymer coating that can be used in deriving <25 nm diameter water-soluble functionalnanoparticles. The advantage of this coating method is that it is applicable to various hydrophobic metal andsemiconductor nanoparticles, provides different chemical functionality and surface charge to the coated particle,induces minimum cytotoxicity, and can be used in deriving a bifunctional cellular nanoprobe for simultaneousimaging and surface enhanced Raman spectroscopy based detection.

Introduction

Metal and semiconductor nanoparticles have size and shapedependent tunable optical properties1 useful for various excitingapplications that includes quantum dot based fluorescenceimaging for biomedical diagnostic;2 noble metal nanoparticlebased dark field imaging,3 ultrasensitive detection,4 and photo-thermal therapy;5 and composite nanoparticle based multimodalimaging and detection.6 Although synthetic methods are welladvanced to prepare high quality nanoparticles of different sizesand shapes,7 prior to application, they need to be transformedinto functional nanoparticles via appropriate coating andconjugation chemistry.8 Unfortunately, current coating methodssuch as thiol based small molecule coating, lipid coating, silicacoating, and coating by various polymers have many limitationsin deriving a wide range of functional nanoparticles.2,8,9 Onemajor limitation is the poor colloidal stability of weaklyadsorbed coating (shell) that often desorbs by another competi-tive molecule or due to the reaction of surface bound functionalgroup (e.g., chemisorbed thiol reacts with maleimide basedreagent) during conjugation chemistry.9 Among those coatings,silica coating, ligand bridging, and some polymer coatings areproved to be most powerful as they introduce the cross-linkedshell structure that protects the nanoparticle from adversechemical and physical environments during conjugation chem-istry or physiological conditions.8a,c,d,f,i However, these cross-linked coatings often produce coated particles of larger overalldiameters (>10 nm) where coating contributes to a 5-50 timesincrease in particle diameter.8d-f Such particles which are largerin size than proteins have limitations in subcellular imaging,protein imaging/dynamics studies, and other applications.2 Thusresearchers are focusing on new coating methods that canproduce colloidally stable and water-soluble functional nano-particles of smaller size.10 In another direction, various com-posite nanoparticle based multimodal imaging and detection areunder intense investigation, but their larger diameter (in therange of 50 nm to a few micrometers) again limits many

interesting applications.6 For example, the quantum dot (QD)-iron oxide nanocomposite is useful for fluorescence andmagnetic resonance imaging (MRI) as well as for magneticseparation, and the iron oxide-gold nanocomposite is useful formagnetic separation and surface enhanced Raman (SERS) baseddetection; however, their larger diameter limits cellular deliveryand subcellular targeting.6c,d Herein we report a <25 nm diameterstable bifunctional cellular nanoprobe with a cross-linkedcoating. The nanoprobe consists of a 2-10 nm diameter Au/Ag/Au-Ag core, coated with an imidazole based cross-linkedpolymeric shell and functionalized with a bioaffinity peptide.In a nanoprobe labeled cell, the Au/Ag/Au-Ag core is detectedvia scattering based dark field imaging and the imidazole coatingis detected via SERS. The coating method is also extended toCdSe-ZnS based quantum dots.

The bifunctional nanoprobe reported here consists of animidazole based cross-linked polymer coating. This coatingmethod has been developed to convert 2-10 nm diameterhydrophobic nanoparticles into 10-20 nm diameter stable andwater-soluble coated nanoparticles. Imidazolium salts andimidazole derivatives have been widely used as room temper-ature ionic liquids, green solvents for organic synthesis, a sourceof carbene precursors, and a stabilizer for nanoparticle11,12

Recently, imidazole based polymeric microparticles have beensynthesized and used as metal ion adsorbents and heterogeneousorganometallic catalysts.13 We have used imidazole basedpolymer coatings on different inorganic nanoparticles and foundthat this coating is useful in deriving various water-solublefunctional nanoparticles.

Experimental Section

General. Gold(III) chloride, silver acetate, tetrabutylammo-nium borohydride, Igepal CO-520, imidazole, imidazole-4 aceticacid sodium salt, L-histidine, L-histidinol dihydrochloride, 1,4-dibromo-2,3-butanediol, 2,4,6-tris(bromomethyl)mesitylene, andN-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate(SMCC) were purchased from Sigma-Aldrich and used as-received without further purification. TAT peptide with thesequence CGRKKRRQRRR (MW-1499.8) was purchased fromGL Biochem (Shanghai) Ltd. with 97% purity.

* To whom correspondence should be addressed. E-mail: [email protected].

† Centre for Advanced Materials.‡ Department of Materials Science.§ Jubilant Chemsys Ltd.

J. Phys. Chem. C 2009, 113, 21484–2149221484

10.1021/jp905685a 2009 American Chemical SocietyPublished on Web 12/08/2009

Page 2: Imidazole Based Biocompatible Polymer Coating in Deriving

Synthesis of Hydrophobic Gold and Silver Nanoparticles.The synthetic method was followed from our earlier methodwith some modifications.7e The metal salt (30 mg of AuCl3 or17 mg of Ag-acetate) was dissolved in 9 mL of toluene in thepresence of 260 µL of octylamine. Next, 100 µL of oleic acidwas added to the same solution. Finally, a tetrabutyl ammoniumborohydride solution (50 mg dissolved in 1 mL toluene and100 µL oleic acid mixture: CAUTION! hydrogen gas evolutionupon oleic acid addition) was injected into the metal saltsolution. The colored solution (pink for Au and yellow for Ag)appeared immediately. Next, 20 mL of anhydrous ethanol wasadded to precipitate the particles. Particles were isolated bycentrifuging at 6000 rpm for 3-4 min and dissolved in 10 mLof cyclohexane.

Synthesis of Hydrophobic ZnS Capped CdSe QuantumDots. CdSe quantum dots were synthesized by a high temper-ature method using carboxylate precursors of Cd in octadecene.7b

CdSe nanoparticles were purified from free ligands and cappedby ZnS shell at 200 °C in octadecene via the alternate injectionof Zn stearate in octadecene and elemental S dissolved inoctadecene. Afterwards, synthesized particles were purified fromfree ligands using the standard precipitation-redispersionprocedures and finally dissolved in Igepal-cyclohexane reversemicelles for polymer coating.

Polymer Coating Procedure. 20 mL of Igepal-cyclohexanereverse micelle was prepared by mixing 2 mL of Igepal into 18mL of cyclohexane, and this solution was used to preparesolutions of 2,4,6-tris(bromomethyl)mesitylene, 1,4-dibromo-2,3-butanediol, and hydrophobic nanoparticles. Typically, 2.0mg (0.05 mmol) of 2,4,6-tris(bromomethyl)mesitylene wasdissolved in 1 mL of the reverse micelle solution. In a separatevial, 12 mg (0.5 mmol) of 1,4-dibromo-2,3-butanediol wasdissolved in 1 mL of the reverse micelle solution. Solutions ofthe imidazole derivative or their mixtures were prepared bydissolving ∼8-20 mg (∼0.05-0.1 mmol) of them in 0.1-0.50mL of water.

For the polymer coating, 10 mL of the purified hydrophobicnanoparticle (QD/Au/Ag) solution was prepared in Igepal-cyclo-hexane reverse micelles. The particle concentration was adjustedusing the absorbance value at the first absorption maxima forCdSe-ZnS (QD) and the plasmon absorbance value for Au andAg (at 520 and 410 nm respectively), using a 1 cm optical pathlength. The absorbance was ∼0.5 for QD and ∼1.0-2.0 forAu/Ag. Next, the aqueous solution of imidazole or the mixture

of the two imidazole derivatives was mixed into it. Then, thereverse micelle solution of 2,4,6-tris(bromomethyl)mesityleneand 1,4-dibromo-2,3-butanediol were introduced and an opticallyclear homogeneous solution was obtained. In case of L-histidinol,0.1-0.3 mL of water was added further if the solution is notoptically clear. The reaction was continued for 4-12 h. Thebest combination and conditions for producing water-solublenanoparticles is shown in Table 1 and the Supporting Informa-tions. After the reaction, particles were precipitated by addinga minimum of ethanol and washed with ethanol 3-4 times.Finally particles were solubilized in water/ethanol. If the solutionwas not optically clear a drop of dilute NaOH (for carboxylatefunctionalized particle) or formic acid (for amine functionalizedparticle) was added to make a clear solution.

Nanoparticle Enlargement by Silver Coating. 10 mL ofpolymer coated Au nanoparticles was prepared in water withan Au concentration of ∼1 mM. Next, the solution was mixedwith a AgNO3 solution to adjusted it to a final concentrationbetween 5-10 mM. Then an equivalent amount (with respectto AgNO3) of ascorbic acid was added in a dropwise mannerto reduce the silver ion. The color changed from pink to paleyellow/brown yellow in 1-10 min, depending on the amountof silver.4c Particle solutions were then preserved for use.

Peptide Conjugation. 2 mL of amine functionalized polymercoated particle (coating that uses L-histidine) solutions wereprepared in phosphate buffer of pH 7.0, mixed with a SMCCsolution (1 mg dissolved in 1 mL of dimethylformamide), andincubated for 1 h.8k Next, nanoparticles were separated fromfree reagents using Sephadex G25 collum and then mixed with100 µL of TAT peptide solutions (2 mg TAT peptide dissolvedin 1 mL phosphate buffer solution) and incubated at 4 °C forovernight. Then the solution was allowed for overnight dialysis(using a membrane with MWCO 14 000) to separate particlesfrom free peptide. After dialysis the particles were diluted withtris buffer of pH 7.0 and preserved at 4 °C.

Cell Labeling. HepG2 cells were subcultured in 24 well cellculture plates overnight so that cells were attached to the wellplate. In some of the well plates, a glass slide was put at thebottom so that cells can grow and attach on those slides. Next,they were mixed with 100 µL of TAT functionalized QD/Au/Ag solution and kept in an incubation chamber for 1-2 h. Next,they were washed with PBS buffer solution 3 times, and finally,PBS buffer or cell culture medium was added for the imagingstudy. For bright field and fluorescence imaging, the cell culture

TABLE 1: Summary of Particle Size, Surface Charge, and Colloidal Stability of Polymer Coated Particles of the Best Qualityin Aqueous Solutions of Varied pH

nanoparticle,polymer forming

monomers,a TEM size(nm) before and after coatingb

surface charge,DLS size (nm) inacidic solution of

pH ) 3-4 (stability)

surface charge,DLS size (nm) inHEPES buffer of

pH ) 5.5 (stability)

surface charge,DLS size (nm) in

phosphate buffer ofpH ) 7.0 (stability)

surface charge,DLS size (nm) inborate buffer of

pH ) 9.5 (stability)

Au, imidazole-4-aceticacid, 3 ( 2, 3 ( 2

+20 mV, ---(aggregate in 15 min)

-5 mV, ---(aggregate in 15 min)

-12 mV, 18 ( 5(stable for 2-3 months)

-20 mV, 15 ( 5(stable for 2-3 months)

Ag, imidazole-4-aceticacid, 2.5 ( 2, 2.5 ( 2

+12 mV, ---(aggregate slowly in 15 min)

-4 mV, ---(aggregate in 15 min)

-10 mV, 15 ( 6(stable for 2-3 months)

-30 mV, 9 ( 4(stable for 2-3 months)

QD(green), imidazole-4-aceticacid, 3 ( 1, 3 ( 1

+15 mV, ---(aggregate in 15 min)

-5 mV, ---(aggregate in 15 min)

-12 mV, 22 ( 6(stable for 2-3 months)

-21 mV, 15 ( 6(stable for 2-3 months)

Au, histidine, 3 ( 2, 3 ( 2 +30 mV, 14 ( 4(stable for 2-3 days)

immediate precipitation -5 mV, 20 ( 6(stable for a month)

-26 mV, 8 ( 3(stable for 2-3 months)

Ag, histidine, 2.5 ( 2, 2.5 ( 2 +20 mV, 15 ( 4(stable for 2-3 days)

immediate precipitation - 5 mV, 22 ( 7(stable for a month)

-36 mV, 18 ( 5(stable for 2-3 months)

Au-Ag(enlarged), histidine,10 ( 3, 10 ( 3

+20 mV, ---(slowly aggregates)

immediate precipitation -10 mV, 22 ( 7(stable for a month)

-20 mV, 21 ( 7(stable for a month)

a In all cases 1,4-dibromo-2,3-butanediol and 2,4,6-tris(bromomethyl)mesitylene were used in addition. b About 100-200 particles weremeasured to estimate the particle size.

Imidazole Based Biocompatible Polymer Coating J. Phys. Chem. C, Vol. 113, No. 52, 2009 21485

Page 3: Imidazole Based Biocompatible Polymer Coating in Deriving

plates were directly used. However, for dark field imaging andsurface enhanced Raman study, cells attached to the glass slideswere used. Typically, the glass slides with cells attached wereplaced inverted on another larger glass slide so that cells stayedbetween the two slides. The samples were then used for theimaging/detection study.

MTT Assay. HepG2 and NIH3T3 cells were trypsinized andresuspended in DMEM culture medium. The cells were seededin a 96-well, flat bottom microplate in 100-200 µL of fullDMEM culture medium and kept overnight at 37 °C and 5%CO2. The particles of different concentration were loaded toeach well and each concentration has 6 duplications. Afterincubation for 48 h, 20 µL of MTT solution (5 mg/mL) wasadded to each well 4 h before the end of the incubation. Themedium was discarded and the produced formazan was dis-solved with 200 µL of DMSO. The plates were read withabsorbance at 550 nm. The optical density is directly correlatedwith cell quantity, and cell viability was calculated by assuming100% viability in the control set without any nanoparticles.

Instrumentation. UV-visible absorption spectra were re-corded using an Agilent 8453 spectrophotometer in a 1-cmquartz cell. Fluorescence spectra were measured using aFluoromax-4 spectrofluorometer (Horiba Jobin Yvon) fluores-

cence spectrometer. The quantum yield (QY) was measuredusing integrated fluorescence intensity of the CdSe-ZnS (QD)and reference (flourescein, QY ) 97%). An FEI Technai G2transmission electron microscope was used for transmissionelectron microscopy (TEM) studies. Samples were prepared byplacing a drop of the diluted particle solution on a carbon-coatedcopper grid. For the polymer staining experiment, a dried sampledeposited in the grid was immersed into a 1% phosphomolybdicacid solution for 10 s and then air-dried. Dynamic light scattering(DLS) particle size and surface charge were analyzed by aZetaPALS zeta potential analyzer (Brookhaven InstrumentCorporation). Histidine and the imidazole-4-acetic acid basedpolymer were also studied via DLS to understand their overallsize and molecular weight. A MALDI-TOF mass spectrometer(Voyager-DE PRO) was used for molecular weight determina-tion with cinapinic acid as the matrix.

Cell imaging was performed using an Olympus microscopeIX81 with DP70 digital camera. Confocal fluorescence imagingwas performed using an Olympus Fluoview 300 confocal laserscanning system with 488 nm laser excitation. Dark fieldimaging was performed using an inverted Olympus 71 micro-scope with a dark field condenser (U-DCW) and an oil Irisobjective. In this setup, the condenser delivers a narrow beam

SCHEME 1: Polymerization Chemistry (1a) and Reversed Micelle Based Coating Strategy (1b)

21486 J. Phys. Chem. C, Vol. 113, No. 52, 2009 Jana et al.

Page 4: Imidazole Based Biocompatible Polymer Coating in Deriving

of white light and oil immersion objective collects only thescattered light from the sample. The image was captured usinga colored camera (model DP71), and Q-capture software wasused for image acquisition and for adjusting the white lightbalance. The Raman spectrum was measured using RenishawinVia Raman system using 633 nm excitation, 10 mW laserpower, and 10 s integration time.

Results

Polymer Coating. The polymerization strategy is shown inparts 1a and 1b of Scheme 1. We exploited the reaction ofimidazole nitrogen groups with alkyl bromide.13 Two types of

bromides such as 1,4-dibromo-2,3-butanediol and 2,4,6-tris-(bromomethyl)mesitylene were used in optimum ratio wheredibromobutanol introduces water-soluble alcohol groups andtribromomesitylene cross-links the polymer. The nature ofimidazole dictates the particle surface charge and chemicalfunctionality. The polymer coating was performed in Igepal-cy-clohexane reverse micelle medium.14 Igepal is a nonionicsurfactant and produces 1-10 nm size spherical reverse micelles,depending on the water-surfactant molar ratio.14a The advantageof the reverse micelle is that it can solubilize both thehydrophobic nanoparticle and polar or nonpolar polymer precur-sor via their polar micelle interface and polymerization can beinitiated from homogeneous solution.14 The polymerizationworks at room temperature, and the reaction time was optimizedbetween 4-12 h depending on the monomers used. Afterpolymerization, particles were precipitated by ethanol addition(which breaks the reverse micelle), separated, washed, anddissolved in water. This coating method has been extended todifferent hydrophobic nanoparticles as well as to differentimidazole monomers (Table 1 and Figures 1-5). Although themethod works well for gold, silver, and ZnS capped CdSe (QD)and produces water-soluble particles, it did not work for ironoxide nanoparticles (produces water insoluble particles). Dif-ferent imidazole monomers such as imidazole, imidazole-4-acetic acid, L-histidine, L-histidinol, and their mixtures weretested and it was found that imidazole-4-acetic acid andL-histidine produce water-soluble particles that are the moststable. The reaction conditions were optimized by varying themolar ratio of different reactants and other reaction conditions,in order to get the most stable colloidal solution (Table 1 andthe Supporting Information). The possibility of particle ag-gregation during polymer coating was minimized by examiningdifferent experimental conditions and finding conditions thatproduce water-soluble nanoparticles of smaller size. It was foundthat 5-10 mol % of 2,4,6-tris(bromomethyl)mesitylene is theoptimum concentration; a higher concentration makes insolubleparticles due to excessive cross-linking, and a lower concentra-tion produces particles of poor colloidal stability. A higheramount (40-50 mol %) of 1,4-dibromo-2,3-butanediol was usedin order to get good water solubility of coated particle.

Original hydrophobic nanoparticles are completely insolublein water, but they become water-soluble after polymer coating.This good water solubility suggests that particle do not segregatebefore polymerization. This was confirmed from systematictransmission electron microscopic (TEM) study of coated

Figure 1. TEM image of polymer coated gold nanoparticles preparedwith varying amounts of polymer forming precursors. (a,b) Moreprecursors, 15% 2,4,6-tris(bromomethyl)mesitylene and different reac-tion times (6 h for a and 12 h for b) were used to produce these partiallysoluble particles. Here polymer shells are visible as light contrast,although not very uniform. (c) Less precursors with high resolutionwhere the polymer shell is invisible. (d) Less precursor but the polymeris stained with phosphomolybdic acid; where the light gray colorsurrounding the dark colored particle shows the phosphomolybdic acidstained polymer. In this study histidine, 1,4-dibromo-2,3-butanediol,and 2,4,6-tris(bromomethyl)mesitylene were used as the polymercoatings.

Figure 2. TEM image of polymer-coated Ag and QD where thepolymer is stained with phosphomolybdic acid. The light gray colorsurrounding the dark colored particle shows the phosphomolybdic acidstained polymer. In this study histidine, 1,4-dibromo-2,3-butanediol,and 5 mol % of 2,4,6-tris(bromomethyl)mesitylene were used as thepolymer coatings.

Figure 3. Representative MALDI-mass spectra of the histidine basedpolymer.

Imidazole Based Biocompatible Polymer Coating J. Phys. Chem. C, Vol. 113, No. 52, 2009 21487

Page 5: Imidazole Based Biocompatible Polymer Coating in Deriving

nanoparticle (Figures 1 and 2 and the Supporting Information).When more monomers were used or the reaction was continuedfor a longer time, a thick polymer shell was formed along withextensive particle aggregation. These thick shells are observedunder TEM (Figure 1a). However, coatings produced inoptimized conditions (that provide soluble nanoparticle) are toothin to observe under TEM. (Figure 1b,c), unless staining wasused (Figures 1d and 2 and the Supporting Information). Thisresult indicates that thin shells of <8 nm are necessary to producewater-soluble nanoparticles with good colloidal stability andwithout significant particle aggregation. The TEM study alsoshows that Au, Ag, and CdSe-ZnS particles coated with thinpolymer shells are ∼2-5 nm in diameter and enlarged Au-Agwas ∼10 nm in diameter; they are reasonably isolated from eachother with minimum aggregation and do not change theirdiameter appreciably after coating (Table 1 and the SupportingInformation). When TEM particle diameters are compared withthe diameters obtained from dynamic light scattering (DLS)results, it shows that DLS diameters are about 10-15 nm largerfor all cases than the respective TEM diameters, indicating thatthe polymer coating is about 5-8 nm thick (Table 1). This wasfurther confirmed from TEM observations of coating thicknessafter phosphomolybdic acid staining (Figures 1 and 2 and theSupporting Information). These results show that the polymeris coated on the particle surface, but they are not as uniform aswhat is shown in Scheme 1.

We also did a control DLS experiment and MALDI massspectrum (Table 1 and Figure 3) of the polymer alone, to

understand the size/molecular weight of the coated polymer.The DLS study shows that the polymer is 4-7 nm in size, andassuming a spherical morphology, the molecular weight of thepolymer ranges between ∼25 000 and 40 000. The MALDImass spectrum shows strong peaks at ∼35 000 and a weakerpeak at ∼38 000 suggesting that polymer molecular weight ispredominantly 35 000. The presence of more than one peak iseither due to polymer polydispersity, the clustering effectbetween polymers, or multiple charging of polymers.15 TheNMR study of coated particles shows that the original hydro-phobic surfactant from the particle surface is completely replacedby polymer (Figure 4 and the Supporting Information).

Property of Coated Nanoparticles. Aqueous dispersion ofpolymer coated particles and their respective optical propertiesare shown in Figures 5 and 6. The comparison of the opticalproperty before and after coating shows that it does not changeappreciably, indicating that the particle size remains the sameafter coating, which is further supported by the transmissionelectron microscopic (TEM) study. The fluorescence quantumyield (QY) for quantum dots also remains same (20-30%) anddoes not quench after the polymer coating. The temperaturestability of polymer coated particle was tested in the 4-40 °Crange and showed no sign of aggregation and no appreciablechange of optical properties even after long-term (>10 days)preservation (Supporting Information). This temperature stabilitytest shows that coated particles should be stable during cellulargrowth temperature (37 °C) and other conditions.

Figure 4. Proton NMR spectra of the histidine based polymer and the same polymer coated with Au nanoparticles. Peak positions are labeledaccording to the different types of protons shown (labeled as 1, 2, 3, 4, 5, Ha, and Hb) in the polymer structure above. In this study histidine,1,4-dibromo-2,3-butanediol, and 5 mol % of 2,4,6-tris(bromomethyl)mesitylene were used as the polymer coatings.

21488 J. Phys. Chem. C, Vol. 113, No. 52, 2009 Jana et al.

Page 6: Imidazole Based Biocompatible Polymer Coating in Deriving

The colloidal stability of the polymer coated particle wasdetermined in different aqueous buffer solutions of varying pH.It showed that colloidal stability depends on the nature ofimidazole monomers used for polymer coating that introducedifferent surface functional groups around the particles. Table1 summarizes the surface charge, particle size, and colloidalstability of the particle after coating with different imidazolebased polymers. It shows that coatings that uses L-histidine andimidazole-4-acetic acid produce particles that are stable incommon buffer for more than a month. Most of the coatedparticles become positively charged and precipitate in acidicmedium due to the protonation of carboxylic groups (fromimidazole-4-acetic acid or histidine) and manifest the cationicimidazolium salt property of polymer. The imidazole-4-aceticacid based coating stabilizes the particle as it introduces negativesurface charge to the coated particle. Interestingly, histidine withboth primary amine and carboxylate introduces tunable particlesurface charge from positive to negative, depending on thesolution pH (Table 1). The DLS particle size modulates withsolution pH which reflects the pH dependence of colloidalstability. For example, histidine coated particles at pH 9.5 aresmaller but the size increases at pH 7.0 due to partial aggregationof the particle.

A cytotoxicity study was conducted to understand the effectof coating on toxicity (Figure 7). Cell survival was >75% for48 h incubation of 1-20 nM (nano molar) particle solution(which is the common concentration used for cell labeling8k),although survival decreases at higher concentrations of QD andAg which is possibly due to the toxic effect of these particles.

Functionalization and Labeling Application. An importantadvantage of this coating is that the coated particle can be furtherfunctionalized with the desired chemical or biochemical ofinterest using conventional bioconjugation chemistry. We have

functionalized the primary amine terminated Au, Ag, and QDwith TAT peptide using N-succinimidyl 4-(maleimidomethyl)-cyclohexanecarboxylate (SMCC), a conventional reagent forcovalent linkage between amine functionalized particle/biomol-ecule and thiol bearing biomolecule/particle.8a,k In present caseprimary amine functionalized particle (with histidine coating)was first reacted with succinimidyl group of SMCC, producingmaleimide bearing particle that is further reacted with thiol groupof cysteine-TAT. The TAT functionalized particle retained theoptical property of respective nanoparticle and possess goodcolloidal stability. This result shows that polymer coatingprovides good protection of nanoparticle toward adverse chemi-cal (e.g., bioconjugation) and physical environment (e.g.,chromatographic separation, buffer stability).

The TAT peptide functionalized particles were used forcellular labeling applications. It is well-known that TATenhances the particle uptake to cell,16 and we also observed asimilar result (Figures 8 and 9). Labeling of TAT-QD wasobserved with fluorescence microscopic imaging while labelingof TAT-Au/Ag was observed via dark filed microscopicimaging. Intense fluorescence from labeled QD and intensescattered light from labeled Ag/Ag was observed from cell.Control samples that have no TAT on their surface do not showsuch a colored image, indicating that TAT enhances the cellularuptake of particle.

Multifunctional Property. An additional advantage of thispolymer coating is that the coating introduces a surface enhancedRaman (SERS) based chemical signature that can be used fordetection of nanoprobes after its labeling to the target. In orderto get a good SERS signal, the Au/Ag particles need to beenlarged, as the SERS signal increases with particle size.17 Inthe control experiment, we found that polymer coated small Au/Ag gives a very weak SERS signal (or no signal at all) but thesignal becomes intense after particle enlargement (Figure 8d).The particle enlargement involves a simple seeding growthmethod, where the coated Au/Ag particle act as a seed whichgrows upon deposition of silver via the reduction of free silverion by ascorbic acid.17b The vibration SERS signals arecharacterized as imidazole ring breathing (1280 cm-1), ringstretching (1300 and 1375 cm-1), and ring CdC stretching (1550and 1645 cm-1), all of which are coming due to coated polymer(Figure 3d).18 When this enlarged particle was functionalizedby TAT and used for cell labeling, the particle acts for bothdark field imaging and SERS based detection (Figure 8c,d).Development of this type of nanoprobe for simultaneousimaging and detection (SERS based) is applicable to Au/Ag(not for QD) as the surface plasmon of Au/Ag is responsiblefor the electromagnetic enhancement mechanism of SERS.17

Discussion

Imidazole derivatives have been used earlier as stabilizersfor different nanoparticles11,12 and for synthesis of polymericmicroparticles.13 However, in the present case, we used theirpolymeric form in providing a cross-linked shell structure fornanoparticles. This cross-linked shell converts hydrophobicnanoparticles into hydrophilic nanoparticles with good colloidalstability. The surface charge and chemical functional groups ofcoated particles can be varied by using different imidazolemonomers, and amine/carboxylate groups can be used for furtherfunctionalization. Particularly significant is the histidine andimidazole-4-acetic acid coated particle that produces primaryamine and carboxylate terminated nanoparticles, respectively.The adsorption of polymer on the particle surface was evidentfrom the replacement of the hydrophobic surfactant by the

Figure 5. Water-soluble nanoparticles prepared from respectivehydrophobic nanoparticles via imidazole based polymer coating. (a,b)Absorption spectra of Ag (0.2 mM) and Au (1 mM) nanoparticles before(dotted line) and after (continuous line) polymer coating. (c) Successiveabsorption spectra of polymer coated Au-Ag nanoparticles duringparticle enlargement of Au via Ag deposition. Inset shows a typicaldigital picture of a stable colloidal solution.

Imidazole Based Biocompatible Polymer Coating J. Phys. Chem. C, Vol. 113, No. 52, 2009 21489

Page 7: Imidazole Based Biocompatible Polymer Coating in Deriving

polymer and observation of imidazole ring breathing and ringstretching modes in the SERS spectrum. Multiple binding byimidazole groups present in the polymer enhances the bindingstrength and thus stabilizes the coated particle, similar to thiolbased molecules.8j,10

The presented coating method exhibits several advantagesover existing coatings. This coating provides a cross-linked shellwhich is different from conventional thiol based coatings9 butsimilar to silica8i and polyacrylate8k coatings. The cross-linked

shell provides better protection of nanoparticles during conjuga-tion chemistry and other adverse conditions.8i,k This coatingmethod is simpler than silica/polyacrylate based coatings.Although the silica coating is very sensitive to experimentalconditions8i and the polyacrylate coating needs an initiator orhigh temperature,8k the presented coating occurs at roomtemperature by a simple mixing of reactants. Biocompatibilityprovides another advantage of this coating. Although the silicacoating is probed to be nontoxic,19 the biocompatibility of the

Figure 6. (a) UV-visible absorption spectra of green emitting CdSe-ZnS in toluene (continuous line) and the same sample in water after polymercoating (dotted line). The inset is the digital image of the aqueous polymer coated particle solution. (b) Fluorescence spectra of the polymer coatedCdSe-ZnS solution of two different colors, prepared from the respective hydrophobic CdSe-ZnS via imidazole based polymer coatings.

Figure 7. Cytotoxicity study of various polymer coated particles in NIH3T3 and HepG2 cell line via MTT assay.

Figure 8. Dark field imaging and surface enhanced Raman spectroscopic (SERS) study of noble metal nanoparticle labeled HepG2 cell. (a)Polymer coated Au particle, (b) TAT functionalized Au particle, (c) TAT functionalized Au-Ag particle, and (d) SERS of histidine based polymercoated particle (black Au, blue Au-Ag, pink Au-Ag-TAT, and green Au-Ag-TAT after labeling in HepG2 cell.

21490 J. Phys. Chem. C, Vol. 113, No. 52, 2009 Jana et al.

Page 8: Imidazole Based Biocompatible Polymer Coating in Deriving

polyacrylate coating strongly depends on the nature of precursormonomers used.8k The biocompatibility of the presented coatingis due to the use of biomolecules and nontoxic precursors usedin the coating processes.

The presence of a Raman signal is another advantage of thiscoating that offers multifunctionality of Au/Ag based nano-probes. Usually Raman probes need to be attached by additionalsteps via chemisorption or conjugation steps.17 In contrast, herethe coating itself provides a Raman signal for Au/Ag basednanoprobes which can be used for SERS based detections. Thenanoprobe developed here has advantages over other multi-functional probes.6 The size of the nanoprobe is small (<25 nm),which minimizes the steric interaction during biolabeling stepsand also has the option to enlarge the particle after labeling.17b

In addition, the synthetic approach in producing multifunctionalnanoprobes is straightforward compared to other cases thatinvolves coating followed by conjugation/adsorption of theRaman probe or involvement of more than one type ofnanoparticle.6

Conclusion

In conclusion we developed an imidazole based coating forhydrophobic nanoparticles to convert them into water-solubleand functionalized nanoparticles. This coating has severaladvantages over conventional thiol based coatings and polymercoatings. The coating method is applicable to metal andsemiconductor nanoparticles, provides various chemical func-tionality and surface charge to the coated particle, inducesminimum cytotoxiciy, and protects nanoparticles from adversephysical and chemical environments, and the derived nanoprobesoffer SERS based detection options. A peptide based functionalnanoprobe has been derived from this coating and used as acellular labeling reagent, demonstrating that a variety of otherfunctional nanoprobes can be synthesized from this coatedparticle.

Acknowledgment. The authors thank Dr. Nihar R. Jana ofNational Brain Research Centre (NBRC), Manesar, India, forproviding the cellular imaging facility. This work is supportedby the DST (SR/S5/NM-47/2005), Government of India. A.S.and S.B. acknowledge CSIR, India for providing fellowship.

Supporting Information Available: Table showing differentconditions used for coating, NMR spectra of polymers of coatedparticles, and the TEM of coated particle. This material isavailable free of charge via the Internet at http://pubs.acs.org.

References and Notes

(1) (a) Alivisatos, A. P. Science 1996, 271, 933–937. (b) Link, S.; El-Sayed, M. A. J. Phys. Chem. B 1999, 103, 4212–4217. (c) Murphy, C. J.;Jana, N. R. AdV. Mater. 2002, 14, 80–82.

(2) (a) Medintz, I. H.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H.Nat. Mater. 2005, 4, 435–446. (b) Michalet, X.; Pinaud, F. F.; Bentolila,L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir,S. S.; Weiss, S. Science 2005, 307, 538–544.

(3) (a) Orendorff, C. J.; Sau, T. K.; Murphy, C. J. Small 2006, 2, 636–639. (b) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. J. Am.Chem. Soc. 2006, 128, 2115–2120.

(4) (a) Rosi, N. L.; Mirkin, C. A. Chem. ReV. 2005, 105, 1547. (b)Jana, N. R. Analyst 2003, 128, 954–956. (c) Jana, N. R.; Ying, J. Y. AdV.Mater. 2008, 20, 430–434.

(5) (a) Loo, C.; Lowery, A.; Halas, N.; West, J.; Drezek, R. Nano Lett.2005, 5, 709–711. (b) Norman, R. S.; Stone, J. W.; Gole, A.; Murphy, C. J.Sabo-Attwood. Nano Lett. 2008, 8, 302–306.

(6) (a) Wang, D.; He, J.; Rosenzweig, N.; Rosenzweig, Z. Nano Lett.2004, 4, 409–413. (b) Sathe, T. R.; Agrawal, A.; Nie, S. Anal. Chem. 2006,78, 5627–5632. (c) Stoeva, S. I.; Lee, J.-S.; Smith, J. E.; Rosen, S. T.;Mirkin, C. A. J. Am. Chem. Soc. 2006, 128, 8378–8379. (d) Selvan, S. T.;Patra, P. K.; Ang, C. Y.; Ying, J. Y. Angew. Chem., Int. Ed. 2007, 46,2448–2452. (e) Heitsch, A. T.; Smith, D. K.; Patel, R. N.; Ress, D.; Korgel,B. A. J. Solid State Chem. 2008, 181, 1590–1599. (f) Park, J.-H.; onMaltzahn, G.; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J. Angew Chem. Int.Ed. 2008, 47, 1–6. (g) Kim, J.; Kim, H. S.; Lee, N.; Kim, T.; Kim, H.; Yu,T.; Song, I. C.; Moon, W. K.; Hyeon, T. Angew Chem. Int. Ed. 2008, 47,8438–8441.

(7) (a) Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem.Soc. 1993, 115, 8706–8707. (b) Li, J. J.; Wang, Y. A.; Guo, W.; Keay,J. C.; Mishima, T. D.; Johnson, M. B.; Peng, X. J. Am. Chem. Soc. 2003,125, 12567–12575. (c) Pradhan, N.; Goorskey, D.; Thessing, J.; Peng, X.J. Am. Chem. Soc. 2005, 127, 17586–17587. (d) Sun, S.; Murray, C. B.;Weller, D.; Folks, L.; Moser, A. Science 2000, 287, 1989–1992. (e) Jana,N. R.; Peng, X. J. Am. Chem. Soc. 2003, 125, 14280–14281. (f) Shevchenko,E. V.; Talapin, D. V.; Schnablegger, H.; Kornowski, A.; Festin, O.;Svedlindh, P.; Haase, M.; Weller, H. J. Am. Chem. Soc. 2003, 125, 9090–9101. (g) Hyeon, T.; Lee, S. S.; Park, J.; Chung, Y.; Na, H. B. J. Am.Chem. Soc. 2001, 123, 12798–12801. (h) Jana, N. R.; Chen, Y.; Peng, X.Chem. Mater. 2004, 16, 3931–3935. (i) Jana, N. R. Small 2005, 1, 875–882.

(8) (a) Parak, W. J.; Gerion, D.; Zanchet, D.; Woerz, A. S.; Pellegrino,T.; Micheel, C.; Williams, S. C.; Seitz, M.; Bruehl, R. E.; Bryant, Z.;Bustamante, C.; Bertozzi, C. R.; Alivisatos, A. P. Chem. Mater. 2002, 14,2113–2119. (b) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.;Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759–1762. (c) Guo,W.; Li, J. J.; Wang, Y. A.; Peng, X. J. Am. Chem. Soc. 2003, 125, 3901–3909. (d) Yu, W.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson,J. P.; Ge, N.; Paele, F.; Bruchez, M. P. Nat. Biotechnol. 2003, 21, 41–46.(e) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. Nat.Biotechnol. 2004, 22, 969–976. (f) Pellegrino, T.; Manna, L.; Kudera, S.;Liedl, T.; Koktysh, D.; Rogach, A. L.; Keller, S.; Radler, J.; Natile, G.;Parak, W. J. Nano Lett. 2004, 4, 703–707. (g) Nikolic, M. S.; Krack, M.;Aleksandrovic, V.; Kornowski, A.; Forster, S.; Weller, H. Angew. Chem.,Int. Ed. 2006, 45, 6577–6580. (h) Dubois, F.; Mahler, B.; Dubertret, B.;Doris, E.; Mioskowski, C. J. Am. Chem. Soc. 2007, 129, 482–483. (i) Jana,N. R.; Earhart, C.; Ying, J. Y. Chem. Mater. 2007, 19, 5074–5082. (j)Nandanan, E.; Jana, N. R.; Ying, J. Y. AdV. Mater. 2008, 20, 2068–2073.(k) Yei, Y.; Jana, N. R.; Tan, S.; Ying, J. Y. Bioconjugate Chem. 2009, 20,1752–1758.

(9) (a) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem.Res. 2000, 33, 27–36. (b) Aldana, J.; Lavelle, N.; Wang, Y.; Peng, X. J. Am.Chem. Soc. 2005, 127, 2496–2504.

(10) (a) Susumu, K.; Uyeda, H. T.; Medintz, I. L.; Pons, T.; Delehanti,J. B.; Mattoussi, H. J. Am. Chem. Soc. 2007, 129, 13987–13996. (b) Liu,W.; Howrath, M.; Greytak, A. B.; Zheng, Y.; Nocera, D. G.; Ting, A. Y.;Bawendi, M. G. J. Am. Chem. Soc. 2008, 130, 1274–1284. (c) Smith, A. M.;Nie, S. J. Am. Chem. Soc. 2008, 130, 11278–11279.

(11) (a) Welton, T. Chem. ReV. 1999, 99, 2071–2083. (b) Canal, J. P.;Ramnial, T.; Dickie, D. A.; Clyburne, J. A. C. Chem. Commun. 2006, 1809–1818.

(12) (a) Dupont, J.; Fonseca, G. S.; Umpierre, A. P.; Fichtner, P. F. P.;Teixeira, S. R. J. Am. Chem. Soc. 2002, 124, 4228–4229. (b) Itoh, H.; Naka,K.; Chujo, Y. J. Am. Chem. Soc. 2004, 126, 3026–3027. (c) Ryu, H. J.;Sanchez, L.; Keul, H. A.; Raj, A.; Bockstaller, M. R. Angew. Chem., Int.Ed. 2008, 47, 7639–7643.

(13) (a) Park, K. H.; Ku, I.; Kim, H. J.; Son, S. U. Chem. Mater. 2008,20, 1673–1675. (b) Zhang, Y.; Zhao, L.; Patra, P, K.; Hu, D.; Ying, J. Y.Nano Today 2009, 4, 13–20.

(14) (a) Lipgens, S.; Schubel, D.; Schlicht, L.; Spilgies, J.-H.; Ilgenfritz,G. Langmuir 1998, 14, 1041–1049. (b) Moffat, B. A.; Reddy, G. R.;McConville, P.; Hall, D. E.; Chenevert, T. L.; Kopelman, R. R.; Philbert,M.; Weissleder, R.; Rehemtulla, A.; Ross, B. D. Mol. Imag. 2003, 2, 324–332.

(15) (a) Mazarin, M.; Viel, S.; Breton, B. A.; Thevand, A.; Charles, L.Anal. Chem. 2006, 78, 2758–2764. (b) Schriemer, D. C.; Li, L. Anal. Chem.2006, 68, 2721–2725.

Figure 9. Confocal microscopic images of the HepG 2 cell afterlabeling with polymer coated QD and TAT peptide functionalizedpolymer quoted QD (TAT-QD). Cells were incubated with QD solutionfor 1 h, and then free QD was washed prior to imaging.

Imidazole Based Biocompatible Polymer Coating J. Phys. Chem. C, Vol. 113, No. 52, 2009 21491

Page 9: Imidazole Based Biocompatible Polymer Coating in Deriving

(16) (a) Josephson, L.; Tung, C.-H.; Moore, A.; Weissleder, R. Bio-conjugate Chem. 1999, 10, 186–191. (b) Derfus, A. M.; Chan, W. C. W.;Bhatia, S. N. AdV. Mater. 2004, 16, 961–966. (c) Chen, F.; Gerion, D. NanoLett. 2004, 4, 1827–1832. (d) Santra, S.; Yang, H.; Stanley, J. T.;Hollowway, P. H.; Moudgil, B. M.; Walter, G.; Mericle, R. A. Chem.Commun. 2005, 3144–3146.

(17) (a) Jana, N. R.; Pal, T. AdV. Mater. 2007, 19, 1761–1765. (b) Maran,M. M.; Jana, N. R. J. Raman Spectrosc. 2007, 38, 1326–1331.

(18) (a) Jeong, H.; Lee, B.-J.; Cho, W. J.; Ha, C.-S. Polymer 2000, 41,5525–5529. (b) Schrekker, H. S.; Gelesky, M. A.; Stracke, M. P.; Schrekker,C. M. L.; Machado, G.; Teixeira, S. R.; Rubim, J. C.; Dupont, J. J. ColloidInterface Sci. 2007, 316, 189–195.

(19) Selvan, S. T.; Tan, T. T.; Ying, J. Y. AdV. Mater. 2005, 17,1620–1625.

JP905685A

21492 J. Phys. Chem. C, Vol. 113, No. 52, 2009 Jana et al.