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Phase Transfer and Surface Functionalization of Hydrophobic Nanoparticle using Amphiphilic Poly(amino acid) Koushik Debnath, Kuheli Mandal , and Nikhil R. Jana Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00282 • Publication Date (Web): 29 Feb 2016 Downloaded from http://pubs.acs.org on March 5, 2016

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Phase Transfer and Surface Functionalization of Hydrophobic Nanoparticle using Amphiphilic Poly(amino acid) Koushik Debnath, Kuheli Mandal and Nikhil R. Jana* Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata-700032, India *Corresponding author. E-mail: [email protected]. Telephone: +91-33-24734971. Fax: +91-33-24732805.

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ABSTRACT: Functionalization of nanoparticle with chemical and biochemical is essential for their biomedical and other application. However, most of the high quality nanoparticles are hydrophobic in nature due to surfactant capping and their conversion into water soluble functional nanoparticle via appropriate coating and conjugation chemistry is extremely critical issue. Here we report amphiphilic poly(amino acid)-based one pot coating and conjugation approach that can transform hydrophobic nanoparticle into water soluble nanoparticle functionalized with primary amine, thiol and biomolecule. We have designed amphiphilic polyaspartimide that can anchor hydrophobic nanoparticle through octadecyl groups, leaving the polar polyethylene glycol and aspartimide groups exposed out words. The aspartimide group is then reacted with primary amine containing chemical/biomolecule with the formation of water soluble functional nanoparticle. This approach has been extended to different hydrophobic nanoparticle and biomolecule. Presented approach has advantages over existing approaches as coating and functionalization can be performed in one pot and functional nanoparticles have < 12 nm hydrodynamic size, high colloidal stability and biocompartibility. Developed approach can be used to derive biocompatible nanobioconjugate for various biomedical applications.

Keywords: nanoparticle, quantum dot, coating, bioconjugation, biopolymer, bioimaging, fluorescent probe

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INTRODUCTION Semiconductor nanocrystal or quantum dot (QD) and other nanoparticles are emerging as new class of cellular and subcellular imaging probes.1-7 Compared to molecular probes these nanoparticle based probes have strong absorption and emission, broad excitation window (particularly for QD), low photo bleaching problem and options for unlimited functionalization.3,5 Most of the high quality nanoparticles are synthesized in organic phase, capped with hydrophobic surfactant and soluble in organic solvent but insoluble in water.8-13 Such organic phase synthesis offers the advantage in fine tuning of nucleationgrowth kinetics for controlling nanoparticle size and shape. Examples include iron oxide nanoparticle,10,11 QD composed of ZnS capped CdSe,8 doped semiconductor nanoparticle,12 gold nanoparticle,9 silver nanoparticle9 and fluorescent carbon nanoparticle.13 These hydrophobic nanoparticles need to be transformed into water soluble nanoparticle functionalized with desired chemical/biomolecule on their surface for different biomedical applications. Thus coating and functionalization approaches have been developed to produce functional nanoparticle.1,5,7,14,15 There are two principle surface chemistry/coating approach that transform hydrophobic nanoparticle into water soluble functional nanoparticle. In the first approach, hydrophobic ligands on the nanoparticle surface are completely replaced by hydrophilic molecule/polymer and then used for functionalization via conjugation chemistry.16-24 Thiol based molecules and polymers are most successfully used for this ligand exchange. This approach is simple and produces water soluble nanoparticle of smaller hydrodynamic size. However, nanoparticles demonstrate poor colloidal stability during conjugation chemistry as adsorbed ligand can be easily replaced by other thiols or

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molecules.14 This problem can be partially solved using multidentate thiols24 or via crosslinking of surface molecules.14 In the second approach amphiphilic molecule/polymer is used to coat the original nanoparticle where hydrophobic groups of molecule25 and polymer14,26-38 anchor with hydrophobic ligands on the nanoparticle surface, exposing the hydrophilic groups outward. Resultant hydrophilic nanoparticles are functionalized with molecule of interest via known conjugation chemistry. This process is most successfully applied using commercially available amphiphilic polymaleic anhydride and used in deriving various nanobioconjugates.26-31,38 Most significant advantage of this approach is that nanoparticle can retain the optical property and process lead to colloidally stable nanoparticle without significant aggregation. However, hydrodynamic size of hydrophilic nanoparticle increases significantly due to high molecular weight of polymer used. In addition ampliphilic nature of polymer can induces non-specific interaction with biological interface if the hydrophobic functional groups are exposed outwaord.7 These study show that preferable requirements for advanced coating include cross-linked robust shell around nanoparticle, appropriate coverage of nanoparticle for minimizing nonspecific interaction, use of biocompatible molecule/polymer as coating materials and easier conjugation steps.14 We work on coating and functionalization of nanoparticle for transforming as synthesized nanoparticle into cellular imaging nanoprobe.7,14 Toward this goal we have synthesized multidentate thiol polymer,20,23 modified silica coating for making thin silica shell,19 developed polyacrylate coating,14 polyimidazole coating,14 and adapted amphiphilic polymaleic anhydride coating.14 Among them polyacrylate coating appears most successful in producing water soluble functional nanoparticle with high colloidal

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stability under physiological condition.14 However, hydrodynamic size of nanoparticle increases significantly (~ 5-10 times as compared to 2-6 nm inorganic core) after this coating that limits subcellular targeting application and in some cases the coating introduces cytotoxicity.7 Here we report amphiphilic polyaspartic acid-based one pot coating and functionalization approach of hydrophobic nanoparticle. Polyaspartic acid is a poly(amino acid) that has been transformed into functional polymer for drug delivery carrier39-42 and coating material for nanoparticle.23,35,36 Although amphiphilic polyaspartic acid has been reported as coating material for nanoparticle,35,36 it is limited to few selected nanoparticle and functionalization of such coated nanoparticle is largely unexplored. Here we show that this polymer can be used for coating and functionalization for wide variety of hydrophobic nanoparticles. Presented approach has three distinct advantages over reported amphiphilic polymer coating methods and polyaspartic acid, in particular. First, functionalization of nanoparticle can be adapted in one pot approach during the coating step. Second, coated nanoparticles have high water solubility and hydrodynamic size of functional nanoparticle is relatively small (9-12 nm) as compared to widely used polymaleic anhydride and other polymer coatings. Third, synthesized polymer is biocompatible as it is made of biopolymer and resultant functional nanoparticle can be used for in vitro/in vivo applications.

EXPERIMENTAL SECTION

Materials. L-Aspartic acid, mesitylene, phosphoric acid, octadecylamine, O,O′-bis(2aminopropyl)

polypropylene

glycol-block-polyethylene

glycol-block-polypropylene

glycol (PEG-diamine), poly(maleic anhydride alt-1-octadecene) (Mn--30,000-50,000,

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PMA), arginine, spermine, glucosamine hydrochloride, iron(III) chloride, stearic acid, tetramethylammonium hydroxide, 1-octadecene, 4-methylmorpholine N-oxide, silver acetate, octylamine, oleic acid, tetrabutylammonium bromide, cadmium oxide, trioctylphosphine oxide, selenium, trioctylphosphine, zinc stearate, sulfur, 5,5' dithiobis2-nitro benzoic acid (DTNB) and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich and used as received. Instrumentation. All UV-visible spectra were measured on a Shimadzu UV-2550 UVvisible spectrophotometer using a quartz cell of 1 cm path length. Fluorescence spectra were measured on a SynergyMx (BioTek). The MTT assay was performed using a Synergy TM MX multi-mode microplate reader. NMR spectra were measured with an FT-NMR Bruker (DPX-500 MHz) instrument. Cellular images were captured using an Olympus IX 81 with DP 70 digital camera. Dynamic light scattering (DLS) and zeta potential studies were performed using a model NanoZS (Malvern) instrument. Fourier transform infrared (FTIR) spectra were measured with a Nicolet 6700 FT- IR instrument (Thermo Scientific) using KBr plates. Mass spectra were measured with a Bruker ultraflextreme MALDI-Mass spectrometer equipped with a 337 nm nitrogen laser and 2,5-dihydroxybenzoic acid as matrix. Synthesis of hydrophobic nanoparticle. Hydrophobic γ-Fe2O3 was synthesized using high temperature organometallic approach as described earlier.10 In brief iron stearate was heated to 300 ºC in octadecene in presence of oleylamine. Hydrophobic silver nanoparticle was synthesized using our reported method by borohydride reduction of silver

acetate,

dissolved

in

toluene-dodecylamine

mixture.9

Hydrophobic quantum dot with CdSe core and ZnS shell was synthesized using reported

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method.8 At first CdSe is synthesized at 280 ºC and then ZnS shelling is performed at 200 ºC. All these nanoparticles were purified from free surfactants by standard methods that include acetone/ethanol induced precipitation and chloroform induced redispersion. Finally, chloroform solution of nanoparticle was prepared as stock solution. Synthesis of octadecylamine and polyethylene glycol conjugated polyaspartimide (ODA-PSI-PEG). Polyaspartimide (PSI) was synthesized by our previously reported method with some modification.42 Briefly, 3 g of L-aspartic acid was suspended in 10 mL mesitylene under inert condition, mixed with 165 µL phosphoric acid (88 %) and heated to 150 °C for 4 h. White residue was collected at room temperature and dissolved in dimethylformamide and then excess water was added to precipitate the polysuccinimide. The

precipitate

was

washed

with

water

to

remove

phosphoric

acid

and

dimethylformamide and then washed with methanol for several times. Finally, solid polysuccinimide was dried in vacuum. Next, 250 mg PSI was dissolved in 15 mL dry dimethylformamide, mixed with 135 mg octadecylamine and heated at 70 °C for 24 h under inert atmosphere. The solution was cooled to room temperature and mixed with 600 µL PEG-diamine and further heated at 80 °C under inert atmosphere for 24 h. The resultant ODA-PSI-PEG was collected by the addition of diethyl ether. The precipitate was washed with methanol– diethyl ether mixture (1:1) for several times and dried under vacuum. Coating and bioconjugation of hydrophobic nanoparticle. About 0.2 mL chloroform solution of nanoparticle and 0.2 mL chloroform solution of PSI-ODA-PEG were mixed and sonicated for 15 min. Next, this mixed chloroform solution was drop wise added into 2.0 mL hot (50-60 °C) aqueous solution within 5 min under vigorous stirring condition.

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The composition of aqueous solution was varied by using bicarbonate solution (pH ~10) or biocarbonate solution of biomolecule (e.g. arginine, spermine, glucosamine and cysteamine). The chloroform was evaporated within 10 min and nanoparticle was transferred into water with the resultant formation of aqueous nanoparticle solution. The solution was cooled to room temperature and kept for another 2 h. During this time succinimide group reacts with primary amine or hydrolyses. The solution was then dialyzed against distilled water using dialysis membrane (MWCO 12,000). Coating of hydrophobic nanoparticle by PMA. At first 40 mg poly(maleic anhydride alt-1-octadecene) was dissolved in one mL chloroform through sonication. Next, one mL chloroform solution of hydrophobic nanoparticle was mixed well and sonicated for 30 min. Well sonicated solution was then kept in air for overnight for evaporation of chloroform. After complete evaporation of chloroform, bicarbonate (pH 10) solution of PEG-diamine is added and kept for another 2 h under stirring condition. Basic solution pH and longer time helps ring opening reaction of maleic anhydride with primary mine groups of PEG-diamine. The colloidal solution is then dialyzed to remove free reagents. Functionalization test: a)

Phenanthrenequinone

test

for

arginine.

Ethanol

solution

of

9,10-

phenanthrenequinone (150 µM) was prepared. Next, 50 µL of arginine functionalized nanoparticle was taken in a vial and mixed with 150 µL of 9,10-phenanthrenequinone solution followed by addition of 25 µL NaOH solution (2.0 M). Next, the mixture was incubated at 60 °C for 3 hrs. Next, 100 µL of this solution was mixed with 100 µL HCl (1.2 N) and allowed to stand for one hour at room temperature in dark condition. After that fluorescence was measured at an excitation wavelength of 312 nm.

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b) Anthrone test. Anthrone solution (0.2 wt %) was prepared in 80 % conc. H2SO4. Next, 2 mL anthrone solution was mixed with 200 µL solution of glucosamine functionalized nanoparticle and heated in water bath for 15 min. Next, the mixture was cooled in ice cooled water and absorption spectra were measured. c) Fluorescamine test. At first acetone solution of fluorescamine (one mg/mL) and aqueous solution of functionalized nanoparticle in borate buffer solution of pH 9.5 were prepared separately. Next, equal volume (0.2 mL) of fluorescamine solution and nanoparticle solution were mixed well and fluorescence was measured at an excitation wavelength of 400 nm. d) Thiol test. Acetone solution of 5,50 -dithiobis-2-nitrobenzoic acid (DTNB) (0.5 mg/mL) and aqueous solution of functionalized nanoparticle in tris buffer solution of pH 8.8 were separately prepared. Next, 0.1 mL of DTNB solution was added to one mL nanoparticle solution and mixed well. Next, the thiol characteristic absorbance peak at 410 nm was measured in UV spectrometer. Cell labeling study of functional nanoparticle. The Chinese Hamster Ovary (CHO) cell line was cultured on 24-well plates using 0.5 mL of Dulbecco’s Modiﬁed Eagle’s Medium (DMEM) medium with 10 % (v/v) fetal bovine serum (FBS). After overnight, cells were attached on culture plate. Next, 100 µL of functional QD was added. After 3 h of incubation at 37 °C, cells were gently washed twice with phosphate buffered saline (PBS) and fresh DMEM medium was added. Cells were then imaged under ﬂuorescence microscope using blue excitation. Cell viability study by MTT assay: Trypsinized CHO cells were resuspended in DMEM culture medium. The CHO cells were seeded to 24-well plates with 500 µL DMEM in

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each plate. Next, 200 µL of each functional nanoparticle solution was added and incubated at 37 °C and under 5 % CO2. After incubation for 24 h, 50 µL of MTT solution (5 mg/mL) was added to each well and incubated for 4 h. The supernatant medium was discarded and 500 µL of sodium dodecyl sulfate solution (8 g dissolved in 30 mL waterDMF mixture with 1:1 volume ratio) was added. The plates were kept for 2 h and then the absorbance was recorded at λmax 550 nm. The optical density was directly correlated with cell quantity and cell viability was calculated by assuming 100 % viability for the control set without any sample.

RESULTS Design and synthesis of amphiphilic poly(amino acid). Chemical structure of amphiphilic poly(amino acid) is shown in Scheme 1. It has polyaspartimide (PSI) structure derived from aspartic acid. PSI has polysuccinimide backbone with some of the succinimide groups are opened with octadecylamine (ODA) and PEG-diamine. Coating mechanism of resultant ODA-PSI-PEG polymer around hydrophobic nanoparticle surface is shown in Scheme 1. The octadecyl group intercalates with the hydrophobic surfactant layer around hydrophobic nanoparticle and thus would coat the nanoparticle. In contrast polar succinimide and PEG groups would expose outside. After coating, the succinimide groups are used for bioconjugation via ring opening reaction with primary amines of desired biomolecule.23 Succinimide is also reacted with cystamine to generate thiol functionalized nanoparticle. Similarly reaction with PEG-diamine introduces PEG and primary amine functional groups where PEG minimize nonspecific interaction of

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nanoparticle and primary amine can be used for functionalization via conventional conjugation chemistry. Amphiphilic ODA-PSI-PEG is synthesized following our reported method.42 In brief PSI is prepared via phosphoric acid catalyzed thermal polycondensation of aspartic acid. Next, some of the succinimide groups of PSI are reacted with ODA and PEGdiamine in two successive steps with the resultant formation of ODA-PSI-PEG. The molar ratio of PSI, ODA and PEG-diamine are appropriately adjusted in such a way that degree of substitution of octadecylamine and PEG is in the range of 10-20 mole %. The reaction condition has been adjusted so that molecular weight of final polymer remain < 30 KD (Supporting Information, Figure S1) and succinimide groups remain intact (do not hydrolyze) so that they can be used for conjugation chemistry during the coating step. One pot coating, phase transfer and functionalizaton of hydrophobic nanoparticle using amphiphilic poly(amino acid). One pot coating and functionalization of hydrophobic nanoparticle has been developed using the ODA-PSI-PEG based amphiphilic poly(amino acid). We have used three different hydrophobic nanoparticles for this experiment. The nanoparticles include γ-Fe2O3, Ag and ZnS capped CdSe quantum dot (QD) of 4-6 nm size (Figure 1). These hydrophobic nanoparticle are selected as they are commercially available and widely used as optical probe. These nanoparticles are synthesized using well established method.8-10 Nanoparticles are purified after synthesis and separated from free surfactants via standard method. Finally, nanoparticle solutions are prepared in chloroform. Details of one pot coating, phase transfer and functionalization is shown in Scheme 2 and results are summarized in Figure 1, 2 and Table 1. An aqueous solution of

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chemical/biomolecule is prepared in bicarbonate buffer of pH 10.0 and kept under stirring and warm (50-60 °C) condition. The composition of aqueous solution is varied by using different biomolecule (e.g. arginine, spermine, glucosamine) or chemical such as cysteamine or simply bicarbonate buffer. Next, chloroform solution of a mixture of ODA-PSI-PEG and hydrophobic nanoparticle is drop wise added into this solution. Warm condition offers rapid evaporation of chloroform and nanoparticle is transferred into aqueous phase via polymer coating. The solution is cooled to room temperature and kept for another 2 h. Basic solution pH and longer time helps ring opening reaction of succinimide with primary mine groups of biomolecule/cysteamine. The colloidal solution is then dialyzed to remove free reagents and directly used for characterization and application. Fluorescence quantum yield of QD has been measured using quinine sulfate as standard. Various control experiments have been performed in order to confirm the role of ODA-PSI-PEG in transforming hydrophobic nanoparticle into water soluble nanoparticle and to minimize nanoparticle aggregation during this coating and phase transfer processes. (Supporting Information, Table S1 and Figure S2-S7) If ODA-PSI-PEG is not used during coating stage, nanoparticle becomes water insoluble. If chloroform is evaporated first from the mixture of nanoparticle and ODA-PSI-PEG followed by mixing with basic solution of functional molecule, then hydrodynamic size of polymer coated nanoparticle becomes larger due to extensive particle aggregation. (Supporting Information, Figure S3) In addition if room temperature (25 °C) is used for amphiphilic poly(amino acid) coating as described in Scheme 2 the chloroform evaporation takes longer time (>15 min) and resultant hydrodynamic size becomes larger due to particle

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aggregation. (Supporting Information, Figure S4) It is well known that hydrophobic nanoparticles are soluble in organic solvents such as chloroform, toluene, cyclohexane and if the solvent is evaporated they can be again solubilized in same organic solvent as long as they are capped with hydrophobic surfactant. However, they can never be solubilized in water. Thus water solubility of these hydrophobic nanoparticles indicates that their surface is modified by ODA-PSI-PEG. FTIR study confirms the presence of ODA-PSI-PEG in coated nanoparticle, suggesting that polymer is anchored with nanoparticle as shown in Scheme 1. (Supporting Information, Figure S7) We have done additional control experiments to minimize by-products such as empty micelles and encapsulation of multiple nanoparticle in a single micelle. In particular we have varied the nanoparticle to polymer weight ratio, determined the minimum concentration for complete phase transfer and then used this condition for nanoparticle phase transfer. (Supporting Information, Figure S2) Thus unnecessary use of excess polymer minimizes the formation of empty micelle. TEM-based monitoring of coated nanoparticle shows mostly isolated nanoparticle, suggesting that conditions used in Scheme 2 provide minimum multiparticle encapsulations in a single micelle. We have also tried to extend this approach for larger size nanoparticles and found that it has also worked for 25 nm γFe2O3 nanoparticle. (Supporting Information, Figure S6) Functionalization condition has been optimized in terms of solution pH, reaction temperature and reaction time. It is known that succinimide ring opening by primary amine is favored at higher temperature and basic solution pH. Considering the fact that many biomolecules are unstable under high temperature we have used moderate temperature of 50-60 °C. We have used bicarbonate buffer of pH 10 and 2 h reaction time

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as optimum reaction condition for conjugation chemistry. Following this approach we have successfully synthesized polymer coated nanoparticle functionalized with primary amine, thiol, PEG, arginine, spermine and glucosamine. Primary amine and PEG functionalization are achieved by succinimide ring opening reaction with PEG-diamine. Thiol functionalization is achieved by succinimide ring opening reaction with cysteamine. Different biofunctionalization is achieved by succinimide ring opening reaction with arginine, spermine and glucosamine. Functionalization of each type has been confirmed using well known methods.4346

(Figure 3 and Supporting Information, Figure S8) 9,10-Phenanthrenequinone-based

test has been used for determination of arginine functionalization where arginine produces a blue fluorescent product after reacting with 9,10-phenanthrenequinone.43 Figure 3a shows the appearance of blue fluorescence after reaction with 9,10phenanthrenequinone which confirms that arginine is successfully attached with the nanoparticle surface. Anthrone test has been used for confirmation of glucosamine functionalization via formation of blue-green coloration by the reaction between anthrone and furfural that is produced from carbohydate.44 Figure 3b shows the appearance of such color in anthrone test, confirming that nanoparticle is successfully functionalized with glucosamine. DTNB test has been used for thiol functionalization of nanoparticle.45 DTNB forms yellow colour after reacting with thiol and similar absorption band is also observed for thiol functionalized nanoparticle. (Figure 3c) Functionalization of nanoparticle with PEG-diamine and spermine produces nanoparticle terminated with primary amine. Thus testing of primary amine has been used to confirm the functionalization with PEG-diamine and spermine. Fluorescamine test has been used for

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detection of primary amines.46 Fluorescamine reacts with primary amine to form blue fluorescent product. Figure 3d shows that PEG-diamine and spermine functionalized nanoparticle produce strong blue emission after reaction with fluorescamine, suggesting that nanoparticle is functionalized with PEG-diamine and spermine. Property of functional nanoparticle. Property of functional nanoparticles has been extensively studied in order to judge if they can be used in various biomedical applications. Nanoparticle should have good colloidal stability under physiological condition so that they interact with bioenvironment without precipitation. We have tested the colloidal stability in different buffer solution, high salt concentration and cell culture media and compared with the widely used PMA-based coating approach. (Figure 4 and Supporting Information, Table S1 and Figure S5) Result shows that nanoparticles are stable under those conditions for weeks/months and this colloidal stability is comparable to the widely used PMA coating approach. Hydrodynamic size of nanoparticles has been investigated by dynamic light scattering and compared with the hydrodynamic size of polymer before and after forming micelle. (Figure 4 and Supporting Information, Figure S6) Results show that size of functional nanoparticle ranges between 9-12 nm. Considering the 3-5 nm hydrodynamic size of the presented polymer having molecular weight of 25-30 KD, it can be assumed that only a monolayer of polymer is capped around 4-6 nm nanoparticle surface. In contrast PMA coating approach produces particle with larger hydrodymamic size --- typically in the range of 10-58 nm. (Figure 4 and Supporting Information, Table S1) Surface charge of nanoparticles has been determined using zeta potential measurements. (Table 1 and Supporting Information, Table S1) All the functional

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nanoparticles show negative surface charge in the studied pH range of 4.5, 7.4 and 10.0. However, the extent of anionic charge depends on the nature of functionalization and solution pH but does not significantly depend on the nature of core nanoparticle. Glucosamine, cysteamine and PEG functionalized nanoparticles show charge in the range of -13 mV to -27 mV. Presence of dissociated carboxylate groups is responsible for such anionic surface charge. Arginine functionalized nanoparticle show charge in the range of -10 mV to -12 mV. Relatively lower value is due to the presence of protonated guanidine group that partially balance the carboxylate anions. In contrast spermine functionalized nanoparticle shows varying surface charge from -2 mV to -23 mV as the pH increases from 4.5 to 10.0. Low surface charge at pH 4.5 can be explained by the presence of protonated primary and secondary amines which partially balance the anionic charge generated by carboxylate. As the pH increases the protonation state of amines decreases that results the increased anionic surface charge. Biomedical application potential of functional nanoparticle has been explored in order to demonstrate the potential of this coating and functionalization approach. PEG, SH and glucose functionalized nanoparticles have low non-specific interaction with cell and do not label cells. In contrast arginine and spermine functionalized nanoparticle can label cells due to cationic functional groups (protonated primary/secondary amine) on their surface. Figure 5 shows representative result using functional QD and CHO cells where fluorescence property of QD has been used to study the cell–nanoparticle interaction. Typically, cells are incubated with functional QD for 3 h and then washed cells are imaged under fluorescence microscope. Results show that PEG functionalized QD does not label cells but spermine and arginine functionalized QD label cells. This

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result indicates that functional nanoparticle can be used for specific cell labeling application. Cytotoxicity of functional nanoparticles has been studied via MTT assay in order to demonstrate that polymer-based coating and functionalization of nanoparticle does not introduce additional toxicity. Results are summarized in Figure 6, showing that all functional nanoparticle are reasonably less toxic in the concentrations higher than labeling concentration.

DISCUSSION Functionalization of nanoparticle is a critical issue for their application. Although many methods are developed for coating and functionalization, improved/simplified methods are still necessary. Present method focus on three aspect of coating and conjugation of nanoparticle. First, coating polymer is designed from biomolecule so that it does not introduce additional toxicity. The cytotoxicity study proves that our coating approach is in fact biocompatible. Second, functionalization is developed during coating step via one pot approach that simplifies the functionalization. In conventional approach hydrophobic nanoparticle is converted into water soluble nanoparticle and then linked with desired molecule via conjugation chemistry. In contrast presented approach uses reactive functional group (succinimide) of polymer for conjugation and does not require additional step and conjugation reagent. Third, attempts have been made to keep the hydrodynamic size small. The hydrodynamic size of presented functional nanoparticles is 9-12 nm which are relatively small than conventionally used polymer coated nanoparticles. In particular the hydrodynamic size is > 15 nm for well known polymaleic

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anhydride-based coating of nanoparticle and > 30 nm for polyacrylate coated nanoparticle which are widely used in deriving functional nanoparticle.14 We have demonstrated the functionalization of nanoparticle with primary amine, thiol, PEG and different biomolecules. There are several reasons for these functionalization studies. Primary amine and thiol functionalized nanoparticles are routinely used for biofunctionalization of nanoparticle using commercially available chemicals and protocols.14 Interest on PEG functionalization is due to their known property to minimize non-specific binding interaction.7 However, main interest was that if biofunctionalization can be achieved during phase transfer step via one pot approach which can greatly simplify the bioconjugation steps. We have convincingly demonstrated that different hydrophobic nanoparticle can be bioconjugated during coating step and they can be used as cell imaging probe. However, the proposed method has following limitations that need to remember. First, conjugation condition requires higher temperature and high pH and many biochemicals may not survive under such condition. Second, conjugation requires higher concentration of biomolecule. Considering the fact that many biochemicals are costly this approach may not be suitable. Third, coated nanoparticle are inherently anionic is nature due to carboxylate groups of polymer. This anionic charge can induce electrostatic repulsion with cellular environment and inhibit interaction with cell unless anionic charge is partially neutralized by cations. For example arginine and spermine functionalized nanoparticle label cells as these groups offer positive charge and balances some of the anions. CONCLUSION

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We have designed and synthesized amphiphilic polyaspartimide having octadecylamine and polyethylene glycol functional group. This polymer can be used for coating and functionalization of hydrophobic nanoparticle via one pot approach. The polymer anchor with hydrophobic nanoparticle surface through octadecylamine groups and out wards exposed aspartimide group is then reacted with primary amine containing chemical/biomolecule with the formation of water soluble functional nanoparticle. Following this approach we have transformed hydrophobic iron oxide/quantum dot/silver nanoparticle into water soluble nanoparticle functionalized with primary amine, thiol and biomolecule. This approach can be extended to other hydrophobic nanoparticle and biomolecule.

ASSOCIATED CONTENT Supporting Information. Details of characterization of polymer, polymer coated nanoparticle, colloidal stability study of functional nanoparticles and comparative study using PMA coating. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected]

Notes The authors declare no competing financial interests. ACKNOWLEDGEMENT

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The authors acknowledge DST, government of India for financial assistance. (No. SB/S1/IC-13/2013) K.D. acknowledges IACS for providing research fellowship and K.M. acknowledges CSIR, India for research fellowship.

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Table 1. Property of different functional nanoparticles derived from hydrophobic nanoparticle using amphiphilic poly(amino acid)-based coating and functionalization. Nanoparticle

γ-Fe2O3

Ag

QD

Conjugated biomolecule/ chemical arginine spermine glucosamine cysteamine, SH PEG, NH2 arginine spermine glucosamine cysteamine, SH PEG, NH2 arginine spermine glucosamine cysteamine, SH PEG, NH2

Hydrodynamic size at pH 4.5 7.4 10.0

Surface charge at pH 4.5 7.4 10.0

Application

9-12 nm

-10 -3 -18 -18 -27

-10 -16 -27 -21 -30

-11 -18 -23 -22 -32

bioprobe bioprobe bioprobe functionalization functionalization

9-12 nm

-11 -3 -16 -13 -28

-10 -14 -26 -22 -30

-12 -22 -23 -24 -34


9-12 nm

-11 -2 -16 -16 -27

-9 -16 -24 -20 -33

-10 -22 -22 -23 -35


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Scheme 1. Synthesis approach of amphiphilic poly(amino acid), coating principle on hydrophobic nanoparticle surface and functionalization of coated nanoparticle via reaction between succinimide group of polymer and chemical/biomolecule.

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Scheme 2. One pot coating and functionalization approach that involves drop wise addition of chloroform solution of mixture of hydrophobic nanoparticle and polymer into hot aqueous solution of chemical/biomolecule at basic pH. Chloroform rapidly evaporates, nanoparticle is transferred into water via polymer coating and chemical/biomolecule reacts with coated polymer. Finally solution is dialyzed and aqueous solution of functional nanoparticle is formed.

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a)

d) γ-Fe2O3

30

γ-Fe2O3

30

Number %

Number %

γ-Fe2O3

15

0

15

0

3 4 5 6 7 8 9

3 4 5 6 7 8 9

Size (nm)

b)

Size (nm) e)

50

CdSe-ZnS

25

γ-Fe2O3

γ-Fe γ-Fe 2O 3 3 2O

50

Number %

Number %

CdSe-ZnS

25

0

0

5

5 6 7 8 9 10 11

6

c)

7

8

9 10

Size (nm)

Size (nm)

f)

Ag

Ag

γ-Fe γ-Fe 2O 3 3 2O

30

Number %

30

Number %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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15

0

15

0

2 3 4 5 6 7

2

Size (nm)

3

4

5

Size (nm)

6

7

Figure 1. TEM images of as synthesized hydrophobic γ-Fe2O3, CdSe-ZnS and Ag nanoparticle (a,b,c) and same nanoparticles after coating with amphilic poly(amino acid) (d,e,f). Respective size distribution histograms are shown using more than 100 particles. .

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1.0

a)

chloroform water γ-Fe2O3

Absorbance

2.5 2.0

in CHCl3

1.5

chloroform water

b) γ-Fe2O3 in H 2O

Ag in CHCl3

Absorbance

3.0

0.5

1.0

Ag in H 2O

0.5 0.0 300

400

500

600

700

0.0 300

800

c)

chloroform water

1.5

QD in CHCl3

1.0

QD in H2O

0.5 0.0 300

400

500

600

700

800

Fluorescence intensity (a.u)

2.0

400

500

600

700

800

Wavelength (nm)

Wavelength (nm)

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60000

d)

chloroform water

40000 QD in CHCl3

20000

0 500

Wavelength (nm)

550

600

QD in H 2O

650

700

Wavelength (nm)

Figure 2. UV-visible absorption/fluorescence spectra and digital images of γ-Fe2O3 (a), Ag (b) and QD (c,d) before and after polymer coating, showing that optical property of the nanoparticles is retained after polymer coating.

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Figure

3.

Evidence

of

surface

functionalization

Page 32 of 36

of

nanoparticle.

a)

9,10-

Phenanthrenequinone test for detection of arginine. The appearance of blue fluorescence after reaction with 9,10-phenanthrenequinone confirms that arginine is successfully attached with the nanoparticle surface. b) Anthrone test for detection of glucosamine functionalization of nanoparticle. The appearance of blue-green color confirms that nanoparticle is successfully functionalized with glucosamine. c) DTNB test for detection of thiol functionalization of nanoparticle. Formation of yellow colour after reacting with thiol confirms thiol functionalization of nanoparticle. d) Fluorescamine-based primary amine test for confirmation of PEG and spermine functionalization of nanoparticle. Appearance of strong blue emission after reaction with fluorescamine suggests that nanoparticle is functionalized with PEG and spermine.

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a)

γ-Fe2O3

QD

PMA Poly(amino acid)

20

10

20

10

0

0 0

20

40

60

Size (nm)

80

0

100

20

40

60

80

100

Size (nm)

Ag PMA Poly(amino acid)

20

10

Number (%)

50

30

Poly (amino acid)(control)

40 30 20 10 0

0 0

b)

PMA Poly(amino acid)

30

Number (%)

Number (%)

30

Number (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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20

40

60

80

100

0

20

40

60

Size (nm)

Size (nm)

Poly(amino acid) coated

PMA coated

AgPEG

γ-Fe2O3PEG

QDPEG under UV

AgPEG

γ-Fe2O3PEG

80

100

QDPEG under UV

Figure 4. a) Dynamic light scattering (DLS)-based size distribution of poly (amino acid) and PMA coated nanoparticles. All the coated nanoparticles are synthesized using Scheme 2 and functionalized with PEG. The size of poly (amino acid) below its critical micelle concentration is shown as control. b) Colloidally stable nanoparticle coated with poly(amino acid) or PMA and functionalized with PEG taken in phosphate buffer of pH 7.4 having 0.1 M NaCl.

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QD-PEG

BF

50 microns

50 microns

F

50 microns

50 microns

merged

F

50 microns

merged

50 microns

50 microns QD-arginine

QD-arginine

F

50 microns QD-spermine

QD-spermine

QD-arginine

BF

QD-PEG

QD-PEG

QD-spermine

BF

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merged

50 microns

Figure 5. Fluorescence microscopic image of CHO cells labeled with functional nanoparticle. Cells are incubated with nanoparticle for 3 h and then washed cells are imaged under blue excitation. Results show that spermine and arginine functionalized nanoparticle label cells but PEG functionalization inhibits cell labeling.

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100

80

60

% Viability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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40

20

0

Figure 6. MTT-based cytotoxicity data of functional nanoparticles. Cells are incubated with nanoparticle with 0.5 mg/mL (5 times as compared to labeling concentration) of final concentration for 24 h and then used for cytotoxicity study.

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Table of Contents (TOC)

+

NH NH N O O x O n-x-y NH NH O

3

y

H3C

amphiplilic polyaspartimide

O

3

C18H37

39

hydrophobic nanoparticle

O

O

O

One pot coating, bioconjugation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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O

CH3 O

γ-Fe2O3

Ag

H2N CH3

QD under UV

QD

nanobioconjugate

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