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Hyperbranched polyglycerol modified fluorescent nanodiamond for biomedical research
Hyperbranched polyglycerol modified fluorescent nanodiamond for biomedical research Jean-Paul Boudou, Marie-Odile David, Vandana Joshi, Housam Eidi, Patrick A. Curmi PII: DOI: Reference:
S0925-9635(13)00138-6 doi: 10.1016/j.diamond.2013.06.019 DIAMAT 6148
To appear in:
Diamond & Related Materials
Received date: Revised date: Accepted date:
17 April 2013 23 June 2013 29 June 2013
Please cite this article as: Jean-Paul Boudou, Marie-Odile David, Vandana Joshi, Housam Eidi, Patrick A. Curmi, Hyperbranched polyglycerol modified fluorescent nanodiamond for biomedical research, Diamond & Related Materials (2013), doi: 10.1016/j.diamond.2013.06.019
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ACCEPTED MANUSCRIPT Hyperbranched polyglycerol modified fluorescent nanodiamond for biomedical research Jean-Paul Boudou*,¥, Marie-Odile David, Vandana Joshi, Housam Eidi, and Patrick A. Curmi*
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Institut National de la Santé et de la Recherche Médicale, UMR829, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université Evry-Val d’Essonne, Evry 91025, France
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* Corresponding authors:
Jean-Paul Boudou: Laboratoire Aimé Cotton, CNRS, UPR3321, Université Paris-Sud, 91405 Orsay cedex, France, Tel : +33 1 47 09 31 34, +33 6 44 26 42 78, Email: [email protected]
Present address: LAC, CNRS, UPR3321, Université Paris-Sud, 91405 Orsay cedex, France, [email protected].
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¥
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Patrick A. Curmi: LSABNP, INSERM, UMR829, Université Evry-Val d’Essonne, Evry 91025, France, Tel : +33 1 69 47 03 23, Fax : +33 1 69 47 02 19, Email: [email protected]
Abstract
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The aim of the present work was to functionalize fluorescent nanodiamond by covalent
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grafting with hyperbranched polyglycerol. Fluorescent nanodiamond, derived from high pressure high temperature diamond microdiamond, was oxidized then thermally reacted with
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pure glycidol in the absence of catalyst. Thermal polymerization of glycidol was notably faster on the nanodiamond surface as a result of a surface initiation of the isothermal ring opening polymerization. Interestingly, the aqueous dispersion of the resulting nanoparticles
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appeared stable at high ionic strength. Furthermore, the fluorescent nanodiamond grafted with hyperbranched polyglycerol displayed several hydroxyl end–groups which could be further derivatized by carboxylation or carbamatization and subsequently conjugated with protein linked via an amide bound. Notably, nanodiamonds retain their unique fluorescent characteristics. This work suggests that fluorescent nanodiamond coated with hyperbranched glycidol could be promising in biomedical research where aqueous dispersion of fluorescent nanoparticles stable in physiological medium are in high demand to label, track and quantify biomolecules. Keywords: Nanodiamond Fluorescence Polyglycerol Bioconjugation Biomolecule labeling
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1. Introduction
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In recent years many papers have appeared dealing with the surface chemistry of different
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types of nanodiamonds [e.g.1,2,3,], but fewer papers have been devoted to the covalent grafting of polymeric spacers on nanodiamonds for improving their dispersion stability in water and avoiding aggregation of the nanocrystals in physiological buffer solutions as well as
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hypertonic aqueous media needed in selected biochemical assays. It has been shown that
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polyether backbone polymers grown in situ can cover densely the nanodiamond surface [4,5,6]. The resulting nanoparticle can be dispersed stably in physiological solution and shows
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an excellent biocompatibility. Nanodiamonds thus appear eligible to the emerging area of the
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polyglycerol systems. Their green synthesis, biocompatibility, very low toxicity and high functionality make them very valuable intermediates for biomedical research and
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pharmaceutical development [e.g. 7,8,9]. In the present work, we prepared a new fluorescent nanohybrid via the very simple and
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industrially scalable thermal treatment with glycidol of a homemade NIR fluorescent nanodiamond (FND) [10,11]. We further showed that the resulting material is adapted to physiological medium and detectable in cultured cell using fluorescence microscopy. The modified fluorescent nanodiamond was covalently conjugated with a model protein. 2. Experimental Section 2.1. Synthesis 2.1.1. Products
Reagent grade chemicals were purchased from Sigma–Aldrich and used as received. Fluorescent nanodiamond was prepared from a synthetic micron diamond powder [10,11].
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ACCEPTED MANUSCRIPT After electron irradiation (10 MeV, 5 x 1019 e-/cm2), the sample was annealed at 800 °C under vacuum. To convert microdiamond into smaller particles, we first used nitrogen jet milling
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autogeneous micronization. A nanomilling procedure was then used to convert the jet milling
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product into a mixture of fluorescent nanodiamond and contaminant. The purification was
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done as previously described with HF/HNO3 at 150 °C for 48 h [10]. The product was subsequently treated in aqueous solution of NaOH (50%, v/v) or perchloric acid (70%, v/v) at 190 °C for 48 h to improve the cleaning and to saturate the surface with surface oxygen
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groups. The product of the treatment with NaOH was named FND–NaOH, and the product of
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the treatment with HClO4 was named FND-HClO4—in short FND. This last product was modified with hyperbranched polyglycerol. The NaOH hydrolysis was done for a better
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understanding of the surface oxide groups introduced by the hot acid treatments.
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2.1.2. Procedure
A hyperbranched polyglycerol (HPG) shell was grown on nanodiamond surface based on the
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method proposed by Lukehart et al. [12] and by Komatsu et al. [4,5] In this method, the surface groups (active hydrogen–containing species) from the diamond initiate the glycidol
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neutral ring opening polymerization (ROP) during heating. The diamond material and conditions for polymerization being either a cubic–diamond nanocrystal (derived from high pressure high temperature diamond) in the absence of catalyst at 140 °C for 20 h [5], or detonation nanodiamond, either in the absence (at 50 °C) or the presence of catalyst (tertiary amine) at 75 °C for 24 h [12]. In this study, the neutral ROP was initiated by surface anhydride groups, carboxyl and hydroxyl groups, previously introduced on fluorescent cubic– diamond nanocrystals (FND). The experimental conditions used here were similar to those used by Zhao et al. [4] but importantly much less drastic (2 h) to prevent autopolymerization of glycidol. The procedure is different from the surface glycidol initiated anionic ROP
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ACCEPTED MANUSCRIPT [12,13,14]. The reaction resulted in HPG grafted FND and free HPG which could be removed by washing with methanol.
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FND powder (5 mg) was dispersed in glycidol (3 mL) by means of an ultrasonic bath at room
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temperature. The mixture was placed in 2 mL screw top vials with Teflon septa. Headspace
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was swept with argon and the vials were placed in a preheated oven at 140 °C and left at this temperature for either 2 or 20 h. The resulting gel was diluted with methanol (40 mL) in an
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ultrasonic bath, and the product (FND–HPG) was isolated by 15000 g centrifugation and washed free of unbound polymer (HPG) by three repeated dispersions in methanol and
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15000 g centrifugation cycles. These three methanol washing cycles were followed by three repeated dispersion in water and 15000 g centrifugation cycles. The final FND–HPG pellet
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Ultra–4 centrifugal filter units.
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was finally dispersed in water, and then washed with a high volume of water on Amicon
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2.1.3. Derivatization and protein grafting:
FND-HPG-COOH was obtained by the reaction of FND-HPG with succinic anhydride in pyridine under argon at 70 °C for 1 h. The reaction mixture was centrifuged at 15000 g and
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the pellet washed several times by dispersion in ethanol and centrifugation at 15000 g. Final FND-HPG-COOH pellet was dispersed in water, and then washed with water on Amicon Ultra-4 centrifugal filter units. FND–HPG hydroxyl end–groups were activated using 1,1'– Carbonyldiimidazole (CDI) [15]. FND–HPG was first dried, and then immersed in a 0.05 to 0.25 M solution of CDI in anhydrous THF for 12 h under an argon atmosphere. After activation, FND–HPG–CDI was isolated by centrifugation with THF and water. Finally, it was washed with water on Amicon Ultra-4 centrifugal filter units. Coupling Reaction between FND–HPG–CDI and bovine serum albumin (BSA), taken as a model protein, was carried out by stirring 100 g of BSA together with 10 mg of FND–HPG–CDI in borate buffer (pH 8.5) under argon at room temperature for 48 h. FND–HPG–BSA was then isolated by sequential
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ACCEPTED MANUSCRIPT centrifugation at 15000 g for 5 min in water, and then washed with water on Amicon Ultra–4 centrifugal filter units.
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2.2. Attenuated Total Reflectance–Fourier transform infrared spectroscopy (ATR–FTIR)
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Spectra were acquired by means of ATR–FTIR spectrometry. The FND and FND derivative dispersions in water were dried under an argon flow on a PIKE Technologies GladiATR with a single–reflection (angle of incidence 45°) diamond crystal plate isothermally heated at
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75 °C. FTIR spectra were collected on a Thermo Nicolet 380 spectrometer equipped with a DTGS temperature–stabilized coated detector (4000 to 600 cm−1, spectral resolution: 4 cm−1,
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200 accumulations). ATR–FTIR peak frequencies can be shifted due to the environment, and peak intensities depend on the ATR mode. Loss of hydration water and increased salt
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interaction with the dry film may cause of the band position. For instance, the asymmetric
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stretching vibration of carboxylate groups is typically 10–40 cm-1 lower in water than in solid
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[16]. Bearing in mind these possible shifts, we made the structural assignment of the more critical FTIR bands based on published data [17,18].
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2.3. Dynamic light scattering and zeta potential measurement Nanoparticles were dispersed in water. The pH and the conductivity of the dispersion were controlled by an autotitrator Delsa™Nano AT and zeta potential measurements were made using a Delsa Nano C Particle Size apparatus, (Beckman Coulter) equipped with two 658 nm laser diode sources (30 mW power each) and a temperature controller (from 15 to 90 °C). Size distribution was measured by dynamic light scattering at a scattering angle of 165°. Scattering data were collected for 70 individual measurements at a constant scattering angle and averaged for each sample. They were fitted using volume-weighted cumulative analysis to estimate the diffusion coefficient of the micelles in solution. The particle zeta potential was determined by measuring the electrophoretic movement of charged particles under an applied
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2.4. Epifluorescence microscopy and microspectrofluorimetry
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Fluorescence images were taken and spectroscopic measurements made using a Zeiss Axioplan 2 microscope with a 63x, 1.4 NA oil immersion objective. A 532 nm laser beam (200 mW power) was used as the illuminating source and was guided to the microscope by an
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optic fiber. Fluorescence images were obtained with a Princeton Instruments EMCCD Camera Rolera em–c², with typical exposure times from 100 to 500 ms. Spectra were acquired
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by focusing the fluorescent nanodiamond emission from the microscope onto an Acton SP2150i spectrometer (Princeton instruments), and detected with a PIXIS–100B–eXcelon
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CCD camera (Princeton instruments).
The fluorescent nanodiamonds were observed either in water or in fixed HeLa cells, taken as
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model biological cell. HeLa cells were grown on sterile glass coverslips in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 5% (v/v) fetal bovine serum (FBS),
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2 mM L-glutamine and 1% antibiotics (penicillin and streptomycin). Cells were maintained in a humidified 5% CO2 atmosphere at 37 °C. Twenty–four hours after the beginning of the culture, FND-HPG nanodiamond dispersion (0.2 g/L in pure water) was added to the cells in the culture medium at a fifty–fold dilution (10 L of FND–HPG nanodiamond stock in 500 µL final volume of DMEM for a 3.6 cm2 cell culture dish). Cells were cultivated for an additional 24 hours, and then washed three times with phosphate buffered saline (PBS) to remove unincorporated nanodiamonds. The cells were then fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at 37°C. The glass coverslips were then mounted on microscope glass slides and processed for fluorescence microscopy. Fluorescence images were analyzed for fluorescence intensity using the ImageJ [19].
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ACCEPTED MANUSCRIPT 2.5. SDS-PAGE analysis One-dimensional electrophoresis was performed on 4% polyacrylamide gels [20]. The gels
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were fixed and stained with silver and then dried. To assess the covalent HPG-BSA binding to
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the surface of nanodiamond, we compared the migration of BSA alone and of FND–HPG
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processed for BSA coupling through carbonyldiimidazole reaction. BSA or the nanodiamondHPG-BSA complex was boiled 2 min in SDS-rich loading buffer (10% Glycerol, 60 mM
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Tris/HCl pH 6.8, 2% SDS, 0.01% bromophenol blue, 1.25% beta-mercaptoethanol).
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3. Results and Discussion
3.1. Fluorescent nanodiamond preconditioning before reaction with glycidol
FND-NaOH
FND-HClO4
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Figure 1. ATR–FTIR of of FND–NaOH and FND–HClO4 (FND). : non– carboxylic hydroxyl group, : carboxylic group, : cyclic carbonyl group, : ether group, W: water.
The initial fluorescent nanodiamond [10,11] was further oxidized with hot aqueous concentrated perchloric acid (FND–HClO4) to introduce additional oxygen groups as a surface site for the in situ growing of the hyperbranched polyglycerol chains. ATR–FTIR spectrum of FND–HClO4, named in short FND (Figure 1), shows two major peaks: one 7
ACCEPTED MANUSCRIPT around 3400 cm-1 (this broad peak can be assigned to water as well as alcohol hydroxyl groups) and the other one around 1800 cm-1 (this marked peak can be assigned to carbonyl
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asymmetric stretching of cyclic groups, such as carboxylic anhydride or –lactone). Smaller
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bands at ca. 1264 and 1109 cm-1 would correspond to the C–O–C stretching vibrations.
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Evidence of a contribution of anhydride type groups to the peak at ca. 1800 cm-1 is provided by the complete disappearance of this peak and the appearance of new peaks at ca. 1760 and 1373 cm-1 after hydrolysis in hot aqueous concentrated NaOH and exhaustive washing with
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water (FND-NaOH). On nanodiamond surfaces where functional groups are directly attached
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to the c–diamond lattice, the carboxylic carbonyl peak would occur at abnormally high frequency (ca. 1760 cm-1, instead of 1700 to 1730 cm-1 for saturated aliphatic acids or sp2
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shell of detonation nanodiamond) [12,21] as a result of the known effect of ring strain (here,
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the c diamond {100} lattice plane) on the attached carbonyl frequencies [22,23,24,25]. This IR band position shift illustrates that functional groups added to a heterogeneous surface are
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not passive guests. Surface coverage and electronic support effects could alter the vibrational, rotational and energy levels of the surface bonds, and subsequently changes the IR band
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frequencies.
These considerations support the fact that the FND ATR-FTIR spectrum contains carboxyl group hidden peaks in the two broad bands, ca. 1800 and 1260 cm-1. These surface oxide groups would be responsible for the strong nanodiamond surface electronegativity at neutral pH (zeta potential around – 50 mV). 3.2. Pure glycidol polymerization in the absence of nanodiamond Before combining the preconditionned nanodiamond sample (FND) with glycidol, we observed the uncatalyzed thermal polymerization of pure glycidol to test the polymerization and ATR–FTIR protocols. This polymerization was slow since, even after 2 hours at 140 °C,
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ACCEPTED MANUSCRIPT the product still appeared as a translucent liquid with a FTIR spectrum similar to that of non heated glycidol. However, after 20 hours, the product turned to a translucent viscous liquid
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which displayed characteristic peaks of a hyperbranched polyglycidol FTIR spectrum
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(Figure 2). These observations showed the strong dependency of the non catalytic
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polymerization on time. After a treatment at 120 °C for 18 h, Weiss et al. [26] found a mean number molecular weight (Mn) of 1268 g.mol–1. It means a very small nanoparticle [27].
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G
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G-2h
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Figure 2. ATR–FTIR of glycidol: before (G) and after heating at 140 °C for 2 or 20 h. : ether–type C–O bonds
3.3. Hyperbranched polyglycerol growing from the fluorescent nanodiamond surface Thermal polymerization of glycidol loaded with nanodiamond was notably faster. A light grey gel was formed after 2 h. The presence of a HPG shell around nanodiamond (FND–HPG) was evidenced by a neutral zeta potential (at pH 5–9, 20–30 °C, < 50 mS.cm-1) and a characteristic ATR–FTIR spectrum (Figure 3). Figure 3 shows the IR spectra of FND before and after 2 and 20 h polymerization reaction. We observed that peaks due to the HPG polymer are superimposed with those of the FND oxygen surface groups. FND–HPG IR bands of ether
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ACCEPTED MANUSCRIPT bonds, possibly formed by reaction of glycidol epoxide with FND tertiary alcohol groups, are not discernible because these hypothetical ether groups are hidden by the polymer backbone
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made of sequential ether–linkages. On the other hand, IR peaks, clearly visible at ca. 1780
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and 1250 cm-1, correspond to an ester bond formed by the reaction of glycidol or polyglycidol
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with carboxylic groups present at the surface. It is known that glycidol hydroxyl group reaction with anhydride groups can be induced merely by heating at 120°C [28]. The subsequent nascent carboxylic groups react with the epoxide groups giving ester linkages.
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Then, the epoxide groups react with nascent or existing hydroxyl groups producing an ether
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linkage.
FND-HPG- 2 h FND-HPG- 20 h
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Wavenumber, cm-1
Figure 3. ATR–FTIR of FND and FND–HPG obtained by the reaction of glycidol with FND at 140 °C for 2h or 20 h. : ester group.
Dispersion stability was studied by centrifugation at room temperature in salty water. We showed that the largest part of FND–HPG remains in the supernatant after dispersion in high ionic strength water (1 M NaCl) and centrifugation at 4500 g for 5 min which otherwise causes an immediate precipitation of uncoated nanodiamond. The dispersion stability in salty
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ACCEPTED MANUSCRIPT water can be explained by steric hindrance provided by hyperbranched polymer covering the surface of the diamond nanocrystal.
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Fluorescence intensity, a.u.
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Figure 4. Fluorescence emission spectra of isolated nanodiamonds (laser excitation at 532 nm—emission long pass 600 nm, the cut–off below 600 nm is thus due to filtering). a: Fluorescence image of FND dispersed in water with the spectrum of a selected nanodiamond (b); c: FND–HPG in HeLa cells seen by phase (left) and epifluorescence microscopy with the spectrum of a selected nanodiamond (d).
Fluorescence of FND-HPG was easily observed by epifluorescence microscopy in pure water and in fixed cells (Figure 4). Interestingly, the shape of the FND–HPG emission spectrum under 532 nm illumination in these two conditions was about the same as that of the FND dispersion in pure water which indicates that the HPG grafting procedure does not influence the optical properties of NV-rich nanodiamond. Size fractionation of FND and FND–HPG dispersions, either in pure water (neutralized with PBS buffer), or salty water (by addition of NaCl), were made by centrifugation at 15000 g.
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ACCEPTED MANUSCRIPT FND–HPG was identified in the fractions, both by microspectrofluorimetry and by the measurement of a low zeta potential. Polydispersity index (PDI), defined as the ratio between
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the mean value of DLS intensity and number distribution at 25 °C, was relatively narrow for
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FND in pure neutral water: about 1.6, and very broad for FND–HPG: 9.7 in pure water and
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11.1 in salty buffer (Figure 5). It is known that for typical addition polymerization, values of the PDI can range around 10 to 20. In the case of thermally induced glycidol ROP, high PDI (5 to 7) was reported by Weiss et al. [24]. The authors showed that the polymer molecular
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weights, as well as the PDI, increase with temperature and time. 12
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FND intensity, water (0.3 mS/cm), ZP = - 48.7 mV FND-HPG intensity, water (0.5 mS/cm), ZP = - 0.2 mV FND-HPG intensity, salty water (28.6 mS/cm), ZP = - 0.7 mV FND number, water (0.3 mS/cm), ZP = - 48.7 mV FND-HPG number, water (0.5 mS/cm), ZP = - 0.2 mV FND-HPG number, salty water (28.6 mS/cm), ZP = - 0.7 mV
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Distribution,%
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Figure 5. Number and intensity distributions of the hydrodynamic diameter of the initial fluorescent nanodiamond (FND) dispersed in pure water and of the same grafted with hyperbranched polyglycerol (FND–HPG) and dispersed in neutral pure or salty water.
The broad FND–HPG PDI forbids any estimation of a mean shell thickness by a plain difference between the core–shell and the core diameters, measured either by DLS or by electron microscopy imaging. These parameters can only be determined after an optimal size fractionation by chromatography as done by Komatsu et al. [4]. It is the reason why it was not our intention here to determine them, but to reduce the FND–HPG PDI first.
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ACCEPTED MANUSCRIPT However, an inaccurate prediction of the thickness of the ND shell (8.5 nm) can be made by difference between the particle diameters corresponding to the mode of the DLS intensity
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distribution before and after HPG grafting (Figure 5).
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Further studies to decrease the thickness and lower the polydispersity involve the optimization
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of a controlled polymerization of glycidol with supplementary size fractionation. The controlled polymerization should be made at lower temperature with optimal nanodiamond
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initiator surface groups (such as amine groups) and subsequent slow addition of the monomer to the reaction [5,29,30].
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3.4. Chemical end–group derivatization of hyperbranched polyglycerol
FND-HPG-COOH
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Wavenumber, cm-1
Figure 6. ATR–FTIR of FND–HPG–COOH obtained by succinylation of FND–HPG. : ester group, : carboxylic/carboxylate groups
Once grafted with HPG, the fluorescent nanoparticled display several hydroxyl end–groups which can easily be derivatized by carboxylation or carbamatization. ATR–FTIR of FND– HPG–COOH (Figure 6), obtained by succinylation of FND–HPG, shows a near complete
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ACCEPTED MANUSCRIPT disappearance of CO stretching vibration of primary and secondary alcohols at ca. 1050 and 1100 cm-1 (1074 and 1117 cm-1 on the present spectrum), correlated with the appearance of
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carboxyl group (at 1416, 1588 and 1732 cm-1). This evolution of surface functionality was
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parallel with a decrease of the zeta potential from neutral to ca. - 45 mV in pure water at
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25°C.
ATR–FTIR spectrum of FND–HPG–CDI (Figure 7), obtained by reaction of FND–HPG with
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1,1'–carbonyldiimidazole, shows the disappearance of the OH band (3401 cm-1) and the
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FND-HPG
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Figure 7. ATR–FTIR of FND-HPG after hydroxyl CDI activation. : carbamate group, : ester group.
appearance of several new peaks related to the imidazole carbamate. Indeed, on the FND– HPG–CDI spectrum, a very strong peak at ca. 1765 cm-1 can be assigned to the C=O bond stretching of the carbamate group. In addition, a low intense peak at ca. 3138 cm-1 is attributed to the C–H stretching mode of imidazole group (i.e., C2–H, C3–H and C5–H). The peak at ca. 1471 cm-1 can be assigned to deformation vibration of the C–H groups. Nevertheless, the absence of an OH stretching at 3400 cm-1 and the appearance of a large 14
ACCEPTED MANUSCRIPT number of new peaks after activation are consistent with a significant fraction of the OH functions reacting with imidazole carbamate.
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3.5 Fluorescent nanodiamond-protein conjugation
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After reaction of the stable activated FND–HPG–CDI species with BSA and subsequent exhaustive washing, we observed the complete disappearance of the carbamate band at ca. 1765 cm-1 (attributed to the stretching mode of the C=O group in imidazole carbamate)
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(Figure 8), which indicate that the activated sites either formed an amide band or were hydrolyzed. On the resulting product, part of the activated OH from the FND–HPG
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nanoparticle were conjugated with BSA molecules, while other were restored to hydroxyl groups when not engaged with BSA bonding. In the region around 1650 cm -1 we found the
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amide I band which mainly consists of C=O stretching vibrations. The amide II (coupling of
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bending vibrations of N–H and stretching vibrations of C–N) band was observed around
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1530 cm-1. ATR-FTIR spectrum alone provides first clear indications for covalent attachment by primary amide bounds between BSA and FND–HPG–CDI. By coupling BSA with FNDHPG-CDI a certain number of hydroxyl groups are restored by hydrolysis. The other ends
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groups are conjugated with the protein. These observations are corroborated by the fact that BSA physical adsorption onto polyglycerol is improbable [e.g. 31]. To further document the covalent binding of BSA to FND–HPG, together with covalent attachment of HPG to nanodiamond, the FND–HPG–BSA product was submitted to SDS– PAGE electrophoresis. We then compared its migration and silver staining pattern with that of BSA alone and of FND–HPG–CDI (Figure 9). It is worthy to note that in these harsh conditions, any adsorbed BSA, HPG or HPG–BSA complex detach from the nanodiamond surface. The results show that BSA alone appears with a major band at 67 kDa together with minor contaminant proteins that migrate at higher apparent molecular weights. When the
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FND-HPG-CDI-BSA
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Figure 8. ATR–FTIR of FND–HPG after hydroxyl CDI activation followed by coupling with bovine serum albumin and hydrolysis of imidazole carbamate derivates. : carbamate group, : ester group, : amide group, : ether–type C–O bonds.
BSA band at 67 kDa. (Lane 2). This clearly reflects a covalent binding of BSA with
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nanodiamond thanks to the HPG shell. The fact that BSA is distributed as a smear instead of a definite band is due to the polydispersity of the FND–HPG–BSA product. To further document this result, we washed the FND–HPG–BSA material with methanol to ascertain the absence of a putative HPG adsorption on the diamond surface (Lane 4). The results confirm the presence of the HPG–BSA product covalently grafted on diamond as attested by a continuous BSA smear (Lane4). As a control we also checked that the FND–HPG–CDI product is not stained by the silver staining procedure (Lane 7).
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67 kDa. 1
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Figure 9. SDS-PAGE analysis of FND-HPG-BSA. Lane 1: 1ug BSA alone. Lane 2: FND-HPG-BSA complex after extensive pure water washing. Lane 3: supernatant of lane 2. Lane 4: FND-HPG-BSA complex after extensive serial washing with pure water and methanol. Lane 5: supernatant of lane 4. Lane 6: empty. Lane 7 FND-HPG-CDI: control pellet. Lane 8: supernatant of Lane 7.
We have demonstrated that the product is adapted for protein grafting using BSA as a protein
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model. HPG and the HPG-BSA grafted products are both stable in saline solution (responding to one of our main objective) and we can easily observe by fluorescence microscopy and
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spectrometry that the polymeric shell do not impede the optical properties of nanodiamond. In a next study, the homogeneous protein grafting rate necessary for bio-application will be
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adjusted by controlling the pH and the time of the reaction between FND–HPG–CDI and bovine serum albumin. 4. Conclusions
In summary, these results demonstrate the applicability of hyperbranched polyglycerol for the functionalization of fluorescent nanodiamond. The hyperbranched polyglycerol modified nanodiamond can form stable dispersion in high ionic strength aqueous solution while retaining the exceptional optical properties of the initial fluorescent nanodiamond in water as well as in physiological medium and cultured cells. Finally, it is shown that fluorescent nanodiamond grafted with hyperbranched polyglycerol can be further derivatized by carboxylation or carbamatization and subsequently covalently conjugated with bovine serum
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ACCEPTED MANUSCRIPT albumin, taken as a model protein. This later paves the way for molecular recognition or targeting in biological systems.
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Acknowledgements
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Nano in vivo) and by the NeRF IdF (Grant Nano in Brain).
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The work described in this paper has been in part supported by C’Nano IdF (Grant AAP2010
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Prime Novelty Statement
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We report for the first time on the polymer and protein grafting on Red/NIR fluorescent nanodiamond surface via in situ glycidol polymerization. The fluorescent nanohybrid dispersion is stable in physiological media and can be conjugated with protein. Such product should be useful
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for biomedical research.
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Graphical abstract
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Highlights
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►Fluorescent nanodiamonds can be conjugated with hyperbranched polyglycerol.
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►Aqueous dispersion of the fluorescent nanohybrid is stable at high ionic strength.
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►Hydroxyl end groups can be used for post-modifications and protein grafting.
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S0925-9635(13)00138-6 doi: 10.1016/j.diamond.2013.06.019 DIAMAT 6148
To appear in:
Diamond & Related Materials
Received date: Revised date: Accepted date:
17 April 2013 23 June 2013 29 June 2013
Please cite this article as: Jean-Paul Boudou, Marie-Odile David, Vandana Joshi, Housam Eidi, Patrick A. Curmi, Hyperbranched polyglycerol modified fluorescent nanodiamond for biomedical research, Diamond & Related Materials (2013), doi: 10.1016/j.diamond.2013.06.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Hyperbranched polyglycerol modified fluorescent nanodiamond for biomedical research Jean-Paul Boudou*,¥, Marie-Odile David, Vandana Joshi, Housam Eidi, and Patrick A. Curmi*
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Institut National de la Santé et de la Recherche Médicale, UMR829, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université Evry-Val d’Essonne, Evry 91025, France
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* Corresponding authors:
Jean-Paul Boudou: Laboratoire Aimé Cotton, CNRS, UPR3321, Université Paris-Sud, 91405 Orsay cedex, France, Tel : +33 1 47 09 31 34, +33 6 44 26 42 78, Email: [email protected]
Present address: LAC, CNRS, UPR3321, Université Paris-Sud, 91405 Orsay cedex, France, [email protected].
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¥
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Patrick A. Curmi: LSABNP, INSERM, UMR829, Université Evry-Val d’Essonne, Evry 91025, France, Tel : +33 1 69 47 03 23, Fax : +33 1 69 47 02 19, Email: [email protected]
Abstract
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The aim of the present work was to functionalize fluorescent nanodiamond by covalent
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grafting with hyperbranched polyglycerol. Fluorescent nanodiamond, derived from high pressure high temperature diamond microdiamond, was oxidized then thermally reacted with
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pure glycidol in the absence of catalyst. Thermal polymerization of glycidol was notably faster on the nanodiamond surface as a result of a surface initiation of the isothermal ring opening polymerization. Interestingly, the aqueous dispersion of the resulting nanoparticles
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appeared stable at high ionic strength. Furthermore, the fluorescent nanodiamond grafted with hyperbranched polyglycerol displayed several hydroxyl end–groups which could be further derivatized by carboxylation or carbamatization and subsequently conjugated with protein linked via an amide bound. Notably, nanodiamonds retain their unique fluorescent characteristics. This work suggests that fluorescent nanodiamond coated with hyperbranched glycidol could be promising in biomedical research where aqueous dispersion of fluorescent nanoparticles stable in physiological medium are in high demand to label, track and quantify biomolecules. Keywords: Nanodiamond Fluorescence Polyglycerol Bioconjugation Biomolecule labeling
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1. Introduction
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In recent years many papers have appeared dealing with the surface chemistry of different
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types of nanodiamonds [e.g.1,2,3,], but fewer papers have been devoted to the covalent grafting of polymeric spacers on nanodiamonds for improving their dispersion stability in water and avoiding aggregation of the nanocrystals in physiological buffer solutions as well as
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hypertonic aqueous media needed in selected biochemical assays. It has been shown that
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polyether backbone polymers grown in situ can cover densely the nanodiamond surface [4,5,6]. The resulting nanoparticle can be dispersed stably in physiological solution and shows
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an excellent biocompatibility. Nanodiamonds thus appear eligible to the emerging area of the
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polyglycerol systems. Their green synthesis, biocompatibility, very low toxicity and high functionality make them very valuable intermediates for biomedical research and
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pharmaceutical development [e.g. 7,8,9]. In the present work, we prepared a new fluorescent nanohybrid via the very simple and
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industrially scalable thermal treatment with glycidol of a homemade NIR fluorescent nanodiamond (FND) [10,11]. We further showed that the resulting material is adapted to physiological medium and detectable in cultured cell using fluorescence microscopy. The modified fluorescent nanodiamond was covalently conjugated with a model protein. 2. Experimental Section 2.1. Synthesis 2.1.1. Products
Reagent grade chemicals were purchased from Sigma–Aldrich and used as received. Fluorescent nanodiamond was prepared from a synthetic micron diamond powder [10,11].
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ACCEPTED MANUSCRIPT After electron irradiation (10 MeV, 5 x 1019 e-/cm2), the sample was annealed at 800 °C under vacuum. To convert microdiamond into smaller particles, we first used nitrogen jet milling
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autogeneous micronization. A nanomilling procedure was then used to convert the jet milling
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product into a mixture of fluorescent nanodiamond and contaminant. The purification was
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done as previously described with HF/HNO3 at 150 °C for 48 h [10]. The product was subsequently treated in aqueous solution of NaOH (50%, v/v) or perchloric acid (70%, v/v) at 190 °C for 48 h to improve the cleaning and to saturate the surface with surface oxygen
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groups. The product of the treatment with NaOH was named FND–NaOH, and the product of
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the treatment with HClO4 was named FND-HClO4—in short FND. This last product was modified with hyperbranched polyglycerol. The NaOH hydrolysis was done for a better
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understanding of the surface oxide groups introduced by the hot acid treatments.
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2.1.2. Procedure
A hyperbranched polyglycerol (HPG) shell was grown on nanodiamond surface based on the
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method proposed by Lukehart et al. [12] and by Komatsu et al. [4,5] In this method, the surface groups (active hydrogen–containing species) from the diamond initiate the glycidol
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neutral ring opening polymerization (ROP) during heating. The diamond material and conditions for polymerization being either a cubic–diamond nanocrystal (derived from high pressure high temperature diamond) in the absence of catalyst at 140 °C for 20 h [5], or detonation nanodiamond, either in the absence (at 50 °C) or the presence of catalyst (tertiary amine) at 75 °C for 24 h [12]. In this study, the neutral ROP was initiated by surface anhydride groups, carboxyl and hydroxyl groups, previously introduced on fluorescent cubic– diamond nanocrystals (FND). The experimental conditions used here were similar to those used by Zhao et al. [4] but importantly much less drastic (2 h) to prevent autopolymerization of glycidol. The procedure is different from the surface glycidol initiated anionic ROP
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ACCEPTED MANUSCRIPT [12,13,14]. The reaction resulted in HPG grafted FND and free HPG which could be removed by washing with methanol.
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FND powder (5 mg) was dispersed in glycidol (3 mL) by means of an ultrasonic bath at room
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temperature. The mixture was placed in 2 mL screw top vials with Teflon septa. Headspace
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was swept with argon and the vials were placed in a preheated oven at 140 °C and left at this temperature for either 2 or 20 h. The resulting gel was diluted with methanol (40 mL) in an
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ultrasonic bath, and the product (FND–HPG) was isolated by 15000 g centrifugation and washed free of unbound polymer (HPG) by three repeated dispersions in methanol and
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15000 g centrifugation cycles. These three methanol washing cycles were followed by three repeated dispersion in water and 15000 g centrifugation cycles. The final FND–HPG pellet
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Ultra–4 centrifugal filter units.
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was finally dispersed in water, and then washed with a high volume of water on Amicon
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2.1.3. Derivatization and protein grafting:
FND-HPG-COOH was obtained by the reaction of FND-HPG with succinic anhydride in pyridine under argon at 70 °C for 1 h. The reaction mixture was centrifuged at 15000 g and
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the pellet washed several times by dispersion in ethanol and centrifugation at 15000 g. Final FND-HPG-COOH pellet was dispersed in water, and then washed with water on Amicon Ultra-4 centrifugal filter units. FND–HPG hydroxyl end–groups were activated using 1,1'– Carbonyldiimidazole (CDI) [15]. FND–HPG was first dried, and then immersed in a 0.05 to 0.25 M solution of CDI in anhydrous THF for 12 h under an argon atmosphere. After activation, FND–HPG–CDI was isolated by centrifugation with THF and water. Finally, it was washed with water on Amicon Ultra-4 centrifugal filter units. Coupling Reaction between FND–HPG–CDI and bovine serum albumin (BSA), taken as a model protein, was carried out by stirring 100 g of BSA together with 10 mg of FND–HPG–CDI in borate buffer (pH 8.5) under argon at room temperature for 48 h. FND–HPG–BSA was then isolated by sequential
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ACCEPTED MANUSCRIPT centrifugation at 15000 g for 5 min in water, and then washed with water on Amicon Ultra–4 centrifugal filter units.
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2.2. Attenuated Total Reflectance–Fourier transform infrared spectroscopy (ATR–FTIR)
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Spectra were acquired by means of ATR–FTIR spectrometry. The FND and FND derivative dispersions in water were dried under an argon flow on a PIKE Technologies GladiATR with a single–reflection (angle of incidence 45°) diamond crystal plate isothermally heated at
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75 °C. FTIR spectra were collected on a Thermo Nicolet 380 spectrometer equipped with a DTGS temperature–stabilized coated detector (4000 to 600 cm−1, spectral resolution: 4 cm−1,
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200 accumulations). ATR–FTIR peak frequencies can be shifted due to the environment, and peak intensities depend on the ATR mode. Loss of hydration water and increased salt
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interaction with the dry film may cause of the band position. For instance, the asymmetric
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stretching vibration of carboxylate groups is typically 10–40 cm-1 lower in water than in solid
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[16]. Bearing in mind these possible shifts, we made the structural assignment of the more critical FTIR bands based on published data [17,18].
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2.3. Dynamic light scattering and zeta potential measurement Nanoparticles were dispersed in water. The pH and the conductivity of the dispersion were controlled by an autotitrator Delsa™Nano AT and zeta potential measurements were made using a Delsa Nano C Particle Size apparatus, (Beckman Coulter) equipped with two 658 nm laser diode sources (30 mW power each) and a temperature controller (from 15 to 90 °C). Size distribution was measured by dynamic light scattering at a scattering angle of 165°. Scattering data were collected for 70 individual measurements at a constant scattering angle and averaged for each sample. They were fitted using volume-weighted cumulative analysis to estimate the diffusion coefficient of the micelles in solution. The particle zeta potential was determined by measuring the electrophoretic movement of charged particles under an applied
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ACCEPTED MANUSCRIPT electric field. Scattered light was detected at a 90° angle and a temperature of 25 °C. Zeta potential values were calculated from measured velocities.
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2.4. Epifluorescence microscopy and microspectrofluorimetry
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Fluorescence images were taken and spectroscopic measurements made using a Zeiss Axioplan 2 microscope with a 63x, 1.4 NA oil immersion objective. A 532 nm laser beam (200 mW power) was used as the illuminating source and was guided to the microscope by an
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optic fiber. Fluorescence images were obtained with a Princeton Instruments EMCCD Camera Rolera em–c², with typical exposure times from 100 to 500 ms. Spectra were acquired
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by focusing the fluorescent nanodiamond emission from the microscope onto an Acton SP2150i spectrometer (Princeton instruments), and detected with a PIXIS–100B–eXcelon
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CCD camera (Princeton instruments).
The fluorescent nanodiamonds were observed either in water or in fixed HeLa cells, taken as
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model biological cell. HeLa cells were grown on sterile glass coverslips in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 5% (v/v) fetal bovine serum (FBS),
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2 mM L-glutamine and 1% antibiotics (penicillin and streptomycin). Cells were maintained in a humidified 5% CO2 atmosphere at 37 °C. Twenty–four hours after the beginning of the culture, FND-HPG nanodiamond dispersion (0.2 g/L in pure water) was added to the cells in the culture medium at a fifty–fold dilution (10 L of FND–HPG nanodiamond stock in 500 µL final volume of DMEM for a 3.6 cm2 cell culture dish). Cells were cultivated for an additional 24 hours, and then washed three times with phosphate buffered saline (PBS) to remove unincorporated nanodiamonds. The cells were then fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at 37°C. The glass coverslips were then mounted on microscope glass slides and processed for fluorescence microscopy. Fluorescence images were analyzed for fluorescence intensity using the ImageJ [19].
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ACCEPTED MANUSCRIPT 2.5. SDS-PAGE analysis One-dimensional electrophoresis was performed on 4% polyacrylamide gels [20]. The gels
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were fixed and stained with silver and then dried. To assess the covalent HPG-BSA binding to
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the surface of nanodiamond, we compared the migration of BSA alone and of FND–HPG
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processed for BSA coupling through carbonyldiimidazole reaction. BSA or the nanodiamondHPG-BSA complex was boiled 2 min in SDS-rich loading buffer (10% Glycerol, 60 mM
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Tris/HCl pH 6.8, 2% SDS, 0.01% bromophenol blue, 1.25% beta-mercaptoethanol).
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3. Results and Discussion
3.1. Fluorescent nanodiamond preconditioning before reaction with glycidol
FND-NaOH
FND-HClO4
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+W
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Wavenumber, cm-1
Figure 1. ATR–FTIR of of FND–NaOH and FND–HClO4 (FND). : non– carboxylic hydroxyl group, : carboxylic group, : cyclic carbonyl group, : ether group, W: water.
The initial fluorescent nanodiamond [10,11] was further oxidized with hot aqueous concentrated perchloric acid (FND–HClO4) to introduce additional oxygen groups as a surface site for the in situ growing of the hyperbranched polyglycerol chains. ATR–FTIR spectrum of FND–HClO4, named in short FND (Figure 1), shows two major peaks: one 7
ACCEPTED MANUSCRIPT around 3400 cm-1 (this broad peak can be assigned to water as well as alcohol hydroxyl groups) and the other one around 1800 cm-1 (this marked peak can be assigned to carbonyl
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asymmetric stretching of cyclic groups, such as carboxylic anhydride or –lactone). Smaller
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bands at ca. 1264 and 1109 cm-1 would correspond to the C–O–C stretching vibrations.
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Evidence of a contribution of anhydride type groups to the peak at ca. 1800 cm-1 is provided by the complete disappearance of this peak and the appearance of new peaks at ca. 1760 and 1373 cm-1 after hydrolysis in hot aqueous concentrated NaOH and exhaustive washing with
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water (FND-NaOH). On nanodiamond surfaces where functional groups are directly attached
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to the c–diamond lattice, the carboxylic carbonyl peak would occur at abnormally high frequency (ca. 1760 cm-1, instead of 1700 to 1730 cm-1 for saturated aliphatic acids or sp2
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shell of detonation nanodiamond) [12,21] as a result of the known effect of ring strain (here,
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the c diamond {100} lattice plane) on the attached carbonyl frequencies [22,23,24,25]. This IR band position shift illustrates that functional groups added to a heterogeneous surface are
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not passive guests. Surface coverage and electronic support effects could alter the vibrational, rotational and energy levels of the surface bonds, and subsequently changes the IR band
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frequencies.
These considerations support the fact that the FND ATR-FTIR spectrum contains carboxyl group hidden peaks in the two broad bands, ca. 1800 and 1260 cm-1. These surface oxide groups would be responsible for the strong nanodiamond surface electronegativity at neutral pH (zeta potential around – 50 mV). 3.2. Pure glycidol polymerization in the absence of nanodiamond Before combining the preconditionned nanodiamond sample (FND) with glycidol, we observed the uncatalyzed thermal polymerization of pure glycidol to test the polymerization and ATR–FTIR protocols. This polymerization was slow since, even after 2 hours at 140 °C,
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ACCEPTED MANUSCRIPT the product still appeared as a translucent liquid with a FTIR spectrum similar to that of non heated glycidol. However, after 20 hours, the product turned to a translucent viscous liquid
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which displayed characteristic peaks of a hyperbranched polyglycidol FTIR spectrum
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(Figure 2). These observations showed the strong dependency of the non catalytic
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polymerization on time. After a treatment at 120 °C for 18 h, Weiss et al. [26] found a mean number molecular weight (Mn) of 1268 g.mol–1. It means a very small nanoparticle [27].
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G
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G-2h
Wavenumber, cm-1
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G - 20 h
Figure 2. ATR–FTIR of glycidol: before (G) and after heating at 140 °C for 2 or 20 h. : ether–type C–O bonds
3.3. Hyperbranched polyglycerol growing from the fluorescent nanodiamond surface Thermal polymerization of glycidol loaded with nanodiamond was notably faster. A light grey gel was formed after 2 h. The presence of a HPG shell around nanodiamond (FND–HPG) was evidenced by a neutral zeta potential (at pH 5–9, 20–30 °C, < 50 mS.cm-1) and a characteristic ATR–FTIR spectrum (Figure 3). Figure 3 shows the IR spectra of FND before and after 2 and 20 h polymerization reaction. We observed that peaks due to the HPG polymer are superimposed with those of the FND oxygen surface groups. FND–HPG IR bands of ether
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ACCEPTED MANUSCRIPT bonds, possibly formed by reaction of glycidol epoxide with FND tertiary alcohol groups, are not discernible because these hypothetical ether groups are hidden by the polymer backbone
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made of sequential ether–linkages. On the other hand, IR peaks, clearly visible at ca. 1780
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and 1250 cm-1, correspond to an ester bond formed by the reaction of glycidol or polyglycidol
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with carboxylic groups present at the surface. It is known that glycidol hydroxyl group reaction with anhydride groups can be induced merely by heating at 120°C [28]. The subsequent nascent carboxylic groups react with the epoxide groups giving ester linkages.
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Then, the epoxide groups react with nascent or existing hydroxyl groups producing an ether
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linkage.
FND-HPG- 2 h FND-HPG- 20 h
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FND
Wavenumber, cm-1
Figure 3. ATR–FTIR of FND and FND–HPG obtained by the reaction of glycidol with FND at 140 °C for 2h or 20 h. : ester group.
Dispersion stability was studied by centrifugation at room temperature in salty water. We showed that the largest part of FND–HPG remains in the supernatant after dispersion in high ionic strength water (1 M NaCl) and centrifugation at 4500 g for 5 min which otherwise causes an immediate precipitation of uncoated nanodiamond. The dispersion stability in salty
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ACCEPTED MANUSCRIPT water can be explained by steric hindrance provided by hyperbranched polymer covering the surface of the diamond nanocrystal.
a
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Fluorescence intensity, a.u.
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Figure 4. Fluorescence emission spectra of isolated nanodiamonds (laser excitation at 532 nm—emission long pass 600 nm, the cut–off below 600 nm is thus due to filtering). a: Fluorescence image of FND dispersed in water with the spectrum of a selected nanodiamond (b); c: FND–HPG in HeLa cells seen by phase (left) and epifluorescence microscopy with the spectrum of a selected nanodiamond (d).
Fluorescence of FND-HPG was easily observed by epifluorescence microscopy in pure water and in fixed cells (Figure 4). Interestingly, the shape of the FND–HPG emission spectrum under 532 nm illumination in these two conditions was about the same as that of the FND dispersion in pure water which indicates that the HPG grafting procedure does not influence the optical properties of NV-rich nanodiamond. Size fractionation of FND and FND–HPG dispersions, either in pure water (neutralized with PBS buffer), or salty water (by addition of NaCl), were made by centrifugation at 15000 g.
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ACCEPTED MANUSCRIPT FND–HPG was identified in the fractions, both by microspectrofluorimetry and by the measurement of a low zeta potential. Polydispersity index (PDI), defined as the ratio between
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the mean value of DLS intensity and number distribution at 25 °C, was relatively narrow for
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FND in pure neutral water: about 1.6, and very broad for FND–HPG: 9.7 in pure water and
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11.1 in salty buffer (Figure 5). It is known that for typical addition polymerization, values of the PDI can range around 10 to 20. In the case of thermally induced glycidol ROP, high PDI (5 to 7) was reported by Weiss et al. [24]. The authors showed that the polymer molecular
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weights, as well as the PDI, increase with temperature and time. 12
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FND intensity, water (0.3 mS/cm), ZP = - 48.7 mV FND-HPG intensity, water (0.5 mS/cm), ZP = - 0.2 mV FND-HPG intensity, salty water (28.6 mS/cm), ZP = - 0.7 mV FND number, water (0.3 mS/cm), ZP = - 48.7 mV FND-HPG number, water (0.5 mS/cm), ZP = - 0.2 mV FND-HPG number, salty water (28.6 mS/cm), ZP = - 0.7 mV
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Distribution,%
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Figure 5. Number and intensity distributions of the hydrodynamic diameter of the initial fluorescent nanodiamond (FND) dispersed in pure water and of the same grafted with hyperbranched polyglycerol (FND–HPG) and dispersed in neutral pure or salty water.
The broad FND–HPG PDI forbids any estimation of a mean shell thickness by a plain difference between the core–shell and the core diameters, measured either by DLS or by electron microscopy imaging. These parameters can only be determined after an optimal size fractionation by chromatography as done by Komatsu et al. [4]. It is the reason why it was not our intention here to determine them, but to reduce the FND–HPG PDI first.
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distribution before and after HPG grafting (Figure 5).
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Further studies to decrease the thickness and lower the polydispersity involve the optimization
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of a controlled polymerization of glycidol with supplementary size fractionation. The controlled polymerization should be made at lower temperature with optimal nanodiamond
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initiator surface groups (such as amine groups) and subsequent slow addition of the monomer to the reaction [5,29,30].
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3.4. Chemical end–group derivatization of hyperbranched polyglycerol
FND-HPG-COOH
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FND-HPG
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Figure 6. ATR–FTIR of FND–HPG–COOH obtained by succinylation of FND–HPG. : ester group, : carboxylic/carboxylate groups
Once grafted with HPG, the fluorescent nanoparticled display several hydroxyl end–groups which can easily be derivatized by carboxylation or carbamatization. ATR–FTIR of FND– HPG–COOH (Figure 6), obtained by succinylation of FND–HPG, shows a near complete
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ACCEPTED MANUSCRIPT disappearance of CO stretching vibration of primary and secondary alcohols at ca. 1050 and 1100 cm-1 (1074 and 1117 cm-1 on the present spectrum), correlated with the appearance of
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carboxyl group (at 1416, 1588 and 1732 cm-1). This evolution of surface functionality was
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parallel with a decrease of the zeta potential from neutral to ca. - 45 mV in pure water at
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25°C.
ATR–FTIR spectrum of FND–HPG–CDI (Figure 7), obtained by reaction of FND–HPG with
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1,1'–carbonyldiimidazole, shows the disappearance of the OH band (3401 cm-1) and the
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FND-HPG
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FND-HPG-CDI
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Figure 7. ATR–FTIR of FND-HPG after hydroxyl CDI activation. : carbamate group, : ester group.
appearance of several new peaks related to the imidazole carbamate. Indeed, on the FND– HPG–CDI spectrum, a very strong peak at ca. 1765 cm-1 can be assigned to the C=O bond stretching of the carbamate group. In addition, a low intense peak at ca. 3138 cm-1 is attributed to the C–H stretching mode of imidazole group (i.e., C2–H, C3–H and C5–H). The peak at ca. 1471 cm-1 can be assigned to deformation vibration of the C–H groups. Nevertheless, the absence of an OH stretching at 3400 cm-1 and the appearance of a large 14
ACCEPTED MANUSCRIPT number of new peaks after activation are consistent with a significant fraction of the OH functions reacting with imidazole carbamate.
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3.5 Fluorescent nanodiamond-protein conjugation
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After reaction of the stable activated FND–HPG–CDI species with BSA and subsequent exhaustive washing, we observed the complete disappearance of the carbamate band at ca. 1765 cm-1 (attributed to the stretching mode of the C=O group in imidazole carbamate)
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(Figure 8), which indicate that the activated sites either formed an amide band or were hydrolyzed. On the resulting product, part of the activated OH from the FND–HPG
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nanoparticle were conjugated with BSA molecules, while other were restored to hydroxyl groups when not engaged with BSA bonding. In the region around 1650 cm -1 we found the
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amide I band which mainly consists of C=O stretching vibrations. The amide II (coupling of
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bending vibrations of N–H and stretching vibrations of C–N) band was observed around
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1530 cm-1. ATR-FTIR spectrum alone provides first clear indications for covalent attachment by primary amide bounds between BSA and FND–HPG–CDI. By coupling BSA with FNDHPG-CDI a certain number of hydroxyl groups are restored by hydrolysis. The other ends
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groups are conjugated with the protein. These observations are corroborated by the fact that BSA physical adsorption onto polyglycerol is improbable [e.g. 31]. To further document the covalent binding of BSA to FND–HPG, together with covalent attachment of HPG to nanodiamond, the FND–HPG–BSA product was submitted to SDS– PAGE electrophoresis. We then compared its migration and silver staining pattern with that of BSA alone and of FND–HPG–CDI (Figure 9). It is worthy to note that in these harsh conditions, any adsorbed BSA, HPG or HPG–BSA complex detach from the nanodiamond surface. The results show that BSA alone appears with a major band at 67 kDa together with minor contaminant proteins that migrate at higher apparent molecular weights. When the
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FND-HPG-CDI-BSA
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FND-HPG-CDI
Wavenumber, cm-1
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Figure 8. ATR–FTIR of FND–HPG after hydroxyl CDI activation followed by coupling with bovine serum albumin and hydrolysis of imidazole carbamate derivates. : carbamate group, : ester group, : amide group, : ether–type C–O bonds.
BSA band at 67 kDa. (Lane 2). This clearly reflects a covalent binding of BSA with
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nanodiamond thanks to the HPG shell. The fact that BSA is distributed as a smear instead of a definite band is due to the polydispersity of the FND–HPG–BSA product. To further document this result, we washed the FND–HPG–BSA material with methanol to ascertain the absence of a putative HPG adsorption on the diamond surface (Lane 4). The results confirm the presence of the HPG–BSA product covalently grafted on diamond as attested by a continuous BSA smear (Lane4). As a control we also checked that the FND–HPG–CDI product is not stained by the silver staining procedure (Lane 7).
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67 kDa. 1
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Figure 9. SDS-PAGE analysis of FND-HPG-BSA. Lane 1: 1ug BSA alone. Lane 2: FND-HPG-BSA complex after extensive pure water washing. Lane 3: supernatant of lane 2. Lane 4: FND-HPG-BSA complex after extensive serial washing with pure water and methanol. Lane 5: supernatant of lane 4. Lane 6: empty. Lane 7 FND-HPG-CDI: control pellet. Lane 8: supernatant of Lane 7.
We have demonstrated that the product is adapted for protein grafting using BSA as a protein
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model. HPG and the HPG-BSA grafted products are both stable in saline solution (responding to one of our main objective) and we can easily observe by fluorescence microscopy and
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spectrometry that the polymeric shell do not impede the optical properties of nanodiamond. In a next study, the homogeneous protein grafting rate necessary for bio-application will be
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adjusted by controlling the pH and the time of the reaction between FND–HPG–CDI and bovine serum albumin. 4. Conclusions
In summary, these results demonstrate the applicability of hyperbranched polyglycerol for the functionalization of fluorescent nanodiamond. The hyperbranched polyglycerol modified nanodiamond can form stable dispersion in high ionic strength aqueous solution while retaining the exceptional optical properties of the initial fluorescent nanodiamond in water as well as in physiological medium and cultured cells. Finally, it is shown that fluorescent nanodiamond grafted with hyperbranched polyglycerol can be further derivatized by carboxylation or carbamatization and subsequently covalently conjugated with bovine serum
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ACCEPTED MANUSCRIPT albumin, taken as a model protein. This later paves the way for molecular recognition or targeting in biological systems.
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Acknowledgements
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Nano in vivo) and by the NeRF IdF (Grant Nano in Brain).
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The work described in this paper has been in part supported by C’Nano IdF (Grant AAP2010
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Prime Novelty Statement
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We report for the first time on the polymer and protein grafting on Red/NIR fluorescent nanodiamond surface via in situ glycidol polymerization. The fluorescent nanohybrid dispersion is stable in physiological media and can be conjugated with protein. Such product should be useful
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for biomedical research.
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Graphical abstract
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ACCEPTED MANUSCRIPT
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Highlights
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►Fluorescent nanodiamonds can be conjugated with hyperbranched polyglycerol.
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►Aqueous dispersion of the fluorescent nanohybrid is stable at high ionic strength.
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►Hydroxyl end groups can be used for post-modifications and protein grafting.
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