One-Pot Synthesis of Highly Dispersible Fluorescent Nanodiamonds

Subscriber access provided by NEW MEXICO STATE UNIV

Article

One-pot synthesis of highly dispersible fluorescent nanodiamonds for bioconjugation Daiki Terada, Shingo Sotoma, Yoshie Harada, Ryuji Igarashi, and Masahiro Shirakawa Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00412 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 5, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 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

Bioconjugate Chemistry

One-pot synthesis of highly dispersible fluorescent nanodiamonds for bioconjugation Daiki Terada†, Shingo Sotoma¶, Yoshie Harada¶, Ryuji Igarashi†,§,‡,•,* and Masahiro Shirakawa†,* †

Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8519, Japan ¶ Institute for Protein Research (IPR), Osaka University, 3-2 Yamadaoka, Suita-shi, Osaka 565-0871, Japan § PRESTO, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan ‡ QST Future Laboratory, National Institute for Quantum and Radiological Science and Technology, Anagawa 4-9-1, Inageku, Chiba 263-8555, Japan. •

National Institute for Radiological Sciences, National Institute for Quantum and Radiological Science and Technology, Anagawa 4-9-1, Inage-ku, Chiba 263-8555, Japan.

ABSTRACT: Fluorescent nanodiamonds (FNDs) have been attracting much attention as promising therapeutic agents and probes for bioimaging and nanosensing. For their biological applications, several hydrophilizing methods to enhance FND colloidal stability have been developed to suppress their aggregation and the nonspecific adsorption to biomolecules in complex biomedical environments. However, these methods involve several complicated synthetic and purification steps, which prohibit the use of FNDs for bioapplications by biologists. In this study, we describe a simple one-pot FND hydrophilization method that comprises coating of the surface of the nanoparticles with COOH-terminated hyperbranched polyglycerol (HPG-COOH). HPG-COOH-coated FNDs (FND-HPG-COOHs) were found to exhibit excellent dispersibility under physiological conditions despite the thinness of 5-nm HPG-COOH layer. Biotinylated FND-HPG-COOHs specifically captured avidin molecules in the absence of nonspecific protein adsorption. Moreover, we demonstrated that FND-HPG-COOHs conjugated with antibodies can be used to selectively target integrins in fixed HeLa cells. In addition, intracellular temperature changes were measured via optically detected magnetic resonance using FND-HPG-COOHs conjugated with mitochondrial localization signal peptides. Our one-pot synthetic method will encourage the broad use of FNDs among molecular and cellular biologists, and pave the way for extensive biological and biomedical applications of FNDs.

INTRODUCTION Fluorescent nanodiamonds (FNDs) containing nitrogenvacancy color centers (NVCs) are promising probes for bioimaging1–3 and nanosensing4 because of their biocompatibility5–8 and excellent photostability. These nanoparticles display neither photobleaching nor photoblinking9,10. NVCs within FNDs emit fluorescence in the near-infrared 650–900 nm range, which is an ideal wavelength band for in vivo optical imaging2. Additionally, the most prominent feature of FNDs is their suitability as nanometer-sized sensors. In particular, by measuring the spin state-dependent photoemission of their NVCs, FNDs can be used for sensing various physical parameters, such as temperature1,11,12, magnetic fields13–16, and electric fields17,18. Therefore, through the use of FNDs, promising approaches become possible for detecting and measuring a variety of extra- and intracellular events and parameters, including, for instance, thermophoresis1, chemical events4, membrane potentials, and ion concentrations19–21. However, such biomedical applications are limited by the following two undesirable traits of FNDs: (1) aggregation under physiological conditions and (2) difficulty in functionalizing their surface. An approach frequently used to overcome these limitations involves coating the FNDs with silica22–27 or polyethylene glycol (PEG)28,29. However, the procedures to realize these

modifications involve several complex chemical and physical steps aimed at the functionalization of the surface of FNDs and at the purification of the obtained particles. Furthermore, silica and PEG coatings do not suppress aggregation30 and nonspecific biomolecule adsorption29,31,32. The non-covalent adsorption29,32,33 of proteins, such as bovine serum albumin (BSA) and human serum albumin, on the surface of FNDs is implemented to avoid FND aggregation and nonspecific molecule adsorption. Although the non-covalent modification of FNDs through electrostatic33 and hydrophobic interactions34 is a convenient approach, it potentially limits further FND chemical modifications because the stability of the molecular interactions on the nanoparticle surface depends on solution conditions. Hyperbranched polyglycerol (HPG)-coated FNDs are nontoxic, biocompatible, and characterized by extremely high solubility, even in physiological conditions35. HPG comprises many hydroxy (–OH) terminal groups, which are more hydrophilic than the ether groups of PEG chains. Moreover, the branched polyglycerol chains in HPG effectively cover the hydrophobic surface of FNDs. Therefore, coating FNDs with HPG enhances the hydrophilicity of the surface of FNDs, and drastically suppresses nonspecific adsorption of biomolecules such as proteins31. As a method for a chemical coupling with HPG, cross-linker based on, for instance, N-

ACS Paragon Plus Environment

Bioconjugate Chemistry 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

hydroxysuccinimide (NHS)-ester reaction or Michael addition are used for bioconjugation with HPG-coated FNDs (FNDHPGs) to keep the higher hydrophilicity36. Commonly used cross-linkers require additional reaction steps to convert the somewhat inert –OH terminal groups present in the HPG chains into moieties possessing high reactivity, such as the carboxy (–COOH), amino (–NH2), or thiol (–SH) groups. Although carboxylation using succinic anhydride is frequently performed for this purpose37, it requires organic base catalysts such as pyridine and, notably, additional reaction and purification steps to obtain carboxylated FND-HPGs (FND-HPGCOOHs) (Scheme 1a). In this study, we demonstrate a simple, one-pot synthetic method to obtain FNDs coated with COOHterminated HPG that enables the generation of hydrophilic particles (FND-HPG-COOHs) that can undergo bioconjugation reactions more quickly and easily than FND-HPGs (Scheme 1b). In our method, to avoid having to use an organic base catalyst, the –OH groups in the HPG chains were carboxylated using succinic anhydride at high temperatures. Thus, the FND-HPG-COOHs were used for biotin–avidin conjuga-

tion. FND-HPG-COOHs were also conjugated with integrin antibodies to stain fixed cells. Furthermore, FND-HPGCOOHs were conjugated with mitochondrial localization signal peptides and introduced into live cells. Subsequently, optically detected magnetic resonance (ODMR) measurements were performed on NVCs within FNDs to evaluate the temporal temperature stability of these live cells.

Scheme 1. (a) Conventional and (b) one-pot synthesis of an HPG-coated fluorescent nanodiamond with terminal carboxy groups (FND-HPG-COOH).

Figure 1. Characterization of FND-COOHs (blue) and FND-HPG-COOHs (red). FT-IR spectra (a), zeta potentials (b), size distribution by percentage (c), and dispersibility in phosphate-buffered saline (PBS) (d). RESULTS AND DISCUSSION To confirm the synthesis of FND-HPG-COOHs, the characteristics of the obtained particles were investigated by FT-IR spectroscopy (Fig. 1). In particular, significantly enhanced – OH, –CH, and C–O–C vibrations (3300, 2915-2865, and 11001037 cm−1, respectively) were observed in the spectrum of the obtained particles compared (Fig. 1a, red spectrum) to that of untreated FND-COOHs (Fig. 1a, blue spectrum), indicating that an HPG layer had grown on the surface of FNDs and that

the -OH groups in HPG had been replaced by -COOH groups. The surface characteristics of the obtained particles were investigated by performing zeta potential measurements. The absolute value of the negative zeta potential was measured to be lower for FND-HPG-COOHs obtained after the one-pot treatment than for FND-COOHs (Fig. 1b). However, the zeta potential of the obtained particles was not positive, in contrast to what had been previously reported for FND-HPG37. This


Page 2 of 8

Page 3 of 8 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

Bioconjugate Chemistry result strongly supports the presence of -COOH groups on the surface of the obtained particles and, thus, the successful synthesis of FND-HPG-COOHs. Additionally, we evaluated the thickness of the HPG-COOH layer on the FND surface. For this purpose, the hydrodynamic particle sizes of FND-HPGCOOHs and FND-COOHs were measured by dynamic light scattering. According to the results obtained, the size distributions were 48.60 ± 14.58 nm and 57.55 ± 15.39 nm, respectively (Fig. 1c). These values indicate that the mean thickness

of the HPG-COOH layer was approximately 5 nm. The layer was as thin as ~16 nm by our method using even 100-nm FNDs (see Supporting Information S1). Surprisingly, despite its thinness, the HPG-COOH layer confers adequate dispersibility to the FNDs (Fig. 1d). Unlike FND-COOH, FND-HPGCOOHs were dispersed in phosphate-buffered saline (PBS) under physiological condition over 2 hours. Hence, our onepot method is suitable for the generation of water-dispersible nanodiamonds having -COOH groups on their surface.

Figure 2. (a) Schematic depiction of specific and nonspecific binding of avidin to an FND-HPG-biotin particle. Although avidin specifically binds biotin with high affinity, the hydrophobicity of the surface of nanodiamonds causes avidin to bind nanodiamonds nonspecifically. (b, c) Fluorescence emission data as a way to probe avidin binding specificity to FND-HPG-biotins (b) and FND-biotins (c).

The biotin–avidin system is widely used for the bioconjugation of fluorescent reagents with macromolecules. Using this system, FND-HPG-COOHs too can be conjugated specifically to a variety of biomoecules31,36, such as proteins, antibodies, and nucleic acids. We evaluated the efficiency and specificity of the bioconjugation of biotinylated FND-HPG-COOHs (FND-HPG-biotins) and avidin under physiological buffer conditions. For this purpose, FND-HPG-COOHs and FNDCOOHs (as a control) were modified with an amine-containing biotin derivative by NHS/WSC coupling to prepare the corresponding biotinylated FNDs (FND-HPG-biotins and FNDbiotins, respectively). The binding efficiency of FND-HPGbiotins to avidin was evaluated by comparing the fluorescence emission intensity of FND-HPG-biotins with that of FNDHPG-COOHs, after both species were treated with fluoresceinlabeled avidin (Fig. 2a). Measurements indicated that, following biotinylation, the binding efficiency of FND-HPG-COOHs was enhanced more than 83-fold (Fig. 2b). However, biotinylation enhanced the binding efficiency of FND-COOHs no more than 3.2-fold. Therefore, the HPG-COOH coating enhanced the avidin binding specificity of the FNDs at least 25 times.

method confers onto the FND-based particles adequate aqueous dispersibility and bioconjugation specificity under physiological conditions. Thus, the evidence indicates that FNDHPG-COOHs obtained by our method have a high potential as fluorescent probes for molecular and cellular imaging. To demonstrate this potential, we prepared a conjugate of FNDHPG-COOH and anti-integrin β3 antibodies (FND-HPGantibody), stained fixed HeLa cells with 4.8 µg/mL of FNDHPG-antibody, and performed fluorescence imaging of the fixed cells (Fig. 3a–c). Notably, integrins are highly expressed on tumor cells (such as U87MG glioblastoma cells), and are thus known to be one of the most important biomolecular targets for drug delivery systems (DDS) and oncotherapy38,39. As a result of the staining experiment, FND-HPG-antibodies (Fig. 3c, red bright spots) were specifically localized in the lamellar cytoplasm, where focal adhesions containing integrin β3 are also known to localize40 (Fig. 3c). On the other hand, significantly fewer red bright spots were observed in cells treated with 4.8 µg/mL of FND-HPG-COOHs without integrin β3 antibodies (Fig. 3d–f). These results indicate that the thin layer of HPG-COOH drastically suppressed nonspecific adsorption of cellular components, even in the solution supplemented with a variety of biomacromolecules typical of fixed cells.

Taken together, the aforementioned results indicate that the 5nm HPG-COOH layer obtained through our one-pot synthetic



Figure 3. Bright-field and confocal fluorescence images of surface-modified FNDs in fixed HeLa cells: (a–c) FND-HPG-Integrin β3 antibodies and (d–f) FND-HPG-COOHs. (a, d) fluorescence images. (b, e) Bright-field images. (c, f) Merged fluorescence and bright-field images. FNDs (red bright spots) were indicated by white dashed circles. Nonspecific FND-HPG-COOH absorption (d) was much fewer than that of FND-HPG-Integrin β3 antibodies (a). Importantly, FNDs can be used not just for fluorescence imaging but also for the nanoscale sensing of intracellular physical parameters, such as electric and magnetic fields, and temperature by ODMR techniques1,11–18. By using the characteristic as a temperature sensor, we used FND-HPG-COOHs synthesized through the one-pot method to monitor the temporal temperature stability of HeLa cells. For this purpose, FNDHPG-COOHs were conjugated with mitochondrial localization signal peptides (MLS; MSVLTPLLLRGLTGSARRLPVPRAKIHC)41 and introduced into HeLa cells by electroporation (see Supporting Information S3). Mitochondria have the key role in ATP production by consuming energy, and therefore they also produce excess enthalpy. Recently, a question of the “spatial variety of mitochondrial temperature” has been a hotly debated topic42,43. However, few experimental results of temporal temperature stability have been reported previously, despite the temporal

a

b

stability may affect the results of the spatial variety. For this reason, we chose mitochondria as a thermosensing target. We conjugated FND-HPG-COOH with MLS peptides (FND-HPGMLS), and treated HeLa cells with 45 µg/mL of FND-HPGMLS by electroporation. Then, ODMR frequency spectra were recorded in the absence of an external magnetic field at 37 ºC in living HeLa cells (Fig. 4a and b) and fit to multiple Cauchy functions (Fig. 4c, see also Supporting Information S4). The temporal temperature changes in cells were estimated from the best fit parameter of the spectral fit. As a typical result, the mean temperatures and measurement errors at each sampling time point were 0.12 ± 0.96, -0.71 ± 1.14, and 0.58 ± 1.67 K, respectively (Fig. 4d). Therefore, the temperature changes were smaller than 1 K and within the measurement error, indicating that FND-HPG-COOHs synthesized through our onepot method can be successfully used to conduct temperature measurements.

c

d

Temperature change HKL

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

Page 4 of 8

4 2 0 -2 -4 0

10

20 30 40 Time HminL

50

60

Figure 4. Typical results of temperature monitoring experiments conducted in a cell. Bright field (a) and fluorescence (b) images, typical ODMR spectrum (c, at 8 min; red solid line represents the fit curve) and temporal temperature changes (d) are shown. Temperature changes were obtained at 8, 28 and 50 min by measuring D-value changes of nitrogen-vacancy color centers in a nanodiamond (indicated by the red arrow). The temperature change at each point was obtained by averaging for 15 min. The temporal mean temperature was defined as temperature changes 0 K. The errors were estimated from the best fit parameters of optically detected magnetic resonance spectra.


Page 5 of 8 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


CONCLUSIONS In the present study, we showed that FND-HPG-COOHs prepared through the proposed one-pot synthetic method present a typically 5-nm-thin HPG-COOH layer on their surface and display excellent aqueous dispersibility. Biotinylated FNDHPG-COOHs were found to bind to avidins specifically. Moreover, particles obtained conjugating FND-HPG-COOH with antibodies were found to work well in experiments aimed at molecular labeling in fixed cells. In addition, we also demonstrated that FND-HPG-COOHs prepared by the mentioned method can be used to conduct temperature measurements in live cells. In summary, our one-pot synthetic method provides an easy-to-use technique that allows the bioconjugation of FNDs to be performed and renders new bioapplications of FNDs possible not only to chemists but also to bioresearchers. In particular, FND-HPG-biotins should be immediately available as a highly specific and effective DDS platform for oncotherapy through use of the biotin–avidin system44. In conclusion, we believe that our one-pot method paves the way to the wide and practical use of FNDs in the life sciences and clinical fields.

EXPERIMENTAL PROCEDURES Materials and Reagents. Type Ib diamond particles synthesized by HPHT method, Micron+MDA (100 nm particle size) and MD50 (50 nm particle size) were purchased from Element Six and Tomei Diamond Co. Ltd, respectively. All results of the experiments were independent of the difference in suppliers. 4% formaldehyde in phosphate-buffered saline (PBS), succinic anhydride, methanol, and glycine were purchased from Wako Pure Chemical Ind. 1Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC) was purchased from Dojindo Laboratories. Bovine serum albumin (BSA) (CAT. No. A3294), glycidol, and Nhydroxysulfosuccinimide (sulfo-NHS) were purchased from Sigma-Aldrich. Triton x-100 was purchased from Nacalai Tesque. Anti-integrin β3 chain monoclonal antibody (CAT No. M031-0) and fluorescein-labeled avidin (Avidin D) were purchased from Funakoshi Co., Ltd. Fetal bovine serum (FBS), PBS, Dulbecco’s modified Eagle’s medium (DMEM), trypsin, and EZ-Link™ amine-PEG2-biotin were purchased from Thermo Fisher Scientific Inc. Instruments. Fourier transform infrared (FT-IR) spectra were obtained on an ALPHA (BRUKER) spectrometer with a resolution of 4 cm−1. Zeta potentials and the diameters of samples were measured using a Malvern Zeta sizer Nano instrument. Time-dependent changes in the dynamic light scattering intensities of samples in PBS were determined using a Wyatt DynaPro NanoStar instrument. Fluorescence spectra were recorded using a FP8300ST Spectrofluorometer (JASCO). Confocal fluorescence experiments were conducted on a laser confocal scanning microscope (Carl Zeiss LSM710/780 ZEN). An Optima TM TLX Ultracentrifuge (BECKMAN COULTER) was used for nanodiamond purification. Production of fluorescent nanodiamonds.

A nanodiamond powder was irradiated with a 2-MeV proton beam at a dose of 1018 cm-2 for 50 h, followed by thermal annealing in vacuum at 800 °C for 2 h and oxidation in air at 550 °C for 2h. Synthesis of modified nanodiamonds. Carboxylated FNDs (FND-COOHs). Fluorescent nanodiamonds were treated with an H2SO4:HNO3 (9:1 v/v) mixture at 70 °C for 3 days, 0.1 M NaOH solution for 2 h at 90 °C, and a 0.1 M HCl solution n for 2 h at 90 °C, as previously reported.36 One-pot synthesis of FND-HPG-COOHs. FNDs-COOH (15 mg) in milli-Q (ultrapure water) was washed with glycidol three times for solvent replacement, dispersed in 4 ml of glycidol, sonicated, and stirred at 140 °C for 1 h under an argon atmosphere. After the reaction, 290 mg of succinic anhydride was directly added to the reaction pot, and the mixture was stirred at 140 °C for 1 h under an argon atmosphere. The reactant gel was diluted with milli-Q : methanol (1 : 1, v/v) and centrifuged at 45 krpm for 30 min to collect ND-HPGCOOH. FND-HPG-COOH particles were then washed three times by centrifugation to remove free polyglycerol. Synthesis of FND-biotins and FND-HPG-biotins. 1 ml of 10 mg/ml FNDs-COOH was made to react with 12 mg of sulfoNHS and 16 mg of WSC for 30 min to activate the surface carboxyl groups. The particles were then washed with milli-Q three times to remove unreacted reagents. 30 µL of 10 mg/ml NH2-biotin were then added, and the resulting mixture stirred for 2 h in ambient conditions. Next, the mixture was made to react with 50 µL of 15 mg/ml glycine for 30 min to quench the rest of activated carboxyl groups. Each sample was washed with milli-Q by centrifugation, yielding FND-biotins and FND-HPG-biotins. Synthesis of FND-HPG-Integrin β3 antibodies and mitochondrial localization signal peptide. FNDs were modified with HPG-COOH according to the one-pot synthesis method (see Supporting Information SI). Following modification, 2 ml of FND-HPG-COOHs (4.8 mg/ml) were activated by SulfoNHS/WSC in milli-Q for 30 min at room temperature and subsequently washed with milli-Q twice. The activated FNDs (48 µg) were made to react with integrin β3 antibody (100 µg) in PBS or mitochondrial localization signal peptide (150 µg) in milli-Q at 4 °C overnight. Afterwards, 1 µl of glycine (15 mg/ml) was added to the mixture to quench the rest of the activated COOH groups. Test for biotin–avidin interaction. FND-COOH and FND-biotin, and FND-HPG-COOH and FND-HPG-biotin particles were dispersed in milli-Q at a concentration of 5 mg/ml and 0.5 mg/ml, respectively, and made to react with 1 µL of fluorescein-labeled avidin for 2 h at room temperature. The FND-based particles were collected by centrifugation and repeatedly washed until the fluorescein signal at 495 nm — recorded in the supernatant using a fluorescent spectrometer — became negligibly low. After the washing process, the samples were suspended in PBS and monitored using the fluorescence spectrometer.



Cell culture. HeLa cells were cultured in DMEM and 10% FBS in 5% CO2 at 37 °C. Once they reached ~95% confluence, the cells were treated with trypsin and reseeded in cell culture dishes in order to observe and grow cells for imaging. Confocal fluorescence microscopy imaging. HeLa cells were seeded and incubated in the culture medium to ~70% confluence. To produce formaldehyde-fixed cells, HeLa cells were washed with PBS and fixed in 4% formaldehyde in PBS pH 7.0 for 10 min at room temperature. Next, the fixed HeLa cells were washed with PBS then treated with 0.5% triton x-100 in PBS for 5 min at room temperature to remove additional cellular membrane lipids. After this treatment, the fixed HeLa cells were washed with PBS twice and incubated with 1% BSA for 5 h to block nonspecific binding of the antibodies. FNDs dispersed in PBS with 0.1% BSA (4.8 µg/ml) were added to the fixed cells. After incubation overnight, the fixed cells were subsequently washed with PBS three times. The excitation wavelength of the nanodiamonds is 561 nm, and the emission wavelength region is from 619 nm to 717 nm. Temperature measurement in living cells. FND-HPG-COOHs conjugated with mitochondrial localization signal peptides (FND-HPG-MLSs) were introduced into HeLa cells by electroporation using NEON Transfection System (ThermoFisher). ODMR spectra of the FND-HPG-MLSs were measured using home-made ODMR equipment reported previously2. Temperature dependent D-values were obtained from the best fit to the spectra using mutually conjugated two Cauchy distribution functions, which represent spin excitation energies to |+1> and |-1> from |0>, respectively. ∆D ~ 75kHz/K was used as an approximation of temperature dependence of D-values. This approximation has no significant effect on the analyses of temperature evaluation in the error range of our measurement.

version of the manuscript. This work was supervised by R.I., Y.H. and M.S.

ACKNOWLEDGMENT This research was supported by World Premier International Research Center Initiative (WPI), MEXT Japan, JSPS KAKENHI Grant Numbers 26119001, 26286028, 26220602, Grant-in-Aid for Young Scientists, AMED Core Research for Evolutionary medical Science and Technology (AMED CREST, Grant Number JP16gm0510004), Japan Science and Technology Agency under Core Research for Evolutional Science and Technology (JST CREST, Grant Number JPMJCR1333, Japan), Precursory Research for Embryonic Science and Technology (PRESTO, Grant Number JPMJPR14F1), Japan Society for Promotion of Science under the Funding Program for Next-Generation World Researchers (NEXT Program), Sasakawa Scientific Research Grant.

REFERENCES (1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

ASSOCIATED CONTENT Supporting Information. Corrected supplementary information. This material is available free of charge via the Internet at http://pubs.acs.org.

(9)

AUTHOR INFORMATION (10)

Corresponding Author * To whom correspondence should be addressed: [email protected] or [email protected]. Author Contributions D.T. performed the surface modification of nanodiamonds, characterization of the modified nanodiamonds, all the cell experiments and ODMR measurements with R.I. S.S. devised the procedure of the one-pot synthesis of ND-HPG-COOH. R.I. designed and built the measurement system and analysis software. D.T. and R.I. analyzed the ODMR measurement data. D.T. and R.I. wrote the main manuscript text and prepared the figures. All authors have given approval to the final

(11)

(12)

(13)

Kucsko, G.; Maurer, P. C.; Yao, N. Y.; Kubo, M.; Noh, H. J.; Lo, P. K.; Park, H.; Lukin, M. D. Nanometre-Scale Thermometry in a Living Cell. Nature 2013, 500 (7460), 54–58. Igarashi, R.; Yoshinari, Y.; Yokota, H.; Sugi, T.; Sugihara, F.; Ikeda, K.; Sumiya, H.; Tsuji, S.; Mori, I.; Tochio, H.; et al. RealTime Background-Free Selective Imaging of Fluorescent Nanodiamonds in Vivo. Nano Lett. 2012, 12 (11), 5726–5732. Chang, Y.-R.; Lee, H.-Y.; Chen, K.; Chang, C.-C.; Tsai, D.-S.; Fu, C.-C.; Lim, T.-S.; Tzeng, Y.-K.; Fang, C.-Y.; Han, C.-C.; et al. Mass Production and Dynamic Imaging of Fluorescent Nanodiamonds. Nat. Nanotechnol. 2008, 3 (5), 284–288. Rendler, T.; Neburkova, J.; Zemek, O.; Kotek, J.; Zappe, A.; Chu, Z.; Cigler, P.; Wrachtrup, J. Optical Imaging of Localized Chemical Events Using Programmable Diamond Quantum Nanosensors. Nat. Commun. 2017, 8, 14701. Vaijayanthimala, V.; Cheng, P. Y.; Yeh, S. H.; Liu, K. K.; Hsiao, C. H.; Chao, J. I.; Chang, H. C. The Long-Term Stability and Biocompatibility of Fluorescent Nanodiamond as an in Vivo Contrast Agent. Biomaterials 2012, 33 (31), 7794–7802. Liu, K.-K.; Cheng, C.-L.; Chang, C.-C.; Chao, J.-I. Biocompatible and Detectable Carboxylated Nanodiamond on Human Cell. Nanotechnology 2007, 18 (32), 325102. Mohan, N.; Chen, C. S.; Hsieh, H. H.; Wu, Y. C.; Chang, H. C. In Vivo Imaging and Toxicity Assessments of Fluorescent Nanodiamonds in Caenorhabditis Elegans. Nano Lett. 2010, 10 (9), 3692–3699. Schrand, A. M.; Huang, H.; Carlson, C.; Schlager, J.; Hussain, S. M.; Dai, L. Are Diamond Nanoparticles Cytotoxic ? J. Phys. Chem. B 2007, 111, 1–7. Fu, C.-C.; Lee, H.-Y.; Chen, K.; Lim, T.-S.; Wu, H.-Y.; Lin, P.K.; Wei, P.-K.; Tsao, P.-H.; Chang, H.-C.; Fann, W. Characterization and Application of Single Fluorescent Nanodiamonds as Cellular Biomarkers. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (3), 727–732. Yu, S.-J.; Kang, M.-W.; Chang, H.-C.; Chen, K.-M.; Yu, Y.-C. Bright Fluorescent Nanodiamonds: No Photobleaching and Low Cytotoxicity. 2005, 17604–17605. Acosta, V. M.; Bauch, E.; Ledbetter, M. P.; Waxman, A.; Bouchard, L. S.; Budker, D. Temperature Dependence of the Nitrogen-Vacancy Magnetic Resonance in Diamond. Phys. Rev. Lett. 2010, 104 (7), 1–5. Tzeng, Y. K.; Tsai, P. C.; Liu, H. Y.; Chen, O. Y.; Hsu, H.; Yee, F. G.; Chang, M. S.; Chang, H. C. Time-Resolved Luminescence Nanothermometry with Nitrogen-Vacancy Centers in Nanodiamonds. Nano Lett. 2015, 15 (6), 3945–3952. Balasubramanian, G.; Chan, I. Y.; Kolesov, R.; Al-Hmoud, M.; Tisler, J.; Shin, C.; Kim, C.; Wojcik, A.; Hemmer, P. R.; Krueger, A.; et al. Nanoscale Imaging Magnetometry with Diamond Spins under Ambient Conditions. Nature 2008, 455 (7213), 648–651.


Page 6 of 8

Page 7 of 8

Bioconjugate Chemistry (14)

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

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

(27)

(28)

(29)

Maze, J. R.; Stanwix, P. L.; Hodges, J. S.; Hong, S.; Taylor, J. M.; Cappellaro, P.; Jiang, L.; Dutt, M. V; Togan, E.; Zibrov, A. S.; et al. Nanoscale Magnetic Sensing with an Individual Electronic Spin in Diamond. Nature 2008, 455 (7213), 644–647. Le Sage, D.; Arai, K.; Glenn, D. R.; Devience, S. J.; Pham, L. M.; Rahn-Lee, L.; Lukin, M. D.; Yacoby, A.; Komeili, A.; Walsworth, R. L. Optical Magnetic Imaging of Living Cells. Nature 2013, 496 (7446), 486–489. Barry, J. F.; Turner, M. J.; Schloss, J. M.; Glenn, D. R.; Song, Y.; Lukin, M. D.; Park, H.; Walsworth, R. L. Optical Magnetic Detection of Single-Neuron Action Potentials Using Quantum Defects in Diamond. Proc. Natl. Acad. Sci. 2016, 113 (49), 14133. Iwasaki, T.; Naruki, W.; Tahara, K.; Makino, T.; Kato, H.; Ogura, M.; Takeuchi, D.; Yamasaki, S.; Hatano, M. Direct Nanoscale Sensing of the Internal Electric Field in Operating Semiconductor Devices Using Single Electron Spins. ACS Nano 2017, 11 (2), 1238–1245. Dolde, F.; Fedder, H.; Doherty, M. W.; Nöbauer, T.; Rempp, F.; Balasubramanian, G.; Wolf, T.; Reinhard, F.; Hollenberg, L. C. L.; Jelezko, F.; et al. Electric-Field Sensing Using Single Diamond Spins. Nat. Phys. 2011, 7 (6), 459–463. Steinert, S.; Ziem, F.; Hall, L. T.; Zappe, A.; Schweikert, M.; Götz, N.; Aird, A.; Balasubramanian, G.; Hollenberg, L.; Wrachtrup, J. Magnetic Spin Imaging under Ambient Conditions with Sub-Cellular Resolution. Nat. Commun. 2013, 4. McGuinness, L. P.; Hall, L. T.; Stacey, A.; Simpson, D. A.; Hill, C. D.; Cole, J. H.; Ganesan, K.; Gibson, B. C.; Prawer, S.; Mulvaney, P.; et al. Ambient Nanoscale Sensing with Single Spins Using Quantum Decoherence. New J. Phys. 2013, 15. Hall, L.; Hill, C. D.; Cole, J. H.; Städler, B.; Caruso, F.; Mulvaney, P.; Wrachtrup, J.; Hollenberg, L. C. L. Monitoring Ion-Channel Function in Real Time through Quantum Decoherence. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (44), 18777–18782. Chu, Z.; Zhang, S.; Zhang, B.; Zhang, C.; Fang, C.-Y.; Rehor, I.; Cigler, P.; Chang, H.-C.; Lin, G.; Liu, R.; et al. Unambiguous Observation of Shape Effects on Cellular Fate of Nanoparticles. Sci. Rep. 2014, 4, 4495. Duffy, E.; Mitev, D. P.; Thickett, S. C.; Townsend, A. T.; Paull, B.; Nesterenko, P. N. Assessing the Extent, Stability, Purity and Properties of Silanised Detonation Nanodiamond. Appl. Surf. Sci. 2015, 357, 397–406. Rehor, I.; Slegerova, J.; Kucka, J.; Proks, V.; Petrakova, V.; Adam, M. P.; Treussart, F.; Turner, S.; Bals, S.; Sacha, P.; et al. Fluorescent Nanodiamonds Embedded in Biocompatible Translucent Shells. Small 2014, 10 (6), 1106–1115. Rehor, I.; Lee, K. L.; Chen, K.; Hajek, M.; Havlik, J.; Lokajova, J.; Masat, M.; Slegerova, J.; Shukla, S.; Heidari, H.; et al. Plasmonic Nanodiamonds: Targeted Core-Shell Type Nanoparticles for Cancer Cell Thermoablation. Adv. Healthc. Mater. 2015, 4 (3), 460–468. Prabhakar, N.; Näreoja, T.; von Haartman, E.; Karaman, D. Ş.; Jiang, H.; Koho, S.; Dolenko, T. a; Hänninen, P. E.; Vlasov, D. I.; Ralchenko, V. G.; et al. Core-Shell Designs of Photoluminescent Nanodiamonds with Porous Silica Coatings for Bioimaging and Drug Delivery II: Application. Nanoscale 2013, 5 (9), 3713–3722. Bumb, A.; Sarkar, S. K.; Billington, N.; Brechbiel, M. W.; Neuman, K. C. Silica Encapsulation of Fluorescent Nanodiamonds for Colloidal Stability and Facile Surface Functionalization. J. Am. Chem. Soc. 2013, 135 (21), 7815– 7818. Zhang, B.; Li, Y.; Fang, C. Y.; Chang, C. C.; Chen, C. S.; Chen, Y. Y.; Chang, H. C. Receptor-Mediated Cellular Uptake of Folate-Conjugated Fluorescent Nanodiamonds: A Combined Ensemble and Single-Particle Study. Small 2009, 5 (23), 2716– 2721. Chang, B. M.; Lin, H. H.; Su, L. J.; Lin, W. Der; Lin, R. J.; Tzeng, Y. K.; Lee, R. T.; Lee, Y. C.; Yu, A. L.; Chang, H. C.

(30)

(31)

(32)

(33)

(34)

(35)

(36)

(37)

(38)

(39)

(40)

(41)

(42)

(43)

(44)

Highly Fluorescent Nanodiamonds Protein-Functionalized for Cell Labeling and Targeting. Adv. Funct. Mater. 2013, 23 (46), 5737–5745. Neburkova, J.; Vavra, J.; Cigler, P. Coating Nanodiamonds with Biocompatible Shells for Applications in Biology and Medicine. Curr. Opin. Solid State Mater. Sci. 2017, 21 (1), 43–53. Sotoma, S.; Igarashi, R.; Iimura, J.; Kumiya, Y.; Tochio, H.; Harada, Y.; Shirakawa, M. Suppression of Nonspecific ProteinNanodiamond Adsorption Enabling Specific Targeting of Nanodiamonds to Biomolecules of Interest. Chem. Lett. 2015, 44 (3), 354–356. Liu, W.; Yu, F.; Yang, J.; Xiang, B.; Xiao, P.; Wang, L. 3D Single-Molecule Imaging of Transmembrane Signaling by Targeting Nanodiamonds. Adv. Funct. Mater. 2016, 26 (3), 365– 375. Wu, Y.; Ermakova, A.; Liu, W.; Pramanik, G.; Vu, T. M.; Kurz, A.; McGuinness, L.; Naydenov, B.; Hafner, S.; Reuter, R.; et al. Programmable Biopolymers for Advancing Biomedical Applications of Fluorescent Nanodiamonds. Adv. Funct. Mater. 2015, 25 (42), 6576–6585. Kong, X. L.; Huang, L. C. L.; Hsu, C. M.; Chen, W. H.; Han, C. C.; Chang, H. C. High-Affinity Capture of Proteins by Diamond Nanoparticles for Mass Spectrometric Analysis. Anal. Chem. 2005, 77 (1), 259–265. Zhao, L.; Takimoto, T.; Ito, M.; Kitagawa, N.; Kimura, T.; Komatsu, N. Chromatographic Separation of Highly Soluble Diamond Nanoparticles Prepared by Polyglycerol Grafting. Angew. Chemie - Int. Ed. 2011, 50 (6), 1388–1392. Sotoma, S.; Iimura, J.; Igarashi, R.; Hirosawa, K.; Ohnishi, H.; Mizukami, S.; Kikuchi, K.; Fujiwara, T.; Shirakawa, M.; Tochio, H. Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes. Nanomaterials 2016, 6 (4), 56. Boudou, J. P.; David, M. O.; Joshi, V.; Eidi, H.; Curmi, P. A. Hyperbranched Polyglycerol Modified Fluorescent Nanodiamond for Biomedical Research. Diam. Relat. Mater. 2013, 38, 131–138. Zhan, C.; Gu, B.; Xie, C.; Li, J.; Liu, Y.; Lu, W. Cyclic RGD Conjugated Poly(Ethylene Glycol)-Co-Poly(Lactic Acid) Micelle Enhances Paclitaxel Anti-Glioblastoma Effect. J. Control. Release 2010, 143 (1), 136–142. Zhao, L.; Xu, Y. H.; Akasaka, T.; Abe, S.; Komatsu, N.; Watari, F.; Chen, X. Polyglycerol-Coated Nanodiamond as a Macrophage-Evading Platform for Selective Drug Delivery in Cancer Cells. Biomaterials 2014, 35 (20), 5393–5406. Ko, H. Y.; Choi, K. J.; Lee, C. H.; Kim, S. A Multimodal Nanoparticle-Based Cancer Imaging Probe Simultaneously Targeting Nucleolin, Integrin Αvβ3and Tenascin-C Proteins. Biomaterials 2011, 32 (4), 1130–1138. Rizzuto, R.; Nakase, H.; Darras. B.; Francke, U.; Fabrizi, G. M.; Mengel, T.; Walsh, F.; Kadenbach, B.; DiMauro, S.; Schon, E. A.; A gene specifying subunit VIII of human cytochrome c oxidase is localized to chromosome 11 and is expressed in both muscle and non-muscle tissues. J. Biol. Chem. 1989, 264 (18), 10595-10600 Kiyonaka, S.; Kajimoto, T.; Sakaguchi, R.; Shinmi, D.; OmatsuKanbe, M.; Matsuura, H.; Imamura, H.; Yoshizaki, T.; Hamachi, I.; Morii, T.; Mori. Y.; Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells. Nat. Methods 2013, 10 (12), 1232-1238 Baffou, G.; Rigneault, H.; Marguet, D.; Jullien, L.; A critique of methods for temperature imaging in single cells. Nat. Methods 2014, 11 (9), 899-901 Brannon-Peppas, L.; Blanchette, J. O. Nanoparticle and Targeted Systems for Cancer Therapy. Adv. Drug Deliv. Rev. 2012, 64 (SUPPL.), 206–212.



Page 8 of 8

Table of Contents


8

One-Pot Synthesis of Highly Dispersible Fluorescent Nanodiamonds

Recommend Documents