surface engineering of detonation nanodiamonds via ultrasonication-promoted ceric ammonium nitrate treatment

Diamond & Related Materials 104 (2020) 107738

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Disaggregation, stabilization, and innovative functionalization/surface engineering of detonation nanodiamonds via ultrasonication-promoted ceric ammonium nitrate treatment

T

Daniel Itshak-Levya,b, L.L. Israela,b, B. Schmerlinga, S. Kannanb,c, H. Sadea,b, S. Michaelib,c, ⁎ Jean-Paul Lellouchea,b, a b c

Department of Chemistry, Bar-Ilan University, Ramat Gan 5290002, Israel Institute of Nanotechnology & Advanced Materials (BINA), Bar-Ilan University, Ramat Gan 5290002, Israel Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel

A R T I C LE I N FO

A B S T R A C T

Keywords: Functional nanodiamonds Disaggregation Nanodiamond surface functionalization/ engineering

We present a novel method for aqueous effective disaggregation, dispersion, and stabilization of detonation nanodiamonds (NDs) that also allows easy further second-step nanodiamond (ND) functionalization/surface engineering through lanthanide-based coordination chemistry. This method includes ultrasonic irradiation of NDs in the presence of a strong mono-electronic ceric ammonium nitrate (CAN, [Ce(IV)(NH4)2(NO3)6]) oxidant. The resulting CAN-treated NDs are positively charged with lanthanide [CeLn]3/4+ complexes/cations, enabling an anti-aggregation effect together with the ability to be further surface-modified through [CeLn]3/4+ ligand exchange (lanthanide coordinative chemistry). Therefore, this method produces ~10 nm-sized CAN-modified nanoparticles (NDs-CAN NPs) that are highly positively charged (ξ potential maximal value: +45.7 mV & average zeta potential: +34.6 mV). The obtained ND surface modification by [CeLn]3/4+ complexes/cations enabled an organic-type coordination attachment of various different organic molecules. This innovative way of dealing with the well-known ND aggregation phenomenon enables a novel way for the development of a wide range of biomedical, imaging, and diagnostic-related ND-based applications.

1. Introduction Nanodiamonds' (NDs) remarkable properties, especially chemical stability, nontoxicity, and potential surface functionalization, have led researchers to view them as most promising materials in the nanomedical field as high-load nanoscale carriers for different types of molecules such as drugs, proteins, antibodies, enzymes, isotopes, or DNA sequences [1–3]. Chow et al. successfully used NDs conjugated with the cancer drug doxorubicin (NDX) as an effective drug delivery system. Compared to the treatment with doxorubicin alone, the efficiency of the drug-diamond conjugate was enhanced since NDX NPs penetrated cells and prevented them from expelling the drug, thereby lowering its usual toxicity, and enabling the application of higher doses of NDX, leading to the significant reduction of the tumors [4]. Most synthetic NDs are produced by the detonation of mixtures of explosives, generally RDX ((O2NNCH2)3) and TNT (C6H2(NO2)3CH3) in a closed chamber under a N2, CO2, and H2O-based atmosphere. After detonation, diamond-containing soot is purified by oxidation to remove non-diamond allotropes

⁎

of carbon, followed by acidic treatment to remove non‑carbon impurities, to obtain 4–8 nm nanoparticles (NPs) of diamond (nanodiamonds, NDs), or ultra dispersed diamonds (UDDs). The composition of NDs differs depending on the detonation process and the purification steps, though, generally, the core of obtained NDs is pure diamond with some nitrogen-vacancy centers (NVC) created by the internalization of nitrogen during the detonation process. The NVC phenomenon enables the fluorescence feature of corresponding NDs (FNDs). ND surfaces contain several organic groups, mostly oxygen-containing functional groups such as carbonyl (]O) and carboxyl (COOH) groups, which enables the covalent tailoring of the NDs surface [5]. Although the attractive properties of NDs are well known, a crucial problematic aggregation phenomenon hinders their use. Indeed, NDs form strong aggregates of 100–500 nm size, which further agglutinate into tight clusters of several microns when dried or dispersed into a wide variety of media. The mechanism behind aggregation is not yet fully known, but might be linked to high surface energy, hydrophobicity, π-π interactions, and electrostatic interactions [6]. Xu et al.

Corresponding author at: Department of Chemistry, Bar-Ilan University, Ramat Gan 5290002, Israel. E-mail address: [email protected] (J.-P. Lellouche).

https://doi.org/10.1016/j.diamond.2020.107738 Received 28 October 2019; Received in revised form 28 January 2020; Accepted 30 January 2020 Available online 30 January 2020 0925-9635/ © 2020 Elsevier B.V. All rights reserved.

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Scheme 1. General ND disaggregation procedure.

magnetic NPs by using the strong lanthanide-based complex Ceric Ammonium Nitrate [CAN, CeIV(NH4)2(NO3)6] oxidant under ultrasonication. The observed resulting NP water-based dispersions were stable, with highly positive NP zeta potential of +45.7 mV and NP DLS size of 6.61 ± 2.04 nm. Quite interestingly, the obtained Ce(III/IV) cations/complexes-doped surface of such NPs was highly positively charged, promoting a quite effective repulsive interaction between NPs [15]. We developed a similar oxidative process to adapt the different reactivity parameters of the previous iron oxide NPs (IONPs)-based research to novel NDs (time and power of sonication, CAN: NDs weight ratio, etc.). We propose here a similar methodology, that combines ultrasonication, the use of a lanthanide-based mono-electronic strong oxidant (CAN), and excluding centrifugation to produce long-lasting stable dispersions of NDs in water. We believe that treating NDs with CAN while simultaneously disaggregating them by ultrasonication will prevent functional ND reaggregation, by positively charging their surface and producing dispersions that are stable over time, thanks to the strong repulsive interactions between the positively charged nanoparticles thus obtained, providing NDs with anti-aggregating ability (Scheme 1). For their basic characterization, Fourier-transform infrared spectroscopy (FTIR) was first used for a qualitative study of the generated NDs-CAN surface, while the basic NP composition (core versus surface features) was further analyzed by inductively coupled plasma (ICP), thermogravimetric analyses (TGA), elemental analyses, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). XPS and XRD were also used to define the correct oxidation number of surface doping Ce+3/+4 cations/complexes. In our previous work (on maghemite-based IONPs), the mode of attachment of elemental Ce onto the surface of the maghemite NPs proved to be a combination of doping within the Fe atoms lattice and coordinative bonding with functional electronically rich groups present on the IONP surface, resulting in a combination of both Ce+3 and Ce+4 cations/complexes. This same oxidative doping process was also conducted with previously oxygenated NDs (ONDs) to see if a higher degree of ND oxidation influences the attachment/doping level of cerium-based cations/complexes. Moreover, we also tested the ability of existing surface doping Ce(III/ IV) complexes/cations formed on the surface of the NDs-CAN particles as linkers for further functionalization of NDs-CAN particles through lanthanide coordinative chemistry. Different types of molecules were chosen as conjugates, namely, 25 kDa branched polyethylenimine (bPEI), hyaluronic acid (HA), and antibacterial ampicillin were incubated with NDs-CAN and were analyzed for full characterization. Our current research seeks to present a rapid nanofabrication methodology to produce stable dispersions of NDs-based NPs, which will facilitate the functionalization of various types of organic molecules, for use in numerous biomedical applications.

suggest that the unusual tightess of ND aggregates is due to the covalent bonding between the organic impurities present on their surface and the formation of CeOeC ethereal linkages between interacting particles [7]. Existing methods for dispersing nanodiamonds include physical methods, such as milling with zirconium beads, [8] sonication, [9] combined thermal treatment for graphitization followed by oxidation of the surface, [7] or size-exclusion centrifugation, and chemical methods through the use of surfactants. Those methods have serious limitations: milling-based techniques use beads larger than the desired ND size, leaving the researchers unsure why size reduction occurred [6,8]. Disaggregating NDs by milling uses zirconia beads to crush the diamonds, but since diamonds are harder than zirconia, the product is contaminated with zirconia debris and particles from the milling machine [6]. Sonochemistry methods for disaggregation rely on the fact that accoustic waves pass through the medium containing NDs, inducing cavitation. The generated cavities, which are vapor-filled bubbles, collide with the ND aggregates, provoking shock-induced implosions that release relatively huge amounts of energy that can disrupt the existing interactions between the corresponding particles. Indeed, ND sonication produces disaggregation, but results in a nonhomogeneous size of such treated ND particles; and the strong horn-ultrasonication needed for the task can also contaminate the corresponding medium with particles from the horn itself [9]. Size-exclusion centrifugation might be used to circumvent this problem in order to produce homogenous dispersions of selected rare small-sized NDs present among the aggregates, but only in small yields, since most of the aggregates precipitate. The graphitization-oxidation method resulted in 50 nm-sized NPs, but also created even tighter aggregates, thereby harming the homogeneity of the dispersion [10]. Above all, an efficient disaggregating method should take into consideration the stability over time of these dispersions by preventing dispersed NDs from reforming aggregates. For the functionalization of ND surfaces, the various methods that are currently used are mainly based on organic covalent functionalization of the functional groups present on NDs surfaces. For example, NDs can be oxidized through acidic treatment (HNO3, H2SO4) in order to enrich ND surfaces with carboxylic groups, which can then be modified through organic chemistry to obtain the desired functionalization [11]. Nevertheless, these methods have a few significant drawbacks, namely, they are time-consuming (up to 10 h for reactions), require additional passivation steps, and make use of toxic reagents such as fluorine gas or sulfuric acid [12–14]. In addition, and quite relevantly, most current research concerning ND functionalization ignores the ND aggregation phenomenon, meaning that the researches were conducted on ND aggregates of variable sizes. The applicative potential of NDs lies in the ability to produce stable ND dispersions with appropriate surface modifications. Previous researches in our laboratory attempted to solve the aggregation issue of maghemite-based 2

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five cycles (4000 rpm, 18 °C, 5 min). We named this sample NDs-CANAmpicillin, and a similar nondoped NDs-ampicillin sample was prepared/tested for comparison purposes.

2. Materials and methods 2.1. Cerium-modification of NDs — NDs-CAN NPs

2.3.4. Toxicity test (MTT assays) 0.5 · 106 THP1 monocytes) human acute monocytic leukemia cell line (maintained in 4 ml in Roswell Park Memorial Institute medium (RPMI) were incubated with 1, 10, 50, 100, 200 μg of the NDs-CAN nanoparticles for 48 h. After 48 h, MTT reagent — thiazolyl blue tetrazolium bromide (from Sigma-Aldrich®) was added to the cell culture dish (working concentration 0.5 mg·ml−1) and incubated for 4 h at 37 °C. Following the incubation, the cells were pelleted, the supernatant was removed by centrifugation at 1500 rpm/10 min, the pellet was dissolved in 100 μl of DMSO, and read in 96 well plate at 570 nm/ 690 nm for formazan and the background, respectively, using a Synergy H1-ELISA reader. The THP1 cells were incubated for 48 h with different concentrations of both pristine NDs and Ce-modified NDs-CAN NPs.

The NDs/FNDs used in this paper were nonfunctional detonation nanodiamonds (NDs), purchased from Sigma-Aldrich®. The characteristics of NDs as reported by the manufacturer are: diamond nanopowder, < 10 nm particle size (TEM), ≥95% trace metals basis, spherical. 10 mg of NDs/FNDs were dispersed in 30 ml of double-distilled water (ddH2O) in a three-necked sonication vessel and irradiated in an ultrasonicator for 60 min (25% amplitude). During ultrasonication, an aqueous solution of CAN oxidant (10 mg/ml) was slowly added using a syringe. The nonbound CAN was then washed with ddH2O by pouring the solution into a centrifugal filter tube (100,000 kDa), and centrifuging for five cycles (4000 rpm, 18 °C, 5 min). The solution was then placed in a centrifuge for 10 min (11,000 rpm, −2 °C). After centrifugation, the supernatant was decanted into a vial and redispersed in a sonication bath for 5 min. We termed the nanomaterial sample obtained NDs-CAN/FNDs-CAN.

2.3.5. Methods X-ray photoelectron spectroscopy (XPS), FTIR, inductively coupled plasma atomic emission spectroscopy (ICP-AES), thermogravimetric analyses (TGA), and X-ray powder diffraction (XRD) analyses required the preparation of dry powder samples (by freezing with liquid nitrogen and lyophilization using a Free Zone 2.5 l benchtop freeze dry system, Labconco, Kansas City, MO, USA). High-power ultrasonication reactions were conducted using a high-power ultrasonicator (Sonics®, Vibra cell, 750 W, power modulator at 25%) equipped with a titanium horn under an argon atmosphere. Samples for low- and high-resolution TEM analyses were prepared by spreading a small drop of aqueous ND-based nanoparticle dispersions on amorphous carbon-coated copper grids (Formvar carbon 400 mesh grids, SPI® Supplies West Chester, PA, USA) followed by air-drying. Transmission electron microscopy at low and high resolutions (TEM/HR-TEM) made use of both JEM 1400 and 2100 (JEOL USA Inc.) microscopes (200 kV acceleration voltage, 2x2k and 4x4k CCD cameras, respectively). FT-IR spectra were recorded using a Bruker TENSOR 27 spectrometer (Diffuse Reflectance Accessory EasyDiff, PIKE Technologies, 4 cm−1 resolution). DLS (hydrodynamic NP average diameter) and zeta-potential measurements were taken for 0.2 mg/ml aqueous solutions (pH 7) using a Zetasizer Nano-ZS device (Malvern Instruments Ltd., UK), employing a nominal 5 mW HeeNe laser (operating wavelength: 633 nm, 20C, triplicate measurements). ICP-AES elemental analyses were carried out using an ULTIMA 2 spectrometer (HORIBA, Jobin Yvon Inc.). Surface-sensitive X-ray photoelectron spectroscopy (XPS) analyses were conducted using a Kratos Axis HS apparatus equipped with Kratos “Vision 2” package software. NP samples deposited on a double-sided carbon-based self-adhesive tape (complete coverage) were loaded in an ultra-high vacuum chamber (5 × 10−10 Torr). Thermogravimetric analyses (TGA) were performed using a TGA/DSC1 analyzer (N2 atmosphere at a heating rate of 10 °C min−1, Mettler-Toledo, OH, USA). Dry NP samples (8–10 mg) were used for TGA thermograms and corresponding weight loss derivative function graphs in a 25–850 °C temperature range. An X-ray diffraction (XRD) pattern was performed with a Bruker AXS D8 ADVANCE Series II diffractometer (Germany) equipped with a LynxEye detector (reflection θ–θ geometry, Cu Kα radiation, divergence slit 0.6 mm, anti-scattering slit 8 mm). The resulting diffraction data were collected in the angular range of 10° < 2θ < 80°, step size 0.02°, with a step time of 3 s/step.

2.2. Cerium-modification of oxidized NDs The oxidation of NDs was achieved following Krueger et al. [8] 10 mg of detonation nanodiamonds were placed in a furnace at 620 °C for 2 h (ONDs). The resulting ONDs were dispersed in 30 ml of doubledistilled water (ddH2O) in a three-necked sonication vessel and irradiated in an ultrasonicator for 60 min (25% amplitude). During ultrasonication, an aqueous solution of CAN oxidant (10 mg/ml) was slowly added using a syringe. The nonbound CAN was then washed with ddH2O by pouring the solution into a centrifugal filter tube (100,000 kDa), and centrifuging for five cycles (4000 rpm, 18 °C, 5 min). The solution was then placed in a centrifuge for 10 min (11,000 rpm, −2 °C). After centrifugation, the supernatant was decanted into a vial and redispersed in a sonication bath for 5 min. We termed the nanomaterial sample obtained ONDs-CAN. 2.3. Second-step functionalization — nanoparticle surface engineering using lanthanide cation/complex coordinative chemistry 2.3.1. 25 kDa b-polyethylenimine 10 mg of 25 kDa branched polyethylenimine (PEI) purchased from Sigma-Aldrich® was dissolved in 30 ml of ddH2O. The solution was mixed using a VortexGenie2 automatic stirrer. 3 ml of the solution was added to 15 ml of NDs-CAN aqueous solution (1.06 g/l). The solution containing both NDs-CAN and PEI was mixed for 24 h. To remove the nonattached bPEI, the resulting greyish composite was washed and centrifuged five times with ddH2O (11,000 rpm, −2 °C, 10 min). We termed the sample NDs-CAN-PEI. As reference and for reactivity comparison, a second similar solution was prepared following the same experimental procedure, but with non‑cerium-functionalized nanodiamonds (NDs-PEI). 2.3.2. Hyaluronic acid 5 ml of a 0.18 mg/ml aqueous solution of hyaluronic acid purchased from Sigma-Aldrich® (HA Mw = 799.641 g/mol) was added to 5 ml of NDs-CAN aqueous solution (1.06 g/l). The solution was mixed for 24 h and the excess of HA was then washed with ddH2O by pouring the solution into a centrifugal filter tube (100,000 kDa) and centrifuging for five cycles (4000 rpm, 18 °C, 5 min). We termed the sample obtained named NDs-CAN-HA, and a nondoped NDs-HA sample was prepared for comparison purposes.

3. Results 3.1. ND disaggregation and stability

2.3.3. Ampicillin 27 mg of ampicillin purchased from Sigma-Aldrich® was added to 9 ml of NDs-CAN solution (1 mg/ml), and mixed for 48 h. Cleaning was done using a centrifugal filter tube (100,000 kDa) and centrifuging for

The hydrodynamic size and zeta potential data of the particles are shown in Fig. 1. The starting material (pristine NDs) was the most aggregated sample, with a high dispersity of diameters going from 800 nm 3

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Fig. 1. DLS hydrodynamic sizes of NPs (a) pristine NDs, NDs-CAN, NDs-CAN-Ampicillin (b) pristine NDs, ONDs, ONDs-CAN and (c) FND, FND-CAN.

to several microns (1–8 μm). After the disaggregation, the NDs-CAN particles' hydrodynamic size was reduced to about 50 nm with homogeneous distribution (Fig. 1a). The heating-oxidation of pristine NDs (OND, Fig. 1b) also produced disaggregation, resulting in ~2 μm particles, which were further reduced to ~50 nm after this disaggregation process. A similar decreasing size effect is seen for FNDs from ~5 μm to ~122 nm (Fig. 1c). The real sizes of obtained NPs are shown by TEM micrographs of NDs-CAN (Fig. 2a, b, c). The pristine NDs (Fig. 2e) form large clusters (hundreds of nanometers long), while the CAN-modified NDs are more evenly dispersed and less aggregate, and individual ~10 nm-sized particles can be readily observed in TEM images (Fig. 2a, b, c). High surface potential values (Fig. 3) are due to high electrostatic repulsion between positively charged particles. CAN-modification of the NP surface caused their surface potential to immediately increase to ~+40 mV after the process and stabilize at ~+36 mV over the next 2 days (stored on a shelf at room temperature). While water-dispersion of pristine NDs results in turbid, greyish unstable solutions in which ND aggregates quickly sink, the resulting NDs-CAN aqueous dispersion remained clear and stable (Fig. 4). Neither sedimentation nor aggregation were detected when a sample of NDs-CAN remained untouched on a

shelf for several months (Fig. 5).

3.2. Assessment of NDs and NDs-CAN composition and surface functional groups We used thermogravimetric analyses (Fig. 6) to compare the temperature-dependent mass decrease of lanthanide-modified NDs (NDsCAN) to pristine NDs from room temperature up to 800 °C. The curve of pristine NDs (Fig. 6, blue line) shows a high rate of weight loss from 25 °C to ~150 °C, that can be linked to water evaporation, and a slow rate of weight loss from ~150 °C to 800 °C, which corresponds to organic impurities present on the ND surface. The TGA curve of the NPs resulting from our process (NDs-CAN, Fig. 6, red line) shows the same high rate of water weight loss from 25 °C to ~150 °C, followed by an even higher rate of weight loss from ~150 °C to ~350 °C, which corresponds to the loss of organic functional groups. In general, the total decrease in weight was higher for chemically modified NDs. The difference between the residual weights of modified and nonmodified NDs was found to be 13% for CAN-modified NDs. Corresponding to the TGA results, the ICP and elemental analyses, as well, showed an increase of the percentage of cerium and nitrogen in NDs-CAN, originating from 4

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Fig. 2. TEM images of NDs-CAN NPs: (a) low magnification (200 nm), (b)–(d) high magnification (20 & 50 nm), and (e) pristine NDs (2 μm).

series of multiplets with 8 identifiable peaks. Six peaks can be correlated to Ce+4 (denoted as v, v″, v‴, u, u′, and u‴, with binding energies of 887.48, 893.82, 906.22, 908.46, 903.55, and 921.5 eV, respectively) and two extra peaks (denoted u′ and v″, with binding energies of 903.6 and 912.2 eV, respectively) that can be correlated to Ce+3. Moreover, the obtained XRD pattern corresponds to the presence of 2 main crystalline phases (Fig. 8), i.e., a CeO2 tetragonal phase (Fig. 8, blue line), [18] and a nano-cubic diamond (Fig. 8, green line) [19]. Eight reflections at the Bragg angles of 28.3, 32.8, 47.0, 55.8, 58.5, 68.7, 75.9, and 78.2° correspond to the planes of the tetragonal crystal structure of CeO2 [18], while the reflections in the range of 42° to 45.5° correspond to the major reflections at 43.9° and 75.2° in the diamond cubic

the CAN reagent (Table 1). The XPS quantification of the surface showed the presence of 12.66% Ce oxide, and the corresponding survey scans showed only peaks relating to carbon, oxygen, and cerium. This XPS analysis of NDs-CAN was used to define the type of Ce-complexes/ oxidation status formed on the ND surface. The peak-fitted deconvolution spectra of Ce 3d levels are shown in Fig. 7. The Ce element exists in two main oxidation states (Ce+4 and Ce+3), and each shows characteristic XPS spectra features. Ce (IV) oxide is composed of two multiplets for a total of 6 identifiable peaks corresponding to the 3d5/2 and 3d3/2 spin-orbit splitting, as seen in the spectrum of CeO2 (Fig. 7a), while Ce (III) oxide has four identifiable peaks (Fig. 7b) [16–18]. The Ce 3d spectrum obtained from NDs-CAN powder (Fig. 7c) shows two

Fig. 3. Zeta potential measurement of 1 mg/ml NP solutions (pH 7): pristine NDs (blue), NDs-CAN (pink), and NDs-CAN-Ampi (ampicillin, green). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 5

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Fig. 4. Aqueous solution of 1 mg/ml pristine NDs (right) in water vs. NDs-CAN (left).

Fig. 5. Stability over time of NDs-CAN in water (1 mg/ml).

structure [19]. The theoretical Cubic Ce2O3 phase, that is described in Fig. 8 ([19], red line), shows a mismatch of this phase compared to the main Ce(IV) oxide phase in the sample, though with such large peaks we cannot dismiss the existence of Ce as Ce+3. 3.3. Second-step functionalization Second-step functionalization consisted of a further attachment to the obtained CAN-modified NDs (NDs-CAN). Fig. 9 shows the thermogravimetric analyses comparing fully functionalized NPs prepared with pristine NDs to fully functionalized NPs prepared with previously CANmodified NDs (NDs-CAN). In all three graphs (a, b, and c) the curve corresponding to previously CAN-modified NDs shows the same pattern, i.e., a high rate of weight loss, which is explained by (a) the evaporation of water absorbed on the surface until ~150 °C and (b) decomposition of organic material until ~300 °C. The curves corresponding to not previously Ce-modified NDs (NDs-CAN) show less weight loss. There is a 6% weight loss difference between the PEIcovered diamonds prepared with pristine NDs (NDs-PEI) and the PEIcovered diamonds prepared with previously CAN-modified NDs (NDs-

Fig. 6. TGA of pristine NDs (blue) vs. CAN-modified NDs (red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Table 1 Composition comparison of pristine NDs, NDs-CAN, and NDs-CAN surface.

Nitrogen (% wt.) Cerium (% wt.) Oxygen (% wt.) ⁎

Pristine NDs

NDs-CAN

NDs-CAN surface

1.5 0 11.79

3.5 0.53 11.95

n/a 12.66⁎ 17.23

The value of 12.66% refers to the relative amount of Ce oxide.

Fig. 8. X-ray diffraction pattern of NDs-CAN. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

Fig. 7. XPS Ce 3d spectra of Ce(III) oxide (a), Ce(IV) oxide (b) compared to the Ce 3d XPS spectrum obtained for NDs-CAN (c).

CAN-PEI). Moreover, the weight loss percentage for the NDs-PEI is similar to that for the pristine NDs (Fig. 6). It indicates that more PEI is bound to NDs-CAN, while very little PEI, if any, is bound to nonmodified NDs. We see the same pattern for the functionalization of NDsCAN with HA (13% weight loss) and with ampicillin (15% weight loss). The ampicillin presence is also supported by the following FTIR spectra (Fig. 10), which show the resemblance between spectra of pristine NDs and NDs-Ampicillin (where antibiotic was mixed with nonCAN-modified NDs), on the one hand, and, on the other, the spectra of ampicillin and NDs-CAN-Ampicillin (where antibiotic was mixed with

Fig. 9. TGA graphs of pristine NDs vs. (a) HA, (b) PEI, (c) ampicillin.

CAN-modified NDs-CAN NPs). All spectra contain peaks between 1300 cm−1 and 1900 cm−1, which correspond to the several carbonyl groups present in both ampicillin and pristine NDs. The spectra of both the pristine NDs and the NDs-Ampicillin showed characteristic peaks of NDs, namely, CeO bending peak at 1100 cm−1, and CeO stretching at 1600 cm−1, due to several oxygen-containing groups on the ND surface. 7

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3.4. Biocompatibility assessment (MTT assays) Both pristine and chemically modified NDs showed a dose-dependent toxicity with a significant cell viability decrease at the 100–200 μg·ml−1 levels. But no difference was seen between the toxicity data obtained from both pristine and modified-NDs, while surfaceengineered NDs-CAN NPs proved to be slightly less toxic. 4. Discussion To now, the functionalization and disaggregation of NDs were treated separately. Here we present a process to obtain a stable dispersion of easily functionalizable 10–20 nm-sized NDs-CAN particles. The results show 95% disaggregation, as the size of both NDs and FNDs decreased from several microns to a few nanometers. The combination of 3 disaggregation factors enabled a smooth disaggregation process without damaging the integrity of the NDs or the FNDs, which retained the optical properties that make them excellent candidates for many biomedical applications. The resulting hydrophilic NDs/FNDs-CAN NPs were stable: an NDs-CAN sample dating from last year is still dispersed and stable to now. Compared to existing methods, our nanofabrication process is rapid and simple. In contrast, the milling process by itself takes up to 3 h, [6,8] and the graphitization-oxidation method involves several hours of heating and cooling. Our method requires only 1 h of ultrasonication with oxidative CAN, 5 cleaning rounds of 5 min centrifugal ultrafiltration, and 15 min excluding centrifugation (for a total of 100 min) to change pristine highly aggregated NDs to 10–20 d·nm NPs that can easily be functionalized through ligand-exchange reactions, only requiring mixing NDs-CAN NPs with the desired molecule in an incubator. In order to avoid reaggregation of NDs, NPs produced by existing methods need to include an extra step of functionalization with some antiaggregating agents, generally surfactants to ready the molecule to be functionalized through covalent bonding which includes additional chemical reactions. As expected, the antiaggregation stability of NDs-CAN is due to the formation of Ce complexes [Ce+3/+4Ln] on their surface. In the presence of an overabundance of oxygen, CAN exchanges its nitrate ligands with oxygen-containing groups present on the surface of NDs through coordination chemistry to form Ce oxide. The charge of [Ce+3/+4Ln] complexes on the NDs surface provides the NDs with an antiaggregation capability, due to the strong electrostatic repulsion between positively charged particles. It should be emphasized that in this paper the resulting surface-engineered diamond NPs were not separated into primary grains of nanodiamonds, but into specific “diamond nano-aggregates,” since their measured size is under a hundred nanometers. Therefore, we still refer to them as “nanodiamonds,” word although this term should not be confused with “monodispersed nanodiamond particles.” Interestingly, as regards NDs-CAN, both XPS and XRD analyses showed that the elemental Ce was predominantly found as Ce+4 cation while, in previous research where CAN was used to disaggregate maghemite NPs, both Ce+3 and Ce+4 were found on the surface of such modified IONPs. This sheds light on the mode of attachment of the Ce element onto the NPs surface. The electron configuration [Xe]4f1 5d1 6s2 of Ce4+ is the most stable form, gaining noble gas configuration after losing 4 electrons. Ce3+ appears when there is a depletion of oxygen-containing groups or when the Lewis acid acting as the ligand is not oxygen. Consequently, the maghemite-CAN NPs Ce exhibit two types of attachment: formation of Ce (IV) oxide with oxygen containing impurities on the maghemite surface through coordination, and creating Ce3+ defects in the crystal structure of the iron oxide. This observation is supported by the fact that in the same experiment on maghemite NPs, the loading step of CAN was enhanced by increasing the amount of oxygen-containing impurities by previously diluting CAN in acetone. The previous oxidation of the NDs did not result in higher loads of Ce. It produced an extra disaggregation level (hydrodynamic

Fig. 10. FTIR absorbance spectra of ampicillin, pristine NDs, ampicillin attached to NDs with and without CAN (NDs-Ampicillin and NDs-CANAmpicillin, respectively).

The spectra of ampicillin and NDs-CAN-Ampicillin showed several similar peaks characteristic of ampicillin, e.g., the peak at ~695 cm−1 from the carbonyl of the lactam group, the peak at ~1025 cm−1 that can be attributed to the contribution of both the phenyl group and the amide group from the lactam, the peak at ~1385 cm−1 corresponding to the geminal dimethyl group, and the characteristic amide peak at ~1650 cm−1 [20]. The pristine-NDs did not bind to the ampicillin molecules, so the resulting spectrum resembles pristine-NDs. The NDsCAN surface is covered with ampicillin, so the resulting spectrum of NDs-CAN-Ampicillin resembles the spectrum of ampicillin, with some differences due to absorbance of NDs-CAN particles. The HR-TEM images (Fig. 11) of the resulting NDs-CAN-PEI show strong contrast between Ce located on the NDs (red arrows), and the NDs (yellow arrows). Recognizable branched-PEI (blue arrows) chains are observed around and between the NDs-CAN domains.

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Fig. 11. HRTEM images of NDs-CAN-PEI. NDs are indicated by yellow arrows, cerium, by red arrows, and PEI, by blue arrows. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

size of 70 d·nm), perhaps because the oxidation of NDs gives NPs a negative surface potential (−22.4 mV), producing electrostatic repulsion between negatively charged particles. This was sufficient to separate some of the aggregates, resulting in ~300 d·nm. In this case, ultrasonication acts on already reduced NPs, leading to NPs smaller in size than non-oxidized NDs. FNDs were disaggregated without damaging the optical properties that make them excellent candidates for many biomedical applications, such as imaging or mapping. The resulting NDs-CAN-conjugate NPs remained stable, keeping a highly positive or highly negative surface potential. The zeta-potential decreased to −22 mV with ampicillin, because of the various Lewis acid functions (amine, carboxylate, for example) of ampicillin shielding the Ce positive charge. A negative surface potential is not considered unstable, since only the charge itself defines the stability, not the sign of its charge. In order to check if our process of disaggregation through ultrasonic irradiation with CAN induces or increases the citotoxicity of the resulting modified NDs, an MTT viability assay was performed (Fig. 12). The citotoxicity results clearly show that our ND surface engineering process did not increase the toxicity of such modified NDs. The process even slightly decreased toxicity. Surprisingly, though, our starting material (NDs from Sigma-Aldrich®) was more toxic than any toxicitytested NDs source that one could find and check from the corresponding literature. The overwhelming majority of papers on both in vitro and in vivo studies that examined the cell viability of NDs concluded that NDs were biocompatible and nontoxic [3,21–23]., so our results do not cast doubt on the biocompatibility feature of modified NDs. The original idea of functionalizing NDs surface through coordinative chemistry using lanthanide complexes was proven to be

Fig. 12. Cytotoxicity evaluation after 48 h of incubation with NDs-CAN or pristine NDs in human monocytic cell line (THP-1).

sound. Our method produces stable dispersions of NDs/FNDs in a short nanofabrication time, with antiaggregation and antibacterial properties. A patent has been registered for the nanofabrication process [24]. We suggest that by removing the aggregation obstacle from the application of NDs, we will allow research to advance NDs applications more quickly and in a simpler manner.

CRediT authorship contribution statement Daniel Itshak-Levy: Investigation, Writing - original draft, Writing - review & editing. L.L. Israel: Methodology. B. Schmerling: Formal analysis, Methodology. S. Kannan: Investigation, Resources. H. Sade: 9

Diamond & Related Materials 104 (2020) 107738

D. Itshak-Levy, et al.

Writing - original draft, Writing - review & editing, Formal analysis, Validation. S. Michaeli: Investigation, Resources. Jean-Paul Lellouche: Project administration, Supervision, Conceptualization, Methodology.

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Declaration of competing interest All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version. This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue. The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript Acknowledgements We thank Drs. Yosef Gofer and Michal Eigenberg from the Chemistry Department of Bar-Ilan University for their valuable assistance regarding XPS analyses. Moreover, we also thank the anonymous Journal reviewers for their constructive comments that greatly improved our paper. We would also like to show our gratitude to our student interns Ethan S. Ohayon and Benjamin D. Zaionz (Unversity of Waterloo, Canada) for whom we anticipate a great future scientific career. References [1] R.J. Narayan, W. Wei, C. Jin, M. Andara, A. Agarwal, R.A. Gerhardt, et al., Microstructural and biological properties of nanocrystalline diamond coatings, Diam. Relat. Mater. (2006), https://doi.org/10.1016/j.diamond.2006.08.024. [2] L. Moore, J. Yang, T.T.H. Lan, E. Osawa, D.K. Lee, W.D. Johnson, et al., Biocompatibility assessment of detonation nanodiamond in non-human primates and rats using histological, hematologic, and urine analysis, ACS Nano (2016), https://doi.org/10.1021/acsnano.6b00839. [3] V.N. Mochalin, O. Shenderova, D. Ho, Y. Gogotsi, The properties and applications of nanodiamonds, Nat. Nanotechnol. (2012), https://doi.org/10.1038/nnano.2011. 209. [4] Y. Kuo, T.Y. Hsu, Y.C. Wu, H.C. Chang, Fluorescent nanodiamond as a probe for the intercellular transport of proteins in vivo, Biomaterials (2013), https://doi.org/10. 1016/j.biomaterials.2013.07.043. [5] K. Iakoubovskii, M.V. Baidakova, B.H. Wouters, A. Stesmans, G.J. Adriaenssens, A.Y. Vul’, et al., Structure and defects of detonation synthesis nanodiamond, Diam. Relat. Mater. (2000), https://doi.org/10.1016/S0925-9635(99)00354-4. [6] A. Krüger, F. Kataoka, M. Ozawa, T. Fujino, Y. Suzuki, A.E. Aleksenskii, et al., Unusually tight aggregation in detonation nanodiamond: identification and

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