Fructose-Coated Nanodiamonds: Promising Platforms for Treatment of

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Fructose-coated nanodiamonds: promising platforms for treatment of human breast cancer Jiacheng Zhao, Haiwang Lai, Hongxu Lu, Christopher Barner-Kowollik, Martina Heide Stenzel, and Pu Xiao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00754 • Publication Date (Web): 21 Jul 2016 Downloaded from http://pubs.acs.org on July 22, 2016

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Fructose-coated nanodiamonds: promising platforms for treatment of human breast cancer Jiacheng Zhao,a,b Haiwang Lai,a,c Hongxu Lu,a,c Christopher Barner-Kowollik,d Martina H. Stenzel,*,a,c and Pu Xiao*,a,c a

Centre for Advanced Macromolecular Design, bSchool of Chemical Engineering and cSchool of

Chemistry, The University of New South Wales, Sydney, Australia; dPreparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie (ITCP), Karlsruhe Institute of Technology (KIT), Engesserstr. 18, 76131 Karlsruhe, Germany and Institut für Biologische Grenzflächen, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76297 Eggenstein-Leopoldshafen, Germany

Corresponding Authors: [email protected]; [email protected]

Abstract: Well-defined

carboxyl

end-functionalized

glycopolymer

poly(1-O-methacryloyl-2,3:4,5-di-O-

isopropylidene-β-D-fructopyranose) has been prepared via reversible addition-fragmentation chain transfer polymerization and grafted onto the surface of amine-functionalized nanodiamonds via a simple conjugation reaction. The properties of the nanodiamond-polymer hybrid materials are investigated using infrared spectroscopy, thermogravimetric analysis, dynamic light scattering, and transmission electron microscopy. The dispersibility of the nanodiamonds in aqueous solutions is significantly improved after the grafting of the glycopolymer. More interestingly, the cytotoxicity of amine-functionalized nanodiamonds is significantly decreased after decoration with the glycopolymer even at a high concentration (125 µg/mL). The nanodiamonds were loaded with doxorubicin to create a bioactive drug delivery carrier. The release of doxorubicin was faster in media of pH 5 than that in pH 7.4. The nanodiamond drug delivery systems with doxorubicin are used to treat breast cancer cells in 2D and 3D models. Although the 2D cell culture results indicate that all nanodiamonds-doxorubicin complexes are significantly less toxic than free doxorubicin, the glycopolymer-coated nanodiamondsdoxorubicin show higher cytotoxicity than free doxorubicin in the 3D spheroids after treatment for 8

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days. The enhanced cytotoxicity of Poly(1-O-MAFru)62-ND-Dox in 3D spheroids may result from the sustained drug release and deep penetration of these nanocarriers, which play a role as “Trojan Horse”. The massive cell death after eight-day incubation with Poly(1-O-MAFru)62-ND-Dox demonstrates that glycopolymer-coated nanodiamonds can be promising platforms for breast cancer therapy.

Keywords: nanodiamond, polymer, RAFT polymerization, doxorubicin, human breast cancer

Introduction: Nanodiamonds have been emerging as ideal candidates for various biomedical applications1-7 due to their diverse advantages including non-toxicity, high biocompatibility, excellent optical properties, large surface areas, tuneable surface structures, non-bleaching fluorescence and many other properties.3,6,8 However, unmodified crude nanodiamonds normally tend to agglomerate and precipitate in solution, preventing their homogeneous surface modification and circumscribe their applications in, for instance, the area of drug delivery.9 Several efforts have been devoted to overcome this drawback and to facilitate their surface modification such as the beads-assisted sonication process9 and wetsirred-media-milling process10. These deagglomeration processes are normally conducted before or during the surface modification of nanodiamonds with the addition and assistance of other compounds (e.g. zironia beads9). Interestingly, the grafting of various polymers onto the surface of nanodiamonds can also reduce the tendency to agglomerate, thus increasing their dispersion stability in organic solvents, e.g. by coating with V-shaped polymer brushes of poly(t-butyl methacrylate)-b-poly(glycidyl methacrylate)-b-polystyrene11 or long alkyl chains.12 Furthermore, to adapt the nanodiamond systems for biomedical applications, it is important to improve their dispersibility in aqueous solutions. It has been reported that the dispersibility of nanodiamonds in water was significantly enhanced after the grafting of polyoxyethylene onto the nanodiamond surface.13 More interestingly, nanodiamonds with surface grafted water soluble poly(oligo(ethylene glycol) methyl ether methacrylate) through a “grafting-from” approach exhibited ACS Paragon Plus Environment

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better dispersion in aqueous solutions and can act as a promising carrier for the anti-cancer drug cisplatin.14 The hydrophilic polymer surface promoted the cellular uptake and thus enhanced the cytotoxicity to ovarian cancer cells.14 Even though a high grafting density can be achieved in the “grafting-from” approach, characterization of the surface-grafted polymers can be difficult.15 Recently, we employed a light-triggered strategy to functionalize nanodiamonds with well-defined polymers prepared through reversible addition-fragmentation chain transfer (RAFT) polymerization.15 Even though there are a few reports on the grafting of polymers on the surface of nanodiamonds,11,13,15-19 the use of these core-shell structures in biomedical applications is, to the best of our knowledge, still in its infancy with only few reports13,19-21 in the literature. Moreover, the grafting of fructose-based glycopolymer to nanodiamond surface has never been reported. Fructose-based polymers have recently shown to enhance the cellular uptake by breast cancer cells, thanks to the overexpression of GLUT-5 receptors on the surface.20,21 These glycopolymer-micelles normally exhibit excellent breast cancer cell-specific uptake. Therefore, it will be interesting to investigate the decoration of nanodiamonds with fructose-based glycopolymer on their cellular uptake by breast cancer cells. Herein, carboxyl end-functionalized poly(1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-β-Dfructopyranose) is prepared via RAFT polymerization and grafted onto the surface of aminefunctionalized nanodiamonds through a simple conjugation reaction. The properties of the nanodiamond-polymer

hybrid

materials

are

investigated

using

infrared

spectroscopy,

thermogravimetric analysis, dynamic light scattering, and transmission electron microscopy. Moreover, the anti-cancer drug doxorubicin is loaded to the prepared nanodiamond-polymer hybrid materials to demonstrate the ability of the nanoparticles to transport therapeutic payload into cells and across multicellular tumour spheroids.


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Experimental Materials Nanodiamonds(ND; < 10 nm particle size, Aldrich), N, N’-dicyclohexylcarbodiimide (DCC; Aldrich), 4-dimethylaminopyridine (DMAP; 94%, Aldrich), trifluoroacetic acid (TFA; 99%, Aldrich), and dichloromethane (DCM; anhydrous, >99.8%, Aldrich) were used as received. 1, 4-dioxane (99%, Ajax Finechem) was purified by reduced-pressure distillation. 2, 2’-Azobisisobutyronitrile (AIBN) was recrystallized twice from methanol before use. Amine-functionalized nanodiamonds (ND-NH2) were prepared as reported previously.22 The RAFT agent 4-cyanopentanoic acid dithiobenzoate (CPADB) was synthesized according to a literature procedure.23

Synthesis of Poly(1-O-MAipFru)62 The synthesis of 1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose followed a similar procedure as reported previously.21 The polymerization of the glycomonomer was described as follows: in a Schlenk tube, 1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose (4 g, 12.2 mmol), AIBN (2.6 mg, 1.62 × 10-2 mmol) and CPADB (35 mg, 0.12 mmol) were dissolved in 1, 4-dioxane (8.5 mL). Then the tube was degassed by three freeze-pump-thaw cycles. The polymerization was carried out at 70 ℃ and stopped at 7 h by cooling the solution in ice water (conversion: 62%). The polymer solution was poured into a large excess of diethyl ether for precipitation. The viscous polymer was dried under vacuum for 24 h. After reaction, the polymer solution was dialyzed against deionized water for two days (MWCO 3500).

Size exclusion chromatography (SEC) The molecular weight and dispersity Ð of the prepared polymers were analyzed via size exclusion chromatography (SEC). A Shimadzu modular system comprising a SIL-10AD auto-injector, ACS Paragon Plus Environment

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DGU-12A degasser, LC-10AT pump, CTO-10A column oven and a RID-10A refractive index detector was used. A 5.0-µm bead-size guard column (50 × 7.8 mm) followed by four 300 × 7.8 mm linear columns (500, 103, 104, and 105 Å pore size, 5 µm particle size) were employed for analysis. N, Ndimethylacetamide [DMAc; HPLC grade, 0.05% w/v 2,6-di-butyl-4-methylphenol (BHT) and 0.03% w/v LiBr] with a flow rate of 1 mL/min at 50 ℃ was used as mobile phase. 50 µL of polymer solution with a concentration of 2 mg/mL in DMAc was used for every injection. The calibration was performed using commercially available narrow-polydispersity PMMA standards (0.5-1000 kDa, Polymer Laboratories).

Grafting of polymer chains to the nanodiamond surface ND-NH2 (50 mg) was added to dry dichloromethane (DCM, 2 mL) and sonicated for 1 hour before introducing Poly(1-O-MAFru)62 (500 mg), N, N’-dicyclohexylcarbodiimide (DCC, 50 mg, 0.24 mmol), and 4-dimethylaminopyridine (DMAP, 30 mg, 0.24 mmol). The solution was stirred for 72 hours followed by centrifugation to isolate the nanodiamonds. The obtained nanodiamonds were then washed by consecutive washing/centrifugation cycles with DCM and then acetone for 5 times before drying under vacuum. The deprotection of the block copolymers (to removal of isopropylidene groups) was carried out under acidic conditions followed by dialysis against MQ to remove TFA.

Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) The infrared spectra of nanodiamonds were measured using ATR-IR (BRUKER, IFS 66/s).


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Thermogravimetric Analysis (TGA) Thermogravimetric analysis was performed on a Perkin-Elmer Thermogravimetric Analyzer (Pyris 1 TGA). Pre-dried samples were heated from room temperature to 800 ℃ at a constant temperature increase of 20 ℃/min using air as the furnace gas.

Dynamic Light Scattering (DLS) The hydrodynamic diameter Dh was determined using a Malvern Zetaplus particle size analyser (laser, angle = 173°) at a nanodiomand concentration of 100 µg/mL. Samples were prepared in deionized water and sonicated for 30 min prior to the measurements. The ζ potential determinations were based on electrophoretic mobility of the nanoparticles in the aqueous medium, which was performed using folded capillary cells in automatic mode.

Transmission Electron Microscopy (TEM) The TEM micrographs were obtained using a JEOL1400 transmission electron microscope comprising of a dispersive X-ray analyzer and a Gatan CCD facilitating the acquisition of digital images. The measurement was conducted at an accelerating voltage of 80 kV. The samples were prepared by casting the solution (100 µg mL-1) onto a copper grid. The grids were dried by air before measurement.

High-Performance Liquid Chromatography (HPLC) The high-performance liquid chromatography (HPLC) system is a Shimadzu modular system consisting of a SPD-20A UV−vis detector, DGU-20A3 degasser, LC-20AD pump (1 mL min-1), SIL20A HT auto sampler, and a Cole-Palmer single compact HPLC column heater (30 °C). The column


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was a Phenomenex Viva C18 (250 × 4.6 mm) with a pore size of 5 µm. The injection volume of each sample was 20 µL. A 22:78 mixture of acetonitrile (HPLC grade) and Milli-Q water (pH = 3.1 adjusted by phosphoric acid) was used as the mobile phase. A range of Dox solutions with different and known concentrations was prepared in the same solvent and used to prepare standard curve (Figure S1). The elution time of Dox was 21.5 min.

Doxorubincin (Dox) Loading Dox was loaded onto NDs (i.e. polymer-coated NDs or ND-NH2) according to the previously reported procedure.24 Briefly, NDs were dispersed in MQ water with a concentration of 1 mg/mL followed by ultrasonication for 1 hour. Then 1 mL NDs suspension (1 mg mL-1) was mixed with 1 mL Dox aqueous solution (200 µg/mL) to produce an ND-Dox complex solution. The pH of solution was adjusted with 1 mM NaOH to reach around 7.4. Dox was subsequently physically adsorbed on the surface of NDs to form purple Dox-NDs complexes. Unloaded Dox was removed by centrifugation. The amount of free Dox in the supernatant was analyzed by HPLC. The Dox loading efficiency (DLE) was analyzed via comparing the amount of Dox absorbed to ND with the initial amount of Dox added via HPLC. Drug loading efficiency (DLE) was calculated according to the following equation: DLE = (Dox added initially – Dox in supernatant after centrifugation)/(Dox added initially) × 100%.

Doxorubincin (Dox) Release from Nanodiamonds Dox, adsorbed to NDs at an initial Dox concentration of 0.1 mg mL-1, was desorbed in PBS (pH = 7.4) or phosphoric acid solution (pH = 5) at 37 °C, respectively. The percent of Dox desorption is based on the amount of Dox initially adsorbed onto the NDs and the amount of Dox released in PBS or phosphoric acid solution. Centrifugation for 30 min to pellet the ND-drug complex was performed. The adsorption solution was replaced with PBS and phosphoric acid solution. The ND-drug pellet was ACS Paragon Plus Environment

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suspended by gentle inversion and subsequently incubated at 37 °C. Dox in the supernatant was measured by HPLC after 2, 4, 6, and 9 hours and then in daily intervals. After each measurement, the supernatant was discarded and the ND-drug pellet was replaced with fresh PBS or phosphoric acid solution, subsequently inverted, and then incubated as demonstrated above.

WST-1 Assay for Cytotoxicity Human breast cancer MCF-7 and MDA-MB-231 cells were seeded in 96-well plates (4000 cells per well for MCF-7 Cells and 8000 cells per well for MDA-MB-231 cells) with 200 µL of culture medium Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2.2 g/L NaHCO3, 10% (v/v) foetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin in the incubator (5% CO2/95% air atmosphere at 37 °C) for 24 h. The sample solution to be tested was sterilized via UV irradiation (20 min) before the solution was serially halved via dilution in sterile Milli-Q water. The nanodiamond solutions were then loaded into the plate at 100 µL per well. After incubation for 72 h, supernatant was discarded and the plates were washed once with PBS. Then 100 µL of fresh warm medium with 5 µL WST-1 were added to each well and incubated for 2 hours in the incubator. The absorbance of each well was read on a Bio-Rad BenchMark microplate reader at 440 nm with a reference wavelength of 650 nm. The date was analyzed and plotted with Graphpad Prism 6.0.

3D Multicellular Tumor Spheroids (MCTS) Formation The MCTs were prepared as follows: MCF-7 cells were suspended in DMEM media at a density of 7.5 × 103 cells/mL. 200 µL of the cell suspension was seeded into each well of ultralow attachment 96-well plate (Corning) and incubated for 3 days.


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Acid phosphatase (APH) assay The MCTs were incubated with ND-Dox solution in 96 well suspension culture plates. After 4 and 8 days, MCTs and entire supernatant were transferred into U shape 96-well microplates with a pipettor and then centrifuge for 5 min at RT at 400×g to spin down spheroids, clusters and single cells. The spheroid pellet was washed by carefully replacing 170 µL of the supernatant with PBS. A final volume of 100 µL was obtained by repeating centrifugation and discard supernatant. Then 100 µL of APH assay buffer was added to each well and incubated for 90 min at 37 ℃. After incubation, supplement each well with 10 µL of 1 N NaOH and transfer the supernatant to standard flat-bottomed 96-well microplates. The absorption at 405 nm within 10 min was measured by a microplate reader.

Laser Scanning Confocal Microscopy Cells (MCF-7 cells) were seeded in 35 mm Fluorodishes (World Precision Instruments) at a density of 1 × 105 cells per well in 2 mL cell growth medium and cultured for 2 d. Free Dox, NH2-ND-Dox, and Poly(1-O-MAFru)62-ND-Dox were loaded to the cells at a Dox concentration of 100 µg/mL and incubated in the incubator for 12 h. After incubation, the cells were washed thrice with PBS and observed under a Zeiss LSM 780 laser scanning confocal microscope system. The Zeiss LSM 780 system was consisted of an argon laser (excitation wavelengths: 448 nm) connected to a Zeiss Axio Observer.Z1 inverted microscope (Air 20×/0.8 NA objective). The Zen2012 imaging software (Zeiss) was used for imaging acquisition and processing.


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RESULTS AND DISCUSSION Synthesis and coating of fructose-based glycopolymer on the nanodiamonds The synthesis of Poly(1-O-MAipFru) was carried out via RAFT polymerization (Scheme 1).25 Isopropylidene-protected

glycomonomer

(i.e.

1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-β-D-

fructopyranose) was polymerized at 70 ℃ in the presence of 4-cyanopentanic acid dithiobenzoate (CPADB) as a RAFT agent. The conversion of the monomers (62%) was tracked by 1HNMR. The degree of polymerization (DP) was calculated by the following equation: DP = [monomer]/[CTA] × c , where [Monomer] and [CTA] are the concentrations of the monomer and CTA, respectively; and c is the monomer conversion. The ratio of monomer to RAFT agent is 100 for Poly(1-O-MAFru)62. Thus, DP of Poly(1-O-MAFru) is 62.

Scheme 1. Synthesis and Preparation of ND-Poly(1-O-MAFru)62.

The SEC traces of Poly(1-O-MAFru)62 is shown in Figure 1. The molecular weight of the polymer (i.e. 10590 g/mol for Poly(1-O-MAFru)62) showed a unimodal distribution. The low dispersity index (Ð < 1.1) of the polymer indicates that the polymerization was conducted in a controlled manner.


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Normalized Response

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1.0

Poly(1-O-MAipFru)62

0.8

PDI = 1.08

0.6 0.4 0.2 0.0 3.0

3.2

3.4

3.6

3.8

4.0

4.2

4.4

LogMW

Figure 1. SEC curve of Poly(1-O-MAFru)62.

Amine-functionalized nanodiamonds (ND-NH2), which were prepared as reported earlier,22 were coated with polymer as shown in Scheme 1. The reaction was carried out under stirring at ambient temperature for 72 h. After the reaction, the solution was centrifuged and washed with DCM and then acetone for 5 cycles to ensure the removal of excess polymer. The deprotection of the glycopolymers was carried out under acidic conditions followed by dialysis against MQ to remove TFA. The functionalized nanodiamonds were then lyophilized for characterization. The FTIR spectra of ND-NH2 and polymer-functionalized NDs are demonstrated in Figure 2. The amino groups of ND-NH2 were observed at 3500-3000 cm-1 (N-H stretch) and 1630 cm-1 (N-H bend), which are in agreement with literature.26 After conjugation with Poly(1-O-MAipFru)62, the absorption at 1630 cm-1 disappeared confirming the successful reaction between surface bound amino functionalities and the terminal carboxyl groups of the polymer (Figure 2). As expected, a new absorption band of the resulting amide group at 1670 cm-1 appeared after reaction. In addition, the characteristic absorption bands of fructose at 1000-1200 cm-1 in the spectrum of ND-Poly(1-OMAFru)62 further confirm the successful coating of the glycopolymer onto nanodiamonds.


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Figure 2. FTIR spectra of ND-NH2, Poly(1-O-MAipFru)62, and ND-Poly(1-O-MAFru)62.

The quantification of polymers conjugated to the surface of nanodiamonds was carried out via thermogravimetric analysis (TGA). As demonstrated in Figure 3, Poly(1-O-MAFru)62 degraded rapidly once the temperature increases above 250 °C. However, the majority of the weight loss of ND-NH2 took place above 500 °C, which is consistent with the previously reported result.27 The weight loss correlating to the surface-grafted polymer of ND-Poly(1-O-MAFru)62 is around 15%. The amount of grafted polymer chains were then calculated based on the weight loss percentage using the method reported previously.22 The surface density of polymer chains ND-Poly(1-O-MAFru)62 are 0.17 molecules/nm2. Interestingly, the thermo-stability of nanodiamonds was enhanced by surface decoration with the polymer strands, which is consistent with reports in which PMMA/Multiwall carbon nanotubes nanocomposite exhibited higher thermo-stability than the matrix alone.28 The starting temperature for the bulk degradation of ND-Poly(1-O-MAFru)62 is close to 600 °C,at which residual product of degraded polymer started to decompose further. It seems that the conjugated polymers on the surface degraded first, followed by the decomposition of the nanodiamonds (Figure S2). The bulk degradation of the nanodiamonds started only after the complete degradation of coated polymers. The higher stability of the modified nanodiamonds could therefore be due to residual charring product of ACS Paragon Plus Environment

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degraded polymers on the surface, which partly isolated the nanodiamonds from heat, thus contributing to higher thermo-stability. As a control experiment, the physical adsorption of polymers by nanodiamonds was investigated by mixing nanodiamonds with polymers under stirring for three days. As shown in Figure 3, the enhanced thermal-stability of nanodiamonds as observed by surface conjugated polymer was not observed when the polymers were simply absorbed only. Since covalent linking between polymers and nanodiamonds is absent, the degraded polymers are easily removed by the air flow during the TGA measurement and have no capacity to act as thermal insulating layers.

1.0 0.8 Weight (%)

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0.6 0.4 ND-Poly(1-O-MAFru)62

0.2

ND-NH2 Poly(1-O-MAipFru)62

0.0

ND-NH2 + Poly(1-O-MAipFru)62

0

100 200 300 400 500 600 700 800 Temperature/oC

Figure 3. TGA analysis of ND-NH2, ND-Poly(1-O-MAFru)62, Poly(1-O-MAipFru)62 and ND-NH2/ Poly(1-O-MAipFru)62 mixtures.

Suspension stability of polymer-coated nanodiamonds in water The suspension stability of nanodiamonds in aqueous solution is an important parameter for drug delivery applications.29 Precipitation and aggregation of nanodiamonds in the blood stream prior to reaching the specific target will lead to failure of the system and cause side effects. The sizes of the nano carrier also play an important role in cellular uptake. Nanoparticles with a size of 50 nm are reported to have the highest cellular uptake.21,30 Therefore, surface stabilization is necessary to prolong


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the circulation time and inhibit aggregation of nanodiamonds in aqueous solution. Herein, the size and stability of polymer-grafted nanodiamonds were investigated using DLS and TEM. The unmodified nanodiamonds have a size of 10 nm. However, agglomeration of nanodimonds in acqueous solution was usually observed due to the intrinsic hydrophobicity of nanodiamonds. Surface modification of nanodiamonds with polymers is an effective method to enhance the dispersibility and stability in solution. As shown in Table 1, the DLS results illustrate that amino-functionalized nanodiamonds (ND-NH2) have a size of 169 nm, which is much higher than the size of polymer-coated nanodiamonds [i.e. ND-Poly(1-O-MAFru)62: 80 nm] (Table 1). In addition, polymer-coated nanodiamonds also exhibit lower PDIs than ND-NH2, which indicates that surface coating with polymers significantly enhances the dispersibility of nanodiamonds in water. The size and dispersibility of nanodiamonds are further confirmed by TEM. As illustrated in Figure 4, the results are in good agreement with those obtained from DLS. The sizes of ND-Poly(1-O-MAFru)62 is much smaller than that of unmodified ND-NH2 and aggregation was significantly reduced. The excellent dispersibility of polymer-coated nanodiamonds is ascribed to the high surface charge of these nanodiamonds. Specifically, hydrophilic Poly(1-O-MAFru)62 coated nanodiamonds exhibit great dispersibility in water which can be attributed to the fact that the grafted polymers led to an increased zeta-potentials (i.e. 46.4 mV for ND-Poly(1-O-MAFru)62; Table 1) compared to that of ND-NH2 (23.2 mV; the value is consistent with other reports.26), which enhances the stability in aqueous solution.31

Table 1. Size and zeta potential values of nanodiamonds measured by DLS. Nanodiamonds ND-NH2 ND-Poly(1-O-MAFru)62

Dh (nm) 169 80


PDI 0.72 0.14

Zeta Potential (mV) 23.2 46.4

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Figure 4. TEM images of nanodiamonds. (A) ND-NH2; (B) ND-Poly(1-O-MAFru)62.

The storage suspension stability of nanodiamonds in water was also tested (Figure 5A and 5B): after storage at room temperature for 72 hours, no precipitation was observed in the aqueous solution of ND-Poly(1-O-MAFru)62 as demonstrated in Figure 5. In contrast, amino-functionalized nanodiamonds (ND-NH2) settled out of the solution due to aggregation. These results highlight that polymer conjugation endows the ND-based materials with considerably enhanced dispersibility and suspension stability. Furthermore, the suspension stability of nanodiamonds in PBS solution was also evaluated (Figure 5C and 5D). Similar results were observed as well, which confirms the higher dispersibility of glycopolymer-functionalized nanodiamonds in both aqueous and physiological solutions.


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Figure 5. Stability of nanodiamonds in Solution. (A) NH2-ND in MQ water; (B) ND-Poly(1-OMAFru)62 in MQ water; (C) NH2-ND in PBS; (D) ND-Poly(1-O-MAFru)62 in PBS. (Storage at room temperature for 72 hours, concentration: 100 µg mL-1); [the permission to the use of the University of New South Wales UNSW Australia’s logo (for print and electronic use) has been granted by UNSW Australia].

Toxicity of polymer-coated nanodiamonds The cytotoxicity of nanodiamonds was subsequently tested as nanoparticles can cause possible adverse effects like lipid peroxidation of the plasma membrane, inflammation and genotoxicity.32 The proliferation of breast cancer cells MCF-7 in contact with nanodiamonds was investigated at various concentrations over a period of 24 h (Figure 6). Polymer-coated nanodiamonds did not show any toxicity to cells while amino-functionalized nanodiamonds significantly inhibited cell proliferation at high concentrations (above 31 µg/mL). Surface functionalization therefore enhances not only the suspension stability in solution, but also has beneficial effects in the quest to reduce cytotoxicity.


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MCF-7 cells 150

Cell survival rate (%)

ND-NH2 ND-Poly(1-O-MAFru)62

125

****

****

****

100 75 50 25

12 5. 00

62 .5 0

31 .2 5

15 .6 3

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3. 91

0 1. 95

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Concentration ( g/mL) Figure 6. Cytotoxicity of ND-NH2 and ND-Poly(1-O-MAFru)62 after 24 hours. Data represent means ± SD, n=4. ****, significant difference, P < 0.0001

Doxorubicin loading and releasing The use of nanodiamonds as drug delivery platforms for doxorubicin (Dox) has been widely investigated.24,33 However, studies regarding surface decoration of nanodiamonds with ligands to achieve active targeting (such as fructose which has high affinity to GLUT 5 transporters that overexpressed by breast cancer cells) are scarce. Compared with free Dox, which can be actively effluxed from cancer cells by drug transporter proteins like MDR1 and ABCG2,34 nanodimonds-Dox complex formed by physical adsorption with sodium hydroxide chemical treatment, has the capacity to enhance sustained release in vitro and in vivo.24 Herein, the effect of surface decoration on drug release


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was investigated as well. The Dox loading was carried out using a 5:1 ND/Dox ratio, which was the optimized ratio to form stable complexes as reported.24 The Dox loading efficiency (Table 2) of polymer-coated diamonds and unmodified nanodiamonds are close to 90%, which exhibit excellent Dox loading capacity. The driving force for the physical adsorption of Dox on the surface of nanodiamonds may be ascribed to the electrostatic interaction. As shown in Table S1, the zeta potential values of NH2-ND-Dox (9.5 mV) and Poly(1-O-MAFru)62-ND-Dox (18.3 mV) are much smaller than those of ND-NH2 (23.2 mV) and ND- Poly(1-O-MAFru)62 (46.4 mV) without loaded Dox. The adsorption of Dox on the surface of nanodiamonds resulted in reduced surface charge. Interestingly, after the loading of Dox onto ND-Poly(1-O-MAFru)62, the system still exhibited excellent suspension stability. But NH2-ND-Dox precipitated in aqueous condition after 12 hours of storage (Figure S3 in the SI).

Table 2. Dox loading efficiency of NH2-ND-Dox and Poly(1-O-MAFru)62-ND-Dox measured by HPLC. Nanodiamonds NH2-ND-Dox Poly(1-O-MAFru)62-ND-Dox

ND/Dox (m/m) 5/1 5/1

Drug loading efficiency (%) 88 85

The release of Dox from various nanodiamond drug delivery systems was demonstrated in Figure 7. ND-Dox systems at initial Dox concentrations of 0.1 mg/mL were desorbed in PBS (pH = 7.4) and phosphoric acid solution (pH = 5) at 37 °C. The percentage of Dox desorption is based on the amount of Dox initially adsorbed onto the NDs and the amount of Dox released in medium. Before each measurement, the suspension was centrifuged for 30 min to pellet the ND-Dox complex. The quantity of Dox in the supernatant was measured by HPLC after various time intervals. An initial large bolus of Dox was released within a few hours in both systems upon placement in both PBS (pH = 7.4) and phosphoric acid solution (pH = 5), which was commonly observed in many controlled release ACS Paragon Plus Environment

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formulations.35 The amount of Dox released from nanodiamond drug delivery systems in phosphoric acid solution (pH = 5) during initial 9 hours is around 3-fold higher than that in PBS (pH =7.4). The protonation of the drug at pH5 reduces the compatibility of the drug with the surrounding matrix, thus pushing out the drug. After an initial burst release, the release of Dox slowed down in both PBS and phosphoric acid solution. These results indicate that ND-Dox complexes are relatively stable in PBS solution while rapid desorption of Dox took place in an acidic environment.

100 80 Dox Desorption (%)

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

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60 40 20

Poly(1-O-MAFru)62-ND-Dox @ pH = 7.4 Poly(1-O-MAFru)62-ND-Dox @ pH = 5 NH2-ND-Dox @ pH = 7.4

0

NH2-ND-Dox @ pH = 5

0

20

40

60

80

100 120 140 160

Time (hours)

Figure 7. Drug release profile of Poly(1-O-MAFru)62-ND-Dox and NH2-ND-Dox in PBS buffer solution (pH = 7.4) and phosphoric acid solution (pH = 5) at 37℃.

Cytotoxicity of ND-Dox complex in 2D breast cancer cell culture models The cytotoxicity of Free Dox, NH2-ND-Dox, and Poly(1-O-MAFru)62-ND-Dox on breast cancer cell lines (MCF-7 and MDA-MB-231 cells) are shown in Figure 8. Compared with free Dox, nanodiamond drug (Dox) delivery systems resulted in less cell death in both MCF-7 and MDA-MB231 cells, which is in agreement with earlier results using LT2M and 4T1 cells.24 This is not surprising as Dox needs to be released from the drug carrier to cause cell death by intercalation with DNA, which inhibits DNA replication and cell growth. Compared with nanodiamond based nanocarriers, free Dox


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can cause cell death instantly after internalization, thus exhibiting higher cytotoxicity at the same incubation period. NH2-ND-Dox exhibited higher cytotoxicity than polymer-ND-Dox after 72 hours incubation, probably due to the inherent toxicity of the drug carrier (i.e. ND-NH2) itself.

Cell survival rate (%)

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Figure 8. Cytotoxicity of Free Dox, NH2-ND-Dox and Poly(1-O-MAFru)62-ND-Dox after 72 hours.

Cytotoxicity of ND-Dox complex in 3D breast cancer cell culture models


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3D spheroids which mimic in vivo tumor tissues are a versatile tool for studying penetration of nanoparticles and drugs.36,37 Compared with 2D cell culture models, the treatment of 3D spheroids is significantly affected by the penetration and drug release rate of drug delivery systems.38,39 Since Dox was reported to be actively effluxed from tumor cells by many drug transporter proteins, including MDR1 et al,34 Dox-ND complex might exhibit different therapeutic efficacy in 2D and 3D models. To test the cytotoxicity of drug delivery systems in 3D models, the cell viability of MCF-7 cells was evaluated on Day 4 and Day 8 respectively. As shown in Figure 9, after incubation for 4 days, free Dox treated MCF-7 spheroids exhibited higher cytotoxicity than other groups. Around 85% MCF-7 cells were killed during the initial 4-day treatment of free Dox (Table 3), compared with control group. The cell death of NH2-ND-Dox treated spheroids is slightly higher than that of Poly(1-O-MAFru)62-ND-Dox treated spheroids after 4 four-day incubation. These results are consistent with those of 2D monolayer models after three-day treatment. However, during the period from day 4 to day 8, Poly(1-O-MAFru)62-ND-Dox treated MCF-7 spheroids exhibited higher cell death, compared with the period from day 0 to day 4 [Figure 9(B)]. More than 90% cells of MCF-7 spheroids were killed by incubation with Poly(1-O-MAFru)62-ND-Dox from Day 4 to Day 8. NH2-ND-Dox caused around 66% cell death of MCF-7 cells while free Dox exhibited limited cytotoxicity to cells since the incubation from Day 4. These results suggest that Doxnanodiamonds complex prolonged the effective time of Dox. Significant cell death of MCF-7 cells took place after 4-day incubation with Dox-nanodiamonds complex while instant drug efficacy was observed when MCF-7 spheroids were treated with free Dox. Interestingly, the drug efficacy of free Dox could not persist after the initial 4-day treatment and no significant cell death was obtained during the period from Day 4 to Day 8. However, Poly(1-O-MAFru)62-ND-Dox exhibited accelerated antitumor efficiency from Day 4 to Day 8. The cell viability of MCF-7 spheroids treated by Poly(1-O-


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MAFru)62-ND-Dox is significant lower than that treated by free Dox after 8 days [Figure 9 (D)], suggesting an improved therapeutic efficacy by Poly(1-O-MAFru)62-ND-Dox than free Dox. Compared with the reduced cytotoxicity in 2D cell culture modes, Poly(1-O-MAFru)62-NDDox exhibited enhanced therapeutic efficacy in 3D spheroids than free Dox. The different cytotoxicity results may be ascribed to the structural difference between two cell culture models. In 2D cell culture models, cells are cultured on the plastic flask surface in a monolayer structure. For the 3D models, cells are suspended in the medium in the form of multicellular spheroids and drugs have to be delivered to the core of spheroids to achieve successful therapy in 3D models. In former study, we found that penetration of nanoparticles in 3D models depended more on transcellular transport than diffusion through ECM between cells and fast release of Dox from uncrosslinked micelles during penetration would kill peripheral cells and cause cessation of drug delivery.38 Therefore, an ideal drug nanocarrier should exhibit deep penetration as well as sustained drug release in 3D spheroid models. To investigate the penetration of free Dox and Dox-ND complex, confocal microscopy was used to investigate the location of Dox and nanocarriers. As shown in Figure 10, green fluorescence represents Dox while red fluorescence represents nanodiamonds. After 12-hour incubation, both NH2-ND-Dox and Poly(1-OMAFru)62-ND-Dox exhibited deeper penetration than free Dox. The reduced penetration of free Dox may be ascribed to the partial death of peripheral cells, which were caused by the internalized Dox. Interestingly, the deeper penetration of NH2-ND-Dox and Poly(1-O-MAFru)62-ND-Dox didn’t result in higher cytotoxicity than free Dox after four-day incubation in 3D spheroids. This is due to the sustained releasing profiles of NH2-ND-Dox and Poly(1-O-MAFru)62-ND-Dox, which is shown in Figure 7. Though the amount of NH2-ND-Dox and Poly(1-O-MAFru)62-ND-Dox inside the spheroids are high, the released free Dox was limited during the initial four days. However, after 8 days, most of the Dox were released from the surface of nanodiamonds and massive cell death was observed in NH2-ND-Dox and Poly(1-O-MAFru)62-ND-Dox treated spheroids. Therefore, the role of NH2-ND-Dox and Poly(1-O-


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MAFru)62-ND-Dox played in 3D models is like “Trojan Horse”. Release of Dox was triggered after nanocarriers have penetrated deeply inside the 3D spheroids. In contrast, transcellular transport of drugs was inhibited in free Dox treated spheroids and drugs were delivered via diffusion. The difficulty for free Dox to penetrate the cell barriers caused limited cell death during the period from Day 4 to Day 8. In addition, Poly(1-O-MAFru)62-ND-Dox which has active ligands (fructose moieties) and good particle size, can deeply penetrate into the inner part of MCF-7 spheroids and release Dox in a sustained manner afterwards. NH2-ND-Dox which has higher drug loading efficiency and faster Dox release rate than Poly(1-O-MAFru)62-ND-Dox in acidic condition, may have killed the cells instantly once they were internalized by peripheral cells. In addition, ND-NH2 which is toxic to cells at high concentration (Figure 6) might accumulate in cells and cause cell death in the first few days. Therefore, around 25% cells were killed during the incubation with NH2-ND-Dox after 4 days. However, the poor size (Table 1 and Figure 4) and suspension stability of ND-NH2 (Figure 5) as well as the fast release of Dox in acidic condition hindered the cellular uptake and penetration of NH2-ND-Dox, thus less cell death was caused compared with Poly(1-O-MAFru)62-ND-Dox from day 4 to day 8. These results indicate that surface decoration of nanodiamonds using specific polymers can significantly increase the suspension stability and biocompatibility of ND-Dox as well as tuning the drug releasing rate. The excellent therapeutic efficacy of Poly(1-O-MAFru)62-ND-Dox in MCF-7 3D spheroids suggests that polymer-diamonds hybrid materials may be promising platforms for breast cancer therapy.


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0.3

(B)

Day 4 Day 8

0.2

0.1

DD N 2-

Fr u) 6 Po ly (1 -O -M A

N

H

2-

N

D

D -D

ox

ox

O X

0.0 Fr ee

Optical density (a.u.)

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62

2

ox N D -D

Po ly (1 -O -M A

Fr u) 6

2-

N D -D 2-

N H

Fr ee

D O

ox

X

Optical density (a.u.)

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Figure 9. Cell viability of MCF-7 spheroids incubated with Free Dox, NH2-ND-Dox and Poly(1-OMAFru)62-ND-Dox. (A) Cell viability after 4-day and 8-day treatment, compared with control groups; (B) cell viability change during the period from Day 4 to Day 8. (C) cell viability after 4 days treatment; (D) cell viability after 8 days treatment. Data represent means ± SD, n=10. **, significant difference, P < 0.01 ***, significant difference, P < 0.001; ****, significant difference, P < 0.0001.


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Table 3. Cell death percentage of MCF-7 Spheroids incubated with Free Dox, NH2-ND-Dox and Poly(1-O-MAFru)62-ND-Dox during the period from Day 0 to Day 8. Drug Delivery System Free Dox NH2-ND-Dox Poly(1-O-MAFru)62-ND-Dox

Cell death percentage of MCF-7 Spheroids Day 0 ~ Day 4 Day 4 ~ Day 8 - 85% + 11% - 25% - 66% + 4% - 96%

Figure 10. Cellular uptake of (A) Free Dox; (B) NH2-ND-Dox; (C) Poly(1-O-MAFru)62-ND-Dox by MCF-7 spheroids after 12 hours incubation; Green fluorescence represents Dox; Red fluorescence represents nanodiamonds. The images were merged with DIC (Differential interference contrast) images. Scale bars (confocal microscopic images) = 100 µm.


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Conclusions To summarize, the development of nanodiamond-based drug delivery system is a valuable strategy to improve the therapeutic efficiency of Dox. In this study, poly(1-O-methacryloyl-2,3:4,5-diO-isopropylidene-β-D-fructopyranose), which has carboxyl groups at the end of polymer chains, was prepared via RAFT polymerization and grafted onto the surface of amine-functionalized nanodiamonds. The resulting polymer coated ND displayed better dispersibility in aqueous solutions compared to naked ND. Loading of the anti-cancer drug doxorubicin resulted in formation of a drug delivery system that could successfully deliver the drug into breast cancer cell. Although nanodiamonds-Dox complexes were significantly less toxic than free doxorubicin in 2D models, the polymer-coated nanodiamonds-Dox showed higher efficiency than free doxorubicin in the 3D spheroid models after treatment for 8 days. The results suggest that polymer-diamonds hybrid materials can be promising platforms for breast cancer therapy.

ASSOCIATED CONTENT Standard curve of Dox solution measured by HPLC (Figure S1); TGA curves of ND-NH2, Poly(1-OMAipFru)62, and ND-Poly(1-O-MAFru)62 (Figure S2).

Corresponding Authors *E-mail: [email protected]; [email protected]

Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS: J. Z. acknowledges the China Scholarship Council (CSC) for scholarship support. P. X. acknowledges funding from the Australian Research Council’s Discovery Early Career Researcher Award (DE140100318). C.B.-K. and M. H. S. acknowledge support for this project from the German Research Council (DFG). C.B.-K. acknowledges continued support from the Karlsruhe Institute of Technology (KIT) in the context of the STN and BIFTM program.


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REFERENCE (1) Terranova, M. L.; Orlanducci, S.; Rossi, M.; Tamburri, E. Nanoscale 2015, 7, 5094. (2) Passeri, D.; Rinaldi, F.; Ingallina, C.; Carafa, M.; Rossi, M.; Terranova, M. L.; Marianecci, C. J. Nanosci. Nanotechnol. 2015, 15, 972. (3) Hong, G.; Diao, S.; Antaris, A. L.; Dai, H. Chem. Rev. 2015, 115, 10816. (4) Moosa, B.; Fhayli, K.; Li, S.; Julfakyan, K.; Ezzeddine, A.; Khashab, N. M. J. Nanosci. Nanotechnol. 2014, 14, 332. (5) Zhu, Y.; Li, J.; Li, W.; Zhang, Y.; Yang, X.; Chen, N.; Sun, Y.; Zhao, Y.; Fan, C.; Huang, Q. Theranostics 2012, 2, 302. (6) Mochalin, V. N.; Shenderova, O.; Ho, D.; Gogotsi, Y. Nat. Nano 2012, 7, 11. (7) Zhao, L.; Xu, Y.-H.; Qin, H.; Abe, S.; Akasaka, T.; Chano, T.; Watari, F.; Kimura, T.; Komatsu, N.; Chen, X. Adv. Funct. Mater. 2014, 24, 5348. (8) Krueger, A.; Lang, D. Adv. Funct. Mater. 2012, 22, 890. (9) Liang, Y.; Ozawa, M.; Krueger, A. ACS Nano 2009, 3, 2288. (10) Hsin, Y. L.; Chu, H.-Y.; Jeng, Y.-R.; Huang, Y.-H.; Wang, M. H.; Chang, C. K. J. Mater. Chem. 2011, 21, 13213. (11) Cheng, J.; He, J.; Li, C.; Yang, Y. Chem. Mater. 2008, 20, 4224. (12) Krueger, A.; Boedeker, T. Diamond Relat. Mater. 2008, 17, 1367. (13) Cha, I.; Hashimoto, K.; Fujiki, K.; Yamauchi, T.; Tsubokawa, N. Mater. Chem. Phys. 2014, 143, 1131. (14) Huynh, V. T.; Pearson, S.; Noy, J.-M.; Abboud, A.; Utama, R. H.; Lu, H.; Stenzel, M. H. ACS Macro Lett. 2013, 246. (15) Wuest, K. N. R.; Trouillet, V.; Goldmann, A. S.; Stenzel, M. H.; Barner-Kowollik, C. Macromolecules 2016, 49, 1712. (16) Steenackers, M.; Lud, S. Q.; Niedermeier, M.; Bruno, P.; Gruen, D. M.; Feulner, P.; Stutzmann, M.; Garrido, J. A.; Jordan, R. J. Am. Chem. Soc. 2007, 129, 15655. (17) Wang, H.-D.; Yang, Q.; Niu, C. H. Diamond Relat. Mater. 2010, 19, 441. (18) Hutter, N. A.; Steenackers, M.; Reitinger, A.; Williams, O. A.; Garrido, J. A.; Jordan, R. Soft Matter 2011, 7, 4861. (19) Zhang, X.; Fu, C.; Feng, L.; Ji, Y.; Tao, L.; Huang, Q.; Li, S.; Wei, Y. Polymer 2012, 53, 3178. (20) Dag, A.; Callari, M.; Lu, H.; Stenzel, M. H. Polym. Chem. 2016, 7, 1031. (21) Zhao, J.; Babiuch, K.; Lu, H.; Dag, A.; Gottschaldt, M.; Stenzel, M. H. Chem. Commun. 2014, 50, 15928. (22) Huynh, V. T.; Pearson, S.; Noy, J.-M.; Abboud, A.; Utama, R. H.; Lu, H.; Stenzel, M. H. ACS Macro Lett. 2013, 2, 246. (23) Mitsukami, Y.; Donovan, M. S.; Lowe, A. B.; McCormick, C. L. Macromolecules 2001, 34, 2248. (24) Chow, E. K.; Zhang, X. Q.; Chen, M.; Lam, R.; Robinson, E.; Huang, H.; Schaffer, D.; Osawa, E.; Goga, A.; Ho, D. Sci Transl Med 2011, 3, 73ra21. (25) Gregory, A.; Stenzel, M. H. Prog. Polym. Sci. 2012, 37, 38. (26) Zhang, X.-Q.; Chen, M.; Lam, R.; Xu, X.; Osawa, E.; Ho, D. ACS Nano 2009, 3, 2609. (27) Krüger, A.; Liang, Y.; Jarre, G.; Stegk, J. J. Mater. Chem. 2006, 16, 2322. (28) Grumelard, J.; Taubert, A.; Meier, W. Chem. Commun. 2004, 1462. (29) Wang, Y.; Zheng, Y.; Zhang, L.; Wang, Q.; Zhang, D. J. Control. Release 2013, 172, 1126. (30) Jiang, W.; KimBetty, Y. S.; Rutka, J. T.; ChanWarren, C. W. Nat. Nano 2008, 3, 145.


29

Biomacromolecules

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

(31)

Page 30 of 31

Hanaor, D.; Michelazzi, M.; Leonelli, C.; Sorrell, C. C. J. Eur. Ceram. Soc. 2012, 32,

235. (32) Panessa-Warren, B. J.; Warren, J. B.; Wong, S. S.; Misewich, J. A. J. Phys.-Condes. Matter 2006, 18, S2185. (33) Moore, L.; Chow, E. K.; Osawa, E.; Bishop, J. M.; Ho, D. Adv. Mater. 2013, 25, 3532. (34) Sharom, F. J. Pharmacogenomics 2008, 9, 105. (35) Huang, X.; Brazel, C. S. J. Control. Release 2001, 73, 121. (36) Minchinton, A. I.; Tannock, I. F. Nat. Rev. Cancer. 2006, 6, 583. (37) Ma, H.-l.; Jiang, Q.; Han, S.; Wu, Y.; Cui Tomshine, J.; Wang, D.; Gan, Y.; Zou, G.; Liang, X.-J. Mol. Imaging. 2012, 11, 487. (38) Lu, H.; Utama, R. H.; Kitiyotsawat, U.; Babiuch, K.; Jiang, Y.; Stenzel, M. H. Biomater. Sci. 2015, 3, 1085. (39) Jiang, Y.; Lu, H.; Dag, A.; Hart-Smith, G.; Stenzel, M. H. J. Mater. Chem. B 2016, 4, 2017.


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For Table of Contents Use Only Fructose-coated nanodiamonds: promising platforms for treatment of human breast cancer Jiacheng Zhao,a,b Haiwang Lai,a,c Hongxu Lu,a,c Christopher Barner-Kowollik,d Martina H. Stenzel,*,a,c and Pu Xiao*,a,c


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Fructose-Coated Nanodiamonds: Promising Platforms for Treatment of

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