PaclitaxelâNanodiamond Nanocomplexes Enhance Aqueous

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Paclitaxel-nanodiamond nanocomplexes enhance aqueous dispersibility and drug retention in cells Dae Gon Lim, Ju Hyun Jeong, Hyuk Wan Ko, Eunah Kang, and Seong Hoon Jeong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b08079 • Publication Date (Web): 22 Aug 2016 Downloaded from http://pubs.acs.org on August 28, 2016

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ACS Applied Materials & Interfaces

Paclitaxel-nanodiamond nanocomplexes enhance aqueous dispersibility and drug retention in cells Dae Gon Lim1, Ju Hyun Jeong1, Hyuk Wan Ko1, Eunah Kang2*, Seong Hoon Jeong1* 1

2

College of Pharmacy, Dongguk University-Seoul, Gyeonggi, Republic of Korea

School of Chemical Engineering and Material Science, Chung-Ang University, Seoul, Republic of Korea

* To whom correspondence should be addressed. Seong Hoon Jeong, PhD College of Pharmacy Dongguk University – Seoul Goyang, Gyeonggi 410-820, Republic of Korea Tel: 82) 10-5679-0621 E-mail: [email protected]

Eunah Kang, PhD School of Chemical Engineering and Material Science Chung-Ang University 221 Heukseok-Dong, Dongjak-Gu, Seoul, Republic of Korea Tel: 82)-2-820-6684 E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT

Nanodiamonds (NDs) with 5 nm crystalline structures have been recognized as emerging carbon delivery vehicles due to their biocompatible inertness, high surface to volume ratio, and energy absorbance properties. In this study, carboxylated nanodiamond (ND-COOH) was reduced to hydroxylated nanodiamond (ND-OH) for stable and pH-independent colloidal dispersity. The poorly water soluble paclitaxel (PTX) was physically loaded into ND-OH clusters, forming amorphous PTX nanostructure on the interparticle nanocage of ND substrate. Stable physical PTX loading onto ND substrate with stable colloidal stability showed enhanced PTX release. ND-OH/PTX complexes retained the sustained release of PTX by up to 97.32 % at 70 hr, compared with the 47.33% release of bare crystalline PTX. Enhanced PTX release from ND substrate showed low cell viability in Hela, MCF-9, and A549 cancer cells due to sustained release and stable dispersity in a biological aqueous environment. Especially, the IC50 values of ND-OH/PTX complexes and PTX in Hela cells were 0.037 µg/mL and 0.137 µg/mL, respectively. Well dispersed cellular uptake of suprastructure ND-OH/PTX nanocomplexes was directly observed from the TEM images. ND-OH/PTX nanocomplexes uptaken into cells might provide convective diffusion with high PTX concentration, inducing initial necrosis. This study suggests that poorly water soluble drugs can be formulated into a suprastructure with ND and acts as a highly concentrated drug reservoir directly within a cell.

Keywords: Nanodiamond, drug delivery, cellular uptake, paclitaxel, hydroxylated, carboxylated, colloidal stability


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1. Introduction Nanodiamonds (NDs) have been recognized as new emerging carbon materials in biomedical applications, such as imaging1-2, drug delivery platforms3, and interfacing enhancers in dental resin.4 Especially, NDs as molecule carriers of drug delivery platform are interconnected to the new interface of proteins5-7, genes8, and small chemical drugs.9 The advantages of NDs are attributed to their biocompatible inertness, high surface to volume ratio, and strong moleculeabsorbing capability, enabling them to provide stable hybrid complexes. Moreover, the carbon surfaces of NDs, which are covered by a variety of oxygen-containing functional groups, have led to the incorporation of biologically active compounds and drugs, serving as the basis of covalent functional groups.10 The detonated NDs with a 5 nm crystalline structure spontaneously form 10~100 nm-sized clusters, called agglutinates, not a single crystalline particle in an aqueous solution. ND agglutinates could facilitate the loading of therapeutic agents on the surface of or in the internal nano-scaled pores within ND clusters through non-covalent interactions.11 ND delivery platforms provide the spatial substrate of hydrophobic hydration via the hydrogen bonds, electrostatic interaction12-13, and non-covalent interaction of van der Waals14 and π-π stacking.15 Moreover, the high surface to volume ratio offers a spatial reservoir for physical nanocage or intense charge density for stable electrostatic interaction, providing a stable agglutinate of nano-dispersion in an aqueous solution. Poorly water-soluble paclitaxel (PTX), an antineoplastic agent, has been delivered in the form of nanocrystals16, micelles17, and polymeric nanoparticles.18-19 PTX has demonstrated impressive clinical activity against ovarian, breast, and non-small cell lung carcinomas, and AIDS-related Kaposi’s sarcoma and it acts by stabilizing abnormal microtubule structures.20 Due to the low intrinsic solubility of PTX of 0.30 µg/mL in water21, it shows poor oral systemic availability.22 The representative administration as an intravenous infusion, Taxol®, is a formulation of PTX (6



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mg/mL) in a 50:50 ratio of Cremophor EL and ethanol.23 However, Cremophor EL has severe adverse reactions, such as hypertension, nephrotoxicity, and neurotoxicity. Thus, there is still room to enhance the solubility increase of PTX for developing a PTX formulation. In particular, hydrotropic agents and hydrotropic polymer have elevated PTX delivery through high loading efficacy24 by incorporating PTX as an amorphous form. Moreover, the crystalline PTX alignment has been produced by PTX solvation with an organic solvent in the solid state.25 Nanocrystalline PTX can also be formulated with the top-down method of the milling process, which minimizes the size within the matrix of a stabilizer.26 Nanocrystalline PTX formed from wet milling was used for the treatment of hyperthermic intraperitoneal chemotherapy, providing a high local concentration in a peritoneal tumor.27 The nanocrystalline form of hydrophobic PTX was stably dispersed in an aqueous solution with therapeutic efficacy.28 The nanocrystalline drug also has advantages of minimized toxic excipients and high solubility with increased surface area. Nanocrystalline drugs have been known to provide drug tracking within cell cytosol and highly effective drug delivery with a highly concentrated platform.27 Nanoscaled PTX in an aqueous environment may be achieved by concentrated incorporation on an ND platform. Previous studies of the ND/hydrophobic drug complex showed the chemically conjugated29 or physical adsorption form.30 The release of the adsorbed hydrophobic drug seemed to be dependent on the surface properties of ND, including pH dependence or charge interaction, rather than a simple concentration gradient. Highly purified NDs with acidwashing possesses the exterior of a carboxyl end group, possessing strong negative zeta potential.31 Molecular simulation suggested that the hydroxyl group on ND obtained thermodynamically stable colloidal properties compared with other functional groups32 and was able to engage with hydrophobic PTX in an aqueous solution. Together with ND surface


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modification, however, formulation of the PTX-concentrated form has been less exploited on an ND delivery platform. In this study, the nanocrystalline PTX and ND-OH/PTX nanocomplexes were prepared with the solvent exchange method. These groups were used to investigate whether nanoscaled dispersion in an aqueous environment enhances drug delivery efficiency. The carboxyl surface of nanodiamond (ND-COOH) was modified to a hydroxyl end group to ensure stable colloidal dispersion with pH independence. Hydroxylated ND encases the PTX molecules that were grown on the ND nanocage of interparticle nanosubstrates. The physical properties of waterinsoluble PTX in ND complexes were examined, as the PTX was loaded into ND-OH agglutinates. Cell viability and cellular uptake were investigated for the therapeutic efficacy after the treatment of ND-OH/PTX nanocomplexes in Hela, MCF-9, and A549 cells.

2. Experimental method 2.1. Materials The commercial products of carboxylated ND and PTX were kindly supplied by Nanoresource Co., Ltd. (Seoul, Korea) and Samyang Biopharmaceuticals (Seoul, Korea), respectively. Fluorescein isothiocyanate (FITC)-labeled Annexin V and propidium iodide (PI) were purchased from Novus Biologicals (CO, USA). Ethanol and acetonitrile (ACN) were purchased from Avantor Performance Materials (PA, USA). All other reagents were of an analytical grade.

2.2. Preparation of ND-OH particles The reduction of ND-COOH was carried out with the addition of NaBH4 to generate the hydroxyl end group on the ND substrates (Figure 1a). The ND-COOH (50 mg) was sonicated



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and dispersed in ethanol (100 mL) and mixed with NaBH4 (100 mg). The mixture was heated at 60 ºC while stirring for 24 hr. After the reduction, the resulting ND-OH was centrifuged and the supernatant was removed to separate ND-OH at the bottom. The remaining ND-OH was washed with ethanol (50 mL × 2) and distilled water (50 mL × 3). The product of ND-OH particles was dispersed in distilled water and lyophilized using a Lyopride 20R lyophilizer (Ilshin Biobase, Gyeonggi, Korea) for further study.

2.3. Preparation of ND-OH/PTX complexes and nanocrystalline PTX The dispersion of ND-OH (2 mg) in distilled water (10 mL) was prepared with bath sonication until aggregates were not observed with the naked eye. The PTX stock solution was prepared in ethanol with the concentration of 2 mg/mL. ND-OH/PTX complexes and nanocrystalline PTX were prepared with probe sonication (150 W) for 3 min using a VCX-500 ultrasonic processor (Sonics & Materials, USA). A PTX stock solution of 50 µL was added dropwise into 1 mL of ND-OH dispersion or water to produce PTX-only nanocrystals and ND-OH/PTX complexes with a 2:1 weight ratio (Figure 2a). The dispersions were gently stirred at 50oC to remove residual ethanol, allowing PTX complete solvent exchange. For solid state analysis, nanocrystalline PTX and ND-OH/PTX complexes were lyophilized and further desired concentration of ND-OH/PTX complexes on the basis of PTX (assumed with loading efficiency 100%) was redispersed in PBS for release study and cell viability.

2.4. Characterization of ND-OH, ND-OH/PTX complexes, and nanocrystalline PTX The reduced ND-COOH into hydroxyl groups, named ND-OH, and ND-COOH, were characterized with XPS. The XPS measurement was performed using a Multilab ESCA 2000


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system (VG Microtech, UK), which was equipped with a Mg Kα X-ray source (1253.6 eV) and a hemispherical analyzer. The spectra were obtained with the absolute energy resolution of 0.1 eV. The morphology of ND-OH, nanocrystalline PTX, and the ND-OH/PTX complexes were examined with transmission electron microscopy (TEM). The dispersion of ND-OH, nanocrystalline PTX, and ND-OH/PTX in deionized water was casted dropwise on the lacy carbon grid. The excess water was wiped and the grid allowed to dry overnight. High resolutionTEM observation was performed using a JEM-3010 (JEOL, Japan) with an acceleration voltage of 300 kV. To characterize the dispersive properties of the ND-OH in aqueous environment, the particle size and zeta potential were measured using a Zetasizer Nano ZS90 (Malvern Instruments, UK). The dispersions of ND-OH, ND-COOH, nanocrystalline PTX, and ND-OH/PTX complexes were prepared in distilled water at a concentration of 0.1 mg/mL. For the measurement of zeta potentials, sample solutions were titrated using MPT-2 autotitrator with 0.1 M sodium hydroxide and hydrochloric acid (Sigma Aldrich, USA) at pH range from 4.0 to 10.0. Each measurement was carried out after equilibration time of 2 min at 25 ºC. A disposable sizing cuvette (Sarstedt, Germany) for the hydrodynamic radius and a disposable capillary cell (Malvern Instruments, UK) for zeta potential were used with volume of 1 mL. All measurements were performed at a fixed angle of 90°. Average particle size, polydispersity index (PDI), and zeta potential were calculated from the five measurements. The PXRD (powder X-ray diffraction) analysis was carried using a D8 ADVANCE with DAVINCI (Bruker AXS Inc., Germany) equipped with Cu Kα radiation and a high speed LynxEye detector. The spectra of ND-COOH, ND-OH, nanocrystalline PTX, and ND-OH/PTX complexes were obtained over a 2θ range of 4−40° with an increment of 0.02° at a rate of



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6°/min. A total of 10 mg of powder was used to fill the powder sample holder for the measurement. Each spectrum was analyzed using DIFFRAC plus Eva (Bruker AXS Inc., Germany).

2.5. PTX release from ND-OH/PTX complexes PTX release from nanocrystalline PTX and ND-OH/PTX complexes was carried out to evaluate the drug release profiles. Suspensions of nanocrystalline PTX and ND-OH/PTX complexes (1 mL, 20 µg/mL) were placed into a dialysis membrane (Spectra Dialysis membrane MWCO 50kD; Spectrum Laboratories, USA), and the membrane bag was placed in 50 mL of PBS (phosphate buffered saline, pH 7.4) in a centrifuge tube. The tubes were continuously shaken at 120 rpm and thermostated at 37 ± 0.5°C (SI-300R; Jeio Tech, Korea). At predetermined time intervals of 0.5, 2, 5, 10, 22, 46, and 70 hr, samples of nanocrystalline PTX and ND-OH/PTX complexes were withdrawn. The dispersions within a dialysis bag were centrifuged at 14,000 rpm for 15 min to separate unreleased PTX. The quantitative amount of PTX in the supernatant was analyzed using an HPLC system (1100 Series; Agilent Technologies, USA) equipped with a diode array detector at an ultraviolet wavelength of 230 nm UV absorbance spectra. PTX solubility in ACN was ~20 mg/mL. For the analysis, calibration curve from 0.2 µg/mL to 200 µg/mL of PTX was obtained with good linearity and accuracy, which was ranged within PTX solubility under the mobile phase condition of 40:60 = ACN:water.33 The retention time of PTX was 30.3 min with an analysis time of 40 min, using an Eclipse Plus C18 column (4.6 × 150 mm, 5 µm, Agilent Technologies, USA). The released amount was plotted against time. Statistical analysis was performed using


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the student’s t-test and analysis of variance (one-way ANOVA, Dunnett’s multiple comparison test) (SigmaStat 3.5; Systat Software, USA) with the chosen significance level of P ≤ 0.05.

2.6. Cell viability test The cytotoxic effects of ND-OH, nanocrystalline PTX, and the ND-OH/PTX complexes were assessed by using crystal violet staining. A549, MCF7, and Hela cells were seeded with a cell density of 1 × 104 cells per well into a six-well plate. After 24 hr, the ND-OH, nanocrystalline PTX, and ND-OH/PTX complexes were added to the wells with indicated PTX concentrations (from 0.005 to 10 µg/mL) and incubated for 72 h at 37 °C under the ambient conditions of 5% CO2 and 100% humidity. After incubation, the medium was removed, and the cells were washed with PBS twice, and then stained with 0.05 % crystal violet (Sigma Aldrich, USA) for 30 min at room temperature. The cells were washed with tap water twice and air-dried. For the detection of OD values, 100 µL of 1 % sodium dodecyl sulfate (SDS) was added per well. The absorbance was read at 570 nm with a microplate reader (BioTek, Germany). All experiments were carried out in triplicate.

2.7. Transmission electron microscopy imaging The cellular uptake of PTX was visualized using transmission electron microscopy (TEM). Hela cells were seeded with a cell density of 1 × 106 cells per well into a six-well plate. After 24 hr of cell seeding, nanocrystalline PTX and ND-OH/PTX complexes were added to the wells with 1 (PTX) and 2/1 (ND-OH/PTX) µg/mL concentrations. The Hela cells were incubated for 6 hr at 37°C in DMEM medium under of 5% CO2 and 100% humidity. After treatment, the cells were washed with PBS twice and fixed with 2.5% glutaraldehyde in 100 mM of phosphate buffer



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at pH 7.0 overnight. The cells were then post-fixed in 1% osmium tetroxide, dehydrated through a series of ethanol concentrations, and treated with 50% and 100% propylene oxide. Resin infiltration was performed with 2:1 and 1:2 mixes of propylene oxide:spur resin for 1 hr and with 100% spur resin overnight. After infiltration, the resin was changed and the sample was moved into embedding molds. The sample was polymerized at 60 °C for 24 hr. After embedding and sectioning, TEM observation was performed using a JEM-1010 (JEOL, Japan) with an acceleration voltage of 80 kV.

2.8. Flow cytometry The Hela cells for flow cytometry were seeded and plated with the TEM experiment methods (6, 12 hr). The cells for flow cytometric analysis were washed twice with cold PBS after cell harvesting. The washed cells were resuspended in 100 µL of binding buffer (10 mM pH 7.4 HEPES buffer, 140 mM NaCl, 2.5 mM CaCl2) and incubated with 5 µL of FITC conjugated with Annexin V and propidium iodide (PI) for 10 minutes in dark conditions. Flow cytometry was conducted to compare the cellular efficacy of ND-OH/PTX complex and nanocrystalline PTX by using FITC-labeled Annexin V and PI. The untreated Hela cells were used as a control group. FITC fluorescence was measured using BD FACSAria™ III. The fluorescence from FITClabeled Annexin V was observed using a 530/30 nm band pass filter, and the fluorescence from propidium iodide was observed through a 585/42 nm band pass filter by plotting a log of fluorescence intensity versus the number of events. The cell population was gated, and a total of 50,000 events were recorded in the gated region per sample. The data was analyzed using BD FACSDiva software. Cells without treatment with PTX were used as a negative control.


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3. Results and Discussion 3.1. Characterization of nanodiamonds The feature of ND dispersions at various pH conditions was characterized with hydrodynamic diameters and zeta potentials. Figure 1b shows the change of zeta potential of ND agglutinates depending on pH. The zeta potentials of ND-COOH were negative in the pH ranges from 4 to 10. The zeta potentials of ND-COOH at pH 4 and pH 10 were -6.8 ± 1.8 mV and –36.9 ± 0.3 mV, respectively. ND-COOH dispersion showed a significant decrease in zeta potential as the pH increased from 4 to 10. The carboxyl groups were deprotonated at an increased pH, causing the ND surfaces to be negatively charged. Based on the XPS results, the starting material, NDCOOH, contains rich carboxyl groups with high density. Hydroxylated ND (ND-OH) produced by the reduction of ND-COOH showed stable zeta potentials in the wide pH ranges compared with those of ND-COOH. The zeta potentials of ND-OH were stable from -51.4 ± 0.9 mV to 44.5 ± 0.7 mV as the pH changed from 4 to 10. The zeta potentials of ND-COOH and ND-OH were –22.5 ± 0.3 mV and -42.7 ± 1.2 mV at pH 7, respectively. Both ND-COOH and ND-OH showed negative zeta potentials at pH 7, suggesting good colloidal stability. The consistent negative zeta potentials of ND-OH at various pHs suggest strong colloidal stability compared with that of ND-COOH. Figure 1c shows the size distribution of ND agglutinates measured by dynamic laser light scattering. The diameters of the dispersed ND-OH and ND-COOH were also dependent on the environmental pH. The Z-average sizes of ND-COOH were stable at 105.2 ± 1.6 nm at pH 7. However, a decrease in pH caused significantly increased aggregates of ND-COOH up to 2,122 ± 297 nm. The diameter of ND-COOH was 935.52 nm at pH 6, showing a dramatic increase of ND aggregates compared with that at pH 7. Hydroxylated ND, reduced from the ND-COOH,



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showed less particle size change with regard to pH environment. The Z-average sizes of ND-OH ranged from 140.0 ± 7.3 nm at pH 4 to 52.6 ± 0.3 nm at pH 10, showing less pH sensitivity and high dispersion stability. Significant changes in the particle size of ND-COOH between pH 5.0 and pH 6.0 originated from the ionization of the carboxyl group on ND-COOH. The dissociation constants (pKa) of carboxylic acids, such as acetic acid, propionic acid, and valeric acid, were approximately 5.0.34 The ionization extent of the carboxyl groups determined the strength of the inter-particle interaction. When the carboxyl groups were ionized, the ND-COOH particles had a negative charge and repulsion between the particles became significantly increased, resulting in a reduced particle size and augmented absolute zeta potential. Regarding ND-OH, the pKa values of alcohol and phenol were reported to be 14–16 and 10, respectively.35 Therefore, ND-OH was not ionized, showing stabilized particle size compared with that of ND-COOH.

Figure 1. Schematic pictures of reduction process of hydroxylated nanodiamond (ND-OH) from carboxylated nanodiamond (ND-COOH) (a). Z-average size (b) and zeta potential (c) of NDCOOH and ND-OH agglomerate at various pH conditions. XPS spectra obtained from ND-


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COOH and ND-OH. C1s core-level spectra of ND-COOH (d), O1s core-level spectra of NDCOOH (e), C1s core-level spectra of ND-OH (f), and O1s core-level spectra of ND-OH (g).

The reduction of ND-COOH was characterized with XPS to compare hydroxylated NDs after the reaction with NaBH4. XPS analyses of ND-COOH and ND-OH nanoparticles were carried out to confirm the functionalization of the ND surface into the hydroxyl group. Figure 1d, 1e, 1f, and 1g show XPS scans of ND-COOH and ND-OH with the C1s and O1s bands. The peaks of ND-COOH and ND-OH were deconvoluted into component peaks using Gaussian fitting. The C1s peak of ND-COOH was deconvoluted to six binding energies at 282.6 eV (sp2), 283.5 eV (sp3), 284.6 eV (C-C), 286.0 eV (C-O), 287.5 eV (C=O), and 289.0 eV (-COOH) (Figure 1d). The C1s peak of ND-COOH also showed a shoulder near 282.6 eV and 283.5 eV due to the sp hybridization structures of carbon on the diamond surface. The deconvoluted peak at 289.0 eV in ND-COOH presented strong intensity compared with that of ND-OH. This indicates that the carboxyl group of ND-COOH presented high purity. The C1s peak of ND-OH was also deconvoluted to six binding energies (Figure 1f). However, the peak at 282.6 eV (sp2) disappeared compared with that of ND-COOH. The disappearance of the sp2 peak indicates the reduction of the carbonyl functional group and the increase of the hydroxyl functional group. A strong peak of ND-OH at 286.0 eV (C-O) appeared, while a reduced peak of ND-OH at 287.5 eV (C=O) was presented, indicating that the hydroxyl group on the ND surface was mainly presented. The O1s peaks of ND-COOH and ND-OH were deconvoluted with two conventional binding energies at 531.2 eV (C=O) and 533.3 eV (C-O-C/C-OH), as shown in Figure 1e and 1g. The deconvoluted peak at 531.2 eV in NDs-OH presented weak intensity compared with that of ND-COOHs. The peak of ND-OH at 531.2 eV was attributed to the carbonyl group (C=O),



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showing a weak contribution. The binding energy of the ND-OH peak at 533.3 eV originated from the hydroxyl group (C-O-C/C-OH), indicating that the reduction of the carboxyl group had been completed (Figure 1g). It was noted that the O1s peak of ND-COOH showed a large area of carbonyl deconvolution at 531.2 eV (C=O) of the binding energies, indicating the high density of carboxyl functional groups on the ND surface. Overall, the XPS analyses presented that hydroxyl groups were produced from the reduction of the carboxyl groups on the ND surface.

3.2. Characterization of ND-OH/PTX nanocomplexes and nanocrystalline PTX


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Figure 2. Schematic pictures of formation of ND-OH/Paclitaxel (ND-OH/PTX) nanocomplex and crystalline PTX. Green drops indicate concentrated PTX in ethanol solution (a). Dispersion stability of ND-COOH, ND-OH, nanocrystalline PTX and ND-OH/PTX complex in distilled water and DMEM buffer (b). TEM image of ND-OH agglutinates with their individual sizes ranging from 4 to 5 nm in diameter (c). TEM images of ND-OH/PTX complex (d) and paclitaxel crystal (e). PXRD spectra of anhydrous PTX powder, lyophilized PTX, ND-OH/PTX complex, and ND-OH particles (f). Z-average size and zeta potential of ND-OH, ND-OH/PTX complex, and paclitaxel (g). In vitro drug release profiles of crystalline PTX and ND-OH/PTX complex (h).

PTX dissolved in ethanol with a high concentration of 2 mg/mL was introduced under sonication to the dispersion of hydroxylated ND or water (Figure 2a). After lyophilization, the solid states and morphology of ND-OH, ND-OH/PTX nanocomplexes, and nanocrystalline PTX were examined with X-ray diffraction and TEM. The agglutinate size of ND-OH ranged from 50 nm to 100 nm, consistent with the DLS results. The individual 5 nm sized ND did not present a separately dispersed state (Figure 2c). Depending on the environmental pH condition, a stabilized particle size existed as a form of agglutinate. The rod shape of nanocrystalline PTX was observed from the TEM image (Figure 2e). The average diameter and length of nanocrystalline PTX were 820 nm and 520 nm, respectively. The observation of rod-shaped nanocrystalline PTX was consistent with those of previous studies.26, 36-37 Nanocrystalline PTX was formed as PTX in ethanol was dropped into the water. During the precipitation and the solvent exchange, PTX solidified into a rod-shaped structure. In contrast, the morphology of NDOH/PTX nanocomplexes showed that PTX was incorporated into the spaces between the NDs,



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without any observation of rod-like nanocrystalline PTX structures (Figure 2d, Figure S1). The ND-OH/PTX nanocomplexes were composed of a PTX-rich region and a ND-rich region. During the solvent exchange and nanoprecipitation, PTX was randomly solidified and caged within the interparticle nanocage of the NDs. It was speculated that no formation of PTX nanocrystalline on the presence of ND was induced by hydrogen bonding on the hydroxylated NDs in the aqueous solution. The hydroxylated ND as stable colloids may play a role as a surfactant to prevent the growth of sole PTX crystalline, and as substrate to be entrapped within nanocage of ND. Thus, the interspace of ND-OH aggregates may become a substrate platform for amorphous PTX to be solidified into the nanoscale. Simple physical adsorption induced by ππ stacking between NDs and drugs might be also partial factor for PTX to be loaded on the ND surface, as previously reported.38 Our study showed the solvent exchange of PTX during nanoprecipitation allowed it to be loaded with a highly concentrated PTX amorphous form into the interparticle nanocage of the ND-OH. Figure 2f shows the examination of the crystal structures of ND-OH/PTX nanocomplexes, ND-OH, anhydrous PTX, and nanocrystalline PTX, measured with PXRD. The PXRD spectra of anhydrous PTX powder showed peaks at 2θ = 5.6o, 9.1o, 10.4o, 12.7o, and 21.1 o.25 Freeze-dried nanocrystalline PTX exhibited the distinct appearance of PXRD peaks compared with anhydrous PTX, indicating that a polymorphic transformation occurred during solidification and the solvent exchange process under probe sonication. Five new peaks at 6.2 o, 9.8 o, 11.2 o, 13.3 o, and 16.7o appeared in the nanocrystalline PTX, which were nonexistent with peaks in the anhydrous PTX. These peaks were reported as specific peaks of PTX dihydrate. Polymorphic transformation occurred during the dissolving and freeze drying processes of anhydrous PTX.39 ND-OH did not exhibit any significant peaks in the 2θ range of 4o to 40o. To be specific, ND-OH/PTX complex


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showed a similar PXRD peak pattern to freeze dried PTX. It showed a significantly reduced peak intensity compared with nanocrystalline PTX and anhydrous PTX. The reduced PXRD peak of the ND-OH/PTX nanocomplexes indicates that most complexed PTX with ND-OH existed as an amorphous form and a small amount of PTX existed as a dihydrate form. The dispersions of ND-OH, nanocrystalline PTX, and ND-OH/PTX nanocomplexes were examined in an aqueous solution with DLS and zeta potentiometer analysis. Figure 2g shows that the Z-averages of ND-OH, nanocrystalline PTX, and ND-OH/PTX complexes were 61.56 ± 5.32, 95.92 ± 7.42, and 5033 ± 224 nm, respectively, at pH 7. The zeta potentials of ND-OH, nanocrystalline PTX, and ND-OH/PTX complexes were -42.73 ± 2.43, -33.02 ± 2.82, and 0.08 ± 0.24 mV, respectively. The fast formation of crystalline PTX was shown in an aqueous solution up to 5 µm due to its strong hydrophobicity, resulting in physical adhesion between nanocrystalline PTX, as displayed in the TEM image. The incorporation of PTX into ND-OH caused a 55.82% increase in diameter compared with that of ND-OH. This result showed that hydroxylated NDs provided a platform for the success of the delivery carrier, forming stable colloidal properties of ND-OH/PTX nanocomplexes within 100 nm in diameter. Under the same formulation, the introduction of ND-OH led to the formation of PTX nanocomplexes with stable colloidal properties. In addition, ND-OH/PTX complex showed impressive dispersion stability after complex formation (Figure 2b). Nanocrystalline PTX showed a much higher Z-average size than ND-OH/PTX complex and precipitated within 12 hr of storage in DMEM buffer solution. ND-OH/PTX complexes maintained a suspension state in the buffer solution and showed a similar Z-average size until 3 days. ND-COOH maintained a clear suspension state in distilled water, while it precipitated in the buffer solution.



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3.3. PTX release in vitro To evaluate in vitro drug release profiles of PTX, samples from nanocrystalline PTX and NDOH/PTX nanocomplexes were taken after predetermined time intervals and quantitative amounts of PTX were analyzed with HPLC. After the HPLC analysis, the ratio of released PTX was calibrated by subtracting the released amount from the initial amount of PTX. The ratios of released PTX at ND-OH/PTX nanocomplexes and nanocrystalline PTX were 97.32 ± 6.33 and 47.30 ± 2.79 %, respectively, after 70 hr. The released amount from ND-OH/PTX nanocomplexes was constantly higher than that from nanocrystalline PTX over the time period (Figure 2h). The release of the initial burst was 38.31 ± 2.90% and 16.69 ± 1.16% at 0.5 h in the ND-OH/PTX nanocomplexes and nanocrystalline PTX, respectively. Dissolved PTX in the suspension samples caused an initial burst as exposed to the release medium. The released amount of nanocrystalline PTX was only 30.61% after the initial bust, indicating that nanocrystalline PTX might be aggregated and not capable of a sustained drug release reservoir. In contrast, ND-OH/PTX nanocomplexes showed 59.01% of the released amount after the initial burst. The accumulative amount released consistently increased over time, providing sustained release. This result supports that ND incorporation with PTX offers a drug reservoir for a prolonged time. Dispersion stability might affect the release amount in the PTX release test. The difference of cumulative released amounts between ND-OH/PTX and nanocrystalline PTX increased as time progressed. As the diffused amount was dependent on the free PTX concentration in the donor chamber, the precipitation of PTX was an important factor for the release test. This result indicates that ND-OH prevents PTX from precipitation or aggregation through adsorption. When free PTX in the donor chamber diffused to the receptor chamber, adsorbed PTX in ND-OH,


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functioning as a reservoir and solubility enhancer, was released to the donor chamber, maintaining a high free PTX concentration. PTX is a poorly water soluble drug with a low solubility of 10.8 µg/mL in water23, which requires the delivery carrier in vivo. In the solubility measurement, there was no significant difference of PTX solubility between ND-OH/PTX nanocomplexes and nanocrystalline PTX in the saturated solution. This result indicated that ND did not increase the intrinsic solubility of PTX, but increased the dispersibility of PTX through the adhesion of non-dissolved PTX, promoting PTX as an amorphous form. The sustained PTX release from ND with hydroxyl end groups on the surface indicated that ND retained a significant amount of PTX stably through adsorption during the solvent exchange formulation. ND incorporation also altered the polymorphic crystallization of PTX into an amorphous form. This result suggested that NDOH/PTX nanocomplexes have the advantage of releasing adsorbed drugs consistently, which was supported by the TEM images.

3.4. Cell viability test The cell viability of ND-OH, PTX, and ND-OH/PTX on the A549, MCF-7, and Hela carcinoma cells was analyzed by MTS assay (Figure 3). ND-OH did not show significant cytotoxicity on all the cell lines during 72 h of incubation. In contrast, ND-OH/PTX and PTX showed significant concentration-dependent reduced cell viability after 72 h of incubation.



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Figure 3. Cytotoxicity evaluation of ND-OH/PTX complex compared with crystalline PTX and ND-OH using MTS assay performed with A549 (a), Hela (b), and MCF-9 cell line (c). Cell viability comparison between cell lines at 0.05 µg/mL (d).

The cell viabilities of ND-OH/PTX and PTX in the A549 cell line changed from 90.82% to 18.87% and from 108.09% to 35.93% as the PTX concentration changed from 0.005 µg/mL to 10 µg/mL, respectively. The cell viabilities of ND-OH/PTX and nanocrystalline PTX in the Hela cell line changed from 98.68% to 16.88% and from 110.18% to 14.51% as the PTX concentration changed from 0.005 µg/mL to 10 µg/mL, respectively. The cell viabilities of NDOH/PTX and PTX in the MCF-9 cell line changed from 101.50% to 62.96% and from 103.85% to 65.02% as the PTX concentration changed from 0.005 µg/mL to 10 µg/mL, respectively. NDOH/PTX and PTX showed a plateau beyond 1 µg/mL due to the following mechanism: PTX acts by inhibiting the formation of microtubule structures during the mitosis process and inducing cell


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death and did not affect the non-division of cells.20 Though MCF-7 is also sensitive cells to PTX, A549 cell line showed lower cell viability, compared to MCF-7, as reported by Liebmann, et al.40. MCF-7 cells that express the multi-drug resistance phenotype might be resistant to high concentration (over 1000 nM) of PTX, showing plateau without critical decrease in cell viability. Cell viability of MCF-7 at 1 µg/mL (1.1 µM) and 10 µg/mL (11.7 µM) showed 51.6% and 62.9% with the treatment of ND-OH/PTX complexes, respectively. This moderate cell viability of MCF-7 is consistent with delivery of PTX and ceramide to MCF-7.41 ND-OH/PTX showed a lower IC50 value than PTX in this experiment. The IC50 values of NDOH/PTX and PTX in A549 cells were 0.068 µg/mL and 0.168 µg/mL, respectively. NDOH/PTX showed a 60% smaller IC50 value than PTX in the A549 cells. The IC50 values of NDOH/PTX and PTX in Hela cells were 0.037 µg/mL and 0.137 µg/mL, respectively. The IC50 value of ND-OH/PTX was 73% smaller than that of PTX in the Hela cells. The IC50 values of ND-OH/PTX and PTX in MCF-7 cells were not calculated due to their high cell viability. The highest difference in cell viability was observed in all cell lines in 0.05 µg/mL (Figure 3d). The cell viabilities of ND-OH/PTX and PTX were 63.59 ± 4.18% and 94.79 ± 5.03% in A549 cells, 57.17 ± 3.18% and 82.75 ± 4.27% in MCF-7 cells, and 39.06 ± 2.36% and 93.25 ± 4.69% in Hela cells. The differences of cell viability might be attributed to the precipitation of unbounded PTX and different cellular uptake systems. The precipitation of PTX reduced its efficacy, as precipitated PTX crystal could not penetrate the cell membrane. ND-OH prevents PTX precipitation or aggregation through adsorption and improves the efficacy of PTX cellular uptake.

3.5. Transmission electron microscopy imaging



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Figure 4. Cellular uptake of ND-OH/PTX complexes and nanocrystalline PTX. TEM images of ND-OH/PTX complex in Hela cells with 6,000× (a), 100,000× (b), and 200,000× (c). TEM images of PTX nanoparticle in Hela cell with 6,000× (d), 100,000× (e), and 200,000× (f). Nanocrystalline PTX indicated with red arrows. ND-OH around PTX nanoparticles indicated with dark blue arrows. Red boxed areas are magnified in next figure. N: nucleus, ER: endoplasmic reticulum, and MVB: multivesicular body.

The mechanism of cellular uptake between ND-OH/PTX and nanocrystalline PTX was examined by TEM analysis (Figure 4). The images showed that the ND-OH/PTX and


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nanocrystalline PTX were taken up within the Hela cells after 12 hr. During the treatment of NDOH/PTX nanocomplexes, it was observed that internalized ND-OH/PTX nanocomplexes were dispersed with a size of 50–100 nm in the Hela cells. ND-OH/PTX complexes were localized at the nucleus, vesicles, endoplasmic reticulum, and cytoplasm (Figure 4b and 4c) within a cell. However, rod-shaped nanocrystalline PTX existed as a form of aggregation in the Hela cell (Figure 4d, 4e, and 4f) or existed with an adherent form on the cellular membrane. Because the aggregation size of nanocrystalline PTX was large (from 500 nm to 700 nm), cellular uptake was less effective. ND-OH/PTX complexes entered cells via endocytosis and showed more efficient uptake compared with that of nanocrystalline PTX. Multiple vesicles containing ND-OH/PTX nanocomplexes were observed in ND-OH/PTX treated Hela cells, proving that ND-OH/PTX nanocomplexes were not disassociated and performed as a convective drug reservoir. NDOH/PTX complexes possess the ability to shuttle their PTX reservoir in and out of the cells through the process of endocytosis, resulting in improved drug efficacy.42 Vesicles containing PTX were not observed in crystalline PTX-treated Hela cells. Instead, aggregated crystalline PTX disrupted the cell membrane and diffused into the cytoplasm (Figure S2). This phenomenon supports that the uptake of crystalline PTX was conducted by passive diffusion. ND-OH/PTX nanocomplexes with suppressed crystalline PTX were effectively distributed with less aggregation and enhanced dispersion within Hela cells, indicating stable colloidal complexes of ND incorporating PTX were appropriate for effective cellular uptake and for sustained release without the disassociation of PTX nanocomplexes. Similar result was reported as ND suppressing doxorubicin crystal formation. Doxorubicin is an anticancer drug with a crystalline morphology. When doxorubicin was mixed with ND, doxorubicin was absorbed onto the ND surface as an amorphous form.3



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3.6. Flow cytometry Annexin V-FITC and PI staining assay were performed using flow cytometry to determine whether apoptosis and necrosis were induced by crystalline PTX and ND-OH/PTX nanocomplexes at the Hela cells. The Hela cells were exposed to 1 µg/mL of PTX or ND-OH PTX with a 2:1 weight ratio for 6 hr (Figure 5) and 12 hr (Figure S3).

Figure 5. Flow cytometric analysis of apoptosis from Hela cell. Fluorescence intensity of propidium iodide (PE-A) plotted over Annexin V-FITC (FITC-A) (a). Histogram showing number of cells with fluorescence intensity of PE-A (b). PI+ indicates positively propidium iodide stained cell.

After the treatment, the samples stained with Annexin V and PI were analyzed by flow cytometry (Figure 5a). The fluorescence increase in the window of PI and Annexin V staining


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(Annexin V+/PI+ group, Q2) suggests late stage apoptosis cell death. The Annexin V-/PI+ cell group (Q1), Annexin V-/PI- cell group (Q3), and Annexin V+/PI- cell group (Q4) were identified as necrotic, healthy, and early apoptotic cells, respectively. ND-OH/PTX treated cells for 6 hr showed the percentages of 14.9% Q1, 1.7% Q2, 82.7% Q3, and 0.7% Q4. Annexin V negative/PI positive intensity was significantly increased for the ND-OH/PTX treated group compared with the untreated group (4.0% Q1, 0.4% Q2, 95.6% Q3, and 0.0% Q4) and the ND-only treated group (5.8% Q1, 0.3% Q2, 93.3% Q3, and 0.6% Q4). This result suggested that the major cell death was caused by necrosis. The crystalline PTX-treated group showed 10.0% Q1, 1.7% Q2, 87.6% Q3, and 0.9% Q4. PTX has been known to induce mitotic arrest and to precede apoptotic/necrotic cell death.43 It was assumed that PTX induced cell death occurs via two processes: apoptosis at low concentrations and necrosis at high concentrations.44 Nanocrystalline PTX and ND-OH/PTX (1 µg/mL) induced major cell death through necrosis, since a higher concentration than IC50 was employed. Moreover, ND-OH/PTX nanocomplexes with stable dispersion might promote the uptake of PTX into the cell and induce the necrosis pathway due to direct convective local high concentration. The PI positive populations in ND-OH/PTX, PTX, ND-OH, and the negative control were 16.6%, 11.5%, 6.1%, and 4.4%, respectively. The NDOH/PTX treated group showed significantly increased PI positivity in Hela cells compared with the PTX-treated cell group (Figure 5b). The apoptosis of cells was a time-dependent cell death procedure. The ND-OH/PTX treatment, which provided high local PTX concentration within a cell, induced necrosis as a predominant cell death pathway for a relatively short exposure time.45 Our study traced the loci of ND-OH/PTX complexes within a cell. Stable dispersion of NDOH/PTX complexes showed more homogenous distribution within a cell, probably providing effectively convective diffusion of drug. Once considered NDs as biomaterials in vivo, the fate of



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NDs in vivo has been interested and tracked in several studies. It is generally accepted that surface properties of NDs can reduce phagocytic attack, increasing the ND circulation in vivo.46 The study by Marcon et al. reported that cytotoxicity was not shown to 50 µg/mL of ND in HEK293 cells, while the embryotoxicity for carboxylated ND was expressed for both gastrulation and neurulation.47 Radioactive

18

F-labeled ND was used for ND biodistribution in

vivo by Rojas et al.48 Prefiltered NDs showed a highly excreted distribution into the urinary tract, indicating that the controlled size of ND aggregates are critical factor for long circulation, biodistribution, and excretion. Moreover, the long term toxicity study of fluorescence ND in rats was investigated by administration of subcutaneous, intradermal, and intraperitoneal injections for a dose per week.46 After sacrifice of 12 weeks, dark carbon-laden cells in the tissue morphology were observed in the dermis with no tissue damage, inflammation, or necrosis in the surrounding cells. Encouraging ND applications in vivo have revealed in a manner, yet realistic ND applications as therapeutic exogenous biomaterials should be carried in further extensive studies. Considering that biodegradable polymeric and protein based drug delivery systems are clinically available, ND as a drug delivery carrier may not be significant. ND itself is energy absorbing material and fluorescence ND with N-vacancy site has high photostability, compared to organic fluorophores. Thus, as unique features of NDs are combined with the techniques of conventional drug delivery, potential multifunctions employed by intrinsic properties of ND will give an impact in bioimaging, therapy, and further bioelectric interfaces, together with drug delivery. Our study suggested that ND-OH/PTX complexes have realistic potentials that can be employed as a drug delivery carrier.


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4. Conclusion In this study, ND-COOH was reduced to ND-OH to improve colloidal dispersion in a pHindependent manner. Reduced ND with a hydroxyl group showed a stable agglutinate size, regardless of pH change and even in buffered physiological media. A PTX supra-nanostructure was formed by the solvent exchange method as nanocomplexes within interparticle nanocage ND-OH. ND-OH/PTX nanocomplexes showed reduced PTX crystallinity and particle aggregation. Through thermodynamically enhanced colloidal dispersion, ND-OH/PTX nanocomplexes maintained sustained release, showing a lower IC50 value compared with that of crystalline PTX. The direct visualization of ND-OH/PTX nanocomplexes within a Hela cell proved that the PTX suprastructure with ND was taken up into cells, probably providing convective diffusion with a high concentration within a cell. The localized high concentration of ND-OH/PTX nanocomplexes might provide initial necrosis, corresponding to the FACS analysis. This study suggests that poorly water soluble drugs can be formulated into a suprastructure with ND substrates and play a role in a highly concentrated drug reservoir directly within a cell. This can be succeeded by the surface modification of ND, which possesses stable colloidal dispersion properties. The ND surface modification and formulation can increase the potential of new effective drug delivery vehicles for future therapeutics.

Supporting Information TEM image of ND-OH/PTX nanocomplexes with low magnification, membrane disruption by PTX nanocrystaline in Hela cell, and flow cytometric analysis result of apoptosis from Hela cell for 12 hr.



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Acknowledgements This research was supported by the Bio & Medical Technology Development Program of the NRF,

funded

by the

Korean

government,

MSIP

(NRF-2014M3A9A9073811),

and

Basic Science Research Program through the National Research Foundation of Korea(NRF), fun ded by the Ministry of Science, ICT & Future Planning (NRF-2015R1C1A2A01053307).


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Graphical Abstract 71x35mm (300 x 300 DPI)


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PaclitaxelâNanodiamond Nanocomplexes Enhance Aqueous

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