Cancer-Cell-Specific Mitochondria-Targeted Drug Delivery by Dual

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Cancer-Cell-Specific Mitochondria-Targeted Drug Delivery by DualLigand-Functionalized Nanodiamonds Circumvent Drug Resistance Miu Shan Chan, Ling Sum Liu, Hoi Man Leung, and Pik Kwan Lo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15954 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 15, 2017

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ACS Applied Materials & Interfaces

Cancer-Cell-Specific Mitochondria-Targeted Drug Delivery by Dual-Ligand-Functionalized Nanodiamonds Circumvent Drug Resistance Miu Shan Chan, Ling Sum Liu, Hoi Man Leung, and Pik Kwan Lo* Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong, Hong Kong SAR and Key Laboratory of Biochip Technology, Biotech and Health Care, Shenzhen Research Institute of City University of Hong Kong, Shenzhen 518057, China. KEYWORDS nanodiamonds, peptides, mitochondria, targeted delivery, drug resistance

ABSTRACT We demonstrate a nanotechnology approach for the development of cancer-cellspecific sub-cellular organelle-targeted drug nanocarriers based on photostable nanodiamonds (ND) functionalized with folic acid and mitochondrial localizing sequence (MLS) peptides. We showed that these multifunctional NDs not only distinguish between cancer cells and normal cells, and transport the loaded drugs across the plasma membrane of cancer cells, but also selectively deliver them to mitochondria and induce significant cytotoxicity and cell death compared with free Dox localized in lysosomes. Importantly, the cellular uptake of Dox was

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dramatically increased in a resistant model of MCF-7 cells, which contributed to the significant circumvention of P-glycoprotein-mediated drug resistance. Our work provides a novel method of designing nanodiamond-based carriers for targeted delivery and for circumventing drug resistance in doxorubicin-resistant human breast adenocarcinoma cancer cells. 1. INTRODUCTION In the clinical treatment of diseases, theranosis that combines the implementation of therapy and diagnosis simultaneously is of great important.1 For successful theranosis, the effective delivery of sufficient amount of drug to the targeted disease site with observable imaging signals is highly necessary. Many chemotherapy agents such as doxorubicin (Dox) generally act on both healthy and diseased cells due to their poor selectivity. Their clinical applications are restricted to induce drug resistance in certain diseased cells and dose-related toxicity, such as myelosuppression and cumulative cardiotoxicity.2-4 Thus, the development of new drug delivery systems to improve the therapeutic efficacy is currently a topic of intense interest.5-8 These new systems would facilitate selective delivery of sufficient quantity of pharmaceutical substances not only to specific intracellular organelles of cancer cells which are in a diseased state, but also circumvent drug resistance and facilitate intracellular tracking.9-11 Several nanoparticle-based vehicles such as quantum dots,12 magnetic nanoparticles13 and gold nanoparticles 14 have been considered as versatile drug carriers for cancer therapy. However, they generally have restricted to clinical applications due to photoblinking, photobleaching, size- and shaped-dependent fluorescence, and intrinsic toxicity.15-16 To address these problems, nanodiamond (ND) has recently emerged as a promising platform for different biology applications, owning to its unique features including biocompatibility, non-cytotoxicity, functionalization versatility, high affinity to biomolecules.17-19 The nitrogen-vacancy (NV)


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centers in ND are non-photoblinking and non-photobleaching, which exhibit a maximum fluorescence emission at ~ 700 nm and a reasonable lifetime of about 19 ns. These enable longterm in vivo particle tracking and in vitro fluorescence cell labeling/imaging.20-21 These combined properties make them the best candidate for drug delivery.22-23 So far, the development of multifunctional NDs as targeted delivery systems with simultaneous capabilities in cancercell-specific organelle-targeting, imaging, therapeutic activity in addition to circumvention of drug resistance has not yet to be reported.24-25 To achieve this, we herein demonstrated the construction of a versatile ND system that incorporates with receptor-binding ligands, intracellular organelle targeting moieties and chemotherapeutic for multi-modal imaging, targeting and therapeutic applications (Figure 1). Two targeting ligands including mitochondrial localizing sequence (MLS) peptide and folic acid (FA) have been simultaneously attached to ND surface for cancer-cell-specific mitochondriatargeted delivery. Dox is successfully loaded onto these dually-ligand-functionalized NDs which selectively transport across the cell membrane of MCF-7/HeLa cells to the mitochondria. Interestingly, Dox targeted to mitochondria indeed induces remarkable toxicity and cell death in live cells compared with free Dox in lysosomes. With the multifunctional ND nanocarriers, the cellular uptake of Dox is dramatically enhanced, facilitating significant improvement of cellkilling capability to Dox-resistant cancer cells. Our results provide rationale to explore the use of nanodiamond-based

delivery

systems

for

cancer-cell-specific

organelle-targeting

and

circumventing drug resistance in doxorubicin-resistant human breast adenocarcinoma cancer cells.


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Figure 1. Synthetic scheme of MLS-NDs, MLS-FA-PEGylated-NDs and Dox-loaded MLS-FA-PEGylated-NDs, (a) i. EDC/sulfo-NHS, 2 h, ii. MLS + PEGylated FA, overnight, (b) Doxorubicin, 6 h, (c) i. EDC/ sulfo-NHS, 2 h, ii. MLS, overnight, (d) i. EDC/sulfo-NHS, 2 h, ii. PEGylated FA, overnight.

2. EXPERIMENTAL SECTION Materials and Reagents. Monocystalline Diamond (ND) power with MSY 0-0.05micro was purchased from Microdiamant. Sulfuric acid, nitric acid, perchloric acid, sodium hydroxide, hydrochloric glutaraldehyde, Ferrocyanide,

acid,

1-ethyl-3-(-3-dimethylaminopropyl)

paraformaldehyde, Uranyl

acetate,

cacodylate

buffer,

carbodiimide osmium

N-hydroxysulfosuccinimide,

hydrochloride,

tetroxide,

doxorubicin

Potassium and

3-[4,5-

dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were used as purchased from Aldrich. Spurr low viscosity epoxy embedding kit was form Polysciences, Inc.. Mitochondria signaling peptide was purchased by First Base. PEG-amine was purchased from J & K. FA-PEGamine was purchased from Nanocs. MES Buffered Saline Packs was purchased from Thermo Fisher Scientific. Fetal bovine serum (FBS), phosphate buffered saline (PBS), Dulbecco’s


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Modified Eagle Medium (DMEM), penicillin streptomycin solution, trypsin and organelles trackers/markers were purchased from Invitrogen. Instruments. The physical properties of raw ND and modified ND were characterized by Fourier Transform Infra-Red Spectrometer (PE spectrum 2000) and Dynamic Light Scattering Particle Size Analyzer (Malvem Zetasizer Nano ZS). UV/Vis measurement was measured by UV-VD (Agient 8453). Confocal fluorescence experiment was conducted on Laser Confocal Scanning Microscope (Leica TCS SP5) with 63× magnification. Bio Tek Powerwave XS microplate reader was used for the MTT studies. BD FACSCantoTM II flow cytometer was used for fluorescence-activated cell sorting (FACS) studies. TEM images were obtained from a Philips Technai 12. XPS analysis was performed on PHI5802 system (Physical Electronics) with Al K (alpha) monochromated X-ray source operating at 58.7eV Pass energy and 0.25eV eV/step. Synthesis of functionalized NDs. Carboxyl-NDs 50 mg of raw nanodiamonds were acidified with sulfuric acid, nitric acid and perchloric acid in ratio 1:1:1 at 110 oC for overnight. After the reaction, the acidified NDs were washed with D.I. water for three times, and then refluxed with 0.1 M NaOH at 110 oC for overnight. Finally, the alkaline-treated NDs were further refluxed with 0.1 M HCl. After the reaction, the carboxyl-NDs were washed with D.I water for a few times and stored at 4 oC. MLS-NDs 5 mg of the acidified NDs were activated by EDC/Sulf-NHS in MES buffer at pH = 6.0 for 1 h at r.t. The activated NDs were washed with PB buffer at pH = 7.04, then reacted with excess MLS


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in PB buffer at pH = 7.04 at r.t. for 2 h and then at 4 oC for overnight. The MLS-NDs were finally washed with D.I. water for few times and stored at 4 oC. FA-PEGylated-NDs 5 mg of the acidified NDs were activated by EDC/Sulf-NHS in MES buffer at pH = 6.0 for 1 h at r.t. The activated NDs were washed with PB buffer at pH = 7.04, then reacted with excess FAPEG in PB buffer at pH = 7.04 at r.t. for 2 h and then at 4 oC for overnight. The FA-PEGylatedNDs were finally washed with D.I. water for few times and stored at 4oC. MLS-FA-PEGylated-NDs 5 mg of the acidified NDs were activated by EDC/Sulf-NHS in MES buffer at pH = 6.0 for 1 h at r.t. The activated NDs were washed with PB buffer at pH = 7.04, then reacted with excess MLS/FA-PEG (3:1) in PB buffer at pH = 7.04 at r.t. for 2 h and then at 4 oC for overnight. The MLS-FA-PEGylated -NDs were finally washed with D.I water for few times and store at 4oC. Confocal Fluorescence Microscopy Imaging. For specific organelle targeting: Cells were seeded and cultured in 35 mm glass bottom dishes overnight. Corresponding samples were added and incubated for several hours. After being washing with buffer for few times, cell imaging was were ready. For the colocalization study, 3 µL of 0.5 mM of the relevant tracker was added and incubated with cell samples for ~ 30 min. The excitation wavelength of nanodiamond is 633 nm and the emission wavelength region is above 700 nm. The excitation wavelength of organelle trackers including mitotracker green and lysotracker is 488 nm and the emission wavelength region is from 500 to 550 nm. The excitation wavelength of doxorubicin is 488 nm and the emission wavelength region is from 580 to 650 nm. For folic acid study: HeLa or A 549 cell samples was seeded on glass bottom culture plate with or without folic acid pre-treating, are then


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treated 10 µg of corresponding ND samples for 2 h in incubator with 5% CO2 at 37 oC. Subsequently, all cell samples were washed with 1 × PBS for three times and then ready for imaging. Cell Cultivation. HeLa, regular MCF-7 or MCF-7/ADR cells were cultured in DMEM and 10 % FBS supplemented with 1% penicillin and streptomycin in 5 % CO2 environment at 37 °C. Once they reached up to ~ 95% confluence, they were treated with a standard trypsin-based technique and then reseeded in confocal imaging dishes with concentration of ~ 4 × 104 cell L-1. Cells were incubated in the culture media at a concentration to allow 70% confluence. Different samples were added to the cells with 70 % confluence and then incubated for additional 12 h before performing further characterizations. TEM Imaging. 4 × 104 HeLa cells were seeded on the cover slip, cultured overnight, and then treated with the MLS-NDs for 12 h. After rinsing with PBS for few times, the cell samples were placed onto a glass vial containing primary fixation with 2.5 % of glutaraldehyde and 2 % of paraformaldehyde in 0.1 M cacodylate buffer at pH 7.2 at r.t. for 2 h. The secondary fixation with 1% of OSO4 and 1.5 % of Potassium Ferrocyanide in 0.1 M cacodylate buffer in dark for 2 h at r.t. was then performed. After each fixation, the samples were rinsed with 0.1 M cacodylate buffer three times for 5 min, then dehydrated with ethanol from 30% to 100% and further with 100% acetone. The dehydrated cell samples were infiltrated with acetone and Spurr’s resin from ratio 3:1 to 0:1. The infiltrated samples were then embed in plastic mold and baked in oven at 70 o

C for 2 days. Before TEM imaging, the baked sample were undergone ultrathin sectioning and

collected on the grid. The collected samples were stained with 5% uranyl acetate solution and then conducting imaging.


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Drug Loading. MLS-FA-PEGylated-NDs (~ 4 mg) was sonicated under 100 W for 30 min in D.I. water. Doxorubicin solution of ~100 µg was then added and shaken at 4°C for 48 h to facilitate doxorubicin binding to NDs by physical adsorption. The suspension was then centrifuged at 10000 rpm for 15 min. The concentration of doxorubicin adsorbed on NDs was determined by analyzing the UV/Vis absorbance of doxorubicin at 480 nm before and after the adsorption reaction. The amount of doxorubicin loaded onto MLS-FA-PEGylated NDs and its loading efficiency were calculated as shown below: The mass of Dox on MLS-FA-PEGylated NDs =

×

MTT assay. Cytotoxicity assay was used to determine the viability of cells as a function of incubation time and drug concentrations. 1 × 104 cells were seeded and cultured overnight on 96 wells plate in 37 oC with 5% CO2. The corresponding samples were then added and incubated for additional 24 h or 48 h. 0.5 mg/mL MTT solution was added to the sample-treated cell samples togather with fresh medium and then incubated for additional 2 h at 37 °C. After incubation, the medium was removed and the solution of DMSO and ethanol (1:1) was added. The absorbance at 570 nm was measured using a microplate reader. XPS analysis. XPS analysis was conducted on PHI5802 system (Physical Electronics) with Al K (alpha) monochromated X-ray source operating at 58.7eV Pass energy and 0.25eV eV/step. The ND samples were dropped on the silicon wafer and let it dry under vacuum. Surface depth of 1020 nm for analysis was chosen. 3. RESULTS AND DISCUSSION


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To develop a multifunctional ND system, suitable surface modifications on ND from a chemistry point of view is of great importance. In the design, a covalent approach was used to link multiple biomolecules to the nanodiamonds via amino-reactive NHS chemistry. Firstly, the commercially available NDs were cleaned with strong acid mixture H2SO4-HNO3-HClO4 (1:1:1,vol/vol/vol) and then oxidized to become carboxyl-NDs (Scheme S1). The Fourier Transform Infrared spectroscopy (FTIR) spectrum of the oxidized NDs exhibits the O-H stretching vibration at 3410 cm-1 and the corresponding C=O stretching vibration of the carboxyl group at 1629 cm-1 (Figure S1). The carboxyl groups were then converted into the amine-reactive sulfo-NHS ester intermediates

using

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide

(EDC)

and

N-

hydroxysulfosuccinimide (Sulfo-NHS) in 2-(N-morpholino)ethanesulfonic acid (MES, 10 mM) buffer at pH 6.01. To develop NDs as sub-cellular organelle-targeted systems, it is necessary for the targeting ligands acting as directing devices to the sites of interest. In this regard, we modified NDs with peptide tags which get trafficked by the cell’s internal machinery to the organelle

in

question.

A

cell-permeable,

mitochondria-targeted

peptide

(NH2-

MLSLRQSIRFFKPATRTLCSSRYLL) is used to covalently couple to NHS-activated NDs in 10 mM phosphate buffer solution at pH 7.4 in order to generate MLS-NDs. Subsequently, the cellular uptake of carboxyl-NDs, NHS-NDs and MLS-NDs was evaluated via a laser confocal fluorescence microscopy in HeLa and/or MCF-7 cells after incubating for 24 h. Carboxyl-NDs and NHS-NDs showed very little uptake in HeLa cells while they were localized in lysosomes (Figure 2a-b). On the other hand, a certain amount of MLS-NDs were taken up by both cell lines and their fluorescence signals were colocalized with MitoTracker Green, but not with Hoechst or LysoTracker Green (Figures 2c and S2). Transmission electron microscopy (TEM) imaging of


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ND-treated HeLa cells also clearly revealed the targeting process in cellular environment as a function of time. During incubation, we observed that most of the MLS-NDs were firstly distributed nearby the mitochondria after 1 h while they were then entering the mitochondria after incubating for 3 h. Finally, a substantial amount of MLS-NDs were passed through the double-membrane of mitochondria and localized inside the mitochondria (Figure 2d). It is believed that MLS-ND was firstly taken up via endocytosis, then released to cytoplasm after endosome escaping and finally targeted to mitochondria.26 These results clearly indicated that the differences observed in cellular distribution are attributable to the surface bioconjugation of NDs with specific sequences of peptides. In principle, this peptide-modification approach can be seamlessly adapted to generate a new class of NDs targeting to a large variety of intracellular sub-organelles for different purposes.


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Figure 2. Intracellular distribution of modified NDs. Confocal fluorescence images of (a) carboxyl-NDs, (b) NHSNDs and (c) MLS-NDs which were incubated with HeLa cells for 12 h before staining with (i) LysoTrackers Green (ii) MitoTracker Green and (iii) Hoechst (Scale bar is 10 m). (d) TEM images of MLS-NDs incubated with HeLa cells as a function of incubation time after the fixation treatment (Scale bar is 200 m).

To explore the specific adsorption and cellular uptake of NDs, specific receptor binding ligands (RBLs) are also conjugated to the surface of peptide-functionalized NDs.27-29 Folic acid (FA) has proven to be an efficient targeting molecule as folate receptors are over-expressed on the surfaces of most cancer cells.30 For good targeting efficiency, RBLs need to be easily accessible. Thus, a polyethylene glycol (PEG) chain (~ 5000 Da) is used to expose the folic acid moieties far away from the densely packed ND surface. The co-existence of PEGylated FA and MLS ligands onto ND’s surface was achieved by mixing amino-functionalized MLS and aminofunctionalized PEGylated FA (3:1) to sulfo-NHS-activated NDs in phosphate buffer at pH 7.4, resulting in MLS-FA-PEGylated-NDs. From XPS analysis, we found that the ratio of different elements such as C, N, O and S on nanodiamond samples listed in Table S1-2 is in a good agreement with our expected. Particularly, the ratio of MLS to PEGylated FA on MLS-FAPEGylated-NDs is found to be ~ 6:1 (see SI). To investigate the accessibility of both FA and MLS peptide, confocal fluorescence studies were conducted to examine the cellular selectivity and localization of dual-ligand-functionalized NDs. We found that MLS-FA-PEGylated-NDs could bind strongly to HeLa and/or MCF-7 cell’s surfaces through specific folate ̶ folate receptor interaction, be efficiently taken up via FA receptor-mediated endocytosis and selectively localized in mitochondria (Figure 3a). On the other hand, no such cellular uptake observations result in A549 cells (Figure S3). In addition, no significant uptake difference between FAPEGylated-NDs and MLS-FA-PEGylated-NDs affinities when folate-receptor positive tumor cells were pretreated with free FAs, which have already saturated the FA-binding sites (Figure 3b-c).

31

On the other hand, FA-PEGylated-NDs (without MLS) were only localized in


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lysosomes (Figure 3d). It is clearly revealed that the localization differences noted are obviously attributable to the presence of MLS as mitochondria targeting moiety. In addition, MLS-NDs (without PEGylated FA) only showed an effective uptake after incubating for 3-4 h while MLSFA-PEGylated-NDs exhibited effective cellular uptake after incubating for 1-2 h in MCF-7 cells due to the strong interaction between the FA ligand and FA receptors on cell’s surface (Figure S4a). These results clearly indicated that there is no competition between FA and MLS on MLSFA-PEGylated-NDs and they are fully accessible to favor the selective recognition of FAreceptor positive tumor cells and mitochondrial localization. The amount of MLS-NDs taken up by both HeLa and MCF-7 cell lines also increases as a function of nanodiamond’s concentration (Figure S4b). In general, NDs showed lower uptake efficiency in HeLa cells than that in MCF-7 cells when its concentration and incubation time are the same. We believe that dual-ligand functionalization could be an effective strategy to design and construct the ND platform for biomedical applications in the areas of drug delivery, thermometry and bio-imaging in specific sub-cellular organelles of cancer cells.32-35


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Figure 3. Cellular uptake of FA-PEGylated-NDs and MLS-FA-PEGylated-NDs by HeLa and A549. (a) Confocal fluorescence images of MLS-FA-PEGylated-NDs incubated with HeLa or MCF-7 cells for 2 h before staining with MitoTracker Green. Confocal fluorescence images showed the uptake efficiency after incubating of HeLa cells with (b) FA-PEGylated-NDs + free FA and (c) MLS-FA-PEGylated-NDs + free FA. (d) Confocal fluorescence images of FA-PEGylated NDs incubated with HeLa or MCF-7 cells for 2 h before staining with lysosome (Scale bar is 10 m).

The formation of these modified NDs is confirmed by dynamic light scattering (DLS) (Table S3) and FTIR analysis. It is indicated that carboxyl-NDs in water form average particle sizes of nearly 36.72 nm with narrow size distribution, indicating its good dispersity. MLS-NDs and MLS-FA-PEGylated-NDs form average cluster size of nearly 476.1 and 278.8 nm respectively. Conjugation with peptide and/or PEGylated-FA increased the average cluster size by a few times than the unmodified one, suggesting that biomolecules conjugation in buffer system makes nanodiamonds more prone to aggregation. However, the cellular uptake of modified NDs is still high because the particle’s size distribution is quite broad and its polydispersity index (PDI) is larger than 1, indicating all particles are not in the same size (Table S4). In general, there is a wide range of sizes obtained, e.g. MLS-ND: 342-1106 nm; MLS-FAPEGylated-ND: 396.1-712.4 nm. Thus, smaller sizes of modified nanodiamonds could still be effectively taken up by cells without any aggregation problem. Compared with carboxyl-ND, MLS-ND and MLS-FA-PEGylated-ND showed a zeta potential of 7.46 and 11.1 mV respectively, which had higher zeta potentials than carboxyl-NDs (-93 mV) in deionized water due to the coating of positively-charged MLS molecules. The physicochemical properties of PEGylated FA and/or MLS conjugated NDs were examined using FTIR (Figure S1). The conjugation of amino-functionalized PEGylated FA onto carboxyl-ND was confirmed by the presence of NH–CO stretching vibration at 1631 cm-1. Comparing carboxyl-NDs and FAPEGylated-NDs shows a new peak of 1704 cm-1 in FA-PEGylated-NDs, which is corresponding to the carbonyl group (C=O) of -COOH of folic acid. The –NH– peaks shifted from 3390 cm-1 for amino-functionalized PEGylated FA to 3427 cm-1 for the FA-PEG-ND conjugates. These


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results suggested that a biocompatible and specifically targeted FA-PEGylated-NDs was successful synthesized via the covalent attachment of functional PEG polymer brushes. The formation of MLS-NDs was confirmed by the presence of peptide bond which exhibits a number of IR-active amide bands. The FTIR spectrum of MLS-NDs exhibits the C=O stretching vibration of the amide at 1643 cm-1 (amide I) and the N–H bending and C–N stretching vibration at 1534 cm-1 (amide II). These arise from the amide bonds that link the amino acids. Comparing carboxyl-NDs and MLS-NDs shows new peaks in MLS-NDs at 2847 cm-1, 2869 cm-1, 2929 cm-1 and 2959 cm-1, corresponding to signals from the symmetric CH2 stretching, symmetric CH3 stretching, asymmetric CH2 stretching and asymmetric CH3 stretching respectively of substitutes of amino acids. The co-existence of MLS and PEGylated FA onto the MLS-FA-PEGylated-NDs was also confirmed by the presence of typical NH–CO stretching vibration of amide bond at 1633 cm-1, the C=O stretching of -COOH of folic acid at 1695 cm-1, the N–H bending and C–N stretching vibration of peptide bond at 1539 cm-1 (amide II) and the symmetric CH2 stretching, symmetric CH3 stretching, asymmetric CH2 stretching and asymmetric CH3 stretching of substitutes of amino acids from 2840 to 2958 cm-1 region. Collectively, these results indicated that the MLS and PEGylated FA were successfully attached on carboxyl NDs. As NDs have already shown potential applications for delivery applications,36-39 the capability of ND platform for drug loading and cancer-cell-specific mitochondrial delivery was further investigated. Recently, it is showed that Dox exclusively transported to mitochondria of diseased cells would induce reactive oxygen species (ROS) generation and also facilitate damage of the components in respiration chain. Thus, targeting anticancer drug e.g. Dox to mitochondria would allow escaping from efflux of drug in order to significantly induce cellular apoptosis.40 In this study, Dox is considered as a drug of mitochondria damaging. The amount of Dox adsorbed


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and release afterwards was calculated by determining the absorption maximum of Dox at 488 nm (Figure S5). It is found that the percentage of Dox loaded on 1 mg of MLS-FA-PEGylated-ND is 75% µg (Table S5). We also found that ~ 15 - 18% of loaded Dox molecules start to release after 2 h under different pH values of solution (Figure S6). In addition, higher incubation temperature (e.g. under pH 7.4 at 37 °C) results in significant enhancement of drug release (~ 40%). Confocal fluorescence imaging of 10 µg of Dox-loaded, MLS-FA-PEGylated-NDs in HeLa and regular MCF-7 cells exhibits overlapping of fluorescence signals of NDs (red) and Dox (blue) (Figure 4a) in addition to those fluorescence signals of mitotracker (green) and NDs (red) (Figure 4b). These results indicated the selective delivery of Dox to mitochondria. On the other hand, similar concentrations of free Dox were only localized in lysosomes (Figure 4c). A published literature reported that free Dox has been located in nuclear of HeLa cells after treatment for 5h.25 However, we found that the majority localization of Dox is highly depended on its dosage. In general, Dox molecules are taken up by cells via endocytosis pathway and localized in lysosomes. If high concentration of Dox molecules is used, some of them may be released from lysosomes, get into cytoplasm and then enter nuclear subsequently to kill tumor cells. In our work, we would like to take advantages of nanodiamond as carrier to deliver sufficient amounts of Dox molecules (< 1 µM) to mitochondria to kill tumor cells instead of delivering a high dosage of Dox to nuclear to do the same task. Thus, our results suggested that intracellular delivery of Dox could be regulated via multifunctional ND platform. As expected, it would be highly helpful to transport chemotherapeutic agents that are not cell permeable and directed them to specific intracellular organelles via such a nanodiamond-based delivery system. This is the first example of the use of dual-labeled nanodiamonds for cancer-cell-specific uptake and targeted delivery of drug molecules to mitochondria.


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Figure 4. Intracellular delivery of Dox via multifunctional ND platform. Confocal fluorescence images of (a-b) Dox-loaded, MLS-FA-PEGylated-NDs and (c) free Dox incubated with HeLa and regular MCF-7 for 12 h before staining with different organelle trackers. (Scale bar is 10 m).

MTT assay was used to determine the viability of cells as a function of nanodiamond’s concentration, incubation time and drug concentrations. MTT results indicated that MLS-FAPEGylated-NDs exhibited low cytotoxicity to the regular MCF-7 cells even up to concentration of 0.15 mg/mL, confirming their biocompability (Figure 5a). To make a comparison of cell viability with/without the nanodiamond carriers, the optimized amount of Dox used has been initially evaluated. We found that all of the cells were completely dead after one day when the concentration of Dox was above 2 µM (Figure 5c). In this regard, we decided to load < 1 µM Dox onto the modified ND samples for mitochondria-targeted drug delivery purposes. In comparison with unloaded Dox localized in lysosomes, Dox targeted to mitochondria usually induce a higher percentage of cell death even under the same concentrations of drug loading as a function of incubation time (Figure 5b). It is concluded that differences obtained are highly attributable to the change in cellular distribution of Dox from lysosome to mitochondria by multifunctional nanodiamond-based vehicles, leading to a significant toxicity and enhanced cellular apoptosis. Therefore, we strongly believe that a large amount of loaded Dox molecules were firstly carried by MLS-FA-PEGylated-NDs, selectively entered into mitochondria within 12 h before they gradually detached and released to kill tumor cells in mitochondria. These studies not only emphasize the utility of dual-functionalized NDs as outstanding drug nanocarriers and delivery vehicles for targeted delivery, but also reveal their therapeutical applications in some organelle-related diseases.


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Figure 5. Cell viability studies of MCF-7 cells in the presence of (a) MLS-FA-PEGylated NDs as a function of incubation time and concentration. (b) free Dox and MLS-FA-PEGylated NDs as a function of incubation time and drug loading concentration, (c) Dox as a function of incubation time and concentration.

Since multifunctional ND systems are taken up by cells and improve drug retention, we hypothesized that the MLS-FA-PEGylated NDs could efficiently circumvent drug resistance in the MCF-7/ADR adenocarcinoma cell model, which overexpresses P-gp. Flow cytometry and MTT assay were performed to explore the ability of the multifunctional ND-based delivery systems to circumvent drug resistance in MCF-7/ADR in terms of enhanced cellular Dox uptake and accumulation in specific sub-cellular organelle. MCF-7/ADR cells were incubated overnight with 2 µM of free Dox or Dox-loaded, functionalized ND samples for flow cytometry analysis. It is found that MCF-7/ADR cells treated with Dox-loaded, MLS-FA-PEGylated-NDs exhibited much stronger fluorescence at 585 ± 42 nm which represents the Dox signals than those without


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treatment (no NDs) or those treated with free Dox and Dox-loaded, carboxyl-NDs (Figure 6a and S7). The amount of free Dox was not enough to induce cell death in MCF-7/ARD cells even up to 10 µM of drug (Figure 6b-c) because unloaded Dox itself easily diffuses or transports out of MCF-7/ARD cells via P-gp transporter protein channel. There is no chance for them to reach to the nuclear to kill the tumor cells. On the other hand, loaded Dox molecules could be successfully delivered to MCF-7/ADR cells with the help of MLS-FA-PEGylated-ND carriers. We found that the relative number of viable MCF-7/ADR cells was dramatically reduced after incubating for 2 days when Dox concentration was above 4 µM (see Figure 6b, red curve). Since this is a multidrug-resistant MCF-7 human breast cancer cell line, any Dox released from the ND carriers before reaching mitochondria should be pumped out and thus could not migrate into nucleus to kill tumor cells. These results strongly suggest that loaded Dox molecules were carried by MLS-FA-PEGylated-NDs, selectively entered into mitochondria of MCF-7/ADR cells before they gradually detached and released in mitochondria to kill tumor cells. Thus, delivering drug via multifunctional ND system not only produces enhanced efficacy relative to unloaded drug or drug localized in lysosomes, but also circumvent drug resistance in MCF-7/ADR cells.


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Figure 6. (a) The normalized mean fluorescence signals of Dox in MCF-7/ADR cells after incubating with different samples, e.g. i. no Dox, ii. Free Dox, iii. Dox-loaded, carboxyl-NDs, and iv.

Dox-loaded, MLS-FA-

PEGylated-NDs. Excitation was at 488 nm while collection was from 543-627 nm. (b) MTT analysis of free Dox and Dox-loaded, MLS-FA-PEGylated-NDs incubated with MCF-7/ADR cells for 2 days as a function of drug concentrations. Cell viability in terms of live cell percentage was shown. (c) Confocal fluorescence imaging showed the mitochondrial-targeting ability of MLS-FA-PEGylated in MCF-7/ADR cell lines (Scale bar is 10 µm).

4. CONCLUSIONS In overall, we presented the first example of the use of nanodiamond-based vehicles that were dually functionalized with targeting ligands for multi-modal imaging, mitochondrial targeting and circumventing drug resistance. Functionalization of NDs with FA and MLS not only allows them distinguish between cancer cells that overexpress folate receptor and normal cells that do


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not, but also drastically alter its intracellular localization to mitochondria. In fact, this system can load with anti-cancer drugs via physical adsorption. Subsequently, these drug molecules could be effectively delivered to mitochondria and induce remarkable cytotoxicity and cell death in both HeLa and regular MCF-7 cells compared with free drugs delivered to lysosomes. With the multifunctional ND delivery system, the cellular uptake of Dox was dramatically enhanced, which contributed to the significant improvement of cell-killing ability to Dox-resistant MCF-7 cells. This work provides a novel strategy of designing nanodiamond-based carriers for cancercell-specific mitochondria-targeted delivery and for circumventing drug resistance in doxorubicin-resistant human breast adenocarcinoma cancer cells.

Conflict of Interest: The authors declare no competing financial interest.

ASSOCIATED CONTENT Supporting Information. Construction and characterizations of functionalized NDs, FTIR spectra, cellular uptake results and confocal fluorescence imaging results. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Pik Kwan Lo [email protected] ACKNOWLEDGMENT


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This work was supported by National Science Foundation of China 21304077, 21574109 and Hong Kong Research Grants Council (ECS) 21300314 and CityU 9680104, 7004398, 7004655. We would like to thank Prof. Paul K Chu in the Department of Physics and Materials Science at City University of Hong Kong for providing the MCF-7/ADR cells. REFERENCES (1) Koo, H.; Huh, M. S.; Sun, I.; Yuk, S. H.; Choi, K.; Kim, K.; Kwon, I. C. In Vivo Targeted Delivery of Nanoparticles for Theranosis. Acc. Chem. Res. 2011, 44, 1018-1028. (2) Cortés-Funes, H.; Coronado, C. Role of Anthracyclines in the Era of Targeted Therapy. Cardiovasc. Toxicol. 2007, 7, 56-60. (3) Gottesman, M. M. Mechanisms of Cancer Drug Resistance. Annu Rev Med. 2002, 53, 615– 627. (4) Pastan, I.; Gottesman, M. M. Multidrug Resistance. Annu Rev Med. 1991, 42, 277–286. (5) Cui, W.; Li, J.; Decher, G. Self-Assembled Smart Nanocarriers for Targeted Drug Delivery. Adv. Mater. 2016, 28, 1302–1311. (6) Calderón, M.; Sosnik, A. Polymeric Soft Nanocarriers as Smart Drug Delivery Systems: State-of-the-art and Future Perspectives. Biotechnol. Adv. 2015, 33, 1277–1278. (7) Linko, V.; Ora, A.; Kostiaine, M. A. DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices. Trends Biotechnol. 2015,33, 586-594. (8) Karimi, M.; Ghasemi, A.; Zangabad, P. S.; Rahighi, R.; Basri, M. M. S.; Mirshekari, H.; Amiri, M.; Pishabad, Z. S.; Aslani, A.; Bozorgomid, M.; Ghosh, D.; Beyzavi, A.; Vaseghi, A.;


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TOC


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Cancer-Cell-Specific Mitochondria-Targeted Drug Delivery by Dual

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