Anticancer Effect of Î±-Tocopheryl Succinate Delivered by Mitochondria

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Anticancer Effect of #-Tocopheryl Succinate Delivered by Mitochondria-Targeted Mesoporous Silica Nanoparticles Qiuyu Qu, Xing Ma, and Yanli Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13974 • Publication Date (Web): 23 Nov 2016 Downloaded from http://pubs.acs.org on December 3, 2016

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ACS Applied Materials & Interfaces

Anticancer Effect of α-Tocopheryl Succinate Delivered by Mitochondria-Targeted Mesoporous Silica Nanoparticles Qiuyu Qu,† Xing Ma,† Yanli Zhao*,†,‡ †

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical

Sciences, Nanyang Technological University, 21 Nanyang Link, 637371 (Singapore) ‡

School of Materials Science and Engineering, Nanyang Technological University, 50

Nanyang Avenue, 639798 (Singapore)

KEYWORDS: Cancer therapy; Mesoporous silica nanoparticles; Mitochondria; Targeted delivery; α-Tocopheryl succinate

ABSTRACT: Mitochondria targeted mesoporous silica nanoparticles (MSNPs) having an average diameter of 68 nm were fabricated and then loaded with hydrophobic anticancer α-tocopheryl succinate (α-TOS). The property of targeting mitochondria was achieved by the surface functionalization of triphenylphosphonium (TPP) on MSNPs, since TPP is an mitochondria-targeting ligand. Intracellular uptake and mitochondria targeting of fabricated MSNPs were evaluated in HeLa and HepG2 cancerous cell lines as well as HEK293 normal cell line. In addition, various biological assays were conducted with the aim to investigate the effectiveness of α-TOS delivered by the functional MSNPs, including studies of cytotoxicity, mitochondria membrane potential, intracellular adenosine triphosphate (ATP) production and apoptosis. Based on these experiments, high anticancer efficiency of α-TOS delivered by 1 ACS Paragon Plus Environment


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mitochondria targeted MSNPs was demonstrated, indicating a promising application potential of MSNP-based platform in mitochondria targeted delivery of anticancer agents.

Introduction The proliferation of cells highly depends on the energy supply by adenosine triphosphate (ATP) that is mostly generated in mitochondria, the critical organelle in both normal cells and cancerous cells. However, mitochondria in cancerous cells act quite different from that in normal cells. One of the major differences is that the production efficiency of the ATP molecule in normal cells is higher as compared with that in cancerous cells. The reason behind is that the energy production in cancerous cells relies on glycolysis process, which is called the “Warburg effect”, one of the indicators of cancer.1,2 Paradoxically, cancerous cells consume more energy to maintain a high rate of cell dividing, which means that cancerous cells require more amount of mitochondria than normal cells.3 In addition, mitochondria in cancerous cells are more sensitive to mitocans, i.e., agents with therapeutic effects to mitochondria. Therefore, targeting mitochondria has drawn more attention in cancer 4-8 therapy. In light of the importance of mitochondria, several mitocans have been developed.9,10

α-Tocopheryl succinate (α-TOS) is one of the most effective mitocans in inducing the apoptosis. As one form of vitamin E analogues, α-TOS is an inhibitive competitor of ubiquinone, displacing and preventing ubiquinone from binding to succinate dehydrogenase (SDH) in order to result in the disorder in the electron transportation chain and further induce the mitochondria destabilization and intrinsic apoptosis.11-14 More importantly, it was found that α-TOS could selectively kill cancerous cells with little or no side effects to normal 2 ACS Paragon Plus Environment

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However, the main drawback preventing α-TOS from its wide application in clinical is its hydrophobic property. In most studies regarding the anticancer effect of α-TOS in vitro and in vivo, organic solvents like dimethyl sulfoxide (DMSO) and ethanol are applied to dissolve α-TOS, making the experiments difficult to avoid side effects. Therefore, it is essential to develop α-TOS delivery systems to overcome the side effects. Up to date, there only limited discussions regarding the delivery of α-TOS based on nanocarriers. In this work, applying mesoporous silica nanoparticles (MSNPs) in the delivery of α-TOS to mitochondria in cancerous cells was made for the first time. MSNPs have been extensively investigated concerning the application of drug delivery.16-22 With the merits of high surface area, high pore volume, and tunable pore and particle sizes, MSNPs could be impressively loaded with drug molecules having different molecule sizes.23-25 In addition, MSNPs were reported to be biocompatible.26 Compared with other delivery systems such as polymers, MSNPs are easy to fabricate and commercially low cost. Moreover, MSNPs are also known as effective delivery carriers for hydrophobic drug molecules, making them more suitable for delivering α-TOS.27,28 Various mitochondria targeting ligands have been employed, including chemical compounds such as triphenylphosphonium (TPP) and peptides like XJB peptides and Szeto-Schiller (SS) peptides.29-32 Having three phenyl groups, TPP is normally positively charged, thus attracting negatively charged mitochondria. In addition, TPP with sufficient lipophilicity can facilitate the process of trans-membrane. The attractive performance of TPP in targeting mitochondria has been fully developed.33-35 In this work, TPP was utilized in the surface functionalization to obtain mitochondria-targeted MSNPs. The mitochondria-targeted 3 ACS Paragon Plus Environment


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MSNPs were characterized and further loaded with α-TOS. After that, the anticancer efficiency of α-TOS-loaded MSNPs was conducted against HeLa and HepG2 cancer cell as well as HEK293 normal cell line. The aim of the current work is to improve biological activity of α-TOS by applying simple mitochondria-targeted MSNP delivery system with efficiency (Figure 1).

Figure 1. Schematic diagram of α-TOS delivery by mitochondria-targeted MSNPs.

Experimental Section Fabrication of MSNPs. MSNPs with amine surface functionalization were synthesized following the conventional sol-gel method,36 using cetyltrimethylammonoium bromide (CTAB, 500 mg), tetraethylorthosilicate (TEOS, 2.0 mL), 3-aminopropyltriethoxysilane (APTES, 0.5 mL), and NaOH aqueous solution (2.0 M, 1.2 mL). After the nanoparticle formation, condensed HCl (37%) was employed for the removal of CTAB surfactant, and the obtained product was named MSNPs-NH2.

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The functionalization of TPP on MSNPs was carried out before the CTAB removal in order

to

give

selective

N-hydroxysulfosuccinimide

functionalization (NHS,

35

mg)

on

the

outer

surface.

Generally,

and

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide (EDC, 25 mg) were added to TPP solution (50 mg) in deionized water (5 mL). The activation of the carboxylic acid on TPP was carried out by stirring the mixture solution for 2 h. After that, the TPP solution was added into the CTAB-containing MSNPs-NH2 (10 mg) in deionized water (10 mL). The mixture was then reacted for 48 h. The removal of CTAB from the formed product was carried out using a similar method described above, affording MSNPs-PPh Fluorescein isothiocyanate 3.

(FITC)

tagged

MSNPs

(MSNPs-NH2-FITC

and

MSNPs-PPh3-FITC) were prepared by adding FITC/APTES solution to TEOS/CTAB solution. The CTAB removal and TPP conjugation procedures were similar to the method described above. α-TOS loading. At first, the α-TOS stock solution was prepared in 95 % ethanol at a concentration of 2 mg/mL. MSNPs-NH2 (2.0 mg) and MSNPs-PPh3 (2.0 mg) were suspended in the α-TOS stock solution (2 mg/mL), and then reacted for 24 h. After that, α-TOS loaded MSNPs, i.e., MSNPs-NH2 (α-TOS) and MSNPs-PPh3 (α-TOS), were collected by centrifugation at 10000 rpm for 5 min. In order to eliminate free α-TOS molecule, α-TOS loaded MSNPs were re-collected after re-suspending them in methanol. This methanol washing was conducted quickly, so that the washing process would not significantly influence the loading capacity of these MSNPs. MTT cytotoxicity assay. HeLa, HepG2 and HEK293 cells were cultured in a good

condition at first. Then, different cells in 96-well plant at 1 × 104 cells per well were 5 ACS Paragon Plus Environment


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cultured for 24 h. In the meantime, the culture media with different concentrations of MSNP samples were prepared. After that, old media were replaced with fresh ones containing different MSNPs. Old media were again replaced by fresh media containing (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) after further 24 h

incubation. After another 4 h incubation, the media with MTT were replaced with DMSO (100 µL). Then, the absorbance of tested wells at λ = 560 nm was measured. The percentage of the cell viability was calculated according to the difference between measured intensities of the test well and the control well without the sample treatment. Confocal laser scanning microscopy. Different cells were cultured on cover slides in at 1 × 105 cells per well for 24 h. After that, these cells were treated with MSNPs-NH2-FITC, MSNPs-PPh3-FITC, MSNPs-NH2 (α-TOS) and MSNPs-PPh3 (α-TOS) for 24 h, respectively. The mitochondria staining was carried out based on the protocol provided by Invitrogen. Generally, staining solution was prepared with a proper concentration (50 nM) and placed in the dark. Then, the old culturing solution was replaced with a fresh one having mitochondria staining solution (50 nM), and cells were cultured in the incubator for additional 45 min. Following the washing process, stained cells were treated by formaldehyde (4.0 %). The cells were washed with phosphate-buffered saline (PBS) before the observation by a confocal microscopy. The protocol provided by Invitrogen was used for the lysosome tracing. Briefly, cultured cells in plastic-bot-tomedm-dishes (35 mm) were treated with MSNPs for 4h, 8h and 12h, respectively. Then, old media were replaced with fresh ones containing Lyso-Tracker (50 and cells were cultured in the incubator for another 30 min before directly observing by 6 ACS Paragon Plus Environment

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confocal microscopy. The fluorescence intensity of FITC, Lyso-Tracker Red, and

Mito-Tracker Deep Red was collected at λ = (520 ± 10), (610 ± 10), and (620 ± 10) nm channels under excitations at λ = 488 nm, 543 nm, and 594 nm, respectively. Flow cytometry. The cellular uptake of different MSNPs (with FITC functionality) was studied in HeLa and HepG2 cells by using flow cytometry. Briefly, HeLa and HepG2 cells in 6-well plate at 1 × 105 cells per well were incubated for 24 h. Then, old media were replaced by fresh ones having different MSNP samples, and cells were further incubated for 24 h. After that, old media were removed and cells were washed with fresh media. Cells were harvested in 2.0 mL tube with the treatment of trypsin and collected by centrifugation. In order to eliminate free MSNPs, cells were re-collected by centrifugation in a new 2.0 mL tube. Finally, these cells were ready for flow cytometry study. Mitochondria isolation. Mitochondria isolation was conducted in HeLa, HepG2 and HEK293 cells. The isolation process was carried out based on the mitochondria isolation kit. Basically, different cells cultured in T-75 cell culture flask with 1 × 107 cell density were treated by trypsin. Then, cells were pelleted by centrifuging them in a tube at 850× g. The packed cells were treated by the cell-rupturing species (800 µL) from the isolation kit. The tube containing cells was vortexed for 10 seconds. The tube was added with mitochondria isolation reagent (10 µL). After that, the isolation reagent (800 µL) was added into the cell-containing tube, and the tube was gently shaken for a few times. Under the centrifugation at 700× g, the supernatant was moved to a new 2.0 mL tube. The fluorescence intensity excited at 488 nm was measured. The UV absorption intensity of α-TOS was measured by using NanoDrop spectrophotometer. 7 ACS Paragon Plus Environment


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ATP level detection. The ATP level detection in HeLa and HepG2 cells was carried out using ATP detection kit, and the detection process was conducted following the protocol provided. Generally, HeLa and HepG2 cells were cultured in a 6-well plate at 1 × 105 cells per well for 24 h. The medium was replaced by fresh one having different testing samples. The cells were treated by the cell lysis buffer (200 µL) after additional 24 h incubation, and the cell debris containing ATP was obtained. The luminescence intensities after the treatment with the reaction substrate provided in the detection kit were observed. The ATP level was determined by the calculation based on a standard curve obtained by measuring several ATP solutions with different concentrations of. Rotenone was employed as a positive control. Detection of mitochondria membrane potential. The mitochondria membrane potential of HeLa and HepG2 cancerous cells were detected by applying TMRE mitochondrial membrane potential assay kit. The detection process was carried out following procedures recommended by Abcam. Basically, HeLa and HepG2 cells seeded in a 6-well plate at 1 × 105 cells per well were incubated for 24 h, respectively. The media were replaced by fresh ones having different MSNP samples. The medium was replaced again with fresh one having TMRE (200 nM) after further 24 h incubation. The fluorescence of TMRE excited at 549 nm was measured. In addition, the mitochondria membrane potential was also detected by using flow cytometry method. Generally, HeLa and HepG2 cells were cultured in a 6-well plate at 1 × per well for 24 h. Then, old media were replaced by fresh ones having MSNP samples. After further incubation for 24 h, the media were again replaced by fresh media containing TMRE (200 nM). Cells were treated with trypsin and collected by centrifugation. After washing with


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PBS for two times and harvesting again in 2.0 mL tube, cells were ready for flow cytometry study. Mitochondrial oxidative phosphorylation inhibitor (FCCP) was set as a positive Caspase study. The activities of caspase 9 and caspase 3 in HeLa and HepG2 cells were detected by using Caspase 9 Assay Kit (Colorimetric, ab65608) and Caspase 3 Assay Kit (Colorimetric, ab39401) provided by Abcam, respectively. The detection procedures were carried out following the protocol from Abcam. Briefly, HeLa and HepG2 cells in a 6-well plate at 1 × 105 cells per well were cultured for a period of 24 h. The medium was replaced by new one having MSNP testing samples. Cells were treated by trypsin and pelleted in 2.0 mL tube. Then, pelleted cells were re-suspended in cold cell lysis buffer (50 µL). The cells lysis solution was centrifuged at 10,000× g for 1 min, and the supernatant was moved to a fresh tube for the assay detection. The caspase reaction buffer was prepared according to the provided protocol. The supernatant was added into 96-well plate by 50 µL per well. Then, reaction buffer (50 µL) along with the substrate (5 µL, 4 mM) was added into the tested well. The well was gently mixed, and incubated at 37 °C for 1.5 h. Finally, the optical intensity (405 nm) of each tested well was measured by micro-plate reader.

Results and Discussion Fabrication and characterizations of functional MSNPs. Amine group functionalized MSNPs (MSNPs-NH2) and TPP functionalized MSNPs (MSNPs-PPh3) were fabricated by following surfactant-directed method. The surfactant applied here is CTAB. The surface functionalization of amine group on MSNPs was obtained by adding APTES during the 9 ACS Paragon Plus Environment


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formation of mesoporous channels. The TPP group was functionalized on the surface of MSNPs-NH2 through the conjugation between the amino group of MSNPs-NH2 and acid of TPP. In order to track these MSNPs in vitro, FITC conjugated MSNPs (MSNPs-NH2-FITC and MSNPs-PPh3-FITC) were fabricated based on the co-condensation method, which showed yellow color.

Figure 2. (a) TEM image and (b) FE-SEM image of the obtained MSNPs-PPh3.

Figure 2 shows field emission electron microscopy (FE-SEM) and transmission electron microscopy (TEM) images of synthesized MSNPs-PPh3, presenting regular spherical structures. The average diameter of obtained nanoparticles was around 68 nm, which was measured based on the calculation of over 100 nanoparticles from TEM images. Dynamic light scattering (DLS) method was also applied in characterizing the size of nanoparticles. Larger size of 115 nm was indicated from DLS experiments possibly because of inevitable swelling effect of nanoparticles. FT-IR spectroscopy and Zeta potential measurements were used to characterize the successful surface functionalization of amine and TPP groups. As shown in Figure S1a, the 10 ACS Paragon Plus Environment

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peak at 1560 cm-1 in the FT-IR spectrum of MSNPs-NH2 clearly indicates the presence of groups on the nanoparticles. But, no such peak observed in the FT-IR spectrum of MSNPs-PPh3 means that no obvious amino groups were remained on MSNPs-PPh3, the successful reaction between carboxylic acid groups and amino groups. In addition, one clear peak at 700 cm-1 in the FT-IR spectrum of MSNPs-PPh3 is assigned to the C-H on the phenyl group, further supporting the functionalization of the TPP group on the surface. Apart from FT-IR spectra, Zeta potential of the nanoparticle surface charge was measured in PBS

to

further

confirm

the

TPP

functionalization.

On

account

of

different

protonation/deprotonation environments, the amino groups on MSNPs-NH2 showed a charge at acidic conditions and a negative charge under basic conditions (Figure S1b). On the other hand, the surface charge of MSNPs-PPh3 was always positive under both acidic and conditions, meaning that the amino groups were significantly consumed during the As such, the reaction between the amino group and TPP to afford the TPP surface was successfully achieved. In order to prove the repeatability of the fabrication process, five independent experiments for the preparations of MSNPs-NH2 and MSNPs-PPh3 were conducted with the same amounts of precursors. The surface charge measurements on these five groups of nanoparticles indicate repeatable functionalization of -NH2 and TPP groups. Powder X-ray diffraction (XRD) analysis was then carried out and the results indicate well-ordered hexagonal structure of inner channels for the obtained MSNPs-PPh3. In addition, high Brunauer-Emmett-Teller (BET) surface area of 721 m2/g for MSNPs-PPh3 was observed from the N2 adsorption/desorption measurement (Figure S2). MCM-41 type mesoporous structure of MSNPs-PPh3 was confirmed by the obtained type IV BET isotherm curve.



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α-TOS loading and intracellular uptake. The loading of α-TOS was conducted by suspending synthesized nanoparticles (MSNPs-NH2 and MSNPs-PPh3) in α-TOS solution (1 mg/mL in 95 % ethanol). The entrapment efficiencies of α-TOS in MSNPs-NH2 and MSNPs-PPh3 were calculated to be 7.46 % and 7.30 %, respectively. Thus, the loading capacity of α-TOS was 3.73 wt% and 3.65 wt% for MSNPs-NH2 and MSNPs-PPh3 respectively, indicating no obvious influence of the TPP surface functionalization on the loading capacity of the nanoparticles. The loading capacity was calculated according to the UV intensity difference at 284 nm between stock α-TOS solution and supernatant solution after the α-TOS loading. In addition, the successful α-TOS loading was characterized by the BET surface area studies (Figure S2). Lowered surface areas of MSNPs-NH2 (α-TOS) and MSNPs-PPh3 (α-TOS) compared with original MSNPs were observed due to the α-TOS loading in the pores of MSNPs. The in vitro α-TOS release profile from MSNPs-PPh3 (TOS) was obtained in cell culture medium as well (Figure S3), indicating that α-TOS could be released upon time.


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Figure 3. Cellular uptake of MSNPs-NH2-FITC and MSNPs-PPh3-FITC in (a) HeLa and (b) HepG2 cells. The cell incubation time for the nanoparticle uptake study was 24 h. FL-1: FITC fluorescence.



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Figure 4. Confocal microscope images of HepG2 cells treated with (a) MSNP-PPh3-FITC (50 µg/mL) and (b) MSNP-NH2-FITC (50 µg/mL) after indicated incubation hours. Scar bar: 20 µm. The prerequisite of a proper drug delivery carrier is its good intracellular uptake. Thus, intracellular uptake test of MSNPs-NH2 and MSNPs-PPh3 with the FITC functionality was performed by applying flow cytometry method using HeLa cervical cancer cell line and HepG2 liver hepatocellular carcinoma cell line. As shown in Figure 3, both MSNPs-NH2-FITC and MSNPs-PPh3-FITC showed high FITC fluorescence intensity in HeLa and HepG2 cells, indicating their successful intracellular uptake as well as low cytotoxicity as drug delivery platforms. Sufficient research has shown that MSNPs could be intracellularly internalized through the process of endocytosis in mammalian cells.37,38 In order to further investigate the 14 ACS Paragon Plus Environment

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internalization of MSNPs-NH2 and MSNPs-PPh3, the endocytosis process after the nanoparticle uptake was studied. It is believed that MSNPs would be initially trapped by lysosomes after the cellular uptake, and such trapping could influence the delivery efficiency. Therefore, time-dependent endocytosis of these MSNPs was conducted using confocal laser scanning microscopy (CLSM) with the staining of lysosomes by Lyso-Tracker Red in HepG2 cells (Figure 4). According to the obtained images (Figure 4), after initial 4 h incubation, clear green spots corresponding to the fluorescence of the FITC functionality on MSNPs-NH2-FITC and MSNPs-PPh3-FITC could be observed, further indicating their successful intracellular uptake and low cytotoxicity. In addition, no clear yellow spots appeared in the merged images, meaning no overlapping between these MSNPs and lysosomes at this stage. Thus, these MSNPs were not yet trapped by lysosomes after the incubation of initial 4 h. After 8 h incubation, obvious yellow spots appeared in the cases of MSNPs-NH2-FITC and MSNPs-PPh3-FITC, indicating that these MSNPs and lysosomes were co-localized and these MSNPs were trapped by lysosomes. After further incubation, however, no clear yellow color could be observed in the case of MSNPs-PPh3-FITC, proving that MSNPs-PPh3-FITC could escape from lysosomes at the time of 12 h incubation (Figure 4a). Such lysosome escape can be possibly attributed to the facilitation of cross membrane process due to the TPP surface functionality of MSNPs-PPh3. For the case of MSNPs-NH2-FITC after 12 h, distinct yellow color could still be observed (Figure 4b), meaning that MSNPs-NH2-FITC was trapped by lysosomes as before. Therefore, it could be concluded that MSNPs-PPh3 presents quick endocytosis process. Similar results were also obtained by using HeLa cells.39 15 ACS Paragon Plus Environment


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Mitochondria targeting. The study of mitochondria targeting by MSNPs-PPh3 was carried out using CLSM (Figure 5). Different cancer cell lines (HeLa and HepG2 cell lines) were treated with MSNPs-PPh3-FITC, and mitochondria within cells were stained by Mito-Tracker Red. As seen from the obtained CLSM images, these MSNPs and mitochondria were indicated as green and red dots, respectively. In the case of treating HeLa cells with MSNPs-NH2-FITC, no obvious yellow dots representing the co-localization of green and red dots could be found in the merged image. In contrary, distinct yellow dots could be observed in the merge image when using MSNPs-PPh3-FITC, indicating the successful co-localization between MSNPs-PPh3-FITC and mitochondria, and further proving the evident ability of MSNPs-PPh3 in targeting mitochondria of HeLa cells. The overlapping coefficient index of confocal images was calculated as well. The index for merged images in Figure 5a was 0.408 for MSNPs-NH2-FITC and 0.662 for MSNPs-PPh3-FITC, indicating that MSNPs with TPP functionalization present good overlapping effect between the nanocarriers and mitochondria. Similar mitochondria targeting effect of MSNPs-PPh3 could be found in HepG2 cells as well. Interestingly, when treating HEK293 normal cells with MSNPs-PPh3-FITC, no obvious co-localization could be obtained (Figure S4). Such observation could be probably due to different mitochondria membrane activity and potential, since mitochondria in cancer cells usually have higher activity and membrane potential than normal cells. Furthermore, it was found that mitochondria in normal cells could not be well stained. Since the staining of mitochondria is based on highly negatively charged membrane potential, this observation indicates that mitochondria in normal cells were less active than that in cancerous cells. 16 ACS Paragon Plus Environment

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To further validate the successful targeting to mitochondria in cancer cells, the fluorescence and UV detections of isolated mitochondria from treated HeLa, HepG2 and HEK293 cells were conducted. Mitochondria were isolated from cells after the treatment of different MSNPs. As shown in Figure 6a, higher FITC fluorescence intensity was observed in the isolated mitochondria population from both HeLa and HepG2 cells treated with MSNPs-PPh3-FITC, directly proving the mitochondria targeting ability of MSNPs-PPh3. In the case of mitochondria from HEK293 normal cells, however, no obvious FITC intensity difference was found, showing no specific mitochondria targeting effect of MSNPs-PPh3 in HEK293 cells. Apart from the fluorescence detection, the UV intensity detection was also performed, since the loaded α-TOS has a strong UV absorption peak at 284 nm. Here, the UV test was conducted after cells were treated with α-TOS loaded MSNPs. As seen from Figure 6b, obviously higher UV intensity in mitochondria was found in the cases of HeLa and HepG2 cancer cells treated with MSNP-PPh3 (α-TOS), and no obvious UV intensity difference was observed from HEK293 normal cells. Thus, it could be concluded that MSNPs-PPh3 has an excellent capability in targeting mitochondria of cancer cells.



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Figure 5. Confocal microscope images of (a) HeLa cells and (b) HepG2 cells after treating with MSNPs-NH2-FITC and MSNP-PPh3-FITC (50 µg/mL). Scar bar: 20 µm.


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Control MSNPs-NH2-FITC MSNPs-PPh3-FITC

HeLa

HepG2

HEK293

Control MSNPs-NH2 (α-TOS) MSNPs-PPh3 (α-TOS)

HeLa

HepG2

HEK293

Figure 6. (a) FITC fluorescence and (b) UV absorption intensities of isolated mitochondria originated from HeLa, HepG2 and HEK293 cells without and with indicated treatments.

Cytotoxicity and mitochondria alterations. In order to investigate the anticancer efficacy of α-TOS loaded MSNPs and the cytotoxicity of MSNP-based delivery platforms, the MTT assay was carried out in HeLa and HepG2 cancerous cells as well as HEK293 normal cells 19 ACS Paragon Plus Environment


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(Figure 7). Higher intensity of MTT assay (cell survival ratio) indicates higher cellular activity and lower cytotoxicity.

From the obtained data in Figure 7a, it could be noted that all tested cells showed high cell viability (above 80 %) even at 100-200 mg/mL concentrations of MSNPs-NH2 and MSNPs-PPh3, meaning that these MSNP-based nanocarriers have low cytotoxicity toward both cancerous cells and normal cells. MSNPs-PPh3 (α-TOS) presented high anticancer efficiency at concentrations from 25 to 200 mg/mL in both HeLa and HepG2 cancerous cells, since cell survival ratios in this concentration range were much lower than that in the case of MSNPs-NH2 (α-TOS). The half maximal inhibitory concentration (IC50) values for MSNPs-PPh3 (α-TOS) in HeLa and HepG2 cells were calculated to be 45.8 mg/mL and 51.6 mg/mL, respectively. This observation can be reasonably explained by the fact that α-TOS is a kind of agent particularly effective to mitochondria. Such active effect can be enhanced by targeted delivery of α-TOS to mitochondria using the MSNPs-PPh3 nanocarrier, increasing the concentration of α-TOS in mitochondria at the same time. For the test in HEK293 normal cells, however, high cell viability was still obtained in the whole range of concentrations, indicating that both MSNP nanocarriers and α-TOS loaded MSNPs showed no obvious cytotoxicity toward normal cells. Therefore, it could be concluded that the anticancer efficacy of α-TOS can be significantly enhanced by mitochondria targeted MSNPs-PPh3, and more importantly, α-TOS loaded MSNPs-PPh3 showed no obvious cytotoxicity to normal cells.


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Cell

MSNPs -NH2

MSNPs MSNPs-NH2 MSNPs-PPh3 Rotenone -PPh3 (α-TOS) (α-TOS)

Figure 7. (a) Cells viability of HeLa, HepG2 and HEK293 cells and (b) intracellular ATP level of HeLa and HepG2 cells after the treatment of MSNPs-NH2, MSNPs-PPh3, MSNPs-PPh3 (α-TOS), and MSNPs-NH2 (α-TOS) for 24 h, respectively. 21 ACS Paragon Plus Environment


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Since α-TOS has a therapeutic effect to mitochondria, the function of mitochondria should be affected when α-TOS is delivered into mitochondria. Therefore, alterations of mitochondria resulted from delivered α-TOS were investigated. The distinct function of mitochondria is the ATP production, which is the most commonly used form of energy to supply cellular activities. In cancerous cells, the energy supply is more important than that in normal cells because of the long lasting cell proliferation of cancerous cells, leading to the fact that cancerous cells are more sensitive toward cellular ATP level alteration. As such, we investigated intracellular ATP levels before and after the treatment of different MSNPs. The relative results obtained were calculated based on a standard curve of ATP solutions with various concentrations (Figure S5). Rotenone was used as the positive control. In the case of testing MSNPs without the α-TOS loading (Figure 7b), no obvious decrease of intracellular ATP level was observed, further demonstrating the low cytotoxicity of MSNP-based nanocarriers. Interestingly, more obvious decrease of the ATP level was observed from MSNPs-PPh3 (α-TOS) than that of MSNPs-NH2 (α-TOS), which could be resulted from the successful delivery of α-TOS to mitochondria by MSNPs-PPh3. Thus, the active α-TOS was successfully delivered to mitochondria by MSNPs-PPh3 for decreasing intracellular ATP level. One of the important features of mitochondria is its highly negatively charged membrane that gives high mitochondria membrane potential for controlling the ion transportation, metabolism as well as cell death.3,40-42 Such high membrane potential, on the other hand, is the symbol of active mitochondria.43 Thus, the detection of mitochondria membrane potential was conducted in HeLa and HepG2 cells in order to measure the activity of mitochondria 22 ACS Paragon Plus Environment

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after the treatment of MSNPs-PPh3 (α-TOS). Active mitochondria with high membrane potential could be stained by an experimental TMRE kit, giving strong fluorescence intensity. From the results shown in Figure 8, compared with control cells, no obvious influence on the mitochondria membrane potential was observed when treating cells with MSNPs-NH2 and MSNPs-PPh3, again showing the low cytotoxicity of these MSNPs. While in the event of testing MSNPs loaded with α-TOS, lowered mitochondria membrane potential of both HeLa and HepG2 cells treated with MSNPs-PPh3 (α-TOS) was observed than that with MSNPs-NH2 (α-TOS), indicating that the function of α-TOS was achieved by using mitochondria targeted MSNPs-PPh3. In other words, α-TOS could be successfully delivered to mitochondria by MSNPs-PPh3, resulting in the accumulated concentration of α-TOS in mitochondria as well as the lowered mitochondria membrane potential.

HeLa

HepG2

HEK293



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Figure 8. (a) Mitochondria membrane potential (TMRE fluorescence intensity) detected after treating HeLa cells with different MSNPs (50 µg/mL) and FCCP for 24 h. (b,c) Flow cytometry studies of mitochondria membrane potential on HepG2 and HeLa cells, respectively. FL-1: Fluorescence of TMRE.

Apoptosis study. Apoptosis is a biomedical event that occurs in the process of programmed cell death in multicellular organisms, which can lead to various cellular changes in physical morphology, nuclear fragmentation, global mRNA decay and so on.44,45 It is essential to know that the apoptosis process is highly regulated, since it is unstoppable in both cancerous 24 ACS Paragon Plus Environment

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and normal cells. Thus, it is generally accepted that inducing the apoptosis in cancerous cells is one of the methods to achieve the cancer treatment. More importantly, the cascade apoptosis process is closely related to the function of mitochondria. For example, it was proven that the primary signal molecule of apoptosis process, cytochrome c, is released from mitochondria because of the formation of mitochondrial apoptosis-induced channel.46 It was also proven that α-TOS is an effective apoptosis inducer.14,47 Therefore, it is necessary to investigate the apoptosis caused by the dysfunction of mitochondria that is resulted from α-TOS delivered by MSNPs-PPh3. During the cascade apoptosis process, featured measurements commonly involve the activity detections of caspase 9 that is one of the initiator caspases, and caspase 3 that is subsequently activated by caspase 9. Here, the activity of caspase 9 and caspase 3 was detected in HeLa and HepG2 cancerous cells after the treatment of different MSNPs at different time points for 12h, 18h, and 24 h (Figure 9 and Figure S6). FCCP was set as a positive control. Higher optical intensity at 405 nm corresponds to higher activity of caspase. As shown in Figure 9, the treatment by MSNPs-NH2 and MSNPs-PPh3 showed almost no influence on activities of both caspase 9 and caspase 3 in HeLa and HepG2 cells, further proving the low cytotoxicity of these MSNPs. In the case of testing α-TOS loaded MSNPs in HeLa and HepG2 cells, however, MSNPs-PPh3 (α-TOS) presented higher activities of both caspase 9 and caspase 3, indicating higher activity of apoptosis process induced by MSNPs-PPh3 (α-TOS). Thus, it could be concluded that the property of hydrophobic α-TOS to effectively induce the apoptosis can be enhanced when delivered by MSNPs-PPh3 to mitochondria.



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MSNPs -NH2

MSNPs MSNPs MSNPs Control -PPh3 -NH2 -PPh3 (α-TOS) (α-TOS)

Cell

MSNPs -NH2


Cell

Cell

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MSNPs -NH2


MSNPs -NH2


Figure 9. Activity of caspase 9 and caspase 3 in HeLa and HepG2 cells after the treatment of different MSNPs for 24 h. Control: FCCP. O.D.: optical density.

Conclusions In summary, mitochondria targeted MSNPs (MSNPs-PPh3) were successfully fabricated by the surface functionalization of TPP. The property of targeting mitochondria was demonstrated by CLSM study in HeLa and HepG2 cancerous cell lines. Hydrophobic anticancer agent, α-TOS, was successfully loaded in designed MSNPs for the first time. The synthesized MSNPs showed good cellular uptake and low cytotoxicity against HeLa and HepG2 cells, and quick endocytosis and lysosome escape capability. More importantly, α-TOS loaded MSNPs showed no obvious toxicity to HEK293 normal cells. The 26 ACS Paragon Plus Environment

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hydrophobicity of α-TOS was overcome by using the MSNPs-PPh3 nanocarrier. Anticancer effects of α-TOS including inducing the cytotoxicity and mitochondria dysfunctions as well as apoptosis were achieved and further enhanced by the targeted delivery using the MSNPs-PPh3 nanocarrier to mitochondria. As a result, the present research broadens the application of MSNPs as drug delivery platforms, demonstrating its promising potential in overcoming the hydrophobicity of drug molecules as well as targeted mitochondrial sub-cellular drug delivery.

ASSOCIATED CONTENT Supporting Information: This material is available free of charge via the Internet at http://pubs.acs.org. FT-IR, surface charge measurements, BET isotherms, release profile, confocal images, and activities of cell caspase 9 and caspase 3.

AUTHOR INFORMATION Corresponding Author * Email: [email protected].

ACKNOWLEDGMENT

This work is supported by the Singapore Academic Research Fund (AcFR) Tier 1 (RG112/15), and the NTU-Northwestern Institute for Nanomedicine.

REFERENCES 27 ACS Paragon Plus Environment


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Table of Contents Graphic


Anticancer Effect of Î±-Tocopheryl Succinate Delivered by Mitochondria

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