Folic acid-conjugated TiO2-doped mesoporous carbonaceous nanocomposites loaded with Mitoxantrone HCl for chemo-photodynamic therapy.

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Photochemical & Photobiological Sciences

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with Mitoxantrone HCl for chemo-photodynamic therapy Zhi Li, Ya Ou-yang, yang liu, Yi-qiu Wang, Xia-li Zhu, Zhen-zhong Zhang* School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, PR China Corresponding

author:

Zhen-zhong

Zhang,

Tel:

86-371-67781890

Email:

Published on 06 May 2015. Downloaded by Dalhousie University on 19/05/2015 01:34:00.

[email protected] Absract: Recently, porous carbons have showed great potential in many areas. In this study, TiO2-doped mescoporous carbonaceous (TiO2@C) nanoparticles were obtained by a simple one-pot hydrothermal treatment, folic acid (FA) was conjugated to TiO2@C through an amide bond, then Mitoxantrone HCl (MTX) was adsorbed onto TiO2@C-FA and obtained a drug delivery system, TiO2@C-FA/MTX. TiO2@C-FA/MTX showed a much faster MTX release at pH 4.5 than at pH 6.0 and pH 7.4. Furthermore, compared with free MTX, these drug delivery system showed a dose-dependent cytotoxicity by varing the irradiance, afforded higher antitumor efficacy in cultured PC3 cells in vitro. The ability of TiO2@C-FA/MTX to combine the chemotherapy with photodynamic activity enhanced cancer cell killing effect in vitro, demonstrating that TiO2@C-FA/MTX had a great potential for cancer therapy in the future. Key words: Porous carbons, TiO2, Folic acid, Drug delivery, chemo-photodynamic therapy

Photochemical & Photobiological Sciences Accepted Manuscript

10.1039/C5PP00097A Folic acid-conjugated TiO2-doped mescoporous carbonaceous nanocompositesDOI:loaded


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1. Introduction Porous carbons have have great potential applications in many areas, such as green energy storage devices 1, physical adsorbents2, elctrochemical devices 3 and gas storage materials4, owing to their important properties: high chemical stability, low cost, high surface area, large pore volume, as well as good electronic conductivity and high storage


capacity5. However, the employment of porous carbons for drug delivery is still at early stage of development 6, 7. porous non-carbonaceous materials are well-known drug delivery media8-11, silica-based mesoporous materials have been successfully employed for a multitude of drugs9. However, It has been reported that chronic exposure to silica can weaken the immune system 12, crystalline silica can be cytotoxic and can cause increased granulation in the pulmonary13. Porous activated type of carbon was traditionally employed as emergency medicine for the treatment of drug overdose or toxic chemical ingestion for both infants and adults without any sign of toxicity14, it can be inferred that porous carbon will be acceptable to human physiology. Pore structure of porous materials ranges from microporous (pore diameter < 2 nm), mesoporous (2 nm ～ 5nm and 10 ～ 50 nm) to macroporous (pore diameter > 50 nm)15. Among the porous materials, mesoporous/ macroporous materials exhibit low mass density, high surface area and large pore volume. Compared with either a solely mesoporous equivalent, the interconnected macropore and mesopore channels may allow the dissolution media to easily penetrate into the particles and facilitate drug dissolution, more drug can be loaded into the materials due to their higher surface area and pore volume 16-18. Titanium dioxide (TiO2) has been widely studied as photocatalysts because of their exceptional optical properties, chemical stability, non-toxicity and low cost19-21. In recent years, TiO2 was noticed as a potential photosensitizer in the field of photodynamic therapy (PDT), the electrons in the valence band of TiO2 can be excited to the conduction band by ultraviolet (UV), thus resulting in the photo induced hole-electron pairs. These photo induced electrons and holes can react with surrounding H2O and O2 molecules and generate various reactive oxygen species (ROS), which can kill cancer cells 22-24. However,


DOI: 10.1039/C5PP00097A

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3.2 eV; rutile: 3.0 eV), and low quantum efficiency, which greatly reduces the photocatalytic activities of TiO2. TiO2 can only be excited by UV light which is harmful and hinders its practical applications25, 26, because UV radiation can damage the molecular basis of life. The biological important nucleic acids, proteins, and lipids are susceptible to UV induced photodamage 27.


Fortunately, there are a number of empirical ways to develop visible-light-sensitive photocatalysts of TiO2 by means of physical and chemical processes. For example, CdS quantum-dots-doped TiO2 nanocomposites can exhibit a very high photodynamic efficiency under visible-light irradiation and used as a new "photosensitizer" for cancer cell treatment 28.

In

this

paper,

TiO2-doped

mescoporous

carbonaceous

(TiO 2@C)

nanoparticles were obtained by a simple one-pot hydrothermal treatment 29,

30

,

amine-functionalized TiO2@C (TiO2@C-NH ) was obtained by APTES reaction, after that folic acid (FA) was conjugated on TiO2@C-NH through reaction with carboxyl to form amide bonds in the presence TiO2@C-FA ( Fig.1.) and their potential in PDT under visible light source was studied. FA was widely used as targeting molecule for many cancers targeting therapy 31. So FA was linked to the drug delivery to obtain an active tumor targeting effect to PC3 cells32, 33. Mitoxantrone (MTX), a first line cancer chemotherapeutic, is a minimally toxic type II topoisomerase inhibitor that disrupts DNA synthesis and DNA repair in both healthy and cancer cells34. In this study, MTX was adsorbed in porous carbons, achieving high loading efficiency and getting a system for combination chemotherapy with PDT. 2.1. Materials Glucose anhydrouse was purchased from Tianjin Fengchuan Chemicals Co. Ltd. Folic acid (FA),

Titanium

tetrachloride

Dicyclohexylcarbodiimide

(TiCl4),

(DCC)

were

(3-Aminopropyl) gotten

from

triethoxysilane

Aladdin

Chemistry

and Co.

N,N’Ltd.

Mitoxantrone HCl (MTX, 20120503, purity98) was from Beijing Yi-He Biotech Co. Ltd. Sulforhodamine B(SRB), RPMI-1640 cell culture medium, penicillin, streptomycin, fetal bovine

serum

(FBS)

were

bought

from

Gibco

Invitrogen.

1,1'-dioctadecyl-3,3,3',3'-tetramethyl indotricarbocyanine Iodide (DiR) was supplied by


DOI: 10.1039/C5PP00097A practical application of pure TiO2 has been restricted due to its wide band gap (anatase:


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DOI: 10.1039/C5PP00097A

2.2. Synthesis of TiO2@C Glucose was selected as the carbon source and TiCl4 as the Titanium source. By a simple one-pot hydrothermal treatment according to the steps in the literature29, TiO2-doped mescoporous carbonaceous materials were obtained. Briefly, 1g Glucose dissolved in ethanol- acetate mixture ( ethanol : acetate = 1:2, 30mL), then added dropwise with TiCl4


(2mL). After stirring at 140℃ for 10h, the mixture was evaporated and dried in vacuum at 60 ℃ for 24h, then purified by repeated rinsing with ethanol and deionized water several times and filtrations. The final products(TiO2@C) were dried in vacuum at 60℃ for 24h. 2.3. Conjugation of FA to TiO2@C and Characterization TiO2@C (10mg) was added to ethanol (10mL) and stired well, APTES (100µL) was added into the mixture, stirred at room temperature for 12h, then purified by repeated rinsing with ethanol and deionized water several times and filtrations. The resulting solid products were dried in vacuum at 60 ℃ for 24h. Finally, the above product (50mg), FA (5mg) and DCC (10mg) were added to pyridine (5mL), then stirred at room temperature in the dark for 24h, the mixture was evaporated and repeated rinsing with ethanol and deionized water several times and filtrations, finally dialyzed by a membrabe (MWCO=10,000, sepectrum laboratories Inc) for 48h to remove free FA, DCC and pyridine. The resulting product (TiO2@C- FA) was dried in vacuum at 40 ℃ for 24h.TEM (Tecnai G2 20, FEI, HK) was used for morphological of TiO2@C-FA. The optical properties of TiO2@C-FA were characterized using an ultra-violet-visible (UV-vis) spectrometer (Lambda 35, Perkin-Elmer, USA). Energy dispersed X-ray (EDAX, philips XL30) spectrometer was used to indicate the element combination. FT-IR spectra were recorded on a Nicoleti S10 spectrometer (Thermo). 2.4. MTX adsorption onto TiO2@C-FA and determination of MTX loading First, a high concentration (250µg/mL) of MTX solution was prepared by dissoving 1.25mg MTX in 5mL deionized water. Then, 12.5mg TiO2@C-FA was added to deionized water (5ml) and sonicated for 10 min. After that, 0.1～1 mL TiO2@C-FA solution was added into 1mL MTX solution and stirred overnight at room temperature, then stored at room temperature for 24h, the nanosuspension was separated by centrifugation to remove free


Beyotime institute of biotechnology Co. Ltd.

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sonicating 5 min then was stored at 4℃ until used. At the same time, the concentrations of free MTX in supernatant were determined by high performance liquid chromatography (HPLC,1100 Agilent, USA) with the following conditions: an Eclipse XDB-C18 column (150mm×4.6mm, 5.0µm); mobile phase acetate ( sodium acetate: 18mg/mL, acetic acid glacial: 9.8µL/mL) /methanol 64:36; column temperature 30;detection wave length 627nm;


flow rate 1.0ml/min. DLS (Zetasizer NanoZS-90, Malvern, UK) was used for characterizing particle size, zeta potential of TiO2@C-FA/MTX. 2.5. Release determination of MTX For release study, TiO2@C-FA /MTX was placed into dialysis bags, which were dialyzed in 20mL acetate buffer (pH 4.5), 20mL acetate buffer (pH 6.0), and 20mL phosphate buffered saline (pH7.4), respectively.The release assay was performed at 37.0±0.5℃ with a stirring rate of 100r/min.3mL solution was drawn from acetate solution at various time points, being replaced by the same volume of fresh solution.The concentration of MTX released from TiO2@C-FA /MTX into solution was quantified using HPLC under the above chromatographic conditions. 2.6. Cell experiments PC3 human prostate cancer cell line was obtained from Chinese Academy of Sciences Cell Bank (Catalog No.TCHu158). Cells were cultured in normal RPMI-1640 culture medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in 5% CO2 at 37℃ in a humidified incubator. In vitro PDT treatment. To investigate the cancer cell phototoxicity of MTX, TiO2@C, TiO2@C-FA and TiO2@C-FA /MTX under 532nm laser irradiation, PC3 cells were incubated with those formulations at the fixed concentrations (TiO2@C and TiO2@C-FA concentration: 30µg /mL, MTX concentration: 2.28µg /mL) for 8h, and then exposed to an 532nm laser at irradiance of 0mW/cm 2, 200mW/cm 2 or 300mW/cm 2 for 1.5min, the samples were further incubated for 24h. Finally, a standard cell viability assay using SRB


DOI: 10.1039/C5PP00097A MTX and the resulting TiO2@C-FA/MTX nanosuspension redispersed in water after


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DOI: 10.1039/C5PP00097A

Cellular uptake. DiR was widely use as flourescent markers of nanoparticles, in order to study the nanoparticles uptake in vitro and in vivo. DiR in ethanol solution (2mg/mL) was added to TiO2@C-FA/MTX nanosuspension, then stirred at 0 ℃ in the dark for 2h, after rotated separated, the precipitate was purified by repeated rinsing with deionized water


and filtrations to remove the excess DiR to obtain TiO2@C-FA/MTX–DiR complex. Intra cellular uptake of TiO2@C/MTX-DiR and TiO2@C-FA /MTX-DiR were performed with PC3 cells. PC3 cells were seeded at 1×106 cells per well on glass cover slips in 6-well plates, then treated with TiO2@C-FA/ MTX and TiO2@C/MTX (TiO 2@C-FA concentration: 30µg /mL and MTX concentration:2.98µg /mL) for 1h. After washing three times with PBS, the cells were imaged by Flurescence Mcroscope (Zeiss LSM 510), and the intensity of fluorescence was measured by flow cytometry using an exitation wavelength of 748nm and an emission wavelength of 780nm. Intra cellular ROS detection. ROS generation inside cells was detected using DCFH-DA Reactive Oxygen Species Assay Kit35. PC3 cells were seeded in confocal dishes at a density of 1×106 cells per well on glass cover slips in 6-well plates. Following incubation with or without TiO2@C-FA/ MTX(TiO2@C-FA concentration: 30µg /mL and MTX concentration:2.98µg /mL) for 1h,DCFH-DA was loaded into the cells. After 30 min incubation, cells were washed twice with PBS and then exposed to 532nm irradiation for 1min at the irradiance of 200 mW/cm 2. After irradiation, fluorescence images of treated cells were acquired using Flurescence Mcroscope. Detection of DNA fragmentation. The cells treated with TiO2@C-FA/ MTX(TiO2@C-FA concentration: 30µg /mL and MTX concentration:2.98µg/mL) for 8h, and then a 200mW/cm 2 of 532nm laser was delivered for 5min. After that, the cells were collected. 50µL of 1% low-melting agrose was mixed with 50µL of the cell suspension (1 × 105 cells/mL). The mixture was immediately spread onto a frosted slide, which was previously covered with 1% normal-melting agarose and kept at 4 ℃ to solidify. The solidified gel containing the cells was treated under dim light with a solution ( 2.5M NaCl, 0.1M EDTA,100M Tris, pH10, 1%(v/v) TritonX-100, 10% (v/v) DMSO) at 4℃ for 1.5h, then the


was conducted.

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neutralized using 0.4M Tris HCl solution (pH7.5). DNA was stained with PI (1:10000) and captured at 100× magnification under a fluorescence microscope (Olympus, Japan).Cells containing damage DNA have an appearance of a comet with a bright head and tail, meanwhile, undamaged DNA appears as an intact nucleus with no tail.


3. Results and discussions 3.1.Synthesise and characterization of TiO2@C- FA FT-IR results showed that FA conjugated to TiO2@C was confirmed by the strong amide bond ( ～ 1626cm -1), and benzene, conjugated double absorption ( ～ 1576cm -1)36,37. A very strong absorbance in the range of 450～500 cm -1, can be attributed to Ti-O stretching vibration38 (Fig.2A.). The elemental analysis using EDAX indicated the presence of Ti, C, and O in the nanoparticles, which reconfirmed that these nanoparticles were TiO2-doped carobn materials ( Fig.2C). As seen in UV-VIS spectrum of TiO2@C- FA (Fig.2B.), an absorption peak at 282nm was observed in TiO2@C- FA, this absorption peak was not observed in spectrum of TiO2@C, also indicating that FA was linked to TiO2@C. The optical

properties

of

TiO2@C-FA

and

TiO2@C

were

characterized

using

an

ultra-violet-visible (UV-vis) spectrometer and the concentration of our samples was very low (50μg/mL), when the concentration was increased into 250μg/mL, absorption spectra of TiO2@C could be found from 500-600nm. (Fig.2B.). The micro structure and morphology of TiO2@C-FA were examined by the transmission electron microscopy (TEM) in Fig. 2C, the results showed that TiO2@C-FA was porous nanoparticles, and these nanoparticles has a large number of macroporous and certain a mount of mesopores.The high-magnification TEM image shown in Fig.2C highlighted the surface of a nanoparticles, where lots of “black dots” exist, while the no “black dots” on the surface of TiO2@C was found, indicating that FA was linked to the surface of TiO2@C. 3.2. Preparation and characterization of TiO2@C-FA/MTX To determine the adsorption equilibrium level of MTX embedding onto TiO2@C-FA, different feed ratios of MTX/ TiO2@C-FA were performed, indicating that MTX embedding ratioincrease from 3.8% to 96.5% ( weight ratio of embedding MTX/ MTX dosage) with the


DOI: 10.1039/C5PP00097A solidified gel was conducted at 25V and 300mA for 25min. The slides were then


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TiO2@C-FA /MTX showed an average size distribution around 615 nm (Fig.3C) and a zeta potential around-14.2mV (Fig.3D).To investigate the release kinetics of active drug from nanoparticle-drug system, we incubated the nanoparticles in buffers with various pH values. As seen in Fig.3E, MTX release from TiO2@C-FA was strongly dependent on the pH of the medium.The cumulative release amount of MTX could reach up to 89 % after 24h at pH 4.5,


much higher than that at pH 6.0 or 7.4, which was 62%, 34%, respectively. 3.3. Cellular uptake and intracellular PDT To explore the difference in uptake of drug- nanoparticle system by PC3 cells, we labeled these nanoparticles with DiR and tracked their internalization into cells through colocalization of DiR-signal (red fluorescence). The results showed that the uptake of TiO2@C-FA/MTX- DiR was faster than that of TiO2@C/MTX- DiR. Compared with TiO2@C/MTX- DiR groups, larger increase in

flourescence that was evenly distributed

throughout the cells was found in TiO2@C-FA/MTX- DiR groups (Fig.4A), the intensity of fluorescence,

measured

by

flow

cytometry,

was

also

revealed

that

more

TiO2@C-FA/MTX- DiR nanoparticles were uptaked than TiO2@C/MTX- DiR nanoparticles (Fig.4B). TiO2@C-FA/MTX delivery system could effectively enhance uptake of MTX by PC3 cells. The dark cytotoxicity study of TiO2@C-FA on PC3 cells was carried out at different concentrations of TiO2@C-FA in order to determine the systemic toxicity of the blank drug carrier. As shown in Fig.5A., cell viability remained above 90% even at the concentration up to 100 µg/ml, this result showed that TiO2@C-FA itself possessed low toxicity without light irradiation to PC3 cells. In order to investigate the PDT efficiency of TiO2@C-FA/MTX in cancer cells, PC3 cells were incubated with TiO2@C-FA, MTX, TiO2@C-FA/MTX at the fixed concentrations of TiO2@C-FA (30µg /mL ) and MTX (2.28µg /mL) for 8h, and then exposed to an 532nm laser with irradiance of 0 mW/cm 2, 200mW/cm 2 or 300mW/cm 2 for 1.5min. As shown in Fig.5B., cells cultured with culture medium only possessed low toxicity, cell viability remained above 90% even with irradiance of 300mW/cm 2 for 1.5min, this results indicated that the light alone do not cause cytotoxicity. Cells cultured with MTX possessed


DOI: 10.1039/C5PP00097A increased amount of TiO2@C-FA (Fig.3A). Dynamic light scattering analysis of

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statistically differences ( p>0.05 ), these rusults indicated laser irradiation did not effect the cytotoxicity of MTX. A laser irradiance-dependent cytotoxicity of TiO2@C-FA and TiO2@C-FA/MTX was shown. The cell viability with TiO2@C-FA at irradiance of 0 mW/cm 2, 200 mW/cm 2 and 300mW/cm 2 decreased to above 99%, 65% and 57%, respectively. The results

suggested

that

532nm

laser

irradiation

enhanced

the

cytotoxicity

of


[email protected] cell viability with TiO2@C-FA/MTX with irradiance of 0 mW/cm 2, 200 mW/cm 2 and 300 mW/cm 2 decreased to above 51%, 24% and 11%, respectively, these rusults indicated that this drug delivery system combinated chemotherapy with PDT. It was also found that TiO2@C-FA/MTX had lower cell viability on PC3 cells than TiO2@C/MTX at all irradiance, suggesting that TiO2@C-FA/MTX delivery system could delivery more drug into tumor cells. 3.4. Intracellular ROS production and DNA fragmentation caused under 532nm laser irradiation Whether the TiO2@C-FA/MTX nanoparticles generate ROS in the PC3 cells were investigated under the 532nm laser irradiating. ROS productions were observed in PC3 cells incubated with TiO2@C-FA/MTX by using probe DCFH-DA. Fig. 6 showed the fluorescence micrographs of illuminated PC3 cells incubated with DCFH-DA probe in medium with or without TiO2@C-FA/MTX for 1h, revealing that only trace green fluorescence in cells treated with probe alone was observed, while strong fluorescence evenly distributed throughout cells treated with TiO2@C-FA/MTX was found, indicating that generation of intracellular ROS was induced by TiO2@C-FA/MTX under 532nm laser irradiation. PC3 cells treated with TiO2@C-FA/MTX and TiO2@C-FA/MTX/ 532nm laser emerged DNA fragmentations as revealed by electrophoretic analysis (Fig. 7a and 7b), while no DNA fragmentation was present in total cells from control group or the group to treat with 532nm laser alone (Fig. 7c and 7d). In this study, the images were analyzed by comet assay software project. Cells containing damaged DNA appeared as a comet with bright head and tail, whereas those containing undamaged DNA appeared as an intact nucleus with no tail. Compared with control group, the content of tail DNA in TiO2@C-FA/MTX


10.1039/C5PP00097A cytotoxicity, while, the cell viability from irradiance 0 mW/cm 2 to 300 mW/cm 2 DOI: were no


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the content of tail DNA in TiO2@C-FA/MTX /532nm laser group was 33.6±8.2%. There is a significant

difference

of

the

tail

DNA

between

TiO2@C-FA/MTX

and

TiO2@C-FA/MTX/532nm laser groups (p≤0.01). 4. Discussion In this paper, glucose was selected as the carbon source. TiO2-doped porous carbon


materials were prepared through a one-pot solvothermal method coupling the growth of TiO2 and carbonization of glucose. Hydrolysis of TiCl4 accelerated the carboniazation of glucose. We could not prepare porous carbon using only glucose with the same condition at 140℃. The mesoporous materials has been successfully employed for drug delivery 39. However, because of sterical hindrance, mesoporous materials with small mesopores are not suitable for loading of large molecular drugs and the drug-loading capacity is a little limited16. mesoporous/ macroporous materials may allow the dissolution media to easily penetrate into the particles and more drug can be loaded into the materials40. In this study, the micro structure of TiO2@C-FA was examined by the TEM, it can be observed that TiO2@C-FA was meso–macroporous materials. Because of their porous structure, TiO2@C-FA nanoparticles exhibit excellent absorbencies for MTX, Fig.3B showed that when TiO2@C-FA (12.5mg) was forced to deionized water (5mL) containing MTX(1.25mg), the MTX had been taken up completely after 24h. The pH-responsive system is of particular interest for cancer therapy, because extracallular tumor and endosome are more acidic than normal tissues 41. Zhu et al42 demonstrated that pH may play an important role in the drug release mesoporous carbon nanospheres. The drug may be able to remain inside the mesoporous carbon nanospheres at physiological pH but be efficiently released in the acidic environment in tumors.In this paper, we found that MTX release from TiO2@C-FA was also strongly dependent on the pH of the medium. The cumulative release amount of MTX at pH 4.5 was much higher than that at pH 6.0 or 7.4.These nanoparticles release anticancer drug in a pH dependent manner might reduce the side effects of active drug, and improve its efficacy. The PDT efficiency of TiO2@C-FA was carried out on PC3 cells. TiO2@C-FA had a little


10.1039/C5PP00097A group was 16.9±9.2%, which could be attributed to the therapeutic effect of MTX, DOI: however,

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that cellular uptake of TiO2@C-FA/MTX- DiR was faster than that of TiO2@C/MTX- DiR. This is probably due to the FA- targeted delivery system that can be transferred into cells faster, and as a result, its effects are signicantly better than the other groups. PDT involves three components: a photosensitizer (PS), a special wavelength of drug-activating light and oxygen. A major advantage of PDT over conventional chemotherapy is that PS itself is


minimally toxic in the absence of light. Furthermore, compared with radiotherapy, the activating light is non-ionizing and hence its effect on tissues without PS drug is not harmful43, 44. In this study, TiO2@C-FA itself possesses low toxicity without light irradiation to PC3 cells. TiO2@C-FA/MTX induced generation of intracellular ROS under 532nm laser irradiation. Comet assay indicated TiO2@C-FA/MTX under 532nm laser caused more damage to PC3 cells than the other groups. The DNA damage result was consistent with the phototoxicity assay, and also revealed the synergistic enhancement effects of MTX and photodynamic therapy to PC3 cells. So TiO2@C-FA/MTX has photocytotoxicity under 532nm laser irradiation and can be used as cancer therapeutic photosensitizer. In summary, a FA functionalized TiO2-doped mescoporous carbonaceous nanoparticles, TiO2@C-FA was successfully synthesized and characterized. These nanoparticles showed low cytotoxity without irradiation. MTX loaded TiO2@C-FA showed excellent PDT and high anticancer efficacy, these results indicated that there is a great potential of TiO2@C-FA/MTX forapplication in cancer therapy.

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DOI: 10.1039/C5PP00097A


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Fig.1. A schematic illustration of TiO2@C-FA/MTX nanocomposite preparation



DOI: 10.1039/C5PP00097A


A

C

C

B b

a

(cm-1 ) (nm)


Page 15 of 20 Photochemical & Photobiological Sciences

DOI: 10.1039/C5PP00097A

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Fig.2. Characterization of TiO2@C-FA A) FT-IR spectrum of a: TiO2@C-FA, b: TiO2@C; B) UV spectrum of a: TiO2@C-FA （50μg/mL）, b: TiO2@C（50μg/mL）; C) a, b and c: TEM image of TiO2@C-FA, d: TEM image of TiO2@C, e: EDAX Spectrum image of TiO2@C-FA


A

B

D

C

E


DOI: 10.1039/C5PP00097A

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DOI: 10.1039/C5PP00097A

A

B

Fig.4. Uptakes of TiO2@C-FA/MTX and TiO2@C /MTX nanocomposite in cancer cells (TiO2@C and TiO2@C-FA concentration: 30µg /mL, MTX concentration:2.98µg /mL). Cellular uptake



Fig.3. Characterization of TiO2@C-FA/MTX A) MTX embedding at different feeding amouts of TiO2@C-FA; B) 1: Photos of TiO2@C-FA (2.5mg/mL) in water 2: Photos of MTX (0.25mg/mL) in water 3: TiO2@C-FA /MTX redispersed in water after sonicating 5 min; C) and D) Dynamic light scattering analysis of TiO2@C-FA/MTX showing an average size distribution around 615nm and a zeta potential around -14.2mV; E) The pH-dependent cumulative MTX release from [email protected] rates and amounts of MTX released were strongly dependent on the pH of the medium. Data were presented as mean± standard deviation(n =3).


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microscopy (A) and flow cytometry (B) using an exitation wavelength of 748nm and an emission wavelength of 780nm. .


A

B

Concentration of TiO2@C-FA (μg /mL)

Fig.5. a) Cytotoxicity of TiO2@C-FA on PC3 cells without irradiation b) Relative viabilities of PC3 cells treated with MTX, TiO2@C, TiO2@C-FA, TiO2@C-FA/MTX with or without laser irradiation. PC3 cells were incubated with those formulations at the fixed concentrations (TiO2@C and TiO2@C-FA concentration: 30μg /mL, MTX concentration:2.28μg /mL) for 8h, and then exposed to an 532nm laser for 1.5min, the samples


DOI: 10.1039/C5PP00097A was performed by staining the nanoparticels with DiR and performed by fluorescence


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were further incubated for 24h.

a

DOI: 10.1039/C5PP00097A

b

Fig.6. Detection of intracellular reactive oxygen production (ROS) by DCFH-DA staining in PC3 cells.Cells were exposed to 532nm irradiation for 1min at the irradiance of 200 mW/cm2. After irradiation, fluorescence images of treated cells were acquired using Flurescence Mcroscope. a: incubated with TiO2@C-FA/MTX; b: incubated without TiO2@C-FA/MTX (TiO2@C-FA concentration: 30µg /mL and MTX concentration:2.98µg /mL).


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a

c

b

d

Fig.7. Image of single cell gel electrophoresisi for DNA damage a) cell treated with TiO2@C-FA/MTX for 8h then exposed to 532nm laser for 1min at the irradiance of 200 mW/cm2; b) cell treated with TiO2@C-FA/MTX for 8h; c) untreated cells; d) cells exposed to 532nm laser alone for 1min at the irradiance of 200 mW/cm2. (TiO2@C-FA concentration: 30µg /mL, MTX concentration:2.98µg /mL)



DOI: 10.1039/C5PP00097A

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