nanomaterials Article

Targeting and Photodynamic Killing of Cancer Cell by Nitrogen-Doped Titanium Dioxide Coupled with Folic Acid Jin Xie, Xiaobo Pan, Mengyan Wang, Longfang Yao, Xinyue Liang, Jiong Ma, Yiyan Fei, Pei-Nan Wang and Lan Mi * Department of Optical Science and Engineering, Shanghai Engineering Research Center of Ultra-Precision Optical Manufacturing, Green Photoelectron Platform, Fudan University, 220 Handan Road, Shanghai 200433, China; [email protected] (J.X.); [email protected] (X.P.); [email protected] (M.W.); [email protected] (L.Y.); [email protected] (X.L.); [email protected] (J.M.); [email protected] (Y.F.); [email protected] (P.-N.W.) * Correspondence: [email protected]; Tel.: +86-21-6564-2092 Academic Editor: Luigi Pasqua Received: 27 April 2016; Accepted: 1 June 2016; Published: 14 June 2016

Abstract: Titanium dioxide (TiO2 ) has attracted wide attention as a potential photosensitizer (PS) in photodynamic therapy (PDT). However, bare TiO2 can only be excited by ultraviolet illumination, and it lacks specific targeting ligands, which largely impede its application. In our study, we produced nitrogen-doped TiO2 and linked it with an effective cancer cell targeting agent, folic acid (FA), to obtain N-TiO2 -FA nanoconjugates. Characterization of N-TiO2 -FA included Zeta potential, absorption spectra and thermogravimetric analysis. The results showed that N-TiO2 -FA was successfully produced and it possessed better dispersibility in aqueous solution than unmodified TiO2 . The N-TiO2 -FA was incubated with human nasopharyngeal carcinoma (KB) and human pulmonary adenocarcinoma (A549) cells. The KB cells that overexpress folate receptors (FR) on cell membranes were used as FR-positive cancer cells, while A549 cells were used as FR-negative cells. Laser scanning confocal microscopy results showed that KB cells had a higher uptake efficiency of N-TiO2 -FA, which was about twice that of A549 cells. Finally, N-TiO2 -FA is of no cytotoxicity, and has a better photokilling effect on KB cells under visible light irradiation. In conclusion, N-TiO2 -FA can be as high-value as a PS in cancer targeting PDT. Keywords: titanium dioxide nanoparticle; photodynamic therapy; cancer cell targeting; folic acid

1. Introduction Photodynamic therapy (PDT) based on the photochemical reactions of photosensitizer (PS) is a noninvasive and mild medical treatment for cancer diseases [1–4]. Titanium dioxide (TiO2 ) nanoparticles (NPs) have been widely used as photocatalysts, photochromics and photovoltaics due to their excellent photoreactivity [5–7]. Recently, TiO2 has attracted more attention as a potential PS in PDT due to its high chemical stability, excellent oxidation capability and low toxicity [8–10]. Under ultraviolet (UV) irradiation, TiO2 can be activated and generate cytotoxic reactive oxygen species (ROS). However, UV light is also harmful to normal cells or tissues and has a limited penetration distance in tissues. To enhance the visible light absorption of TiO2 , various attempts have been made by dye-adsorbed [11,12] or doping methods [13,14]. Moreover, a major challenge for the application of TiO2 in PDT is its lack of specific targeting ligands. To improve the cancer cellular uptake of TiO2 NPs, specific ligands, such as monoclonal antibodies, peptides and aptamers binding to specific proteins or surface antigens, could be coupled with TiO2 NPs. For example, it has been found that folate receptor (FR) is overexpressed on the surfaces of many human tumor cells, whereas it expresses little on the Nanomaterials 2016, 6, 113; doi:10.3390/nano6060113

www.mdpi.com/journal/nanomaterials

Nanomaterials 2016, 6, 113

Nanomaterials 2016, 6, 113

2 of 9

2 of 9

surface of normal cells [15,16]. Folic acid (FA) has high affinity to FR, which can serve as a good tumor marker for to targeting treatment in PDT. themarker use of for FA to target FR over-expressed cells affinity FR, which can serve as a Although good tumor targeting treatment in PDT. tumor Although were studied depth various as ZnO [17]inand Au nanorods [18], the use ofin FA to for target FR nanomaterials over-expressedsuch tumor cellsnanoparticles were studied depth for various FA nanomaterials functionalizedsuch TiO2asNPs with enhanced visible light absorption has not been explored. Recently, ZnO nanoparticles [17] and Au nanorods [18], FA functionalized TiO2 NPs the with Lee Wen-Chien team light modified FA onhas TiOnot used the obtained under enhanced visible absorption beenand explored. Recently, theFA/TiO Lee Wen-Chien 2 NPs, 2 as a PS team UVmodified light [19].FA However, a photokilling allunder cells, so is not[19]. encouraged on TiO2 UV NPs,light and itself used can the have obtained FA/TiO2 as on a PS UVitlight However,in UV light itself Herein, can havewea reported photokilling on all cells, it is not encouraged in PDT application. PDT application. a promising PS,so nitrogen-doped TiO2 with FA on its surface Herein, we reported a promising PS, nitrogen-doped TiO 2 with FA on its surface (N-TiO 2 -FA). The (N-TiO2 -FA). The prepared N-TiO2 -FA NPs were characterized by Zeta potential, absorption spectra N-TiO2-FA analysis. NPs were characterized by nasopharyngeal Zeta potential, absorption spectra andprepared thermogravimetric In this study, human carcinoma (KB) cellsand and thermogravimetric analysis. In this(A549) study, cells human nasopharyngeal carcinoma cells and cell human human pulmonary adenocarcinoma were used as FR-positive and(KB) FR-negative lines, pulmonaryThe adenocarcinoma (A549)ofcells were used as FR-positive and FR-negative cell lines, respectively. targeting efficiency N-TiO 2 -FA for different cells was studied by laser scanning respectively. The targeting efficiency of N-TiO 2-FA for different cells was studied by laser scanning confocal microscopy. Finally, the cytotoxicity and photokilling effect of N-TiO2 -FA under visible light confocalwere microscopy. Finally, the cytotoxicity and photokilling effect of N-TiO2-FA under visible irradiation researched. light irradiation were researched. 2. Results 2. Results 2.1. Preparation and Characterization of N-TiO2 -FA 2.1. Preparation and Characterization of N-TiO2-FA Figure 1 shows the synthesis procedure of N-TiO2 -FAs. The nitrogen-doping TiO2 (N-TiO2 ) Figure 1 shows the synthesis procedure of N-TiO 2-FAs. The nitrogen-doping TiO2 (N-TiO2) NPs were first obtained by calcination of pure TiO 2 NPs in ammonia atmosphere for higher visible NPs were firstin obtained by of calcination 2 NPs in ammonia atmosphere for higher visible light absorbance the range 400–550 of nmpure [20].TiO Using amino silanization method [21], the N-TiO2 light absorbance in the range of 400–550 nm [20]. Using amino silanization method [21], the N-TiO2 NPs were modified to obtain positively charged N-TiO2 -NH2 NPs, whose surfaces were full of the NPs were modified to obtain positively charged N-TiO2-NH2 NPs, whose surfaces were full of the protonated amino groups (-NH3 + ).+ FA and N-Hydroxysuccinimide (NHS) were reacted to form protonated amino groups (-NH3 ). FA and N-Hydroxysuccinimide (NHS) were reacted to form NHS-FA, then FA was conjugated with N-TiO2 -NH2 following a standard procedure [22]. It should be NHS-FA, then FA was conjugated with N-TiO2-NH2 following a standard procedure [22]. It should noted that FA has α- and γ-carboxylic acids and both of them could react with NHS. In such a reaction, be noted that FA has α- and γ-carboxylic acids and both of them could react with NHS. In such a the reaction, γ-carboxylic acid is primarily due to its higher reactivity [23]. the γ-carboxylic acid isconjugated primarily conjugated due to its higher reactivity [23].

Figure 1. Synthesis procedure of N-TiO2-folic acid (FA) nanoparticles (NPs). Figure 1. Synthesis procedure of N-TiO2 -folic acid (FA) nanoparticles (NPs).

of 10 μg·mL−1 was shown in Figure 2A. It can be seen that the subtracting spectrum was very similar with the spectrum of 1 μg·mL−1 FA solution, which indicated that FA was successfully linked with the NPs. As shown in Figure 2B, the absorption spectra of N-TiO2 and N-TiO2-FA NPs were both in the visible region between 400 and 550 nm. The absorption of N-TiO2 was similar to the previous report due to nitrogen-doping [20]. N-TiO2-FA NPs demonstrated enhanced Nanomaterials 2016, 6, 113 3 of 9 absorbance respect to N-TiO2, which may be attributed to the surface modifier. The higher absorption of N-TiO2-FA might lead to higher photoactivity under visible light. The The absorbanceanalysis spectra (TGA) of N-TiO -FA air-dried and N-TiO solutions were measured and NPs analyzed. thermogravimetric of2 the N-TiO 2-NH 2, and N-TiO2-FA were 2 -NH22, N-TiO The absorption spectrum of N-TiO -FA subtracting that of N-TiO -NH with the same concentration shown in Figure 2C. The weight losses at 800 °C of N-TiO2, N-TiO 2 2 2-NH 2 2, and N-TiO2-FA NPs were ´1 was shown in Figure 2A. It can be seen that the subtracting spectrum was very similar of 10 µg¨ 2.26, 5.75,mL and 11.87 wt %, respectively. The weight loss of N-TiO2-NH2 NPs with respect to N-TiO2 ´1 FA solution, which indicated that FA was successfully linked with with the spectrum of 1 µg¨ was 3.49 wt %, which wasmL due to the amino silane coupling agent. It could be estimated that the the NPs. As shown in Figure 2B, theN-TiO absorption spectra N-TiO NPs were both in conjugated amount of -NH2 on one 2 NP was 205 of byN-TiO assuming N-TiO 2 NPs spheres with 2 and 2 -FAwere the region 400 and 550 The absorption N-TiO the2 previous the visible diameter of 10between nm. The weight lossnm. of N-TiO 2-FA NPs of with respect N-TiOto 2-NH was 6.12 2 wastosimilar report to nitrogen-doping N-TiO2 -FAofNPs enhancedamount absorbance respect to wt %, due which was due to the [20]. disappearance FA.demonstrated Thus, the conjugated of FA on one N-TiO which may be attributed toabout the surface modifier. The higher absorption of can N-TiO N-TiO22, NP was further estimated 193. According to the TGA result, one conclude that 2 -FA might lead to higher photoactivity underagents visiblewere light.conjugated The thermogravimetric analysis (TGA) of the air-dried almost all of the amino coupling with FA. N-TiO were shown in Figure weightmeasurement. losses at 800 ˝ CIt of The surface electrical property samples was studied by 2C. ZetaThe potential is 2 , N-TiO 2 -NH 2 , and N-TiO 2 -FAofNPs N-TiO NPs were 2.26, 5.75, and 11.87 wt respectively. The well known that when theN-TiO absolute of Zeta potential is above 30%, mV, the NPs can beweight stably 2 , N-TiO 2 -NH 2 , and 2 -FAvalue loss of N-TiO to N-TiO wt %,2 which was due to the dispersed in 2 -NH the solvent [24].respect The Zeta potential of N-TiO was −10.4 ± 0.6 mV,amino whichsilane was 2 NPs with 2 was 3.49 coupling agent. It could be estimated that the conjugated amount of -NH on one N-TiO NP was 205 negatively charged and with poor dispersibility. Capping with cationic amino groups, the Zeta 2 2 by assuming N-TiO spheres with the2 was diameter 10 nm. weight loss of N-TiO potential of the positively charged N-TiO 2-NH +20.6of ± 2.3 mV.The As FA conjugated on N-TiO 2 NPs were 2 -FA2 NPs respect N-TiOnegatively -NH was 6.12 wt %, which was due to the disappearance of FA. Thus, the NPs,with N-TiO 2-FA to became charged (−27.4 ± 1.2 mV) and more stably dispersed in solution 2 2 conjugated of FA on one N-TiOthat was further estimated about 193. with According the TGA at pH = 7. amount These results confirmed FA was successfully conjugated N-TiOto 2 NPs and 2 NP result, one can conclude that almost allNPs of the agents were conjugated with FA. implied that the obtained N-TiO 2-FA hadamino good coupling dispersibility.

Figure 2. (A) Comparison of the absorption spectrum of 1 μg·mL−1 folic acid (FA) solution and the Figure 2. (A) Comparison of the absorption spectrum of 1 µg¨ mL´1 folic acid (FA) solution and subtraction spectrum of N-TiO2-FA subtracting N-TiO2-NH2; (B) Absorption spectra of N-TiO2 and the subtraction spectrum of N-TiO2 -FA subtracting N-TiO2 -NH2 ; (B) Absorption spectra of N-TiO2 N-TiO2-FA NPs; (C) Thermogravimetric analysis of the air-dried N-TiO2, N-TiO2-NH2 and and N-TiO2 -FA NPs; (C) Thermogravimetric analysis of the air-dried N-TiO2 , N-TiO2 -NH2 and N-TiO2-FA NPs. N-TiO2 -FA NPs.

2.2. Cellular Uptake The surface electrical property of samples was studied by Zeta potential measurement. It is well The cellular of unmodified TiOpotential 2 NPs is inefficient due their poor while known that when uptake the absolute value of Zeta is above 30 mV,tothe NPs candispersibility, be stably dispersed N-TiO 2 -FA NPs with improved dispersibility can enter cells. According to our previous result, TiO2 in the solvent [24]. The Zeta potential of N-TiO2 was ´10.4 ˘ 0.6 mV, which was negatively charged NPswith could enter cells non-specifically [24].cationic It wasamino reported that the theZeta negatively charged TiO2 NPs and poor dispersibility. Capping with groups, potential of the positively entered HeLa by various pathways such as caveolin-mediated endocytosis, clathrin-mediated charged N-TiOcells -NH was +20.6 ˘ 2.3 mV. As FA conjugated on N-TiO NPs, N-TiO -FA became 2 2 2 2 endocytosis, and micropinocytosis. When cells were incubated in 4 °C, there were still some TiO2 negatively charged (´27.4 ˘ 1.2 mV) and more stably dispersed solution at pH = 7. These results NPs entering HeLa cells successfully [24]. In this study, KB cells as the FR-positive cancer cellsthat andthe A549 cells as confirmed that FA was conjugated with N-TiO implied obtained 2 NPs and the FR-negative cancer cells were cultured to investigate the cellular uptake of N-TiO 2 -FA with the N-TiO2 -FA NPs had good dispersibility. −1 same condition. After 40 min incubation with culture medium containing 50 μg·mL N-TiO2-FA, 2.2. Cellular Uptake higher reflection intensity of internalized N-TiO2-FA than A549 cells as shown in KB cells exhibited Figure Moreover, it of canunmodified be seen that some of isN-TiO 2-FA NPs were around the cell membranes The3A. cellular uptake TiO NPs inefficient due to their poor dispersibility, while 2

N-TiO2 -FA NPs with improved dispersibility can enter cells. According to our previous result, TiO2 NPs could enter cells non-specifically [24]. It was reported that the negatively charged TiO2 NPs entered HeLa cells by various pathways such as caveolin-mediated endocytosis, clathrin-mediated endocytosis, and micropinocytosis. When cells were incubated in 4 ˝ C, there were still some TiO2 NPs entering HeLa cells [24]. In this study, KB cells as the FR-positive cancer cells and A549 cells as the FR-negative cancer cells were cultured to investigate the cellular uptake of N-TiO2 -FA with the same condition. After 40 min incubation with culture medium containing 50 µg¨ mL´1 N-TiO2 -FA, KB cells

Nanomaterials 2016, 6, 113

4 of 9

exhibited higher reflection intensity of internalized N-TiO2 -FA than A549 cells as shown in Figure 3A. 4 of 9 Moreover, it can be seen that some of N-TiO2 -FA NPs were around the cell membranes of A549, instead of A549, inside.instead This suggests thatThis the suggests cellular uptake of cellular TiO2 -FAuptake NPs was more effective for FR-positive KB of of inside. that the of TiO 2-FA NPs was more effective cells. The average amount of N-TiO -FA NPs located in KB, A549 and FA-pretreated A549 cells 2 for FR-positive KB cells. The average amount of N-TiO2-FA NPs located in KB KB,orA549 and with increasing incubation time were quantified by analyzing the reflection intensity of N-TiO 2 FA-pretreated KB or A549 cells with increasing incubation time were quantified by analyzing -FA the NPs insideintensity individual cells. As shown in Figure 3B, the amounts of endocytosed N-TiO2 -FAamounts NPs in KB reflection of N-TiO 2-FA NPs inside individual cells. As shown in Figure 3B, the of cells were about twice that in A549 cells and FA-pretreated cells. For the FA-pretreated KB cells, the endocytosed N-TiO 2-FA NPs in KB cells were about twice that in A549 cells and FA-pretreated cells. receptors of KB cells were loaded FA, amount N-TiO2 -FA FA-pretreated KB For the FA-pretreated KBoccupied cells, thebyreceptors of so KBthe cells wereofoccupied by inloaded FA, so the cells was similar with those of A549 cells. This result also confirmed that FA was successfully linked amount of N-TiO2-FA in FA-pretreated KB cells was similar with those of A549 cells. This result with confirmed N-TiO2 NPs andFA thewas N-TiO could target FR-positive cancer cells with high efficiency. 2 -FA NPs linked also that successfully with N-TiO 2 NPs and the N-TiO2-FA NPs could Figure FR-positive 3B showed that the amount of N-TiO -FA in FA-pretreated cells decreased by around target cancer cells with high 2efficiency. Figure 3(B)A549 showed that the amount of 10% compared with non-pretreated A549 cells. Therefore, it can be concluded that FA can only block N-TiO2-FA in FA-pretreated A549 cells decreased by around 10% compared with non-pretreated a small part of uptakeitof N-TiO for A549 cells. alsoa incubated and A549 cells 2with 2 -FA NPsthat A549 cells. Therefore, can be concluded FA can onlyWe block small part KB of uptake of N-TiO -FA N-TiO 40 min that the amounts N-TiO cellsN-TiO were similar to those of FA-pretreated 2 for 2 inwith NPs for A549 cells.and Wefound also incubated KB andof A549 cells 2 for 40 min and found that the ones. Thisofresult proved that the coupled FA on N-TiO2 NPs improved the This uptake efficiency ofthat NPsthe for amounts N-TiO 2 in cells were similar to those of FA-pretreated ones. result proved FR-positive cells. coupled FA cancer on N-TiO 2 NPs improved the uptake efficiency of NPs for FR-positive cancer cells. Nanomaterials 2016, 6, 113

Figure Figure 3. 3. (A) (A) Confocal Confocal microscopic microscopic images images of of human human nasopharyngeal nasopharyngeal carcinoma carcinoma (KB) (KB) and and human human 2-FA (red) for 40 min. Scale bar is 50 μm. pulmonary adenocarcinoma (A549) cells treated with N-TiO pulmonary adenocarcinoma (A549) cells treated with N-TiO2 -FA (red) for 40 min. Scale bar is 50 µm. (B) (B) Reflection Reflection intensity intensity of of internalized internalized N-TiO N-TiO22-FA -FA in in KB KB (black), (black),FA-pretreated FA-pretreated KB KB (red), (red), A549 A549 (blue) (blue) cells A549 (green) (green) as as aa function function of of incubation incubation time. time. cells and and FA-pretreated FA-pretreated A549

2.3. 2.3. Cytotoxicity Cytotoxicity of of N-TiO N-TiO22-FA -FA The cell viability viability assays assaysofofKB KBand and A549 cells were measured the cells incubated The cell A549 cells were measured afterafter the cells werewere incubated with −1 N-TiO2-FA for 1 h in order to evaluate its cytotoxicity. As shown in Figure 4, with 50–200 μg·mL ´ 1 50–200 µg¨ mL N-TiO2 -FA for 1 h in order to evaluate its cytotoxicity. As shown in Figure 4, all the all the surviving of the treated cells were than It canthat be nitrogen-doping, concluded that surviving fractionsfractions of the treated cells were greater than greater 90%. It can be90%. concluded nitrogen-doping, surface-modification with FA has little influence on cytotoxicity. surface-modification or coupling with FAorhascoupling little influence on cytotoxicity. These results also showed These results also showed that the cytotoxicity of the N-TiO 2-FA NPs is quite low and negligible, that the cytotoxicity of the N-TiO2 -FA NPs is quite low and negligible, indicating that N-TiO2 -FA NPs indicating that N-TiO2-FA are and wellofbiocompatible, safe in dark, and of therapy application are well biocompatible, safeNPs in dark, therapy application value. value.

Nanomaterials 2016, 6, 113

5 of 9

Nanomaterials 2016, 6, 113 Nanomaterials 2016, 6, 113

5 of 9 5 of 9

−1 Figure 4. The The dark dark cytotoxicity cytotoxicity of ofN-TiO N-TiO2-FA -FA withthe theincubation incubationconcentrations concentrationsofof50–200 50–200µg¨ μg·mL Figure 4. mL´1 2 2-FAwith −1 Figure 4. The dark cytotoxicity of N-TiO with the incubation concentrations of 50–200 μg·mL on KB and and A549 A549cells. cells.The Thecontrol controlgroups groupsofof untreated cells were also shown comparison. Data on KB untreated cells were also shown for for comparison. Data are on KB and A549 cells. The control groups of untreated cells were also shown for comparison. Data are expressed as mean ± SD = 4). expressed as mean ˘ SD (n =(n4). are expressed as mean ± SD (n = 4).

2.4. Targeting and Photokilling Effect on Cancer Cells 2.4. 2.4. Targeting Targeting and and Photokilling Photokilling Effect Effect on on Cancer Cancer Cells Cells The photokilling effects were measured under visible-light irradiation (400–575 nm) of the The photokilling effects were under visible-light irradiation (400–575 nm) nm) of theoflight effects weremeasured measured visible-light (400–575 −2. The light The dosephotokilling 190 ´ J·cm surviving fractions ofunder KB and A549 cellsirradiation in dependence on the kindsthe of 2 . The dose 190 J¨ cm surviving fractions of KB and A549 cells in dependence on the of −2. The surviving fractions of KB and A549 cells in dependence on the kinds light dose 190 J·cm of photosensitizer samples were shown in Figure 5. N-TiO2-NH2 showed a weak and kinds similar photosensitizer samples were shown in Figure 5. N-TiO -NH showed a weak and similar photokilling 2 2 photosensitizer samples shown Figure N-TiOfractions. 2-NH2 showed a weak and similar photokilling effect on bothwere cell lines within about 80% 5. survival N-TiO2-FA showed a higher effect on botheffect cell lines withcell about 80% survival fractions. N-TiO a higher photokilling 2 -FA showed photokilling on both lines with about 80% survival fractions. N-TiO showed a higher photokilling effect on A549 cells with 68.9% cell viability which is due to its2-FA better dispersibility effect on A549effect cells with 68.9%cells cell viability which is viability due to itswhich better dispersibility with thedispersibility comparison photokilling on A549 with 68.9% cell is due to its better with the comparison of N-TiO2-NH2. However, N-TiO2-FA exhibited an excellent photokilling effect of N-TiO -NH2 . However, N-TiO exhibited an excellent photokilling effect on KB cells with the 2comparison 2 -FA with of N-TiO 2-NH 2. However, N-TiO2-FA exhibited an excellent photokilling effect on KBthe cells with the cell viability dropping to 34.5%. Compared with N-TiO2-NH2, the N-TiO2-FA cell viability dropping to 34.5%. Compared N-TiOCompared demonstrated the 2 -NH2 , the N-TiON-TiO 2 -FA NPs on KB cells with thethe celltargeting viability specificity droppingwith to 2-NH2, the N-TiO2-FA NPs demonstrated for34.5%. FR and exhibitedwith a better photokilling effect on targeting specificity for FR and exhibited a better photokilling effect ona FR-positive tumor cells, which NPs demonstrated the targeting specificity for FR and exhibited better photokilling effect on FR-positive tumor cells, which we can attribute to the coupling with FA. we can attribute to the coupling with FA. FR-positive tumor cells, which we can attribute to the coupling with FA.

Figure 5. Photokilling effects of N-TiO2-FA (green) and N-TiO2-NH2 (blue) with the concentration of Figure 5. Photokilling effects of N-TiO 2-FA (green) and N-TiO2-NH2 (blue) with the concentration of −1 on A549 and KBof cells. The control group irradiated (red) were 200 μg·mL Figure 5. Photokilling effects N-TiO (green) and(black) N-TiO2and -NHlight with thegroup concentration of 2 -FA 2 (blue) −1 on A549 and KB cells. The control group (black) and light irradiated group (red) were 200 μg·mL also shown foron comparison. Data are The expressed mean(black) ± SD (nand = 4).light irradiated group (red) were 200 µg¨ mL´1 A549 and KB cells. controlasgroup also shown shown for for comparison. comparison. Data Data are are expressed expressed as as mean mean ˘ ± SD also SD (n (n ==4). 4).

3. Discussion 3. Discussion 3. Discussion Wang et al. [25] evaluated the effectiveness of nano-TiO2-based PDT with UV light on glioma. Wang et al. [25] evaluated the effectiveness of nano-TiO2-based PDT with UV light on glioma. TheyWang conducted theevaluated experiment on female BALB/c-nude mice with the treatment solutions et al. [25] the effectiveness of nano-TiO2 -based PDT with UV light on glioma. They conducted the experiment on female BALB/c-nude mice with the treatment solutions −1 TiO2 and the dosage of TiO2 in mice was about 10 mg·kg−1. You et al. [26] containing 200 μg·mL They conducted the experiment on female BALB/c-nude mice with the treatment solutions containing −1. You et al. [26] containing ´200 μg·mL−1 TiO 2 and the dosage of TiO2 in mice was about 10 in mg·kg 1 TiO 2 with dosage vitro mg·kg−1 in conducted therapy using TiO 200 µg¨ mLsonodynamic dosage of TiO micethe was aboutof 10100 mg¨μg·mL kg´1 .−1−1You et al.and [26]5 conducted 2 and the 2 in 2 with the dosage of 100 μg·mL in vitro and 5 mg·kg−1be in conducted sonodynamic therapy using TiO −1 ´ 1 ´ mice. Considering these reports, solution concentration 200 μg·mL in this study sonodynamic therapy using TiO2 the with the dosage of 100 µg¨of mL in vitro−1and 5 mg¨ kg 1could in mice. mice. Considering these reports, the solution concentration of 200 μg·mL in this study could be therapeutic vivoreports, and the dosage concentration of NPs should be µg¨ decided to could the weight of the Considering in these the solution of 200 mL´1according in this study be therapeutic therapeutic in vivo and the dosage of NPs should be decided according to the weight of the experiment animals. in vivo and the dosage of NPs should be decided according to the weight of the experiment animals. experiment animals. Compared much more more common common gold gold nanorods nanorods [27], [27], the the cetyltrimethylammonium cetyltrimethylammonium Compared with with the the much Compared with theinitial muchstabilizer more common gold nanorods [27],inevitable the cetyltrimethylammonium bromide (CTAB) as the of gold nanorods exhibits toxicity in in biological biological bromide (CTAB) as the initial stabilizer of gold nanorods exhibits inevitable toxicity bromide (CTAB) as the initial stabilizer of gold nanorods exhibits inevitable toxicity in issues [28]. Additionally, the nitrogen-doped TiO2 particles can be prepared fruitfully by biological an easily issues [28]. Additionally, the nitrogen-doped TiO2 particles can be prepared an easily operative method with much lower cost. Moreover, the nitrogen-doped TiO2fruitfully particles by possess the operative method with much lower cost. Moreover, the nitrogen-doped TiO2 particles possess the

Nanomaterials 2016, 6, 113

6 of 9

issues [28]. Additionally, the nitrogen-doped TiO2 particles can be prepared fruitfully by an easily operative method with much lower cost. Moreover, the nitrogen-doped TiO2 particles possess the photosensitizing properties in visible light, while gold nanorods were often used as the carrier of the tradition photosensitizers [29,30]. Therefore, nitrogen-doped TiO2 nanoparticles were of high value in therapy applications. 4. Materials and Methods 4.1. Materials Anatase TiO2 NPs (Sigma-Aldrich Inc., St. Louis, MO, USA) with a nominal diameter less than 15 nm were used in this study. An activated silane coupling agent 3-aminopropyltriethoxysilane (APTES, 99%, Aladdin Inc., Shanghai, China) was used for positive charge modification of TiO2 NPs. Other chemical agents involved in positive charge modification include Dimethylformamide (DMF, 98%, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), 2-(9H-fluoren-9-ylmethoxycarbonylamino)oxyacetic acid (Fmoc-Aoa, Chem-Impex International, Inc., Wood Dale, IL, USA), N,N-Diisopropylethylamine (DIPEA, 99.5%, Sigma-Aldrich Inc., St. Louis, MO, USA), (Benzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP, 98%, EMD Chemicals, Inc., Gibbstown, NJ, USA), piperidine (ě99.5%, Sigma-Aldrich Inc., St. Louis, MO, USA), and ammonium solution (25%–28%, Tongsheng Inc., Yixing, China). The chemical agents used in the conjugation of folic acid on TiO2 NPs were folic acid (FA, ě97%, Sigma-Aldrich), dimethyl sulfoxide (DMSO, ě99.7%, Sigma-Aldrich), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 99%, Sigma-Aldrich), and N-Hydroxysuccinimide (NHS, 99%, Sigma-Aldrich). 4.2. Preparation of N-TiO2 -FA The anatase TiO2 NPs were first calcinated in ammonia atmosphere to obtain nitrogen-doping TiO2 (N-TiO2 ) NPs as described in our previous work [20]. The N-TiO2 nanoparticles were modified with APTES to obtain positively charged N-TiO2 -NH2 NPs following the procedure in our previous work [24]. In brief, 0.7 mL APTES, 0.94 mg Fmoc-Aoa (1 mM), 3.122 mg PyBOP (2 mM) and 1 µL DIPEA (0.5 mM) were dispersed in 2.3 mL DMF, followed by stirring for 12 h at room temperature. Then 0.7 mg TiO2 , 0.7 mL ammonia water and 3.3 mL DMF were added into the stirred solution. The produced suspension was sonicated for 20 min and stirred for another 24 h before adding 70 µL redistilled piperidine. The positively charged N-TiO2 -NH2 was obtained by stirring the reaction solution for 12 h. Subsequently, FA (0.011 mM), EDC (0.003 mM) and NHS (0.003 mM) with a mole ratio of 10:3:3 were placed in a flask and dissolved in 20 mL DMSO to prepare NHS-FA, and then 1 mg N-TiO2 -NH2 was dispersed in it, followed by an ultrasonic dispersion for 15 min. The mixture solution was stirred to react at room temperature for 12 h. The N-TiO2 -FA sample was separated from the reaction mixture by centrifugation at 8000 rpm for 30 min and washed by DMSO once and by deionized water twice to remove the excess FA, EDC and NHS. 4.3. Characterization of Samples The Zeta potentials of N-TiO2 -FA, N-TiO2 -NH2 , as well as bare TiO2 NPs dissolved in deionized water with the concentration of 50 µg¨ mL´1 were measured by Zetasizer Nano ZS90 (Malvern Instruments; Worcestershire; UK) at 25 ˝ C and pH = 7. Absorption spectra of the samples were measured using a spectrometer (Shimadzu, UV3101pc; Japan). Thermogravimetric analysis (TGA) for the air-dried N-TiO2 , N-TiO2 -NH2 and N-TiO2 -FA NPs was performed on a SDT Q600 (TA Instrument, New Castle, DE, USA) from 20 to 800 ˝ C at a heating rate of 10 ˝ C¨ min´1 under N2 flow. 4.4. Cell Culture KB and A549 cells were cultured in RPMI-1640 medium (Roswell Park Memorial Institute 1640, Gibco, New York, NY, USA) with 10% (v/v) fetal bovine serum (Sijiqing Inc., Zhejiang, China) in

Nanomaterials 2016, 6, 113

7 of 9

a humidified standard incubator with a CO2 atmosphere at 37 ˝ C. The cells were seeded in Petri dishes or 96-well plates according to experimental needs. All experiments were performed after the cells have reached 80% confluence with normal morphology. 4.5. Cellular Uptake Study of N-TiO2 -FA KB or A549 cells in Petri dishes were incubated with RPMI-1640 medium containing 50 µg¨ mL´1 TiO2 -FA for 8, 16, 24, 32, or 40 min. Then, the medium containing surplus TiO2 NPs was removed and changed to fresh culture medium. For comparison, a dish of KB cells and a dish of A549 cells were treated with 500 µg¨ mL´1 FA in RPMI-1640 medium for 2 h before being treated with N-TiO2 -FA NPs by the same method. Cells in each experiment were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) solution for 10 min and were washed three times using PBS before observation by a laser scanning confocal microscope (LSCM, Olympus, FV300/IX 71, Tokyo, Japan), which is equipped with a 488 nm Ar+ continuous laser excitation (Coherent, Santa Clara, CA, USA) and a water-dipping objective (60ˆ, NA = 1.2). The cellular uptake of N-TiO2 -FA and N-TiO2 NPs were observed by the LSCM. The reflection images of the TiO2 NPs were obtained through a channel of the microscope without filter. The differential interference contrast (DIC) micrographs were observed simultaneously in a transmission channel to demonstrate the cell morphology and identify the borders of the cells. The quantitative study of N-TiO2 -FA NPs was analyzed using an open source software ImageJ to evaluate the reflection intensity of NPs inside individual cells. At least 50 cells were analyzed for each condition. 4.6. Measurements of Photokilling Effect and Cytotoxicity KB or A549 cells grown in 96-well plates (1 ˆ 105 cells per well) were incubated with TiO2 -FA NPs dispersed in RPMI-1640 medium with different concentrations between 50 and 200 µg¨ mL´1 for 1 h in the dark. Then the medium was removed and replaced by fresh culture medium. To study the photokilling effect, the cells were irradiated by an 18-W tungsten halogen lamp. Two pieces of quartz lens were used to obtain a concentrated parallel light beam and two filters were used to obtain a light beam of 400–575 nm. The visible-light illumination dose for cells was 190 J¨ cm´2 . After irradiation, the cells were incubated in the dark for 24 h before further study. The cytotoxicity examinations were carried out with the same procedure as the photokilling effect but without irradiation. The cell viability assays were conducted by a modified MTT method using WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H tetrazolium, monosodium salt, Beyotime, Jiangsu, China). Each well containing 100 µL culture medium was added with 10 µL of WST-8. The cells were incubated at 37 ˝ C with 5% CO2 for 2 h and the absorbance was measured at 450 nm using a microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). To diminish the influence of the NPs, the absorbance values at 450 nm before adding WST-8 dyes were also measured as a reference. Cells incubated in RPMI-1640 medium without any treatment were used as the control group. Each experiment was conducted and measured independently for at least three times. 5. Conclusions In this study, we have successfully produced N-TiO2 -FA nanoconjugates. Zeta potential results showed that N-TiO2 -FA had a negative Zeta potential and possessed good dispersibility. The absorption spectra and TGA result showed that FA was connected to the surface of N-TiO2 NPs. The incubation experiment results showed that N-TiO2 -FA had higher uptake efficiency in FR-positive KB cells than FR-negative A549 cells, as well as the FA-pretreated cells. Finally, N-TiO2 -FA had no obvious cytotoxicity, but had a significant photokilling effect on FR-positive KB cells with visible light illumination. Therefore, N-TiO2 -FA can be of high value as a PS for cancer targeting PDT.

Nanomaterials 2016, 6, 113

8 of 9

Acknowledgments: The authors are grateful to the support of National Natural Science Foundation of China (11574056, 31500599, 61505032). Author Contributions: Jin Xie performed the experiments and drafted the manuscript; Xiaobo Pan and Mengyan Wang contributed by assisting in experimental setup; Longfang Yao and Xinyue Liang contributed the cell samples preparation; Jiong Ma, Yiyan Fei and Pei-Nan Wang contributed instruction of data collection and interpretation; Lan Mi designed the experiments, contributed as the advisor to the research, and revised and edited the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

References 1. 2. 3. 4.

5.

6. 7. 8.

9.

10. 11.

12. 13. 14.

15.

16. 17.

Dougherty, T.J.; Gomer, C.J.; Henderson, B.W.; Jori, G.; Kessel, D.; Korbelik, M.; Moan, J.; Peng, Q. Photodynamic therapy. J. Natl. Cancer Inst. 1998, 90, 889–905. [CrossRef] [PubMed] Dolmans, D.E.; Fukumura, D.; Jain, R.K. Photodynamic therapy for cancer. Nat. Rev. Cancer 2003, 3, 380–387. [CrossRef] [PubMed] Kamkaew, A.; Lim, S.H.; Lee, H.B.; Kiew, L.V.; Chung, L.Y.; Burgess, K.; Burgess, K. BODIPY dyes in photodynamic therapy. Chem. Soc. Rev. 2013, 42, 77–88. [CrossRef] [PubMed] Lin, J.; Wang, S.J.; Huang, P.; Wang, Z.; Chen, S.H.; Niu, G.; Li, W.W.; He, J.; Cui, D.X.; Lu, G.M. Photosensitizer-loaded gold vesicles with strong plasmonic coupling effect for imaging-guided photothermal/photodynamic therapy. ACS Nano 2013, 7, 5320–5329. [CrossRef] [PubMed] Pelaez, M.; Nolan, N.T.; Pillai, S.C.; Seery, M.K.; Falaras, P.; Kontos, A.G.; Dunlop, P.S.M.; Hamilton, J.W.J.; Byrne, J.A.; O’shea, K. A review on the visible light active titanium dioxide photocatalysts for environmental applications. Appl. Catal. B 2012, 125, 331–349. [CrossRef] Shi, H.; Magaye, R.; Castranova, V.; Zhao, J.S. Titanium dioxide nanoparticles: A review of current toxicological data. Part. Fibre Toxicol. 2013, 10. [CrossRef] [PubMed] Bai, Y.; Mora-Seró, I.; De Angelis, F.; Bisquert, J.; Wang, P. Titanium dioxide nanomaterials for photovoltaic applications. Chem. Rev. 2014, 114, 10095–10130. [CrossRef] [PubMed] Ma, W.; Yi, S.G.; Lin, L.W.; Huang, H.; Peng, T.; Liu, Z.T.; Liu, Z.P.; Pei, D.N.; Miao, X.Y.; Wen, Y. Anticancer Effect of Photodynamic Therapy with Photosan-Loaded Titanium Dioxide Nanoparticles on Panc-1 Pancreatic Cancer Cells In Vitro. Sci. Adv. Mat. 2016, 8, 1145–1153. [CrossRef] Zhang, S.C.; Yang, D.J.; Jing, D.W.; Liu, H.W.; Liu, L.; Jia, Y.; Gao, M.H.; Guo, L.J.; Huo, Z.Y. Enhanced photodynamic therapy of mixed phase TiO2 (B)/anatase nanofibers for killing of HeLa cells. Nano Res. 2014, 7, 1659–1669. [CrossRef] Yin, Z.F.; Wu, L.; Yang, H.G.; Su, Y.H. Recent progress in biomedical applications of titanium dioxide. Phys. Chem. Chem. Phys. 2013, 15, 4844–4858. [CrossRef] [PubMed] Pan, X.; Xie, J.; Li, Z.; Chen, M.; Wang, P.N.; Chen, L.; Mi, L. Enhancement of the photokilling effect of aluminum phthalocyanine in photodynamic therapy by conjugating with nitrogen-doped TiO2 nanoparticles. Colloids Surf. B 2015, 130, 292–298. [CrossRef] [PubMed] Tokuoka, Y.; Yamada, M.; Kawashima, N.; Miyasaka, T.; Miyasaka, T. Anticancer effect of dye-sensitized TiO2 nanocrystals by polychromatic visible light irradiation. Chem. Lett. 2006, 35, 496–497. [CrossRef] Li, Z.; Pan, X.; Wang, T.; Wang, P.N.; Chen, J.Y.; Mi, L. Comparison of the killing effects between nitrogen-doped and pure TiO2 on HeLa cells with visible light irradiation. Nanoscale Res. Lett. 2013, 8. [CrossRef] [PubMed] Janczyk, ´ A.; Wolnicka-Głubisz, A.; Urbanska, K.; Stochel, G.; Macyk, W. Photocytotoxicity of platinum (IV)-chloride surface modified TiO2 irradiated with visible light against murine macrophages. J. Photochem. Photobiol. B 2008, 92, 54–58. [CrossRef] [PubMed] Parker, N.; Turk, M.J.; Westrick, E.; Lewis, J.D.; Low, P.S.; Leamon, C.P. Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal. Biochem. 2005, 338, 284–293. [CrossRef] [PubMed] Hilgenbrink, A.R.; Low, P.S. Folate receptor-mediated drug targeting: From therapeutics to diagnostics. J. Pharm. Sci. 2005, 94, 2135–2146. [CrossRef] [PubMed] Hu, Z.; Li, J.; Li, C.; Zhao, S.; Li, N.; Wang, Y.; Wei, F.; Chen, L.; Huang, Y. Folic acid-conjugated graphene–ZnO nanohybrid for targeting photodynamic therapy under visible light irradiation. J. Mater. Chem. B 2013, 1, 5003–5013. [CrossRef]

Nanomaterials 2016, 6, 113

18.

19. 20. 21. 22. 23. 24. 25. 26.

27.

28. 29. 30.

9 of 9

Huang, P.; Bao, L.; Zhang, C.; Lin, J.; Luo, T.; Yang, D.; He, M.; Li, Z.; Gao, G.; Gao, B. Folic acid-conjugated silica-modified gold nanorods for X-ray/CT imaging-guided dual-mode radiation and photo-thermal therapy. Biomaterials 2011, 32, 9796–9809. [CrossRef] [PubMed] Lai, T.Y.; Lee, W.C. Killing of cancer cell line by photoexcitation of folic acid-modified titanium dioxide nanoparticles. J. Photochem. Photobiol. A 2009, 204, 148–153. [CrossRef] Li, Z.; Mi, L.; Wang, P.N.; Chen, J. Study on the visible-light-induced photokilling effect of nitrogen-doped TiO2 nanoparticles on cancer cells. Nanoscale Res. Lett. 2011, 6, 1–7. [CrossRef] [PubMed] Ye, L.; Pelton, R.; Brook, M.A. Biotinylation of TiO2 nanoparticles and their conjugation with streptavidin. Langmuir 2007, 23, 5630–5637. [CrossRef] [PubMed] Hermanson, G. Bioconjugate Techniques; Acad Press: San Diego, CA, USA; New York, NY, USA; Boston, MA, USA, 1996; pp. 173–177. Yoo, H.S.; Park, T.G. Folate-receptor-targeted delivery of doxorubicin nano-aggregates stabilized by doxorubicin–PEG–folate conjugate. J. Control. Release 2004, 100, 247–256. [CrossRef] [PubMed] Xie, J.; Pan, X.; Wang, M.; Ma, J.; Fei, Y.; Wang, P.N.; Mi, L. The role of surface modification for TiO2 nanoparticles in cancer cells. Colloids Surf. B 2016, 143, 148–155. [CrossRef] [PubMed] Wang, C.; Cao, S.; Tie, X.; Qiu, B.; Wu, A.; Zheng, Z. Induction of cytotoxicity by photoexcitation of TiO2 can prolong survival in glioma-bearing mice. Mol. Biol. Rep. 2011, 38, 523–530. [CrossRef] [PubMed] You, D.G.; Deepagan, V.G.; Um, W.; Jeon, S.; Son, S.; Chang, H.; Yoon, H.I.; Cho, Y.W.; Swierczewska, M.; Lee, S.; et al. ROS-generating TiO2 nanoparticles for non-invasive sonodynamic therapy of cancer. Sci. Rep. 2016, 6. [CrossRef] [PubMed] Wang, N.; Zhao, Z.; Lv, Y.; Fan, H.; Bai, H.; Meng, H.; Long, Y.; Fu, T.; Zhang, X.; Tan, W. Gold nanorod-photosensitizer conjugate with extracellular pH-driven tumor targeting ability for photothermal/photodynamic therapy. Nano Res. 2014, 7, 1291–1301. [CrossRef] Khlebtsov, N.; Dykman, L. Biodistribution and toxicity of engineered gold nanoparticles: A review of in vitro and in vivo studies. Chem. Soc. Rev. 2011, 40, 1647–1671. [CrossRef] [PubMed] Jang, B.; Choi, Y. Photosensitizer-conjugated gold nanorods for enzyme-activatable fluorescence imaging and photodynamic therapy. Theranostics 2012, 2, 190–197. [CrossRef] [PubMed] Jang, B.; Park, J.Y.; Tung, C.H.; Kim, I.H.; Choi, Y. Gold nanorod´photosensitizer complex for near-infrared fluorescence imaging and photodynamic/photothermal therapy in vivo. ACS Nano 2011, 5, 1086–1094. [CrossRef] [PubMed] © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).