Enhanced Photogeneration of Reactive Oxygen Species and Targeted

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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 19483−19493

Enhanced Photogeneration of Reactive Oxygen Species and Targeted Photothermal Therapy of C6 Glioma Brain Cancer Cells by Folate-Conjugated Gold−Photoactive Polymer Nanoparticles Jaber Keyvan Rad,† Ali Reza Mahdavian,*,† Samideh Khoei,‡ and Sakine Shirvalilou‡ †

Polymer Science Department, Iran Polymer & Petrochemical Institute, P.O. Box 14965/115, Tehran 1497713115, Iran Medical Physics Department, School of Medicine, Iran University of Medical Sciences, Tehran 1449614525, Iran

‡

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S Supporting Information *

ABSTRACT: Tumor-selective photodynamic therapy is a successful method for ablation of malignant and cancerous cells. Herein, we introduce the design and preparation of functionalized acrylic copolymer nanoparticles with spiropyran (SP) and imidazole groups through a facile semicontinuous emulsion polymerization. Then, Au3+ ions were immobilized and reduced on their surface to obtain photoresponsive gold-decorated polymer nanoparticles (PGPNPs). The prepared PGPNPs were surface-modified with folic acid as a site-specific tumor cell targeting agent and improve intracellular uptake via endocytosis. Fourier transform infrared spectroscopy and energy dispersive X-ray spectroscopy analyses, UV−vis spectroscopy, scanning electron microscopy, and high-resolution transmission electron microscopy images were employed to characterize their spectral and morphological properties. Fluorescence microscopy images and inductively coupled plasma analysis demonstrated the cell line labeling capability and improved targeting efficiency of folate-conjugated PGPNPs (FA-PGPNPs) toward rat brain cancer cells (C6 glioma) with 71.8% cell uptake in comparison with 28.8% for the nonconjugated ones. Nonpolar SP groups are converted to zwitterionic merocyanine isomers under UV irradiation at 365 nm and their conjugation with Au nanoparticles exhibited enhanced photogeneration of reactive oxygen species (ROS). These were confirmed by intracellular ROS analysis and cytotoxicity evaluation on malignant C6 glioma cells. Owing to the strong surface plasmon resonance absorption of gold nanoparticles, FAPGPNPs provided elevated local photothermal efficiency under near-IR irradiation at 808 nm. The prepared multifunctional FAPGPNPs with a comprehensive integration of prospective materials introduced promising nanoprobes with targeting ability, enhanced tumor photodynamic therapy, cell tracking, and photothermal therapy. KEYWORDS: gold nanoparticles, spiropyran-acrylic, folic acid, cell targeting, photodynamic, photothermal therapy

1. INTRODUCTION The unique properties and tremendous progress in nanotechnology have drawn much attention, in many research fields for versatile applications including biology, optical device, and catalysis.1,2 Hybrid nanocomposites, owing to the possibility of combining the diverse physical, chemical, and functionalities of different individual components, show greater potential advantages over single component nanoparticles (NPs).3,4 Photochromic spiropyran (SP) upon external stimuli such as light or heat undergoes reversible isomerization between a three-dimensional inert, nonfluorescent, closed ring colorless SP form and a planar, zwitterionic, open ring, fluorescent, and colored merocyanine (MC) form.5−7 The reversible changes in physical and chemical properties of these molecular species, especially high brightness and photostability compared with conventional organic dyes have led to a great interest for the development of sensing, tracking, and labeling nanoscale devices with light.8−11 A deficiency for employment of SP− MC photoisomerization in biological systems originates from poor penetration of UV light into tissues. It has been shown © 2018 American Chemical Society

that near-infrared (NIR) two-photon excitation at 780 nm with deeper penetration into tissues converts SP to MC and addresses to the aforementioned problem.12,13 The versatile and tunable photoswitching of SP groups upon external stimulants (e.g., UV or visible light and heat) depends critically on the surrounding medium and some properties such as flexibility, polarity, molecular packing, charge, pH, orientation, and their mutual interactions.7,14−16 In polar medium like water, open-ring MC is the preferred form and results in negative photochromism.17 Therefore, subsequent relatively rapid hydrolysis of MC in physiological conditions will significantly limit its exploitation in the aqueous media.18 Giordani and co-workers studied the effect of SP cytotoxicity on three different cellular models and they showed its non-toxic effect at low concentrations.19 Nilsson et al. investigated biological activity of SP and their results demonstrated that the Received: April 1, 2018 Accepted: May 22, 2018 Published: May 22, 2018 19483

DOI: 10.1021/acsami.8b05252 ACS Appl. Mater. Interfaces 2018, 10, 19483−19493

Research Article

ACS Applied Materials & Interfaces

surface of tumor cells. Kang et al.51 studied the effect of folatefunctionalized polyamidoamine (PAMAM) dendrimers for the delivery of antisense oligonucleotides (ASODNs, as an inhibitor for the cell growth) to rat C6 glioma cells. They found that folate-functionalized PAMAM dendrimers containing ASODNs show more cell uptake (97.36%) in comparison with the nonfolate ones (94.10%) and ASODNs alone (0.73%). In another study, the cytotoxic effect of folate-functionalized silica NPs loaded with curcumin was investigated on prostate cancer cells line (PC3) and normal cells line (PrEC, prostate epithelial cell).52 Cytotoxicity experiments demonstrated that folate-functionalized NPs were specifically effective for targeting and killing of PC3 cells, while minor cytotoxicity to PrEC cells was observed. To the best of our knowledge, there is no report on the use of photochromic species like SP as the photosensitizer or amplifier for ROS generation in photodynamic therapy. Here and for the first time, SP−MC photoisomerization under UV irradiation is introduced as a probe for the enhanced photodynamic therapy by using of photoresponsive golddecorated polymer NPs and their capability in the elevated ROS generation will be investigated. To improve the efficacy of therapeutic means by targeting and selective internalization to cancerous cells, the obtained photoresponsive gold-decorated polymer NPs (PGPNPs) were functionalized with FA (FAPGPNPs) through L-cysteine linkages. These multifunctional NPs were assessed for cell viability by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Then, they were triggered by UV irradiation at 365 nm to study their therapeutic role for rat brain cancer cells (C6 glioma) because of the enhanced photogeneration of ROS and even for their labeling due to the fluorescence emission of the generated MC isomers. However, the applicability of these NPs in PTT by illumination with continuous-wave NIR laser (808 nm), with respect to the presence of several active species in FA-PGPNPs, was investigated and the results revealed their targeting and multidisciplinary advanced potentiality in such NPs.

nonpolar SP form has good cell membrane penetrating characteristic without cytotoxicity.20 In contrast, the MC form reveals poor passing through the cells membrane (because of its zwitterionic nature) and high cytotoxic effect, which is attributed to the intermolecular interactions and/or generation of reactive oxygen species (ROS) in the cytoplasm. Unique properties of noble metal NPs and especially gold NPs (AuNPs), including the surface plasmon resonance (SPR), efficient light-to-heat conversion, ease of synthesis, functionalization, chemically inertness, biocompatibility, and their biological applications like photodynamic therapy, have drawn much attention in recent years.21−24 Besides, photothermal therapy (PTT) is an alternative method for exploitation of AuNPs, which can provide high temperatures around NPs under visible to NIR laser irradiation and cause thermal ablation of cancer cells with their consequent death.25,26 The strong SPR band is the result of interaction between electromagnetic fields and conduction band electrons in noble metals and semiconductors that involves collective oscillation of the conduction electrons on the NPs surface.27 The SPR band could be tunable by controlling the NP size and shape, aggregation, refractive index, and polarity of the surrounding medium.28,29 It has been reported that the overlap between dipolar SPR band of metal NPs and absorption band of dyes may result in strong coupling and greatly improves their absorption coefficient by localized electric field, and thus, singlet oxygen photogeneration is remarkably enhanced.30−32 The MC isomer is susceptible to triplet-singlet intersystem crossing (ISC) process because of its zwitterionic character.33 Therefore, AuNPs−MC interactions can lead to plasmonenhanced fluorescence and also may show an improved ability to produce cytotoxic ROS because of the efficient ISC.3,34,35 Photodynamic therapy (PDT) is known as a minimally invasive and important cancer therapy technique which incorporates photosensitizers to absorb and transfer visible light energy to the nearest molecular oxygen.36−40 Incomplete reduction of oxygen generates ROS (oxidative radicals) to jeopardize cancer cells viability and treats various diseases. ROS are toxic and would destroy malignant tissue or cancer cells, and the photoinduced tumor destruction efficiency depends on the localization and concentration of photosensitizer in tumor cells, type of photosensitizer, and time of irradiation.39,41,42 Li et al. prepared silica-coated gold nanorods accompanied by indocyanine green (ICG) and investigated their potent for singlet oxygen generation.32 They found that the overlap between exciton absorption of ICG and SPR of Au nanorods greatly enhanced ROS generation by the localized electric field. The extent of internalization of gold−polymer nanocomposite particles into the normal cells and tumor cells are both identical. To achieve effective intracellular uptake at a tumor site and increase the residence time for a therapeutic purpose, active targeting agents must be employed.43,44 Folic acid (FA) is known as one of the promising active targeting agents for cancer cells and is transferred into the cells cytosol through folate-receptors binding sites on the surface of cell lines.44−47 Overexpression of folate-receptor on cancerous tissues (e.g., cancers of breast, ovaries, endometrium, kidneys, colon, brain, and myeloid cells of hematopoietic origin) provides a unique opportunity to make FA as a suitable targeting agent to deliver hybrid gold−polymer NPs into such sites.48−50 Also, stability, inexpensiveness, and nonimmunogenic nature are other reasons to use FA as a susceptible targeting agent because of its high affinity in binding to the

2. EXPERIMENTAL SECTION 2.1. Materials. 2,3,3-Trimethylindolenine, L-cysteine, N-vinylimidazole (N-VIM), gold(III) chloride trihydrate (HAuCl4·3H2O), cetyltrimethylammonium bromide (CTAB as a cationic surfactant and shape-directing agent for AuNPs), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), N,N′-dicyclohexyl carbodiimide (DCC), 4(dimethylamino)pyridine (DMAP), sodium borohydride (NaBH4), ethylenediamine tetraacetic acid, and MTT were purchased from Sigma-Aldrich. 2,2-Azobis[2-(2-imidazoline-2-yl)propane]dihydrochloride (VA-044) (as initiator) from Wako Pure Chemical Industries Ltd. (Osaka, Japan) was used. All of the solvents, 2-hydroxy5-nitrobenzaldehyde, methyl methacrylate (MMA), hydrochloric acid (HCl, 37%), triethylamine, 2-bromoethanol, acryloyl chloride, ammonia solution (25%), sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, dichloromethane (DCM), tetrahydrofuran (THF), and dimethyl sulfoxide (DMSO) were supplied by Merck Chemical Co. THF, DMSO, and DCM were dried over sodium, calcium chloride, and molecular sieve, respectively, and distilled off before use. Other solvents and all reagents were used without further purification. Deionized (DI) water was used in the polymerization recipe. 2.2. Preparation of Photoresponsive NPs Containing N-VIM (PNPsIm). To prepare photoresponsive functionalized polymer NPs with N-VIM, SP ethyl acrylate (SPEA, as the photochromic monomer) was synthesized primarily according to our previously reported procedure.53 In an emulsion polymerization recipe, 35 mL of aqueous solution of 150 mg of CTAB and 30 mg of VA-044 was prepared and 19484


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Figure 1. Synthesis of FA-Lcys. allowed to adhere overnight at 37 °C in 5% CO2. The medium was then replaced with the same volume of Ham’s F-12 containing different concentrations (0−10 mg/mL) of PGPNPs and FA-PGPNPs and afterward, cytotoxicity was assessed in 2 and 24 h incubation at 37 °C for 4 h in the dark after addition of PGPNPs and FA-PGPNPs. Then, the cells were washed twice with PBS, and 100 μL of MTT (5 mg/mL in PBS) was added to each well and the plates were incubated at 37 °C for 4 h in the dark. The medium was removed and 200 μL of DMSO was added to dissolve the blue formazan crystals for 20 min. The absorbance was read on an ELISA Reader at 540 nm. The relative cell viability was calculated by dividing the mean optical density value of the control group, and the average value was obtained from six parallel samples. The effect of UV irradiation was investigated after treating of C6 glioma cells with the aforementioned NPs in a range of concentrations from 0 to 10 mg/mL for 2 and 24 h at 37 °C in 5% CO2 and then exposing them to UV lamp (365 nm) with a 10 cm distance for 3 min. Immediately after UV irradiation and illumination with green light (532 nm) for 90 s, the cell viability was measured using the technique mentioned before. All experiments were performed in triplicate. 2.6. Cell Uptake of PGPNPs and FA-PGPNPs. Six-well plates were seeded with 1 × 105 C6 glioma cell line per well and incubated for 24 h at 37 °C. On the basis of the in vitro cytotoxicity results, the cells were then exposed to a dispersion of 0.01 mg/mL of the prepared samples (PGPNPs and FA-PGPNPs) for 2 h. After loading, the culture medium was removed by washing with PBS for five times to remove nonabsorbed NPs. The cells were trypsinized and centrifuged at 10 000 rpm for 5 min and then the supernatant was removed and 0.5 mL of aqua regia [1:3 (v/v) HNO3/HCl] was added to dissolve AuNPs of the internalized NPs into the cells. The concentration of uptaken and dissolved Au3+ per well was determined and averaged over the three replicates by inductively coupled plasma−optical emission spectrometry (ICP−OES) analysis. For fluorescence cellular imaging, 5 × 104 C6 cells/well were cultured into 6-well plates and 0.01 mg of NPs was added to the medium during the first 24 h, and then, C6 cells were washed twice with PBS, fixed with formaldehyde, and finally used for fluorescence imaging. 2.7. Intracellular ROS Detection. ROS amounts were evaluated by a fluorescent probe (DCFH) to detect ROS generation rate as a commonly employed fluorescent detection method, and this fluorescent probe was obtained by activation of DCFH-DA.34 C6 cells were cultured in Ham’s F-12 medium (10% FBS), trypsinized, counted, and re-suspended in fresh complete media at a density of 3 × 104 cells/mL. Next, they were seeded into 24-well plates and incubated for 24 h at 37 °C, and then were treated with 0.01 mg/mL of the PGPNPs and FA-PGPNPs in PBS. After a 2 h incubation period, the cells were loaded with an ROS detector probe by replacing the culture media with PBS, containing 5 μM DCFH-DA for 30 min at 37 °C in dark, and nonincorporated DCFH-DA was removed through washing with PBS three times. Afterward, half of the wells were exposed to UV irradiation (365 nm, 12 W) at a distance of 10 cm for 3 min and then illuminated with a green light at 532 nm for 90 s. Fluorescence emissions at 530 nm (excitation at 485 nm) were measured at 1 min intervals for a total of 5 min. In each analysis, two types of control sample were used: negative control (non-UV irradiated cells) and positive control (UV-irradiated cells at 365 nm). 2.8. Laser Irradiation and Photothermal Study of PGPNPs and FA-PGPNPs. Photothermal properties of the prepared PGPNPs

the solution pH was set at 4. Then, it was poured into a three-necked round-bottom reactor, stirred, and deaerated with nitrogen gas. The solution temperature reached to 50 °C and the polymerization was started by adding monomers simultaneously; that is, 100 mg of SPEA in 10 mL of DI water and 2.8 mL of MMA from two separate dropping funnels into the reactor within 15 min. The emulsion polymerization continued at 50 °C for 45 min until the conversion reached to above 50%. Next, 0.1 mL of N-VIM and 0.8 mL of MMA were mixed and added in three portions within 5 min and the polymerization continued at 50 °C for 3 h until 95% monomer conversion was attained with coagulation below 1 wt %. 2.3. Preparation of PGPNPs. Deposition of Au on the surface of photoresponsive acrylic NPs functionalized with N-VIM was carried out by a metal−ligand formation approach. Aqueous stock solution (65 mL) of HAuCl4·3H2O (3 g/L) as the source of Au3+ was added to 35 mL of the preformed PNPsIm latex with stirring overnight at room temperature. The chelated Au3+ ions to N-VIM on the surface of functionalized latex NPs were reduced by addition of 23 mL NaBH4 stock solution (1 g/L) droopingly (over vigorously stirring within a 30 min period) and continued for 1 h at room temperature. The color of the reaction mixture turned from yellow to bright purple, indicating the formation of AuNPs during the progress of Au3+ reduction. 2.4. Preparation of Conjugated FA with PGPNPs (FAPGPNPs). To modify PGPNPs with FA, L-cysteine was selected as a proper interface. The process of conjugation of γ-carboxylic acid group of FA with amine group in L-cysteine has been shown schematically in Figure 1. FA (250 mg, 0.57 mmol) and 18 mg of DMAP (0.15 mmol) were dissolved in 30 mL of anhydrous DMSO by continuous stirring at room temperature for 60 min. Then, 118 mg of DCC (0.57 mmol) was dissolved in 10 mL of dry DCM and it was added to the above solution, and the mixture was vigorously stirred for another 60 min. Next, a solution of 69 mg of L-cysteine (0.57 mmol) in 10 mL of DCM was added to the above solution within 45 min dropwise. The stirring continued for 24 h at room temperature to obtain conjugated folate with L-cysteine. After 24 h, 100 mL of DCM was added to the reaction mixture, and it was centrifuged at 8000 rpm to remove unreacted compounds in the supernatant. For more purification, the precipitates were redispersed in DCM and centrifuged twice. Finally, the obtained FA-Lcys was dissolved (low solubility) in 40 mL of DI water, centrifuged (8000 rpm), and dried in vacuum oven at 40 °C for 24 h to give 235 mg of FA-Lcys in 76% yield. The prepared FA-Lcys (150 mg) was dissolved in 20 mL of DI water, and it was neutralized by addition of 1% (v/v) ammonia solution. The obtained FA-Lcys solution was poured dropwise into the previously prepared PGPNPs dispersion (30 mL) and the mixture was stirred at room temperature for 24 h. Then, the obtained mixture was introduced into the dialysis bag (Mw cut-off: 12 400 Da) and dialyzed against phosphate-buffered saline (PBS, pH = 7.4, 1 L). The external solution was replaced every 12 h during 60 h to eliminate excess surfactants, unreacted FA-Lcys, free AuNPs, and short polymeric chains. Finally, the purified FA-PGPNPs were obtained by freezedrying of the dialyzed product. 2.5. In Vitro Cytotoxicity Evaluation of PGPNPs and FAPGPNPs. The cytotoxicity of PGPNPs and FA-PGPNPs to C6 glioma cell line (rat brain cancer cell) was assessed by the MTT cell viability assay. In summary, C6 glioma cells were seeded into a 96-well microplate at a density of 5 × 104 cells/well in 100 μL of Ham’s F-12 medium, supplemented with 10% fetal bovine serum (FBS), and 19485


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Figure 2. Synthetic route for the preparation of FA-PGPNPs. and FA-PGPNPs were assessed by 808 nm continuous-wave NIR laser with a power density of 0.8 W/cm2 and a spot size of 2 cm2 (radius ≈ 0.8 cm). For this reason, C6 glioma cells were seeded into a 6-well microplate at a density of 1 × 106 cells/well in 2 mL of Ham’s F-12 medium, supplemented with 10% FBS for overnight. Then, the medium was replaced with the same volume of Ham’s F-12 containing (0.1 mg/mL) of PGPNPs and FA-PGPNPs. After 24 h, the cells were washed with PBS and trypsinized. Finally, the cell suspension was transferred into the microtubes and they were exposed to laser irradiation. During NIR irradiation, the temperature changes were monitored using an infrared camera (Testo, Germany) every 1 min for 14 min. 2.9. Characterization. Characterization of the synthesized products were carried out by a FT-IR BRUKER-IFS48 spectrophotometer (Germany), using KBr pellets. To confirm the attachment and determination of AuNPs amount and also conjugation of FA to PGPNPs, energy dispersive X-ray spectroscopy (EDX; INCA model, Oxford Instron, England) was used (analysis was done without coating of the samples with gold). Spectroscopic absorption properties of the nanocomposites were investigated by UV−vis analysis via a ShimadzuUV2550 UV−vis spectrophotometer (Japan). Size and morphology of the nanocomposite particles were studied by a scanning electron microscope (SEM), Tescan Vega II (Czech Republic). For SEM analysis, a drop of the diluted latex was placed on a sample holder and dried under vacuum at 25 °C. Then, they were put under vacuum, evacuated, and a layer of gold was deposited under flushing with argon by using EMITECH K450x sputter-coater (England). Particle size and size distribution was measured by using Malvern Zetasizer ZEN3600 dynamic light scattering (DLS, UK). AuNPs morphologies were detected using a Philips CM-30 high-resolution transmission electron microscope (HR-TEM; The Netherlands) at 200 kV. The samples for TEM analysis were prepared by adding dropwise the diluted aqueous dispersion of FA-PGPNPs onto a 400-mesh copper grid coated with carbon and then dried at 30 °C for 15 min. A UV lamp (365 nm), CAMAG 12VDC/VAC (50/60 Hz, 14VA, Switzerland) was used to stimulate changes in structure, nature, and absorption bands of the photochromic SP groups. The excitation light source for PDT was a 532 nm solid-state laser (MC2000) with a power density of 1.8 W/ cm2 and was employed as a green illumination source. Phase contrast bright-field and fluorescence images of the NPs-cell were taken with a fluorescence microscope with UV light (330−380 nm) (Olympus CK2; Olympus Optical Co., Tokyo, Japan). ROS were measured with the aid of a Synergy HT Microplate Reader (BioTek Instruments) at 37 °C, and the measurements were conducted using a 485 nm excitation and a 528 nm emission. ICP−OES simultaneous (Arcos

EOP model, Spectro Co., Germany) was employed to measure the content of uptaken Au3+ moieties (in PGPNPs and FA-PGPNPs) into the C6 glioma cells.

3. RESULTS AND DISCUSSION Emulsion polymerization is one of the promising methods for preparation of functionalized NPs. Here, the modified photoresponsive polymer NPs containing imidazole groups were prepared through semicontinuous emulsion polymerization in a way that these functionalities would be located in the outer layer of NPs (Figure 2). This will facilitate the adsorption of gold ions on the surface of these NPs to conduct nucleation and formation of AuNPs. Embodiment of SP moieties in the acrylic polymer matrix will result in their protection and obviates the concern about their degradation, release, and direct contact with biological systems and cells. Photochromic SP groups convert to the zwitterionic MC isomers by UV irradiation at 365 nm, which is suitable for ISC and consequent producing of ROS. Moreover, the attached AuNPs to the surface of polymer particles play a synergistic role to increase ROS by localized electric field, because of effective interaction between SPR band of AuNPs and dipolar zwitterion MC isomers. However, the presence of AuNPs will facilitate binding to FA for targeting toward cancerous cells because of the existence of folate receptors on these cells. The synthesis and performance of the obtained FA-PGPNPs will be discussed comprehensively in the following. 3.1. Synthesis and Characterization of PGPNPs and FA-PGPNPs. For conjugation of FA to the AuNPs, L-cysteine as a proper and biocompatible interface was employed. Lcysteine with terminal NH and SH groups is able to bridge between FA and AuNPs. First of all, L-cysteine was condensed with carboxylic acid groups in FA and the obtained amide product was centrifuged and redispersed in water and DCM (2 times in row) to remove unreacted ingredients; and the reaction was followed by Fourier transform infrared spectroscopy (FTIR; Figure S1, Supporting Information). The appearance of stretching vibrations of S−H at 2550 cm−1 and the broad peak of hydroxyl group of −COOH at 3340 cm−1 are good indications for the synthesis of FA-Lcys. 19486


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ACS Applied Materials & Interfaces Table 1. Weight Percent of the Elements in the Precursors and Products from EDX Analysisa sample L-cysteine

a

FA-Lcys

PGPNPs

element

Theo

Meas

Theo

Meas

C N O S Au

31.59 12.27 28.04 28.10

30.58 12.14 29.08 28.20

50.77 21.54 21.52 6.17

50.27 21.27 22.55 5.91

Theo

FA-PGPNPs Meas

Theo

Meas

1.08 2.52

41.09 23.84 32.01 0.94 2.12

41.62 22.12 33.13 3.98

3.13

“Theo” and “Meas” represent “theoretical” and “measured” quantities, respectively.

Figure 3. UV−vis analysis of the diluted PNPsIm (a), PGPNPs (b), and FA-PGPNPs (c) dispersions up to 0.2 wt % before ( UV irradiation at 365 nm. The inset picture in (a) shows color changes during SP to MC isomerization.

) and after (

)

in nitrogen contents in FA-Lcys relative to L-cysteine are in accordance with the theoretical values and imply the progress of reaction (Figure S3, Supporting Information). Appearance of gold element in the EDX spectrum for PGPNPs depicts the attachment of AuNPs onto the surface of modified polymer particles. The conjugation of FA-Lcys to PGPNPs was approved by the presence of 0.94 wt % S in FA-PGPNPs. By means of measuring the sulfur weight percent in FA-Lcys and FAPGPNPs, the amount of conjugated FA-Lcys to PGPNPs was estimated to about 15.91 wt %. The combination EDX distribution map of nitrogen and gold in the prepared PGPNPs sample has been represented in Figure S4 (Supporting Information). The combined map shows the presence of nitrogen (from N-VIM and SPEA) and gold from AuNPs and also well distribution of these atoms in the aforementioned nanocomposite particles. Photochromic and plasmonic properties of the functionalized photoresponsive NPs with SP, that is, PNPsIm, PGPNPs, and FA-PGPNPs dispersions, were investigated by UV−vis spec-

After the preparation of PGPNPs and FA-PGPNPs, they were dialyzed against buffer saline. The purified dispersions were freeze-dried and their FTIR spectra were recorded (Figure S2, Supporting Information). The presence of characteristic peaks of SPEA and PMMA approve the incorporation of employed photochromic dye into PGPNPs. The comparison between FTIR spectra of PGPNPs and FA-PGPNPs demonstrates that the appeared peaks at 3450 and 1600 cm−1 are related to the hydroxyl and imine groups in FA, respectively. Another approval for the preparation of FA-PGPNPs returns to the elimination of S−H vibration at 2550 cm−1 in FA-PGPNPs, which reveals the reaction between thiol groups and AuNPs. EDX analysis was employed to determine composition and the purity of FA-PGPNPs and also the estimation of the gold content in the prepared sample (Table 1). A comparison between theoretical and measured weight percents of sulfur and nitrogen elements in L-cysteine and FA-Lcys confirms the successful synthesis of FA-Lcys as the precursor for the next step. A decrease of 22.29 wt % in sulfur and 9.13 wt % increase 19487


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attached to the surface of primary photoresponsive polymer NPs. These morphological studies reveal that the exploited strategy in preparation of FA-PGPNPs has been accomplished successfully. 3.2. Cytotoxicity Evaluation. Cytotoxic effects of the PGPNPs and FA-PGPNPs on the growth of C6 rat glioma cells were evaluated using an MTT assay. Cell viability after treatment with FA-PGPNPs at the maximum concentration of 0.1 mg/mL for 2 and 24 h remained over 80 and 63%, respectively (Figure 5a). At higher concentrations, a sudden decrease in cell viability was observed and no difference was found in cellular viability between PGPNPs and FA-PGPNPs (p > 0.1). The results demonstrate that both NPs have little cytotoxicity and lay in the region of acceptable domain for in vitro or in vivo studies below 0.1 mg/mL concentration. The inhibitory concentration (IC50) values for PGPNPs and FA-

troscopy (Figure 3). It should be noted that these studies were carried out before and after UV irradiation at 365 nm. UV−vis spectra of the prepared polymer NPs revealed that the added photoactive SPEA has been incorporated into the NPs and it has undergone photoisomerization during irradiation at 365 nm. Appearance of the new absorption band at 500−650 nm (λmax 565 nm) after UV irradiation confirms SP to MC isomerization (Figure 3a). Figure 3b demonstrates a peak at λmax of 523 nm before irradiation at 365 nm, attributing to the surface plasmon absorption of AuNPs in PGPNPs with a spherical morphology22,54,55 (approved by HR-TEM analysis in the following) and confirms binding of these AuNPs to the surface of previously prepared PNPsIm. After UV irradiation at 365 nm, a 10 nm red shift in λmax from 523 to 533 nm and a slight increase in the intensity was observed, which reveal the interaction between gold and dipolar zwitterionic MC isomer (Figure 3b). It is expected that the hydrophilic nature of dipolar MC form would urge its migration to the surface of polymer particles in the aqueous media. As a result, the overlap between absorption bands of AuNPs and MC isomers will consequate in their strong dipolar interactions and the observed red shift (Figure S5, Supporting Information). Absorption bands of FAPGPNPs in the range of 500−650 nm is similar to PGPNPs ones (Figure 3c), but the appearance of absorptions at 342 and 381 nm denotes the attachment of FA to the gold surface through L-cysteine interface.56 Figure 4 demonstrates SEM, DLS, and HR-TEM micrographs of the prepared FA-PGPNPs. The SEM image shows

Figure 4. SEM (a) and HR-TEM with different magnifications (b,c) images of FA-PGPNPs. The light regions in TEM micrographs (yellow arrows) are related to the existing polymer substrate and the dark regions (red arrows) are related to AuNPs. The inset diagram in (a) shows DLS analysis of the FA-PGPNPs sample.

that the NPs are spherical and in the range of 40−60 nm, but AuNPs are not detectable. Besides, the DLS analysis reveals narrow size distribution with unimodal pattern (polydispersity index of 1.11), and it is in good agreement with SEM results. In HR-TEM micrographs, it is evident that the spherical AuNPs have been formed with 10 ± 2 nm diameter and they have been

Figure 5. Cytotoxic evaluation of PGPNPs and FA-PGPNPs samples for C6 rat glioma cells. Viability assay after 2 and 24 h incubation at 37 °C for 4 h in the dark (a); and after 2 and 24 h incubation and subsequent 3 min UV irradiation at 365 nm for PGPNPs (b) and FAPGPNPs (c). 19488


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ACS Applied Materials & Interfaces PGPNPs (right after 24 h) were 0.255 ± 0.05 and 0.184 ± 0.03 mg/mL, respectively. Thus, the MTT assay results revealed that the attached FA has affected the C6 cell growth and inhibitory activity remarkably because of the selective and strong interaction between FA-PGPNPs and folate receptors on the tumoral cells surface mainly. As shown in Figure 5b,c, the mitochondrial activity was reduced in a dose-dependent manner after exposure to UV irradiation (365 nm) for 3 min and significant differences was observed between the treated cells with these NPs before and after UV exposure. Obviously, PGPNPs and FA-PGPNPs caused about 1.1−1.2 fold and 1.3−1.7 fold intensification of cell inhibition after UV irradiation, respectively, in comparison with the control samples (not illuminated) at the concentrations of 0.001−0.1 mg/mL for 2 and 24 h. As a result, the cell inhibition without UV irradiation greatly depends on cellular uptake of the NPs. Comparatively for the same concentrations (0.01 and 0.1 mg/mL after 24 h), FA-PGPNPs demonstrated 11% more cell inhibition relative to PGPNPs. This cell inhibitory returns to the targeting effect of FA toward the folate-receptor on C6 glioma cells which results in more cellular uptake and consequent increased cell inhibitory (approved by ICP−OES analysis in the following section). After UV irradiation, 26% cell destruction was observed for the treated C6 cells with 0.1 mg/mL FA-PGPNPs in comparison with nonirradiated ones. This probably refers to the enhanced ROS photogeneration caused by strongly coupled interaction between MC isomers and the plasmonic resonance of AuNPs and will be discussed in Section 3.4. 3.3. Cellular Uptake of the NPs. Uptake of PGPNPs and FA-PGPNPs into cancer cells is an essential condition for noninvasive therapy and negligible dark-toxicity. The concentrations of internalized prepared NPs into the cells were evaluated by measuring the gold content using ICP−OES spectroscopy. Table 2 reveals the amounts of Au3+ ions in

two folds. This interaction was not observed in nonconjugated NPs (PGPNPs) as the control ones. Another confirmation for assembly between FA-PGPNPs sample and C6 rat glioma cell lines was observed by fluorescence microscopy, as fluorescence property of the internalized NPs after UV irradiation at 365 nm enables monitoring of the uptaken NPs into those cells. The untreated cells showed no fluorescence, and the treated ones with PGPNPs showed weak fluorescence emission (Figure 6). This

Table 2. Theoretical and Measured Amounts of Au3+ Ions in the Prepared Samples sample control PGPNPs FA-PGPNPs internalized PGPNPs internalized FA-PGPNPs

theoretical (ppm)

measured (ppm)

0.00 0.796 0.504

0.00 0.59 0.42 0.17 0.30

Figure 6. Phase contrast bright-field (left) and fluorescence microscopy images (right) of C6 glioma cells with control (top) and those treated with 0.01 mg/mL of PGPNPs (middle) and FAPGPNPs (bottom). Scale bar is 200 μm.

cell uptake (%)

illustrates that the NPs have not achieved to internalize into the C6 cells efficiently. However, FA-PGPNPs were actively entered into the cells, started to aggregate, and showed strong fluorescence emission. These results together with the cytotoxicity evaluation information suggest that the developed FA-PGPNPs can specifically target the cancerous C6 glioma cells via the folate receptor-mediated pathway, which is essential for being employed as a high-tech nanoprobe in targeted PDT and PTT treatment and effective eradication of malignant tissues like C6 cells. 3.4. Intracellular ROS Detection and Analysis. Generation of intracellular ROS can be detected by the oxidationsensitive dye, DCFH, as a fluorescent probe. In the presence of ROS, the trapped nonfluorescent DCFH inside the cells is rapidly oxidized to highly fluorescent dichlorofluorescein (DCF) with emission at 528 nm that is directly proportional to the extent of generated ROS. It is noteworthy that none of AuNPs, SP, or MC moieties have fluorescence emission at this wavelength. The DCF assay can detect all kinds of ROS and is considered as a reliable method for evaluating phototoxicity of the employed NPs.25,34,38,42 Therefore, extra fluorescence emission of DCF, caused by the presence of PGPNPs and

28.8 71.4

PGPNPs and FA-PGPNPs after dissolving in aqua regia from control wells (untreated) and treated wells with PGPNPs and FA-PGPNPs. The comparison between theoretical and measured amount of Au3+ ions in the PGPNPs and FAPGPNPs evidently endorses the C6 cell internalization of AuNPs. Accordingly, the cell uptake for PGPNPs and FAPGPNPs samples were found to be 28.8 and 71.4%, respectively. It appears that the conjugation of FA (as the targeting agent) to the NPs has increased the cellular uptake for the FA-PGPNP sample. Therefore, FA has provided a very high uptake and shows its targeting role for C6 rat glioma cell lines efficiently. The folate receptor is overexpressed on the surface of many tumoral cells, and the presence of FA at the surface of prepared NPs (FA-PGPNPs) can guarantee their adsorption and internalization into such cancerous cells up to more than 19489


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Figure 7. Energy diagram for AuNPs surface plasmon−MC resonance coupling in singlet oxygen generation (a), and fluorescence emission intensity of DCF at 528 nm vs time, with and without UV irradiation (365 nm, 3 min) in C6 glioma cells for the control sample and in the presence of 0.01 mg/mL FA-PGPNPs and PGPNPs samples (b).

generation are a good candidate for selective targeted photodynamic therapy in cancer cells. 3.5. PTT Studies. Because of the strong SPR absorption band of AuNPs,1 FA-PGPNPs may offer remarkably improved photothermal cancer cell killing efficacy upon continuous-wave NIR laser as the excitation source. Temperature increase in human cells over 37 and 43 °C leads to fever and cell death, respectively. The temperature increase of the culture induced by the NIR laser irradiation has been demonstrated in Figure 8. Continuous-wave laser exposure time in the spot region for reaching to 43 °C, as a result of the light-to-heat conversion, in the cell suspension without NPs treatment took 14 min and for those containing FA-PGPNPs and PGPNPs were just 2 and 3 min, respectively. Temperature measurement during NIR laser exposure for both of the cell-treated PGPNP and FA-PGPNP samples within 14 min revealed a noticeable elevation from 25 to 76 and 86 °C, respectively, whereas the C6 glioma cells with cell culture medium (as control) displayed no obvious temperature change. This suggests that AuNPs in the prepared NPs could act as an effective photothermal agent. In addition, after 14 min of NIR laser exposure FA-PGPNPs demonstrate more 10 °C increment relative to PGPNPs, which refers to more AuNP concentration and aggregation in C6 glioma cells because of the presence of the FA targeting agent. Moreover, AuNPs conjugated with FA make these NPs a useful multifunctional nanoprobe in targeted PTT and concentrating in cancer cells with efficient ROS generation upon UV irradiation. NIR images of cell culture have been shown (Figure 9) for control sample and those containing PGPNPs and FA-PGPNPs before and after 10 min irradiation with 808 nm laser. The temperature elevation, specifically in the presence of FA-

FA-PGPNPs, can be ascribed to the photogeneration of cytotoxic ROS. By green light illumination at 532 nm, the valence electrons in the prepared FA-PGPNPs can absorb energy and excite from the singlet ground state (S0) to the singlet excited state (S1) (Figure 7a). Then, S1 may reach to the triplet excited state (T1) by ISC and this subsequently leads to the transforming of the ground state molecular oxygen (3O2) to the singlet oxygen (1O2) through energy transfer from T16 state of FA-PGPNPs. Figure 7b shows the variations in fluorescence intensity within each 1 min interval and during 5 min in the presence of PGPNPs and FA-PGPNPs, with and without UV irradiation for 3 min (365 nm) and subsequent green light exposure (532 nm). Without UV irradiation, FA-PGPNPs demonstrated more fluorescence emission in comparison with nonconjugated ones (PGPNPs), which indicates highly localized plasmonic field of AuNPs and more concentration and accumulation of these NPs in the C6 glioma cells. The fluorescence intensity of DCF increased with a higher slope for those including FA-PGNPs after UV irradiation than the others, which depicts the generation of more ROS there. Also after 3 min of UV irradiation, 1.56- and 1.83-fold increase in fluorescence emission was observed for PGPNPs and FAPGPNPs, respectively, relative to the samples without UV irradiation. Photogenerated intracellular ROS like singlet oxygen will increase biological damages in the exposed cells via gold-enhanced singlet oxygen photogeneration.32,57 This improvement in ROS generation can be attributed to the enhanced interaction between dipolar moment of zwitterionic MC isomer and plasmonic field of AuNPs because of good overlap between MC absorption and SPR of AuNPs and this was previously approved by UV−vis studies too (Figure S5, Supporting Information). Results of intracellular ROS analysis demonstrate that FA-PGPNPs with the enhanced ROS 19490


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PGPNPs reveals the efficient role of these NPs in targeted PTT.

4. CONCLUSION In this study, preparation and characterization of golddecorated and folate-conjugated photoresponsive polymer NPs (FA-PGPNPs) were reported by means of a two-step synthetic route and facile semicontinuous emulsion polymerization. The NPs were modified with FA for site-specific targeting and improvement of therapeutic efficiency. These NPs are potent to be employed in efficient targeted photodynamic and photothermal therapies. AuNPs were included as light-to-heat and singlet oxygen generation source together with SPEA to enhance ROS photogeneration and fluorescing agent and they can be exploited in cell-tracking because of the presence of photoactive moieties. Chemical structures of the prepared samples were characterized by FTIR and EDX analyses. UV−vis analysis showed that the SPR absorption band of the AuNPs overlapped appropriately with the absorption band of MC isomer (λmax of 565 nm) and this resulted in their efficient dipolar interactions and led to 10 nm in SPR red shift. Spherical morphology of the NPs with an average particle size of 40−60 nm and attached AuNPs with 10 ± 2 nm in diameter were approved by SEM and HR-TEM micrographs. The MTT assay revealed increment in cell destruction of the treated C6 rat glioma cancer cells with FAPGPNPs under UV irradiation at 365 nm. On the other hand, these NPs induced enhanced generation of ROS, as an efficient method for cell death and cancer therapy. Fluorescence images and ICP−OES spectroscopy confirmed a significant internalization of folate-conjugated PGPNPs into C6 glioma cells (due to the presence of folate receptors) with 71.4% cellular uptake relative to 28.8% for nonconjugated ones. Moreover, the C6 cell culture medium in the presence of PGPNPs and FAPGPNPs was exposed to continuous-wave NIR laser (808 nm) and the results exhibited remarkable photothermal capacity

Figure 8. Photothermal capacities of AuNPs upon continuous-wave NIR irradiation with 808 nm laser (0.8 W/cm2) for 14 min in control ); and with 0.01 mg/mL PGPNPs ( ) and FA( ) samples in cell culture medium. The upper images PGPNPs ( show the NIR images of the cell culture containing FA-PGNPs in different irradiation time.

Figure 9. NIR images of cell culture for the control sample and those incubated with PGNPs and FA-PGNPs before and after 10 min irradiation with 808 nm laser (0.8 W/cm2). 19491


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New Tools for Live-Cell Imaging. J. Am. Chem. Soc. 2007, 129, 3524− 3526. (9) Zhu, M.-Q.; Zhang, G.-F.; Hu, Z.; Aldred, M. P.; Li, C.; Gong, W.-L.; Chen, T.; Huang, Z.-L.; Liu, S. Reversible Fluorescence Switching of Spiropyran-Conjugated Biodegradable Nanoparticles for Super-Resolution Fluorescence Imaging. Macromolecules 2014, 47, 1543−1552. (10) Wang, Y.; Hong, C.-Y.; Pan, C.-Y. Spiropyran-Based Hyperbranched Star Copolymer: Synthesis, Phototropy, FRET, and Bioapplication. Biomacromolecules 2012, 13, 2585−2593. (11) Keyvan Rad, J.; Mahdavian, A. R.; Khoei, S.; Esfahani, A. J. FRET-Based Acrylic Nanoparticles with Dual-Color Photoswitchable Properties in DU145 Human Prostate Cancer Cell Line Labeling. Polymer 2016, 98, 263−269. (12) Zhu, M.-Q.; Zhang, G.-F.; Li, C.; Aldred, M. P.; Chang, E.; Drezek, R. A.; Li, A. D. Q. Reversible Two-Photon Photoswitching and Two-Photon Imaging of Immunofunctionalized Nanoparticles Targeted to Cancer Cells. J. Am. Chem. Soc. 2011, 133, 365−372. (13) Belfield, K. D.; Liu, Y.; Negres, R. A.; Fan, M.; Pan, G.; Hagan, D. J.; Hernandez, F. E. Two-Photon Photochromism of an Organic Material for Holographic Recording. Chem. Mater. 2002, 14, 3663− 3667. (14) Keyvan Rad, J.; Mahdavian, A. R. Photoswitchable Dual-Color Fluorescent Particles from Seeded Emulsion Polymerization and Role of Some Affecting Parameters on FRET Process. Eur. Polym. J. 2017, 88, 56−66. (15) Abdollahi, A.; Keyvan Rad, J.; Mahdavian, A. R. StimuliResponsive Cellulose Modified by Epoxy-Functionalized Polymer Nanoparticles with Photochromic and Solvatochromic Properties. Carbohydr. Polym. 2016, 150, 131−138. (16) Sharifian, M. H.; Mahdavian, A. R.; Salehi-Mobarakeh, H. Effects of Chain Parameters on Kinetics of Photochromism in AcrylicSpiropyran Copolymer Nanoparticles and Their Reversible Optical Data Storage. Langmuir 2017, 33, 8023−8031. (17) Keyvan Rad, J.; Mahdavian, A. R. Preparation of Fast Photoresponsive Cellulose and Kinetic Study of Photoisomerization. J. Phys. Chem. C 2016, 120, 9985−9991. (18) Stafforst, T.; Hilvert, D. Kinetic Characterization of Spiropyrans in Aqueous Media. Chem. Commun. 2009, 287−288. (19) Movia, D.; Prina-Mello, A.; Volkov, Y.; Giordani, S. Determination of Spiropyran Cytotoxicity by High Content Screening and Analysis for Safe Application in Bionanosensing. Chem. Res. Toxicol. 2010, 23, 1459−1466. (20) Nilsson, J. R.; Li, S.; Ö nfelt, B.; Andréasson, J. Light-Induced Cytotoxicity of a Photochromic Spiropyran. Chem. Commun. 2011, 47, 11020−11022. (21) Pellegrotti, J. V.; Cortés, E.; Bordenave, M. D.; Caldarola, M.; Kreuzer, M. P.; Sanchez, A. D.; Ojea, I.; Bragas, A. V.; Stefani, F. D. Plasmonic Photothermal Fluorescence Modulation for Homogeneous Biosensing. ACS Sensors 2016, 1, 1351−1357. (22) Oo, M. K. K.; Yang, Y.; Hu, Y.; Gomez, M.; Du, H.; Wang, H. Gold Nanoparticle-Enhanced and Size-Dependent Generation of Reactive Oxygen Species from Protoporphyrin IX. ACS Nano 2012, 6, 1939−1947. (23) Pissuwan, D.; Cortie, C. H.; Valenzuela, S. M.; Cortie, M. B. Functionalised Gold Nanoparticles for Controlling Pathogenic Bacteria. Trends Biotechnol. 2010, 28, 207−213. (24) Chadwick, S. J.; Salah, D.; Livesey, P. M.; Brust, M.; Volk, M. Singlet Oxygen Generation by Laser Irradiation of Gold Nanoparticles. J. Phys. Chem. C 2016, 120, 10647−10657. (25) Wang, B.; Wang, J.-H.; Liu, Q.; Huang, H.; Chen, M.; Li, K.; Li, C.; Yu, X.-F.; Chu, P. K. Rose-Bengal-Conjugated Gold Nanorods for in Vivo Photodynamic and Photothermal Oral Cancer Therapies. Biomaterials 2014, 35, 1954−1966. (26) Wang, X.; Wang, C.; Cheng, L.; Lee, S.-T.; Liu, Z. Noble Metal Coated Single-Walled Carbon Nanotubes for Applications in Surface Enhanced Raman Scattering Imaging and Photothermal Therapy. J. Am. Chem. Soc. 2012, 134, 7414−7422.

according to the strong SPR absorption, contributed by the AuNPs. These multifunctional smart nanoprobes could be exploited in biological systems for promising targeted photodynamic and PTT in cancer cell ablation with less nonspecific damages and selectively cell line labeling.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b05252. Comparative FT-IR spectra of L-cysteine and FA-Lcys; FTIR spectra of the prepared PGPNPs and FA-PGPNPs samples; EDX spectra for L-cysteine, FA-Lcys, PGPNPs, and FA-PGPNPs samples; combination EDX distribution map of nitrogen (red spots) and gold (green spots) in the prepared PGPNPs and absorption spectra of PNPsIm and PGPNPs before and after UV irradiation at 365 nm and reversible resonance coupling between dipolar MC isomer and surface plasmon resonance in gold nanoparticles (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +9821 4478 7000. Fax: +9821 4478 7023. ORCID

Ali Reza Mahdavian: 0000-0002-9224-1324 Sakine Shirvalilou: 0000-0003-3213-8824 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We wish to express our gratitude to Iran Polymer and Petrochemical Institute (IPPI) for the financial support of this work (grant no. 24794101).

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REFERENCES

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