Enhanced Photogeneration of Reactive Oxygen Species and Targeted

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Biological and Medical Applications of Materials and Interfaces

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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05252 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 22, 2018

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ACS Applied Materials & Interfaces

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 Rad1, Ali Reza Mahdavian1*, Samideh Khoei2, Sakine Shirvalilou2

1.

Polymer Science Department, Iran Polymer & Petrochemical Institute, P.O. Box: 14965/115, Tehran, Iran Tel: +9821 4478 7000, Fax: +9821 4478 7023; Email: [email protected]

2.

Medical Physics Department, School of Medicine, Iran University of Medical Sciences, Tehran, Iran

Keywords: gold nanoparticles, spiropyran-acrylic, folic acid, cell targeting, photodynamic, photothermal therapy.

*

Corresponding author

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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. FTIR and EDX analyses, UV-Vis spectroscopy, SEM and HR-TEM 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 folateconjugated PGPNPs (FA-PGPNPs) toward rat brain cancer cells (C6 glioma) with 71.8% cell uptake in comparison with 28.8% for the non-conjugated ones. Nonpolar SP groups are converted to zwitterionic merocyanine (MC) 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 multi-functional FA-PGPNPs with a comprehensive integration of prospective materials introduced promising nanoprobes with targeting ability, enhanced tumor photodynamic therapy, cell tracking, and photothermal therapy.


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1 Introduction The unique properties and tremendous progress in nanotechnology have drawn much attentions, in many research fields for versatile applications including biology, optical device, and catalysis1,2. 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 3,4. Photochromic spiropyran upon external stimuli such as light or heat undergoes reversible isomerization between a three-dimensional inert, non-fluorescent, closed ring colorless spiropyran (SP) form and a planar, zwitterionic, open ring, fluorescent, and colored merocyanine (MC) form5– 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 development of sensing, tracking and labeling nanoscale devices with light8–11. A deficiency for employment of SP-MC photoisomerization in biological systems originates from poor penetration of UV light into tissues. It has been shown that near-IR (NIR) two-photon excitation at 780 nm with deeper penetration into tissues converts SP to MC and addresses to the aforementioned problem12,13. The versatile and tunable photoswitching of spiropyran 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 interactions7,14–16. In polar medium like water, open-ring MC is the preferred form and results in negative photochromism17. Therefore, subsequent relatively rapid hydrolysis of MC in physiological conditions will significantly limit its exploitation in the aqueous media18.



Giordani and coworkers studied the effect of spiropyran cytotoxicity on three different cellular models and they showed its non-toxic effect at low concentrations19. Nilsson et al. investigated biological activity of spiropyran and their results demonstrated that the nonpolar SP form has good cell membrane penetrating characteristic without cytotoxicity 20. In contrast, MC form reveals poor passing through the cells membrane (due to its zwitterionic nature) and high cytotoxic effect, which is attributed to the intermolecular interactions and/or generation of reactive oxygen species in the cytoplasm. Unique properties of noble metal nanoparticles (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 many attentions in recent years21–24. Besides, photothermal therapy (PTT) is an alternative method for exploitation of gold nanoparticles, which can provide high temperatures around nanoparticles under visible to near-IR (NIR) laser irradiation and cause thermal ablation of cancer cells with their consequent death25,26. 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 nanoparticles surface

27

. SPR band could be tunable by controlling nanoparticles 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 nanoparticles 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 enhanced30–32. MC isomer is susceptible to triplet-singlet intersystem crossing (ISC) process because of its zwitterionic character33. Therefore, AuNPs-MC interactions can lead to plasmon-enhanced


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fluorescence and also may show an improved ability to produce cytotoxic reactive oxygen species (ROS) due to 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 oxygen36–40. Incomplete reduction of oxygen generates ROS (oxidative radicals) to jeopardize cancer cells viability and treats various diseases. Reactive oxygen species 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 generation32. 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. In order to achieve effective intracellular uptake at a tumor site and increase 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 lines44–47. 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 nanoparticles into such sites48–50. Also, stability, inexpensiveness and non-immunogenic nature are other reasons to use FA as a susceptible targeting agent due to its high affinity in binding to the surface of tumor cells. Kang et al.51 studied the effect of folate-functionalized 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 non-folate ones (94.10%) and ASODNs alone (0.73%). In another study, cytotoxic effect of folate-functionalized silica nanoparticles 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 nanoparticles 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 spiropyran as 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 gold-decorated polymer nanoparticles and their capability in the elevated ROS generation will be investigated. In order to improve the efficacy of therapeutic means by targeting and selective internalization to cancerous cells, the obtained PGPNPs were functionalized with folic acid (FA-PGPNPs) through L-cysteine linkages. These multifunctional nanoparticles were assessed for cell viability by 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 nanoparticles in photothermal therapy 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 nanoparticles.


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2 2.1

Experimental 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 shapedirecting agent for AuNPs), 2'-7'-dichlorofluorescein diacetate (DCFH-DA), N,N′-dicyclohexyl carbodiimide (DCC), 4-(dimethylamino) pyridine (DMAP), sodium borohydride (NaBH4), ethylenediamine

tetraacetic

acid

(EDTA)

and

3-[4,5-dimethyl-thiazol-2-yl]-2,5-

diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. 2,2-Azobis[2-(2imidazoline-2-yl) propane] dihydrochloride (VA-044) (as initiator) from Wako Pure Chemical Industries Ltd. (Osaka, Japan) was used. All of the solvents, 2-hydroxy-5-nitrobenzaldehyde, methyl methacrylate (MMA), hydrochloric acid (HCl, 37%), triethylamine, 2-bromoethanol, acryloyl chloride (AC), 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 nanoparticles containing N-VIM (PNPsIm)

To prepare photoresponsive functionalized polymer nanoparticles with N-VIM, spiropyran ethyl acrylate (SPEA, as the photochromic monomer) was synthesized primarily according to our previously reported procedure53. In an emulsion polymerization recipe, a 35 mL aqueous solution of 150 mg CTAB and 30 mg VA-044 was prepared and 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; i.e.100 mg SPEA in 10 mL DI water and 2.8 mL MMA from two separate dropping funnels into the reactor within 15 min. The emulsion polymerization continued at 50°C for 45 minutes until the conversion reached to above 50%. Next, 0.1 mL N-VIM and 0.8 mL MMA were mixed and added in 3 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 photoresponsive gold-decorated polymer nanoparticles (PGPNPs)

Deposition of Au on the surface of photoresponsive acrylic NPs functionalized with N-VIM was carried out by a metal-ligand formation approach. 65 mL aqueous stock solution of HAuCl4.3H2O (3 g/L) as the source of Au3+ was added to 35 mL of the pre-formed 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 1h at room temperature. The color of reaction mixture turned from yellow to bright purple, indicating the formation of AuNPs during the progress of Au3+ reduction.

2.4 Preparation of conjugated folic acid with PGPNPs (FA-PGPNPs) In order to modify PGPNPs with folic acid, L-cysteine was selected as a proper interface. The process of conjugation of γ-carboxylic acid group of folic acid with amine group in L-cysteine has been shown schematically in Figure 1. 250 mg Folic acid (0.57 mmol) and 18 mg DMAP (0.15 mmol) were dissolved in 30 mL anhydrous DMSO by continuous stirring at room temperature for 60 min. Then, 118 mg DCC (0.57mmol) was dissolved in 10 mL 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 L-cysteine (0.57 mmol) in 10 mL DCM was added to the above solution within 45 min 8 ACS Paragon Plus Environment

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dropwise. The stirring continued for 24 h at room temperature to obtain conjugated folate with Lcysteine. After 24h, 100 mL 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 DI water, centrifuged (8000 rpm) and dried in vacuum oven at 40°C for 24h to give 235 mg FA-Lcys in 76% yield.

Figure 1. Synthesis of FA- Lcys

150 mg of the prepared FA-Lcys was dissolved in 20 mL 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 gold nanoparticles and short polymeric chains. Finally, the purified FA-PGPNPs were obtained by freeze-drying of the dialyzed product. 2.5

In-vitro cytotoxicity evaluation of PGPNPs and FA-PGPNPs

The cytotoxicity of PGPNPs and FA-PGPNPs to C6 glioma cell line (rat brain cancer cell) were assessed by MTT cell viability assay. In summary, C6 glioma cells were seeded into a 96-well 9 ACS Paragon Plus Environment


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 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 to 10 mg/mL) of PGPNPs and FA-PGPNPs and afterward, cytotoxicity was assessed in 2 and 24 hours incubation at 37°C for 4 h in the dark after addition of PGPNPs and FA-PGPNPs. Then, the cells were washed twice with phosphate-buffered saline (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 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 (OD) 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 nanoparticles 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-centimeter distance for 3 min. Immediately after UV irradiation and illumination with green light (532 nm) for 90 sec, the cell viability was measured using the technique mentioned before. All the experiments were performed in triplicate. 2.6

Cell uptake of PGPNPs and FA-PGPNPs

6-Well plates were seeded with 1×105 C6 glioma cell line per well and incubated for 24 h at 37°C. Based on 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 non-absorbed nanoparticles. The cells were trypsinized and centrifuged at 10,000 rpm for 5 min and then the supernatant was


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removed and 0.5 mL of aqua regia (1:3 (V/V) HNO3:HCl)was added to dissolve AuNPs of the internalized nanoparticles 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 nanoparticles were 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-DA34. 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-hour incubation period, the cells were loaded with ROS detector probe by replacing the culture media with PBS, containing 5 µM DCFH-DA for 30 minutes at 37°C in dark, and non-incorporated DCFH-DA was removed through washing with PBS for 3 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 90s. Fluorescence emissions at 530 nm (excitation at 485 nm) were measured at 1 min intervals for a total of 5 minutes. In each analysis, two types of control sample were used: negative control (nonUV 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 and FA-PGPNPs were assessed by 808 nm continuous-wave near-IR (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 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 (EDX) spectroscopy (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 Shimadzu-UV2550 UV-Vis Spectrophotometer (Japan). Size and morphology of the nanocomposite particles were studied by 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 12 ACS Paragon Plus Environment

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using a Philips CM-30 high-resolution transmission electron microscopy (HR-TEM) (The Netherlands) at 200 kV. The samples for TEM analysis were prepared by dropping 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 spiropyran groups. The excitation light source for PDT was a 532 nm solid-state laser (MC2000) with power density of1.8 W/cm2 and was employed as a green illumination source. Phase contrast bright-field and fluorescence images of the nanoparticles–cell were taken with a fluorescence microscope with UV light (330–380 nm) (Olympus CK2; Olympus Optical Co., Tokyo, Japan). Reactive oxygen species (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 nanoparticles. Here, the modified photoresponsive polymer nanoparticles containing imidazole groups were prepared through semi-continuous emulsion polymerization in a way that these functionalities would be located in the outer layer of nanoparticles (Figure 2). This will facilitate the adsorption of gold ions on the surface of these nanoparticles in order to conduct nucleation and formation of AuNPs. Embodiment of spiropyran 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 spiropyran groups convert to the zwitterionic MC 13 ACS Paragon Plus Environment


isomers by UV irradiation at 365 nm, which is suitable for intersystem crossing and consequent producing of reactive oxygen species. 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 gold nanoparticles and dipolar zwitterion MC isomers. However, the presence of AuNPs will facilitate binding to FA for targeting toward cancerous cells due to the existence of folate receptors on these cells. The synthesis and performance of the obtained FAPGPNPs will be discussed comprehensively in the following.

Figure 2. Synthetic route for preparation of FA-PGPNPs


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3.1 Synthesis and characterization of PGPNPs and FA-PGPNPs For conjugation of folic acid to the gold nanoparticles, L-cysteine as a proper and biocompatible interface was employed. L-cysteine with terminal NH and SH groups is able to bridge between FA and gold nanoparticles. 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 FTIR spectroscopy (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. After preparation of PGPNPs and FA-PGPNPs, they were dialyzed against buffer saline. The purified dispersions were freeze-dried and their FT-IR 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 demonstrate that the appeared peaks at 3450 and 1600 cm-1 are related to the hydroxyl and imine groups in folic acid, respectively. Another approval for the preparation of FA-PGPNPs returns to the elimination of S-H vibration at 2550 cm-1 in FAPGPNPs, which reveals the reaction between thiol groups and AuNPs. Energy dispersive X-ray (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. 22.29 wt% decrease in sulfur and 9.13 wt% increase in nitrogen contents in FA-Lcys relative to L-cysteine are in accordance with the theoretical values and imply the progress of reaction



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(Figure S3. supporting information). Appearance of gold element in 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 sulfur weight percent in FA-Lcys and FA-PGPNPs, the amount of conjugated FA-Lcys to PGPNPs was estimated to about 15.91wt%. Table 1. Weight percent of the elements in the precursors and products from EDX analysis*

L-cysteine Theo Meas

FA-Lcys Theo Meas

PGPNPs Theo Meas

FA-PGPNPs Theo Meas

C

31.59

30.58

50.77

50.27

-

41.62

-

41.09

N

12.27

12.14

21.54

21.27

-

22.12

-

23.84

O

28.04

29.08

21.52

22.55

-

33.13

-

32.01

S

28.10

28.20

6.17

5.91

-

-

1.08

0.94

Au

-

-

-

-

3.98

3.13

2.52

2.12

* “Theo” and “Meas” represent “Theoretical” and “Measured” quantities, respectively.

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 nanoparticles with spiropyran, i.e. PNPsIm, PGPNPs, and FA-PGPNPs dispersions, were investigated by UV−Vis spectroscopy (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 nanoparticles revealed that the added photoactive SPEA has been incorporated into the nanoparticles and it has undergone photoisomerization during irradiation at 365 nm. Appearance of the new absorption band at 500650 nm (λmax 565 nm) after UV irradiation confirms SP to MC isomerization (Figure 3a). Figure 16 ACS Paragon Plus Environment

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3b demonstrates a peak at λmax of 523 nm before irradiation at 365 nm, attributing to the surface plasmon absorption of gold nanoparticles in PGPNPs with a spherical morphology22,54,55 (approved by HR-TEM analysis in the following) and confirms binding of these gold nanoparticles to the surface of previously prepared PNPsIm. After UV irradiation at 365 nm, a 10 nm red shift in λmax from 523 nm to 533 nm and a slight increase in the intensity was observed, which reveals the interaction between gold and dipolar zwitterionic MC isomer (Figure 3b). It is expected that the hydrophilic nature of dipolar merocyanine form would urge its migration to the surface of polymer particles in the aqueous media. As a result, the overlap between absorption bands of gold nanoparticles and MC isomers will consequate in their strong dipolar interactions and the observed red shift (Figure S5. supporting information). Absorption bands of FA-PGPNPs 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 interface56.



Figure 3. UV-Vis analysis of the diluted PNPsIm (a),PGPNPs (b) and FA-PGPNPs(c) dispersions up to 0.2 wt% before (

) and after (-

- -) UV irradiation at 365 nm. The inset picture in (a) shows color changes during SP to MC isomerization.

Figure 4 demonstrates SEM, DLS and HR-TEM micrographs of the prepared FA-PGPNPs. SEM image shows that the nanoparticles are spherical and in the range of 40-60 nm, but AuNPs are not detectable. Besides, DLS analysis reveals narrow size distribution with unimodal pattern (polydispersity index (PDI) 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 in diameter and they have been attached to the surface of primary photoresponsive polymer nanoparticles. These morphological studies reveal that the exploited strategy in preparation of FA-PGPNPs has been accomplished successfully.


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Figure 4. SEM (a) and HR-TEM with different magnifications (b and 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 FA-PGPNPs sample.

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 some difference was found in cellular viability between PGPNPs and FA-PGPNPs (p > 0.1). The results



demonstrate that both nanoparticles 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-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 folic acid has affected on C6 cell growth and inhibitory activity remarkably due to the selective and strong interaction between FA-PGPNPs and folate receptors on the tumoral cells surface mainly. As shown in Figure 5b and 5c, the mitochondrial activity was reduced in a dose-dependent manner after exposure to UV irradiation (365 nm) for 3 min and significant differences were observed between the treated cells with these nanoparticles 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 nanoparticles. 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 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 non-irradiated 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.


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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 FA-PGPNPs (c)



3.3

Page 22 of 36

Cellular uptake of the nanoparticles

Uptake of PGPNPs and FA-PGPNPs into cancer cells is an essential condition for non-invasive therapy and negligible dark-toxicity. The concentrations of internalized prepared nanoparticles into the cells were evaluated by measuring gold content using ICP-OES spectroscopy. Table 2 Table reveals the amounts of Au3+ ions in 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 FA-PGPNPs 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 folic acid (as the targeting agent) to the nanoparticles has increased the cellular uptake for FAPGPNPs sample. Therefore, folic acid 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 folic acid at the surface of prepared nanoparticles (FA-PGPNPs) can guarantee their adsorption and internalization into such cancerous cells up to more than 2 folds. This interaction was not observed in non-conjugated nanoparticles (PGPNPs) as the control ones. Table 2. Theoretical and measured amounts of Au3+ ions in the prepared samples

Sample

Theoretical (ppm)

Measured (ppm)

Cell uptake (%)

Control

0.00

0.00

-

PGPNPs

0.796

0.59

-

FA-PGPNPs

0.504

0.42

-

Internalized PGPNPs

-

0.17

28.8

Internalized FAPGPNPs

-

0.30

71.4


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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 nanoparticles after UV irradiation at 365 nm enables monitoring of the uptaken nanoparticles into those cells. The untreated cells showed no fluorescence, and the treated ones with PGPNPs showed weak fluorescence emission (Figure 6). This illustrates that the nanoparticles 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 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.



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 FA-PGPNPs (bottom). Scale bar is 200 µm.

3.4

Intracellular ROS detection and analysis

Generation of intracellular ROS can be detected by the oxidation-sensitive dye, DCFH, as a fluorescent probe. In the presence of ROS, the trapped non-fluorescent 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 nanoparticles25,34,38,42. Therefore, extra fluorescence emission of DCF, caused by the presence of PGPNPs and FA-PGPNPs, can be ascribed to the photogeneration of cytotoxic reactive oxygen 24 ACS Paragon Plus Environment

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species. 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 intersystem crossing (ISC) and this subsequently leads to the transforming of ground state molecular oxygen (3O2) to the singlet oxygen (1O2) through energy transfer from T1state 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 non-conjugated ones (PGPNPs), which indicates highly localized plasmonic field of AuNPs and more concentration and accumulation of these nanoparticles in the C6 glioma cells. The fluorescence intensity of DCF increased with 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 FA-PGPNPs, respectively, relative to the samples without UV irradiation. Photogenerated intracellular reactive oxygen species like singlet oxygen will increase biological

damages

in

the

exposed

cells

via

gold-enhanced

singlet

oxygen

photogeneration32,57.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 surface plasmon resonance 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 generation is a good candidate for selective targeted photodynamic therapy in cancer cells.



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 versus time, with and without UV irradiation (365 nm, 3min) in C6 glioma cells for the control sample and in the presence of 0.01 mg/mL FA-PGPNPs and PGPNPs samples (b)

3.5

Photothermal therapy studies

Due to the strong SPR absorption band of gold nanoparticles1, FA-PGPNPs may offer remarkably improved photothermal cancer cell killing efficacy upon continuous-wave near-infrared (NIR) laser as the excitation source. Temperature raise 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 26 ACS Paragon Plus Environment

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region for reaching to 43°C, as a result of the light-to-heat conversion, in the cell suspension without nanoparticles 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 PGPNPs and FA-PGPNPs samples within 14 min revealed a noticeable elevation from 25 to 76 and 86°C, respectively, while the C6 glioma cells with cell culture medium (as control) displayed no obvious temperature change. This suggests that AuNPs in the prepared nanoparticles could act as an effective photothermal agent. In addition, after 14 min NIR laser exposure FA-PGPNPs demonstrate more 10°C increment relative to PGPNPs, which refers to more AuNPs concentration and aggregation in C6 glioma cells because of the presence of FA targeting agent. Moreover, AuNPs conjugated with folic acid make these nanoparticles as useful multifunctional nanoprobe in targeted photothermal therapy and concentrating in cancer cells with efficient ROS generation upon UV irradiation.



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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 with0.01 mg/mL PGPNPs (

) and FA-PGPNPs (

) samples in cell culture

medium. The upper images show the near-infrared images of the cell culture containing FA-PGNPs in different irradiation time.

Near-infrared 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-PGPNPs reveals the efficient role of these nanoparticles in targeted photothermal therapy.


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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)

4

Conclusion

In this study, preparation and characterization of gold-decorated and folate-conjugated photoresponsive polymer nanoparticles (FA-PGPNPs) were reported by means of a two-step synthetic route and facile semi-continuous emulsion polymerization. The nanoparticles were modified with folic acid for site-specific targeting and improvement of therapeutic efficiency. These nanoparticles are potent to be employed in efficient targeted photodynamic and photothermal therapies. AuNPs were included as light-to-heat and singlet oxygen generation 29 ACS Paragon Plus Environment


source together with SPEA toenhance ROS photogeneration and fluorescing agent and they can be exploited in cell-tracking due to 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 gold nanoparticles 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 nanoparticles with average particle size of 40-60 nm and attached gold nanoparticles with 10±2 nm in diameter were approved by SEM and HRTEM micrographs.MTT assay revealed increment in cell destruction of the treated C6 rat glioma cancer cells with FA-PGPNPs under UV irradiation at 365 nm. On the other hand, these nanoparticles 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 non-conjugated ones. Moreover, the C6 cell culture medium in the presence of PGPNPs and FA-PGPNPs was exposed to continuous-wave NIR laser (808 nm) and the results exhibited remarkable photothermal capacity according to the strong SPR absorption, contributed by the gold nanoparticles. These multifunctional smart nanoprobes could be exploited in biological systems for promising targeted photodynamic and photothermal therapy in cancer cell ablation with less non-specific damages and selectively cell line labeling.

5

Acknowledgment

We wish to express our gratitude to Iran Polymer and Petrochemical Institute (IPPI) for financial support of this work (Grant No. 24794101). 6

Supporting Information

This section includes some anlyses data and is available online. 30 ACS Paragon Plus Environment

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7

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Enhanced Photogeneration of Reactive Oxygen Species and Targeted

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