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Anti-TROP2 conjugated hollow gold nanospheres as a novel nanostructure for targeted photothermal destruction of cervical cancer cells

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Nanotechnology Nanotechnology 25 (2014) 345103 (11pp)

doi:10.1088/0957-4484/25/34/345103

Anti-TROP2 conjugated hollow gold nanospheres as a novel nanostructure for targeted photothermal destruction of cervical cancer cells Ting Liu1,4, Jiguang Tian2,4, Zhaolong Chen3, Ying Liang1, Jiao Liu1, Si Liu3, Huihui Li1, Jinhua Zhan3 and Xingsheng Yang1 1

Department of Obstetrics and Gynecology, Qilu Hospital of Shandong University, Jinan, Shandong, People’s Republic of China 2 Department of Orthopedics, Qilu Hospital of Shandong University, Jinan, Shandong, People’s Republic of China 3 Key Laboratory of Colloid and Interface Chemistry, Ministry of Education, Department of Chemistry, Shandong University, Jinan, Shandong, People’s Republic of China E-mail: [email protected] and [email protected] Received 19 April 2014, revised 15 June 2014 Accepted for publication 30 June 2014 Published 7 August 2014 Abstract

Photothermal ablation (PTA) is a promising avenue in the area of cancer therapeutics that destroys tumor cells through conversion of near-infrared (NIR) laser light to heat. Hollow gold nanospheres (HGNs) are one of the few materials that are capable of converting light to heat and have been previously used for photothermal ablation studies. Selective delivery of functional nanoparticles to the tumor site is considered as an effective therapeutic approach. In this paper, we demonstrated the anti-cancer potential of HGNs. HGNs were conjugated with monoclonal antibody (anti-TROP2) in order to target cervical cancer cells (HeLa) that contain abundant trophoblast cell surface antigen 2 (TROP2) on the cell surface. The efficient uptake and intracellular location of these functionalized HGNs were studied through application of inductively coupled plasma atomic emission spectroscopy (ICP-AES) and transmission electron microscopy (TEM). Cytotoxicity induced by PTA was measured using CCK-8 assay. HeLa cells incubated with naked HGNs (0.3–3 nmol L−1) within 48 h did not show obvious cytotoxicity. Under laser irradiation at suitable power, anti-TROP2 conjugated HGNs achieved significant tumor cell growth inhibition in comparison to the effects of non-specific PEGylated HGNs (P < 0.05). γH2AX assay results revealed higher occurrences of DNA-DSBs with anti-TROP2 conjugated HGNs plus laser radiation as compared to treatment with laser alone. Flow cytometry analysis showed that the amount of cell apoptosis was increased after laser irradiation with antiTROP2 conjugated HGNs (P < 0.05). Anti-TROP2 conjugated HGNs resulted in downregulation of Bcl-2 expression and up-regulation of Bax expression. Our study results confirmed that anti-TROP2 conjugated HGNs can selectively destroy cervical cancer cells through inducing its apoptosis and DNA damages. We propose that HGNs have the potentials to mediate targeted cancer treatment. Keywords: photothermal ablation, cervical cancer, TROP2, hollow gold nanospheres (HGNs), targeted cancer treatment, antibody conjugation

4

These authors equally contributed to this article.

0957-4484/14/345103+11$33.00

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© 2014 IOP Publishing Ltd Printed in the UK

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1. Introduction

Trophoblast cell surface antigen 2 (TROP2) is a 36 kDa transmembrane glycoprotein that belongs to the tumor-associated calcium signal transducer (TACSTD) gene family. Overexpression of TROP2 has been observed in numerous types of epithelial carcinomas, while low or restricted expression was found in normal tissues [17–20]. In our former study, we demonstrated that the elevated expression of TROP2 was closely associated with cervical cancer progression and poor prognosis, knocking down of TROP2 mediated by siRNA significantly result in inhibition of tumor growth and invasion [21]. Due to its particular location and overexpression on the tumor cell surface, TROP2 is becoming an attractive target for cancer immunotherapy. In this article, we described the preparation and synthesis scheme of uniformsized HGNs, stabilized with PEG, bio-conjugated with antiTROP2 monoclonal antibody. The uniqueness of the photothermal treatment lies in the selective uptake and destruction of cancer cells while safeguarding the neighboring normal cells, an appreciable advantage over radiation therapy now frequently used.

Cervical cancer is the third most prevalent cancer among women worldwide [1], and it is also the third leading cause of cancer-related mortality [2]. Despite numerous advances in early diagnosis and treatment of cervical cancer in the recent years, there is still an overall trend of decreasing onset ages. Conventional surgical resection of solid tumors is an effective treatment for early-stage patients (I-IIA), but patients with advanced stage (IIB-IV) always develop distant metastasis and are unsuitable for such operational treatments. The 5-year survival rate for patients with stage III is up to 25–35%, but for stage IV patients, the rate is 15% or less [3, 4]. Noble metal nanoparticles have attracted great interests in the field of biomedical applications with their excellent biocompatibility and tunable light absorption properties, and are being widely investigated for potential use in diagnostics and in novel treatment technologies [5–7]. Among various gold nanostructures, hollow gold nanospheres (HGNs) are a novel class of nanostructures characterized by small sizes, spherical shape, hollow interior, and thin but robust wall. The most dramatic merit of HGNs is that the surface plasmon resonance (SPR) absorption can be precisely altered throughout the near-infrared (NIR) regions. In the NIR region, one important biological transparency window is located in 700–900 nm. And in this window of wavelengths, water molecules exhibit minimal absorption and scatter, which contribute to low attenuation of light tissue and thus, enhance light penetration capability with sufficient intensity and spatial precision. Recent research have shown that HGNs could be efficient photothermal agents for the cancer eradication seeing that the light absorbed by SPR can be efficiently transformed into heat and lead to a localized rise of temperature around the gold clusters. Due to the abnormal morphology and circulation system of cancer cells, they are more sensitive to heat than normal cells. When the local temperature is raised above normal physiological conditions at 40 °C to 45 °C, a series irreversible damage will occur by denaturation of proteins and DNA repair deficiency. In addition, the localized hyperthermia modulated by HGNs upon NIR irradiation also triggers the release of anticancer agents. If the heating agent (conjugated nanoparticles) is only targeted for cancer cells expressing certain antigen, possible undesirable damages to the surrounding normal tissues were prevented, or at least, reduced to a minimum [8–10]. HGNs’ versatile surface guaranteed good biocompatibility toward several biological media such as antibodies, peptides, nucleic acids, and DNA. In addition, the molecules repeatedly demonstrated the ability to maintain original biological activity after conjugation in previous studies [11–13]. Earlier studies have showed that conjugation of HGNs with tumor specific antibodies results in active targeting of the nanoparticles to the tumor site through receptor-mediated endocytosis (RME) [14, 15]. Therefore, the conjugated HGNs will selectively annihilate targeted cancer cells through inducing its apoptosis with heat under relatively low power laser exposure while keeping nonspecific injuries to normal cells to a minimum [16].

2. Materials and methods 2.1. Reagents and materials

Gold (III) chloride trihydrate (HAuCl4 · 3H2O, G4022-1g), N(3-dimethylaminopropyl)-N-ethylcarbodiimidehydrochloride (EDC) and N-hydroxy succinimide (NHS) were purchased from Sigma-Aldrich, USA. SH-PEG-COOH (Mw = 5000 Da, Xibao, Shanghai China) were obtained from Shanghai Xibao Medpep Co. Phosphate buffered solution (PBS) (pH 7.4, 0.1 mol L−1) was prepared with 0.1 mol L−1 Na2HPO4, 0.1 mol L−1 KH2PO4 and 0.1 mol L−1 KCl. 2.2. Cell culture

HeLa cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Dulbecco-modified Eagle medium (DMEM) obtained from Gibco Inc. was selected as the growth medium, 10% fetal bovine serum and 1% penicillin-streptomycin from Invitrogen was added. These cells were cultured in the growth medium and placed in a humidified incubator at 37 °C and 5% CO2 atmosphere. Cells were used for the experiments when they were in logarithmic growth phase. 2.3. Synthesis of HGNs

For the synthesis of HGNs, the silver colloids were first prepared. 180 mg of silver nitrate was added to 100 mL of deionized water under rigorous stirring. High-purity nitrogen was passed into the solution in order to create a nitrogen atmosphere, which should be maintained during the reaction. The solution was then rapidly heated to boiling point, whereupon 1 mL of 1% sodium citrate was added. The solution was further heated for 30 min before cooling to room temperature. Hollow gold nanospheres were prepared using the galvanic replacement reaction between HAuCl4 and silver 2

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colloid in an aqueous solution under refluxing condition. 10 ml of the silver colloid was added into a 50 mL flask under magnetic stirring and then heated to 60 °C. Meanwhile, an aqueous solution of 5 ml HAuCl4 (1 mmol L−1, Aldrich) was slowly added to the flask via a syringe pump at a rate of 45 mL h−1 under magnetic stirring. The solution was heated for 30 min and concentrated at 7000 rpm for 5 min. The HGNs was characterized by UV–vis spectrum and TEM.

laser irradiation point to measure peak sample surface temperature. 2.7. In Vitro photothermal therapy

HeLa cells were seeded onto a 96-well plate at a density of 5000 cells per well, and then, incubated with 2 nmol L−1 PEGylated HGNs or immuno HGNs for 3 h. After incubation, the cells were rinsed with PBS to remove the excess unbound HGNs, and fresh culture medium was then added to each well. For the NIR laser irradiation, the cells were exposed with different powers of NIR laser light (808 nm) for 5 min. After exposure to light, cells were incubated for an additional 3 h at 37 °C. Cell viability was measured using the CCK-8 assay.

2.4. Preparation of immuno HGNs

Bi-functional SH-PEG-COOH (Mw = 5000 Da, Xibao, Shanghai China) in lyophilized form under argon protect was used as a linker to conjugate antibodies to HGNs. In the first step, 200 ul of 2 mmol L−1 aqueous solution of SH-PEGCOOH was allowed to react with 200 ul HGNs (10 nmol L−1 in concentration) at 4 °C in the dark for 4 h. The reaction mixture was centrifuged to remove the byproducts and unreacted PEG. Afterward, the carboxyl terminal of PEG was activated with EDC (5 ul, 40 mg ml−1) and NHS (5 ul, 110 mg ml−1) at room temperature for 1 h. Untreated EDC and NHS were removed from the HGNs by discarding of supernatant after centrifugal separation (8000 rpm, 15 min). In the second step, the PEGylated HGNs were mixed with 15 ul 200 ug ml−1 monoclonal anti-TROP2 antibody (Santa Cruz Biotechnolog, CA, USA) over-night at 4 °C under agitation. Conjugate solutions were centrifuged (8000 rpm, 15 min) and re-suspended in phosphate buffered saline (PBS) to remove excess antibody then stored at 4 °C for future use within one week. To detect the cell uptake of HGNs and immuno HGNs, the gold content in the lysis solution was measured by inductively coupled mass spectrometry (ICP-MS). 5 ml of 20% HNO3 was added into each sample to lyse the cells, and the number of HGNs was calculated via the gold mass. The quantitative measurement of HGNs’ cell uptake was achieved by the following formula: number of HGNs in the lysis/ number of cells.

2.8. Apoptosis assay

HeLa cells (1 × 106) both treated with and without conjugated HGNs were irradiated with NIR laser light at power of 5 W cm−2. Three hours after laser treatment, the cells were collected and washed twice with cold PBS, resuspended in 400 uL Annexin V-FITC binding buffer. Cells were stained with 5 uL of Annexin V-FITC and 10 ul propidium iodide (PI) according to the Apoptosis Detection Kit (Jingmei biotech, Shanghai, China) instructions. Then stained cells were subjected to flow cytometry (BD, San Jose, CA, USA) to detect numbers of cell apoptosis. This experiment was conducted three times. 2.9. Evaluation of anti-TROP2 conjugated HGNs’ enhanced photothermal therapy induced DNA double-strand breaks

γH2AX assay was used to determine the effect of immuno HGNs’ induced double-strand breaks (DSBs) in HeLa cells exposed to NIR laser radiation. HeLa cells treated with 2 nmol L−1 anti-TROP2 conjugated HGNs for 3 h, then exposed to NIR laser at 5 W cm−2 for 5 min. Three hours later, the cells were fixed and double-stained with DAPI and anti-phospho (Ser 139)-Histone H2A.X antibody as described before [22]. Immunofluorescence images were captured using a laser scanning confocal microscope (Nikon PCM2000). All red channel images were taken with the He-Ne laser (argon laser blocked).

2.5. Cytotoxicity of HGNs

The cytotoxicity of the HGNs was assessed by CCK-8 assay (Jingmei, biotech, Shanghai, China), which is a quantitative colorimetric method. The cells were seeded onto the 96-well plate at a density of 5000 cells per well in 100 uL medium. The previous medium was replaced respectively with different concentrations of HGNs (0.3, 1, 2, 3 nmol L−1) for different time intervals (3 h, 6 h, 12 h, 24 h, 48 h). 10 μl CCK-8 reagents were added into each well and the plate was left standing for an additional 2 h at 37 °C. The optical density (OD) was measured using a micro-plate reader (Bio-Rad Model 680, Richmond, CA, USA) at 450 nm wavelength.

2.10. Western blot analysis

Three hours after laser treatment, an equivalent amount of the cell protein samples were loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to PVDF membranes using the Bio-Rad electrotransfer system. After blocked by 5% w/v nonfat dried milk for one hour, the membranes were incubated with primary antibodies specific to Bcl-2, Bax (Cell Signaling Technology, Beverly, MA, USA; 1:1000 dilution) overnight at 4 °C. The membranes were incubated with horseradish peroxidaseconjugated goat anti-rabbit secondary antibody (Beijing Zhong Shan Biotech Co. Ltd, BeiJing, China; 1:4000 dilution) for an additional one hour, the protein bands were

2.6. Photothermal investigation of HGNs

Cuvettes that contained 1.5 ml of 2 nmol L−1 HGNs, PEGylated HGNs and immuno HGNs were broadly irradiated under an 808 nm diode laser (B&WTEK Inc., USA) at 2 W cm−2. During irradiation, a thermocouple was inserted into the solution perpendicularly and was positioned 1 cm above the 3

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Figure 1. Conjugation of anti-TROP2 antibody with HGNs through SH-PEG-COOH. (A) TEM image of HGNs. (B) Schematic presentation

of HGNs’ bioconjugation. (C) TEM image of anti-TROP2 conjugated HGNs. (D) UV-visible extinction of HGNs and anti-TROP2 conjugated HGNs.

visualized by enhanced chemiluminescence (Millipore). βactin (Beijing Zhong Shan Biotech Co. Ltd, Beijing, China; 1:1000 dilution) was used as internal control for protein loading and analysis.

antibody followed by two step protocols was shown in figure 1(C). In the first step, bi-functional SH-PEG-COOH (Mw = 5000 g mol−1) was attached to the surface of HGNs via the strong gold-sulfur bonds, and the carboxyl terminal of PEG was activated by EDC and NHS. In the second step, anti-TROP2 antibody was conjugated to the HGNs via the nucleophilic reaction with one of the amino acid side chains of the antibody with the COOH under slightly basic conditions. A red-shift (38 nm) in the localized surface plasmon resonance was detected after conjugation with antibody, a phenomenon that was expected due to the small change of refractive index on the surface of the HGNs (figure 1(D)). Meanwhile, the shape of HGNs remains unchanged after conjugation with antibody based on the transmission electron microscopy (figure 1(B)), but an increase in the hydrodynamic diameter of the HGNs was observed.

2.11. Statistical analysis

SPSS (SPSS Inc., Chicago, IL, USA) 18.0 software was used to perform statistical analysis. Experimental values were conducted in triplicate and expressed as means and standard errors (SE). Differences between each group were analyzed by the Student's t-test and one-way ANOVA. The result of P < 0.05 indicated that the differences were statistically significant.

3. Result 3.2. Distribution and uptake in cancer cells 3.1. Synthesis and characterization of HGNs

The cellular uptake and intracellular location of these functionalized HGNs were assayed by both phase contrast microscopy and TEM. Cells were imaged after incubation with PEGylated HGNs and anti-TROP2 conjugated HGNs for one hour. From figures 2(B) and (C), we could see that both PEGylated HGNs and anti-TROP2 conjugated HGNs were rapidly internalized into the cells after addition, and excess nanoparticles accumulated randomly around the cell

The HGNs synthesized in our experiment have a mean diameter of 51.6 ± 7 nm and wall thickness of 4.67 ± 1.52 nm calculated based on measurements made by TEM. From figure 1(A), we can clearly see the hollow interior and thin but robust shell. UV-vis extinction spectrum revealed that the plasma resonance peak for HGNs was approximately 780 nm (figure 1(D)). Conjugation of HGNs with anti-TROP2 4

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Figure 2. Uptake of HGNs in HeLa cells. (A) Bright-field light microscopy images of HeLa cells. (B) HeLa cells were imaged after

incubation with PEGylated HGNs for one hour. (C) Image of HeLa cells after incubation with anti-TROP2 conjugated HGNs for one hour. (D) Uptake of PEGylated HGNs versus anti-TROP2 conjugated HGNs per cell measured by ICP-MS.

Figure 3. TEM images of HeLa cells incubated with anti-TROP2 conjugated HGNs. The arrows indicated distribution and uptake of antiTROP2 conjugated HGNs in cytoplasm and endosomes.

location of functional HGNs, TEM images were taken after 12 h of HeLa cells incubation with the 2 nmol L−1 antiTROP2 conjugated HGNs (figure 3). The microtomed sample revealed that anti-TROP2 conjugated HGNs were absorbed in HeLa cells and were mainly located at the cytoplasm, in either endosomes or lysosomes. As shown in figure 3(B), functional HGNs were present in the cell cytosols and were embedded inside the cells in aggregated states.

membrane. The numbers of HGNs internalized by the HeLa cells was quantified using ICP-MS. We found that the uptake of both PEGylated HGNs and anti-TROP2 conjugated HGNs increased with increased incubation time during the first 48 h. The peak uptake incubation time for both PEGylated HGNs and immuno HGNs was observed at 24 h. HeLa cells absorbed much more conjugates than PEGylated HGNs at each time interval (figure 2 (D)) (P < 0.05). To determine the 5

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Figure 4. Cytotoxicity and temperature profiles of HGNs. (A) HeLa cells incubated with different concentrations of naked HGNs (0.3, 1, 2 and 3 nmol L−1) at 3, 6, 12, 24 and 48 h, CCK-8 assay was used to determine the cell viability. (B) NIR laser induced temperature change in HeLa cells after incubation with naked HGNs, PEGylated HGNs and anti-TROP2 conjugated HGNs. Data are expressed as mean ± SD of three independent experiments, *p < 0.05.

(figure 5(A)) showed that the 2 nmol L−1 anti-TROP2 conjugated HGNs plus NIR laser radiation (5 W cm−2) have a much stronger effect on reducing HeLa cells viability compared to PEGylated HGNs (P < 0.05); while the cells treated with PEGylated HGNs or NIR laser radiation alone did not induced significantly growth inhibition. Cells that were incubated with 2 nmol L−1 PEGylated HGNs for three hours and irradiated at 5 W cm−2 maintained 68% viability, but the viability of cells that were incubated with anti-TROP2 conjugated HGNs and irradiated decreased to 40%. We further tested whether the laser power density had an impact on cell growth, cells were subjected to 808 NIR laser radiation of 2.5, 5, 10, 15 W cm−2 for five minutes and were harvested three hours after irradiation (figure 5(B)). For the control cells, cell viability did not change much with the variation of laserirradiated power. When combined with 2 nmol L−1 PEGylated HGNs, the same power of laser radiation induced cellular viability were found at 87%, 72%, 54% and 45%, respectively. If 2 nmol L−1 anti-TROP2 conjugated HGNs were added before irradiation, the same doses of irradiation produced cellular viability of 67%, 50%, 36% and 22%, respectively (P < 0.05). The curve of the cells treated with anti-TROP2 conjugated HGNs showed a rapid decrease in cell viability as the laser power increased.

3.3. Cytotoxicity of HGNs

The cytotoxicity of noble metal nanoparticles is a critical concern for any vivo application. We determined the viability of HeLa cells via CCK-8 assay after incubating them with HGNs. HeLa cells were incubated with different concentrations of HGNs (0.3, 1, 2, 3 nmol L−1 respectively) for five different indicated hours (3 h, 6 h, 12 h, 24 h, 48 h). In figure 4(A), we could see that the naked HGNs did not show the obvious cytotoxicity with increased gold concentrations and incubation periods. The cell viability for groups with 3 nmol L−1 reached 90% at 24 h and 87% at 48 h. Therefore, cell survival analysis based on our result indicated that HGNs of either 0.3 or 3 nmol L−1 did not induce remarkable cytotoxicity, so it is safe of HGNs within 3 nmol L−1 for the following photothermal experiments. 3.4. Temperature profiles of HGNs

In order to investigate the photothermal characteristics of HGNs, we used a thermocouple to measure the temperature changes of HGNs under an NIR coherent diode laser (808 nm) radiation. We found that the temperature sharply increased by 24 °C in the first 10 min, after then the rising trend became gentle and finally reached the peak temperature of 52 °C at 16 min (figure 4(B)). The three types of HGNs were normalized to have similar concentrations of 2 nmol L−1, and all showed the ability of elevating temperature, but there is not obvious difference between them (p < 0.05). In contrast, in the absence of HGNs, the temperature change was rendered insignificant (21 °C to 24 °C).

3.6. Morphology of HeLa cells after photothermal therapy

More accurate viability assays were conducted by observing the morphological changes and double Hoechst-PI dyes staining. Figure 5(C) showed that the untreated cells were found to be intact and exhibited normal proliferation behavior, but the cells treated with anti-TROP2 conjugated HGNs plus laser radiation turned rounded, exhibited unclear boundary, membranous vesicles and fragmentation demonstrating drastic apoptotic features. PI dye can only penetrate cells when the cell membrane integrity was lost, so the cells positively stained red with PI are presumably dead. While the living cells were stained positively with Hoechst. For the cells treated with laser radiation alone, there was no remarkable

3.5. In vitro photothermal ablation of cervical cancer cells

HeLa cells were incubated with either PEGylated HGNs or anti-TROP2 conjugated HGNs for three hours, and then exposed to 808 nm NIR laser. The cell viability was assessed using CCK-8 assay within 3 h after irradiation. According to the cytotoxicity data, 2 nmol L−1 HGNs were used for the combination treatments. The combination treatment 6

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HGNs for 3 h, then exposed to 808 nm NIR laser (5 W cm−2), CCK-8 assay was used to determine the cell viability. (B) Effect of laser power density on HeLa cells. Cells viability was measured within 3 h after a 5 min irradiation treatment. (C) Morphological changes of HeLa cells after incubation with anti-TROP2 conjugated HGNs followed by NIR irradiation. (D) Fluorescent images of HeLa cells double-stained with Hoechst-viability and propidium iodide-mortality, there were more dead cells in the anti-TROP2 conjugated HGNs’ group than control groups. These data are expressed as mean ± SD of three independent experiments, p < 0.05.

change in cell viability. However, for the cells treated with anti-TROP2 conjugated HGNs plus laser (5 W cm−2), fluorescent images showed that there were more dead cells than control groups (figure 5(D)).

3.7. Apoptosis detection by flow cytometry

It has been demonstrated that cell apoptosis plays a considerable role on tumor cell growth, so we further assessed the 7

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Figure 6. Flow cytometric analysis of apoptosis in HeLa cells after photothermal treatments. Anti-TROP2 conjugated HGNs subject to laser irradiation induced a significant increase in apoptosis compared to irradiation alone.

possibility of whether immuno HGNs combined with NIR laser radiation induced apoptosis. HeLa cells were treated with 2 nmol L−1 anti-TROP2 conjugated HGNs for three hours and then exposed to NIR laser at power of 5 W cm−2 for five minutes. Three hours later, flow cytometry was used to determine the amount of cell apoptosis occurrence (figure 6). Cells in the right lower and upper quadrants are consider as early and late apoptosis respectively, while those in the left upper quadrants are considered as dead cells. The apoptotic rate was calculated by the sum of proportion of right lower and upper quadrants. Anti-TROP2 conjugated HGNs subject to laser irradiation induced a significant increase in apoptosis (18.3 ± 2.8%) compared to irradiation alone (2.2 ± 1.0%) (P < 0.01). These data indicate that one mechanism of HGNs’ photothermal effect is inducing cell apoptosis.

Western blot. Western blot analysis showed that there was a significant up-regulation of Bax after anti-TROP2 conjugated HGNs’ treatment together with laser radiation. However, the expression of Bcl-2 was significantly down-regulated after combined treatment (figure 8). The result implies that antiTROP2 conjugated HGNs play a role in apoptosis by regulating the expression of the Bcl-2 family of proteins and activating the mitochondrial apoptotic pathway.

4. Discussion Gold nanoparticles possess well-established biocompatibility and biological functions that have been extensively investigated in several malignant tumor studies. Among different types of gold nanoparticles, HGNs is a promising novel nanostructure for cancer diagnosis and therapy. The HGNs in our study were prepared through a classic galvanic replacement reaction between silver colloids and HAuCl4 under refluxing condition. Every step in the synthesis process of HGNs requires careful handling. Flasks and stirs should be repeatedly washed by aqua regia to prevent contamination, and the reaction temperature and the stirring rate should be precisely controlled. The surface modifications of gold nanoparticles are reported to have the ability to reduce the cytotoxicity and enhance the stability with good dispersibility. PEG is a biocompatible polymer widely used in several biological applications. Recently, different molecular weight forms of PEG are used extensively to stabilize gold nanoparticles and extend blood circulation time. Bi-functional thiol-containing PEG used in our study can interact with HGNs to form stable covalent bonds. The other end of PEG was used to conjugate with antibody via the nucleophilic reaction with one of the amino acid side chains of the antibody. In other words, PEG acts as a linker to conjugate the HGNs and antibody.

3.8. Evaluation of the mechanism of cell death

To determine whether NIR laser induced DNA double-strand breaks (DSBs) in cervical cancer cells, we labeled HeLa cells for gamma-H2AX (γH2AX). The assay was used to detect the phosphorylation of histone-H2AX at serine-139 through measuring the discrete nuclear foci using confocal microscopy. The density in the nucleus is proportional to the amount of unrepaired DNA DSBs in the cell. Cells treated with NIR laser radiation alone (5 W cm−2) did not show significant expression of γH2AX, which indicated that the laser irradiation did not induce apparent DNA damage. However, immuno HGNs’ administration together with NIR laser (5 W cm−2) evidently increased the density of the γH2AX foci compared to cells receiving irradiation alone, suggesting that anti-TROP2 conjugated HGNs can significantly induced DSBs in HeLa cells (figure 7). To further explore the underlying molecular mechanisms by which anti-TROP2 conjugated HGNs together with NIR laser radiation induces apoptosis, we investigated the expression of Bcl-2 and Bax by 8

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γH2AX

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Figure 7. DNA double-strand break (DSB) induction in HeLa cells after photothermal treatments. (A) Cells treated with laser irradiation

alone. (B) Cell treated with anti-TROP2 conjugated HGNs plus laser. HGNs’ administration together with NIR laser (5 W cm−2) evidently increased density of γH2AX foci compared to cells receiving irradiation alone. 1

rapidly leak from tumor tissue into blood capillaries. Another factor to be considered is that the size of gold nanoparticles should be small enough to escape capture by macrophages in the reticulo-endothelial system (RES). In order to find the optimal uptake of nanoparticle size, Chithrani et al have investigated the uptake of 14, 50, and 74 nm citrate-coated GNPs in HeLa cells, and it was found that 50 nm sized particles have the most efficient of cellular uptake [24]. The size of HGNs in our study was initially 51.6 ± 7 nm; when conjugated with TROP2 antibody, the size was about 54.7 ± 5.5 nm as determined by TEM, which was around the optimal dimension. The toxicity of gold nanoparticles is another major concern among biological applications. Recent studies show that HGNs with a variety of surface modifiers are not inherently toxic to human cells, and are especially not capable of causing acute cytotoxicity [25]. However, high doses of gold nanoparticles may cause severe side-effects and thus, limit its clinical applications. It is critical to find more efficient locoregional combination treatments without increasing the current toxicity. In our study, we tested the toxicity of different concentrations of HGNs under different incubation time periods. We found that HGNs did not induce remarkable cytotoxicity with an increase of the gold concentration (0.3–3 nmol L−1) and incubation time (3–48 h), but a decrease in cell viability was observed. Learning from that, a relatively low concentration of HGNs (2 nmol L−1) and a short incubation time (3 h) were adopted in the following photothermal treatments to exclude the influence of cytotoxicity of gold on cell viability. In our former study, we confirmed that TROP2 was highly expressed in HeLa cells, and found that the

2

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Figure 8. Western blot of protein expression of Bcl-2, Bax. Lane 1, laser irradiation alone; lane 2, anti-TROP2 conjugated HGNs plus laser. Western blot analysis showed that there was a significant upregulation of Bax and down-regulation of Bcl-2 after anti-TROP2 conjugated HGNs’ treatment with laser irradiation.

The size and surface properties of gold nanoparticles were considered to be one major issue of targeted cellular uptake. Tumor tissues have an enhanced permeability and retention (EPR) effect due to defective tumor vasculature, abnormal endothelial gap junction, and lack of lymphatic drainage [23]. In order to accumulate the maximum amount of gold nanoparticles in tumor tissues, the size of gold nanoparticles should be large enough that they would not 9

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overexpression of TROP2 was closely related with cell proliferation and invasion. Building upon prior knowledge, we explored the possibility of employing TROP2 antibody-conjugated gold nanospheres in current study for the purpose of specifically delivering HGNs to the targeted cancer cells for photothermal therapy. Naked HGNs can accumulate at tumor cells by passive targeting mechanisms, but the presence of a targeting ligand on the surface of the HGNs potentially enhances the avidity of interaction with TROP2 antigen on cervical cancer cells, and further promotes receptor mediated internalization into tumor cells. TEM was used to visualize the intracellular distribution of HGNs located in vesicles in cytoplasm, separate from nucleus. An NIR coherent diode laser (808 nm) was used to investigate the photothermal characteristics of HGNs, and an electronic thermometer was used to measure the temperature changes. It was found that the temperature reached almost 30 °C through light-induced heating. Due to the desired temperature for hyperthermia therapy lies in a narrow range (41–43 °C), lower temperatures are therapeutically ineffective, and higher temperatures may cause adverse side effects. It is crucial to find the optimization of HGNs’ concentration that induced temperatures within that narrow desired range. Once the desired temperature (40 °C) for hyperthermia therapy is reached, irreversible damage was induced in the cancer cells through protein denaturation that occurs due to changes in either enzyme complexes for DNA synthesis and repair or multi-molecular structures of the cytoskeleton and membranes [26]. In our study, it was confirmed that the HGNs mediated photothermal therapy has an effect on HeLa cell viability. HGNs or laser treatment alone had no obvious effect on cancer cells, but laser irradiation in the presence of the HGNs induced cell death. The efficiency of the antiTROP2 conjugated HGNs coupled with laser treatment was evident with 60% mortality rate of HeLa cells. We also investigated the impact of laser power density on cell growth, and found that the combined therapy induced cell mortality function mostly depends on the power of laser irradiation. The amount of generated heat increased along with the increase of laser power. This linear relationship provides a mean of safekeeping due to the fact that keeping the laser at low power density also in turn limits the level of generated heat at work and thus, minimizes the damage to nearby healthy cells. The effect of photothermal therapy was also confirmed by double Hoechst-PI dyes staining. PI dye can only penetrate the dead cells while the living cells could be stained positively with Hoechst. Fluorescent images showed that there were more dead cells in the anti-TROP2 conjugated HGNs’ group than control groups. To determine whether NIR laser induced DNA double-strand breaks (DSBs) in cervical cancer cells, we immuno-fluorescently labeled HeLa cells for gammaH2AX (γH2AX). It was found that the induction of DSBs in anti-TROP2 conjugated HGNs treated cells was higher than cells treated laser alone. We speculated that DNA damage by anti-TROP2 conjugated HGNs plus laser could be involved in the induction of apoptosis in HeLa cells.

Apoptosis is a gene regulated cell death which plays a pivotal role in cancer therapy [27]. Evasion of apoptosis is considered to be one of the major hallmarks of tumor development. In our analysis of the cancer cells death mechanisms mediated by irradiation, dual staining of cells with Annexin V-FITC and PI was used to quantitatively determine the amount of cell apoptosis. One important finding in our study is that the increase of apoptosis results from the use of a combination of anti-TROP2 conjugated HGNs and laser irradiation as measured by flow cytometry. Furthermore, we analyzed two apoptosis-related proteins expression. Bcl-2 and Bax are two critical regulators of cell apoptosis and play pivotal roles in anti-apoptotic and pro-apoptotic respectively [28, 29]. Figure 8 demonstrated the varying expressions of these two regulators under two controlled environments. A significantly elevated expression of Bax and decreased expression of Bcl-2 were found in the anti-TROP2 conjugated HGNs plus laser radiation group in comparison with the group administrated with radiation alone. Therefore, we concluded that laser irradiation induced HeLa cells apoptosis by disrupting the balance between Bcl-2 and Bax. In our study, anti-TROP2 antibody was successfully conjugated with HGNs via the nucleophilic reaction, and the conjugated HGNs maintained the former retention of immunoreactivity. Our results confirmed that the efficiency of conjugated HGNs cellular uptake was enhanced through its specific binding and internalization. The studies also found that anti-TROP2 conjugated HGNs plus laser irradiation are capable to inducing severe cell damage even when low intensity and short time of irradiation were in use. The amount of cellular death can be controlled by laser power density, even when destruction of the cancer cells was not achieved by laser irradiation alone. Furthermore, we hypothesize that HGNs induce cancer cell apoptosis and DNA double-strand breaks to increase irradiation susceptibility. Our study underlines the fact that photothermal therapy using antiTROP2 conjugated HGNs is a promising therapeutic approach for targeted destruction of cervical cancer cells. Future work will also address the stability of HGNs and make them more suitable for in vivo tumor model studies.

Acknowledgments The project was supported by National Natural Science Foundation of China and Science (No. 81372809) and Technology Development Planning of Shandong Province (No. 2011GSF12121). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the Manuscript.

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