doi: 10.1111/j.1349-7006.2008.00827.x Cancer Sci | July 2008 | vol. 99 | no. 7 | 1479–1484© 2008 Japanese Cancer Association

Blackwell Publishing Asia

Increased apoptotic potential and dose-enhancing effect of gold nanoparticles in combination with single-dose clinical electron beams on tumor-bearing miceMeng-Ya Chang,1 Ai-Li Shiau,2 Yu-Hung Chen,1 Chih-Jui Chang,1,5 Helen H-W Chen,3,4,6 and Chao-Liang Wu1,6

1Department of Biochemistry and Molecular Biology, National Cheng Kung University Medical College, 1 Dashiue Road, Tainan 701, Taiwan, 2Department of Microbiology and Immunology, National Cheng Kung University Hospital 138 Sheng-Li Road, Tainan 701, Taiwan, 3Institute of Clinical Medicine, National Cheng Kung University Medical College, Tainan, 701, Taiwan, 4Department of Radiation Oncology, National Cheng Kung University Hospital, Tainan, 701, Taiwan

(Received December 7, 2007/Revised February 27, 2008/Accepted March 10, 2008/Online publication April 11, 2008)

High atomic number material, such as gold, may be used in con-junction with radiation to provide dose enhancement in tumors. Inthe current study, we investigated the dose-enhancing effect andapoptotic potential of gold nanoparticles in combination with single-dose clinical electron beams on B16F10 melanoma tumor-bearingmice. We revealed that the accumulation of gold nanoparticles wasdetected inside B16F10 culture cells after 18 h of incubation, andmoreover, the gold nanoparticles were shown to be colocalizedwith endoplasmic reticulum and Golgi apparatus in cells. Furthermore,gold nanoparticles radiosensitized melanoma cells in the colonyformation assay (P + 0.02). Using a B16F10 tumor-bearing mousemodel, we further demonstrated that gold nanoparticles inconjunction with ionizing radiation significantly retarded tumorgrowth and prolonged survival compared to the radiation alonecontrols (P < 0.05). Importantly, an increase of apoptotic signals wasdetected inside tumors in the combined treatment group (P < 0.05).Knowing that radiation-induced apoptosis has been considered adeterminant of tumor responses to radiation therapy, and thelength of tumor regrowth delay correlated with the extent ofapoptosis after single-dose radiotherapy, these results may suggestthe clinical potential of gold nanoparticles in improving the outcomeof melanoma radiotherapy. (Cancer Sci 2008; 99: 1479–1484)

Radiation dose enhancement by high atomic number (Z)materials has long been investigated. In theory, loading

high Z materials into the tumor could result in greaterphotoelectric absorption within the tumor than in surroundingtissues, and thereby enhance the dose delivered to a tumorduring radiation therapy. At least 20 years ago, it was noted invitro that this effect might be employed to enhance radiotherapyfor cancer.(1) Accumulating studies have demonstrated thedose enhancement caused by high Z materials in kilovoltagebeams(2–4) and in megavoltage beams.(5–8) Moreover, enhancedcell killing was also observed when cells were irradiatedadjacent to high Z materials by kilovoltage X-rays.(9–12) Inclinical practice, electron beams from linear acceleratorshave increasingly taken the place of kilovoltage X-ray beams forskin and subcutaneous tumors because they offer distinctadvantages in terms of dose uniformity in the target volume and inminimizing the dosage to deeper tissues.(13) Although kilovoltagebeams could maximize tumor dose enhancement, it hastechnical restrictions. The use of kilovoltage X-rays producessignificant dose heterogeneity inside the target tumor.(4,14)

To be clinically useful, a radiosensitizer and/or dose enhancershould significantly increase the therapeutic ratio and shouldbe readily available, easily utilized, and non-toxic. Gold (Au;Z = 79) or nanogold (gold nanoparticles, AuNP) showed dose-

enhancing effects in cell experiments,(15) the murine model,(16)

and through Monte Carlo calculations.(17) Gold nanoparticleshave been actively investigated in a wide variety of biomedicalapplications due to their biocompatibility and ease of conju-gation to biomolecules.(18–21) Besides, nanoparticles have theadvantages of small size (1–100 nm) and ability to evade theimmune system,(22,23) and also have been shown to preferentiallyaccumulate in tumors.(24–28)

While previous studies have primarily examined the doseenhancement factor by Au, it is also known that radiation-induced apoptosis is a significant component of radiation-inducedcell death. Consequently, modulating the apoptotic response andthereby the radiosensitivity is of interest.(29–34) Therefore, in thecurrent study, we investigated the dose-enhancing effect andapoptotic potential of gold nanoparticles in combination withsingle-dose clinical electron beams on B16F10 melanomatumor-bearing mice.

Materials and Methods

Preparation of AuNP. AuNP were prepared as previouslydescribed with slight modifications.(35) All glassware used inthese preparations was thoroughly cleaned in aqua regia (3 partsHCl and 1 part HNO3), and all solutions were made using18-MΩ-deionized, 0.22-μm-filtered water. Briefly, 50 mL ofHAuCl4 (1 mM) was reduced with sodium citrate (38.8 mM,5 mL) by boiling with vigorous stirring for 10 min. The resultingburgundy suspension was cooled, sterile-filtered, and storedin glass bottles at room temperature or 4°C. The AuNP werespherical and well-dispersed with an approximate diameter of13 nm confirmed by a transmission electron microscopy. Theparticle concentration was approximately 10.72 nM (180 μg/mL)quantified by a maximum absorption at 520 nm. The preparednanoparticle solution could be concentrated by centrifugationand was stable for at least 6 months.

Cells and mice. Murine B16F10 melanoma cells were culturedin Dulbecco’s modified Eagle’s medium supplemented with50 μg/mL gentamicin, 2 mM L-glutamine, and 10% cosmic calfserum (Hyclone, Logan, UT, USA) at 37°C in an atmosphere of5% CO2. Female C57BL/6 mice (6–8-week-old) were obtainedfrom the Laboratory Animal Center of the National Cheng KungUniversity. Animals were maintained in specific pathogen-free

5Present address: Institute of Molecular and Cell Biology, Tzu Chi University,No 701, Zhongyang Road, Section 3, Hualien, 97004, Taiwan.6To whom correspondence should be addressed. E-mail: wumolbio@mail.ncku.edu.tw; helen@mail.ncku.edu.tw

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animal care facilities under isothermal conditions with regularphotoperiods. All animal experiments were performed followingthe guidelines approved by the Laboratory Animal Care and UseCommittee of the National Cheng Kung University.

Visualization of AuNP. AuNP localization was visualized bysilver enhancement or by fluorescence labeling of AuNP. B16F10melanoma cells were seeded on cover glass at 37°C for 6 h, andthe medium was replaced by culture medium with or without10 nM AuNP or AuNP-Alexa Fluor 594 conjugates (AlexaFluor 594 was purchased from Invitrogen, Eugene, OR, USA).Eighteen hours post-AuNP addition, cells were washed withphosphate-buffered saline (PBS), the localization of AuNP wasdetermined by silver enhancement of AuNP according to themanufacturer’s instructions (Sigma, St Louis, MO, USA), andthe localization of AuNP-Alexa Fluor 594 conjugates wasdetected directly under a microscopy with an Olympus DP1Tdigital camera system (Olympus Optical, Tokyo, Japan).

Live-cell endoplasmic reticulum (ER) and Golgi labeling. ER-TrackerRed dye and BODIPY TR Ceramide Golgi Tracker (MolecularProbes, Carlsbad, CA, USA) were used, respectively, for live-cellER and Golgi labeling according to the manufacturer’s instructions.

Animal studies.Tumors. C57BL/6 mice were inoculated subcutaneously (s.c.)

in the thigh with syngeneic mouse melanoma B16F10 cells(1 × 106) suspended in 0.1 mL of PBS at day 0, and at day 7,

visible nodules developed at all injection sites with approximatetumor volumes of 50–90 mm3. Groups of four to seven tumor-bearing mice were injected intravenously (i.v.) with 200 μL of200 nM AuNP in phosphate buffer (PB), or with 200 μL of PBSvia the lateral tail vein. All mice were monitored for tumorgrowth and survival as previously described.(36)

Irradiation. Approximately 24 h post-AuNP injection, a 1-inchdiameter tumor region of the leg was irradiated with 6 MeVelectrons using a Varian 2100C linear accelerator (Varian MedicalSystems, Palo Alto, CA, USA) under normoxic conditions. Thedelivered dose was 25 Gy per mice. Animals were anesthetizedwith pentobarbital intraperitoneally (90 μg/g body weight).

Biodistribution of AuNP. Groups of three tumor-bearing micewere injected with AuNP or with PBS via the lateral tail vein.Twenty-four hours postinjection, blood and tissues were excised.Samples were pooled and dried at 105°C until the weightremained constant, and then were homogenized into powder. ForAuNP detection, 0.5 g of tissue samples was used. Six mililitersof concentrated HCO3 was added into each sample, andincubated at room temperature until the samples were completelynitrated. After nitration, 2 mL of H2O2 was added. Once thesolution became clear, samples were then filtrated to remove celldebris. The resulting samples were then analyzed for Auconcentration using atomic absorption detection (National SunYat-Sen University, Kaohsiung, Taiwan).

Clonogenic survival. The effectiveness of the combination ofAuNP and ionizing radiation was assessed by clonogenic assays.The B16F10 melanoma cell lines were treated with AuNP(10 nM) for 18 h and then exposed to different doses of ionizingradiation. Briefly, cells were irradiated with using a linearaccelerator (Varian 2100C; Varian, Palo Alto, CA, USA) at roomtemperature in T-25 flasks. After treatment, cells were trypsinizedand counted. Known numbers were then replated and returnedto the incubator to allow macroscopic colony development.Colonies were counted after 7 days, and the plating efficiencyand surviving fraction for given treatments were calculatedbased on the survival of non-irradiated cells treated with thevehicle or AuNP alone.

Detection of apoptosis. Apoptotic activity was analyzed on thebasis of terminal deoxynucleotidyl transferase-mediateddeoxyuridine triphosphate nick end labeling (TUNEL) assay.Tumors from groups of three tumor-bearing mice were excised6 h postradiotherapy,(37) and embedded in OCT compound (SakuraFinetek USA, Torrance, CA, USA). Five-micrometer thick cryostatsections from each representative specimen were obtained andfixed, and then processed with the DeadEnd FluorometricTUNEL System (Promega, Madison, WI, USA) according to themanufacturer’s protocol. The positive TUNEL signals werecounted under a microscopy with an Olympus DP1T digitalcamera system (Olympus Optical). DAPI was used as the nuclearcounter stain. Apoptotic cells were calculated by averaging thenumber of positive TUNEL signals from eight fields withhighest density of TUNEL signals in each section.

Statistical analysis. Data were expressed as mean ± standarderror of the mean (SEM). The survival analysis was done usingthe Kaplan–Meier survival curve and the log-rank test. Otherstatistical differences were assessed with Student’s t-test.Statistical significance was set at P < 0.05.

Results

Visualization of AuNP in B16F10 cells. To detect the AuNPinside cultured cells, the localization of AuNP was visualized bysilver enhancement (Fig. 1a) or by direct observation under afluorescent microscopy (Fig. 1b). Our data demonstrated thatafter 18 h of incubation, AuNP could be detected inside theB16F10 cells. Moreover, we also found that the AuNP did notcolocalize with the cell nucleus (Fig. 1b).

Fig. 1. Visualization of gold nanoparticles (AuNP) inside B16F10 cells.AuNP localization in cells was visualized by silver enhancement or byfluorescence labeling of AuNP. (a) The localization of AuNP wasdetermined by silver enhancement, and (b) the localization of AuNP-.Alexa Fluor 594 conjugates were detected directly under a microscope(bar = 50 μm; original magnification 200×). BF, Bright field, DAPI, 4′-6-diamidino-2-phenylindole.

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AuNP revealed colocalization with ER and Golgi apparatus inB16F10. As the AuNP revealed a non-uniform distribution in thecytoplasm of B16F10 cells, to investigate the possibility that thesubcellular localization of AuNP is in ER or Golgi, live-cell ER orGolgi staining was used in addition to the silver enhancement ofAuNP. Our results indicated that AuNP were localized in ER(Fig. 2a) and Golgi apparatuses (Fig. 2b) in B16F10 20 h afterincubation.

Biodistribution of Au 24 h postintravenous injection of AuNP intumor-bearing mice. To detect the biodistribution of AuNP 24 hpostinjection, blood and tissues were excised and analyzed forAu concentration using the atomic absorption detection(Fig. 3a). The result showed that at 24 h following injection,a notable accumulation of AuNP inside tumor tissues wasdetected. The tumor-to-tumor surrounding muscle gold ratiowas 6.4:1. Nevertheless, higher concentrations of AuNP werealso found in spleen and liver, which indicated that AuNP werealso uptaken by the reticuloendothelial system.

In agreement with this biodistribution data, sliver enhancementof AuNP in the tumor biopsies also revealed the presence ofAuNP inside tumor tissues (Fig. 3b).

AuNP radiosensitized melanoma cells. B16F10 cells weretreated with AuNP and assessed for radiosensitization byclonogenic cell survival immediately after irradiation (Fig. 4).Our result revealed that AuNP radiosensitized B16F10 melanomacells in the colony formation assay.

Fig. 2. Gold nanoparticles (AuNP) colocalized with endoplasmic reticulum(ER) and Golgi in cells. B16F10 cells were cultured with AuNP for 20 h.The localization of AuNP was determined by silver enhancement, andthe (a) ER-Tracker Red dye and (b) BODIPY TR Ceramide Golgi Trackerwere used for live-cell ER and Golgi labeling (bar = 50 μm; oiginalmagnification 200×; BF, bright field).

Fig. 3. Biodistribution of gold nanoparticles (AuNP) in mice. (a)Twenty-four hours after AuNP injection, tissues of tumor-bearing micewere excised, processed, and used for AuNP detection using atomicabsorption detection. (b) Silver staining of AuNP inside a tumor. Twenty-four hours after AuNP injection, tumors were excised and paraffin-embedded. Five-micrometer thick sections from each representativespecimen were obtained and then processed with the silver enhancementkit (bar = 200 μm). PBS, phosphate-buffered saline.

Fig. 4. Gold nanoparticles (AuNP) radiosensitized melanoma cells.B16F10 cells were treated with AuNP (10 nM for 18 h) and assessed forradiosensitization by clonogenic cell survival immediately after irradiation.For the survival curves, each data point represents the average of threeindependent experiments each plated in triplicate ± SD (solid line,control; dotted line, 10 nM AuNP; *P = 0.02).

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Antitumor effects of the combination of AuNP with radiotherapyin tumor-bearing mice. To investigate whether the combination ofAuNP and radiotherapy resulted in better antitumor effects interms of tumor growth and survival than radiation alone, asyngeneic melanoma model was used in the animal study. Ourresult revealed that the tumor growth was both retarded in micereceiving either radiation alone or receiving AuNP followedby radiation (Fig. 5a) compared to the controls with noradiation. More importantly, tumor volume in the combinationtherapy group was significantly smaller compared with that inradiation alone group (P < 0.05), whereas administration ofAuNP or PBS alone did not exert any antitumor effect on tumor-bearing mice (Fig. 5a).

Furthermore, the Kaplan–Meier survival curves of the treatedgroups are illustrated in Figure 5b. The survival of mice with radia-tion and AuNP combination therapy was significantly longerthan that of the radiation alone mice (P < 0.05; log-rank test).

Apoptosis in tumors with AuNP and radiotherapy combinationtherapy. Increasing evidence has indicated the importantcontributing role of apoptosis in radiation-induced cell deathand for apoptosis as a determinant of radiosensitivity.(30–32) In thepresent study, we examined whether apoptosis was associatedwith the antitumor effects of combination therapy. As shown inFigure 6a, the extent of apoptosis observed in TUNEL-stained

cryosections was found higher after a single-dose radiotherapycompared to that in the no radiation controls. Noticeably, thenumber of apoptotic cells detected was significantly higher inthe AuNP and radiation combination group than that in theradiation alone group (P < 0.05). The quantitative results arerepresented in Figure 6b.

Fig. 5. Antitumor effects of the combination treatment of goldnanoparticles (AuNP) and radiotherapy in tumor-bearing mice. C57BL/6mice were inoculated subcutaneously with B16F10 cells (1 × 106) at day0. At day 7, tumor-bearing mice were injected intravenously with 200 μLof 200 nM AuNP, or with 200 μL of phosphate-buffered saline (PBS) 24 hbefore irradiation (25 Gy/mouse). Mice were monitored for (a) tumorgrowth and (b) survival (n = 4–7; *P < 0.05). RT, radiotherapy.

Fig. 6. Apoptotic activity was analyzed by terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling(TUNEL) assay. Tumors were excised from mice 6 h postradiotherapy.Five-micrometer thick representative cryostat sections were obtainedand then processed with the TUNEL System. (a) Positive TUNEL strainingwas observed under a fluorescent microscopy. (b) Quantitative analysis.Apoptotic cells were calculated by averaging the number of positiveTUNEL signals from eight fields with highest density of TUNEL signals ineach section (n = 8; *P < 0.05; original magnification 40×). RT, radiotherapy.

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Discussion

In the present study, our important finding was that incombination of AuNP with clinical electron beams in radiotherapy,an increase of apoptotic potential was observed in TUNEL-stained cryosections of the tumors in mice. Knowing thatradiation-induced apoptosis has been considered a determinantof tumor responses to radiation therapy, and the length oftumor regrowth delay correlated with the extent of apoptosisafter single-dose radiotherapy,(30) this result indicates theclinical potential of AuNP in improving the outcome of cancerradiotherapy.

Using sliver enhancement and fluorescent staining, we wereable to determine the localization of AuNP inside B16F10 cells.We detected the presence of AuNP in cells after 18 h and 42 hof incubation, and furthermore, we found that AuNP werecolocalized with ER and Golgi staining rather than with thenucleus of cells. It has been shown that continuous ER stressresults in apoptotic cell death;(38) therefore, the accumulation ofAuNP in ER and Golgi may also contribute to the increase ofthe apoptotic potential of cells postirradiation.

Recently, considerable investigations have been made inexploring the role of apoptosis in cellular radiation responses.Although the contribution of cellular apoptotic potential to overallradiosensitivity and tumor responses to radiotherapy has beendebated for several years, increasing evidence suggests thatrestoring the tumor apoptotic potential may have considerabletherapeutic impact.

In the current study, we demonstrated the dose-enhancingeffect of AuNP in conjunction with single-dose clinical electronbeams in a B16F10 melanoma tumor-bearing model, in whichthe tumor growth was retarded and the mice survival wasprolonged. These effects were consistent with the results for aprevious mammary tumor model;(16) however, many less AuNPwere injected intravenously into the mice in this study (1 g/kgcompared to 2.7 g/kg). Besides, in the current study, the irradiationtime-point, 24 h post-AuNP injection, was used instead of atime-point of 2 min postinjection. Accumulation of unlabelednanoparticles within tumor occurred through the enhanced

permeability and retention effect, which takes advantage of thepoorly formed tumor vasculature.(24–28) Therefore, the time-pointwe used for radiation after AuNP injection may be of benefit inproducing high tumor to muscle gold ratio. (Fig. 3a and (39)). Inaddition, by using electron beams produced by a linear acceleratorin place of kilovoltage X-rays, higher dose uniformity within thetarget tumor may be achieved.(8)

A recent study observed that the cellular uptake of AuNPpeaked at diameters of 50 nm, in which spherical nanoparticleswith diameters of 14, 30, 50, 74, and 100 nm were used.(40)

Since nanoparticle dose enhancement will be greatest for increasedcellular uptake, 13 nm AuNP used in this study may havegreater benefit than 1.9 nm AuNP used in the previous study,(16)

from a cellular uptake viewpoint.In this study, radiation was given as a large single dose. However,

in clinical radiotherapy, it is common practice to deliver the totaldose as multiple small fractions in order to reduce normal tissuetoxicity. Previous studies suggest that fractionated radiotherapyinduces an accumulation of cell death by apoptosis proportionalto the number of fractions. This may exceed the number ofapoptotic cells induced by a high single dose.(30,41,42) Therefore,using multiple fractions may be of significance in combinationtherapy of AuNP and radiation to induce more apoptotic cellsand thereby improve the therapeutic ratio and survival of tumor-bearing mice.

In conclusion, this study suggests that AuNP and radiotherapycombination therapy may have therapeutic potential for thetreatment of melanoma. Our results demonstrate that intrave-nous injection of AuNP combined with clinical electron beamssignificantly retards the tumor growth and prolongs survival ofmice. Increasing apoptotic potential in tumors may play animportant role in this combination therapy.

Acknowledgments

This work was supported by grant H93-A930 from the Center for FrontierMaterials and Micro/Nano Science and Technology, National ChengKung University, Taiwan. We appreciated the expert technical supportfrom Li-Yao Chang (National Cheng Kung University Hospital, Taiwan).

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