Laser Heating of Gold Nanospheres Functionalized with Octreotide: In Vitro Effect on HeLa Cell Viability

Abstract

Objective: The aim of this study was to assess the effect of laser heating a well-characterized gold nanoparticle (AuNP)-octreotide system on HeLa cell viability, to evaluate its potential as a suitable agent for plasmonic photothermal therapy. Background data: Octreotide is a synthetic peptide derivative of somatostatin with an effect on the survival of HeLa cells. Peptides bound to AuNPs are biocompatible and stable multimeric systems with target-specific molecular recognition. Methods: Octreotide was conjugated to AuNPs (∼20 nm) by spontaneous reaction with the thiol groups. The nanoconjugate was characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), ultraviolet visible spectroscopy (UV-Vis), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. Irradiation experiments were conducted using an Nd:YAG laser pulsed for 5 ns at 532 nm with a repetition rate of 10 Hz for up to 6 min while delivering an average irradiance of 0.65 W/cm². HeLa cells were incubated at 37°C (1) with AuNP-citrate, (2) with AuNP-octreotide, or (3) without nanoparticles. Results: After laser irradiation, the presence of AuNP caused a significant increase in the temperature of the medium (48°C vs. 38.3°C of that without AuNP). The AuNP-octreotide system resulted in a significant decrease in cell viability of up to 6 % compared with the AuNP-citrate system (15.8±2.1%). Two possible mechanisms could be at play: (1) octreotide alone exerts an effect on survival HeLa cells, or (2) the release of heat (∼727°C per nanoparticle) in the membranes or cytoplasm of the cells caused by the interaction between AuNP-octreotide and somatostatin receptors reduced viability. Conclusions: The AuNP-octreotide system exhibited properties suitable for plasmonic photothermal therapy in the treatment of cervical cancer.

Introduction

O ctreotide is a synthetic peptide derivative of natural somatostatin with high metabolic stability. The inhibitory effects of octreotide are mediated by specific interactions with high-affinity somatostatin receptors. Somatostatin exerts a direct effect on survival HeLa and gerbil fibroma cells in culture, and octreotide can inhibit the tumor cell growth in culture and in tumors from animal models.^1
–3

Several studies have demonstrated that conjugating peptides to gold nanoparticles (AuNPs) produces biocompatible and stable multimeric systems with target-specific molecular recognition.^4

–9

Gold nanorods and silica-gold nanoshells have been shown to be useful as photo-absorbing agents in photothermal therapy, because of their strong and tunable linear absorption in the near-infrared (NIR) region where tissues are optically transparent.^10,11 However, compared with nanorods and nanoshells, gold nanospheres are especially interesting because of the ease of their synthesis and bioconjugation. Recently, Elbialy et al.¹² demonstrated irreversible thermal tumor damage in a subcutaneous Ehrlich carcinoma mouse model using spherical AuNPs irradiated with a low-power argon laser (514 nm). Using gold nanospheres in photothermal cancer therapy can also be performed with short NIR laser pulses to generate a second harmonic or a two-photon absorption process.¹³ Second harmonic generation converts the NIR photons into visible photons (400 nm), which are then absorbed by the gold nanospheres through surface plasmon absorption and electron inter-band transition from the d band to the sp band with the consequent conversion of their energy into heat. NIR photons could also be directly absorbed and converted into heat through a nonlinear two-photon absorption process caused by an aggregated alignment of the nanoparticles bound to cancer cells.¹³

Cervical cancer can be considered a superficial tumor in the early stages, such as stage IB1, during which the tumor size is ≤4 cm and restricted to the cervix. For therapeutic purposes, gold nanospheres functionalized with octreotide (AuNP-octreotide) could be administered by intratumoral injection to patients with cervical cancer to achieve a high level of uptake in the tumor by active and passive mechanisms and, therefore, attain adequate conditions for plasmonic photothermal therapy.

The aim of this study was to assess the in vitro effect of laser heating a well-characterized AuNP-octreotide system on HeLa cell viability, to evaluate its potential as a suitable agent for plasmonic photothermal therapy.

Materials and Methods

Synthesis of AuNPs (AuNP solution)

AuNPs were prepared by citrate reduction of tetrachloroauric acid according to the method introduced by Turkevich et al.¹⁴ Before the reduction process, all glassware was cleaned in “aqua regia” (i.e., 3 parts HCl, 1 part HNO₃), rinsed with deionized water, and placed in a dry heat sterilizer (Felisa^(R), Mexico). The AuNP preparation was performed under aseptic conditions in a Good Manufacturing Practice (GMP)-certified facility. An aqueous solution (100 mL, injectable-grade water) of 1.7 mM trisodium citrate dehydrate (Sigma-Aldrich) was heated to boiling and stirred continuously. Next, 0.87 mL of 1% tetrachloroauric acid (HAuCl₄·3H₂O, Sigma-Aldrich) was added quickly, resulting in a change in solution color from pale yellow to black to deep red. The AuNP solution was dialyzed against injectable-grade water (two times with 0.5 L) for 6 h, reduced to 1/20 of the original volume under vacuum and sterilized by membrane filtration (Millipore, 0.22 μm).

Absorption spectra in the range of 400–700 nm were obtained with a PerkinElmer LAMBDA Bio spectrometer using a 1 cm Quartz Cuvette to monitor the characteristic AuNP surface plasmon band at 522 nm (AuNP 23±2 nm, 1.12×10¹³ particles/mL, OD_λ=521=0.75 diluted 1/20).

Conjugation of octreotide to AuNPs

The octreotide acetate peptide (H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr[ol], molecular weight [MW] 1019.24 g/mol) was supplied by ABX GmbH (Radeberg, Germany) with a purity of >98%, as analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectroscopy.

A 50 μM solution of octreotide was prepared using injectable-grade water. Next, 100 μL (3.011×10¹⁵ molecules) of the octreotide solution was added to 1.5 mL (1.68×10¹³ particles) of the AuNP solution followed by stirring for 15 min. The mixture was sterilized by membrane filtration (Millipore, 0.22 μm). Under these conditions, an average of 179 molecules of octreotide were attached to each nanoparticle (23 nm, surface area=1660 nm², 48,900 surface Au atoms) and it was confirmed by titration as previously reported.^4,6 No further purification was required (Fig. 1).

FIG. 1.

Overall scheme of the octreotide peptide conjugated to gold nanoparticles.

Characterization of AuNP-octreotide

Transmission electron microscopy (TEM)

Gold nanoparticles conjugated to octreotide were characterized in size and shape by TEM using a Jeol JEM 2010 high throughput (HT) microscope operating at 200 kV. Samples were prepared for analysis by evaporating a drop of the aqueous product onto a carbon-coated TEM copper grid.

Ultraviolet visible spectroscopy (UV-Vis)

Absorption spectra from 400 to 700 nm were obtained with a PerkinElmer LAMBDA Bio spectrometer using a 1 cm Quartz Cuvette. Nanoconjugates were measured by UV-Vis spectrometry to monitor the shift in the AuNP-octreotide surface plasmon band (522 nm).

Raman spectroscopy

Raman spectra of AuNP-octreotide and octreotide were performed on a MicroRaman OLYMPUS BX 41 spectrometer with a wavelength of 632.817 nm, a D1 filter and a 100 Micro Hole. Ten scans of 60 sec were acquired. Ten microliter aliquots of the sample were deposited and dried on a pretreated cover-glass [prewashed in three steps: 1HNO3:3HCl (v/v) solution, bi-distilled water and injectable water] under nitrogen atmosphere at room temperature in a laminar flow hood.

X-ray photoelectron spectroscopy (XPS)

XPS of AuNP and AuNP-octreotide was performed with a K-Alpha Thermo Scientific spectrometer equipped with an MgK_α X-ray source (1253.6 eV). The source was operated at 10 kV/20 mA and calibrated using Au 4f_7/2 (84.0 eV) and Ag 3d_5/2 (368.2 eV) from foil samples. The samples were introduced into an ultra-high vacuum (UHV) chamber in the spectrometer (5×10⁻⁹ torr) and measured at 24°C (room temperature). The spot size in the beam was 100 μm. The pressure did not change during the analysis, and 20 scans for Au 4f were performed with an energy step size of 0.1 eV. The binding energies were referenced to the C1s peak at 284.3 eV. A Shirley background was subtracted from all spectra for peak fitting with a symmetric Gaussian-Lorentzian sum function (SpecSurf software).

Far-IR

The far-IR spectra of AuNP-octreotide was acquired on a PerkinElmer spectrometer (Spectrum 400) with an ATR platform (Diamond GLADIATR, Pike Technologies) using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy from 190 to 300 cm⁻¹.

Cell culture

HeLa cells of epithelial origin were derived from an epidermoid carcinoma of a human cervix. These cells harbor transcriptionally active sequences from human papilloma virus 18 (HPV18), including the E6 and E7 oncogenes, which cause HeLa cells to progress through the cell cycle without interruption. The cell line was originally obtained from ATCC (Atlanta, GA). The cells were routinely grown at 37°C, with 5% CO₂ and 100% humidity in minimum essential medium eagle (MEM, Sigma-Aldrich Co., Saint Louis, MO) supplemented with 10% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin).

Laser irradiation

All experiments were conducted using an Nd:YAG laser (Minilite, Continuum^®, Photonic Solutions, Edinburgh, U.K.) pulsed for 5 ns at 532 nm (energy=25 mJ/pulse) with a repetition rate of 10 Hz. The per pulse laser power was measured by using a Dual-Channel Joulemeter/Power Meter (Molectron EPM 2000, Coherent). A diverging lens was used in the path of the laser beam such that the well plate was fully covered by the laser (diameter=7 mm, area=0.38 cm²). The irradiance at the well plate was then calculated as the per pulse laser power divided by the laser spot area. Irradiation was performed for 6 min while delivering 0.65 W/cm² of average irradiance.

HeLa cells supplied with fresh medium were incubated in a 96 well plate at a density of 1×10³ cells/well. The cells were cultured for 24 h at 37°C, with 5% CO₂ and 100% humidity. The growth medium was removed, the well plate was placed in a dry block heater at 37°C and the cells were exposed to one of the following treatments (n=5): (1) 100 μL of AuNP-citrate and 100 μL of phosphate-buffered saline (PBS), pH 7 with irradiation (0.65 W/cm²); (2) 100 μL of AuNP-octreotide and 100 μL of PBS pH 7 with irradiation (0.65 W/cm²); (3) 100 μL of distilled water (without nanoparticles), 100 μL of PBS pH 7 with irradiation (0.65 W/cm²); or (4) no treatment. During laser irradiation, the temperature increase was measured using a type k thermocouple (model TP-01) of immediate reaction previously calibrated (probe diameter=0.8 mm). The thermocouple was introduced into the well and the temperature registered each minute using a digital multimeter connected to a computer. After irradiation, the solution of each well was removed and replaced with fresh growth medium.

The percentage of surviving cells in each well was evaluated by the spectrophotometric measurement of cell viability as a function of mitochondrial dehydrogenase activity, which involves cleavage of the tetrazolium ring of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in viable cells to yield purple formazan crystals that are dissolved in acidified isopropanol (MTT kit, Sigma-Aldrich Co., Saint Louis, MO). The resulting absorbance of the purple solution was measured at 595 nm in a microplate absorbance reader (iMark^™, Bio-rad, USA). The absorbance of the untreated cells was considered to be 100% HeLa cell viability.

Statistical analysis

Differences in the in vitro cell data were evaluated with the Student's t-test (significance was defined as p<0.05).

Results

TEM

A mono-dispersed solution of AuNP-citrate was obtained with particles that have an average diameter of 23±2 nm. TEM images of AuNP-octreotide indicated the presence of a dispersed solution with an increased hydrodynamic diameter (25.9±2 nm). A “corona” was observed around the AuNPs because of the poor interaction of the electron beam with the peptide molecules (low electron density), which is in contrast to the strong scattering of the electron beam when it interacted with the metallic nanoparticles (Fig. 2).⁹

FIG. 2.

Transmission electron microscopy image of gold nanoparticle (AuNP)-octreotide.

Far-IR spectroscopy

AuNP-octreotide exhibited a characteristic band at 279±1 cm⁻¹, which was assigned to the Au-S bond (Fig. 3).¹⁵ In agreement with previous reports, at least three main peaks of the ν_Au-S were found in the range of 200–280 cm⁻¹, which were attributed to multiple adsorption sites.^15,16

FIG. 3.

Far-infrared spectrum of gold nanoparticle (AuNP)-octreotide. The characteristic band assigned to the Au-S bond at 279±1 cm⁻¹ is shown.

UV–Vis spectroscopy

The 23 nm AuNP-citrate spectrum showed surface plasmon resonance (SPR) at 522 nm. A red shift to 524 nm was observed with AuNP-octreotide because of changes in the refraction index and the surrounding dielectric medium as a consequence of the interactions between the peptides and the AuNP surfaces.⁹

XP spectroscopy

The AuNP-citrate spectrum (Fig. 4) showed two main peaks that corresponded to the binding energies (B.E., eV) of electrons in the Au 4f orbitals at 87.4 eV (Au4f_5/2) and 83.7 eV (Au4f_7/2), with the characteristic difference of 3.7 eV and an intensity ratio of 3:4 between the two peaks.¹⁷ The deconvoluted AuNP-octreotide spectrum (Fig. 4) displayed a peak at 87.8 eV with a positive shift of 0.4 eV with respect to the Au 4f_5/2 of AuNP-citrate. The peak that correlated with the Au 4f_7/2 orbital appeared at 84.2 eV. The orbital energies of Au-Au or AuNP (Au⁰) and Au-S (Au⁺¹) bonds are related to changes in their oxidative states (Au⁰ to Au⁺¹); therefore, the shift of electron binding energies to higher values is an intrinsic property of the interaction between gold core electrons and the octreotide peptide.¹⁷

FIG. 4.

X-ray photoelectron spectroscopy (XPS) spectra of gold nanoparticle (AuNP)-octreotide and AuNP-citrate. The experimental bands are represented by dotted lines, and the deconvoluted experimental bands are represented by solid lines.

Raman spectroscopy

AuNP functionalized with octreotide yielded a structured Raman spectrum in the 1800–900 cm⁻¹ region (Fig. 5) (Table 1).

FIG. 5.

Raman spectra of gold nanoparticle (AuNP)-octreotide and AuNP-citrate.

Table 1.

Main Raman Vibration Frequencies (cm ⁻¹) for Octreotide and AuNP-Octreotide

Functional group (vibrations)	Octreotide	AuNP-octreotide
C-H (sym stretch) aromatic ring in phenylalanine	1000, 1008	998
CH₃, CH₂: H-C- deformation	1430, 1457	1430, 1463
Amide I, (C=O stretch)	1664, 1650	1645, 1697
Amide II, (N-H bending mostly)	1579	1569, 1583
Amide III, (C-N stretch)	1204	1265
C-O- stretch	1156, 1078, 1029	1076, 1028
C-N (Fermi resonance) (Trp residue)	1341	1379
C=C (indole)	1551	-
HO- alcohol (bend)	1288	1272

Cell viability after laser irradiation

As shown in Fig. 6, the presence of AuNPs caused the temperature of the medium to increase significantly under the laser irradiated conditions described previously (48°C vs. 38.3°C without AuNP, p<0.05). As expected, there was no difference between AuNP-citrate and AuNP-octreotide in the temperature increase, as it is only determined by the size and concentration of AuNPs in the medium.

FIG. 6.

Increase in temperature upon laser heating of gold nanoparticle (AuNP)-citrate, AuNP-octreotide, and the no-nanoparticle control in HeLa cell cultures. (Irradiance 0.65 W/cm²).

AuNP-citrate and AuNP-octreotide significantly inhibited HeLa cell viability with respect to cells treated without nanoparticles during laser irradiation (p<0.05) (Fig. 7). However, the AuNP-octreotide system caused a significant decrease in cell viability (p<0.05) of up to 6 % by the end of treatment (6 min) compared with AuNP-citrate (15.8±2.1 %) (Fig. 7).

FIG. 7.

Effect of laser heating of gold nanoparticle (AuNP)-citrate, AuNP-octreotide, and the no-nanoparticle control on HeLa cell viability. (Irradiance 0.65 W/cm²).

Discussion

TEM and spectroscopy techniques demonstrated that AuNPs were functionalized with octreotide.

Raman spectra showed several well-defined bands that were observed with vibrational frequencies in the region corresponding to the main functional groups seen in the octreotide spectrum, but specific to AuNP-octreotide because of shifts to lower or higher energies and increases in the band intensities (Fig. 3). The 1341 cm⁻¹ band observed in the octreotide spectrum is assigned to the indole group (N1-C8, Fermi resonance) (Trp residue) and in the AuNP-octreotide spectrum the band shifts to higher energy (1379 cm⁻¹) and increases the intensity, which is a marker of the hydrophobicity of the environment of the indole ring (N1-C8) on the AuNP.⁵ The band at 1551 cm⁻¹ indicates that the indole ring also interacts through the pyrrole C2=C3 with the metal surface. Interaction with the benzene ring was detected (1008, 1000 cm⁻¹ in the octreotide spectrum and 998 cm⁻¹ in AuNP-octreotide spectrum).⁵ AuNP interactions with amide groups AI (C=O, stretch), AII (N-H, bend), and AIII (C-N stretch) were also observed (1664, 1579, and 1204 cm⁻¹, respectively).^5,6

In agreement with Petroski et al.,¹⁵ the group vibrations occurring in the far-IR region, such as the Au-S stretch as well as the C-S stretch and C-C-C- or S-C-C deformations, are all within a bond away from the particle surface, or thereabouts. Possibly multiple peaks of the ν_Au-S (range of 200–280 cm⁻¹) reflect local heterogeneities for different kinds of binding sites on the particle surface. A nanoparticle surface contains various types of defect sites, which can include edge, ledge, step, or kink, among others.¹⁵

Thiol groups of the broken octreotide cysteine disulfide bridges are known to be cleaved upon interaction with Au, because the Au–S bond strength approximates the S–S bond strength (∼40 kcal/mol compared with ∼50 kcal/mol) (Fig. 1).¹⁸

Several trials have shown a significant improvement in clinical outcome when radiotherapy, chemotherapy, or both were performed under hyperthermic conditions in patients with advanced cervical cancer.^19

–22 Hyperthermia increases the efficacy of radiotherapy by improving tumor oxygenation and interfering with DNA repair mechanisms.¹⁹ In combination with chemotherapy, hyperthermia increases the drug concentration in the tumor area.²⁰ However, current techniques for hyperthermia induction display low spatial selectivity for the tissues that are heated. Lasers have been used for inducing hyperthermia, and spatial selectivity can be improved by adding AuNPs to the tissue to be treated.^10,11 By exposing nanoparticles to laser irradiation, it is possible to heat a localized area in tumors without any harmful heating of surrounding healthy tissues. Previous studies using AuNP for hyperthermia have demonstrated that the functionalization of AuNPs with probe molecules, such as AuNP-RGD, AuNP-anti-epidermal growth factor receptor (EGFR) or AuNP-anti-Her2, improves the particle accumulation in cell models significantly.^23,24 In this study, we have demonstrated that the conjugation of octreotide to AuNPs significantly reduces HeLa cell viability compared with AuNP-citrate. It is proposed that two mechanisms could be at play: (1) octreotide itself exerts an effect on the viability of HeLa cells, and (2) the release of heat (∼727°C around each nanoparticle²⁵) in the membrane or cytoplasm of HeLa cells, caused by the interaction between AuNP-octreotide and somatostatin receptors, reduces viability.

Because of the nonlinear optical properties of spherical gold nanoparticles,¹³ the AuNP-octreotide system developed in this study could also be applied to plasmonic photothermal therapy using a NIR laser for the treatment of localized cervical cancer because the NIR window is ideally suited for in vivo applications because of the minimal light absorption by hemoglobin and water.¹⁰

Conclusions

TEM and spectroscopy techniques demonstrated that AuNPs can be successfully conjugated to octreotide through interactions with the thiol groups. The conjugation of octreotide to AuNPs significantly reduced HeLa cell viability when compared with AuNP-citrate following laser irradiation. The AuNP-octreotide system exhibited properties suitable for plasmonic photothermal therapy in the treatment of cervical cancer.

Footnotes

Acknowledgments

This study was supported by the Mexican National Council of Science and Technology (CONACYT-SEP-CB-2010-01-150942).

Author Disclosure Statement

No competing financial interests exist

References

Reubi

J. C.

, Maurer

1985. Autoradiographic mapping of somatostatin receptors in the rat central nervous system and pituitary. Neuroscience, 15:1183–1193.

Mascardo

R.N.

, Sherline

1982. Somatostatin inhibits rapid centrosomal separation and cell proliferation induced by epidermal growth factor. Endocrinology, 111:1394–1396,

Evers

, Parekh

, Townsend

C.M.

, Thompson

J.C.

1991. Somatostatin and analogues in the treatment of cancer. Ann. Surg., 213:190–198.

Ocampo–Garcia

, Ferro–Flores

, Morales–Avila

, Ramirez

, de

. 2011. Kit for preparation of multimeric receptor-specific ^99mTc-radiopharmaceuticals based on gold nanoparticles. Nucl. Med. Commun., 32:1095–1104.

Mendoza–Sanchez

, Ferro–Flores

, Ocampo–Garcia

et al. 2010. Lys³-Bombesin conjugated to ^99mTc-labelled gold nanoparticles for in vivo gastrin releasing peptide-receptor imaging. J. Biomed. Nanotechnol., 6:375–384.

Morales–Avila

, Ferro–Flores

, Ocampo–Garcia

et al. 2011. Multimeric system of ^99mTc-labeled gold nanoparticles conjugated to c[RGDfK(C)] for molecular imaging of tumor α(v)β(3) expression. Bioconjugate Chem., 22:913–922.

Luna–Guiterrez

, Ferro–Flores

, Ocampo–Garcia

et al. 2012. ¹⁷⁷Lu-labeled monomeric, dimeric and multimeric RGD peptides for the therapy of tumors expressing αvβ3 integrins. J. Label. Compd. Radiopharm., 50:140–148.

Jimenez–Mancilla

, Ferro–Flores

, Ocampo–Garcia

et al. 2012. Multifunctional targeted radiotherapy system for induced tumors expressing gastrin-releasing peptide receptors. Curr. Nanosci., 18:193–201.

Ferro-Flores

, Ocampo-Garcia

B.E.

, Ramirez

, de

, Gutierrez-Wing

, Arteaga de Murphy

, Santos-Cuevas

C.L.

2010. Gold nanoparticles conjugated to peptides. Colloids in Biothechnology. Fanum

Boca Raton, FL: CRC Press/Taylor & Francis, 231–252.

10.

Hirsch

L.R.

, Stafford

R.J.

, Bankson

J.A.

et al. 2003. Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance. Proc. Natl. Acad. Sci. U.S.A., 100:13,549–13,554.

11.

Huang

, Neretina

, El-Sayed

M.A.

2009. Gold nanorods: from synthesis and properties to biological and biomedical applications. Adv. Mater., 21:4880–4910.

12.

Elbialy

, Abdelhamid

, Youssef

2010. Low power argon laser-induced thermal therapy for subcutaneous ehrlich carcinoma in mice using spherical gold nanoparticles. J. Biomed. Nanotechnol., 6:687–693.

13.

Huang

, Qian

, El-Sayed

I.H.

, El-Sayed

M.A.

2007. The potential use of the enhanced nonlinear properties of gold nanospheres in photothermal cancer therapy. Lasers Surg. Med., 39:747–753.

14.

Turkevich

, Stevenson

P.C.

, Hillier

1951. Nucleation and growth process in the synthesis of colloidal gold. Discuss. Faraday Soc., 11:55–75.

15.

Petroski

, Chou

, Creutz

2009. The coordination chemistry of gold surfaces: formation and far-infrared spectra of alkanethiolate-capped gold nanoparticles. J. Organ. Chem., 694:1138–1143.

16.

Kato

H.S.

, Noh

, Hara

, Kawai

2002. An HREELS study of alkanethiol self-assembled monolayers on Au(111) J. Phys. Chem. B., 106:9655–9658.

17.

Joseph

, Besnard

, Rosenberger

et al. 2003. Self-assembled gold nanoparticle/alkanedithiol films: preparation, electron microscopy, XPS-analysis, charge transport, and vapor-sensing properties. J. Phys. Chem. B., 107:7406–7413.

18.

Whitesides

G.M.

, Laibinis

P.E.

1990. Wet chemical approaches to the characterization of organic surfaces: self-assembled monolayers, wetting, and the physical–organic chemistry of the solid–liquid interface. Langmuir, 6:87–96.

19.

Franckena

, Stalpers

L.J.A.

, Koper

P.C.M.

et al. 2008. Long-term improvement in treatment outcome after radiotherapy and hyperthermia in locoregionally advanced cervix cancer: An update of the Dutch Deep Hyperthermia Trial. Int. J. Radiat. Oncol. Biol. Phys., 70:1176–1180.

20.

Franckena

, de Wit

, Ansink

A.C.

et al. 2007. Weekly systemic cisplatin plus locoregional hyperthermia: an effective treatment for patients with previously irradiated recurrent cervical carcinoma in a previously irradiated area. Int. J. Hyperthermia, 23:443–450.

21.

Franckena

, Lutgens

L.C.H.W.

, Koper

P.C.M.

et al. 2009. Radiotherapy and hyperthermia for treatment of primary locally advanced cervix cancer: results in 378 patients. Int. J. Radiat. Oncol. Biol. Phys., 73:242–250.

22.

Franckena

, Fatehi

, de Bruijne

et al. 2009. Hyperthermia dose-effect relationship in 420 patients with cervical cancer primarily treated with combined radiotherapy and hyperthermia. Eur. J. Cancer, 45:1969–1978.

23.

Huang

, El-Sayed

I.H.

, El-Sayed

. 2010. Applications of gold nanorods for cancer imaging and photothermal therapy. Methods Mol. Biol., 624:343–357.

24.

Huang

, Peng

, Wang

et al. 2010. A reexamination of active and passive tumor targeting by using rod-shaped gold nanocrystals and covalently conjugated peptide ligands. ACS Nano., 4:5887–5896.

25.

Letfullin

R.R.

, Iversen

C.B.

, George

T.F.

2011. Modeling nanophotothermal therapy: Kinetics of thermal ablation of healthy and cancerous cell organelles and gold nanoparticles. Nanomedicine, 7:137–145.