more efﬁcient nir photothermal therapeutic effect from ...the synthesis of gnrs was referred to...

RESEARCH PAPER

More efficient NIR photothermal therapeutic effectfrom intracellular heating modality than extracellularheating modality: an in vitro study

Wenbo Zhou • Xiangshen Liu • Jian Ji

Received: 28 March 2012 / Accepted: 10 August 2012 / Published online: 31 August 2012

� Springer Science+Business Media B.V. 2012

Abstract In this study, efforts were placed in giving

some in vitro key clues to the question on which is more

efficient for the cancer hyperthermia between intracel-

lular and extracellular modalities. Near infrared (NIR)

photothermal responsive gold nanorods (GNRs) were

adopted to cause cellular thermolysis either from inside

or outside of cells. GNRs were synthesized with the size

of 30.4 nm (in length) 9 8.4 nm (in width). Demon-

strated by ICP-MS (inductively coupled plasmon mass

spectroscopy), UV–Vis spectroscopy and transmission

electron microscopy analyses, various cell uptake doses

of nanoparticles were differentiated due to different

molecular designs on GNRs surfaces and different

types of cells chosen (three cancer cell lines and three

normal ones). Under our continuous wavelengths (CW)

NIR irradiation, it resulted that the cells which inter-

nalized GNRs died faster than the cells surrounded by

GNRs. Furthermore, fluorescent images and flow

cytometry data also showed that the NIR photothermal

therapeutic effect was greater when the amount of

internalized GNRs per cell was larger. Generally

speaking, the GNRs assisted intracellular hyperthermia

exhibited more precise and efficient control on the

selective cancer ablation. To a larger degree, such a

relationship between GNRs distribution and hyperther-

mia efficiency might be applied to wider spectra of cell

types and heat-producing nanoparticles, which pro-

vided a promise for future cancer thermal therapeutic

designs.

Keywords NIR � Cell uptake � Gold nanorods �Intracellular � Hyperthermia

Introduction

As one of the modalities for cancer therapy, hyper-

thermia has merely functioned as an adjunct to other

methods like radiotherapy and chemotherapy for a

long time due to its poor therapeutic efficacy (Barlogie

et al. 1979; Wust et al. 2002; Thiesen and Jordan 2008;

Hurwitz 2010). Since the pioneering researches were

initiated by introducing nanotechnology to non-super-

ficial carcinoma thermotherapy (Gilchrist et al. 1957;

Gordon et al. 1979; Shinkai et al. 1995), great

enthusiasm has been injected to new exogenous

methods which can noninvasively convert relatively

benign electromagnetic energy to fatal thermal energy

for cell denaturation. This therapeutic improvement

was achieved by introducing highly stimuli-respon-

sive nanoagents to the tumor tissues or even cells. One

example is the localized heating effect produced by

Electronic supplementary material The online version ofthis article (doi:10.1007/s11051-012-1128-6) containssupplementary material, which is available to authorized users.

W. Zhou � X. Liu � J. Ji (&)

MOE Key Laboratory of Macromolecular Synthesis

and Functionalization, Department of Polymer Science

and Engineering, Zhejiang University, Hangzhou 310027,

People’s Republic of China

e-mail: [email protected]

123

J Nanopart Res (2012) 14:1128

DOI 10.1007/s11051-012-1128-6

http://dx.doi.org/10.1007/s11051-012-1128-6

magnetic nanoparticles in the alternating magnetic

field (Pankhurst et al. 2003; Gazeau et al. 2008), which

has already been confirmed with success both in the

lab studies and clinical trials (Moroz et al. 2002;

Shinkai 2002; Berry and Curtis 2003; Mornet et al.

2004; Campbell 2007; Zhao et al. 2010). Recently,

several other nanomaterials like carbon nanotubes

(Kam et al. 2005; Kim et al. 2007; Chakravarty et al.

2008), fullerenes (Krishna et al. 2010), graphene

(Markovic et al. 2011), gold (Norman et al. 2002;

Hirsch et al. 2003; Huang et al. 2006; Ma et al. 2009;

Chen et al. 2010), and germanium (Lambert et al.

2007) nanocrystals have also been considered for

cancer ablation. One big advantage of these materials

is their special photothermal conversion under the near

infrared (NIR) laser which is famous for its high

spatial penetration through tissues and cells (Weiss-

leder 2001; Kondepati et al. 2008).

In order to minimize side effects toward normal

tissues, it is required that these nanoparticles should be

administrated in the tumors selectively (Kam et al.

2005; Huang et al. 2006; Lambert et al. 2007; Chen

et al. 2010). One example of such modalities was

provided by us recently, using phosphorylcholine (PC)-

modified NIR photothermal responsive gold nanorods

(GNRs) to selectively target to nasopharyngeal cancer

cells and finally cause cell ablation under NIR irradi-

ation (Zhou et al. 2010). Herein, two ways existed to

satisfy this tumor targetability, both of which could be

realized by the surface modification of nanoparticles.

One common way was to endow nanoparticles with

stealth properties by polyethylene glycol (PEG) layers

(Yang et al. 2008; Lipka et al. 2010), which guaranteed

that injected nanoparticles were stable enough to evade

most of immune systems and finally to be accumulated

inside cancerous tissues owning to the so-called

enhanced permeability and retention (EPR) effect

(Baban and Seymour 1998). Compared with the above

passive targeting means, the active targeting one

adopted ligands like antibodies (Sawyers 2008; Chari

2008) and some other small molecules (Setua et al.

2010; Lai et al. 2010) as effective probes for specific

cell recognition and internalization. Although both of

these tumor-targeting modalities were widely used, the

theme on which was more efficient for cell ablation

between intracellular and extracellular thermal thera-

pies was still in hot debate (Yanase et al. 1997; Jordan

et al. 1999; Rabin 2002; Zharov et al. 2003; Sonvico

et al. 2005; Lapotko 2006; Hergt and Dutz 2007;

Gazeau et al. 2008; Prasad et al. 2008; Wen 2009;

Lukianova-Hleb et al. 2010; Zhao et al. 2010). Previ-

ously, such disagreement was mainly focused on

whether the spatial confinement of heat transfer would

cause huge influence toward thermolysis (Rabin 2002;

Szasz et al. 2003; Pitsillides et al. 2003; Zharov et al.

2003; Zharov and Lapotko 2005; Zharov et al. 2005;

Lapotko 2006; Keblinski et al. 2006; Hergt and Dutz

2007; Prasad et al. 2008; Wen 2009; Lukianova-Hleb

et al. 2010), and relative modeling and calculation were

carried out to analyze this problem (Rabin 2002; Szasz

et al. 2003; Pitsillides et al. 2003; Zharov and Lapotko

2005; Zharov et al. 2005; Keblinski et al. 2006;

Purushotham and Ramanujan 2010). In fact, those

newly initiated NIR therapeutic researches seemed not

to pay much attention to this possible difference.

It has been reported that the predominant molecular

targets of hyperthermia could be nucleic acids,

cytoplasmic proteins, and cell membranes (Barlogie

et al. 1979; Hildebrandt et al. 2002). Though these

findings were based on traditional non-specific heating

modality, they implied that different locations of

GNRs would cause different therapeutic outcome. It

intrigued us to consider if the intracellular NIR

photothermal modality was superior to the extracellu-

lar one. In this paper, primary experiments in vitro

were designed to observe the difference in the ablation

effect by locating heat generation from GNRs either

inside cells or in the solution around cells. Since

surface modification was taken to have the pivotal role

to control the locations of GNRs, four different surface

ligands, namely PC, PEG, folic acid (FA) and

galactose (Gals) were adopted to impart GNRs with

various cell uptake abilities among three pairs of cell

types, including nasopharyngeal cancer and normal

cell types (CNE-1 and rhinal epithelia), liver cancer

and normal cell types (HepG2 and L02), and lymph

cancer and normal cell types (8226 and H9). Mea-

surements of the amounts of internalized GNRs were

conducted by transmission electron microscopy

(TEM), UV–Vis spectroscopy, and ICP-MS (induc-

tively coupled plasmon mass spectroscopy). After

these cells were treated with different GNRs, they

were irradiated by NIR lasers for a series of times and

the times when the cells inside the laser spot region

died out were recorded. In these processes, the

intercellular and extracellular therapeutic efficacies

could also be reflected by the fluorescent cell images

and the flow cytometry data. The required irradiation

of 16 J Nanopart Res (2012) 14:1128

123

times for cell ablation in different conditions were

further evaluated. It was hoped that these quantitative

relationships could give some lights to the compara-

tive superiority between intracellular and extracellular

NIR photothermal therapies, which might guide future

NIR hyperthermia strategies.

Experimental section

Materials

11-Mercaptoundecylphosphorylcholine (HS-PC) and

mercaptopolyethylene glycol (HS-PEG, Mw = 2,000)

were synthesized according to the literature (Nagasaki

et al. 1997; Chen et al. 2005). Hydrogen tetrachloroa-

urate hydrate (HAuCl4�4H2O), cetyltrimethylammo-

nium bromide (CTAB, C19H42BrN), silver nitrate

(AgNO3), sodium borohydride (NaBH4), FA and

L-ascorbic acid (AA, C6H8O6) were purchased from

Sinopharm Chemical Reagent Co., Ltd. 12-Mercap-

toundecanoic acid (HS(CH2)10CO3H) was purchased

from J&K. D(?)-Galatosamine hydrochloride were

purchased from BBI. N-hydroxysulfosuccinimide

sodium salt (NHS) and N-(3-dimethylaminopropyl)-N-

ethylcarbodiimide hydrochloride (EDC) were pur-

chased from Shanghai Medpep Co., Ltd. Thiazolyl blue

tetrazolium bromide (MTT, 98 %, TLC) and fluorescein

diacetate (FDA) were from Sigma-Aldrich Inc. Naso-

pharyngeal carcinoma (CNE-1), rhinal epithelia, and

lymphosarcoma (8226) cell lines were provided by Sir

Run Run Shaw Hospital in Hangzhou. Liver cancer cell

lines (HepG2) were provided by the Second Affiliated

Hospital of Zhejiang University College of Medicine.

Liver normal cell lines (L02) were purchased from

Shanghai Institutes for Biological Science, CAS, China.

Human B lymphocyte cell lines were purchased from

Wuhan Boster Biological Technology, Ltd., China. The

RPMI 1640, H-DMEM, and DMEM cell cultivation

media were purchased from GIBCO. Fetal bovine serum

(FBS) was bought from Hangzhou Sijiqing Biological

Engineering Materials Co., Ltd., China.

Characterization

UV–Vis data were measured by a Shimadzu UV-2550

UV–Vis spectrophotometer. FT-IR data were measured

by a Vector 22 Fourier transform infrared spectrometer

from German Bruker Company. Z-potential data were

measured by Malvern Zetasizer3000HSA. ICP-MS

(inductively coupled plasmon mass spectroscopy) data

were measured by a Thermo XSENIES. TEM images

were pictured by a JEOL JEM-1230 transmission

electron microscope. The diode continuous wave laser

with wavelength of 808 nm was from Hi-Tech Opto-

electronics Co., Ltd., China. The stained cells were

imaged by the Axiovert 200 inverted microscope of

Carl Zeiss with the laser source from Bio Red

Company. Flow cytometry analyses were conducted

on the FCM FACScan from BD Biosciences.

Syntheses of GNRs@PC, GNRs@PEG,

GNRs@FA, and GNRs@Gals

The synthesis of GNRs was referred to the methods by

Murphy et al. (2005) and Nikoobakht and El-Sayed

(2003). Firstly, the gold seeds were prepared in

36.45 g l-1 CTAB solution through the reduction of

0.085 g l-1 HAuCl4 by 0.0132 g l-1 NaBH4. After

4–6 h reaction, 1.5 ml of the above solution were

added into 50 ml growth solution of 36.45 g l-1

CTAB, 0.17 g l-1 HAuCl4, 0.017 g l-1 AgNO3, and

0.09715 g l-1 AA. The mixed solution was stirred for

4–6 min, and then kept still overnight at room

temperature to finally generate GNRs@CTAB. Next,

GNRs@CTAB were purified by being centrifuged at

12,000 rpm for 10 min, decanted, and resuspended in

triply distilled water. This procedure was repeated

another time before this solution and the free HS-PC

solution were mixed at the molar ratio of 1:1 (Au

atom:PC molecule). After ligand exchange overnight,

the CTAB molecules on the surfaces of GNRs were

substituted by HS-PC. The final solution was centri-

fuged at 12,000 rpm for 10 min, and the precipitates

were redispersed in triply distilled water. The

GNRs@PC solution was filtered via a PES 0.22-lm

filtration membrane to clear up the aggregations and

bacteria before cellular experiments. The existence of

PC on the gold surface was measured by FT-IR

(m(–P=O) = 1258 cm-1, m(PO–CH2–) = 1089 cm-1)

and Z-potential (?1.1 mV). The synthesis of

GNRs@PEG was similar as above, in which the

same molar amount of HS-PC was replaced by

HS-PEG. The existence of PEG was measured by

FT-IR (m(–O–CH2–)) = 1051 cm-1) and Z-potential

(?3.2 mV). In the synthesis of GNRs@FA, one fourth

of added HS-PC amount was replaced by 2-mercap-

toethylamine. After this reaction over night, the same

J Nanopart Res (2012) 14:1128 of 16

123

molar amount of FA was added to react with the amino

groups. The existence of FA was measured by FT-IR

(m(C=C) = 1607 cm-1 and 1486 cm-1, m(–CO–NH–)

= 1694 cm-1 and 1571 cm-1) and Z-potential

(?18.64 mV). In the synthesis of GNRs@Gals, one-

fourth of added HS-PC amount was replaced by

12-mercaptoundecanoic acid. After this reaction over

night, the same molar amount of D(?)-galatosamine

hydrochloride was added to react with the carboxyl

groups. The existence of Gals was measured by FT-IR

(m(–CO–NH–) = 1631 cm-1, m(–O–CH2–) = 1049 cm-1)

and Z-potential (?26.88 mV).

Cell cultivation

The rhinal epithelia cells were primarily cultured with

Dulbecco’s Modified Eagles Medium with high glu-

cose (H-DMEM, GIBCO) plus 10 % fetus bovine

serum in an initial density of 600,000 cells ml-1 in

96-well or 6-well microplates. CNE-1 cells, L02 cells,

and H9 cells were primarily cultured with RPMI 1640

plus 10 % fetus bovine serum, 8226 cells, and HepG2

cells were primarily cultured with DMEM plus 10 %

fetus bovine serum in an initial density of 100,000

cells ml-1.

Biostability and cytotoxicity analyses

of GNRs@PC toward different cells

The colloidal stabilities of GNRs@PC, GNRs@PEG,

GNRs@Gals, and GNRs@FA were tested in two cell

cultivation media RPMI 1640 and H-DMEM. The as-

prepared GNRs@PC, GNRs@PEG, GNRs@Gals, or

GNRs@FA was mixed with them to maintain the peak

absorbance of GNRs at 0.2 in the UV–Vis spectra. The

peak wavelength and half-width were monitored for

1 day at 37 �C. Each experimental point during the

UV–Vis measurements was repeated for three times

and the data were represented as mean ± SD

These six cell types were also tested by the

MTT assay for the cytotoxicity of GNRs@PC,

GNRs@PEG, GNRs@FA, and GNRs@Gals toward

them. The cultured cells in the 96-well microplates

were maintained at 37 �C in a humidified atmosphere

of 5 % CO2. When the cell growth reached to nearly

100 % confluence, four kinds of GNRs solutions of

several diluted concentrations were added to the culture

wells to reach a final volume of 100 ll mixed with the

cell cultivation media. The highest concentration of

GNRs in the above mixed media was 0.025 mM. After

12 h, cell viability was determined using the 3-(4,

5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT) assay. When the formation of formazan

dye took place 4 h after adding 10 ll MTT reagent to

the well, the media were replaced by 150 ll DMSO for

dissolving the formazan crystals. After aging for

10 min, the plate was transferred to spectrophotometric

measurements at 550 nm. Each experimental point was

expressed as mean ± SD.

UV–Vis spectroscopic assays of the cell uptake

of GNRs@PC, GNRs@PEG, GNRs@FA,

and GNRs@Gals

The cultured cells in the 96-well microplates were

maintained at 37 �C in a humidified atmosphere of

5 % CO2. When the cell growth reached to nearly

100 % confluence, the cultivation medium in each

well was replaced by fresh one and the GNRs@PC

solution was added into the medium with the final

peak absorbance of GNRs@PC in the NIR spectra

equaling 0.2. The volume of the mixed solution

above cells was kept 100 ll per well. After a serial

of time up to 24 h, the mixed solutions in the

wells were removed out for UV–Vis measurements.

It should be noted that according to the Beer–

Lambert law, the measured absorbance changes of

GNRs at their characteristic NIR peak wave-

length could be transformed to the concentration

changes of gold. The experimental procedures for

GNRs@PEG, GNRs@FA, and GNRs@Gals were

similar except that the same amount of GNRs@PC

was replaced by GNRs@PEG, GNRs@FA, and

GNRs@Gals, respectively. The data were repre-

sented as mean ± SD.

ICP-MS assays of the cell uptake of GNRs@PC,

GNRs@PEG, GNRs@FA, and GNRs@Gals


maintained at 37 �C in a humidified atmosphere of

5 % CO2. When the cell growth reached to nearly

100 % confluence, the cultivation medium above cells

in each well was replaced by fresh one and the

GNRs@PC solution was added with the final peak

absorbance of GNRs@PC in the NIR spectra equaling

0.2. The volume of the mixed solution above cells was

kept 3 ml per well. After 12 h interaction, the cells


123

were washed, trypsinized, and centrifuged to get the

deposits, which were then treated with aqua regia

overnight for dissolving the cells and GNRs@PC

inside. When finally the solutions became transparent,

they were analyzed through ICP-MS for measuring the

amount of gold atoms per cell. The experimental

procedures for GNRs@PEG, GNRs@FA, and GNRs

@Gals were similar except that the same amount of

GNRs@PC was replaced by GNRs@PEG,

GNRs@FA and GNRs@Gals, respectively. Data were

presented as the mean ± SD of three independent

measurements by the equipment.

Fig. 1 The synthetic procedures of four different surface-modified GNRs


123

TEM assays of the cell uptake of GNRs@PC,

GNRs@PEG, GNRs@FA, and GNRs@Gals

The cultured cells were firstly treated as in the ICP-MS

assay. After 12 h interaction, the cells were washed,

trypsinized, and centrifuged to get the precipitates,

which were then fixed with 2.5 % formaldehyde PBS

solution. After 2 h fixation at 4 �C, the samples were

washed with phosophate-buffered saline (0.1 M) for

three times. Then the samples were fixed with 1 %

perosmic oxide for 2 h at 4 �C. After being washed in

water, the samples were dehydrated in an alcohol series,

embedded, and sliced with the thickness between 50 and

70 nm. Finally, the slices were deposited on the copper

grids for TEM analyses. The experimental procedures

for GNRs@PEG, GNRs@FA, and GNRs@Gals

were similar except that the same amount of GNRs@PC

was replaced by GNRs@PEG, GNRs@FA, and

GNRs@Gals, respectively.

NIR irradiation assays


maintained at 37 �C in a humidified atmosphere of 5 %

CO2. When the cell growth reached to nearly 100 %

confluence, the cultivation medium above cells was

replaced by fresh one and the GNR solution was added

into the cultivation medium above cells with the final

NIR peak absorbance of GNRs maintained at 0.2. The

volume of the mixed solution above cells was kept

100 ll per well. After 12 h interaction, the above

medium was removed and 30 ll fresh cultivation

medium was added to cover the cells. The attached

Fig. 2 a FT-IR spectra of five different surface-modified

GNRs. In GNRs@PC, 1258 cm-1 is the characteristic peak

wave number of -P=O and 1089 cm-1 reflects PO–CH2–. In

GNRs@CTAB, the C–N? stretching modes are placed at 910

and 964 cm-1. In GNRs@PEG, 1051 cm-1 is the peak wave

number of C–O–C. In GNRs@Gals, 1049 cm-1 is also the peak

wave number of C–O–C, while the –C=O stretching mode is

placed at 1631 cm-1. In GNRs@FA, the peaks reflecting

benzene are 1607 and 1486 cm-1, while the ones reflecting

–NH–CO– are 1694 and 1571 cm-1. b Z-potential data of these

five GNRs. The positive Z-potential values show that the

surfaces of GNRs are positively charged

Fig. 3 a–e TEM images of GNRs@CTAB, GNRs@PC,

GNRs@PEG, GNRs@Gals and GNRs@FA, respectively.

Close-up shots are inserted which reflect no size changes after

different surface modifications of GNRs. f Collection of

UV–Vis spectra of these five GNRs in the solutions


123

cells at the bottom of each well were irradiated by NIR

lasers of different light powers with a 1 mm focused

spot size. The distance between the NIR laser outlet and

the cells was kept 1 mm. For the extracellular modality,

30 ll mixed medium with the same amount of GNRs as

that inside cells in the intracellular modality was added

instead. After different irradiation time intervals, the

cells were stained with 20 ll 0.033 mg ml-1 fluores-

cein diacetate (FDA) and 100 ll cultivation media for

20 min. Then the stained cells were washed with PBS

solutions for 5–6 times to discard FDA residues

attached to well surfaces. Finally the stained cells were

recorded by the CLSM images.

Due to the unattachable nature of those two lymph

cell types, their NIR effects were studied by flow

cytometry analyses. The cells were at first treated as

the above procedure, followed by staining with FDA

for 20 min. The stained cells were then centrifuged

and resuspended in fresh media. The final cell

solutions were directly moved to flow cytometry

analyses for calculating the number ratios between

stained cells (alive) and unstained cells (dead), which

were reflected by the diagrams of counts and FL1-H

values. The statistic counts in the high fluorescent

region represented the relative number of living cells

while the counts in the low fluorescent region stood for

the number of dead cells.

Results

Preparation and characterization of various

modified GNRs

The GNRs were synthesized through the wet chemical

methods as above. Their average size was 30.4 nm (in

length) 9 8.4 nm (in width), with the aspect ratio at

3.6. Through ligand exchanges, the as-prepared CTAB

bilayers on the GNR surfaces were replaced, respec-

tively, by PC, PEG, FA or Gals (Fig. 1). All of these

four ligands were conjugated to gold surfaces, which

are supported by the FT-IR (Fig. 2a) and Z-potential

data (Fig. 2b). In order to stabilize FA and Gals

modified GNRs, PEG was also partially conjugated

Fig. 4 a–d Optical absorbance changes of longitudinal NIR

peaks and peak half-widths of GNRs@PC, GNRs@PEG,

GNRs@Gals, and GNRs@FA, respectively, in RPMI 1640

and H-DMEM cell cultivation media up to 24 h at ambient

temperature. Each experimental point during the UV–Vis

measurements was repeated three times and the data were

represented as mean ± SD


123

onto the surfaces. Both the TEM images (Fig. 3a–e)

and UV–Vis spectra (Fig. 3f) show that these various

surface modifications did not induce size changes of

GNRs and cause polydisperse distribution or even

aggregation. Further in two cell cultivation media up

to 1 day, all these four kinds of GNRs showed almost

unchanged NIR absorption spectra characteristics like

longitudinal plasmon peak wavelengths and peak

widths (Fig. 4), which fit in with previous reports that

surface modification of PC or PEG could endow

nanoparticles with good colloidal stability (Zhou et al.

2010). Cytotoxicity assays by the MTT method also

reflected that after 12 h interaction with these four

kinds of GNRs, the viabilities of all the six types of

cells did not drop much, which demonstrated very low

cytotoxic effects of these GNRs up to the concentra-

tions of gold atoms of 0.025 mM in the solution

(Fig. 5).

Cell uptake assays

PC modification seemed to universally enhance cancer

cells selective uptake of nanoparticles (our work

recently submitted) while PEG which showed in

stealth property in vivo could hardly induce internal-

ization (Yang et al. 2008; Lipka et al. 2010). Besides,

some small molecules like FA (Setua et al. 2010) and

Gals (Lai et al. 2010) acted as tumor-specific ligands

to certain types of cancer cells. In this study, the Gals

ligand would selectively target to the HepG2 liver

Fig. 5 a–f Viability

changes of six types of cells

in their interactions with

GNRs@PC, GNRs@PEG,

GNRs@Gals, and

GNRs@FA of different gold

concentrations up to

0.025 mM after 12 h. The

data were from MTT assays

which were presented

mean ± SD (n = 3)


123

cancer cell, while the FA ligand would selectively

target to the CNE-1 nasopharyngeal cancer cell. The

above four kinds of GNRs were co-cultured with six

cell types for 12 h and the amounts of GNRs

internalized were measured by three techniques. As

in the TEM images of Fig. 6 (also see enlarged images

from Fig. S3 to Fig. S18), it was found that much more

GNRs@PC were internalized inside two cancer cell

types (HepG2 and CNE-1) than two corresponding

normal cell types (L02 and rhinal epithelia) (Fig. 6a, e,

i, m). GNRs@Gals were selectively internalized by

HepG2 compared with L02 (Fig. 6k, o) while

GNRs@FA were selectively internalized by CNE-1

compared with Rhinal epithelia (Fig. 6d, h). However,

GNRs@Gals could hardly target to nasopharyngeal

cells (Fig. 6c, g), while GNRs@FA could not target to

liver cells (Fig. 6l, p). GNRs@PEG, compared to

other surface modifications, exhibited no selectivity in

cell uptake and could hardly enter any cell type

(Fig. 6b, f, j, n).

Such tendencies could also be reflected by the NIR

absorbance changes of these GNRs in the solution

interacting with different cells. Due to cell uptake, the

concentration of GNRs in the solution which is

proportional to the absorbance of the longitudinal

peak in the NIR region (Fig. 3f) will decrease with

time. After 24 h, the internalized percentages of

GNRs@PC in both two cancer cell types surpassed

50 % (Fig. 7a, c) and the similar results could be found

for GNRs@FA in CNE-1 cancer cells (Fig. 7a) and for

GNRs@Gals in HepG2 cancer cells (Fig. 7c). Mean-

while, the internalized percentages of GNRs@PEG in

all these four cell types were below 10 %. Another

quite different group of cells from the lymph was

further tested in these two assays and the results

followed that more GNRs@PC seemed to be internal-

ized inside cancerous 8226 cells compared with

normal lymph cells H9 (Fig. S1).

These trends could be reflected more quantitatively

by the ICP measurement of the gold content per cell

(Fig. 8, also see the data in Table S1). The numbers of

GNRs@PC in all three cancer cell types surpassed

3.9 9 104 per cell while in normal cells the numbers

dropped to merely about 1.3 9 104. The number of

GNRs@Gals inside HepG2 was 4.9 9 104, which was

much larger than the numbers in corresponding

Fig. 6 TEM images of four

cell types which internalized

different numbers of GNRs

modified by four surface

moieties after 12 h

interactions. The

internalized GNRs were

shown inside red (for

nasopharyngeal cells) and

pink (for liver cells) dottedcircles. (Color figure online)


123

normal ones and the other two cell types. On the

contrary, the number of GNRs@FA inside HepG2 was

much smaller than that inside CNE-1 (6.7 9 104).

GNRs@PEG in the other extreme could hardly be

internalized by all the six cell types, with the numbers

all below 1.5 9 104 per cell. In all, different cell

uptake amounts were achieved by choosing various

surface moieties for GNRs, which paved the way for

clarifying the quantitative relationship between the

locations of GNRs and the NIR therapeutic effects.

NIR laser photothermal assays

After GNRs internalization, all the cells were irradi-

ated under the NIR light and the times needed for cell

ablation in the laser spot region were recorded. The

treated cell samples were then dyed with FDA and

observed under confocal laser scanning microscopy

(CLSM). Firstly, the effect of the locations of GNRs

either inside or outside HepG2 was investigated

toward the ablation time. In the intracellular mode

(Fig. 9a), the cells were incubated with GNRs@PC for

12 h to provide enough time for the cell uptake of

GNR@PC. The NIR irradiation was then carried out

after removing the extracellular GNRs@PC. While in

the extracellular mode (Fig. 9b), the cells were not

treated with GNRs@PC until the NIR irradiation

began to be used. It could be seen from previous

uptake assays that there was few GNRs@PC internal-

ized by cells in such a short time of irradiation. As a

result, the fluorescent images (Fig. 10) showed that the

cell death time when GNRs were placed inside cells

was much shorter than that when GNRs were outside

cells. Under NIR irradiation, the fluorescence on

behalf of the cell viability inside the laser beam spot

disappeared after 130 s in the intracellular mode,

while in the extracellular mode the fluorescence did

not fade until 200 s.

Such ablation efficiency differences could also be

reflected when the cells internalized different amounts

Fig. 7 UV–Vis results of the percentages of GNRs@PC, GNRs@PEG, GNRs@Gals, and GNRs@FA left in the solutions above cells

up to 24 h interaction in CNE-1 (a), rhinal epithelia (b), HepG2 (c), and L02 (d). Data were presented as the mean ± SD (n = 3)


123

of GNRs, as in the CLSM images of Fig. 11 (also see

the exact data in Fig. S2). It was shown that the cell

ablation time differences were directly correlated to

various cell uptake amounts of GNRs, and generally

the death time of cells that internalized more GNRs

was relatively shorter. Under 200 mW NIR irradia-

tion, all the cells that had beyond 4.5 9 104 GNRs per

cell died before 105 s, while such time for most cells

that internalized GNRs less than 1.5 9 104 per cell

was over 180 s. The flow cytometry data of two lymph

cell types also verified this relationship (Fig. 12).

After 240 s, the ablative effect for lymphosarcoma

cells 8226 was prominent that the cell viability ratio

was merely 3.28 %. However, the viability for B

lymphocyte cells H9 was nearly 90 % at the same

condition. In short, it seems that the cell uptake

amount of GNRs is tightly connected with the

eradication time for these cells. Though the relation-

ship might be more complicated than just linear

(Fig. S2B), this result fit in with the tendency found in

the comparison between intracellular and extracellular

modalities in Fig. 10.

Discussion

The above research provides primary evidence that the

intracellular NIR photothermal modality seemed to be

more efficient than the extracellular one. Previously,

this topic on efficiency was still in hot debate, which

was confined by the lack of effective and precise

methods to differentiate the extracellular and intra-

cellular modalities (Shinkai et al. 1995; Jordan et al.

1999; Gazeau et al. 2008). With the advance of

Fig. 8 ICP-MS data of the GNR internalized numbers in different cells. Four types of GNRs with different surface modifications were

tested. Data were shown as the mean ± SD (n = 3)


123

nanotechnology, such researches may be carried out

now by using nano-sized tools like GNRs and different

surface modification moieties. As a result, since

Gordon et al. (1979) firstly postulated that the

intracellular hyperthermia was superior than the

extracellular one, more and more concerns were

focused on this possible difference (Yanase et al.

1997; Jordan et al. 1999; Rabin 2002; Zharov et al.

2003; Sonvico et al. 2005; Lapotko 2006; Hergt and

Dutz 2007; Gazeau et al. 2008; Prasad et al. 2008;

Wen 2009; Lukianova-Hleb et al. 2010; Zhao et al.

2010). However, it is still lack of a definite experi-

mental evidence on which is better by comparing these

two modalities directly. Even if some reports have

tried to make clear by using active targeting moieties

or other non-selective targeting ones like cationic

molecules, vast uncertainties exist which obscure the

primary therapeutic results (Yanase et al. 1997;

Sonvico et al. 2005). Other theoretical and experi-

mental studies further complicated this question by

demonstrating that the effective hyperthermia by one

single photothermal responsive gold nanoparticle was

confined to such a small space that the micro-scale or

even nano-scale heating effect was negligible (Pitsil-

lides et al. 2003; Zharov et al. 2003; Keblinski et al.

2006; Avedisian et al. 2009; Gupta et al. 2010).

Instead, the cell ablation was caused by photome-

chanic effect like microbubble expansion or dynamic

flow in pulsed laser mode, which might also do harm

to neighboring normal cells (Zharov and Lapotko

2005; Zharov et al. 2005; Lapotko 2006; Wen 2009;

Fig. 9 Two modes of NIR therapeutic modalities. In mode (a), all

the GNRs were internalized inside cells which were attached at the

bottoms of wells. Under the NIR irradiation, the cells in the laser

spot regions died out, which were shown in the grey color. After

adding FDA, these dead cells could not be dyed as the neighboring

living cells which showed the green color. In mode (b), all the

GNRs were outside cells. Under the NIR irradiation, all the cells

kept alive and could be dyed in green. (Color figure online)

Fig. 10 CLSM images of

HepG2 after a series of NIR

irradiation times. In the

intracellular mode, the cells

were incubated with

GNRs@PC for 12 h before

irradiation, while in the

extracellular mode, the cells

were not treated with

GNRs@PC before, but

irradiated once the

GNRs@PC were added. The

white dotted circles mean

the laser beam spot areas,

while the red lines are the

watershed after which the

cells in the spot areas began

to die. (Color figure online)


123

Lukianova-Hleb et al. 2010). This also held true for

magnetic nanoparticles, which were used in large

amounts experimentally in order to overcome the

limited heating ability per particle (Szasz et al. 2003;

Wilhelm et al. 2007; Hergt and Dutz 2007;

Prasad et al. 2008). These reports seemed to reflect

that the therapeutic effects in between intracellular and

extracellular modalities did not vary much.

However, it was found that the predominant

molecular targets of hyperthermia could be nucleic

acids, cytoplasmic proteins and cell membranes

(Barlogie et al. 1979; Hildebrandt et al. 2002). This

Fig. 11 CLSM images of

four cell types at different

NIR irradiation times after

different GNRs treatments.

The NIR light irradiation

areas were shown inside the

white dotted circles as in the

images. The yellow linesdivided the times when cells

kept alive and when cells

began to die. (Color figure

online)


123

indicated that as a method of hyperthermia, there was

no need to heat the whole cell for necrosis, but certain

sensitive organelles of the cell. In our study, the

GNRs in most conditions were localized in the

endosomes or lysosomes (Fig. 6 and enlarged images

from Fig. S3 to Fig. S18), in which there existed many

hydrolytic enzymes that once were released to the

cytoplasm, would cause fatal effect to the functional

proteins contained (Alberts et al. 2008). Under the

relatively moderate CW NIR laser which might

not cause photomechanical effects, the confine

ment of heat became the dominant factor to bring

distinct therapeutic effect in our results. Other authors

have also reported similar results of selective heat

attack on other subunits like DNA (Stehr et al. 2008;

Hrelescu et al. 2010; Takeda et al. 2011), mitochon-

dria (Tong and Cheng 2009), and cell membrane

(Zharov et al. 2003; Zharov and Lapotko 2005;

Zharov et al. 2005). Based on this, it is reasonable to

have the quantitative relationship between the cell

uptake amount of GNRs and the cell death time under

the NIR irradiation. In all, it is believed that such a

kind of NIR hyperthermia modality may control

therapeutic effects more precisely and safely, thus

not only decreases unnecessary damage toward nor-

mal tissues and cells, but also enhances ablative

efficiency compared with the extracellular way.

Conclusion

In summary, GNRs with different surface molecular

modifications were firstly prepared. The existence of

these ligands was detected by FT-IR and Z-potential

analyses. Six types of cells with three totally different

cancer cells and their corresponding normal ones were

then adopted to exhibit different cell uptake levels of

GNRs, which were reflected by TEM, UV–Vis and

ICP-MS techniques. Under the CW NIR irradiation, the

difference in the therapeutic effect due to different

GNR locations was discovered, in which two following

results should be intensified: (1) the NIR photothermal

therapeutic effect was greater when the GNRs were

inside cells; (2) the NIR photothermal therapeutic effect

was also greater as the internalized amount of GNRs per

cell was larger. So generally speaking, driven by the

NIR photothermal effect, intracellular hyperthermia

seems to exhibit more precise and efficient control on

the selective cancer ablation. It was hoped that this

result would provide a promise for future nanoparticles

assisted NIR photothermal therapeutic designs.

Acknowledgments This work was financially supported from

National Science Fund for Distinguished Young Scholars

(51025312), the Natural Science Foundation of China (50830106,

21174126) and Open Project of State Key Laboratory of

Supramolecular Structure and Materials (sklssm201204).

Fig. 12 Flow cytometry

data of two lymph cell types

treated with GNRs@PC for

12 h and then irradiated with

the NIR laser at 200 mW for

150 and 240 s. The

horizontal coordinate unit

FL1-H represented the

fluorescent intensity. The

shadow areas under the

curves showed the total

numbers of cells tested. The

dots in the M1 region mean

dead cells which were not

dyed, while the dots in the

M2 region mean the living

cells which could be dyed

and detected


123

References

Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P

(2008) Molecular biology of the cell, Chap 12, 5th edn.

Garland Science, New York

Avedisian CT, Cavicchi RE, McEuen PL, Zhou X (2009)

Nanoparticles for cancer treatment role of heat transfer.

Ann NY Acad Sci 1161:62–73

Baban DF, Seymour LW (1998) Control of tumour vascular

permeability. Adv Drug Deliver Rev 34:109–119

Barlogie B, Corry PM, Yip E, Lippman L, Johnston DA, Khalil

K, Tenczynski TF, Reilly E, Lawson R, Dosik G, Rigor B,

Hankenson R, Freireich EJ (1979) Total-body hyperther-

mia with and without chemotherapy for advanced human

neoplasms. Cancer Res 39:1481–1489

Berry CC, Curtis ASG (2003) Functionalisation of magnetic

nanoparticles for applications in biomedicine. J Phys D

36:R198–R206

Campbell RB (2007) Battling tumors with magnetic nanother-

apeutics and hyperthermia: turning up the heat. Nano-

medicine 2:649–652

Chakravarty P, Marches R, Zimmerman NS, Swafford ADE,

Bajaj P, Musselman IH, Pantano P, Draper RK, Vitetta ES

(2008) Thermal ablation of tumor cells with anti body-

functionalized single-walled carbon nanotubes. Proc Natl

Acad Sci USA 105:8697–8702

Chari RVJ (2008) Targeted cancer therapy: conferring speci-

ficity to cytotoxic drugs. Acc Chem Res 41:98–107

Chen SF, Zheng J, Li LY, Jiang SY (2005) Strong resistance of

phosphorylcholine self-assembled monolayers to protein

adsorption: Insights into nonfouling properties of zwitter-

ionic materials. J Am Chem Soc 127:14473–14478

Chen J, Yang M, Zhang Q, Cho EC, Cobley CM, Kim C, Glaus

C, Wang LV, Welch MJ, Xia Y (2010) Gold nanocages: a

novel class of multifunctional nanomaterials for theranos-

tic applications. Adv Funct Mater 20:3684–3694

Gazeau F, Levy M, Wilhelm C (2008) Optimizing magnetic

nanoparticle design for nanothermotherapy. Nanomedicine

3:831–844

Gilchrist RK, Medal R, Shorey WD, Hanselman RC, Parrott JC,

Taylor CB (1957) Selective inductive heating of lymph

nodes. Ann Surg 146:596–606

Gordon RT, Hines JR, Gordon D (1979) A biophysical approach

to cancer treatment via intracellular temperature and bio-

physical alterations. Med Hypotheses 5:83–102

Gupta A, Kane RS, Borca-Tasciuc DA (2010) Local tempera-

ture measurement in the vicinity of electromagnetically

heated magnetite and gold nanoparticles. J Appl Phys

108:064901

Hergt R, Dutz S (2007) Magnetic particle hyperthermia-bio-

physical limitations of a visionary tumour therapy. J Magn

Magn Mater 311:187–192

Hildebrandt B, Wust P, Ahlers O, Dieing A, Sreenivasa G, Kerner

T, Felix R, Riess H (2002) The cellular and molecular basis

of hyperthermia. Crit Rev Oncol Hemat 43:33–56

Hirsch LR, Stafford RJ, Bankson JA, Sershen SR, Rivera B,

Price RE, Hazle JD, Halas NJ, West JL (2003) Nanoshell-

mediated near-infrared thermal therapy of tumors under

magnetic resonance guidance. Proc Natl Acad Sci USA

100:13549–13554

Hrelescu C, Stehr J, Ringler M, Sperling RA, Parak WJ, Klar

TA, Feldmann J (2010) DNA melting in gold nanostove

clusters. J Phys Chem C 114:7401–7411

Huang X, El-Sayed IH, Qian W, El-Sayed MA (2006) Cancer

cell imaging and photothermal therapy in the near-infrared

region by using gold nanorods. J Am Chem Soc

128:2115–2120

Hurwitz MD (2010) Today’s thermal therapy: not your father’s

hyperthermia challenges and opportunities in application

of hyperthermia for the 21st century cancer patient. Am J

Clin Oncol 33:96–100

Jordan A, Scholz R, Wust P, Schirra H, Schiestel T, Schmidt H,

Felix R (1999) Endocytosis of dextran and silan-coated

magnetite nanoparticles and the effect of intracellular

hyperthermia on human mammary carcinoma cells in vitro.

J Magn Magn Mater 194:185–196

Kam NWS, O’Connell M, Wisdom JA, Dai HJ (2005) Carbon

nanotubes as multifunctional biological transporters and

near-infrared agents for selective cancer cell destruction.

Proc Natl Acad Sci USA 102:11600–11605

Keblinski P, Cahill DG, Bodapati A, Sullivan CR, Taton TA

(2006) Limits of localized heating by electromagnetically

excited nanoparticles. J Appl Phys 100:054305

Kim JW, Shashkov EV, Galanzha EI, Kotagiri N, Zharov VP

(2007) Photothermal antimicrobial nanotherapy and nan-

odiagnostics with self-assembling carbon nanotube clus-

ters. Lasers Surg Med 39:622–634

Kondepati VR, Heise HM, Backhaus J (2008) Recent applica-

tions of near-infrared spectroscopy in cancer diagnosis and

therapy. Anal Bioanal Chem 390:125–139

Krishna V, Singh A, Sharma P, Iwakuma N, Wang Q, Zhang Q,

Knapik J, Jiang H, Grobmyer SR, Koopman B, Moudgil B

(2010) Polyhydroxy fullerenes for non-invasive cancer

imaging and therapy. Small 6:2236–2241

Lai CH, Lin CY, Wu HT, Chan HS, Chuang YJ, Chen CT, Lin

CC (2010) Galactose encapsulated multifunctional nano-

particle for HepG2 cell internalization. Adv Funct Mater

20:3948–3958

Lambert TN, Andrews NL, Gerung H, Boyle TJ, Oliver JM,

Wilson BS, Han SM (2007) Water-soluble germanium(0)

nanocrystals: Cell recognition and near-infrared photo-

thermal conversion properties. Small 3:691–699

Lapotko DO (2006) Laser-induced bubbles in living cells.

Lasers Surg Med 38:240–248

Lipka J, Semmler-Behnke M, Sperling RA, Wenk A, Takenaka

S, Schleh C, Kissel T, Parak WJ, Kreyling WG (2010)

Biodistribution of PEG-modified gold nanoparticles fol-

lowing intratracheal instillation and intravenous injection.

Biomaterials 31:6574–6581

Lukianova-Hleb E, Hu Y, Latterini L, Tarpani L, Lee S, Drezek

RA, Hafner JH, Lapotko DO (2010) Plasmonic nanobub-

bles as transient vapor nanobubbles generated around

plasmonic nanoparticles. ACS Nano 4:2109–2123

Ma LL, Feldman MD, Tam JM, Paranjape AS, Cheruku KK,

Larson TA, Tam JO, Ingram DR, Paramita V, Villard

JW, Jenkins JT, Wang T, Clarke GD, Asmis R, Sokolov

K, Chandrasekar B, Milner TE, Johnston KP (2009)

Small multifunctional nanoclusters (nanoroses) for tar-

geted cellular imaging and therapy. ACS Nano

3:2686–2696


123

Markovic ZM, Harhaji-Trajkovic LM, Todorovic-Markovic BM,

Kepic DP, Arsikin KM, Jovanovic SP, Pantovic AC,

Dramicanin MD, Trajkovic VS (2011) In vitro comparison

of the photothermal anticancer activity of graphene nano-

particles and carbon nanotubes. Biomaterials 32:1121–1129

Mornet S, Vasseur S, Grasset F, Duguet E (2004) Magnetic

nanoparticle design for medical diagnosis and therapy.

J Mater Chem 14:2161–2175

Moroz P, Jones SK, Gray BN (2002) Magnetically mediated

hyperthermia: current status and future directions. Int J

Hyperth 18:267–284

Murphy CJ, Sau TK, Gole AM, Orendorff CJ, Gao JX, Gou LF,

Hunyadi SE, Li T (2005) Anisotropic metal nanoparticles:

synthesis, assembly, and optical applications. J Phys Chem

B 109:13857–13870

Nagasaki Y, Ogawa R, Yamamoto S, Kato M, Kataoka K (1997)

Synthesis of heterotelechelic poly(ethylene glycol) ma-

cromonomers. Preparation of poly(ethylene glycol) pos-

sessing a methacryloyl group at one end and a formyl group

at the other end. Macromolecules 30:6489–6493

Nikoobakht B, El-Sayed MA (2003) Preparation and growth

mechanism of gold nanorods (NRs) using seed-mediated

growth method. Chem Mater 15:1957–1962

Norman TJ Jr, Grant CD, Magana D, Zhang JZ (2002) Near

infrared optical absorption of gold nanoparticle aggregates.

J Phys Chem B 106:7005–7012

Pankhurst QA, Connolly J, Jones SK, Dobson J (2003) Appli-

cations of magnetic nanoparticles in biomedicine. J Phys D

36:R167–R181

Pitsillides CM, Joe EK, Wei X, Anderson RR, Lin CP (2003)

Selective cell targeting with light-absorbing microparticles

and nanoparticles. Biophys J 84:4023–4032

Prasad NK, Rathinasamy K, Panda D, Bahadur D (2008) TC-

tuned biocompatible suspension of La0.73Sr0.27MnO3 for

magnetic hyperthermia. J Biomed Mater Res B 85B:

409–416

Purushotham S, Ramanujan RV (2010) Modeling the perfor-

mance of magnetic nanoparticles in multimodal cancer

therapy. J Appl Phys 107:114701

Rabin Y (2002) Is intracellular hyperthermia superior to extra-

cellular hyperthermia in the thermal sense. Int J Hyperth

18:194–202

Sawyers CL (2008) The cancer biomarker problem. Nature

452:548–552

Setua S, Menon D, Asok A, Nair S, Koyakutty M (2010) Folate

receptor targeted, rare-earth oxide nanocrystals for bi-

modal fluorescence and magnetic imaging of cancer cells.

Biomaterials 31:714–729

Shinkai M (2002) Functional magnetic particles for medical

application. J Biosci Bioeng 94:606–613

Shinkai M, Suzuki M, Iijima S, Kobayashi T (1995) Preparation

and characteristics of magnetite-labeled antibody with the

use of poly(ethylene glycol) derivatives. Biotech Appl

Biochem 21:125–137

Sonvico F, Dubernet C, Colombo P, Couvreur P (2005) Metallic

colloid nanotechnology, applications in diagnosis and

therapeutics. Curr Pharm Des 11:2091–2105

Stehr J, Hrelescu C, Sperling RA, Raschke G, Wunderlich M,

Nichtl A, Heindl D, Kurzinger K, Parak WJ, Klar TA,

Feldmann J (2008) Gold nanostoves for microsecond DNA

melting analysis. Nano Lett 8:619–623

Szasz A, Vincze G, Szasz O, Szasz N (2003) An energy analysis

of extracellular hyperthermia. Electromagn Biol Med

22:103–115

Takeda Y, Kondow T, Mafune F (2011) Selective decomposi-

tion of nucleic acids by laser irradiation on probe-tethered

gold nanoparticles in solution. Phys Chem Chem Phys

13:586–592

Thiesen B, Jordan A (2008) Clinical applications of magnetic

nanoparticles for hyperthermia. Int J Hyperth 24:467–474

Tong L, Cheng JX (2009) Gold nanorod-mediated photo-

thermolysis induces apoptosis of macrophages via damage

of mitochondria. Nanomedicine 4:265–276

Weissleder R (2001) A clearer vision for in vivo imaging. Nat

Biotechnol 19:316–317

Wen DS (2009) Intracellular hyperthermia: nanobubbles and

their biomedical applications. Int J Hyperth 25:533–541

Wilhelm C, Fortin JP, Gazeau F (2007) Tumour cell toxicity of

intracellular hyperthermia mediated by magnetic nano-

particles. J Nanosci Nanotech 7:2933–2937

Wust P, Hildebrandt B, Sreenivasa G, Rau B, Gellermann J,

Riess H, Felix R, Schlag PM (2002) Hyperthermia in

combined treatment of cancer. Lancet Oncol 3:487–497

Yanase M, Shinkai M, Honda H, Wakabayashi T, Yoshida J,

Kobayashi T (1997) Intracellular hyperthermia for cancer

using magnetite cationic liposomes: Ex vivo study. Jpn J

Cancer Res 88:630–632

Yang ST, Fernando KAS, Liu JH, Wang J, Sun HF, Liu YF,

Chen M, Huang YP, Wang X, Wang HF, Sun YP (2008)

Covalently PEGylated CNTs with stealth character in vivo.

Small 4:940–944

Zhao LY, Tang JT, Feng SS (2010) Nanothermotherapy by high

performance magnetic nanoparticles. Nanomedicine 5:

1305–1308

Zharov VP, Lapotko DO (2005) Photothermal imaging of

nanoparticles and cells. IEEE J Quantum Elect 11:733–751

Zharov VP, Galitovsky V, Viegas M (2003) Photothermal

effects during selective nanophotothermolysis. Appl Phys

Lett 83:4897–4899

Zharov VP, Letfullin RR, Galitovskaya EN (2005) Microbub-

bles-overlapping mode for laser killing of cancer cells with

absorbing nanoparticle clusters. J Phys D 38:2571–2581

Zhou WB, Shao JY, Jin Q, Wei QS, Tang JG, Ji J (2010)

Zwitterionic phosphorylcholine as a better ligand for gold

nanorods cell uptake and selective photothermal ablation

of cancer cells. Chem Commun 46:1479–1481


123

more efﬁcient nir photothermal therapeutic effect from ...the synthesis of gnrs was referred to...

Documents