Nano Res

1

Near-infrared light-activated cancer cell targeting anddrug delivery with aptamer modified nanostructures

Yu Yang1, Jingjing Liu2, Xiaoqi Sun2, Liangzhu Feng2, Wenwen Zhu2, Zhuang Liu2 (), and Meiwan Chen1 () Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-015-0898-4

http://www.thenanoresearch.com on September 10 2015

© Tsinghua University Press 2015

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Nano Research

DOI 10.1007/s12274-015-0898-4

TABLE OF CONTENTS (TOC)

Near-infrared Light-activated

Cancer Cell Targeting and Drug

Delivery with Aptamer Modified

Nanostructures

Yu Yang1, Jingjing Liu2, Xiaoqi Sun2,

Liangzhu Feng2, Wenwen Zhu2,

Zhuang Liu2*, Meiwan Chen1*

1 State Key Laboratory of Quality

Research in Chinese Medicine,

Institute of Chinese Medical Sciences,

University of Macau, Avenida da

Universidade, Taipa, Macau, China

2 Institute of Functional Nano & Soft

Materials Laboratory (FUNSOM),

Soochow University, Suzhou, Jiangsu

215123, China

Gold nanorods (GNRs) and single-walled carbon nanotubes (SWNTs) as two model systems

have demonstrated excellent NIR-light activated specific cancer cell binding and drug

delivery by modifying NIR-absorbing nano-drug-carriers with ssDNA caged aptamters,

which can be de-hybridized under NIR laser irradiation and then regain their structure and

cell-binding ability. These NIR-activable cancer-targeted drug delivery systems show a

dual-targeting capability, which is realized by exploiting the intrinsic cancer-cell-binding

ability of aptamers, as well as the spatially / temporally controllable laser irradiation (e.g.

focusing laser on the tumor at the best treatment timing), promising for further reducing

non-specific toxicity to normal tissues and enhancing treatment selectivity against tumor

cells.

Provide the authors’ webside if possible.

Zhuang Liu, http://nano.suda.edu.cn/LZ

 

Near-infrared Light-activated Cancer Cell Targeting andDrug Delivery with Aptamer Modified Nanostructures

Yu Yang1, Jingjing Liu2, Xiaoqi Sun2, Liangzhu Feng2, Wenwen Zhu2, Zhuang Liu2(), Meiwan Chen1() 1 State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macau, China 2 Institute of Functional Nano & Soft Materials Laboratory (FUNSOM), Soochow University, Suzhou, Jiangsu 215123, China

 

Received: day month year

Revised: day month year

Accepted: day month year

(automatically inserted by

the publisher)

© Tsinghua University Press

and Springer-Verlag Berlin

Heidelberg 2014

KEYWORDS

NIR-activable, drug

delivery, aptamer, gold

nanorods, single-walled

carbon nanotubes

ABSTRACT

Stimuli-activated targeted delivery systems to provide highly accurate

treatment to tumors have received considerable attention in recent years.

Herein, we design a light-activable cancer targeting strategy that uses a

complementary DNA sequence to hybridize and mask sgc8 aptamers

conjugated on photothermal agents such as gold nanorods (GNRs) or

single-walled carbon nanotubes (SWNTs). Upon exposure to near-infrared

(NIR) laser, the localized photothermal heating on the surface of those

nano-agents would result in the dehybridization of the double strand DNA and

un-caging of the aptamer sequence to allow specific cancer cell targeting.

Utilizing doxorubicin (DOX) loaded SWNTs as a model system, cancer cell

targeted drug delivery that is activated by NIR light is realized. This work

demonstrates the concept of NIR-activable tumor-targeting delivery systems,

achieving controllable cancer cell binding to potentially enable highly specific

and efficient cancer therapy.

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DOI (automatically inserted by the publisher)

Review Article/Research Article Please choose one

1. Introduction

In the past decade, many different types of

drug delivery systems have been explored, aiming

at increasing efficacy of therapeutic agents and

reducing toxicity to normal cells and tissues.1

Compared to the passive tumor targeting via

enhanced permeability and retention (EPR) effect,

the active tumor targeting can be achieved through

modifying targeting ligands including small

molecules2, peptides3, antibodies4 and aptamers5-7

on the surface of nanocarriers, enabling site-specific

targeting of tumor cells, tumor vasculatures, or

tumor microenvironment. Moreover, in order to

provide more accurate treatment to tumors and

improve the efficiency of anticancer therapy,

stimuli-activable targeted delivery systems based

on external physical stimuli including magnetic

field8, ultrasound9,10, temperature11 and light12 have

also been widely investigated13. Such strategies

would allow specific binding of drug carriers to

cancer cells only under certain physical stimuli

locally applied onto the tumor, to further enhance

treatment specificity and minimize non-specific

binding to normal tissues, some of which may

exhibit low levels of receptor expressions.14

Near-infrared (NIR) light, which exhibits little

phototoxicity and high tissue penetration ability,

has been extensively applied not only in biomedical

imaging15-17, but also in phototherapy of cancer18-22.

As a typical class of phototherapy, photothermal

therapy has been widely studied for efficient cancer

treatment through killing cancer cells by

light-generated hyperthermia.2,18,23,24 However, the

use of NIR light to activate cancer cell targeting of

nanoparticles via the photothermal effect has been

rarely explored except a recent work by Kohane et

al. 25 In that work, poly (NIPAAm-co-AAm), a

thermosensitive polymer, was used to cover the

target moieties conjugated on the surface of

silica-gold core-shell nanoparticles. After NIR laser

irradiation, the thermosensitive polymer was

constringed to allow the exposure of target ligands

for specific cell binding. However, photo-activated

cancer cell imaging and therapy has not yet been

demonstrated in their work.

Aptamers are oligonucleic acids which can

diversely and selectively bind to proteins, small

biological molecules and even cells, and have been

widely studied as targeting ligands for cancer

treatment with nanomolar even picamolar scale

dissociation constants (Kd).5-7,26-28 Herein, we

demonstrate a NIR-activable cancer cell targeting

strategy by conjugating aptamer which is caged by

their complimentary sequences, to the surface of

two types of commonly used photothermal agents,

gold nanorods (GNRs) and single-walled carbon

nanotubes (SWNTs). Upon NIR laser irradiation,

the effective photothermal heating localized on the

surface of GNRs or SWNTs could induce

de-hybridization of double-strand DNA and thus

uncaging of aptamer structures, which could then

recognize specific types of cancer cells. After

realizing NIR-activated specific cancer cell binding

with both GNRs and SWNTs, we further

demonstrate NIR-activated specific drug delivery

using SWNTs as a drug carrier for selective killing

of cancer cells under the control of NIR light. Our

strategy is promising for highly specific / selective

cancer treatment under the control of physical

stimuli.

2 Experimental

2.1 Materials.

HAuCl4 was purchased from Sigma-Aldrich

and doxorubicin (DOX) was purchased from Beijing

HuaFeng United Technology Co. Ltd.

Polylactide-poly (ethylene glycol) (PEG) was

obtained from Biomatrik Inc.

poly(maleicanhydride-alt-1-octadecene) (C18PMH),

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

(EDC) were obtained from Sigma-Aldrich.

Thiol-functionalized sgc8 aptamer and

complementary single stranded DNA were

obtained from Takara Biotechnology (Dalian) Co.,

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3Nano Res.

Ltd. Other chemicals were bought from Sinopharm

Group Co. Ltd.

2.2 Preparation of DNA modified GNRs.

GNRs were synthesized by the well-established

seed-mediated growth method 29 and purified by

washing with water for twice by centrifugation. To

confer them excellent physiological stability for

further experiments, the as-prepared GNRs were

coated with mPEG-SH by mixing 10 ml of 4 μM

GNRs with 2.0 μM mPEG-SH at room temperature

for 12 h. Afterwards, PEGylated GNRs (GNRs-PEG)

were collected by centrifugation, and stored at 4 oC

for further use. The molar concentrations of GNRs

were determined by their molar extinction

co-efficient at 808 nm (~1.02 × 109 M−1cm−1).30

To prepare dsDNA, thiol-functionalized sgc8

aptamer in tris(hydroxymethyl)aminomethane-

ethylendiamintetraessigsäure (Tris-EDTA) buffer

was firstly heated at 95 oC for 2 min and then mixed

with its complementary ssDNA and annealed at 37

oC for 1 h. Thereafter, the as-prepared

thiol-functionalized dsDNA was incubated with

GNRs-PEG solution for 24 h under stirring, and

aged overnight using 0.2 M NaCl. Finally, DNA

modified GNRs complexes (GNRs-Apt/DNA) were

collected by centrifugation at 14800 rpm for 5 min

and stored at 4 oC for further use.

2.3 Functionalization of SWNTs with

C18PMH-PEG-NH2.

C18PMH-PEG-NH2 was synthesized according

to our previously used protocol.31 To prepare

SWNT-PEG-NH2, 1 mg of SWNTs were sonicated in

10 mL aqueous solution containing 10 mg of

C18PMH-PEG-NH2 for 60 min and then centrifuged

at 14800 rpm for 30 min to remove any precipitates,

yielding a black suspension of PEGylated SWNTs.

Then, the excess C18PMH-PEG-NH2 was removed

using a membrane filter with mean pore size of 100

nm. The molar concentrations of SWNTs were

determined by their molar extinction co-efficient at

808 nm (7.9 x 106 M cm-1), with the estimated

average molecular weight of nanotubes to be 170

kDa.32

For aptamer conjugation, 8 mL as-prepared

SWNT-PEG-NH2 (300 nM) was mixed with 5.2 mg

of 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic

acid 3-sulfo-N-hydroxysuccinimide ester sodium

salt (sulfo-SMCC) and stirred at room temperature

for 2 h. After removal of excess sulfo-SMCC using

an Amico centrifugal filter device with a molecular

weight cut off (MWCO) of 100 kDa, the obtained

SWNT-PEG-NH2 was mixed with dsDNA prepared

as aforementioned overnight in the presence of 0.l

mM tris(2-carboxyethyl)phosphine (TCEP) to break

disulfide bonds at 4 oC. Finally, the un-reacted

aptamer was removed with the Amico centrifugal

filter device.

2.4 Characterization

Transmission electron microscopy (TEM) images of

as-prepared GNRs and SWNTs were observed

using a transmission electron microscope (Philips

CM300) at an acceleration voltage of 200 kV.

UV-Vis-NIR absorption spectra were recorded with

a UV-Vis-NIR spectrophotometer (PerkinElmer

Lambda 750). Laser irradiation was carried out

using an optical-fiber-coupled power-tunable diode

laser (Hi-Tech Optoelectronics Co., Beijing, China)

Fluorescent emission spectra were obtained with

excitation at 490 nm using a FluoroMax-4

luminescent spectrometer (HORIBA JobinYvon

S.A.S).

2.5 Drug Loading 

DOX loading onto different PEGylated SWNTs

was performed by mixing DOX with PEGylated

SWNTs (0.05 mg/mL) in PBS, (20 mM, pH8)

according to our previously developed procedure19.

For the DOX loading saturation experiment,

SWNT-PEG-Apt/DNA (0.05 mg/mL) was mixed

with various concentrations of DOX (0.1-0.4 mg/mL)

in PBS (20 mM, pH8) under stirring for 24 h. Excess

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4 Nano Res.

unloaded DOX was removed using an Amico

centrifugal filter device with MWCO of 100 kDa

and washed at least three times with water until the

filtrate was free of color. Loading of DOX on other

SWNT-PEG solutions were implemented using the

same protocol.

2.6 Cell culture

Human leukemic lymphoblasts cells

(CCRF-CEM cells) were cultured using RPMI-1640

culture medium supplemented with 10 % fetal

bovine serum (FBS) and 1 % penicillin/streptomycin

at 37 °C within a humidified atmosphere containing

5 % CO2.

2.7 NIR light activated cellular uptake of

GNRs-Apt visualized by dark field imaging

The cellular uptake profiles of various GNRs

into CCRF-CEM cells were evaluated using an

Olympus BX51 optical microscopy with the

dark-field imaging setting. In brief, CCRF-CEM

cells were seeded in 12-well plate at a density of

1×105 cells per well and then incubated with GNRs,

GNRs-Apt/DNA, and GNRs-Apt at 0.25 nM in

terms of GNRs. After being irradiated with an

808-nm NIR laser for 10 min at 0.5 W/cm2, the cells

were incubated for another 2 h. After that, the cells

were collected and washes with fresh cell culture

medium for 3 times followed by being imaged

using the aforementioned dark field imaging

system.

2.8 NIR light activated cellular uptake of

SWNTs-Apt visualized by a confocal laser

scanning microscopy

To explore the cellular uptake profile of various

SWNTs nanoconjugates under the NIR laser

irradiation, the amine groups of SWNT-PEG-NH2

were pre-labeled with FITC during aptamer

conjugation. CCRF-CEM cells were seeded in

12-well plates at a density of 1×105 cells per well and

then treated with FITC-labeled SWNT-PEG,

SWNT-PEG-Apt/DNA, and SWNT-PEG-Apt at 50

nM in terms of SWNTs followed by being irradiated

with an 808-nm NIR laser at the power density of

0.5 W/cm2 for 10 min. Then, the cells were

incubated for 2 h and imaged under a confocal laser

scanning microscopy (Leica SP5II, German).

2.9 NIR-light activated targeted cell killing abilities

of DOX loaded SWNT-Apt nanocomplexes

The in vitro cytotoxicity of various DOX loaded

SWNT nanoconjugated was determined by a

standard methyl thiazolyl tetrazolium (MTT) assay.

In brief, CCRF-CEM cells (1×104 cells per well) were

cultured in 96-well plates for 12 h. Then, the cells

were incubated with free DOX, SWNT-PEG+DOX,

SWNT-PEG-Apt/DNA+DOX, or

SWNT-PEG-Apt+DOX at a concentration of 5

g/mL in terms of DOX, as well as

SWNT-PEG-Apt/DNA and SWNT-PEG-Apt at the

same SWNTs concentration (50 nM) to their

corresponding counterparts. After being irradiated

with an 808-nm laser at a power density of 0.5

W·cm-2 for 10 min, the cells were incubated for 2 h

before being washed and transferred into fresh cell

culture for 24 h of re-incubation. After that, MTT

assay was conducted to determine relative cell

viabilities of different samples. 3. Results and Discussion

The synthesis of aptamer modified

photothermal nanostructures is illustrated in

Scheme 1. Sgc8 (5’-(SH)- TT TTT TTT TTT TTT

TTT TAT CTA ACT GCT GCG CCG CCG GGA

AAA TAC TGT ACG GTT AGA-3’)29, an aptamer

which has high affinity and selectivity to bind the

cell membrane protein tyrosine kinase-7 (PTK7),

was conjugated on the surface of photothermal

nanoparticles. Then, a single-strand DNA (ssDNA)

with the complementary sequence (5’-TCT AAC

CGT ACA GTA TTT T-3’) was added to mask the

sgc8 aptamer via the formation of double-stranded

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5Nano Res.

DNA (dsDNA). Upon NIR laser irradiation, the heat

converted by NIR light would enable the

dehybridization of dsDNA and the subsequent

exposure of the sgc8 aptamer sequence, to allow for

specific cancer cell binding.

Gold nanorods (GNRs) were firstly used as a

photothermal agent in this experiment because of

their high efficiency of photothermal conversion as

well as convenient and adjustable surface

modification. GNRs were synthesized according to

the seed-mediated method.29,33-35 As shown in

Figure 2a&b, the aspect ratio of gold nanorods

(GNRs) was approximately 3 as revealed by

transmission electron microscope (TEM) imaging.

The strong surface plasmon resonance absorption

peak of GNRs in the NIR region was observed from

the UV-vis-NIR spectrum (Figure 2b). As expected,

GNRs displayed remarkable and

concentration-dependent photothermal effect under

irradiation by an 808-nm NIR laser, indicating that

they were effective photothermal agents (Figure 2c).

In the next step, thiol-terminated PEG was

conjugated on the surfaces of GNRs via Au-S bond

to reduce the agglomeration and cytotoxicity of

GNRs. Meanwhile, the thiol-terminated poly-T

chain linked with the 5’-end of sgc8 aptamer, which

could self-hybridize to form the special hairpin

structure, was also conjugated on the surface of

GNRs (GNRs-Apt) via Au-S bond36. Sgc8 aptamer

has high affinity to bind protein tyrosine kinase-7,

which is over-expressed on the membrane of

CCRF-CEM cells (human T lymphoblast cell).

Subsequently, ssDNA with the complementary

sequence was added to hybridize with sgc8

aptamer and mask / cage its cancer cell binding

ability.

GNRs could be imaged under dark-field

microscope due to their strong light scattering by

surface plasmonic resonance.37 Therefore, we

investigated the NIR-activated cancer cells binding

of GNRs-Apt/DNA complexes via dark-field

scattering imaging. As shown in Figure 2d, strong

scattered light signals were observed for

CCRF-CEM cells after incubation with GNRs-Apt

but for those incubated with plain GNRs,

suggesting the high affinity binding of GNRs-Apt to

CCRF-CEM cells. Subsequently, followed by adding

of complementary sequence, the GNRs-Apt was

hybridized with its complementary sequence and

the recognition capability between CCRF-CEM cells

and GNRs-Apt/DNA was vanished. As the result,

rather weak scattered light signals were observed

for CCRF-CEM cells incubated with

GNRs-Apt/DNA (Figure 2d). Interestingly, with the

irradiation of 808 nm laser illuminating at the

power intensity of 0.5 W·cm-2 for 10 min, strong

scattered light signals were observed for cells

incubated with GNRs-Apt/DNA complexes. This

phenomenon could be explained by that GNRs

could convert absorbed NIR light into heat, and

then trigger the de-hybridization of dsDNA by

increasing the local temperature to be above the Tm

of this dsDNA, so as to expose the sgc8 aptamer for

cancer cell targeting. In the meanwhile, weak

signals were also observed when CCRF-CEM cells

were incubated with GNRs under 808 nm NIR laser

because of the photothermally enhanced cellular

uptake of nanoparticles19.

To further prove the enhanced cell binding of

GNRs-Apt/DNA on cells under NIR laser was

indeed owing to the de-hybridization of dsDNA,

GNRs-Apt/DNA complexes were irradiated by the

808-nm NIR laser (0.5 W·cm-2 for 10 min) first before

incubation with CCRF-CEM cells. As shown in

Supporting Figure S1, the strong scattered light

signals from CCRF-CEM cells evidenced that the

photothermal effect could induce dsDNA

dehybridization to release complementary sequence,

exposing sgc8 aptamers to allow binding with cells.

Overall, GNRs-Apt/DNA complexes exhibit NIR

light-controllable cancer cell binding ability, which

may be utilized to enable high efficient cancer

therapy and reduce the normal tissues toxicity.

In order to utilize NIR light activated cancer

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6 Nano Res.

cells targeting to achieve light-controlled cancer

therapy, SWNTs, a commonly used photothermal

agent and drug nanocarrier38,39, was used as another

nano-platform in addition to GNRs in this work.

Firstly, amine-terminated polyethylene glycol

(PEG)-grafted poly (maleic

anhydride-alt-1-octadecene) (C18PMH-PEG-NH2)

polymer was used to modify the surface of SWNTs

to enhance their biocompatibility and prevent their

agglomeration in aqueous solutions. The same

thiol-terminated poly-T chain linked the 5’-end of

sgc8, was introduced on the surface of SWNTs via a

bi-functional linker. TEM image revealed the length

of SWNT-PEG-aptamer conjugates

(SWNT-PEG-Apt) to be in the range from 100 to 300

nm (Figure 3a). With strong NIR absorbance,

SWNTs could also serve as an effective

photothermal agent under NIR laser exposure

(Figure 3b).

Subsequently, a commonly used anti-cancer drug,

doxorubicin (DOX), was loaded onto the outer-wall

of SWNTs via π-π stacking and hydrophobic

interaction owing to their sp2-bonded carbon

surface and high surface area. As shown in figure

3c, the UV-vis-NIR absorption spectra of

SWNT-PEG-DOX complexes demonstrated the

special absorption of DOX at ~490 nm was

enhanced with the increasing of mass of added

DOX, indicating DOX could be effectively loaded

on SWNTs. DOX loaded SWNTs (SWNT-PEG+DOX)

with a mass ratio of 1:1 was used in our

experiments.

We next would like to evaluate the concept of

NIR-activated cancer cell targeting with

SWNT-PEG-Apt/DNA. Since the fluorescence of

DOX was quenched after loading on the surface of

SWNTs (Supporting Figure S2), a fluorescent dye

(FITC) was used to label PEGylated SWNTs to

enable confocal fluorescence imaging of cells. In the

experiment, CCRF-CEM cancer cells were incubated

with SWNT-PEG, SWNT-PEG-Apt, or

SWNT-PEG-Apt/DNA, all of which were

pre-labeled by FITC. Confocal fluorescence images

of those cells were performed with or without

exposing to NIR light. As expected, strong

fluorescence signals were detected from

CCRF-CEM cancer cells with incubation of

SWNT-PEG-Apt but not for those incubated with

SWNT-PEG (Figure 3d). Similar to data with gold

nanorods, after the aptamer was caged by the

complementary ssDNA, the cell binding of

SWNT-PEG-Apt/DNA was greatly reduced. By

comparison, obvious enhanced fluorescence was

seen from CCRF-CEM cancer cells incubated with

SWNT-PEG-Apt/DNA exposed to the NIR laser,

because SWNTs could efficiently convert NIR light

into heat to dehybridize the dsDNA and un-cage

the sgc8 aptamers for cells binding. As expected

and consistent to previous findings (Figure 1d),40

the mild photothermal heating of SWNT-PEG

incubated cells would also slightly enhanced the

cellular uptake of SWNTs, but to a degree much

lower than that of cells incubated with

SWNT-PEG-Apt in dark, or SWNT-PEG-Apt/DOX

with NIR exposure. Those results were further

confirmed by quantitative flow cytometry data.

SWNT-PEG-Apt/DNA under laser irradiation

showed the highest level of cellular binding /

uptake (Supporting Figure S3), owing not only to

the NIR-activated cancer cell binding, but also to

the photothermally enhanced cellular uptake of

nanotubes.19,40

At last, NIR-activated targeted cell killing

ability was demonstrated by evaluating the relative

cell viabilities of CCRF-CEM cells treated by free

DOX, SWNT-PEG+DOX, SWNT-PEG-Apt/DNA,

SWNT-PEG-Apt/DNA+DOX, SWNT-PEG-Apt and

SWNT-PEG-Apt+DOX in the presence or absence of

NIR laser irradiation using the standard MTT assay.

As shown in Figure 4, our results indicated that

SWNT-PEG-Apt and SWNT-PEG-Apt/DNA would

not induce obvious cell death even after 10-min

laser irradiation at 0.5 W/cm2, indicating that

PEGylated SWNTs are not toxic to cells and the

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7Nano Res.

mild heating effect induced by SWNTs would be

tolerable by cells, consistent with our previously

published results. 40,41 Under the tested condition

(relatively low drug concentration and short

incubation time), free DOX showed rather limited

cancer cell killing efficiency regardless of laser

irradiation, so as SWNT-PEG+DOX in dark. The

mild photothermal heating of SWNT-PEG+DOX

could slightly enhance its toxicity against cancer

cells, likely owing to the mild hyperthermia

enhanced endocytosis of nano-carriers. Interesting,

SWNT-PEG-Apt/DNA+DOX, while being non-toxic

to cells in dark, showed rather effective cancer cell

killing ability in the presence of NIR light, which

could uncage the cell-binding aptamer sequence to

allow for the specific binding of drug-loaded

nanotubes with cells. As a positive control,

SWNT-PEG-Apt+DOX without caging aptamer

with the base-pairing ssDNA exhibited high

cytotoxicity to cells under both dark and laser

irradiation conditions.

Our results in this work demonstrate excellent

NIR-light activated specific cancer cell binding and

drug delivery by modifying NIR-absorbing

nano-drug-carriers with caged aptamters. In

comparison with commonly explored conventional

cancer targeting strategies, such NIR-activable

cancer-targeted drug delivery systems show a

dual-targeting capability, which is realized by

exploiting the intrinsic cancer-cell-binding ability of

aptamers, as well as the spatially / temporally

controllable laser irradiation (e.g. focusing laser on

the tumor at the best treatment timing), promising

for further reducing non-specific toxicity to normal

tissues and enhancing treatment selectivity against

tumor cells.

4. Conclusion

In summary, our work using GNRs and SWNTs

as two model systems have demonstrated excellent

NIR-light activated specific cancer cell binding and

drug delivery by modifying NIR-absorbing

nano-drug-carriers with ssDNA caged aptamters,

which can be de-hybridized under NIR laser

irradiation and then regain their structure and

cell-binding ability. In comparison with commonly

explored conventional cancer targeting strategies,

such NIR-activable cancer-targeted drug delivery

systems show a dual-targeting capability, which is

realized by exploiting the intrinsic

cancer-cell-binding ability of aptamers, as well as

the spatially / temporally controllable laser

irradiation (e.g. focusing laser on the tumor at the

best treatment timing), promising for further

reducing non-specific toxicity to normal tissues and

enhancing treatment selectivity against tumor cells.

Acknowledgements

This work was partially supported by the National

Natural Science Foundation of China (51222203,

51132006), the National “973” Program of China

(2011CB911002, 2012CB932601), a Jiangsu Natural

Science Fund for Distinguished Young Scholars, the

Macao Science and Technology Development Fund

(062/2013/A2) and the Research Fund of the

University of Macau

(MYRG2014-00033-ICMS-QRCM,

MRG004/CMW/2014/ICMS).

Electronic Supplementary Material: Supplementary

material (data regarding dark-field images of

CCRF-CEM cells, Fluorescence emission spectra of

DOX before and after loading on the

SWNT-PEG-Apt/DNA, and Flow cytometry assay of

CCRF-CEM cells) is available in the online version of

this article at

http://dx.doi.org/10.1007/s12274-***-****-*

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Schematic 1. Schematic illustration to show NIR-activable targeted platform based on NIR-absorbing nano-carriers conjugated with

ssDNA-caged aptamers.

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11Nano Res.

 

Figure 2. Characterization of GNRs-Apt/DNA and NIR-activable targeted cancer cell binding. a) A TEM image of GNRs in water.

Inset: a photo of as-made GNRs solution in water. b) UV–VIS–NIR spectra of GNRs, GNRs-PEG and GNRs-Apt/DNA. c)

Photothermal heating curves of GNRs with different concentrations under 808-nm light irradiation for 5 min at a power density of 0.7

W·cm-2. d) Dark-field images of CCRF-CEM cancer cells incubated with GNRs, GNRs-Apt and GNRs-Apt/DNA with and without

exposure to 808-nm laser for 10 min. The Scale bar: 100 μm.

 

 

 

 

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12 Nano Res.

 

Figure 3. Characterization of SWNT-PEG-Apt/DNA and NIR-activable targeted cancer cell binding. a) A TEM image of SWNT -PEG.

Inset: a photo of as-made SWNT-PEG solution in water. b) Photothermal heating curves of SWNT-PEG with different concentrations

irradiated with the 808-nm light for 5 min at a power density of 0.7 W·cm-2. c) UV–Vis–NIR spectra of SWNT-PEG-Apt/DNA loaded

with various concentrations of DOX. d) Laser scanning confocal microscope images of CCRF-CEM cells incubated with FITC-labeled

SWNT-PEG, SWNT-PEG-Apt and SWNT-PEG-Apt/DNA with and without exposure to 808-nm NIR light for 10 min. The Scale bar: 50

μm.

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13Nano Res.

Figure 4. Relative viabilities of CCRF-CEM cells after various treatments. In this experiment, CCRF-CEM cells were incubated

with DOX, DOX loaded SWNT-PEG (SWNT-PEG+DOX), SWNT-PEG-Apt, DOX loaded SWNT-PEG-Apt

(SWNT-PEG-Apt+DOX), SWNT-PEG-Apt/DNA, and DOX loaded SWNT-PEG-Apt/DNA (SWNT-PEG-Apt/DNA+DOX) with and

without exposure to the 808-nm laser at 0.5 W/cm2 for 10 min. After further incubation for 2 h, cells were washed and transferred

into fresh cell culture for addition incubation for 24 h before the MTT assay.

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Electronic Supplementary Material

Near-infrared Light-activated Cancer Cell Targeting andDrug Delivery with Aptamer Modified Nanostructures

Yu Yang1, Jingjing Liu2, Xiaoqi Sun2, Liangzhu Feng2, Wenwen Zhu2, Zhuang Liu2(), Meiwan Chen1() 1 State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macau, China 2 Institute of Functional Nano & Soft Materials Laboratory (FUNSOM), Soochow University, Suzhou, Jiangsu 215123, China

Supporting information to DOI 10.1007/s12274-****-****-* (automatically inserted by the publisher)

  

Figure S1. Dark-field images of CCRF-CEM cancer cells. a) GNRs-Apt/DNA was exposed to 808-nm laser for 10 min before being

added into cells. b) CCRF-CEM cancer cells were incubated with GNRs-Apt upon exposure to 808-nm laser for 10 min. Strong signals

were observed for both samples. The Scale bar: 100 μm.

 

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Nano Res.

 

Figure S2. Fluorescence emission spectra of DOX before and after loading on the SWNT-PEG-Apt/DNA . Excitation wavelength =

490 nm.

 

 

 

 

 

Figure S3. Flow cytometry assay of CCRF-CEM cells after different treatment.