A Self‐Assembled DNA Origami‐Gold Nanorod Complex for ......tumor cells. For the origami-drug...

full paperswww.MaterialsViews.com

5134 www.small-journal.com © 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

A Self-Assembled DNA Origami-Gold Nanorod Complex for Cancer Theranostics

Qiao Jiang , Yuefeng Shi , Qian Zhang , Na Li , Pengfei Zhan , Linlin Song , Luru Dai , Jie Tian , Yang Du , * Zhen Cheng , * and Baoquan Ding *

1. Introduction

DNA molecules have been used as building blocks for self-

assembly into nanomaterials with various complex geome-

tries. [ 1 ] Through rational design, DNA strands spontaneously

assemble into desired 2D or 3D shapes. [ 2 ] Particularly, the

origami techniques provide DNA materials with well-defi ned

nanoscale shapes, with uniform sizes, precise spatial address-

ability, and excellent biocompatibility. [ 3 ] With these features,

the DNA nanostructures show great potential for biomedical

applications; various DNA-based biomedical imaging probes

or payload delivery carriers have been developed.

Recently, a series of studies have demonstrated that

DNA polyhedral wireframe nanocages can be employed as

delivery carriers for enhanced transport of their payloads,

for instance, small molecular anticancer drugs, immune

stimulating oligo DNA (CpG sequences), [ 4 ] small interfering

RNA, [ 5 ] and several antigen protein molecules, [ 6 ] either in

vitro or in vivo. DNA origami nanostructures offer more

appealing properties, such as enhanced size and shape control DOI: 10.1002/smll.201501266

A self-assembled DNA origami (DO)-gold nanorod (GNR) complex, which is a dual-functional nanotheranostics constructed by decorating GNRs onto the surface of DNA origami, is demonstrated. After 24 h incubation of two structured DO-GNR complexes with human MCF7 breast cancer cells, signifi cant enhancement of cell uptake is achieved compared to bare GNRs by two-photon luminescence imaging. Particularly, the triangle shaped DO-GNR complex exhibits optimal cellular accumulation. Compared to GNRs, improved photothermolysis against tumor cells is accomplished for the triangle DO-GNR complex by two-photon laser or NIR laser irradiation. Moreover, the DO-GNR complex exhibits enhanced antitumor effi cacy compared with bare GNRs in nude mice bearing breast tumor xenografts. The results demonstrate that the DO-GNR complex can achieve optimal two-photon cell imaging and photothermal effect, suggesting a promising candidate for cancer diagnosis and therapy both in vitro and in vivo.

Cancer Therapy

Dr. Q. Jiang, Dr. N. Li, P. Zhan, L. Song, Prof. L. Dai, Prof. B. DingCAS Key Laboratory of Nanosystem and Hierarchial Fabrication National Center for NanoScience and Technology 11 BeiYiTiao, ZhongGuanCun , Beijing 100190 , China E-mail: [email protected]

Dr. Y. Shi Institute of Genetics and Developmental Biology Chinese Academy of Sciences Beijing 100190 , China

Dr. Q. Zhang, Prof. J. Tian, Prof. Y. Du Institute of Automation Chinese Academy of Science 95 ZhongGuanCun East Road , Beijing 100190 , China E-mail: [email protected]

Prof. Z. Cheng Department of Radiology Stanford University 1201 Welch Road , Lucas Center, P095 , Stanford , CA 94305 , USA E-mail: [email protected]

small 2015, 11, No. 38, 5134–5141

http://doi.wiley.com/10.1002/smll.201501266

www.MaterialsViews.com

5135© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.small-journal.com

for the construction of multifunctional delivery and release

carriers. The multivalent DNA origami nanostructures have

gained special attention because of improved delivery effi -

cacy to targeted cells and reduced susceptibility to nontar-

geted ones. [ 7 ] In particular, hexagonal barrel-shaped DNA

origami has been applied to targeting delivery of molecular

cargoes, which is achieved by the activation and reconfi gura-

tion of the sites specifi cally functionalized DNA aptamers. [ 7 ]

In a CpG DNA sequences transport system, hollow 30-helix

DNA origami tubes have been successfully used as effi cient

nanocarriers and triggered strong immune responses of sple-

nocytes. [ 8 ] Furthermore, triangular and tubular origami were

used for intercalation of the anticancer drug, doxorubicin, and

improved cytotoxicity was achieved against drug-resistant

tumor cells. For the origami-drug complex, the signifi cant

enhancement of cytotoxicity is attributed to increased intra-

cellular internalization of drug with the aid of DNA origami

vehicles. [ 9 ] In another doxorubicin-origami delivery system,

the encapsulation effi ciency and the release rate of the drug

were tunable by controlled design of the origami nanostruc-

ture. Compared to free drug, increased cytotoxicity and lower

intracellular elimination rate were achieved. [ 10 ] DNA origami

has been used to fabricate nanoscale robots that are capable

of dynamically i nteracting with each other in living cock-

roaches ( Blaberus discoidalis ). [ 11 ] In the tumor bearing mice

model, DNA origami served as antitumor drug carriers and

successfully transported the payloads to tumor regions. [ 12 ]

The in vivo biodistribution confi rmed that DNA origami real-

ized the effects of enhanced passive targeting and long-lasting

accumulation at tumor region. Essentially, further experi-

ments demonstrated that the DNA origami-drug delivery

system displayed optimal antitumor effi cacy in vivo without

inducing observable systemic toxicity.

However, the bare DNA nanostructures cannot pro-

vide imaging or therapy functions directly. The construction

of the DNA-based platform for diagnostic or therapeutic

applications is usually achieved by loading the sorts of

molecular cargoes (antibody protein, [ 7 ] fl uorescent probes, [ 13 ]

immunostimulatory oligo DNA, [ 8 ] or drug molecules [ 9,10,12 ]

onto DNA nanostructures. Particularly, in all the reported

DNA-based carrier system, only one small molecular drug,

chemotherapeutics doxorubicin, [ 9,10,12 ] was used. On the

other hand, noble metal nanostructures such as aggregated

gold nanospheres, gold nanorods (GNRs), gold nanoshells

and nanocages, and hollow gold/silver dendrites showed

intriguing optical and photothermal conversion properties

due to localized surface plasmon resonances (LSPRs), which

offer great potential for simultaneous molecular imaging and

photothermal cancer therapy. [ 14 ] Specifi cally, GNRs that can

absorb and scatter strongly in the NIR region are the excel-

lent candidate for fl uorescent visualization and plasmonic

photothermal therapy. [ 14b,h , 15 ] Based on these developments,

we proposed to combine the photothermal effect of GNRs

and optimal delivery effect of DNA origami together to

achieve enhanced therapeutic effi cacy.

We constructed a self-assembled DNA origami-GNR

complex (abbreviated to DO-GNR), which is a dual-func-

tional nanotheranostics, by decorating GNRs onto the surface

of DNA origami. The hybridized nanoparticle/DNA complex

designed as integration of the optical photothermal effects

of GNR with passive tumor targeting and long-lasting accu-

mulation of origami. Moreover, DO-GNRs offer two-photon

imaging and photothermal ablation in one DNA nanoplat-

form, suggesting a promising candidate for cancer diagnosis

and therapy. We used the structured DO-GNR complex to

perform photothermal therapy in vitro and in vivo.

2. Results

2.1. Construction of DO-GNR Complex

The experimental scheme is illustrated in Figure 1 . Triangular

and tubular shaped origami were folded by annealing the

M13mp18 genome DNA strand (scaffold), capture strands,

and staple strands in a ratio of 1:10:10 from 95 °C to room

temperature, according to Rothemund's [ 2a ] and Yan’s work [ 16 ]

with several modifi cations. Each edge of the triangular

shaped origami was ≈120 nm long. The length of tubular ori-

gami was nearly 380 nm. DNA capture strands with carefully

designed sequences were extended from one arm of the tri-

angular DNA template working as binding sites to precisely

organize one GNR (40 × 12 nm) as shown in Figure 1 . The

binding sites were designed to display linear pattern on the

top surface of triangular or tubular DNA templates to match

the shape of GNR. The assembled DNA origami nanostruc-

ture was subsequently purifi ed with a fi lter device to remove

the extra capture and staple stands. Next, the purifi ed DNA

origami and GNRs functionalized with corresponding com-

plementary DNA strands were mixed and annealed from

45 °C to 25 °C in 2 h for 30 cycles. After hybridization, GNR

was organized at the desired binding sites on the DNA

platform. The DO-GNR complex was characterized and

then administrated to human breast tumor cells (MCF7).

After two-photon fl uorescence imaging, the triangle shaped

DO-GNR demonstrated preferable tumor cell accumulation,

subsequently it was used for the investigation of the photo-

thermal ablation effects (Figure 1 ).

2.2. Characterization of DO-GNR Complex

The DNA origami nanostructures were characterized after

GNRs loading. For the purifi cation of assembled struc-

tures, agarose gel was used. The target products were sliced

and extracted from the gel with freeze-squeeze column

(Bio-Rad) at 4 °C. The purifi ed nanostructures were then

characterized by transmission electron microscope (TEM)

and ultraviolet–visible (UV–vis) spectrometer. TEM images

of conjugates of triangular DO-GNR and tubular DO-GNR

complex are shown in Figure 2 a,b and Figures S1 and S2

(Supporting Information). TEM images showed clearly that

GNR was attached on origami template at designed loca-

tion. These results provided direct evidence of the forma-

tion of DO-GNR complex and the morphology of the DNA

nanostructures was retained after assembled of GNRs.

Figure 2 c shows UV–vis extinction spectra of unloaded GNR

(black line), triangle DO-GNR complex (red line), and tube

small 2015, 11, No. 38, 5134–5141



DO-GNR complex (blue line). The three spectra nearly coin-

cide with each other except the conjugates’ small peak at

≈260 nm, indicating the presence of DNA origami.

2.3. Internalization of DO-GNR Complex

The property of strong plasmon resonance in the NIR region

makes GNRs ideal contrast agents for two-photon lumines-

cence (TPL) imaging of live cells. [ 15c ] Effi cient and fast con-

jugation methods of DNA modifi ed GNR have been well

studied. After single strand DNA (ssDNA) modifi cation,

GNRs can be precisely organized on DNA origami template

by addressable DNA hybridization. After incorporation

of GNRs on the DNA origami nanostructures, DO-GNR

complex was incubated with tumor cells and the internaliza-

tion of the origami nanostructures was investigated by TPL.

MCF7 cells were employed as a cellular model for direct

visualization of the internalization of GNRs and DO-GNR

complex conjugates. After 24 h incubation with GNR, tri-

angle DO-GNR complex, or tube DO-GNR complex

(0.1 × 10 −9 m ), the live cells were investigated by two-photon

excitation laser-scanning microscopy after removing excess

nanoparticles and conjugates. The wheat germ agglutinin

conjugates (Alexa Fluor 594 conjugate) staining was used

for labeling cell membrane. The TPL excited by two-

photon laser ( λ max = 750 nm) was visible inside cells treated

with DO-GNR complex and bare GNR, demonstrating

that DO-GNR complex nanostructures and GNR can

enter and accumulate in cells. The enhanced internalization

small 2015, 11, No. 38, 5134–5141

Figure 1. DNA origami-gold nanorod (DO-GNR) theranostic system. A long single-stranded M13mp18 genomic DNA scaffold strand (black) is folded into the triangular or tubular nanostructures with well-defi ned binding sites through the hybridization of rationally designed staple strands and capture strands (red). GNRs (40 × 12 nm) functionalized with corresponding complementary ssDNA strands are assembled at the designated locations on the origami template through DNA hybridization, forming DO-GNR complex. The intracellular uptake of different nanostructures of DO-GNR complex was investigated in MCF7 human breast cancer cells. After two-photon fl uorescence imaging, the triangle shaped DO-GNR complex demonstrated optimal tumor cell accumulation, it was then used for the investigation of the photothermal effects.

Figure 2. Characterization of DO-GNR complex by TEM and UV spectrophotometer. TEM image of a) triangular DO-GNR complex and b) tubular DO-GNR complex (scale bar = 100 nm). The insets show individual structure of DO-GNR NP (scale bar = 50 nm). c) UV spectra of GNRs and two shapes of DO-GNR complex.



by DO-GNR complex can be directly observed ( Figure 3 a

and Figure S3, Supporting Information). In the TPL images,

stronger intensity was detected in tumor cells treated with

DO-GNR complex than with bare GNR. The increased

TPL phenomenon caused by DO-GNR complex was con-

fi rmed by quantitative analysis of these images by using

Image J (Figure 3 b and Figure S3, Supporting Information).

The results demonstrated that DO-GNR complex can accu-

mulate more in tumor cells than bare GNRs. Compared to

GNR, DO-GNR complex showed enhanced cell internali-

zation, which may attribute to the size and shape of assem-

bled triangular and tubular origami template. Particularly,

the triangle shaped origami showed slightly better inter-

nalization effects, therefore it was next used for further

photothermal therapy of tumor cells and nude mice bearing

breast tumors.

2.4. In Vitro Photothermal Therapy of DO-GNR Complex

For the in vitro photothermal therapy, triangle DO-GNR

complex was utilized for photothermal ablation of tumor

cells by NIR laser. The MCF7 cells were treated with

0.1 × 10 −9 m purifi ed triangle DO-GNR complex or GNR

alone for 24 h incubation. After visualization of DO-GNR

complex inside the tumor cells with TPL, the photothermal

therapy was applied by laser confocal microscopy or NIR

laser. After exposure to the two-photon laser, the obvious

photoinduced cell membrane blebbing appeared in the

MCF7 cells treated with DO-GNR complex, indicating the

photothermal damages generated by the DO-GNR complex

( Figure 4 a). Under the same condition, the GNR-treated

cells did not show signifi cant morphological change in the

bright fi eld (Figure 4 a). Exposure to continuous NIR laser

was performed to confi rm the improved photothermolysis

effi cacy of DO-GNR complex. Cell viability assay was con-

ducted after administration with GNR and DO-GNR com-

plex for 24 h, respectively, and then exposure to continuous

NIR laser (12 W cm −2 , 3 min and 6 W cm −2 , 3 min). For the

two groups of GNR and DO-GNR complex, induced tumor

cell death was observed. MCF7 cells treated with DO-GNR

complex displayed signifi cantly lower cell viability compared

to the unloaded GNR-treated ones (Figure 4 b). These results

indicated that enhanced photothermal therapy effi cacy is

presumably due to increased intracellular accumulation of

DO-GNR complex (Figure 3 and Figure S3, Supporting

Information). Together with the TPL property, the DO-GNR

system also provided an appealing candidate for imaging-

guided photothermal therapy.

2.5. In Vivo Photothermal Effects of DO-GNR Complex

For the in vivo photothermal therapy, the MCF7 xenograft

tumor bearing mice were used and were intravenously

injected through tail with 0.9% saline (150 µL), GNRs

(3 × 10 −9 m , 150 µL), and DO-GNR complex (3 × 10 −9 m ,

150 µL), respectively. The NIR irradiation (1.5 W cm −2 ,

8 min) was performed on the tumor xenografts 24 h postin-

jection. The temperature of tumors was measured before and

after NIR laser irradiation. Similar to the saline treatment

in the blank group, DNA origami itself did not negatively

impact on the temperatures of tumor regions (Figure S4,

Supporting Information). As shown in Figure 5 , the ther-

mographic maps demonstrated that the DO-GNR complex

induced the highest increase of temperature compared to

the saline and GNR-treated groups. The results suggested

that DO-GNR complex can increase the optical and pho-

tothermal effects compared with plain GNRs, which was

due to the enhanced tumor passive targeting and long-

lasting properties at tumor region of DNA origami. [ 12 ] After

the NIR laser irradiation and thermal imaging, the images

small 2015, 11, No. 38, 5134–5141

Figure 3. Improved cellular uptake of GNR using DNA origami carriers. a) Two-photon photoluminescence (TPL) images of MCF7 breast cancer cells (scale bars = 20 µm) after incubation with GNRs (0.1 × 10 −9 M ) or DO-GNR complex (0.1 × 10 −9 M ) for 24 h. The two-photon laser excitation wavelengths were taken upon at 750 nm (for UV absorption peak at ≈750 nm), while the lasers power was 2%. The wavelength regions were monitored at 495–540 nm. The WGA was used for cell membrane staining. Additional TPL images are given in the Supporting Information. b) All the TPL images were quantitatively analyzed by Image J for average intracellular fl uorescence intensity. Error bars represent the standard error of the mean of three independent experiments in quadruplicate images of cells (* P < 0.05 and *** P < 0.001).



showed that the tumors with saline treatment and laser

irradiation grew rapidly, indicating that the tumor itself

was not impaired by NIR laser irradiation. In GNR treat-

ment group, the tumors were burned after irradiation and

formed scars on the surface after laser irradiation. For the

nude mice treated with DO-GNR complex, the tumors were

burned obviously after irradiation, leaving the original site

with black scars (Figure S5a, Supporting Information). The

mouse death was recorded and the survival rate was calcu-

lated. Thirty days after NIR irradiation, the mouse survival

rate was highest in the DO-GNR complex treatment group

compared to the saline and GNR-treated ones (Figure S5b,

Supporting Information). The data indicated that DNA ori-

gami facilitated the photothermal effects of GNR on tumors

and therefore effectively prolong survival time of the tumor

bearing mice.

3. Discussion

Nanoscale systems are emerging as a class of cancer diag-

nostic and therapeutic tools. Drug delivery systems have been

widely reported, including the applications of liposomes,

micelles, dendrimers, carbon nanotubes, and nanoparticles. [ 17 ]

Many nanoscale delivery systems have

demonstrated tumor-targeting imaging

and increased antitumor effi ciency, owing

to enhanced permeability and retention

(EPR) effects in tumor regions, and size

and shape-dependent cellular uptake. [ 18 ]

One promising trend is the development

of nanotheranostics, integrating multiple

functional groups with diagnostic and

therapeutic effects at the nanoscale. [ 19 ] In

the present work, GNRs were attached

to DNA origami at designated locations,

forming DO-GNR hybrids complex with

uniform shapes and sizes. With LSPR

peaks in NIR region, DO-GNR complex

exhibited a broad two-photon photolu-

minescence (450–650 nm) when excited

by two-photon laser source. The highly

effi cient TPL excited by NIR laser makes

DO-GNR complex ideal optical agents

for two-photon imaging, which has great

potential in early and noninvasive diag-

nosis of cancers. Two-photon imaging

small 2015, 11, No. 38, 5134–5141

Figure 4. Enhanced photothermal ablation of DO-GNR complex. a) Photothermolysis (750 nm, 2% of lasers power, scanning area 500 × 500 µm 2 , 100 frames) on MCF7 cells incubated 24 h with GNRs (0.1 × 10 −9 M ) or DO-GNR complex (0.1 × 10 −9 M ), scale bars = 20 µm. b) Cell viability of MCF7 cells after administration with medium only as blank, GNRs, and DO-GNR complex, respectively, for 24 h and exposure to continuous red laser at 750 nm (6 W cm −2 , 3 min and 12 W cm −2 , 3 min). Error bars represent the standard error of the mean of three independent experiments in triplicate wells of cells (* P < 0.05 and *** P < 0.001).

Figure 5. Enhanced photothermal generation of DO-GNR complex in vivo. The MCF7 tumor-bearing mice intravenously injected with saline as blank, GNR, and DO-GNR complex were measured after NIR irradiation (1.5 W cm −2 , 8 min). a) The near-infrared thermographic maps of mice after photothermal therapy. b) The enhancement of temperature in tumor tissues after different administration. The error bars represent the standard error of the mean of three independent experiments (* P < 0.05 and *** P < 0.001).



demonstrated that hybrid DO-GNR exhibited much stronger

TPL intensity compared with GNRs. These results indicated

the enhancement of cell accumulation by DO-GNR com-

plex, which was presumably induced by the optimal size

and shape-dependent internalization effect of DNA origami

(Figure S6, Supporting Information). Specifi cally, the trian-

gular DO-GNR complex demonstrated better cellular inter-

nalization and was used in next-step photothermal therapy

in vitro and in vivo.

Integrated with the TPL property, the DO-GNR system

provided an appealing platform for imaging-guided pho-

tothermal therapy. The TPL-guided photothermal ablation

was performed in MCF7 cells and triangular DO-GNR com-

plex demonstrated signifi cant improved photothermolysis

effi cacy compared with GNRs. After exposure to the two-

photon laser, obvious heat-induced cell membrane blebbing

appeared in the MCF7 cells treated with DO-GNR complex,

suggesting the enhanced photothermal damages generated

by the enriched GNRs inside the cells. On the other hand,

GNR and DO-GNR complex induced tumor cell death were

both observed after continuous NIR irradiation. MCF7 cells

treated with DO-GNR complex displayed signifi cantly lower

cell viability compared to the GNRs treated ones, at both

12 W cm −2 and 6 W cm −2 laser exposure. Interestingly, the

hybrid DO-GNR complex required only 6 W cm −2 laser

exposure for 3 min to induce more than 80% cell death

through photothermal ablation, which was about two times

photothermal damage effects compared with GNR-treated

group under the same intensity irradiation. Actually, high-

temperature hyperthermia can generate systemic and local

side effects. [ 20 ] Considering application in vivo and further

clinical use, relative lower power of NIR laser is essential,

which could decrease injure of hyperthermia for surrounding

healthy tissues. [ 20,21 ] These in vitro experiments demonstrated

the enhanced photothermal therapy effi cacy induced by

DO-GNR complex, which is attributed to increased intracel-

lular accumulation of GNRs (Figure 3 and Figure S3, Sup-

porting Information).

The preferable photothermal effects were then con-

fi rmed in the MCF7 xenograft tumor bearing mice. After

NIR laser irradiation at 24 h postinjection intravenously, the

DO-GNR complex induced the highest increase of tempera-

ture with comparison of saline and GNR treatments. The

survival time of DO-GNR complex treated group was also

the longest among the three different groups. The enhanced

heat-generating and improved photothermal therapy in vivo

were realized by DO-GNRs complex, due to the optimal

passive targeting and long-lasting retention effects at tumor

region. [ 12 ] Gold signals detected by inductively coupled

plasma mass spectrometry (ICP-MS) in tumor cells or tumor

tissues treated with DO-GNR were signifi cantly higher than

GNR-treated ones (Figure S7, Supporting Information,

* P < 0.05, *** P < 0.0001). The results of ICP-MS were con-

sistent with the imaging data, revealing that DO-GNR com-

plex can accumulate more in tumor cells than GNRs. The

in vivo data showed DO-GNR enhanced accumulation and

retention in tumors, indicating that the DNA origami carriers

with superior EPR effects induced DO-GNR accumulation

at the tumors. The in vitro and in vivo photothermal therapy

data demonstrated that the hybrid DO-GNR complex was a

promising candidate for cancer therapy.

4. Conclusion

In this work, a self-assembled biological DNA origami-inor-

ganic GNR complex serving as a successful dual-functional

nanotheranostics was reported. The hybrid nanoplatform

integrated the advantages of self-assembly DNA origami

nanostructures and GNRs, demonstrated a promising can-

didate for cancer diagnosis and therapy in vitro and in

vivo. Optimal intracellular TPL of DO-GNR complex was

observed. Signifi cant enhancement of photothermolysis in

vitro was achieved by two-photon laser or NIR laser irradia-

tion compared to GNR. Moreover, the DO-GNR complex

exhibited enhanced antitumor effi cacy compared with GNRs

in nude mice bearing breast tumor xenografts. In this nan-

otheranostic system, the DO-GNR complex integrates pref-

erable delivery capability with two-photon cell imaging and

photothermal ablation therapy.

To further develop a smart nanotheranostic system, DNA

origami nanostructures can serve as the template to integrate

multiple functional elements into one platform. The organiza-

tion of multiple photothermal conversion nanoparticles at pre-

designed locations of origami template could achieve enhanced

photothermal effects. Tumor-targeting biomolecules (such as

peptides and aptamers) and DNA intercalated drugs (doxo-

rubicin) can also be assembled into the same template. The

mechanical reconfi guration of DNA origami through appro-

priate structural design promises controlled release of the

loaded cargos. Using DNA origami as the nanoplatform, combi-

natorial diagnosis and therapy could be realized. After loading

other functional elements, such as iron oxide nanoparticles on

origami template, the hybrid DNA-iron oxide nanocomplex

could play a very important role to understand the distribution

and fate of the formulation via magnetic resonance imaging.

These will result in a multifunctional DNA-based platform,

which will extend the applications of the self-assembled DNA

nanostructures in cancer diagnosis and therapy. Our study here

will not only offer new insights into the understanding of mul-

tifunctional nanotheranostics but also encourage the develop-

ment of DNA origami platform as effi cient and biocompatible

candidates for novel antitumor drug delivery systems.

5. Experimental Section

Materials : All oligonucleotides were purchased from Invitrogen (Shanghai, China). The origami staple strands were all diluted to 100 × 10 −6 M and used without further purifi cation. The 3′-thiol-modifi ed DNA strands were purifi ed by high-performance liquid chromatography and the concentration of each strand was esti-mated by measuring the UV absorbance at 260 nm. M13mp18 phage DNA (N4040S) was purchased from New England Biolabs (USA). Auric acid (HAuCl 4 ), cetyltrimethylammonium bromide (CTAB), sodium borohydride (NaBH 4 ), silver nitrate (AgNO 3 ), and tris(carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (USA).

small 2015, 11, No. 38, 5134–5141



Synthesis of GNRs and Modifi cation of GNRs with Thiolated DNA : Seed-mediated growth was performed to synthesize GNRs according to the method developed by Nikoobakht and El-Sayed. [ 22 ] Next, GNRs were modifi ed with oligo-DNA at low pH value according to the method of Ding. [ 23 ]

DNA Origami Assembling and Purifi cation : Triangle and tube shaped DNA origami structures were assembled according to Rothemund’s and Yan’s work with several modifi cations. See Schemes S1 and S2 (Supporting Information) for the details of ori-gami design scheme. A 1:10:10 molar ratio between the M13mp18 ssDNA (5 × 10 −9 M ), the short staple strands, and capture strands were used. The DNA origami was assembled in 1 × TAE-Mg 2+ buffer (40 × 10 −3 M Tris, 20 × 10 −3 M acetic acid, 2 × 10 −3 M ethylenedi-aminetetraacetic acid (EDTA); 12.5 × 10 −3 M magnesium acetate; pH 8.0) in an Eppendorf thermocycler (Eppendorf China) by slowly cooling from 90 °C to room temperature over 12 h. DNA origami was then fi ltered with 100 kDa MWCO centrifuge fi lters (Amicon, Millipore, USA) to remove extra DNA staple strands.

Self-Assembly of DO-GNR Complex : Purifi ed DNA origami was mixed with GNRs with a ratio of two GNRs for one DNA origami. The mixture was annealed from 45 °C to 25 °C in 2 h for 30 cycles.

Purifi cation of DO-GNR Complex with Agarose Gel Electropho-resis : For DO-GNR complex purifi cation, EtBr-free agarose gel was used (running buffer 0.5 × tris-boric acid-EDTA (TBE) buffer with 11 × 10 −3 M Mg 2+ , loading buffer 50% glycerol, 15 V cm −1 ). Selected bands were cut off and the DO-GNR complex was extracted from the gel with Freeze-Squeeze column (Bio-Rad, USA) at 4 °C.

TEM Characterization of DO-GNR Complex : 7 µL of DO-GNR solution was deposited onto carbon-coated grids and incubated for 10 min. For negative staining of DNA nanostructures, the grid was treated with a drop of 0.7% uranylacetate for 40 s and kept at room temperature for 2 h. TEM imaging was carried out by using a Tecnai G2-20S TWIN, operated at 80 kV in the dark-fi eld mode.

Cell Culture : MCF7 is a human breast adenocarcinoma cancer cell line, which was kindly provided by Prof. Xingjie Liang (NCNST). MCF7 cells were cultured in Dulbecco's Modifi ed Eagle Medium (Hyclone, Thermo Scientifi c) supplemented with 10% fetal bovine serum (Hyclone, Thermo Scientifi c) and with L-glutamine, peni-cillin, streptomycin (GIBICO, Invitrogen). The cells were cultured in an atmosphere of 5% CO 2 at 37 °C.

Cellular Internalization of GNRs and DO-GNR Complex : MCF7 cells were seeded in confocal dishes, cultured overnight, and then incubated with GNRs (0.1 × 10 −9 M ) and purifi ed conjugates of DO-GNR complex (0.1 × 10 −9 M , diluted by culture medium) for 24 h. The wheat germ agglutinin (WGA) conjugates (Alexa Fluor 594 conjugate, red fl uorescence, Invitrogen) was used for cell mem-brane staining. The cells were incubated with WGA (1 × 10 −6 M ) for 5 min at 25 °C. After being washed with phosphate-buffered saline (PBS), the living cells were visualized by two-photon laser confocal fl uorescent microscopy (Olympus, Japan).

Photothermal Ablation of Tumor Cells after Two-Photon Laser Exposure : MCF7 cells were seeded in confocal dishes, cultured overnight, and then incubated with GNRs (0.1 × 10 −9 M ) and puri-fi ed conjugates of DO-GNR complex (0.1 × 10 −9 M , diluted by cul-ture medium) for 24 h. After being washed with PBS, the living cells were exposed under two-photon laser (laser power: 2%; scanning area: 100 × 100 µm 2 , 100 frames).

Cell Viability Assay after NIR Laser Exposure : The photothermal-induced tumor cell death after GNRs or DO-GNR complex

incubation and laser irradiation were assessed with a cell-counting kit (cck-8, Dojindo, Japan) containing a highly water-soluble tetrazolium salt (WST-8) [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetra-zolium, monosodium salt]. After seeded in 96-well plates and cultured overnight, the MCF7 cells were incubated with GNRs and DO-GNR complex for 24 h. Thereafter, the cells were irradiated with NIR laser (750 nm, 4 W or 2 W) for 3 min. Finally, the cells were incubated with fresh serum-free medium containing 0.5 mg mL −1 WST-1 for 1 h at 37 °C for the cytotoxicity assay. Absorb-ance values at 450 nm were measured using a microplate reader (TECAN, infi nite M200, Switzerland).

Establishment of Tumor Xenograft Mouse Model and Photo-thermal Therapy In Vivo : 5- to 6-week-old athymic female BALB/c nude mice were purchased from the Department of Experimental Animals, Peking University Health Science Center, and all animal procedures were performed in accordance with the guidelines of the local Institutional Animal Care and Use Committee Peking Uni-versity (Permit Number: 2011-0039). The subcutaneous tumors were established by injecting 1 × 10 6 MCF7 cells into the right upper fl anks of the BALB/c nude mice. Mice with tumor volumes around 100 mm 3 were randomly divided into different groups (four mice per group) including saline, origami, GNR, and DO-GNR complex groups. For the in vivo photothermal therapy, the MCF7 tumor-bearing mice were intravenously injected with control saline, origami (3 × 10 −9 M , 150 µL), GNR (3 × 10 −9 M , 150 µL), and DO-GNR (3 × 10 −9 M , 150 µL), respectively. 24 h after injection, the NIR irradiation (1.5 W cm −2 , 8 min) was performed on the tumor xenografts. [ 24 ] The temperature of tumors was measured before and after NIR laser irradiation, and the thermographic maps were made. The mouse death from each group was recorded for 30 d and the survival rate was calculated.

Statistical Analysis : One-way analysis of variance (ANOVA) and Turkey multiple comparisons test or Student’s t -test was used to determine the statistical differences. * P < 0.05 and *** P < 0.001 were considered statistically signifi cant. Statistical analysis was conducted using Prism4.0 (San Diego, CA, USA).

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

Q.J. and Y.S. contributed equally to this work. The authors are grateful for the fi nancial support from the National Science Foun-dation of China (21173059, 91127021, 21222311, 21273053, 81227901, and 81470083), the National Basic Research Pro-gram of China (973 Program, 2012CB934000, 2015CB755500, 2014CB748600, and 2011CB707702), the 100-Talent Program of Chinese Academy of Sciences (B.Q.D.), and the Beijing Natural Sci-ence Foundation (L140008).

small 2015, 11, No. 38, 5134–5141


5141© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.small-journal.comsmall 2015, 11, No. 38, 5134–5141

[1] N. C. Seeman , in NanoBiotechnology Protocols (Ed: Sandra J. Rosenthal , David W. Wright) , Springer , Germany 2005 , pp. 143 – 166 .

[2] a) P. W. Rothemund , Nature 2006 , 440 , 297 ; b) D. Han , S. Pal , Y. Liu , H. Yan , Nat. Nanotechnol. 2010 , 5 , 712 ; c) D. Han , S. Pal , J. Nangreave , Z. Deng , Y. Liu , H. Yan , Science 2011 , 332 , 342 ; d) B. Wei , M. Dai , P. Yin , Nature 2012 , 485 , 623 ; e) Y. Ke , L. L. Ong , W. M. Shih , P. Yin , Science 2012 , 338 , 1177 .

[3] a) A. V. Pinheiro , D. Han , W. M. Shih , H. Yan , Nat. Nanotechnol. 2011 , 6 , 763 ; b) J. Li , C. Fan , H. Pei , J. Shi , Q. Huang , Adv. Mater. 2013 , 25 , 4386 ; c) P. F. Zhan , Q. Jiang , Z. G. Wang , N. Li , H. Y. Yu , B. Q. Ding , ChemMedChem 2014 , 9 , 2013 .

[4] J. Li , H. Pei , B. Zhu , L. Liang , M. Wei , Y. He , N. Chen , D. Li , Q. Huang , C. Fan , ACS Nano 2011 , 5 , 8783 .

[5] H. Lee , A. K. Lytton-Jean , Y. Chen , K. T. Love , A. I. Park , E. D. Karagiannis , A. Sehgal , W. Querbes , C. S. Zurenko , M. Jayaraman , Nat. Nanotechnol. 2012 , 7 , 389 .

[6] X. Liu , Y. Xu , T. Yu , C. Clifford , Y. Liu , H. Yan , Y. Chang , Nano Lett. 2012 , 12 , 4254 .

[7] S. M. Douglas , I. Bachelet , G. M. Church , Science 2012 , 335 , 831 .

[8] V. J. Schüller , S. Heidegger , N. Sandholzer , P. C. Nickels , N. A. Suhartha , S. Endres , C. Bourquin , T. Liedl , ACS Nano 2011 , 5 , 9696 .

[9] Q. Jiang , C. Song , J. Nangreave , X. Liu , L. Lin , D. Qiu , Z.-G. Wang , G. Zou , X. Liang , H. Yan , B. Ding , J. Am. Chem. Soc. 2012 , 134 , 13396 .

[10] Y.-X. Zhao , A. Shaw , X. Zeng , E. Benson , A. M. Nyström , B. r. Högberg , ACS Nano 2012 , 6 , 8684 .

[11] Y. Amir , E. Ben-Ishay , D. Levner , S. Ittah , A. Abu-Horowitz , I. Bachelet , Nat. Nanotechnol. 2014 , 9 , 353 .

[12] Q. Zhang , Q. Jiang , N. Li , L. Dai , Q. Liu , L. Song , J. Wang , Y. Li , J. Tian , B. Ding , Y. Du , ACS Nano 2014 , 8 , 6633 .

[13] a) X. Shen , Q. Jiang , J. Wang , L. Dai , G. Zou , Z.-G. Wang , W.-Q. Chen , W. Jiang , B. Ding , Chem. Commun. 2012 , 48 , 11301 ; b) R. Jungmann , M. S. Avendaño , J. B. Woehrstein , M. Dai , W. M. Shih , P. Yin , Nat. Methods 2014 , 11 , 313 .

[14] a) X. Huang , P. K. Jain , I. H. El-Sayed , M. A. El-Sayed , Lasers Med. Sci. 2008 , 23 , 217 ; b) E. B. Dickerson , E. C. Dreaden , X. Huang , I. H. El-Sayed , H. Chu , S. Pushpanketh , J. F. McDonald , M. A. El-Sayed , Cancer Lett. 2008 , 269 , 57 ; c) I. H. El-Sayed , X. Huang , M. A. El-Sayed , Cancer Lett. 2006 , 239 , 129 ; d) J. Song , J. Zhou , H. Duan , J. Am. Chem. Soc. 2012 , 134 , 13458 ; e) K. W. Hu ,

C. C. Huang , J. R. Hwu , W. C. Su , D. B. Shieh , C. S. Yeh , Chem. Eur. J. 2008 , 14 , 2956 ; f) J. Chen , D. Wang , J. Xi , L. Au , A. Siekkinen , A. Warsen , Z.-Y. Li , H. Zhang , Y. Xia , X. Li , Nano Lett. 2007 , 7 , 1318 ; g) C. Loo , A. Lowery , N. Halas , J. West , R. Drezek , Nano Lett. 2005 , 5 , 709 ; h) L. Vigderman , B. P. Khanal , E. R. Zubarev , Adv. Mater. 2012 , 24 , 4811 .

[15] a) X. Huang , I. H. El-Sayed , W. Qian , M. A. El-Sayed , J. Am. Chem. Soc. 2006 , 128 , 2115 ; b) Z. Zhang , L. Wang , J. Wang , X. Jiang , X. Li , Z. Hu , Y. Ji , X. Wu , C. Chen , Adv. Mater. 2012 , 24 , 1418 ; c) H. Wang , T. B. Huff , D. A. Zweifel , W. He , P. S. Low , A. Wei , J.-X. Cheng , Proc. Natl. Acad. Sci. USA 2005 , 102 , 15752 ; d) Z. Y. Xiao , C. W. Ji , J. J. Shi , E. M. Pridgen , J. Frieder , J. Wu , O. C. Farokhzad , Angew. Chem. Int. Ed. 2012 , 51 , 11853 .

[16] a) S. Pal , Z. T. Deng , H. N. Wang , S. L. Zou , Y. Liu , H. Yan , J. Am. Chem. Soc. 2011 , 133 , 17606 ; b) B. Ding , H. Wu , W. Xu , Z. Zhao , Y. Liu , H. Yu , H. Yan , Nano Lett. 2010 , 10 , 5065 ; c) L. A. Stearns , R. Chhabra , J. Sharma , Y. Liu , W. T. Petuskey , H. Yan , J. C. Chaput , Angew. Chem. Int. Ed. 2009 , 121 , 8646 .

[17] a) R. A. Petros , J. M. DeSimone , Nat. Rev. Drug Discov. 2010 , 9 , 615 ; b) A. Z. Wang , R. Langer , O. C. Farokhzad , Annu. Rev. Med. 2012 , 63 , 185 .

[18] a) H. Maeda , J. Wu , T. Sawa , Y. Matsumura , K. Hori , J. Con-trol. Release 2000 , 65 , 271 ; b) A. Albanese , P. S. Tang , W. C. W. Chan , Annu. Rev. Biomed. Eng. 2012 , 14 , 1 ; c) O. C. Farokhzad , R. Langer , ACS Nano 2009 , 3 , 16 .

[19] a) H. K. Patra , N. U. Khaliq , T. Romu , E. Wiechec , M. Borga , A. P. F. Turner , A. Tiwari , Adv. Healthcare Mater. 2014 , 3 , 526 ; b) F. M. Kievit , M. Zhang , Adv. Mater. 2011 , 23 , H217 .

[20] P. Wust , B. Hildebrandt , G. Sreenivasa , B. Rau , J. Gellermann , H. Riess , R. Felix , P. Schlag , Lancet Oncol. 2002 , 3 , 487 .

[21] a) K. Yang , S. Zhang , G. Zhang , X. Sun , S.-T. Lee , Z. Liu , Nano Lett. 2010 , 10 , 3318 ; b) Y.-F. Huang , K. Sefah , S. Bamrungsap , H.-T. Chang , W. Tan , Langmuir 2008 , 24 , 11860 .

[22] B. Nikoobakht , M. A. El-Sayed , Chem. Mater. 2003 , 15 , 1957 . [23] D. Shi , C. Song , Q. Jiang , Z.-G. Wang , B. Ding , Chem. Commun.

2013 , 49 , 2533 . [24] S. S. Su , Y. H. Tian , Y. Y. Li , Y. P. Ding , T. J. Ji , M. Y. Wu , Y. Wu ,

G. J. Nie , ACS Nano 2015 , 9 , 1367 .

Received: May 5, 2015 Revised: June 26, 2015Published online: August 6, 2015

A Self‐Assembled DNA Origami‐Gold Nanorod Complex for ......tumor cells. For the origami-drug...

Documents

Transcript of A Self‐Assembled DNA Origami‐Gold Nanorod Complex for ......tumor cells. For the origami-drug...