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From Detection to Resection: Photoacoustic Tomography and Surgery Guidance with Au@liposome-ICG Nanoparticles in Liver Cancer Tianpei Guan, Wenting Shang, Hui Li, Xin Yang, Chihua Fang, Jie Tian, and Kun Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00065 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 27, 2017

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Bioconjugate Chemistry

From Detection to Resection: Photoacoustic Tomography and Surgery Guidance with Au@liposome-ICG Nanoparticles in Liver Cancer Tianpei Guan1,2,3,4 †, Wenting Shang2, 3†, Hui Li2, 3, Xin Yang2, 3, Chihua Fang1*, Jie Tian2, 3*, Kun Wang2, 3* 1

Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical

University, Guangzhou 510280, China; 2

CAS Key Laboratory of Molecular Imaging, Institute of Automation, Chinese

Academy of Sciences, Beijing 100190, China; 3

4

Beijing Key Laboratory of Molecular Imaging, Beijing 100190, China. Guangdong provincial clinical and engineering center of digital medicine,

Guangzhou 510280, China; †

*

Co-first authors: Tianpei Guan, Wenting Shang. Corresponding co-authors:

Chihua Fang: Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China. Tel: +86 20 61643208. Fax: +86 20 61643208. Email: [email protected]. Jie Tian

([email protected]) and Kun Wang ([email protected]),

CAS Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences, Zhongguancun East Road #95, Haidian Dist, Beijing 100190, China. Tel: +86 10 82618465; Fax: +86 10 62527995 1/31

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Abstract Conventional imaging methods encounter challenges in diagnosing liver cancer that is less than 10 mm and/or without typical hypervascular features. With deep penetration and high spatial resolution imaging capability, the emerging photoacoustic tomography may offer better diagnostic efficacy for non-invasive liver cancer detection. Moreover, near-infrared fluorescence imaging guided hepatectomy was proven to be able to identify nodules at millimeter level. Thus, suitable photoacoustic and fluorescence dual-modality imaging probe may benefit patients in early diagnosis and complete resection. In this study, we fabricated indocyanine green loaded gold nanorod@liposome core-shell nanoparticles (Au@liposome-ICG) to integrate both imaging strategies. These nanoparticles exhibit superior biocompatibility, high stability, and enhanced dual-model imaging signal. Then, we explored its effectiveness of tumor detection and surgery guidance in orthotopic liver cancer mouse models. Histological analysis confirmed the accuracy of the probe in liver cancer detection and resection. This novel dual-modality nano-probe holds promise for early diagnosis and better surgical outcome of liver cancer and has great potential for clinical translation. Keywords: Photoacoustic tomography; fluorescence imaging; Au nanorods; Liver cancer; tumor detection, surgery guidance

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INTRODUCTION

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Liver cancer, the third lethal cancer, results in more than 740,000 deaths yearly

3

worldwide1. Early diagnosis and effective intervention were commonly considered to

4

be the best approaches to achieve long-term survival.2 However, only 10 ~ 20% of

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these patients could be diagnosed early enough to receive effective treatment due to

6

the lack of sensitive diagnosis approaches.3 Conventional imaging methods, such as

7

computed tomography (CT) and magnetic resonance imaging (MRI), encounter

8

challenges in diagnosing liver cancer without typical features (Intense arterial uptake

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and delayed phase wash out) and/or with diameter less than 1 cm4,5. Currently,

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although ablation and transplantation are recommended as the curative treatments for

11

suitable candidates, surgical resection still remains the standard of care because of

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donors scarcity and the eradication ability of surgery4,6. Recently, near-infrared (NIR)

13

fluorescence imaging guided surgery, as a novel operation paradigm with superiority

14

in intraoperative tumor margin delineation and micro-lesions identification7, has

15

garnered great attention of biomedical researchers, as well as been treated as the

16

routine standard of care in some centers.8 As it is approved by Food and Drug

17

Administration (FDA), indocyanine green (ICG) is the most commonly used NIR

18

fluorescent dye in clinical practice. ICG fluorescence imaging could detect more than

19

95% HCC lesions8 and identify new nodules undetectable with conventional

20

preoperative imaging or intraoperative ultrasonography in 40% of patients.9 The

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precision and completeness of resection could be obviously improved by NIR

22

fluorescence imaging guidance.7 However, more accurate preoperative detection with 3/31



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high special resolution imaging technology contributes to earlier diagnosis and more

2

effective treatment. Therefore, a novel imaging technique is urgently needed for early

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diagnosis and surgery guidance.

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Photoacoustic tomography (PAT), characterized by high spatial resolution and deep

5

penetration (up to 7 cm), overcomes the drawbacks of pure optical methods by the

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photoacoustic effect and provides a novel opportunity in early diagnosis with its

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imaging ability.10-13 With particular endogenous or exogenous chromophores,

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photoacoustic imaging provides reliable functional and anatomical information, which

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enhances tumor detection, diagnosis, volumetric calculation, and delineation11, 14-16.

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An ideal probe may facilitate clinical practice from preoperative assessment to

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intraoperative manipulation14,17 and benefit patients with long survival and cost

12

efficient intervention.18,19 Theoretically, ICG is remarkable for both photoacoustic and

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fluorescence imaging with its inherent absorption, emission spectrum, and approved

14

applications in clinical practice.20 However, drawbacks such as aggregation, rapid

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clearance, fluorescence quenching, and low efficiency in converting laser energy to

16

heat limit its potential applications as a dual modality contrast agent.21 A recent report

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demonstrated that ICG photoacoustic signal decreased sharply in human liver

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specimens and did not allow the noninvasive detection of orthotopic liver cancer in

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model mice.22 Therefore, a suitable photoacoustic-fluorescence dual modality probe

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with appropriate fluorescence and photoacoustic imaging capability is urgently

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needed 23,24 for liver cancer detection and treatment.

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Typical approaches to fabricate this type of probe include NIR fluorochrome 4/31


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modification and incorporation of Au nanoparticles with NIR dyes. Au nanorod

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(AuNR) is regarded as an effective photoacoustic probe with its superior properties

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such as low toxicity, good biocompatibility, and easier effusion into the tumor region

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through enhanced permeability and retention (EPR) effect.25-27 Moreover, with an

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anisotropic shape, the absorption peak of AuNR could be shifted from red to the NIR

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region and the photoacoustic signal could be enhanced with its large optical

7

absorption cross section.28 Recently, photoacoustic-fluorescence dual modality

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imaging nanoparticles based on AuNRs and ICG such as liposomal ICG,29 liposomal

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ICG/AuNR hybrids,30 and AuNR-ICG-silica core shell structures21 have been

10

designed. However, probes that qualified for simultaneous fluorescence and

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photoacoustic imaging are limited, and further applications of these probes in liver

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cancer detection and surgery guidance remain unexplored.

13

In this study, we fabricated ICG loaded Au nanorod@liposome core-shell

14

nanoparticles

15

photoacoustic-fluorescence imaging probe in liver cancer detection and resection in

16

liver cancer mouse models. The unique design of Au@liposome-ICG endows its

17

crucial role from diagnosis to intraoperative resection. The synergy between AuNR

18

and ICG develops a stable absorption (795 nm), through which an enhanced PAT

19

signal could be obtain in preoperative liver cancer diagnosis. During the operation, the

20

core-shell structure of Au@liposome-ICG enables ICG molecules to sustainably and

21

stably emit at 840 nm, which facilitates the operation with sufficient imaging time.

22

RESULTS AND DISCUSSION

(Au@liposome-ICG)

and

report

its

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effectiveness

as

a


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Liver cancer is the third lethal cancer.1 Because of the lack of effective diagnosis

2

approaches, only 10–20% of patients with liver cancer are diagnosed early enough to

3

receive effective resection,3 and more than 50% recurred within 2 years after

4

operation.31 To bridge the gaps between early diagnosis and effective resection, we

5

fabricated Au@liposome-ICG via encapsulation of AuNR with liposomal ICG. Figure

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1A depicts the Au@liposome-ICG in vivo. The procedure of Au@liposome-ICG

7

fabrication presented in the dashed. After successful fabrication, the probe was

8

intravenously (i.v.) injected into model mice. This Au@liposome-ICG has the

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capabilities of simultaneous in vivo fluorescence and photoacoustic imaging. With its

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fluorescence-photoacoustic dual-modality imaging capabilities, Au@liposome-ICG

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can function for preoperative detection to intraoperative guidance.

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Synthesis and characterization of Au@liposome-ICG

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With a high aspect ratio, Au nanorods penetrate more easily into the tumor regions

14

because of enhanced permeability and retention (EPR) effect than other Au

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nanoparticles.32 Nanostructures with size from 30 to 150 nm are suitable for passive

16

tumor targeting.33 In this study, we measured the length and width of 30 different

17

nanoparticles from diverse TEM fields with imageJ package for Windows. Then

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aspect ratio of each nanoparticle was calculated according to: aspect ratio = length /

19

width. The AuNR synthesized in this study was 28.4 ± 2.4 nm in length with an aspect

20

ratio of 3.2 ± 0.4. After surface modification with liposomal ICG, the length of

21

Au@liposome-ICG increased to 31.2 ± 4.6 nm (aspect ratio: 3.1 ± 0.2) and the

22

nanoparticles were surrounding with 2 nm thickness liposomal ICG in TEM (Figure 6/31


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1

1B,C；Figure S1).

2 3

Figure 1. Schematic illustration and characterization of Au@liposome-ICG. (A)

4

Schematic diagram of the nanoparticles. (B) TEM images of Au-PEG. (C) TEM of

5

Au@liposome-ICG, the AuNR core was covered by about 2-nm thickness liposomal

6

ICG.

7

In PBS, the hydrodynamic size of Au@liposome-ICG was 78.3 ± 37.2 nm, about 28

8

nm more than that of Au-PEG (50.6 ± 21.8 nm). Consistent with a recent report on

9

liposomal ICG AuNR hybrids, the hydrodynamic size of liposomal ICG could be

10

reduced with the appearance of AuNR (Figure 2A).30 The absorption peaks of

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Au-PEG, liposomal ICG, and Au@liposome-ICG were 735 nm, 800 nm, and 795 nm,

12

respectively (Figure 2B). A 20 nm red shift, caused by interactions among the

13

materials through van der Waals forces,34 was identified for Au@lioposome-ICG 7/31



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when compared with free ICG.

2 3

Figure 2. Hydrodynamic size distribution and absorption spectra of the nanoparticles.

4

(A)

5

Au@liposome-ICG. (B) Normalized absorption spectra of the materials.

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Along with the step by step modification with metoxy-poly(ethylene glycol)-thiol

7

(mPEG-SH) and liposomal ICG, the zeta potential shifted from + 48.90 ± 5.81 mV for

8

Au-CTAB to - 33.80 ± 4.61 mV for Au@liposome-ICG, indicating the high stability

9

of Au@liposome-ICG (< −25 mV)35 (Table S1). The negatively charged surface

10

reduced the toxicity of nanoparticles and facilitated their accumulation into tumor

11

lesions.36,37 After 48 hours of incubation with 50% FBS/PBS solution, the absorbance

12

of free ICG decreased (Figure 3A, black), while the liposomal ICG showed a slight

13

decrease (Figure 3A, green). This serum stability improvement may be caused by the

14

lipid bilayer protecting the ICG molecules from exposure to serum proteins and stray

15

light radiation in the air, through which aggregation and degradation of ICG was

16

reduced.38 As Au-PEG showed stable absorbance in the incubation mixture (Figure

Hydrodynamic

size

distribution

of

Au-PEG,

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liposomal

ICG,

and

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1

3A, blue) and a stable liposomal ICG shell, it is reasonable that Au@liposome-ICG

2

exhibits superior stability (Figure 3A, red; Figure S2). Additionally, aggregation of

3

liposomal ICG in PBS was decreased with the appearance of AuNR in the following

4

six days (Figure S3). Collectively, these data indicated that the aggregation of ICG

5

and liposomal ICG in the circulation system and during storage could be suppressed

6

with AuNR core.

7

The fluorescence signals emitted from free ICG and liposomal ICG decreased by one

8

order after 8 hours of continuous radiation with an 808 nm laser. Interestingly,

9

corresponding signal of Au@liposome-ICG remained stable under the same

10

conditions. The lipid bilayer of liposomal ICG protects ICG molecules from exposure

11

to water, thereby reducing ICG quenching.38 Compared to liposomal ICG,

12

Au@liposome-ICG exhibits superior photostability (Figure 3B, Figure S2). We

13

speculate that it is due to the structure of Au@liposome-ICG. The AuNR core absorbs

14

part of the light, following the damage induced by the light energy, the degradation of

15

ICG molecules loaded in the shell could be reduced. Thus, Au@liposome-ICG

16

exhibits superior photostability under continuous radiation. This superiority indicates

17

that Au@liposome-ICG can offer sustained and steady guidance during hepatectomy,

18

which usually last several hours.39

19

In vitro fluorescence imaging and photoacoustic imaging

20

Free ICG is unsatisfactory in dual-modality imaging because of its fluorescence

21

quenching, fast clearance,40 and low efficiency in light to thermal conversion.21

22

Liposomal ICG provided only 45% of photoacoustic signal when compared to AuNR 9/31



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1

at the same optical density.29 However, more powerful fluorescence could be obtained

2

by combining ICG into lipid membrane38,41. In the current study, the fluorescence

3

signal from liposomal ICG and Au@liposome-ICG saturated when the ICG

4

concentration reach to 7.8 µg/mL, while the corresponding fluorescence intensity

5

from free ICG increased slowly among the tested concentrations. The fluorescence

6

signal emitted from liposomal ICG enhanced at least 10 fold when compared with free

7

ICG at equal ICG concentration (Figure 3C). This enhancement of fluorescence could

8

be explained by the fact that the lipid membrane which embedding ICG could reduce

9

the quenching of it in water.38 However, the tendency of liposome was minimized in

10

Au@liposome-ICG and even disappeared as Au@liposome-ICG concentration

11

increased. Considering the linear correlation between the photoacoustic imaging

12

signal and Au concentration, the photoacoustic signal from Au@liposome-ICG was

13

stimulated compared with that of Au-PEG at equal Au concentrations (Figure 3D). We

14

believe that this phenomenon was caused by some light energy (both excitation and

15

emission) converted to heat by the AuNR core as the absorption spectrum of

16

Au@liposome-ICG overlapped with the ICG excitation (745 nm) and emission

17

spectra (840 nm). This enhanced signal may contribute to higher sensitivity and

18

imaging depth.29,42

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1 2

Figure 3. Stability and imaging capability of the materials. (A) During 48 hours of

3

incubation in 50% FBS/PBS mixture, the minimum absorbance changes of

4

Au@liposome-ICG indicated its superior serum stability. (B) Photostability,

5

Au@liposome-ICG emits a stable fluorescence signal after 240 min of continuous

6

radiation. (C) Fluorescence signal intensities as a function of ICG concentrations. (D)

7

Photoacoustic signal intensities as a function of AuNR concentrations. The inserts in

8

C and D represent corresponding imaging sections acquired at increasing

9

concentrations. Data are presented as mean ± SD.

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Cell viability and bio-distribution of Au@liposome-ICG

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Before the in vivo experiment, we detected the biocompatibility of Au@liposome-ICG

12

on liver cancer cells and hepatic stellate cells, although there was a minor discrepancy

13

among the different types of cells (Figure 4). 11/31



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1 2

Figure 4. Comparison of the viability in different cell lines treated with increasing

3

concentrations of nanoparticles. Hepatic stellate cell line (LX-2) and two different

4

hepatoma cell lines (Huh-7 and Hep G2) were incubated with different concentrations

5

of Au-PEG, liposomal ICG, and Au@liposome-ICG. The cytotoxicity of

6

Au@liposome-ICG was comparable to that of liposomal ICG in all three cell lines,

7

but slightly reduced compared to that of Au-PEG, indicating that the liposomal shell

8

encapsulation decreased cell exposure to redundant CTAB. All cell lines retained 85%

9

viability when the concentration of the three nanoparticles increased to 120 µg/mL. *

10

p < 0.05, ** p < 0.01, *** p < 0.001

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To explore the biodistribution of Au@liposome-ICG, whole body image changes were

12

monitored by IVIS system after intra-venous administration of the nanoparticles in

13

tumor bearing BALB/c mice. Strong fluorescence signals were emitted from whole

14

body, especially the liver, 30 min after administration. Six hours later, a remarkable

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fluorescence signal emitting from the tumor region contributed to clearly

16

distinguishing the tumor and peripheral tissues. Moreover, the intensive fluorescent

17

region from the epigastrium in the first day decreased and narrowed to the

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hypogastrium, tumor region, and spleen in superficial images (Figure 5A).

19

Interestingly, the tumor in PAT, acquired 3 days after administration, demonstrated 12/31


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1

heterogeneous hyperoxia in peripheral tissues surrounding the tumor region (Figure 5

2

B, C). This photoacoustic appearance is consistent with the hyper metabolism of

3

tumor cells and abundant blood supply in vessel networks around the lesion to feed

4

the tumor. It has been reported that 30 –150 nm Au nanoparticles are metabolized and

5

excreted through the liver.36,43 Combined with ex-vivo fluorescence imaging of the

6

harvested organs from the euthanized mice 8 days after administration, we confirmed

7

that Au@liposome-ICG were excreted from the body via the bile system in 8 days

8

(Figure 5D).

9 10

Figure 5. Metabolic profile of Au@liposome-ICG. (A) Au@liposome-ICG

11

accumulated in the subcutaneous tumor 6 hours after tail vein injection and was

12

excreted from the body in 8 days. This provided a more than 6-day-imaging window

13

(day 2 to day 7). (B, C) Photoacoustic tomography at day 3 demonstrated 13/31



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hyper-oxyhemoglobin surrounding the tumor region. (D) Consistent with whole body

2

fluorescence imaging at day 8, no fluorescence signal was emitted from the organs

3

harvested at day 8, except the tumor tissue, indicating the excretion of the

4

nanoparticles. Abbreviations: Li: liver; Hea: heart; Pan: pancreas; Ki: kidney; Lu:

5

lung; Int: intestines; T: tumor

6

Preoperative detection with photoacoustic tomography

7

Using the subcutaneous xenograft mouse model, we hypothesized that most of the

8

Au@liposome-ICG were excreted from the normal liver parenchyma in 2 days,

9

providing a sufficient time window (day 2–7) for preoperative preparation and

10

intraoperative guidance. Generally, this interval is close to the length from diagnosis

11

to operation in clinical practice. To reduce the interference of Au@liposome-ICG in

12

the normal liver parenchyma, we performed PAT 3 days after administration

13

according to the metabolic profile. The nodules, confirmed by bioluminescence

14

imaging (Figure 6A), were not detected by PAT before intravenous injection of the

15

probe (Figure 6B). Three days after administration, Au@liposome-ICG accumulated

16

in the tumor region (Figure 6C). Relative amounts of oxyhemoglobin and

17

deoxyhemoglobin have been used as endogenous chromophores in breast44 and

18

ovarian45 cancer detection with photoacoustic imaging in clinical practice.46 In this

19

study, we found that the location of the tumor was consistent with that observed by

20

MRI. The distribution of Au@liposome-ICG was highly consistent with that of

21

oxyhemoglobin and deoxyhemoglobin in PAT (Figure S4). This hemoglobin state is

22

consistent with the typical features of hypoxemia and abundant blood supply in 14/31


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1

malignancies.47

2 3

Figure

6.

Preoperative

detection

with

photoacoustic

4

Bioluminescence imaging of the intrahepatic nodule. (B, C) Photoacoustic

5

tomography acquired before and 3 days after administration of Au@liposome-ICG,

6

the dashed region indicates the tumor location. (D, E) Fluorescence imaging of the

7

model mice acquired with the IVIS system.

8

Intraoperative fluorescence imaging guided tumor resection

9

After localization by photoacoustic imaging, we evaluated the effectiveness of

10

Au@liposome-ICG as a fluorescence probe for liver resection guidance. The nodules

11

were resected under the guidance of Au@liposome-ICG fluorescence imaging. GFP

12

fluorescence confirmed that the tumor derived from Huh-7-GFP-fLuc cells (Figure

13

7A-D). After resection, the fluorescence imaging between Au@liposome-ICG and

14

GFP was performed again respectively to confirm complete resection (Figure 7E-H). 15/31


tomography.

(A)


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1

Postoperative histology analysis confirmed the tumor specimen and tumor margin

2

(Figure 7I-J). To estimate the effectiveness of this Au@liposome-ICG mediated

3

fluorescence imaging guided resection in safe margin, we calculated the thickness of

4

the normal liver parenchyma surrounding the tumor in histopathology. Consistent with

5

the fluorescence imaging acquired with the IVIS system, the pathological section

6

showing in Figure 7L, indicated that the liver parenchyma was only 0.1 mm in thick.

7

These data demonstrated that the excision could be minimized with the guidance of

8

Au@liposome-ICG-mediated fluorescence imaging. It is critical to avoid some severe

9

postoperative complications due to insufficient remnant liver such as infection, liver

10

dysfunction, or even fatal liver failure.48 Altogether, we believe Au@liposome-ICG

11

could be regarded as a photoacoustic-fluorescence dual modality probe for liver

12

cancer detection and intraoperative resection guidance.

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1 2

Figure. 7 Fluorescence guided liver resection using Au@liposome-ICG. (A) The

3

nodule

4

Au@liposome-ICG-mediated fluorescence imaging allowed the detection of the

5

lesion with hyperfluorescence. (C) Detection of the Green fluorescence protein

6

confirmed that the nodule derived from GFP Huh-7-GFP-fluc cells. (D) When merged

7

with each other, the ROI showed high consistency. The nodule was then resected

8

under the guidance of Au@liposome-ICG-mediated fluorescence imaging. (E) The

9

remnant liver is shown in bright field. With Au@liposome-ICG fluorescence imaging

10

(F), GFP fluorescence imaging (G), and the merged image (H), we believe that the

11

tumor was completely resected. (I-K) show the tumor specimen in bright field as well

is

in

low

contrast

to

the

adjacent

liver

17/31


parenchyma.

(B)


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Page 18 of 31

1

as Au@liposome-ICG and GFP fluorescence imaging using the IVIS system and

2

Leica stereo-microscopy, respectively. Hematoxylin-Eosin staining histologically

3

confirmed the tumor, delineated the tumor margin (yellow), and outlined the specimen

4

in cross section (L).

5

Conclusions

6

NIR fluorescence and PAT have complementary capabilities in preoperative diagnosis

7

and intraoperative surgery navigation. Herein, we report a photoacoustic-fluorescence

8

dual-modality imaging probe that can be used for preoperative detection of liver

9

cancer and intraoperative fluorescence imaging guidance. The probe is stable and

10

nontoxic to cells in vitro at relevant concentrations. With the guidance of

11

Au@liposome-ICG-mediated fluorescence imaging, the tumor could be safely and

12

completely resected. The current work demonstrated promising application of the

13

photoacoustic-fluorescence dualmodality probe in liver cancer diagnosis and

14

treatment. Further studies are warranted to comprehensively assess the sensitivity,

15

specificity, and potential application of targeted molecular imaging for liver cancer

16

detection and resection.

17

EXPERIMENTAL PROCEDURES

18

Preparation of Au nanorods

19

Au nanrods were synthesized via seed method49 with minor modifications. Briefly, for

20

seed solution, 5 mL of an aqueous 0.5 M solution of Hydrogen tetrachloroaurate (III)

21

hydrate

22

cetyltrimethylammonium bromide (CTAB) solution with mild stirring. Then, 0.6 mL

(HAuCl4·3H2O)

was

added

to

1.5

mL

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of

aqueous

0.2

M

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1

of 0.01 M ice-cold sodium borohydride (NaBH4) solution were added promptly with

2

vigorous stirring. For the growth solution, 2 mL of 0.01 M AgNO3 solution was add to

3

200 mL of 0.1 M CTAB solution with slow stirring. After 10 min standing, 10 mL of

4

0.01 M HAuCl4·3H2O solution were added and the solution was incubated for 3 min.

5

Then, 1.6 mL of 0.1 M ascorbic acid (AA) solution were added slowly followed by

6

gently mixing for 5 seconds. At last, 0.5 mL of prepared seed solution was added and

7

keeping undisturbed over night at 30 oC.

8

Fabrication of Au@liposome-ICG

9

Liposomal ICG were prepared by thin film method38 with minor modifications.

10

Briefly, 20 mg phosphatidylcholine and 6 mg cholesterol were dissolved in 15 mL

11

chloroform. Then, 5 mL of indocyanine green (ICG)-methanol solution (containing

12

ICG 0.13 mg) were added to this mixture (lipid-ICG molar ratio, about 250:1),

13

followed by evaporation under pressure for 3 hours at 46°C with the flask wrapped

14

with silver paper on a rotary evaporator. Then, the thin films were hydrated with 2 mL

15

Stroke-physiological saline solution for 6 hours, followed by 30 min sonication at

16

60°C to reduce the size of the ICG-loaded liposome. For Au@liposome-ICG, 120 µL

17

liposomal ICG were mixed with 1.2 mg PEGylated AuNR (PEG: Au molar ratio 1.0)

18

and followed by overnight sonication at room temperature. It has been reported that

19

ICG could be nearly completely incorporated into liposomes (about 97%) at lipid/ICG

20

molar ratio of 250/1.38 Therefore, loading efficiency was not analyzed in this study.

21

Characterization of nanoparticles

22

Absorbance spectra and serum stability were analyzed with ultraviolet-visible 19/31



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1

spectrophotometer (UV-2450, Shimadzu, Japan). Absorbance changes were analyzed

2

continuously for 48 hours to estimate the serum stability of the nanoparticles in 50%

3

fetal bovine serum (FBS) containing 50% phosphate buffer saline (PBS, v/v). For

4

photostability, the micelles in phosphate buffer saline (PBS) were continuously

5

exposed to an 808 nm laser (MDL-H-808, Changchun, China) for 4 hours, and the

6

fluorescence intensity was tested every 30 minutes by pre-clinical in vivo imaging

7

system and its included software (IVIS, PerkinElmer, Waltham, MA, USA). The

8

excitation and emission wavelengths in IVIS were set at 745 nm and 840 nm,

9

respectively. Hydrodynamic size distribution and zeta potential of the particles were

10

measured with the particles in PBS on Malvern Zeta sizer (ZEN 3600, Worcestershire

11

UK). The morphology of the particles was assessed by Transmission Electron

12

Microscopy (TEM, JEOL, JEOL-1011, Japan) after colored by ICG according to

13

Portnoy and colleagues’ description41.

14

Cell cytotoxicity assay

15

Cellular cytotoxicity was analyzed with MTS assay kit (Promega, Madison, WI,

16

USA). Human hepatocellular carcinoma cells (HepG2, Huh-7, Huh-7-GFP-fluc) and

17

hepatic stellate cells (LX2) were obtained from the Academy of Military Medical

18

Sciences (China). Cells were cultured at 37 °C and 5% CO2 in Dulbecco’s modified

19

Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine

20

serum (FBS, Gibco) and 1% penicillin-streptomycin (Promega). After digestion with

21

EDTA- trypsin (Gibco), the harvested cells were seeded in 96-well plates at density of

22

104 cells per well and incubated for 24 hours. Then, the medium was replaced with an 20/31


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1

equivalent volume of fresh medium containing increasing concentrations of the

2

samples to be tested. After 24 hours of incubation, the cells were washed with sterile

3

PBS. Then, 120 µL MTS-complete culture medium (Containing 20 µL MTS) were

4

added to each well. Cell viability was estimated by measuring the absorbance at 490

5

nm using Synergy HT microplate reader (BioTek, Winooski, VT, USA).

6

In vitro fluorescence imaging and photoacoustic tomography

7

For fluorescence imaging, Au@liposome-ICG, liposomal ICG, and free ICG were

8

serially diluted. Then, 200 µL of these aqueous solutions (with equal ICG) were

9

instilled into a 96-well plate. IVIS platform was employed to calculate the average

10

fluorescence intensity of each sample. For photoacoustic imaging, five serial different

11

concentrations of Au@liposome-ICG and Au-PEG (with equal Au) were injected into

12

a commercially available phantom (PELICAN, USA) followed by multi-spectral

13

photoacoustic tomography (MSOT) with MSOT inVision 128 system (iThera Medical

14

GmbH, München, Germany). Sections reconstructed with Back Projection method at

15

795 nm for Au@liposome-ICG and 735 nm for Au-PEG were analyzed with ImageJ

16

package for windows (National Institutes of Health, Bethesda, MD, USA) to calculate

17

the signal intensity.

18

Animal model preparation and ethical statement

19

All animal protocols were approved by the Institutional Animal Care and Use

20

Committee of Zhujiang Hospital of Southern Medical University. Male BALB/c nude

21

mice (4–6 weeks old) were obtained from Vital River Laboratory Animal Technology

22

Co. Ltd (Beijing, China). Subcutaneous and orthotopic xenograft liver cancer models 21/31



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1

were established by subcutaneous or in situ injection of 100 µL Matrigel (BD

2

Biosciences, Franklin Lakes, NJ, USA) containing 2 × 106 Huh-7-GFP-fluc cells.

3

In vivo biodistribution of Au@liposome-ICG

4

The biodistribution profile of Au@liposome-ICG was explored in subcutaneous

5

xenograft models. Briefly, 150 µL of Au@liposome-ICG solution (Au, 1.2 mg/mL;

6

ICG 7.8 µg/mL) was intravenously injected. Then, the models were dynamically

7

monitored by the IVIS system under anesthesia with 2% isoflurane. PAT was

8

performed at 680, 735,750, 780, 795, 810, and 850 nm, and images were acquired

9

when the probes accumulated in tumor regions in IVIS. Photoacoustic sections of

10

Au@liposome-ICG acquired at 780 nm wavelength were employed to merge with

11

oxyhemoglobin and deoxyhemoglobin maps. The animal was euthanized when

12

Au@liposome-ICG was excreted from the body as determined by using IVIS imaging.

13

Fluorescence signals from ex-vivo specimens were also analyzed.

14

Photoacoustic preoperative detection and fluorescence imaging

15

guided tumor resection

16

First, bioluminescence imaging and PAT were performed to localize the nodules. Then,

17

150 µL Au@liposome-ICG (Au, 1.2 mg/mL; ICG 7.8 µg/mL) were intravenously

18

injected into three orthotopic liver cancer model mice. Three days later, PAT was

19

performed again under anesthesia with 2% isoflurane. As the nodules in PAT were

20

indistinct, we performed MR imaging (Bruker, BioSpec 94/30 USR, Karlsruhe，

21

Germany) to localize the nodules in cross-section. Then, NIR fluorescence imaging

22

guided tumor resection was performed after exposing the abdominal cavity under 22/31


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1

intraperitoneal anesthesia with 10% chloral hydrate (4 µL/g). During operation, the

2

potential nodules were identified by an experienced surgeon through visual inspection.

3

Then, potential nodules were identified again with our homemade NIR fluorescence

4

imaging surgery guiding system, and confirmed by the detection of the GFP protein

5

with Leica stereo microscopy (Leica M205 FA; Germany). Then, the tumors were

6

resected under the guidance of NIR fluorescence imaging. Both the remnant liver and

7

ex-vivo tumor specimen were imaged with Au@liposome-ICG and GFP fluorescence

8

imaging. To analyze the resected tumor specimens, Hematein and Eosin (Acros

9

Organics) were employed to stain 8-µm frozen sections cut with a cryostat microtome

10

(Leica CM1950, Germany).

11

Statistical analysis

12

Two-way ANOVA with Bonferroni's multiple comparison was employed to compare

13

cytotoxicity of the materials. Statistical analysis was carried out using the GraphPad

14

Prism 6 (GraphPad Software, Inc., San Diego, CA, USA). P value < 0.05 was

15

considered statistically signiﬁcant.

16

ACKNOWLEDGMENTS

17

The authors thank Xiaoyuan Liang, Ting Ai, Ziyu Han for assistance with data

18

collection. This work was supported by the National Natural Science Foundation of

19

China under Grant Nos. 81227901, 81627805, 61231004, 61671449, 81501540,

20

81527805; The Science and Technology Plan Project of Guangzhou (No.

21

201604020144); Digital Theranostic Equipment Research Special Program of The

22

“13th five-year” National Key Research Plan (No.2016YFC0106500); The United 23/31



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1

Fund of National Natural Science Foundation of China and Government of

2

Guangdong Province (No. U1401254).

3

Supporting Information

4

Zeta potential shifts; lower magnification TEM of Au@liposome-ICG; absorbance

5

and fluorescence intensity compared with corresponding supernatant; hydrodynamic size

6

changes in 6 days; location of the tumor in PAT and MRI.

7

ABBREVIATIONS

8

AuNR

Au nanorods

9

NIR

Near-infrared

10

Au-PEG

PEGylated Au nanorods

11

IVIS

In vivo imaging system and its included software

12

GFP

Green fluorescent protein

13

ICG

Indocyanine green

14

PAT

Photoacoustic tomography

15

FBS

Fetal bovine serum

16

PBS

Phosphate buffer saline

17

TEM

Transmission electron microscopy

18 19 20 21 22 24/31


Page 24 of 31

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1

solution. Langmuir 20, 6414-20.

2 3 4 5

Table of Contents

6

In this study, we fabricated indocyanine green loaded gold

7

nanorod@liposome core-shell nanoparticles (Au@liposome-ICG) to

8

integrate both imaging strategies. These nanoparticles exhibit superior

9

biocompatibility, high stability, and enhanced dual-model imaging signal.

10

Then, we explored its effectiveness of tumor detection and surgery

11

guidance in orthotopic liver cancer mouse models.

12

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