Integrin Î±vÎ²3-Targeted C-Dot Nanocomposites as Multifunctional

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Integrin #v#3-targeted C-dot Nanocomposites as Multifunctional Agents for Cell Targeting and Photoacoustic Imaging of Superficial Malignant Tumors Zhe Liu, Waner Chen, Yihong Li, and Qien Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03927 • Publication Date (Web): 02 Nov 2016 Downloaded from http://pubs.acs.org on November 3, 2016

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Analytical Chemistry

Full Paper

Integrin αvβ3-targeted C-dot Nanocomposites as Multifunctional Agents for Cell Targeting and Photoacoustic Imaging of Superficial Malignant Tumors Zhe Liu,*ab Waner Chen,ab Yihong Liab and Qien Xuab a

Wenzhou Institute of Biomaterials and Engineering, Wenzhou Medical University, Wenzhou 325001, Zhejiang, China b Wenzhou Institute of Biomaterials and Engineering, Chinese Academy of Sciences, Wenzhou 325011, Zhejiang, China *E-mail: [email protected] Keywords: Carbon dots, Nanocomposites, Cancer, Imaging agents, Photothermal therapy

Abstract: With a cocktail formulation of soybean milk as a green carbon source and TTDDA as a capping agent, integrin αvβ3-targeted C-dot nanocomposites (MB-CDs@NH-RGD) have been successfully fabricated via a facile microwaving protocol. Modification of surface-coating and RGD-conjugation endow their superior biocompatibility as well as highly specific targeting profile to αvβ3-overexpressed cell lines of MDA-MB231 and B16 as representative superficial malignant tumors. Meanwhile, the significant photothermal effect has been generated on irradiation of these targeted C-dot nanocomposites by a pulsed laser, which proved their eligibility for potential thermal ablation therapy. In vivo photoacoustic imaging using these C-dot nanocomposites as novel imaging probes verified their excellent targeting sensitivity and contrast enhancement. These exciting evidences imply a promising strategy to utilize them for multifunctional nano-theranostic purposes in combination of precision diagnosis and photothermal treatment against superficial malignant tumors.

1

ACS Paragon Plus Environment


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1. Introduction Superficial malignant tumors (SMTs), such as breast cancers and melanoma, are increasingly aggressive diseases with strong elusiveness and high metastatic potential.[1,2] According to the National Cancer Institute, approximate 90% of surviving rates has been afforded for both breast cancers and melanoma. [3] Thus, effective strategies of precise diagnosis and treatment against superficial malignant tumor lesions are of great significance. To this end, the rapid development of nano-biomaterials as well as multifunctional theranostic agents endows a revolutionary route map. Photoacoustic (PA) imaging provides a brand-new approach to the diagnosis of SMTs with enhanced precision by combining optical high sensitivity and acoustic deep tissue penetration.[4,5] For example, photoacoustic endoscopy has been a powerful tool to detect superficial lesions for clinics.[6,7] On the other hand, photothermal therapy (PTT) or hyperthermia has displayed prevailing advantages in comparison to the traditional chemotherapy and radiotherapy of high risks. [8,9] By irradiating photo-sensitive agents with a pulsed laser, a significant temperature increase can be generated which may be implemented for cancer treatments. Since cancerous cells are more sensitive to heating in contrast to normal cells, the effective cancer damage can be achieved in a specific and selective manner. Moreover, side-effects of radiotherapy or chemotherapy may be avoided or minimized for the sake of patient’s successful treatment and recovery. The participation of various multifunctional agents renders another prerequisite to realize a personalized diagnosis and treatment against SMTs.[10,11] By conjugation of biological ligands on these agents, strong binding capability and specific cell targeting can be achieved in vitro, and real-time molecular imaging may also be accomplished in vivo to monitor the pathological process. In particular, the integrin v family plays an important role in tumor neoangiogenesis, development and metastasis, and they are 2


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generally overexpressed in tumor cell membranes and endothelial cells which demonstrate early-stage signals of tumor generation.[12-14] For this reason, they are widely regarded as specific biomarkers for tumor-targeted diagnosis and therapy.[15,16] Cyclic RGD peptides have been explored as effective binding ligands to αvβ integrin receptors, and numerous RGD-conjugated nanoparticles have been utilized for tumor cell targeting and drug/gene delivery.[17-21] However, it has been rarely studied to fabricate integrin αvβ-targeted nanocomposites as multifunctional agents for PA imaging and PTT combined. It will also be highly valuable to translate these nanocomposites to biomedical applications so that an effective non-invasive theranostic strategy for SMTs may be established. Carbon dots (C-dots) have attracted much attention as imaging materials and drug delivery carriers due to their outstanding properties such as chemical inertness, colloidal stability, ready functionalization and superb biocompatibility. [22-24] Various carbon source materials e.g. milk, honey, citric acid and glucose have been used to fabricate C-dots with bottom-up or top-down protocols.[25,26] In our previous study, non-targeted C-dot nanocomposites have been developed as photothermal agents and cell tracking tracers.[27] Herein, we report the fabrication of integrin αvβ3-targeted C-dot nanocomposites with RGD-conjugation on the surface for specific anchoring to breast cancer and melanoma cells, respectively. It was found that the surface modification of C-dot nanocomposites help improve the biocompatibility and decrease the cytotoxicity. The availability to use them as photothermal agents and simultaneous PA imaging probes has been also investigated. Hence, the as-developed C-dot nanocomposites have exhibited multifunctional utilities for both in vitro cell targeting and in vivo PA imaging, which implied a promising strategy to apply them for early-stage molecular diagnosis and treatment of SMTs with high precision and efficacy. 3



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2. Results and discussion 2.1. Fabrication and characterization of integrin v3-targeted C-dot nanocomposites MB-CDs@NH-RGD Starting from a cocktail formulation of soybean milk, methylene blue (MB) and 4,7,10-trioxa-1,13-tridecanediamine (TTDDA), the non-targeted C-dot nanocomposites MB-CDs@NH2 could be fabricated via a green microwaving protocol in a one-pot manner. Methylene blue as a clinical PA agent was encapsulated along with the C-dots formation. TTDDA as a capping agent is not only responsible for the surface-coating and stabilization of the C-dots, but also attributes to the introduction of amine groups. Followed by a traditional EDC-NHS activation, the RGD-conjugated C-dot nanocomposites

MB-CDs@NH-RGD

could

be

afforded

(Figure

1).

This

straightforward synthetic protocol enabled the multiple functionalization within and on the surface of the C-dot nanocomposites, and in comparison to the layer-by-layer approach, it has improved the tolerance of C-dots for chemical modifications and further expand the versatility of C-dots manipulation.

4


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Figure 1. Illustrative fabrication of integrin αvβ3-targeted C-dot nanocomposites (MB-CDs@NH-RGD) starting from cocktail ingredients of soybean milk, MB and TTDDA via RGD conjugation. In order to characterize the structure of MB-CDs@NH-RGD, transmission electron microscopy (TEM) and FT-IR spectroscopy were performed. As shown in Figure 2a, TEM image displayed that MB-CDs@NH-RGD had a uniform spherical morphology with a diameter of 14.4 ± 4.3 nm. The contrast turned blurry under a high magnification probably due to the carbon composition of MB-CDs@NH-RGD. FT-IR spectra gave the characteristic peaks of hydroxyl and carbonyl groups on MB-CDs@NH-RGD at 3402, 1652 and 1402 cm-1 (Figure 2b). The broad peaks at 3300-3500 cm-1 and the strong peaks at 1590-1650 cm-1 fingerprinted the existence of amine groups and verified the surface coating by TTDDA. The strong peaks at 1020-1220 cm-1 apparently turned weak which confirmed that amine groups had been consumed for RGD conjugation association with the yield of MB-CDs@NH-RGD. Besides that, photoluminescence (PL) and UV-Vis absorbance spectra were acquired to test their optical properties. MB-CDs@NH-RGD showed a gradually decreased red-shift ﬂuorescence intensity at the excitation range of 340-440 nm, which shared similar behavior to C-dots and demonstrated its successful fabrication (Figure 2c).[27] In comparison with the UV-Vis profiles of MB and MB-CDs@NH2, a characteristic absorption peak at 280 nm indicated the formation of C-dots, and the maintenance of encapsulated MB could be identified intact with the presence of another characteristic peak at 665 nm resulted from free MB molecules.[27,28] All these data supported the nanocomposite structure of MB-CDs@NH-RGD which was in good accordance to the fabrication protocol. In addition, MB-CDs@NH-RGD was applied to sessile drop analysis to give a static contact angle of 0 degree which implied them highly hydrophilic and suitable for biomedical applications (Figure S1). 5



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Figure

2.

Characterization

of

integrin

αvβ3-targeted

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C-dot

nanocomposites

(MB-CDs@NH-RGD) by transmission electron microscopy (TEM) (a), FT-IR spectra (b), Photoluminescence (PL) (c) and UV-Vis absorbance spectra (d). 2.2. Evaluations of photothermal effect of MB-CDs@NH-RGD To use them as photothermal agents for PTT, an apparatus with MB-CDs@NH-RGD aqueous solution was applied to photothermal irradiation under an 808 nm pulsed laser, and real-time photothermal images were recorded (Figure 3a and S2). By irradiation within 5 min, a significant temperature increase of up to 14.1 ℃ was observed. In contrast, deionized water and free MB solution did not show obvious photothermal effect, and only negligible temperature increases of 4.8 ℃ and 5.6 ℃ were measured, respectively. Further investigations were carried out with varied encapsulated MB concentrations in MB-CDs@NH-RGD, and close dependency was confirmed that higher MB concentration in MB-CDs@NH-RGD contributed more prominent 6


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temperature increase (Figure 3b). Apart from that, the energy density of laser irradiation was also an influential factor. Varied energy density (1, 2, 3 W/cm2) led to diverse photothermal effect with a tunable temperature increase, and this will provide enough freedom with flexible options for individualized photothermal ablation according to different therapeutic demands (Figure 3c).[29,30]

Figure 3. a) Photothermal effect of integrin αvβ3-targeted C-dot nanocomposites (MB-CDs@NH-RGD, 47 μM) compared to deionized water and free MB solution. All samples were irradiated under an 808 nm laser at an energy density of 3 W/cm2 for 5 min. b) Photothermal analysis at different C-dots concentration (0-47 μM) and c) varied energy density of 1, 2, 3 W/cm2 under an 808 nm laser within 5 min. 2.3. Specific cell targeting of MB-CDs@NH-RGD to MDA-MB231 and B16 tumor cells So as to investigate the availability of C-dot nanocomposites for SMTs cell targeting and

labelling,

non-targeted

(MB-CDs@NH-RGD)

were

probes

(MB-CDs@NH2)

co-incubated

with

and

MDA-MB231

targeted and

probes B16

as

representative SMTs cells in parallel experiments followed by removal of non-binding probes and visualization by confocal laser scanning microscopy (CLSM). Non-targeted 7



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probes will not attach to the cell membranes, and free probes will be removed completely so that no fluorescence can be detected by CLSM. In comparison, targeted probes will tightly bind the cell membranes due to RGD ligand specific recognition with integrin αvβ3 receptors.[31-35] As a result of receptor-mediated endocytosis (RME), targeted probes may be internalized into the cytoplasm, and thus intense fluorescence signals from the targeted probes at dual-channel wavelengths of both C-dots (λEx=405 nm) and MB (λEx=640 nm) can be captured. Therefore, distinct differentiation for SMTs cell targeting and labelling by means of non-targeted and targeted C-dot nanocomposites can be realized efficiently (Scheme 1).

Scheme 1. Principle of integrin αvβ3-targeted C-dot nanocomposites (MB-CDs@NH-RGD) specific binding to integrin αvβ3-overexpressed superficial malignant tumor cell lines, e.g. breast cancer cells MDA-MB231 and melanoma cells B16. Receptor-mediated endocytosis (RME) renders their availability for fluorescent dual-channel cell labelling under confocal laser scanning microscopy (CLSM). 8


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Following this hypothesis, human breast cancer cells MDA-MB231 with positive integrin αvβ3-receptor expression have been studied for in vitro cell experiments. After co-incubation with targeted probes MB-CDs@NH-RGD for 2 h, strong fluorescence intensity was detected within and on the cell membranes at the wavelength of 405 nm and 640 nm (Figure 4). That proved that targeted C-dot nanocomposites MB-CDs@NH-RGD exhibited strong binding capability, and they have been attached to MDA-MB231 cells via interaction between RGD ligands and integrin α vβ3 receptors. A number of nanocomposites have been internalized into the cytosol via RME effect and the cellular morphology could be clearly visualized in a dual-channel manner. In contrast to the non-targeted probes MB-CDs, no apparent fluorescence was captured either inside or on the cell membranes which implied no specific cell targeting took place. Therefore, with these as-fabricated C-dot nanocomposites along with the dual-channel cell imaging technique, cell targeting and labelling can be navigated with high resolution and more precision.

Figure 4. Specific cell labelling and visualization of integrin αvβ3-targeted C-dot nanocomposites (MB-CDs@NH-RGD) to breast cancer cells MDA-MB231 under CLSM at dual-channel wavelength of 405 nm and 640 nm (scale bar: 20 μm). The non-targeted probes MB-CDs were conducted as the control. 9



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Similarly, integrin αvβ3-positive melanoma B16 cells as an ordinary SMTs cell line were also investigated. After co-incubation for 2 h, non-targeted probes MB-CDs showed no binding affinity to B16 cells due to absence of targeting ligands to integrin αvβ3, and thus displayed no fluorescence signal at the dual-channel imaging windows of 405 nm and 640 nm. However, targeted probes MB-CDs@NH-RGD were internalized by integrin αvβ3-mediated endocytosis, and were observed accumulated inside the cells with strong fluorescence signal emission captured by CLSM (Figure 5 and the video clip in the Supporting Information). Moreover, it is noted that the as-developed C-dot nanocomposites provide an opportunity for fluorescent labelling and imaging of multiple biomarkers. In addition to RGD integrin αvβ3 receptors, other binding ligands can also be conjugated on them. By means of C-dot nanocomposites with dual-channel imaging nature, in vitro biological or pathological processes and microcellular manipulations might be visualized in a real-time manner.

Figure 5. a) Specific cell labelling and visualization of integrin αvβ3-targeted C-dot nanocomposites (MB-CDs@NH-RGD) to melanoma cells B16 under CLSM at dual-channel wavelength of 405 nm and 640 nm (scale bar: 20 μm). The non-targeted MB@CDs were conducted as the control.

10


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2.4. Cytotoxicity assay and in vivo photoacoustic imaging with targeted probes of MB-CDs@NH-RGD To use the C-dot nanocomposites for in vivo targeted imaging, their biocompatibility should

be

carefully

taken

into

considerations,

and

the

cytotoxicity

of

MB-CDs@NH-RGD was accordingly evaluated. MDA-MB231 and B16 cell lines were respectively co-incubated with MB-CDs@NH-RGD for 3 h, and then a CCK-8 assay was performed. Cell viability showed that after TTDDA surface-coating and RGD conjugation, targeted probes (MB-CDs@NH-RGD) exhibited relatively enhanced biocompatibility in comparison with the non-targeted probes (MB-CDs) without any further modifications. For MDA-MB231 cells, MB-CDs@NH-RGD showed low cytotoxicity and acceptable viability of 69.2 ± 0.9% even at high probe concentrations of up to 200 μg/mL (Figure 6a). In regard to B16 cells, the cell viability treated with MB-CDs@NH-RGD demonstrated comparable to that of MB-CDs (82.7 ± 10.6% vs. 91.1 ± 0.3%) at the probe concentration of 200 μg/mL (Figure 6b). These findings not only certify the necessity for surface modifications, but also render these nanocomposites competency as a biocompatible nanomaterial for in vivo biomedical applications.

11



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Figure

6.

Cytotoxicity

assay

of

integrin

αvβ3-targeted

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C-dot

nanocomposites

(MB-CDs@NH-RGD) to representative superficial malignant tumor cells: breast cancer cells MDA-MB231 (a) and melanoma cells B16 (b), respectively. Considering that MB encapsulated in the C-dot nanocomposites is a clinical PA agent and these nanocomposites have good biocompatible performance, proof-of-concept in vivo PA imaging was then performed. Targeted probes MB-CDs@NH-RGD were intravenously administered via tail vein of a MDA-MB231 tumor-xenografted nude mouse. PA and ultrasound dual-modal imaging was conducted all through 30 min post-injection. As shown in Figure 7, PA images at different time points obviously demonstrated that targeted probes MB-CDs@NH-RGD have been pumped into the tumor area, and palpable PA signals resulted from their specific accumulation could be observed at 5 min. Afterwards, gradual increases of PA signal intensity indicated their active targeting to integrin αvβ3 receptors overexpressed on the vascular endothelial cells, and the PA signal plateau was maintained till 30 min post-injection. As a consequence, the utilization of targeted nanocomposites MB-CDs@NH-RGD has improved the PA contrast enhancement, and also exhibited excellent sensitivity to image the superficial malignant tumors compared to the traditional ultrasound and fluorescence modalities. These features enable them eligible to be used as PA contrast agents against SMTs lesions, and it is also believed that these molecular probes may serve multifunctional nano-theranostic purposes in combination of SMTs precision diagnosis and treatment.

12


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Figure 7. In vivo photoacoustic imaging of MDA-MB231 tumor-xenografted nude mouse after

intraveneous

administration

of

integrin

αvβ3-targeted

C-dot

nanocomposites

(MB-CDs@NH-RGD, 156 μM, 200 μL). 3. Conclusion In summary, the integrin v3-targeted C-dot nanocomposites MB-CDs@NH-RGD have been successfully developed. Their chemical characterization and optical properties were fully studied. The significant photothermal effect has been observed on irradiation of these targeted C-dot nanocomposites by a pulsed laser, which proved eligibility for potential thermal ablation therapy. In the meantime, with RGD conjugation for specific integrin v3 receptor recognition, these nanocomposites demonstrated excellent cell targeting capability which has been verified at the dual-channel CLSM imaging. It is also valuable to find that targeted nanocomposites MB-CDs@NH-RGD have better biocompatibility and lower cytotoxicity than non-modified probes. The in vivo PA imaging further validated prevailing advantages to use them as novel PA contrast agents. These exciting evidences imply a promising 13



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strategy to utilize them for multifunctional nano-theranostic purposes in combination of precision diagnosis and photothermal treatment against superficial malignant tumors. 4. Experimental Section 4.1. Materials Methylene blue (MB) was purchased from J&K Co. Ltd. without further purification. Soybean

milk

was

obtained

from

Yonho

Food

Co.,

Ltd.

4,7,10-Trioxa-1,13-tridecanediamine (TTDDA) was purchased from Macklin Inc. Cyclic arginine-glycine-aspartic acid (RGD) peptide was obtained from ChinaPetides Co. Ltd. 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), phosphate buffer saline (PBS), paraformaldehyde, penicillin and streptomycin were purchased from Aladdin. Dulbecco modified Eagle medium (DMEM), L15 medium and fatal bovine serum (FBS) were obtained from Sigma-Aldrich. 4.2. Fabrication of C-dot nanocomposites MB-CDs@NH-RGD The synthetic protocol was slightly modified as previously reported. [27] Briefly, MB (50 mg, 0.156 mmol) was dissolved in deionized water (100 mL) as a stock solution. Then a mixture of as-prepared MB solution (2.0 mL), fresh soybean milk (2.0 mL) and TTDDA (1.0 mL) was put into a domestic microwaving synthesizer (Initiator 4.1.2, Biotage, Sweden) and heated to 160 °C for 5 h. When cooled down to room temperature, the solution was centrifuged at 10,000 rpm to remove impurities and large particles, and the supernatant was dialyzed in deionized water through a dialysis membrane (MWCO of 1000) for 24 h. The resulted MB-CDs@NH2 was then covalently

conjugated

activation-conjugation

with protocol

RGD to

peptide afford

via

a

traditional

EDC-NHS

MB-CDs@NH-RGD.

Typically,

MB-CDs@NH2 was reacted overnight with RGD, EDC and NHS at a ratio of 1:2:5 by 14


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magnetic stirring (C-MAG HS7 digital, IKA, Germany) in darkness at room temperature. The excessive EDC and NHS were removed through a dialysis membrane (MWCO of 1000) for 2 h. The targeted C-dot nanocomposites (MB-CDs@NH-RGD) was harvested and stored at 4 °C. 4.3. Characterization The zeta potential of MB-CDs@NH-RGD was measured by a Zetasizer (Nano ZS, Malvern Instrument Inc., Worcestershire, UK), and triplicate measurements were analyzed to give the zeta potential in Mean±SD. Transmission electron microscopy (Tecnai G2 20, FEI, USA) was performed to visualize the morphology of MB-CDs@NH-RGD and calculate their size by randomly selecting 100 particles for statistical analysis. The FT-IR spectroscopy (Bio-Rad FTS-6000, Digilab Division, USA) was applied to identify the characteristic peaks of MB-CDs, MB-CDs@NH2 and MB-CDs@NH-RGD. The photoluminescence (PL) spectra were obtained on a spectrofluorometer (FluoroMax-4C-L, Horiba Scientific Instrument Inc., USA) with progressively longer excitation wavelength from 340 nm to 440 nm in 20 nm increments. The absorption spectra of free MB, MB-CDs and MB-CDs@NH-RGD were acquired on a UV-Vis spectrophotometer (Lambda 25, PerkinElmer, USA). An instrument (Theta Lite, Biolin Scientific, Sweden) for contact angle measurements at the mode of sessile drop analysis was utilized to measure contact angles of MB-CDs, MB-CDs@NH2 and MB-CDs@NH-RGD. A confocal laser scanning microscope (A1 PLUS,

Nikan,

Japan)

was

used

to

visualize

the

endocytosis

of

targeted

MB-CDs@NH-RGD into MDA-MB231 and B16 cell lines, and detect their cell targeting capability at the excitation wavelength of 405 nm and 640 nm, respectively. 4.4. Photothermal imaging and temperature increase analysis The solution of MB-CDs@NH-RGD, free MB and deionized water were added to cuvettes respectively, and they were irradiated with an 808 nm pulsed laser (WG 15



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1533D3, Beijing Energy Optoelectronics Technology Co., China) at 3 W/cm2 for 5 min. The solution temperatures and infrared thermographic maps were recorded at a 30s-interval with an infrared thermal imaging camera (S6, IRS Systems Inc., China). The temperature increase curves were generated in correlation to different C-dot concentrations and varied energy density. 4.5. Cell cultures Human breast cancer cells MDA-MB231 and mouse melanoma cells B16 was purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. MDA-MB231 was cultured with L15 medium supplemented with 15% FBS, 50 IU/mL penicillin and 50 μg/mL streptomycin. B16 was cultured with 1640 medium supplemented with 10% FBS, 50 IU/mL penicillin and 50 μg/mL streptomycin. All of the cells were cultured at 37 ℃ in a humidified environment of 5% CO2. 4.6. In vitro cellular studies MDA-MB231 cells were seeded in 60 mm cell tissue-culture plate at a density of 3×105 per well. After 24 h incubation, the plate was washed twice to remove the non-adherent cells, and then replaced with fresh medium. 100 μL of non-targeted MB-CDs and targeted MB-CDs@NH-RGD (C-dot concentration: 1.0 mg/mL) were respectively added, and after further co-incubation for 2 h, each group of cells was washed with PBS for three times, and supplied with fresh PBS. The same protocol was followed for B16 cells manipulation. Confocal laser scanning microscopy was applied to evaluate the cell targeting capability at the excitation wavelength of 405 nm and 640 nm in a dual-channel manner. 4.7. Cytotoxicity assay Cytotoxicity of MB-CDs and MB-CDs@NH-RGD were studied on MDA-MB231 and B16 cells by means of CCK-8 (cell counting kit-8) assay. This protocol is based on the 16


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transformation of WST-8 to orang formazan crystals by mitochondrial enzyme succinate dehydrogenase with the existence of the electronic coupling agents. Cells were seeded (5×105 mL-1) in 96-well plates and incubated at 37 ℃ with a humidified environment of 5% CO2 for 24 h. The medium was then replaced along with the addition of MB-CDs and MB-CDs@NH-RGD respectively. After further incubation for 48 h, fresh L15/1640 medium (100 μL) containing CCK-8 (10 μL) was added following by the fluorescence analysis on a microplate reader (Varioskan LUX, Thermo, USA) at the wavelength of 450 nm. 4.8. In vivo photoacoustic imaging of integrin v3-targeted MB-CDs@NH-RGD Female tumor-bearing nude mice were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. To generate the MDA-MB231 tumor model, 1.0×106 MDA-MB231 cells in a 100 μL solution were subcutaneously injected into the flank area of each nude mouse. All animal experimental procedures have been approved by the Administrative Panel of Wenzhou Institute of Biomaterials and Engineering, Wenzhou Medical University, and these methods were carried out in accordance with the animal experimental guidelines of Wenzhou Medical University. Animals were used for in vivo experiments when the tumor size reached approximately 100 mm3 after tumor-cell inoculation.

200

μL

of

v3-targeted

integrin

C-dot

nanocomposites

(MB-CDs@NH-RGD, 156 μM) was intravenously injected via the tail vein. The mouse was fully anesthetized with isoflurane (1.5% mixed with oxygen, RWD Life Science, China). An in-vivo PA small-animal imaging system (LAZR, Visualsonics, Canada) with a transducer (LZ400, Visualsonics, Canada) frequency of 30 MHz at a laser wavelength of 680 nm was conducted to capture merged PA-ultrasound images within 30 min post-injection. A defined tumor area of 28.0 mm2 was selected as the region of interest (ROI) for PA imaging and data analysis. PA signal intensity was

17



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analyzed with a built-in software (VevoLAB, Visualsonics, Canada) to display the PA intensity-time histogram. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21575106), the Scientific Research Foundation for Returned Scholars, Ministry of Education of China, Zhejiang Qianjiang Talents Program and Wenzhou Government’s Start-up Fund.

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Table of Contents Figure:

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Integrin Î±vÎ²3-Targeted C-Dot Nanocomposites as Multifunctional

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