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Characterization of tumor-targeting Ag2S quantum dots for cancer imaging and therapy in vivo Haiyan Chen,*†a Bowen Li,†b Min Zhang,†a Kang Sun,c Yiran Wang,c Kerui Peng,c Mengdi Ao,c Yiran Guoa and Yueqing Gu*a Nanomedicine platforms that have the potential to simultaneously provide the function of molecular imaging and therapeutic treatment in one system are beneficial to address the challenges of cancer heterogeneity and adaptive resistance. In this study, Cyclic RGD peptide (cRGD), a less-expensive active tumor targeting tri-peptide, and doxorubicin (DOX), a widely used chemotherapeutic drug, were covalently attached to Ag2S quantum dots (QDs) to form the nano-conjugates Ag2S-DOX-cRGD. The optical characterization of Ag2S-DOX-cRGD manifested the maintenance of QDs fluorescence, which suggested the potential of Ag2S for monitoring intracellular and systemic drug distribution. The low bio-
Received 28th June 2014, Accepted 9th August 2014
toxicity of Ag2S QDs indicated that they are promisingly safe nanoparticles for bio-applications. Further-
DOI: 10.1039/c4nr03613a
more, the selective imaging and favorable tumor inhibition of the nanoconjugates were demonstrated at both cell and animal levels. These results indicated a promising future for the utilization of Ag2S QDs as a
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kind of multi-functional nano platform to achieve imaging-visible nano-therapeutics.
1.
Introduction
Nowadays, cancer, as a significant cause of human morbidity and mortality, remains a difficult disease to treat, although untold money and resources have been invested into the fight against it. In recent years, nanomedicine platforms with the potential to simultaneously provide the function of molecular imaging and therapeutic treatment in one system are intriguing more and more researchers.1,2 Imaging-visible nanotherapeutics with the capability of validating cancer biomarkers in tumor tissue through molecular diagnosis and thereby providing critical information for the timely tailoring of targeting strategy are expected to hold the key to address the challenges of tumor heterogeneity and adaptive resistance.3,4 Over the past decades, there has been a fulminating development of various inorganic nano-platforms such as gold nanoparticles,5 magnetic iron oxide nanoparticles,6 mesoporous silica nanoparticles,7 and quantum dots (QDs),8 based on which assorted biomedical applications have been performed. The primary advantage of these nanomaterials for theranostic
a Department of Biomedical Engineering, School of Life Science and Technology, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia Lane, Gulou District, Nanjing 210009, China. E-mail: [email protected], [email protected]; Fax: +86-25-83271046; Tel: +86-25-83271080 b Department of Bioengineering, University of Washington, Seattle, USA c School of Pharmacy, China Pharmaceutical University, 24 Tongjia Lane, Gulou District, Nanjing 210009, China † These authors contributed equally to this work.
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application is their multi-functional properties that enable the decoration of drugs, affinity ligands, and imaging moieties on the same vehicle for targeted and traceable drug delivery. Typically, QDs, nanocrystals of semi-conducting materials featuring a combination of small size with versatile surface chemistry, have been intensively studied by many researchers because of their characteristic optical advantages,9,10 including a narrow emission band, high quantum yields, very long effective Stokes shifts and high photo-stability, which are favorable for realizing the real-time monitoring of otherwise invisible nano-carriers.11 Near-infrared (NIR) fluorescent quantum dots (QDs) are of particular interest for in vivo imaging and nano-theranostics due to their deep penetration into the body and poor absorbance of their fluorescence by hemoglobin and water in the body. To date, various kinds of NIR QDs, such as CdHgTe,12 CdHgTe/ZnS,13 InAs,14 and PbS,15 have been successfully synthesized for in vivo biomedical applications. However, the potential release of chemical constituents, including cadmium, mercury, lead, tellurium, and selenium, leads these NIR QDs to bear acute and chronic toxicities,16,17 restricting their further applications in bio-systems. Although ternary I–III–VI NIR QDs (Cu–In–Se, CuInS2, AgInS2)18–20 without intrinsic toxicity have recently been reported, it is onerous to tune their chemical composition while controlling their optical properties. Intriguingly, a new type of NIR QDs, Ag2S QDs that are free from toxic metals, such as Pb, Cd or Hg, and bear scant release of Ag ions into the biological surroundings due to their ultralow solubility product constant (Ksp = 6.3 × 10−50), are
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widely considered to have excellent biocompatibility21,22 and have received considerable attention. Recently, Dai’s group opened up the possibilities of using Ag2S QDs for in vivo anatomical imaging and early stage tumor diagnosis.23 More recently, selective in vitro imaging based on Ag2S QDs conjugated with different targeting ligands, such as a vascular endothelial growth factor antibody, has been achieved by Wang et al.24 However, the applications of Ag2S QDs mainly focus on biomedical imaging; research employing Ag2S QDs as drug delivery vehicles for theranostic application has rarely been reported. Thus, our present study investigates this promising possibility. In this study, cyclic RGD peptide (cRGD), a less-expensive active tumor targeting tri-peptide, was employed to replace vascular endothelial growth factor antibody for directing the nanoparticles to tumor sites. The arginine-glycine-aspartic acid (RGD) peptide has been widely used for the delivery of imaging agents and drugs to tumors due to its high affinity with integrin αvβ3, which is found to be highly expressed on tumor vasculature and a variety of tumor cells, including human malignant melanoma, breast cancer and advanced glioblastoma.25–27 In addition, cyclic RGD (cRGD) peptide was proven to be a more efficient tumor-targeting ligand in comparison with linear RGD peptide.28 Herein, Ag2S QDs were first synthesized according to previous work,29 after which doxorubicin and cRGD were covalently attached to the as-prepared Ag2S QDs. To the best of our knowledge, it is the first time that Ag2S QDs have been exploited for simultaneous therapeutic and imaging application. It is known that for cancer therapy, diagnostic imaging before initiating treatment is crucial for better understanding the exact tumor sites and tumor heterogeneity. Different from the conventional materials developed for separate diagnostic or therapeutic application, Ag2ScRGD-DOX, which combines the two features can overcome the unfavorable difference in biodistribution and selectivity between distinct imaging and therapeutic agents.
2. Materials and methods 2.1.
Materials
AgNO3 (A.R), Na2S·9H2O, ethanoic acid 3-mercaptopropionic acid (MPA), sodium hydroxide, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N,N′-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS) were purchased from Aladdin (Shanghai, China). CRGD (cyclo (Arg-Gly-AspDTyr-Lys)) was obtained from Jeer Biotech (Shanghai, China). 3-(4,5-Dimethylthialzol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DMEM, fetal bovine serum (FBS), penicillin, streptomycin, trypsin-EDTA and doxorubicin (DOX) were purchased from commercial sources. Ultrapure Millipore water (18.2 MU) was used. The reagents used in this study were used without further purification. 2.2.
Preparation of Ag2S QDs and its conjugates
2.2.1. Synthesis of MPA stabilized Ag2S QDs. A facile synthesizing method has been developed and will be described
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in the following sections. Typically, MPA was dissolved in 75.0 mL of deoxygenated deionized water. The pH of the solution was adjusted to 7.5 using NaOH and CH3COOH solutions (2.0 M). After the addition of AgNO3 (42.5 mg), the pH was readjusted to 7.5. Then, 25.0 mL of deoxygenated aqueous Na2S solution was slowly added to the reaction mixture under vigorous mechanical stirring to obtain a clear orange-yellow solution, after which the pH was again adjusted to 7.5. The solution was then heated to 90 °C and maintained at this temperature for 5 h. The reaction solution was then cooled to room temperature and stored in dark at 4 °C for further analysis. 2.2.2. Synthesis of Ag2S-cRGD, Ag2S-DOX and Ag2S-DOXcRGD. DOX (1.0 mg) was dissolved in 1.0 mL of pure water. EDC and NHS were utilized as coupling agents to react with Ag2S at room temperature for 4 h. The DOX solution was then added and the mixture was stirred in the dark for 12 h at room temperature. The reaction mixture was then placed in a bag filter for 24 h dialysis. The product (Ag2S-DOX) was stored in the dark at 4 °C. Cyclic RGD peptide (cRGD, 1.0 mg) was immobilized onto Ag2S-DOX using coupling agents (EDC and NHS). EDC (5.0 mg) and NHS (4.6 mg) were added into 5.0 mL of the prepared Ag2S-DOX solution, and the mixture was stirred at room temperature for 4 h. cRGD (3.1 mg) was then added and stirred in the dark for 12 h at room temperature. The reaction mixture was then removed to the bag filter for a 24 h dialysis. The product (Ag2S-DOX-cRGD) was stored in the dark at 4 °C. Ag2S-cRGD was obtained using a similar method, except that DOX was not conjugated. 2.3.
Optical characterization of the probes
The absorption spectra of Ag2S QDs, Ag2S-DOX, and Ag2S-DOXcRGD were acquired using a Lambda 25 UV-Vis spectrophotometer (Perkin Elmer, US). The corresponding fluorescence spectra of the samples were measured at room temperature using an LS 55 Fluorescence Spectrometer (Perkin Elmer, US). 2.4.
Cell studies
2.4.1. Cell culture. The human cell lines U87 (malignant glioma), MCF-7 (breast cancer) and MDA-MB-231 (breast cancer) were obtained from American Type Culture Collection (ATCC, USA). The cell lines were cultured in DMEM medium supplemented with 10% (v/v) calf serum, penicillin (100 U mL−1), and streptomycin (100 mg mL−1). The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. 2.4.2. Cytotoxicity evaluation of Ag2S QDs and Ag2S-cRGD. Cytotoxicity of Ag2S QDs and Ag2S-cRGD in normal human liver cells (L02), glioma cells (U87), and human breast cancer cells (MCF-7) was determined by MTT assay. The cells were seeded in 96-well plates (1 × 104 cells per well) and subsequently incubated for 24 h. After treatment with different concentrations (0.02 μM to 12.5 μM) of Ag2S QDs or Ag2ScRGD, the cells were further maintained at 37 °C for 24 h. Each well was washed three times with PBS ( pH 7.0) and then
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20 μL of MTT solution (5.0 μg mL−1) was added. After another 4 h incubation, the medium containing MTT was carefully removed and DMSO (150 μL) was added to each well. The plates were then subjected to measure the absorbance. Cell viability was calculated using the following formula: Cell viability = (Mean absorbance of test wells − Mean absorbance of medium control wells)/(Mean absorbance of untreated wells − Mean absorbance of medium control well) × 100%. The therapeutic efficacy of Ag2S-DOX and Ag2S-DOX-cRGD was also evaluated using MTT assays following the abovementioned method. The concentration range of the samples was 0.1 μg mL−1 to 12.5 μg mL−1, and the incubation time was set at 8 h. 2.4.3. Cellular uptake of Ag2S-DOX and Ag2S-DOX-cRGD. The targeting ability of Ag2S DOX and Ag2S-DOX-cRGD was evaluated using the two tumor cells lines, U87 and MCF-7 cells. The cells were seeded in laser confocal fluorescence microscope (LCFM) culture dishes with a density of 4 × 105 cells per well and subsequently incubated at 37 °C in a humidified atmosphere containing 5% CO2. When the whole cells reached ∼70%–80% confluency, 100 μL of Ag2S-DOX and Ag2S-DOX-cRGD were added into different dishes, and the dishes were then incubated for 1 h, 2 h or 4 h, followed by incubation with Hoechst 33342 solutions (100 μL; 10 μg mL−1) for 30 min. The information was used to determine the intracellular kinetics of Ag2S-DOX and Ag2S-DOX-cRGD. Subsequently, the cells were washed three times with PBS before examining the probe’s affinity for cancer cells by LCFM (FV1000, Olympus, Japan). The red fluorescence of DOX was captured under 488 nm light excitation. Intracellular uptake of the different probes was determined from the red fluorescence intensity in the region of interest (ROI) using Scion Image software. 2.5.
Animal experiments
2.5.1. Animal subjects and tumor model. Athymic nude mice and normal (Kunming) mice were purchased from Charles River Laboratories (Shanghai, China) for in vivo imaging. All the animal experiments were carried out in compliance with the Animal Management Rules of the Ministry of Health of the People’s Republic of China (Document no. 55, 2001) and the guidelines for the Care and Use of Laboratory Animals of China Pharmaceutical University. Tumor models were established by subcutaneously injecting tumor cells (5 × 106 in 50 mL of PBS) into the axillary fossa of the mice. 2.5.2. Dynamic distribution of Ag2S-cRGD and Ag2S-DOXcRGD in tumor-bearing mice. To investigate the targeting ability of the probes, two types of tumor models were established by the subcutaneous injection of MDA-MB-231 (high αvβ3 receptor expression) tumor cells (∼5 × 106 in 80 μL of PBS) into the axillary fossa of female athymic nude mice (n = 5 for each model). As the tumors grew to a diameter of up to 0.1 cm and 0.6 cm, the models of early-stage and late-stage MDA-MB-231 tumor-bearing mice were established successfully. Ag2S-cRGD (0.2 mL, 10 mg kg−1) and Ag2S-DOX-cRGD (0.2 mL, 10 mg kg−1) were then injected into early-stage and
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late-stage MDA-MB-231 tumor-bearing mice through the vena caudalis. The nude mice were then immobilized on a lucite jig. An NIR imaging system equipped with a 765 nm laser as excitation source was employed to trace the distribution of the probe. The background image of the mice was taken prior to the injection. A series of images were collected at 5 min, 1 h, 2 h, 4 h, 12 h, 24 h and 48 h post-injection. To further compare the targeting ability of the probe for early-stage and late-stage MDA-MB-231 tumors, fluorescence intensity from tumor and major organs (heart, liver, bladder and intestine) at different time points (10 min, 3 h and 8 h) was determined using the region of interest (ROI) functions of Scion Image software. The tumor to normal tissue contrast ratios (T/N) at different time points were also calculated. In each case, the background fluorescence that was measured before the injection of the conjugates was subtracted from the post-injection fluorescence. To further investigate the distribution of Ag2S-DOX-cRGD in tumors, a tumor was removed from the mice body at 5 h postinjection of the probe. The tissues were cut into slices (8 mm thickness) by a freezing microtome (CM1850, Leica). The peripheral slices and the central slices of the tumor were collected and observed by laser confocal fluorescence microscope immediately. Red fluorescence (590 nm) emitted from DOX was collected. 2.5.3. Therapeutic efficacy evaluation of Ag2S-DOX-cRGD. The Ehrlich ascites carcinoma (EAC) tumor model for therapy evaluation was established by subcutaneously inoculating EAC cells (∼8 × 106) into the nether axillary fossa of the mice (n = 32). The mice were investigated after 7 days of inoculations. When the tumor was completely developed, the mice were randomly divided into four groups (n = 8 for each group). Saline (the control group, Group 1), DOX solution (Group 2), Ag2S-DOX (Groups 3) and Ag2S-DOX-cRGD (Group 4) were intraperitoneally injected into the mice. Subsequently, each mouse underwent intraperitoneal injection once every other day for a total of four times. The survival rate of the treated mice was calculated according to the equation: Survival rate = Ns/Nt × 100%, where Ns and Nt represent the number of surviving mice and the number of total mice in each group, respectively. 2.5.4. Ex vivo histopathology. To further investigate the therapeutic effects of Ag2S-DOX-cRGD on EAC tumor-bearing mice, the tumors were excised for histopathologic analysis on the 16th day post-injection. The tissue was fixed with 10% neutral buffered formalin and embedded in paraffin. The sliced tumors (8 mm) were stained with hematoxylin and eosin (H&E) and observed by a BX41 bright field microscopy (Olympus).
3. Results 3.1.
Characterization of Ag2S-DOX-cRGD
In this study, MPA-coated Ag2S NPs were covalently conjugated with DOX, a type of widely used antitumor drug, to realize the
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Fig. 1
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Synthesis routine of the nano-conjugate (Ag2S-DOX-cRGD).
Fig. 2 (a) Absorption spectra and (b) fluorescence spectra of Ag2S QDs, Ag2S-DOX and Ag2S-DOX-cRGD; (c) white light appearance and the fluorescence image of Ag2S-DOX-cRGD aqueous solution.
combination of real time imaging and chemotherapy. CRGD was also covalently immobilized on the surface of MPA-coated Ag2S NPs to enhance their targeting capability to integrin αvβ3 over-expressing tumors. The successful synthesis of Ag2S-DOXcRGD was confirmed by real-color/fluorescence images, absorption spectra and fluorescence spectra. Ag2S NPs, Ag2S-DOX and Ag2S-DOX-cRGD were synthesized following the procedures described in the method section, and the specific synthesis routine is shown in Fig. 1. MPA coated Ag2S QDs were prepared in water by the slow addition of sulfide to a mixture of MPA and silver salt. The color of the solution turned yellow with the addition of sulfide ion, and the resulting colloidal QDs solution had a dark brown color. The favorable optical properties of Ag2S QDs were exhibited in the fluorescence spectrum (Fig. 2B), given that their photoluminescence emission peak at 800 nm was distinctly detected, while the quantum yield of synthesized Ag2S QDs was 37.9%. Furthermore, the emission peak centered at
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800 nm has an impressive full width at half-maximum of 65 nm, indicating a narrow size distribution of the Ag2S QDs (Fig. 2B). The conjugation of Ag2S was also monitored by UV-Vis absorption spectra and fluorescence spectra (Fig. 2). The conjugates, Ag2S-DOX and Ag2S-DOX-cRGD, still exhibited the characteristic absorption peaks of DOX (490 nm) (Fig. 2A). The corresponding fluorescence spectra are shown in Fig. 2B. In addition, Fig. 2B suggests that the fluorescence emitted by Ag2S at 800 nm was not influenced by DOX, enabling its application in NIR fluorescence imaging. 3.2.
Cytotoxicity of Ag2S QDs and Ag2S-cRGD
Ideal contrast agents and drug delivery vehicles should be safe and non-toxic to cells and tissues. It is crucial to conduct a toxicity study for the potential applications of Ag2S QDs and their conjugates. Herein, the cytotoxicity of Ag2S QDs and Ag2S-cRGD was first investigated by MTT assay to evaluate
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Fig. 3 Cell viability of different cell lines (L02, U87, MCF-7) after incubation with Ag2S and Ag2S-cRGD respectively within a wide concentration range (0.2–125.0 μg mL−1).
their biocompatibility. The MTT assay relies on the mitochondrial activity of cells and represents a viable parameter for reporting metabolic activity. In our study, the MTT assay was applied to L02, U87 and MCF-7 cell lines for both Ag2S QDs and Ag2S-cRGD. The results were collected after 12 h and 24 h incubation and are demonstrated in Fig. 3. Under all circumstances, neither Ag2S QDs nor Ag2S-cRGD showed any significant cytotoxicity. The two particles displayed low toxicity to three cell lines even at high doses (125 μg mL−1) with a high cell viability after 24 h incubation (L02: >83%; U87: >81%; MCF-7: >80%). In addition, no obvious difference could be observed between the results of 12 h and 24 h for the two probes, which indicated no serious toxic effect in the long term. The observed ultra-low cytotoxicity in MTT assay could be attributed to the low-toxic metallic constituents of Ag2S QDs as well as their ultralow solubility constant, which determines the ultra-slow release of metallic constitu-
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ents to biological surroundings. Thus, these results suggest that Ag2S QDs are promisingly safe nanoparticles for bio-applications. 3.3. Cellular uptake of Ag2S-DOX and Ag2S-DOX-cRGD in U87 and MCF-7 cells Taking the advantage of red fluorescence emitted by DOX itself, the cellular uptake of Ag2S-DOX and Ag2S-DOX-cRGD was easily visualized. After Ag2S-DOX and Ag2S-DOX-cRGD were incubated with U87 tumor cells at 37 °C, the uptake process was examined by LCFM at 1 h, 2 h and 4 h after incubation (Fig. 4A). With regard to the images of Ag2S-DOX, only a weak red fluorescence could be observed, indicating that only little Ag2S-DOX entered U87 tumor cells. A similar result was also obtained from MCF-7 cell lines, in which the expression of αvβ3 receptors (Fig. 4C) is known to be negative. In contrast, Ag2S-DOX-cRGD in U87 cells, in which the expression of αvβ3
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on the surface of Ag2S can facilitate their cellular uptake by the cells with high expression of αvβ3 receptors. In other words, the affinity of Ag2S NPs to integrin αvβ3 over-expressed tumor cells has been strengthened by cRGD, which is one of the primary aims of this study.
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3.4. Dynamic distribution of Ag2S-cRGD and Ag2S-DOX-cRGD in tumor-bearing mouse models
Fig. 4 (a) Laser confocal fluorescence images of U87 cells after incubation with the probes (Ag2S-DOX and Ag2S-DOX-cRGD) for different time (1 h, 2 h and 4 h); (b) Semi-quantitative analysis of the fluorescence intensity of U87 cells after incubation with the probes (Ag2S-DOX and Ag2S-DOX-cRGD) for different times (1 h, 2 h and 4 h); (c) Laser confocal fluorescence images of MCF-7 cells after incubation with the probes (Ag2S-DOX and Ag2S-DOX-cRGD) for 4 h.
receptors is high, showed strong fluorescence after 2 h incubation. The fluorescence signal detected from the U87 cells could obviously eclipse the one detected from the U87 cells treated with Ag2S-DOX, illustrating that the presence of cRGD
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To better understand its pharmacokinetic behavior, Ag2S-cRGD was injected into early-stage and late-stage MDA-MB-231 tumor-bearing mice through the vena caudalis. A series of in vivo images obtained by NIR imaging system at different time intervals is shown in Fig. 5A, B to illustrate the dynamic behavior of Ag2S-cRGD. The bright fluorescent signal revealed that Ag2S-cRGD initially spread quickly throughout the whole bodies in both the two models, then concentrated in the liver, bladder and tumor site, and finally cleared via the hepatobiliary and renal pathways. In an intuitive manner, the fluorescence signal in both MDA-MB-231 tumor models became distinguishable from the normal tissues at 1 h postinjection, and reached peak intensity at 8 h post-injection. The fluorescence of probes from the tumor site and the liver of late-stage tumor-bearing mice is considerably stronger than that of the early-stage tumor-bearing mice at the same time point, indicating a different status of tumor because the capacity of a well-developed tumor to retain Ag2S-cRGD is higher than that of the tumor in nascent state, and thus the fluorescence intensity exhibited by late-stage tumor would be relatively stronger. To semi-quantitatively analyze the targeting ability of Ag2ScRGD in vivo, the fluorescence intensity was gauged by selecting specific regions of interest (ROIs) from the tumors and normal tissues at different time intervals. In each case, the background fluorescence measured before the injection of Ag2S-cRGD was subtracted. The fluorescence intensities in the ROIs from tumor and normal tissues were averaged and then plotted against time (Fig. 5C). For both the two mouse models, the fluorescence intensity ratio between tumor and normal tissue (T/N) reached a peak at 8 h post-injection of Ag2S-cRGD, whereas the tumor/normal ratio of the late-stage tumor remained a slightly higher than that of the early-stage tumor at all time intervals; these observations are consistent with the conclusion from the observation of fluorescence mapping as described above. Meanwhile, the semi-quantitative fluorescence intensity of the tumor and isolated organs (heart, liver, bladder and intestine) from two Ag2S-cRGD-treated mouse models were compared at different post-injection time points (10 min, 3 h and 8 h, Fig. 5D, E and F). Obviously, the conjugate was prone to arrive at the bladder through the kidney-bladder pathway in the early tumor-bearing mouse. Meanwhile, the conjugate displayed a long time accumulation in liver in the late tumorbearing mouse. Both of the two tumor-bearing mice demonstrated high accumulation of the nanoconjugates in tumors. The distribution of the nano-conjugates (Ag2S-cRGD-DOX) in the MDA-MB-231 tumor-bearing mice and the distribution
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Fig. 5 In vivo fluorescence imaging of Ag2S-cRGD in different MDA-MB-231 tumor-bearing mice including (a) the early stage and (b) the late stage; (c) Relative fluorescence intensity ratios of tumor to normal tissue for Ag2S-cRGD injected MDA-MB-231 tumor-bearing mice; Fluorescence intensity from tumor and major organs (heart, liver, bladder and intestine) was assessed by Scion Image software after selecting ROI and compared at (d) 10 min, (e) 3 h and (f ) 8 h post-injection of the probe.
of the conjugates in tumor tissue were investigated. As shown in Fig. 6A, Ag2S-cRGD-DOX initially distributed in the entire body after being injected into the tumor-bearing mouse via the tail vein. The nano-conjugates arrived at tumor 1 h post-injection, and the bright fluorescent signal that appeared at the tumor lasted for 24 h. Fig. 6B showed a noticeable strong red fluorescence that could be observed in both the peripheral slice and the central slice of the tumor, suggesting the high accumulation of Ag2S-cRGD-DOX in malignant tissues. The semi-quantitative analysis of the biodistribution of the probe is displayed in Fig. 6C. The highly specific accumulation of the probe in the MDA-MB-231 tumor was clearly observed compared with that in other tissues (heart, liver, bladder and intestine), revealing its desirable affinity to tumor sites. Moreover, the high fluorescence intensity of liver and kidney could be attributed to their metabolic roles.
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3.5. Anti-tumor activity evaluation of Ag2S-DOX-cRGD and Ag2S-DOX in cell level The anti-tumor activity of Ag2S-DOX-cRGD and Ag2S-DOX was evaluated through MTT assay, which was performed on U87, MDA-MB-231 and MCF-7 cell lines in this section. A dosedependent reduction in MTT absorbance for all the three cell lines treated with Ag2S-DOX-cRGD and Ag2S-DOX is demonstrated in Fig. 7. In the cases of integrin αvβ3 over-expressing tumor cells, including U87, MDA-MB-231, cells treated with Ag2S-DOX-cRGD exhibited lower cell viability than those treated with Ag2S-DOX, as the maximal inhibition ratio of Ag2S-DOX-cRGD is ∼70%, which is significantly increased from ∼50%, the maximal inhibition ratio of Ag2S-DOX. Therefore, the mediation of cRGD helps in potentiating the therapeutic effect of DOX in pertinent tumor cells.
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3.6. Therapeutic efficacy evaluation of Ag2S-DOX-cRGD in tumor-bearing mice
Fig. 6 (a) In vivo fluorescence imaging of Ag2S-DOX-cRGD in MDA-MB-231 tumor-bearing mice; (b) Strong red fluorescence could be observed in peripheral and central slices of the tumor under LSCM, indicating the high uptake of Ag2S-DOX-cRGD by tumor at 5 h post-injection; (c) Fluorescence intensity from tumor and major organs (heart, liver, bladder and intestine) was assessed by Scion Image software after selecting ROI and compared at 10 min, 3 h and 8 h post-injection of the probe.
The images of mice in the four groups that underwent intraperitoneal injection of saline, DOX, Ag2S-DOX, and Ag2ScRGD-DOX once every other day for four times of total treatment are collected in Fig. 8A. Expectedly, the most conspicuous growth of tumor was observed in the saline group, and even visible necrosis, which is a symptom of a late-stage tumor, appeared among this group after the fourth treatment. By contrast, the tumor grew at a comparatively low speed in the DOX and Ag2S-DOX groups, whereas the most remarkable suppression of tumor deterioration was observed in the Ag2S-DOX-cRGD group. The survival rate of mice from all the four groups during 16 days was also calculated and is shown in Fig. 8B. The survival rate of the saline group started to decline early on the 5th day and only half of the mice were alive on the 16th day. The death of mice, although observed in both the DOX and Ag2S-DOX groups, occurred much later, and over 70% of the mice survived this experiment. Desirably, the Ag2S-DOX-cRGD group manifested a satisfying survival rate of 100%. After treatment, center and peripheral tumor tissues were excised from all the four groups and histological analysis was performed 18 days post-injection (Fig. 9). Only a small number of apoptotic and necrotic tumor cells in the center of tumors from the saline group could be observed due to the pathological process. Distinctively, both the periphery and the center of tumors from the DOX, Ag2S-DOX group and Ag2S-DOX-cRGD group displayed large-area apoptosis and necrosis, whereas the most common cell damage could be observed in Ag2S-DOX-cRGD group, confirming the excellent anti-tumor efficacy of Ag2S-DOX-cRGD in vivo.
Fig. 7 Anti-tumor efficacy of Ag2S-DOX and Ag2S-DOX-cRGD was evaluated on different cell lines (U87, MDA-MB-231 and MCF-7). The concentration range of the samples was 0.1 μg mL−1 to 12.5 μg mL−1 and the incubation time was set as 8 h.
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Fig. 8 (a) Photographs of EAC tumor-bearing mice in the four groups (saline, DOX, Ag2S-DOX, and Ag2S-cRGD-DOX) on different days of treatment (4 d, 8 d, 12 d and 12 d). (b) Survival rate of mice for four groups (saline, DOX, Ag2S-DOX, and Ag2S-cRGD-DOX) within 16 days of treatment.
4.
Fig. 9 Histological analysis of tumor sections excised from the center and peripheral tumor tissues of the mice 16 days after intravenous injection of the different samples (saline, DOX, Ag2S-DOX, and Ag2ScRGD-DOX).
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Discussion
Ever since the concept of “theranostics”, which suggests a combination of diagnostics and therapy, was proposed in 2002,30 nanomedicine platforms that integrate imaging and therapeutic function have attracted considerable attention as the next generation of medicine. Diverse nanoparticles with an imaging function, including magnetic nanoparticles, gold nanoparticles and QDs, have been used as drug carriers that can map the behavior of drug at both subcellular and systemic levels.31,32 Among these nano-contrast agents, QDs—nanometer-size luminescent semiconductor nanocrystals—are particularly appealing as potential molecular imaging probes and small-molecule nano-carriers because of their unique properties such as high brightness and long-term stability.33 With intense study on QDs as well as their wide application in bioimaging, the investigation of QD/drug nanoparticle formulations has begun in recent years due to the advancement of QD synthesis and bioconjugation chemistry for immobilizing drug molecules onto the QD surface.34 Taking the advantage of real-time and quantitative imaging analysis enabled by QDs, the kinetics of drug in vivo as well as the information of targeted lesion sites could be obtained. Expectedly, several successful attempts that employed conventional QDs as drug carriers have been reported.35 For instance, the nanoconjugates of CdSe/CdS/ZnS QDs and DOX were developed by Chakravarthy et al. to target alveolar macrophages for pulmonary treatment.36 A site-specific QD-based platform incorporated with antiretroviral drug saquinavir and the biorecognition
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molecule transferrin has also been established by Mahajan et al. for the treatment of neuro-AIDS and other neurological disorders.37 However, the further steps of these studies toward clinical use are still stymied by the inherent toxicity of QDs and their unclear biological degradation mechanisms in vivo. The appearance of Ag2S QDs with near-infrared fluorescence holds the potential to extricate the development of QDs from such a bottleneck constraint. Exciting progress in theranostic application based on nanostructures, such as gold nanoparticles and up-conversion nanoparticles, have been achieved in recent years38,39 but more effort is still required in the research of Ag2S QDs. Inspired by some previously successful cases, a variety of agents, such as IR825, ZnPc and Nitric Oxide, could be employed to broaden the application of Ag2S QDs. The objective of the present study is to establish multi-functional nanomedicine based on Ag2S QDs for the combination of NIR imaging and chemotherapy. Specifically, Ag2S QDs capitalize on the tumor affinity of cRGD and anti-tumor efficacy of doxorubicin, while achieving a theranostic design to allow the visualization of therapeutic efficacy by taking advantage of noninvasive NIR imaging function of QD self. In addition, certain effort has been devoted for employing Ag2S QDs for in vivo application. However, the active targeting ability of Ag2S QDs is still a problem because this problem has either not been taken into account or this problem has not been solved in an economic manner.24 Herein, cRGD was adopted as a targeting ligand and was shown to improve the cellular uptake of chemical drug (Fig. 4) as well as the in vivo NIR imaging capability of Ag2S QDs (Fig. 5). In this study, this strategy was indicated to be feasible for integrin αvβ3 over-expressing tumor cells, including U87, MDA-MB-231. Furthermore, it is not difficult to expect that a similar strategy will also be effective for other tumors, such as high folate receptor (FR)-expressing tumors, by simply replacing the targeting ligand correspondingly. Subsequent to the conjugation of Ag2S QDs, which have excellent NIR optical characteristics29 and biocompatibility40 with a tumor-targeting ligand, cRGD, and the common chemotherapeutic drug, doxorubicin, the unintrusive optical characteristics of Ag2S QDs were demonstrated by the adsorption and fluorescence spectra, eliminating the concern that the thickness of surface coating on QDs might considerably change their optical and thus negatively influence their imaging function (Fig. 2). In addition, cytotoxicity is another important factor that should be taken into consideration for the biological application of QDs. In a typical case, conventional cadmium-based QDs, which are commonly used for in vitro and in vivo studies, are known to cause acute and chronic toxicities in vertebrates at high dosage as a result of their main constituent elements that include cadmium and selenium.41 Favorably, the MTT assay in this study revealed only partial cytotoxicity of Ag2S QDs and its conjugates (Fig. 3), indicating a significant potential of Ag2S QDs for safe exploitation in further biomedical application. As observed from our result (Fig. 5), the conjugate-cancer accumulation was related to the tumor size. As tumor size
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increased, both the accumulation time of the probe in the tumor and in the entire body became extended. As Gu et al.5 have reported, the nano-conjugate (Au-Met-MPA)-based Au nano-clusters demonstrated peak intensity at 10 h post-injection and maintained their detectable fluorescence for 96 h in an MDA-MB-231 tumor-bearing (late stage) mouse. However, the bright fluorescence signal of Au-Met-MPA reached the maximum at 7 h and then gradually disappeared after 72 h in an MDA-MB-231a tumor-bearing (early stage) mouse. Thus, the biodistribution of Ag2S-cRGD in different stages of tumorbearing mice was consistent with Gu’s report. More importantly, by tracking the behavior of Ag2S-DOX-cRGDs and investigating their therapeutic effect at both the cell (Fig. 7), and animal level (Fig. 8 and 9), Ag2S-DOX-cRGDs were demonstrated to work better than DOX, a widely used chemotherapeutic drug in clinics, as well as Ag2S-DOX, while maintaining the NIR imaging function of Ag2S QDs. According to the principles of tumor-specific personalized medicine, before initiating the cancer treatment, molecular profiling of tumor heterogeneity by imaging is indispensable to verify the cancer biomarkers in the intra-and inter-tumor tissue, which could provide essential information regarding target-specific therapy.42,43 After implementing targeted therapy, the mission of eradicating all cancer cells is still not complete because the tumor may inevitably evolve in response to therapy. Thus, the molecular imaging of the tumor that could be quickly repeated is necessary for direct intelligent adjustment of the targeting strategy for cancer therapy. Consistent with the concept referred to above, combining the diagnostic capability of Ag2S QDs with therapeutic intervention in our study is beneficial for addressing the challenges of cancer heterogeneity and adaptive resistance.
5. Conclusion In summary, the nano-conjugates (Ag2S-DOX-cRGD) based on the as-prepared Ag2S QDs, integrating imaging and therapeutic function, were constructed successfully through a facile synthesis routine. The favorable tumor-targeting efficiency of an inexpensive active tumor-targeting peptide (cRGD) decorated Ag2S QDs were verified both in integrin αvβ3 over-expressing tumor cells and tumor-bearing mice models, including U87 and MDA-MB-231. Furthermore, the effective tumor inhibition of the nanoconjugates was also demonstrated at both the cell and animal level. These results vindicated that Ag2S QDs are promising candidates for designing imaging-visible nanotherapeutics for tumor diagnosis and therapy application.
Acknowledgements The authors are grateful to Natural Science Foundation Committee of China (NSFC 81371684, 61335007, 81220108012, 81171395 and 81328012), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University
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(no. SKLNMZZYQ201403) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions for their financial support.
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Characterization of tumor-targeting Ag2S quantum dots for cancer imaging and therapy in vivo Haiyan Chen,*†a Bowen Li,†b Min Zhang,†a Kang Sun,c Yiran Wang,c Kerui Peng,c Mengdi Ao,c Yiran Guoa and Yueqing Gu*a Nanomedicine platforms that have the potential to simultaneously provide the function of molecular imaging and therapeutic treatment in one system are beneficial to address the challenges of cancer heterogeneity and adaptive resistance. In this study, Cyclic RGD peptide (cRGD), a less-expensive active tumor targeting tri-peptide, and doxorubicin (DOX), a widely used chemotherapeutic drug, were covalently attached to Ag2S quantum dots (QDs) to form the nano-conjugates Ag2S-DOX-cRGD. The optical characterization of Ag2S-DOX-cRGD manifested the maintenance of QDs fluorescence, which suggested the potential of Ag2S for monitoring intracellular and systemic drug distribution. The low bio-
Received 28th June 2014, Accepted 9th August 2014
toxicity of Ag2S QDs indicated that they are promisingly safe nanoparticles for bio-applications. Further-
DOI: 10.1039/c4nr03613a
more, the selective imaging and favorable tumor inhibition of the nanoconjugates were demonstrated at both cell and animal levels. These results indicated a promising future for the utilization of Ag2S QDs as a
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kind of multi-functional nano platform to achieve imaging-visible nano-therapeutics.
1.
Introduction
Nowadays, cancer, as a significant cause of human morbidity and mortality, remains a difficult disease to treat, although untold money and resources have been invested into the fight against it. In recent years, nanomedicine platforms with the potential to simultaneously provide the function of molecular imaging and therapeutic treatment in one system are intriguing more and more researchers.1,2 Imaging-visible nanotherapeutics with the capability of validating cancer biomarkers in tumor tissue through molecular diagnosis and thereby providing critical information for the timely tailoring of targeting strategy are expected to hold the key to address the challenges of tumor heterogeneity and adaptive resistance.3,4 Over the past decades, there has been a fulminating development of various inorganic nano-platforms such as gold nanoparticles,5 magnetic iron oxide nanoparticles,6 mesoporous silica nanoparticles,7 and quantum dots (QDs),8 based on which assorted biomedical applications have been performed. The primary advantage of these nanomaterials for theranostic
a Department of Biomedical Engineering, School of Life Science and Technology, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia Lane, Gulou District, Nanjing 210009, China. E-mail: [email protected], [email protected]; Fax: +86-25-83271046; Tel: +86-25-83271080 b Department of Bioengineering, University of Washington, Seattle, USA c School of Pharmacy, China Pharmaceutical University, 24 Tongjia Lane, Gulou District, Nanjing 210009, China † These authors contributed equally to this work.
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application is their multi-functional properties that enable the decoration of drugs, affinity ligands, and imaging moieties on the same vehicle for targeted and traceable drug delivery. Typically, QDs, nanocrystals of semi-conducting materials featuring a combination of small size with versatile surface chemistry, have been intensively studied by many researchers because of their characteristic optical advantages,9,10 including a narrow emission band, high quantum yields, very long effective Stokes shifts and high photo-stability, which are favorable for realizing the real-time monitoring of otherwise invisible nano-carriers.11 Near-infrared (NIR) fluorescent quantum dots (QDs) are of particular interest for in vivo imaging and nano-theranostics due to their deep penetration into the body and poor absorbance of their fluorescence by hemoglobin and water in the body. To date, various kinds of NIR QDs, such as CdHgTe,12 CdHgTe/ZnS,13 InAs,14 and PbS,15 have been successfully synthesized for in vivo biomedical applications. However, the potential release of chemical constituents, including cadmium, mercury, lead, tellurium, and selenium, leads these NIR QDs to bear acute and chronic toxicities,16,17 restricting their further applications in bio-systems. Although ternary I–III–VI NIR QDs (Cu–In–Se, CuInS2, AgInS2)18–20 without intrinsic toxicity have recently been reported, it is onerous to tune their chemical composition while controlling their optical properties. Intriguingly, a new type of NIR QDs, Ag2S QDs that are free from toxic metals, such as Pb, Cd or Hg, and bear scant release of Ag ions into the biological surroundings due to their ultralow solubility product constant (Ksp = 6.3 × 10−50), are
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widely considered to have excellent biocompatibility21,22 and have received considerable attention. Recently, Dai’s group opened up the possibilities of using Ag2S QDs for in vivo anatomical imaging and early stage tumor diagnosis.23 More recently, selective in vitro imaging based on Ag2S QDs conjugated with different targeting ligands, such as a vascular endothelial growth factor antibody, has been achieved by Wang et al.24 However, the applications of Ag2S QDs mainly focus on biomedical imaging; research employing Ag2S QDs as drug delivery vehicles for theranostic application has rarely been reported. Thus, our present study investigates this promising possibility. In this study, cyclic RGD peptide (cRGD), a less-expensive active tumor targeting tri-peptide, was employed to replace vascular endothelial growth factor antibody for directing the nanoparticles to tumor sites. The arginine-glycine-aspartic acid (RGD) peptide has been widely used for the delivery of imaging agents and drugs to tumors due to its high affinity with integrin αvβ3, which is found to be highly expressed on tumor vasculature and a variety of tumor cells, including human malignant melanoma, breast cancer and advanced glioblastoma.25–27 In addition, cyclic RGD (cRGD) peptide was proven to be a more efficient tumor-targeting ligand in comparison with linear RGD peptide.28 Herein, Ag2S QDs were first synthesized according to previous work,29 after which doxorubicin and cRGD were covalently attached to the as-prepared Ag2S QDs. To the best of our knowledge, it is the first time that Ag2S QDs have been exploited for simultaneous therapeutic and imaging application. It is known that for cancer therapy, diagnostic imaging before initiating treatment is crucial for better understanding the exact tumor sites and tumor heterogeneity. Different from the conventional materials developed for separate diagnostic or therapeutic application, Ag2ScRGD-DOX, which combines the two features can overcome the unfavorable difference in biodistribution and selectivity between distinct imaging and therapeutic agents.
2. Materials and methods 2.1.
Materials
AgNO3 (A.R), Na2S·9H2O, ethanoic acid 3-mercaptopropionic acid (MPA), sodium hydroxide, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N,N′-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS) were purchased from Aladdin (Shanghai, China). CRGD (cyclo (Arg-Gly-AspDTyr-Lys)) was obtained from Jeer Biotech (Shanghai, China). 3-(4,5-Dimethylthialzol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DMEM, fetal bovine serum (FBS), penicillin, streptomycin, trypsin-EDTA and doxorubicin (DOX) were purchased from commercial sources. Ultrapure Millipore water (18.2 MU) was used. The reagents used in this study were used without further purification. 2.2.
Preparation of Ag2S QDs and its conjugates
2.2.1. Synthesis of MPA stabilized Ag2S QDs. A facile synthesizing method has been developed and will be described
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in the following sections. Typically, MPA was dissolved in 75.0 mL of deoxygenated deionized water. The pH of the solution was adjusted to 7.5 using NaOH and CH3COOH solutions (2.0 M). After the addition of AgNO3 (42.5 mg), the pH was readjusted to 7.5. Then, 25.0 mL of deoxygenated aqueous Na2S solution was slowly added to the reaction mixture under vigorous mechanical stirring to obtain a clear orange-yellow solution, after which the pH was again adjusted to 7.5. The solution was then heated to 90 °C and maintained at this temperature for 5 h. The reaction solution was then cooled to room temperature and stored in dark at 4 °C for further analysis. 2.2.2. Synthesis of Ag2S-cRGD, Ag2S-DOX and Ag2S-DOXcRGD. DOX (1.0 mg) was dissolved in 1.0 mL of pure water. EDC and NHS were utilized as coupling agents to react with Ag2S at room temperature for 4 h. The DOX solution was then added and the mixture was stirred in the dark for 12 h at room temperature. The reaction mixture was then placed in a bag filter for 24 h dialysis. The product (Ag2S-DOX) was stored in the dark at 4 °C. Cyclic RGD peptide (cRGD, 1.0 mg) was immobilized onto Ag2S-DOX using coupling agents (EDC and NHS). EDC (5.0 mg) and NHS (4.6 mg) were added into 5.0 mL of the prepared Ag2S-DOX solution, and the mixture was stirred at room temperature for 4 h. cRGD (3.1 mg) was then added and stirred in the dark for 12 h at room temperature. The reaction mixture was then removed to the bag filter for a 24 h dialysis. The product (Ag2S-DOX-cRGD) was stored in the dark at 4 °C. Ag2S-cRGD was obtained using a similar method, except that DOX was not conjugated. 2.3.
Optical characterization of the probes
The absorption spectra of Ag2S QDs, Ag2S-DOX, and Ag2S-DOXcRGD were acquired using a Lambda 25 UV-Vis spectrophotometer (Perkin Elmer, US). The corresponding fluorescence spectra of the samples were measured at room temperature using an LS 55 Fluorescence Spectrometer (Perkin Elmer, US). 2.4.
Cell studies
2.4.1. Cell culture. The human cell lines U87 (malignant glioma), MCF-7 (breast cancer) and MDA-MB-231 (breast cancer) were obtained from American Type Culture Collection (ATCC, USA). The cell lines were cultured in DMEM medium supplemented with 10% (v/v) calf serum, penicillin (100 U mL−1), and streptomycin (100 mg mL−1). The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. 2.4.2. Cytotoxicity evaluation of Ag2S QDs and Ag2S-cRGD. Cytotoxicity of Ag2S QDs and Ag2S-cRGD in normal human liver cells (L02), glioma cells (U87), and human breast cancer cells (MCF-7) was determined by MTT assay. The cells were seeded in 96-well plates (1 × 104 cells per well) and subsequently incubated for 24 h. After treatment with different concentrations (0.02 μM to 12.5 μM) of Ag2S QDs or Ag2ScRGD, the cells were further maintained at 37 °C for 24 h. Each well was washed three times with PBS ( pH 7.0) and then
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20 μL of MTT solution (5.0 μg mL−1) was added. After another 4 h incubation, the medium containing MTT was carefully removed and DMSO (150 μL) was added to each well. The plates were then subjected to measure the absorbance. Cell viability was calculated using the following formula: Cell viability = (Mean absorbance of test wells − Mean absorbance of medium control wells)/(Mean absorbance of untreated wells − Mean absorbance of medium control well) × 100%. The therapeutic efficacy of Ag2S-DOX and Ag2S-DOX-cRGD was also evaluated using MTT assays following the abovementioned method. The concentration range of the samples was 0.1 μg mL−1 to 12.5 μg mL−1, and the incubation time was set at 8 h. 2.4.3. Cellular uptake of Ag2S-DOX and Ag2S-DOX-cRGD. The targeting ability of Ag2S DOX and Ag2S-DOX-cRGD was evaluated using the two tumor cells lines, U87 and MCF-7 cells. The cells were seeded in laser confocal fluorescence microscope (LCFM) culture dishes with a density of 4 × 105 cells per well and subsequently incubated at 37 °C in a humidified atmosphere containing 5% CO2. When the whole cells reached ∼70%–80% confluency, 100 μL of Ag2S-DOX and Ag2S-DOX-cRGD were added into different dishes, and the dishes were then incubated for 1 h, 2 h or 4 h, followed by incubation with Hoechst 33342 solutions (100 μL; 10 μg mL−1) for 30 min. The information was used to determine the intracellular kinetics of Ag2S-DOX and Ag2S-DOX-cRGD. Subsequently, the cells were washed three times with PBS before examining the probe’s affinity for cancer cells by LCFM (FV1000, Olympus, Japan). The red fluorescence of DOX was captured under 488 nm light excitation. Intracellular uptake of the different probes was determined from the red fluorescence intensity in the region of interest (ROI) using Scion Image software. 2.5.
Animal experiments
2.5.1. Animal subjects and tumor model. Athymic nude mice and normal (Kunming) mice were purchased from Charles River Laboratories (Shanghai, China) for in vivo imaging. All the animal experiments were carried out in compliance with the Animal Management Rules of the Ministry of Health of the People’s Republic of China (Document no. 55, 2001) and the guidelines for the Care and Use of Laboratory Animals of China Pharmaceutical University. Tumor models were established by subcutaneously injecting tumor cells (5 × 106 in 50 mL of PBS) into the axillary fossa of the mice. 2.5.2. Dynamic distribution of Ag2S-cRGD and Ag2S-DOXcRGD in tumor-bearing mice. To investigate the targeting ability of the probes, two types of tumor models were established by the subcutaneous injection of MDA-MB-231 (high αvβ3 receptor expression) tumor cells (∼5 × 106 in 80 μL of PBS) into the axillary fossa of female athymic nude mice (n = 5 for each model). As the tumors grew to a diameter of up to 0.1 cm and 0.6 cm, the models of early-stage and late-stage MDA-MB-231 tumor-bearing mice were established successfully. Ag2S-cRGD (0.2 mL, 10 mg kg−1) and Ag2S-DOX-cRGD (0.2 mL, 10 mg kg−1) were then injected into early-stage and
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late-stage MDA-MB-231 tumor-bearing mice through the vena caudalis. The nude mice were then immobilized on a lucite jig. An NIR imaging system equipped with a 765 nm laser as excitation source was employed to trace the distribution of the probe. The background image of the mice was taken prior to the injection. A series of images were collected at 5 min, 1 h, 2 h, 4 h, 12 h, 24 h and 48 h post-injection. To further compare the targeting ability of the probe for early-stage and late-stage MDA-MB-231 tumors, fluorescence intensity from tumor and major organs (heart, liver, bladder and intestine) at different time points (10 min, 3 h and 8 h) was determined using the region of interest (ROI) functions of Scion Image software. The tumor to normal tissue contrast ratios (T/N) at different time points were also calculated. In each case, the background fluorescence that was measured before the injection of the conjugates was subtracted from the post-injection fluorescence. To further investigate the distribution of Ag2S-DOX-cRGD in tumors, a tumor was removed from the mice body at 5 h postinjection of the probe. The tissues were cut into slices (8 mm thickness) by a freezing microtome (CM1850, Leica). The peripheral slices and the central slices of the tumor were collected and observed by laser confocal fluorescence microscope immediately. Red fluorescence (590 nm) emitted from DOX was collected. 2.5.3. Therapeutic efficacy evaluation of Ag2S-DOX-cRGD. The Ehrlich ascites carcinoma (EAC) tumor model for therapy evaluation was established by subcutaneously inoculating EAC cells (∼8 × 106) into the nether axillary fossa of the mice (n = 32). The mice were investigated after 7 days of inoculations. When the tumor was completely developed, the mice were randomly divided into four groups (n = 8 for each group). Saline (the control group, Group 1), DOX solution (Group 2), Ag2S-DOX (Groups 3) and Ag2S-DOX-cRGD (Group 4) were intraperitoneally injected into the mice. Subsequently, each mouse underwent intraperitoneal injection once every other day for a total of four times. The survival rate of the treated mice was calculated according to the equation: Survival rate = Ns/Nt × 100%, where Ns and Nt represent the number of surviving mice and the number of total mice in each group, respectively. 2.5.4. Ex vivo histopathology. To further investigate the therapeutic effects of Ag2S-DOX-cRGD on EAC tumor-bearing mice, the tumors were excised for histopathologic analysis on the 16th day post-injection. The tissue was fixed with 10% neutral buffered formalin and embedded in paraffin. The sliced tumors (8 mm) were stained with hematoxylin and eosin (H&E) and observed by a BX41 bright field microscopy (Olympus).
3. Results 3.1.
Characterization of Ag2S-DOX-cRGD
In this study, MPA-coated Ag2S NPs were covalently conjugated with DOX, a type of widely used antitumor drug, to realize the
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Fig. 1
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Synthesis routine of the nano-conjugate (Ag2S-DOX-cRGD).
Fig. 2 (a) Absorption spectra and (b) fluorescence spectra of Ag2S QDs, Ag2S-DOX and Ag2S-DOX-cRGD; (c) white light appearance and the fluorescence image of Ag2S-DOX-cRGD aqueous solution.
combination of real time imaging and chemotherapy. CRGD was also covalently immobilized on the surface of MPA-coated Ag2S NPs to enhance their targeting capability to integrin αvβ3 over-expressing tumors. The successful synthesis of Ag2S-DOXcRGD was confirmed by real-color/fluorescence images, absorption spectra and fluorescence spectra. Ag2S NPs, Ag2S-DOX and Ag2S-DOX-cRGD were synthesized following the procedures described in the method section, and the specific synthesis routine is shown in Fig. 1. MPA coated Ag2S QDs were prepared in water by the slow addition of sulfide to a mixture of MPA and silver salt. The color of the solution turned yellow with the addition of sulfide ion, and the resulting colloidal QDs solution had a dark brown color. The favorable optical properties of Ag2S QDs were exhibited in the fluorescence spectrum (Fig. 2B), given that their photoluminescence emission peak at 800 nm was distinctly detected, while the quantum yield of synthesized Ag2S QDs was 37.9%. Furthermore, the emission peak centered at
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800 nm has an impressive full width at half-maximum of 65 nm, indicating a narrow size distribution of the Ag2S QDs (Fig. 2B). The conjugation of Ag2S was also monitored by UV-Vis absorption spectra and fluorescence spectra (Fig. 2). The conjugates, Ag2S-DOX and Ag2S-DOX-cRGD, still exhibited the characteristic absorption peaks of DOX (490 nm) (Fig. 2A). The corresponding fluorescence spectra are shown in Fig. 2B. In addition, Fig. 2B suggests that the fluorescence emitted by Ag2S at 800 nm was not influenced by DOX, enabling its application in NIR fluorescence imaging. 3.2.
Cytotoxicity of Ag2S QDs and Ag2S-cRGD
Ideal contrast agents and drug delivery vehicles should be safe and non-toxic to cells and tissues. It is crucial to conduct a toxicity study for the potential applications of Ag2S QDs and their conjugates. Herein, the cytotoxicity of Ag2S QDs and Ag2S-cRGD was first investigated by MTT assay to evaluate
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Fig. 3 Cell viability of different cell lines (L02, U87, MCF-7) after incubation with Ag2S and Ag2S-cRGD respectively within a wide concentration range (0.2–125.0 μg mL−1).
their biocompatibility. The MTT assay relies on the mitochondrial activity of cells and represents a viable parameter for reporting metabolic activity. In our study, the MTT assay was applied to L02, U87 and MCF-7 cell lines for both Ag2S QDs and Ag2S-cRGD. The results were collected after 12 h and 24 h incubation and are demonstrated in Fig. 3. Under all circumstances, neither Ag2S QDs nor Ag2S-cRGD showed any significant cytotoxicity. The two particles displayed low toxicity to three cell lines even at high doses (125 μg mL−1) with a high cell viability after 24 h incubation (L02: >83%; U87: >81%; MCF-7: >80%). In addition, no obvious difference could be observed between the results of 12 h and 24 h for the two probes, which indicated no serious toxic effect in the long term. The observed ultra-low cytotoxicity in MTT assay could be attributed to the low-toxic metallic constituents of Ag2S QDs as well as their ultralow solubility constant, which determines the ultra-slow release of metallic constitu-
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ents to biological surroundings. Thus, these results suggest that Ag2S QDs are promisingly safe nanoparticles for bio-applications. 3.3. Cellular uptake of Ag2S-DOX and Ag2S-DOX-cRGD in U87 and MCF-7 cells Taking the advantage of red fluorescence emitted by DOX itself, the cellular uptake of Ag2S-DOX and Ag2S-DOX-cRGD was easily visualized. After Ag2S-DOX and Ag2S-DOX-cRGD were incubated with U87 tumor cells at 37 °C, the uptake process was examined by LCFM at 1 h, 2 h and 4 h after incubation (Fig. 4A). With regard to the images of Ag2S-DOX, only a weak red fluorescence could be observed, indicating that only little Ag2S-DOX entered U87 tumor cells. A similar result was also obtained from MCF-7 cell lines, in which the expression of αvβ3 receptors (Fig. 4C) is known to be negative. In contrast, Ag2S-DOX-cRGD in U87 cells, in which the expression of αvβ3
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on the surface of Ag2S can facilitate their cellular uptake by the cells with high expression of αvβ3 receptors. In other words, the affinity of Ag2S NPs to integrin αvβ3 over-expressed tumor cells has been strengthened by cRGD, which is one of the primary aims of this study.
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3.4. Dynamic distribution of Ag2S-cRGD and Ag2S-DOX-cRGD in tumor-bearing mouse models
Fig. 4 (a) Laser confocal fluorescence images of U87 cells after incubation with the probes (Ag2S-DOX and Ag2S-DOX-cRGD) for different time (1 h, 2 h and 4 h); (b) Semi-quantitative analysis of the fluorescence intensity of U87 cells after incubation with the probes (Ag2S-DOX and Ag2S-DOX-cRGD) for different times (1 h, 2 h and 4 h); (c) Laser confocal fluorescence images of MCF-7 cells after incubation with the probes (Ag2S-DOX and Ag2S-DOX-cRGD) for 4 h.
receptors is high, showed strong fluorescence after 2 h incubation. The fluorescence signal detected from the U87 cells could obviously eclipse the one detected from the U87 cells treated with Ag2S-DOX, illustrating that the presence of cRGD
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To better understand its pharmacokinetic behavior, Ag2S-cRGD was injected into early-stage and late-stage MDA-MB-231 tumor-bearing mice through the vena caudalis. A series of in vivo images obtained by NIR imaging system at different time intervals is shown in Fig. 5A, B to illustrate the dynamic behavior of Ag2S-cRGD. The bright fluorescent signal revealed that Ag2S-cRGD initially spread quickly throughout the whole bodies in both the two models, then concentrated in the liver, bladder and tumor site, and finally cleared via the hepatobiliary and renal pathways. In an intuitive manner, the fluorescence signal in both MDA-MB-231 tumor models became distinguishable from the normal tissues at 1 h postinjection, and reached peak intensity at 8 h post-injection. The fluorescence of probes from the tumor site and the liver of late-stage tumor-bearing mice is considerably stronger than that of the early-stage tumor-bearing mice at the same time point, indicating a different status of tumor because the capacity of a well-developed tumor to retain Ag2S-cRGD is higher than that of the tumor in nascent state, and thus the fluorescence intensity exhibited by late-stage tumor would be relatively stronger. To semi-quantitatively analyze the targeting ability of Ag2ScRGD in vivo, the fluorescence intensity was gauged by selecting specific regions of interest (ROIs) from the tumors and normal tissues at different time intervals. In each case, the background fluorescence measured before the injection of Ag2S-cRGD was subtracted. The fluorescence intensities in the ROIs from tumor and normal tissues were averaged and then plotted against time (Fig. 5C). For both the two mouse models, the fluorescence intensity ratio between tumor and normal tissue (T/N) reached a peak at 8 h post-injection of Ag2S-cRGD, whereas the tumor/normal ratio of the late-stage tumor remained a slightly higher than that of the early-stage tumor at all time intervals; these observations are consistent with the conclusion from the observation of fluorescence mapping as described above. Meanwhile, the semi-quantitative fluorescence intensity of the tumor and isolated organs (heart, liver, bladder and intestine) from two Ag2S-cRGD-treated mouse models were compared at different post-injection time points (10 min, 3 h and 8 h, Fig. 5D, E and F). Obviously, the conjugate was prone to arrive at the bladder through the kidney-bladder pathway in the early tumor-bearing mouse. Meanwhile, the conjugate displayed a long time accumulation in liver in the late tumorbearing mouse. Both of the two tumor-bearing mice demonstrated high accumulation of the nanoconjugates in tumors. The distribution of the nano-conjugates (Ag2S-cRGD-DOX) in the MDA-MB-231 tumor-bearing mice and the distribution
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Fig. 5 In vivo fluorescence imaging of Ag2S-cRGD in different MDA-MB-231 tumor-bearing mice including (a) the early stage and (b) the late stage; (c) Relative fluorescence intensity ratios of tumor to normal tissue for Ag2S-cRGD injected MDA-MB-231 tumor-bearing mice; Fluorescence intensity from tumor and major organs (heart, liver, bladder and intestine) was assessed by Scion Image software after selecting ROI and compared at (d) 10 min, (e) 3 h and (f ) 8 h post-injection of the probe.
of the conjugates in tumor tissue were investigated. As shown in Fig. 6A, Ag2S-cRGD-DOX initially distributed in the entire body after being injected into the tumor-bearing mouse via the tail vein. The nano-conjugates arrived at tumor 1 h post-injection, and the bright fluorescent signal that appeared at the tumor lasted for 24 h. Fig. 6B showed a noticeable strong red fluorescence that could be observed in both the peripheral slice and the central slice of the tumor, suggesting the high accumulation of Ag2S-cRGD-DOX in malignant tissues. The semi-quantitative analysis of the biodistribution of the probe is displayed in Fig. 6C. The highly specific accumulation of the probe in the MDA-MB-231 tumor was clearly observed compared with that in other tissues (heart, liver, bladder and intestine), revealing its desirable affinity to tumor sites. Moreover, the high fluorescence intensity of liver and kidney could be attributed to their metabolic roles.
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3.5. Anti-tumor activity evaluation of Ag2S-DOX-cRGD and Ag2S-DOX in cell level The anti-tumor activity of Ag2S-DOX-cRGD and Ag2S-DOX was evaluated through MTT assay, which was performed on U87, MDA-MB-231 and MCF-7 cell lines in this section. A dosedependent reduction in MTT absorbance for all the three cell lines treated with Ag2S-DOX-cRGD and Ag2S-DOX is demonstrated in Fig. 7. In the cases of integrin αvβ3 over-expressing tumor cells, including U87, MDA-MB-231, cells treated with Ag2S-DOX-cRGD exhibited lower cell viability than those treated with Ag2S-DOX, as the maximal inhibition ratio of Ag2S-DOX-cRGD is ∼70%, which is significantly increased from ∼50%, the maximal inhibition ratio of Ag2S-DOX. Therefore, the mediation of cRGD helps in potentiating the therapeutic effect of DOX in pertinent tumor cells.
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3.6. Therapeutic efficacy evaluation of Ag2S-DOX-cRGD in tumor-bearing mice
Fig. 6 (a) In vivo fluorescence imaging of Ag2S-DOX-cRGD in MDA-MB-231 tumor-bearing mice; (b) Strong red fluorescence could be observed in peripheral and central slices of the tumor under LSCM, indicating the high uptake of Ag2S-DOX-cRGD by tumor at 5 h post-injection; (c) Fluorescence intensity from tumor and major organs (heart, liver, bladder and intestine) was assessed by Scion Image software after selecting ROI and compared at 10 min, 3 h and 8 h post-injection of the probe.
The images of mice in the four groups that underwent intraperitoneal injection of saline, DOX, Ag2S-DOX, and Ag2ScRGD-DOX once every other day for four times of total treatment are collected in Fig. 8A. Expectedly, the most conspicuous growth of tumor was observed in the saline group, and even visible necrosis, which is a symptom of a late-stage tumor, appeared among this group after the fourth treatment. By contrast, the tumor grew at a comparatively low speed in the DOX and Ag2S-DOX groups, whereas the most remarkable suppression of tumor deterioration was observed in the Ag2S-DOX-cRGD group. The survival rate of mice from all the four groups during 16 days was also calculated and is shown in Fig. 8B. The survival rate of the saline group started to decline early on the 5th day and only half of the mice were alive on the 16th day. The death of mice, although observed in both the DOX and Ag2S-DOX groups, occurred much later, and over 70% of the mice survived this experiment. Desirably, the Ag2S-DOX-cRGD group manifested a satisfying survival rate of 100%. After treatment, center and peripheral tumor tissues were excised from all the four groups and histological analysis was performed 18 days post-injection (Fig. 9). Only a small number of apoptotic and necrotic tumor cells in the center of tumors from the saline group could be observed due to the pathological process. Distinctively, both the periphery and the center of tumors from the DOX, Ag2S-DOX group and Ag2S-DOX-cRGD group displayed large-area apoptosis and necrosis, whereas the most common cell damage could be observed in Ag2S-DOX-cRGD group, confirming the excellent anti-tumor efficacy of Ag2S-DOX-cRGD in vivo.
Fig. 7 Anti-tumor efficacy of Ag2S-DOX and Ag2S-DOX-cRGD was evaluated on different cell lines (U87, MDA-MB-231 and MCF-7). The concentration range of the samples was 0.1 μg mL−1 to 12.5 μg mL−1 and the incubation time was set as 8 h.
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Fig. 8 (a) Photographs of EAC tumor-bearing mice in the four groups (saline, DOX, Ag2S-DOX, and Ag2S-cRGD-DOX) on different days of treatment (4 d, 8 d, 12 d and 12 d). (b) Survival rate of mice for four groups (saline, DOX, Ag2S-DOX, and Ag2S-cRGD-DOX) within 16 days of treatment.
4.
Fig. 9 Histological analysis of tumor sections excised from the center and peripheral tumor tissues of the mice 16 days after intravenous injection of the different samples (saline, DOX, Ag2S-DOX, and Ag2ScRGD-DOX).
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Discussion
Ever since the concept of “theranostics”, which suggests a combination of diagnostics and therapy, was proposed in 2002,30 nanomedicine platforms that integrate imaging and therapeutic function have attracted considerable attention as the next generation of medicine. Diverse nanoparticles with an imaging function, including magnetic nanoparticles, gold nanoparticles and QDs, have been used as drug carriers that can map the behavior of drug at both subcellular and systemic levels.31,32 Among these nano-contrast agents, QDs—nanometer-size luminescent semiconductor nanocrystals—are particularly appealing as potential molecular imaging probes and small-molecule nano-carriers because of their unique properties such as high brightness and long-term stability.33 With intense study on QDs as well as their wide application in bioimaging, the investigation of QD/drug nanoparticle formulations has begun in recent years due to the advancement of QD synthesis and bioconjugation chemistry for immobilizing drug molecules onto the QD surface.34 Taking the advantage of real-time and quantitative imaging analysis enabled by QDs, the kinetics of drug in vivo as well as the information of targeted lesion sites could be obtained. Expectedly, several successful attempts that employed conventional QDs as drug carriers have been reported.35 For instance, the nanoconjugates of CdSe/CdS/ZnS QDs and DOX were developed by Chakravarthy et al. to target alveolar macrophages for pulmonary treatment.36 A site-specific QD-based platform incorporated with antiretroviral drug saquinavir and the biorecognition
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molecule transferrin has also been established by Mahajan et al. for the treatment of neuro-AIDS and other neurological disorders.37 However, the further steps of these studies toward clinical use are still stymied by the inherent toxicity of QDs and their unclear biological degradation mechanisms in vivo. The appearance of Ag2S QDs with near-infrared fluorescence holds the potential to extricate the development of QDs from such a bottleneck constraint. Exciting progress in theranostic application based on nanostructures, such as gold nanoparticles and up-conversion nanoparticles, have been achieved in recent years38,39 but more effort is still required in the research of Ag2S QDs. Inspired by some previously successful cases, a variety of agents, such as IR825, ZnPc and Nitric Oxide, could be employed to broaden the application of Ag2S QDs. The objective of the present study is to establish multi-functional nanomedicine based on Ag2S QDs for the combination of NIR imaging and chemotherapy. Specifically, Ag2S QDs capitalize on the tumor affinity of cRGD and anti-tumor efficacy of doxorubicin, while achieving a theranostic design to allow the visualization of therapeutic efficacy by taking advantage of noninvasive NIR imaging function of QD self. In addition, certain effort has been devoted for employing Ag2S QDs for in vivo application. However, the active targeting ability of Ag2S QDs is still a problem because this problem has either not been taken into account or this problem has not been solved in an economic manner.24 Herein, cRGD was adopted as a targeting ligand and was shown to improve the cellular uptake of chemical drug (Fig. 4) as well as the in vivo NIR imaging capability of Ag2S QDs (Fig. 5). In this study, this strategy was indicated to be feasible for integrin αvβ3 over-expressing tumor cells, including U87, MDA-MB-231. Furthermore, it is not difficult to expect that a similar strategy will also be effective for other tumors, such as high folate receptor (FR)-expressing tumors, by simply replacing the targeting ligand correspondingly. Subsequent to the conjugation of Ag2S QDs, which have excellent NIR optical characteristics29 and biocompatibility40 with a tumor-targeting ligand, cRGD, and the common chemotherapeutic drug, doxorubicin, the unintrusive optical characteristics of Ag2S QDs were demonstrated by the adsorption and fluorescence spectra, eliminating the concern that the thickness of surface coating on QDs might considerably change their optical and thus negatively influence their imaging function (Fig. 2). In addition, cytotoxicity is another important factor that should be taken into consideration for the biological application of QDs. In a typical case, conventional cadmium-based QDs, which are commonly used for in vitro and in vivo studies, are known to cause acute and chronic toxicities in vertebrates at high dosage as a result of their main constituent elements that include cadmium and selenium.41 Favorably, the MTT assay in this study revealed only partial cytotoxicity of Ag2S QDs and its conjugates (Fig. 3), indicating a significant potential of Ag2S QDs for safe exploitation in further biomedical application. As observed from our result (Fig. 5), the conjugate-cancer accumulation was related to the tumor size. As tumor size
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increased, both the accumulation time of the probe in the tumor and in the entire body became extended. As Gu et al.5 have reported, the nano-conjugate (Au-Met-MPA)-based Au nano-clusters demonstrated peak intensity at 10 h post-injection and maintained their detectable fluorescence for 96 h in an MDA-MB-231 tumor-bearing (late stage) mouse. However, the bright fluorescence signal of Au-Met-MPA reached the maximum at 7 h and then gradually disappeared after 72 h in an MDA-MB-231a tumor-bearing (early stage) mouse. Thus, the biodistribution of Ag2S-cRGD in different stages of tumorbearing mice was consistent with Gu’s report. More importantly, by tracking the behavior of Ag2S-DOX-cRGDs and investigating their therapeutic effect at both the cell (Fig. 7), and animal level (Fig. 8 and 9), Ag2S-DOX-cRGDs were demonstrated to work better than DOX, a widely used chemotherapeutic drug in clinics, as well as Ag2S-DOX, while maintaining the NIR imaging function of Ag2S QDs. According to the principles of tumor-specific personalized medicine, before initiating the cancer treatment, molecular profiling of tumor heterogeneity by imaging is indispensable to verify the cancer biomarkers in the intra-and inter-tumor tissue, which could provide essential information regarding target-specific therapy.42,43 After implementing targeted therapy, the mission of eradicating all cancer cells is still not complete because the tumor may inevitably evolve in response to therapy. Thus, the molecular imaging of the tumor that could be quickly repeated is necessary for direct intelligent adjustment of the targeting strategy for cancer therapy. Consistent with the concept referred to above, combining the diagnostic capability of Ag2S QDs with therapeutic intervention in our study is beneficial for addressing the challenges of cancer heterogeneity and adaptive resistance.
5. Conclusion In summary, the nano-conjugates (Ag2S-DOX-cRGD) based on the as-prepared Ag2S QDs, integrating imaging and therapeutic function, were constructed successfully through a facile synthesis routine. The favorable tumor-targeting efficiency of an inexpensive active tumor-targeting peptide (cRGD) decorated Ag2S QDs were verified both in integrin αvβ3 over-expressing tumor cells and tumor-bearing mice models, including U87 and MDA-MB-231. Furthermore, the effective tumor inhibition of the nanoconjugates was also demonstrated at both the cell and animal level. These results vindicated that Ag2S QDs are promising candidates for designing imaging-visible nanotherapeutics for tumor diagnosis and therapy application.
Acknowledgements The authors are grateful to Natural Science Foundation Committee of China (NSFC 81371684, 61335007, 81220108012, 81171395 and 81328012), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University
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(no. SKLNMZZYQ201403) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions for their financial support.
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