Beyond a Carrier: Graphene Quantum Dots as a Probe for

Aug 7, 2017 - Traditional strategy for evaluating the therapeutic response is to monitor the change of tumor size using ultrasonography and magnetic r...
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Beyond a Carrier: Graphene Quantum Dots as a Probe for Programmatically Monitoring Anti-Cancer Drug Delivery, Release, and Response Hui Ding,† Fan Zhang,§ Chaochao Zhao,‡ Yanlin Lv,† Guanghui Ma,*,‡ Wei Wei,*,‡ and Zhiyuan Tian*,† †

School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, P. R. China State Key Laboratory of Biochemical Engineering, Institute of ProcessEngineering, Chinese Academy of Sciences, Beijing 100190, P. R. China § Life Science and Technology Institute, Hebei Normal University of Science and Technology, Qinhuangdao 066004, P. R. China ‡

S Supporting Information *

ABSTRACT: On the basis of the unique physicochemical properties of graphene quantum dots (GQDs), we developed a novel type of theranostic agent by loading anticancer drug doxorubicin (DOX) to GQD’s surface and conjugating Cy5.5 (Cy) dye to GQD though a cathepsin D-responsive (P) peptide. Such type of agents demonstrated superior therapeutic performance both in vitro and in vivo because of the improved tissue penetration and cellular uptake. More importantly, they are capable of functioning as probes for programmed tracking the delivery and release of anticancer drug as well as druginduced cancer cell apoptosis through GQD’s, DOX’s, and Cy’s charateristic fluorescence, respectively. KEYWORDS: graphene quantum dots, cathepsin D-responsive, fluorescence quenching, theranostic

C

Among various candidates for nanocarrier of chemotherapeutic agents, GQDs have emerged as the subject of considerable research owing to their salient features, namely single atomic layer with very small lateral size and oxygen-rich functional groups at the edges, which render them ideal for loading hydrophobic anticancer drugs and good stability in vivo. Another salient property of GQDs is their fluorescence originating from quantum confinement and edge effects, which make GQDs an ideal platform for the traceable delivery of chemotherapeutic agents into cancer cells without the involvement of additional fluorescence labeling.8−12 However, GQDs typically emit blue fluorescence with peak in the region of 450−460 nm, which is more vulnearable to light scattering and cannot penetrate deep issue. Thus, GQDs with shortwavelength fluorescence emission is not suitable for imaging the deep-seated target in the in vivo experiments.13−15 Strategy based on additional conjugation with near-infrared (NIR) dye generally suffers from GQDs-mediated fluorescence quenching because GQDs may function as an efficient quencher. Such a dilemma significantly delays the process of developing GQDs-

ancer, one of the leading causes of death among mankind, has become a vital public health risk worldwide. The primary strategies for cancer treatment and cure include surgery, radiation, and chemotherapy.1,2 For cancer that has spread or metastasized, chemotherapy generally acts as the mainstay treatment in clinic. However, the effectiveness of chemotherapy is often limited by its side effects, such as hematotoxicity and cardiotoxicity, because the chemotherapeutic agents not only kill cancer cells that uncontrollably grow and divide but also induce damage to healthy cells and tissues in the body.3,4 Nanotechnology is emerging as a promising tool for reducing the aforementioned side effects and improving the therapeutic effectiveness in cancer chemotherapy. Specifically, via the enhanced permeability and retention (EPR) effect, the administrated nanosized drug carriers are capable of accumulating in tumors through the leaky blood vessels and an impaired lymphatic drainage in tumor tissues, which is expected to minimize the chemical-induced damage to the healthy cells. Additionally, nanocarriers are typically characterized with ease of functionalization for further incorporation of with fluorescent markers for simultaneous diagnosis and imageguide therapy. Such a theranostic approach is critical in addressing the challenges of cancer heterogeneity and adaptation.5−7 © 2017 American Chemical Society

Received: June 19, 2017 Accepted: August 7, 2017 Published: August 7, 2017 27396

DOI: 10.1021/acsami.7b08824 ACS Appl. Mater. Interfaces 2017, 9, 27396−27401

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ACS Applied Materials & Interfaces

Figure 1. Strategy of GQD-based theranostic agent for programmatically monitoring anticancer drug delivery, release, and response.

Figure 2. In vitro evaluation on 2D cell culture system. (a) Cytotoxicity on 4T1 cells after treatment with different formulations. (b) CLSM images of 4T1 cells incubation with GQD-P-Cyand DOX@GQD-P-Cy. Treatments with additionalcathepsin D inhibitor were used as a control.Scale bar: 10 μm. (c) Linear regression analysis of correlation between cell viability and fluorescence intensity.

based theranostic, which makes the personalized treatment impossible.10,16,17 Every coin has two sides. With rational design, the aforementioned GQDs-mediated fluorescence quenching may be well utilized and dramatically function as the cornerstone of a new type of GQDs-based theranostic platform possessing

salient advantages. Herein, we report a GQD-based theranostic platform capable of functioning as an anticancer drug carrier and a signaler for indicating drug delivery, release, and response by providing distinct fluorescence signals at different stages.4,18−20 As illustrated in Figure 1, the NIR fluorescent molecule Cy was covalently attached to GQD via a cathepsin D 27397

DOI: 10.1021/acsami.7b08824 ACS Appl. Mater. Interfaces 2017, 9, 27396−27401

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Figure 3. In vitro evaluation on 3D cell culture system. (a) CLSM images of the multicellular tumor spheroids incubation with GQD-P-Cy and DOX@GQD-P-Cy. Scale bar: 100 μm. (b) Inhibitory effect on the growth of 4T1 spheroid of different formulations. (c) Images of the multicellular 3D tumor spheroids. Scale bar: 100 μm.

responsive peptide (Phe-Ala- Ala-Phe-Phe-Val-Leu-Cys, FAAFFVLC, P) and the functionalized GQD was then loaded with anticancer drug doxorubicin (DOX) via π−π interaction mechanism. In such a system, both DOX and Cy components are within the Förster proximity of GQD and their fluorescence are significantly quenched. As nanocarrier, GQD with blue fluorescence ferry the DOX and P-Cy into tumor tissue via EPR effect.21−24 After internalization and the subsequent release of DOX from GQD substrate, green fluorescence of the free DOX outside the Förster proximity of GQD is expected inside tumor cells. Additionally, DOX is an effective chemotherapeutic agent to induce apoptosis of cancer cells that are typically charaterized with cathepsin D overexpression. Thus, DOXinduced apoptosis of cancer cells is expected to promote the release of cathepsin D capable of cutting off the peptide spacer between Cy molecules and GQD. As a result, the NIR fluorescence of the released Cy molecule outside the Förster proximity of GQD is restored, which is in turn a sign of the cancer cell death. The feasibility was symmetrically tested on simulated medium in vitro, 2D/3D cell culture system and tumor bearing mouse model, which together supported our DOX@GQD-P-Cy as a versatile theranostic agent for personalized anticancer treatment.10,11,13,25 GQDs were synthesized via a facile chemical oxidation strategy and exfoliation technique using polyacrylonitrile carbon fibers as the raw material. The final product (molecular weight 3000−10000) was filtered using ultrafiltration centrifuge tube and frozen to dry. Transmission electron microscopy (TEM) characterization showed that the obtained GQDs were typically ∼4 nm in size with a relatively narrow polydispersity (Figure S1a). The morphology of GQDs was also characterized

by atomic force microscopy (AFM), which indicated the average height of approximately 2 nm (Figure S1b). Through a linker N-(2-Aminoethyl) maleimide (NAEM) (Figure S2), PCy was successfully conjugated on GQD, which could be verified by increased hydrodynamic size (from 8.5 to 15.6 nm, Figure S1c), increased zeta potential (Table S1), X-ray photoelectron spectroscopy (XPS) analysis (Figure S4), spectroscopic characterization (Figure S5), and decreased carboxyl group (from 3.5 mmolg−1 to 0.8 mmol g−1, Figure S6). Using the supramolecular π−π stacking forces, we then succeed in loading DOX onto GQD-P-Cy with a loading effciency of ∼82.5%, which was determined via HPLC with UV detection at 254 nm.26,27 To evaluate the feasibility of DOX@GQD-P-Cy as a versatile probe, we measured its fluorescence features in vitro. DOX@ GQD-P-Cy exhibited a stable blue fluorescence (λmax = 460 nm) sourced from the GQD component (Figure S1d). Over time, the fluorescence signal of DOX (λmax = 565 nm) gradually increased because of the attenuation of fluorescence quenching when DOX was released from the DOX@GQD-P-Cy drug delivery systems and therefore outside the Förster proximity of GQD. For the evolution of Cy fluorescence, addition of cathepsin D enzyme quickly triggered the cleavage of P peptide sequence and enabled the detachment of Cy dye from the GQD quencher, which consequently relit the fluorescence of Cy dye (λmax = 690 nm) that was quenched when it was within the Förster proximity of GQD. The fluorescence intensity dynamically increased with increasing cathepsin D concentration in the range from 0.2 nM to 0.8 nM (Figure S1e). The recovery of Cy fluorescence could be significantly suppressed in the presence of cathepsin D inhibitor, again confirming the 27398

DOI: 10.1021/acsami.7b08824 ACS Appl. Mater. Interfaces 2017, 9, 27396−27401

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Figure 4. (a) Tumor volumes of mice after treatment. (b) Survival percentage of mice in different treatment groups. (c) TUNEL staining of tumor tissue after treatment with different agents. Scale bar: 50 μm. (d) In vivo fluorescence images of 4T1 tumor-bearing after intravenous injection of DOX@GQD-P-Cy. (e) Linear regression analysis of correlation between relative tumor growth rate and fluorescence intensity.

cutoff function of cathepsin D to the peptide linker (Figure S1f). As cathepsin D production highly correlates to the degree of apoptosis, such a behavior pave the way for the evaluating the degree of apoptosis through the generated Cy fluorescence. We gauged the therapeutic efficacy of DOX@GQD-P-Cy by measuring cell viability with CCK-8 kit (Figure 2a). GQD-P-Cy alone showed almost no cytotoxicity to 4T1 breast cancer cells, indicating its good biocompatibility. Once DOX was added in the culture medium, the cell viability gradually decreased with the increased DOX dose. This cytotoxicity was further enhanced in DOX@GQD-P-Cy group. Taking 2.5 μg/mL dose as an example, cell viability was only 65% after DOX treatment, while the value could be reduced to 30% by DOX@ GQD-P-Cy. Such a good therapeutic efficacy could be attributed to the increased cellular uptake served by GQD carrier which was verified by flow cytometry analysis (Figure S7). To evaluate the capacity as a versatile probe, we acquired the fluorescence signals of treated 4T1 cells by confocal laser scanning microscopy (CLSM) at different channels. As shown in Figure 2b, the GQD-P-Cy exhibited blue fluorescence inside cells, suggesting a favorable internalization. The undetectable fluorescence signal of Cy indicated the alive state of GQD-P-Cy treated cells, again demonstrating its good biocompatibility. In DOX@GQD-P-Cy group, we could clearly observe another two types of intracellular fluorescence. On one hand, DOX began to show its fluorescence signal around 565 nm (displayed as green), indicating it had been released from GQD. On the other hand, these free cytotoxic molecules induced cells to

produce cathepsin D enzyme, which triggered the cleavage of P peptide sequence and enabled the detachment of Cy dye from the GQD quencher. Thus, the fluorescence signal of Cy dye around 690 nm (displayed as red) was observed. In contrast, once cathepsin D inhibitor was added, P peptide bridge survived because of the inhibited activity of cathepsin D enzyme. As a result, the Cy dye remained within the Förster proximity of GQD and kept its fluorescence quenched stated, further supporting the specific reaction of P peptide to the cathepsin D enzyme. More importantly, a good correlation between cell viability and Cy fluorescence intensity was confirmed. When the cellular activity reduced, fluorescence intensity became stronger. On the basis of a linear regression analysis as illustrated in Figure 2c, a mathematical model V = −0.82708F + 66.33658 (correlation coefficient R2 = 0.90608) was eventually obtained, where F represents the fluorescence intensity and V is the cell viability. Thus, this mathematical model unequivocally provided us a new option to quantitatively evaluate the therapeutic efficacy by determine the Cy fluorescence. Taking that 2D cell culture model failed to reflect histological characteristics of the tumor, we next prepared 3D multicellular tumor spheroid (MCTS) model by liquid-overlay culture and continued our evaluations (Figure 3).28,29 Through the blue fluorescence sourced from GQD component, we could clearly observe that GQD-P-Cy had penetrated into the MCTS core area, again confirming the GQD as an idea carrier for anticancer drugs. Similar to the 2D data, the fluorescence signals of DOX 27399

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to the improved tissue penetration and cellular uptake they enabled. In addition to functioning as anticancer drug carriers, DOX@GQD-P-Cy could also serve as probes for programmed tracking the drug delivery, release and drug-induced cancer cell apoptosis. Specifically, the intrinsic blue fluorescence of GQD enabled tracking of the internalization process of drug carriers; the recovery of green fluorescence of DOX component provided a clue to the release of DOX from the carrier; and the change inNIR fluorescence signal of Cy triggered by cathepsin D intrinsically provided a facile and reliable option for real-time precise evaluation of cell apoptosis in chemotherapy. Such a versatile theranostic can fulfill the research and clinic need of monitoring drug delivery, release, and response, which will facilitate the establishment of personalized anticancer treatment.

and Cy could also be observed in DOX@GQD-P-Cy treated MCTS, indicating the released DOX had induced the cell apoptosis in MCTS. During a prolonged period of treatment, DOX@GQD-P-Cy clearly demonstrated its effectiveness to inhibit the MCTS growth over the effect of DOX by itself. At 28th day, the whole spheroid eventually collapsed because of the necrotized cells, demonstrating the potent therapeutic effect of DOX@GQD-P-Cy. The above results gave us a big hope and promoted us to test the feasibility of DOX@GQD-P-Cy in vivo. In comparison with the untreated and GQD-P-Cy-treated group, moderately restricted tumor growth was achieved in the group upon treatment of free DOX. Such efficacy could be significantly improved in DOX@GQD-P-Cy group due to the aforementioned improved advantages regarding cell uptake and tissue penetration. Specifically, in a quantitative comparison, the tumor volume determined 26 days after DOX@GQD-P-Cy treatment was merely about one-third of the counterpart value in the case of free DOX (Figure 4a). Additionally, DOX@ GQD-P-Cy treatment could keep most tumor-bearing mice alive at 30 days, whereas all the model mice died within 24 days in DOX group (Figure 4b). More detailed information about cell apoptosis in different groups was acquired by TUNEL staining of tumor tissue sections (Figure 4c). In agreement with the above results, the DOX@GQD-P-Cy group induced the greatest cell apoptosis, again confirming a very satisfactory therapeutic effectiveness of anticancer drug carrier we developed in the present work. We also evaluated the safety of DOX@GQD-P-Cy. Over the treatment period, the body weight was steady in the DOX@GQD-P-Cy treatment group (Figure S8). In addition, few abnormalities were found in the histological sections of heart, liver, spleen, lung, and kidney (Figure S9), which further confirmed the safety of our DOX@ GQD-P-Cy agents. Traditional strategy for evaluating the therapeutic response is to monitor the change of tumor size using ultrasonography and magnetic resonance imaging. These time-consuming methods fail to provide a timely therapeutic feedback and monitor the therapeutic response at cell level. This problem can be solved by using our DOX@GQD-P-Cy as theranostic agents. During the therapeutic period, cathepsin D enzyme generated from the apoptotic cells are capable of cutting off the P bridge and therefore enable observation of bright NIR fluorescence of Cy dye at tumor site. More interestingly, the fluorescence intensity was found highly correlated to the tumor growth rate. When the tumor growth became slow after treatment, the fluorescence intensity became stronger. Similarly, we could obtain a mathematical model G = −2.39941F+5.30171 (correlation coefficient R2 = 0.9691), where F represents the fluorescence intensity and G is the tumor growth rate (Figure 4d, e).16,30 Thus, the combination of such mathematical model and the acquisition of Cy fluorescence at tumor site, based on DOX@GQD-P-Cy agents, is able to enable real-time noninvasive evaluation of therapeutic response in chemotherapy of cancer, which is expected to provide valuable information for clinicians to tailor a most suitable pharmacotherapy for personalized treatment. In summary, we herein developed a novel type of theranostic agents, DOX@GQD-P-Cy, by loading anticancer drug DOX to GQD’s surface and attaching fluorescent Cy dye to GQD though a cathepsin D-responsive P peptide. Such DOX@GQDP-Cy agents demonstrated superior therapeutic performance in both in vitro and in vivo cancer chemotherapy experiments due

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b08824. Detailed experimental materials and synthesis strategies, additional characterizations methods and results, tumor cell imaging, construct of 3D cell culture model, therapeutic study of tumor-bearing mice, and correlation analysis (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Zhiyuan Tian: 0000-0002-1436-6818 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (21622608, 21373218, and 21573234), and Beijing Talents Fund (2015000021223ZK20).

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