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pH Switchable Nanoassembly for Imaging a Broad Range of Malignant Tumors Ye Liu,†,§ Zhuo Qu,†,§ Hongyan Cao,† Hongyan Sun,† Yuan Gao,*,† and Xingyu Jiang*,†,‡ †
CAS Center for Excellence in Nanoscience, Beijing Engineering Research Center for BioNanotechnology, and CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for NanoScience and Technology, No.11 Zhongguancun Beiyitiao, Beijing 100190, P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *
ABSTRACT: Polymer-based fluorescent nanomaterials have proven to universally image various tumors based on their extremely sharp responsiveness to pH change. Such a property has never been realized in supramolecular systems. We herein design a small molecule (DPP-thiophene-4) that is composed of a diketopyrrolopyrrole (DPP) core and two alkyl chains terminated with quaternary ammonium. DPPthiophene-4 can self-assemble into a nonfluorescent nanoassembly when the pH is >7.0 but reversibly disassembles back to fluorescent monomers when the pH is 10-fold) in lysosome number29 and a derailed endocytosis activity over normal cells.30 Therefore, tumor cells will endocytose many more DPP-thiophene-4 nanoassemblies than normal cells. As the endocytic compartments (such as endosomes/lysosomes) are acidic, tumor cells will produce significantly higher fluorescence intensity relative to normal cells (Figure 2A). In consistent with this hypothesis, DPP-thiophene-4 produces more than 50-fold stronger fluorescence in MCF-7 cells than HUVEC cells (Figure 2B). Furthermore, by using Lyso Tracker Green DND26 as an internal reference of intracellular lysosome location, we find that the red emission signal from DPP-thiophene-4 strongly overlaps with the green signal from Lyso Tracker (Pearson’s correlation value of 0.89) in MCF-7 cells (Figure 2B). In contrast, the overlap between DPP-thiophene-4 and mitochondria-specific dye (MitoTracker Green FM dye) is weaker with a Pearson’s correlation value of 0.54 (Figures S15 and S16). All of these results demonstrate that DPP-thiophene-
Figure 2. Fluorescence intensity of tumor cells (MCF-7) and normal cells (HUVEC). (A) The scheme of DPP-thiophene-4 nanoassemblies taken-up by tumor cells and normal cells. (B) For each cell line, 1 × 106 above 90% vitality cells are cultured with 10 μg/mL DPP-thiophene-4 (red), 50 nM Lyso Tracker Green DND26 (green), or 10 μg/mL Hoechst 33342 (blue) for 20 min. The fluorescence intensity of cells is detected by confocal microscopy (Zeiss) at 543 nm excitation for DPP-thiophene-4, 504 nm excitation for Lyso Tracker Green DND-26, or 352 nm excitation for Hoechst 33342; ** p < 0.01.
4 can respond to the acidity in intracellular lysosomes to produce bright fluorescence in tumor cells. To evaluate the capability of DPP-thiophene-4 on imaging malignant tumors in vivo, we choose six representative malignant tumors to confirm the broad applicability of DPPthiophene-4 on the diagnosis of cancers. Breast cancer has the highest incidence among malignant tumors in females worldwide.31 Colorectal cancer is the third most frequently diagnosed malignant tumor among females and the second among males worldwide.32 Cervical cancer has the second highest incidence among malignant tumors in females, even though human papillomavirus vaccine has a global popularization.33 The incidence of esophageal cancer, melanoma, and laryngeal carcinoma are also high worldwide.34 The incidence of these six types of malignant tumors accounts for around 50% of global new cancer cases. For females, the sum incidence of these malignant tumors is around 70% worldwide.34 It is thus reasonable to choose these six malignant tumors to evaluate the ability of DPP-thiophene-4 to broadly diagnose cancers worldwide. We evaluate the breadth of DPP-thiophene-4 on imaging malignant tumors in vivo (Figure 3A). Each mouse simultaneously receives 10 μg/mL DPP-thiophene-4 in the tumor tissue (red dotted line area in Figure 3B) and nontumor area (blue dotted cycle in Figure 3B). After 5 min, all six tumors turned brightly fluorescent, while nontumor areas remained nonfluorescent (Figure 3B). Further quantitative analysis clearly shows the fluorescence emission intensity in tumors is 18−26-fold than that of within nontumor areas (**, p < 0.01, t test). These results indicate that DPP-thiophene-4 can effectively distinguish between the malignant tumor and the nontumor area in the same mouse in vivo. Moreover, we isolate five mouse organs (heart, liver, spleen, brain, and kidney) and 12448
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Figure 3. In vivo imaging of DPP-thiophene-4 for various tumors. (A) The schematic diagram of DPP-thiophene-4 on imaging tumors in vivo. DPP-thiophene-4 is prepared in neutral aqueous solution (nanoassemblies). Such nanoassemblies will disassemble and produce fluorescence once they are injected into tumors characterized by a slight acid microenvironment. (B) Six malignant tumor models are constructed (breast cancer, colorectal cancer, cervical cancer, esophageal cancer, melanoma, and laryngeal carcinoma). PDX means patient-derived xenograft tumor. The tumors are in situ injected DPP-thiophene-4 (10 μg/mL). As a control, the same quantity DPP-thiophene-4 is injected into the legs which are nontumor tissues. There is the strong fluorescence in tumors (red dotted line area), whereas undetectable fluorescence in nontumor areas (blue dotted cycle). We measure the fluorescence intensity using a small-animal in vivo imaging system (CRI Maestro 2). (C) In all tumor mice, five types of organs (heart, liver, spleen, brain, and kidney) and tumor tissues are isolated. The fluorescence intensity of each organ or tumor tissue is quantified by the small-animal in vivo imaging system (CRI Maestro 2). All values are shown as mean ± SD (n = 6); ** p < 0.01.
the tumor tissue and compare the in vitro fluorescence intensity in these organs and the tumor tissue. The isolated tumor tissue produces bright fluorescence, which is far stronger than that in normal organs (Figure 3C). These results demonstrate that DPP-thiophene-4 selectively can image various malignant tumors, instead of normal organs. We comprehensively investigate the biosafety of DPPthiophene-4 in mouse model. The biosafety requirement is critical for further applications of our probe in the clinical trial. We investigate multiple key parameters, including the fluctuation of body weight, cytotoxicity, hemolysis, pharmacokinetics, biodistribution, histopathologic examination based on immunohistochemical staining, and multiple physiological indicators in blood,35,36 to comprehensively evaluate the biosafety of DPP-thiophene-4. Over 30 days, the mice injected by DPP-thiophene-4 with various quantities (1, 5, and 10 μg) do not show any adverse effects in body weight, which is close to that of the normal mice and mice treated with physiological saline solution (Figure 4A). DPP-thiophene-4 exhibits a satisfactory in vitro cytotoxicity on HeLa and HUVEC cells using the cell counting kit (CCK). Their cell viabilities have not significantly changed after 72 h incubation (Figure 4B). DPPthiophene-4 also has a tolerable cytolysis during a broad range of concentrations (from 0 to 50 μg/mL) via the evaluation of hemolysis against sheep red blood cells (Figure 4C). Furthermore, we use a liquid chromatography-mass spectrometry (LC-MS) system to assess the pharmacokinetics in blood
and urine and the biodistribution in different organs of DPPthiophene-4. After 3 days, the residual DPP-thiophene-4 in blood and urine is below the detection limit of LC-MS in treated mice (Figure 4D,E). DPP-thiophene-4 is also dramatically cleared from multiple organs including heart, liver, spleen, lung, kidney, stomach, intestine, and brain after 3 days, and its residual amount is undetectable after 15 days (Figure 4F). Such an in vivo stay period of DPP-thiophene-4 is reasonable and acceptable compared to some FDA-approved imaging agents such as Gadodutrol (Gd-BT-DO3A), Gadoterate (Gd-DOTA), and Ferumoxtran-10 (AMI-227).37,38 These agents possess a half-time ranging from 36 h to even 1 month. Moreover, the histopathologic analysis of all of these above-mentioned organs is performed. There is no evidence to show the infiltration and necrosis of inflammatory cells in the organs from DPP-thiophene-treated mice. Immunohistochemical staining of organs also has no visible difference between the DPP-thiophene-4 treated mice and normal mice (Figure 4G). The introduction of DPP-thiophene-4 also does not significantly change the physiological indicators in the blood of DPPthiophene-4 treated mice, in comparison with that of normal mice (Figure S17). All of these results prove a satisfactory biosafety of DPP-thiophene-4 in vivo.
CONCLUSIONS In conclusion, we develop a supramolecular fluorogenic nanoassembly which can respond sharply to the pH variance 12449
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Figure 4. Biosafety evaluation of DPP-thiophene-4 in cells and mouse model. (A) Within 30 days, the increase of body weight of normal mice, mice injected with physiological saline solution, and mice injected with DPP-thiophene-4 with various quantities (1 μg, 5 μg, 10 μg). Six mice per group. (B) Either HeLa cells or HUVEC cells (1 × 104 cells/well) are prepared in 96-well plates. The cellular viability is monitored from 0 to 72 h (0, 6, 12, 24, 48, and 72 h) by CCK kit (three measurements). (C) DPP-thiophene-4 with different concentrations (from 0 to 50 μg mL−1) is added into 1 × 106 sheep red blood cells and kept at room temperature for 2 h. The percent hemolysis of red blood cells is calculated: hemolysis % = [sample absorbance − negative control absorbance]/[positive control absorbance − negative control absorbance] × 100%. (D−F) DPP-thiophene-4 (2.5 μg) is intravenously injected per each mice. After 10 min, 1 h, 4 h, 1 day, 3 days, 7 days, and 15 days, the blood and urine samples are collected. Eight organs (heart, liver, spleen, lung, kidney, stomach, intestine, and brain) are isolated at the first, third, seventh, and 15th day after DPP-thiophene-4 injection. DPP-thiophene-4 is quantified via LC-MS system to assess its pharmacokinetics in blood and urine and its biodistribution in various organs. (G) After 3, 7, and 15 days injection of DPPthiophene-4 (2.5 μg), histopathologic slides with H&E staining from eight organs (heart, liver, spleen, lung, kidney, stomach, intestine, and brain) are obtained. Normal mice are used as control. All values are shown as mean ± SD. are executed according to guidelines formulated by the Committee of Welfare and Ethics of Laboratory Animals in Beijing. Cell Culture and the Detection of Fluorescence Intensity. MCF-7 and HUVEC cells are respectively cultured with Dulbecco’s modified Eagle’s medium (GIBCO) supplemented by 10% fetal calf serum (GIBCO) on 6-well cell culture plates (Coring) overnight. DPP-thiophene-4 (10 μg/mL), Lyso Tracker Green DND-26 (50 nM), or Hoechst 33342 (10 μg/mL) is added into cells (5 × 105 cells) for 20 min in a 5% CO2 incubator at 37 °C. The fluorescence intensity of cells is detected by confocal microscopy (Zeiss) at the 543 nm excitation for for DPP-thiophene-4, 504 nm excitation for Lyso Tracker Green DND-26, or 352 nm excitation for Hoechst 33342. Pharmacokinetics and Biodistribution. We intravenously inject 2.5 μg DPP-thiophene-4 into each mice. After 10 min, 1 h, 4 h, 24 h, and 72 h, the blood and urine samples are collected. The supernatant serums are further harvested via centrifuging blood at 3000 rmp 10 min. Eight different organs (heart, liver, spleen, lung, kidney, stomach, intestine, and brain) and tumors are isolated at 24 h and 72 h after DPP-thiophene-4 injection. DPP-thiophene-4 in all samples is quantified via LC-MS system according to the previous report.26 Hemolysis. We wash 106 sheep red blood cells (SRBCs) three times with phosphate buffer saline (PBS) solution. DPP-thiophene-4 with different concentrations (from 0 to 50 μg/mL) is added and kept at room temperature for 2 h. We obtain the supernatant via centrifuging and transfer it into a 96-well microreader plate. The plate is read at 570 nm using a microplate reader. The percent hemolysis of RBCs is calculated: hemolysis % = ([sample absorbance − negative control absorbance]/[positive control absorbance − negative control absorbance]) × 100%. Cytotoxicity. The cytotoxicity is evaluated by the cellular viability by CCK assay. HeLa cells or HUVEC cells (1 × 104 cells/well) are prepared in 96-well plates. After 0, 6, 12, 24, 48, and 72 h incubation, CCK solution (20 μL, 5 mg/mL) is added to each well and incubated
between 6.8 and 7.0 (ΔpH = 0.2). Its pH responsiveness and preparation technology are better than that of traditional small molecular pH sensor and polymer-based transistor-like pH nanoprobe (Table S1). In this narrow pH range, its fluorescence emission shows an off−on pattern which is dependent on the formation of nonfluorescent nanoassembly at neutral and its disassembly to yield fluorescent monomers upon protonation at pH 6.8. This reversible self-assembly behavior is applicable to precisely and broadly image multiple malignant tumors in vivo. Different from most existing tumor reporters, such a ligand-free manner is independent from specific targeting moieties for malignant tumors of interest.39−41 This strategy saves tedious work on conjugating fluorophores with tumor-specific ligands (e.g., metabolic substrate, aptamer, growth factor, or antibody) and would be complementary to traditional methods.42−44 Overall, this study presents an alternative strategy to image a broad range of malignant tumors based on supramolecular self-assembly.
METHODS Materials. All chemical materials in this study are purchased from Sigma-Aldrich Company. Three malignant tumor cell lines (HeLa, MCF7 and A375) are stored and cultured in the laboratory ourselves. Human esophageal squamous carcinoma (ECA-109 cell), human laryngeal carcinoma (Hep2+ human laryngeal carcinoma cells), and human colon cancer (PDX) mice are purchased from Model Animal Research Center of Nanjing University. Bal b/c nude mice 6−8 weeks old are obtained from the Vital River Laboratory Animal Center. Animal experiments are approved by the Animal Ethics Committee of National Center for Nanoscience and Technology. Animal operations 12450
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ACS Nano at 37 °C for another 4 h. The absorbance of each well at 450 nm (620 nm wavelength as reference) is measured by a multimode microplate reader (Tecan infinite 200). The cytotoxicity assays are shown using mean SD (three measurements). Establishment of Tumor Mouse Models and the Detection of Fluorescence Intensity in Mouse Models. We establish the human melanoma model (A375 cells), xenografted cervical carcinoma model (HeLa cells), and human breast cancer model (Mcf-7 cells) via subcutaneously injecting 2 × 107 A375 cells, 2 × 107 HeLa cells, or 2 × 107 Mcf-7 cells into each mouse. After 2−3 weeks, the volume of tumor grows to around 50 mm3. Human esophageal squamous carcinoma (ECA-109 cell), human laryngeal carcinoma (Hep2+ human laryngeal carcinoma cells), and human colon cancer (PDX) mice are purchased from Model Animal Research Center of Nanjing University. For each mouse, only one around 50 mm3 tumor grows in the injecting site of tumor cells or tissue mince. DPP-thiophene-4 (2.5 μg for each mouse) is directly injected into one tumor. After 5 min, the fluorescence intensity from tumor tissue and nontumor area in mice is respectively detected via a small-animal in vivo imaging system (CRI Maestro 2). After finishing the fluorescence detection against the whole mouse, five mouse organs (heart, liver, spleen, brain and kidney) and the tumor tissue are isolated. The fluorescence intensity from each organ or tumor tissue is quantified again via the smallanimal in vivo imaging system (CRI Maestro 2).
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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/acsnano.7b06483. Details of synthesis, Experimental procedures, ultraviolet absorption and fluorescent emission spectra, the imaging of DPP-thiophene-4, isolated organs and tumor tissue (PDF)
AUTHOR INFORMATION Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Yuan Gao: 0000-0001-9714-4219 Xingyu Jiang: 0000-0002-5008-4703 Author Contributions §
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (31500816, 81361140345, 51373043, 21535001, 21675036), the Ministry of Science and Technology of China (2013YQ190467), National Key Research and Development Program (2017YFA0205901), Chinese Academy of Sciences (XDA09030305), and “Hundred Talents Project” of the Chinese Academy of Sciences for financial support. REFERENCES (1) Zhao, Y.; Shi, C.; Yang, X.; Shen, B.; Sun, Y.; Chen, Y.; Xu, X.; Sun, H.; Yu, K.; Yang, B.; et al. pH- and Temperature-Sensitive Hydrogel Nanoparticles with Dual Photoluminescence for Bioprobes. ACS Nano 2016, 10, 5856−5863. (2) Sun, X.; Huang, X.; Guo, J.; Zhu, W.; Ding, Y.; Niu, G.; Wang, A.; Kiesewetter, D. O.; Wang, Z.; Sun, S.; et al. Self-Illuminating 64CuDoped CdSe/ZnS Nanocrystals for In Vivo Tumor Imaging. J. Am. Chem. Soc. 2014, 136, 1706−1709. 12451
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