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Letter
A Monitorable Mitochondria-targeting DNAtrain for Image-guided Synergistic Cancer Therapy Tao Jiang, Lihua Zhou, Haixiang Liu, Pengfei Zhang, Guozhen Liu, Ping Gong, Chunbin Li, Weihong Tan, Jianhai Chen, and Lintao Cai Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01777 • Publication Date (Web): 15 May 2019 Downloaded from http://pubs.acs.org on May 16, 2019
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Analytical Chemistry
A Monitorable Mitochondria-targeting DNAtrain for Imageguided Synergistic Cancer Therapy Tao Jiang,ǂab Lihua Zhou,ǂb Haixiang Liu,bc Pengfei Zhang,*bc Guozhen Liu,e Ping Gong,b,f Chunbin Li,b Weihong Tan,d Jianhai Chen,*a and Lintao Cai*b a. Department of Pharmaceutical Sciences, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, P. R. China. * E-mail:
[email protected] b. Guangdong Key Laboratory of Nanomedicine, CAS Key Laboratory of Health Informatics, Shenzhen Bioactive Materials Engineering Lab for Medicine, Institute of Biomedicine and Biotechnology. Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, P. R. China. * E-mail:
[email protected];
[email protected] c. Department of Chemical and Biological Engineering, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, China. d. Center for Research at Bio/Nano Interface, Department of Chemistry and Department of Physiology and Functional Genomics, Health Cancer Center, UF Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA. e. Graduate School of Biomedical Engineering, ARC Centre of Excellence in Nanoscale Biophotonics, Faculty of Engineering, Australian Centre for NanoMedicine, University of New South Wales (UNSW), Sydney, NSW 2052, Australia. f. Dongguan Key Laboratory of Drug Design and Formulation Technology, Key Laboratory for Nanomedicine, Guangdong
Medical University, Dongguan 523808, China. ABSTRACT: It is highly desirable to realize real-time monitoring of drug delivery/release process in cancer treatment. Herein, a monitorable mitochondria-specific DNAtrain (MitoDNAtrs) was developed for image-guided drug-delivery and synergistic cancer therapy. In this system, mitochondria-targeting Cy5.5 dye served as the “locomotive” guiding DNA “vehicle” selectively accumulated into cancer cells with a detectable manner. More importantly, Cy5.5 showed reactive oxygen species (ROS) generation ability, which made it a promising adjuvant chemotherapy amplifier for cancer theranostics.
Chemotherapy is widely used in cancer treatment.1-3 To achieve better cancer therapeutic efficiencies and minimize side effects, the anticancer drugs are typically loaded into the polymer delivery systems4 or covalently conjugated with tumor-targeting ligands, such as aptamers,5-8 antibodies,9,10 peptides,11,12 etc. Although these strategies have been proven to be effective for cancer-targeting delivery and controllable release only at specific tumor sites. It is highly desirable to realize monitoring of drug delivery/release process in situ to minimize the duration of ineffective courses in cancer therapy.13,14 Mitochondria are critical sub-cellular organelles in eukaryotic cells; they play vital roles in energy production, reactive oxygen species (ROS) generation, and apoptosis.15 Cancer cells often exhibit abnormal mitochondrial functions, such as higher mitochondrial membrane potential changes during energy metabolism and increased oxidative stress. These characteristics provide opportunities to target cancer cells and optimize therapeutic efficiency. Mitochondria have been long recognized as an ideal subcellular target for cancer therapy.16-20 More importantly, mitochondria are susceptible to generate excessive ROS (e.g. singlet oxygen and free radicals), which has inspired the design of photosensitizers with mitochondria-targeting ability for enhanced
photodynamic therapy.21-23 Considerable efforts have recently been dedicated to synergistically amplify the therapeutic effects of chemo-drugs.24,25 A variety of cationic organic fluorescent dyes have been developed to target mitochondria.26-29 Notably, some of them can differentiate between normal and cancers cells based on the differences in mitochondrial quantity and membrane potential.30 Recently, a group of NIRF heptamethine cyanine dyes, which could preferentially accumulate in the mitochondria of tumor cells, have emerged as more promising tools for cancer imaging and targeted therapy.31-33 Moreover, mitochondria-specific nanosystems have been developed for tumor-targeted delivery and synergistically amplified therapy.34-36 DNA is the basic genetic material of organisms. Due to its programmable, periodic, and biodegradable nature, DNA is an attractive structural material for bottom-up construction of drug delivery systems.37-39 Numerous DNA nano-structural drug delivery systems have been constructed.40-43 Beyond pure DNA nanostructures, several hybrid DNA nanostructures have also been developed for drug delivery by integrating DNA subunits with inorganic nanomaterial such as gold nanoparticles or organic materials such as liposome.44-47 Besides, DNA-lipid hybrid nanostructures offer the potential to load anticancer drugs in their hydrophobic sections.48 Zhu
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et al reported the use of aptamer-tethered DNA nanotrains (aptNTrs) as carriers for targeted drug transport in cancer therapy. These DNA nanostructures allowed for high drug payloads.49 Motivated by the previous success, we intend to develop a DNA-based theranostic system that could not only selectively deliver chemo-drugs to tumors in a monitorable manner but also greatly amplify the antitumor efficacy of chemotherapeutic drugs. As shown in Scheme 1, the mitochondria-targeting DNAtrain system (MitoDNAtrs) comprised of three components: (1) Cy5.5, which served as both fluorescent mitochondria-targeting molecules and adjuvants assisting drugs; (2) a long linear DNA covalently attached with Cy5.5, served as effective drug carrying vehicles; (3) Doxorubicin (Dox), which is a widely used anthracycline drug with known fluorescent properties, could be intercalated within the CG sequences of RNA and DNA as a therapeutic agent (the optical properties of Cy5.5 and Dox was shown in Figure S1).
Scheme 1. Schematic illustration of the mitochondriatargeting DNAtrain system (MitoDNAtrs) for monitorable transport of molecular drugs into cancer cells. In this system, DNA served as programmable and biodegradable carriers; Cy5.5, a cationic heptamethine indocyanine dye, not only worked as “lighting” locomotive guiding DNAtrain toward mitochondria and transporting the drugs to cancer cells in a monitorable manner, but also served as adjuvants to enhance the effect of chemotherapy. In order to verify the mitochondria-targeting ability and selective accumulation of Cy5.5 in target cells, cells were incubated with Cy5.5 and imaged using confocal microscopy. (Fig. S2) The results showed that obvious red fluorescence signal arose from incubated cancer cells (MCF-7 cells) instead of normal cells (bEnd.3 cells). The cellular uptake and subcellular co-localization experiments further indicated that Cy5.5 entered cancer cells and further accumulate in the mitochondria. (Fig. S3) The cellular uptake mechanism of Cy5.5 was further investigated, which indicated the whole process was associated with energy-dependent glycolytic metabolic patterns and organic-anion transporting polypeptide (OATP) transporters (Fig. S4 and S5). The results were consistent with previous results on heptamethine indocyanine dyes.50 Furthermore, we wondered whether Cy5.5 dye could guide DNA into the target cells. Cancer cells and normal cells were incubated with Cy5.5-DNA bio-conjugate and imaged using confocal microscopy, respectively. As shown in Figure 1a, strong fluorescence signal appeared in MCF-7 cells while almost no fluorescence was observed in bEnd.3 cells, which demonstrated that Cy5.5-DNA conjugates were effectively taken up by the cancer cells, while significantly fewer conjugates were taken up by the normal cells. Subcellular co-
localization experiments further confirmed that Cy5.5-DNA accumulated in the mitochondria after entering cancer cells (Figure. 1b). The mechanism of cellular uptake of Cy5.5-DNA was further studied, and it showed that the uptake process of Cy5.5-DNA was also energy-dependent (Fig. 1c). Moreover, we observed that (BSP), a competitive inhibitor of OATPs, could suppress the transportation of Cy5.5-DNA into tumor cells, which was consistent with the results of free Cy5.5 (Fig. 1d). All these observations confirmed that Cy5.5 itself could selectively enter the cancer cells and finally located in mitochondria. More importantly, it could guide DNA passing through the cell membrane into the target cells, which inspired us using this biocompatible system to carry drugs for further chemotherapy.
Figure 1. Confocal imaging of Cy5.5-DNA bioconjugate. (a) Differentiation of cancerous MCF-7 cells from normal bEnd.3 cells by Cy5.5-DNA. Scale bar: 40 μm. (b) Subcellular localization study of Cy5.5-DNA in MCF-7 cells. Nucleus was stained with Hochest 33242, Mitochondria was stained with MitoTraker Green and lysosome was stained with LysoTracker Green. Scale bar: 25 μm. (c) The effect of energy metabolism on Cy5.5-DNA uptake. Cells incubated on ice (0 oC) or in pre-heated medium (37 oC) for 30 min before observation by confocal microscopy. Scale bar: 50 μm. (d) The effect of inhibitor of organic-anion-transporting polypeptides (OATPs) on Cy5.5-DNA uptake. sulfobromophthalein (BSP) was used as a competitive inhibitor of organic-anion-transporting polypeptides (OATPs) with concentration of 250 μM for 5min. Scale bar: 50 μm. Previous research has indicated that incubation of Dox with the nucleic acid results in the formation of a reversible physical conjugate causing the fluorescence quenching of Dox as well.51 Dox was loaded on MitoDNAtrs through incubation the Cy5.5-DNA conjugate with Dox in PBS buffer at room temperature. The Dox loading efficiency of MitoDNAtrs was evaluated by agarose gel electrophoresis. The results indicated that the maximal Dox loading for MitoDNAtrs was 7:1. Using confocal laser scanning microscopy, the fluorescence of the Cy5.5 and DOX was measured after incubation of the Cy5.5DNA@DOX with MCF-7 cells in time-course studies. Initially, the Cy5.5-DNA@DOX entered the cells and eventually landed on mitochondria. However, as the fluorescence of Dox could be quenched by DNA, there was no signal from Dox. As the time increased, the fluorescence of Dox emerged prominently when they were released from MitoDNAtrs (Figure 2). Overall Cy5.5-DNA selectively delivered Dox into cancer cells, and the whole delivery process could be monitored by fluorescent signals of Cy5.5 and Dox simultaneously.
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Analytical Chemistry It was well known that heptamethine cyanine dyes were not best candidates for photodynamic therapy due to their low generating ability of reactive oxygen species (ROS).52 However, Cy5.5 showed a certain degree of generation ability of ROS upon NIR light irradiation (Figure. S7), which was limited compared to that of Ce6, a typical photosensitizer (Figure. S8). We questioned whether Cy5.5 dye on the MitoDNAtrs could serve as an adjuvant to enhance the cytotoxicity of Dox. MCF-7 cancer cells and bEnd.3 normal cells were incubated with MitoDNAtrs loaded with different doses of Dox, respectively. NIR light irradiation (660 nm, 0.1 W cm−2) was performed at 12 h post MitoDNAtrs addition, respectively. MCF-7 cancer cells and bEnd.3 normal cells directly incubated with Dox were used as control. The CCK-8 assays as depicted in Figure. 3 revealed that the treatments of MitoDNAtrs to cancer cells without light irradiation and pure light irradiation had negligible interference on the cytotoxicity of Dox. Encouragingly, the antitumor efficacy of Dox was significantly amplified by the treatment of MitoDNAtrs and NIR light irradiation simultaneously. It was noted that the treatments of MitoDNAtrs to normal cells upon exposure to NIR light had negligible interference on the cytotoxicity of Dox. The synergistic antitumor effect of MitoDNAtrs was further studied by double staining with calcein AM and propidium iodide (PI) to visualize living/dead cells (Figure S9 and S10). The results were consistent with those of CCK-8 assay, which further confirmed the selective photoinduced synergistic chemotherapy of MitoDNAtrs and implied that even though the ROS generation ability of Cy5.5 was limited, MitoDNAtrs could serve as both targeting delivery carrier and adjuvant for amplification of antitumor efficacy of Dox.
Figure 2. Confocal laser-scanning microscopy images displaying the time-dependent intracellular behaviours of MitoDNAtrs. MCF-7 cells were incubated with MitoDNAtrs for 1 h, 4 h and 6 h, respectively. The intracellular fluorescence of Dox was gradually enhanced, indicating gradual drug releasing from the MitoDNAtrs. Scale bar: 50 μm. The possible mechanism of the synergistic enhanced antitumor effect of MitoDNAtrs was investigated by expression of a series of related proteins in MCF-7 cells and western blot analysis (Figure 4). As shown in Figure 4b, compared with untreated cells groups, the expression of phosphorylated protein kinase B (also known as p-Akt) was inhibited more heavily when the cells were treated with MitoDNAtrs upon NIR light irradiation. The inhibition of the phosphorylation of protein kinase B could immediately or gradually regulate the expression of apoptosis-related proteins. The down-stream apoptotic proteins were also evaluated by western blotting. Compared to the other groups, the Cy5.5DNA@Dox (+) group led to dramatical suppression of antiapoptotic proteins (procapase3 and Bcl-2) and enhanced
expression of proapoptotic proteins (Figure. 4c). Hence, these results illuminated MitoDNAtrs could magnify the anti-tumor efficacy of Dox via boosting the inhibition of p-Akt, which effectively induced mitochondria-originated apoptosis. The cell apoptosis was also evaluated using Flow cytometry approach (Figure. S11).
Figure 3. Photoinduced synergistic cancer therapy experiments. Cell viabilities of MCF-7 cancer cells (a) and bEnd.3 normal cells (b) after 12 h incubation with MitoDNAtrs loaded with different concentrations of Dox were tested by CCK-8 assay with or without NIR light irradiation. The cells were also incubated with Dox alone as control. “+” represent light irradiation. “-” represent no light irradiation. Data are expressed as means ± SD. Differences among groups were analyzed using one-way ANOVA analysis followed by Tukey’s post-test. Asterisks: differences between Dox alone and other treatments statistically significant. *p < 0.05, **p < 0.01. &: differences between two different treatment are statistically significant, &p < 0.05, &&p
