DNA Tetrahedron Delivery Enhances Doxorubicin-Induced Apoptosis of HT-29 Colon Cancer Cells

DNA Tetrahedron Delivery Enhances Doxorubicin-Induced Apoptosis of HT-29 Colon Cancer Cells As a nano-sized drug carrier with the advantage of modifiability and proper biocompatibility, DNA tetrahedron (DNA tetra) delivery is hopeful to enhance the inhibitory efficiency of nontargeted anticancer drugs. In this investigation, doxorubicin (Dox) was assembled to a folic acid-modified DNA tetra via click chemistry to prepare a targeted antitumor agent. Cellular uptake efficiency was measured via fluorescent imaging. Cytotoxicity, inhibition efficiency, and corresponding mechanism on colon cancer cell line HT-29 were evaluated by MTT assay, cell proliferation curve, western blot, and flow cytometry. No cytotoxicity was induced by DNA tetra, but the cellular uptake ratio increased obviously resulting from the DNA tetra-facilitated penetration through cellular membrane. Accordingly, folic acid-DNA tetra-Dox markedly increased the antitumor efficiency with increased apoptosis levels. In details, 100 μM was the effective concentration and a 6-h incubation period was needed for apoptosis induction. In conclusion, nano-sized DNA tetrahedron was a safe and effective delivery system for Dox and correspondingly enhanced the anticancer efficiency. Keywords: DNA tetrahedron, Doxorubicin, Folic acid, Apoptosis, Colon cancer Background make Dox more specific and effective targeting, more Doxorubicin (Dox) is one of the most widely used anti- easily to be encapsulated, and of an excellent intake cap- neoplastic agent, and numerous clinical studies demon- acity and bio-compatibility. strated that Dox can strikingly hinder the growth of Nano-sized drug carriers, such as liposome and inorganic tumor cells in various cellular growth cycles by inhibit- nanoparticles, may facilitate Dox penetrating the tumor cell ing the synthesis of RNA and DNA [1, 2]. Previous stud- membrane, improving the targeting efficiency [8]. Nonethe- ies suggested that the proliferation of tumor cells were less, liposome-based delivery cannot reduce the side effects effectively blocked in G1 phase, and the metastasis was to normal cells because of the relatively poor targeting; also inhibited by Dox at a certain concentration [3]. In meanwhile, inorganic carriers, such as mesoporous silica addition, effective inhibition can be achieved in a rela- nanoparticles, cannot be completely biodegraded in vivo, tively smaller dose when compared with other antican- hampering the process of further drug uptake and bringing cer drugs [4]. However, Dox usually induced side effects potential bio-toxicity. For these functional delivery systems, resulted from the lack of specific targeting for tumor the complexity of preparation, inhomogenization of nano- cells and nonselective inhibition of DNA and RNA, particles structures, and low encapsulation efficiency which seriously limited the clinical applications [5, 6]. obstacle the clinical expansions [9–11]. As a nano-sized Meanwhile, the low cellular intake capacity reduces the drug carrier with the excellent performance on drug deliv- accumulation of Dox in tumor cells [7]. Therefore, an ef- ery, DNA-based structures, such as DNA tetrahedron ficient delivery system for Dox should be developed to (DNA tetra), may penetrate the membrane via avoiding the incompatibility between electro-negative DNA and plasma membrane [12–17]. Drugs and targeting molecules can be * Correspondence: tsyang_jn@sina.com both covalently attached to DNA tetrahedron. Furthermore, Department of Cardiology, Tangshan Gongren Hospital, Tangshan, Hebei 063000, China easy absorption and biodegradation of DNA tetrahedral Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Zhang et al. Nanoscale Research Letters (2017) 12:495 Page 2 of 7 sequences avoid long-time retention. Guanine-rich aptamer in a humidified atmosphere containing 95% air and 5% drugs, such as AS1411, have been successfully delivered carbon dioxide at 37 °C. into A549 tumor cells and performed as targeting agents and inhibitors [18]. Immune regulatory factors, such as Synthesis and Purification of DNA Tetrahedron CpG and siRNA, can also be delivered to tumor cells via In this investigation, synthesis followed the schematic DNA tetra carrier to regulate immune responses [19]. With procedures in Fig. 1, and the single-strand DNA the advantages of low-toxicity, proper biocompatibility, and (ssDNA) sequences of DNA tetrahedron are provided in adjustable targeting, DNA tetra showed bio-safety and po- Fig. 1 as well. In details, each ssDNA was dissolved in tentials for Dox delivery. 0.5× TE buffer, and the corresponding optical density In this study, functional particles of DNA tetrahedron (OD) value of DNA was determined by UV spectropho- assembled with Dox as anticancer drug, and folic acid as tometer at 260 nm. Additional TE buffer was supple- specific recognition molecules [20], were designed, syn- mented to make four chains at the same concentration. thesized, and characterized. The anticancer efficiency The mixing ratio of four ssDNAs was 1:1:1:1 at 1 μMin was evaluated on colon cancer cells considering signifi- 100 μL. The reaction was performed in a polymerase cantly upregulated folate receptors on the surface of cell chain reaction (PCR) machine with the cycling condi- membrane. Specifically, the level of cellular uptake, the tions: 95 °C, 10 min, naturally cooled to 4 °C. All of the degree of Dox-induced apoptosis, and the inhibition on single-strand DNA were purified by HPLC with 260 nm cellular proliferation were measured on HT-29 cell lines. as characteristic absorption peak. In HPLC spectrum, the peak time of DNA tetra was faster than that of single strand, and product was collected at the corresponding Methods time point. Regents and Equipment DNA oligonucleotide chains were purchased from Synthesis and Characterization of Folic Acid-DNA tetra-Dox TAKARA in Dalian, China. Dulbecco’s modified Eagle’s Free hydrogen groups of Dox and folic acid were modi- medium (DMEM) and fetal bovine serum were pur- fied with azide group and then coupled with 3′-OH of chased from Gibco in NY, USA. Penicillin and strepto- ssDNA via click chemistry reaction [21]. When adding a mycin were purchased from Beyotime biotechnology in different amount of functional group tagged ssDNA, the Shanghai, China. Folic acid, doxorubicin, 3-(4,5-di- ratio of functional groups could be stoichiometrically methyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium brom- controlled via specific hybridization of side chains. For ide (MTT), and agarose were purchased from Sigma- synthesis of folic acid-DNA tetra, the molar ratio of folic Aldrich in MO, USA. All the antibodies were purchased acid and DNA tetrahedron was set as 1:1. For synthesis from Abcam Company in Shanghai, China. Other re- of DNA tetra-Dox, the molar ratio of Dox and DNA agents were purchased from Sinopharm Chemical Re- tetrahedron was set as 4:1. For synthesis of folic acid- gent Co., Ltd., in Shanghai, China. DNA tetra-Dox, the molar ratio of folic acid, DNA tetra- UV-Vis spectrophotometer (Thermo Evolution 201) hedron, and Dox was respectively set as 1:1:3. All the and constant temperature incubator were purchased synthesis were carried out at micromolar level at 37 °C from Thermo Fisher in the USA; centrifugal machine and then stored at 4 °C [22]. (GT10-1) was purchased from Beijing Era Beili In this study, the series of DNA tetra complexes was Centrifuge Co., Ltd.; fluorescence spectrophotometer characterized by polyacrylamide gel electrophoresis (UV-1800) was purchased from Shimadzu Corpor- (PAGE) with 8% separation gel (39% Acr-Bis) to check ation. Confocal laser scanning microscopy (Visitech) the purity and relative molecular sizes. The samples con- was purchased from Leica companies; polymerase sisted of the different DNA tetra structures and 6× load- chain reaction instrument (PCR, T100), protein elec- ing buffer with mixing ratio of 2:1. The samples were trophoresis, and nucleic acid electrophoresis apparatus stained and analyzed after gel electrophoresis for 90 min were purchased from Bio-Rad Company; dynamic at 110 V. To figure out the differences on particle sizes, light particle size analyzer was purchased from Beck- DNA tetra samples were also scanned by dynamic light man Company; cell culture plates with 96-well or 24- scattering (DLS) instruments with dynamic light. well were purchased from Dow Corning Corporation. High-performance liquid chromatography (HPLC, DNA tetra-Facilitated Cellular Uptake Agilent 1200) with C18 column was purchased from For the different coupling structures designed in this Agilent Technologies. research, the uptake rates by HT-29 cells were compared The human colon cancer cell line HT-29 was main- to figure out the drug delivery efficiency. Cellular uptake tained in DMEM supplemented with 10% fetal bovine efficiency was evaluated and quantified utilizing the serum, 100 U/mL penicillin, and 100 μg/mL streptomycin characteristic fluorescence spectrum of Dox, i.e., excitation Zhang et al. Nanoscale Research Letters (2017) 12:495 Page 3 of 7 Fig. 1 Schematic diagram of folic acid-DNA tetra-Dox, DNA tetra-Dox, and folic acid-DNA tetra. The single-strand DNA sequences of DNA tetrahedron were provided. The process of targeting for tumor cells, penetrating through the cellular membrane, and inserting the DNAs were depicted light at 470 nm and emission light at 590 nm. The HT-29 bio-safety, and inhibition efficiency; on the other side, cells at 2 × 10 /mL were seeded in 24-well plates and whether the Dox of DNA tetra-Dox has comparable antitu- cultured for 24 h. Cells were incubated with various DNA mor properties as before or not was also explored. tetra structures at 10 μM for another 24 h. The medium Therefore, cytotoxicity and antitumor efficiency of DNA was then discarded, and the cells were rinsed with PBS tetra complexes were evaluated. Complexes respectively at three times. For cell fixation, 4% paraformaldehyde was 100 μM were incubated with HT-29 cells. Cell samples immediately added at room temperature for a 30-min were collected every 6 h and then detected by MTT assay co-incubation, and the cells were rinsed with PBS three to evaluate the effect of incubation period. Particular times again. At last, the 24-well plates were observed by attentions were paid for the difference on anticancer laser confocal microscope to compare the cellular uptake efficiency resulted from the variations of structures. efficiency based on the light intensity of emission light. Furthermore, concentration effects on inhibition of HT-29 after being treated with folic acid-DNA tetra-Dox were Cytotoxicity and Anticancer Efficiency evaluated at 0–200 μM via MTT assays. Cytotoxicity and anticancer efficiency were evaluated using MTT assay, where a redox reaction occurs between the MTT in DMSO and intracellular succinate Western Blot and Flow Cytometry dehydrogenase. HT-29 cells were seeded in 96-well To characterize the cellular apoptosis induced by Dox culture plates and cultured for 24 h. facilitated by DNA tetra delivery, protein samples from For the cytotoxicity of DNA tetra as drug carrier, treated cells were heated and cracked using pyrolysis medium containing DNA tetra structures at the concen- liquid, analyzed by 12% SDS-PAGE under the condition tration of 0–100 μM was added for another 24- or 48-h of 100 V at constant current to separate samples, and incubation. Then, 100 μL of MTT solution (5 mg/mL) then blotted to 0.22-μm-diameter PVDF membranes for was added to each well, and the mixture was incubated 1 h. The samples were blocked with skimmed milk for at 37 °C for 4 h. The liquid was then removed, and the 1 h. After being washed for three times with PBST, cells were lysed and dissolved with 200 μL DMSO. The samples were incubated overnight with rabbit anti- absorbance of supernatant was measured at 570 nm by caspase-3. Then, the samples were probed with a goat- Microreader. The non-treated HT-29 sample was deemed anti-rabbit IgG secondary antibody for 1 h and then as control group. washed with PBST and imaged via Bio-Rad protein For the DNA tetrahedron coupled with folic acid or Dox, imaging system. Further, GAPDH was chosen as internal on the one side, the research focused on the stability, reference protein since its stable expression in cells. Zhang et al. Nanoscale Research Letters (2017) 12:495 Page 4 of 7 Flow cytometry was further utilized to quantify the monomer size was less than 15 nm. With the increase of levels of apoptosis at the selected concentration and coupling groups, the symmetrical structure of DNA tetra time point via MTT assays. HT-29 cells were incubated was destroyed and the medium of particle sizes of with inhibitors for a period of time and then measured compounds increased significantly. In particular, size via quantitative flow cytometry using Annexin V-PI expansion of folic acid-DNA tetra-Dox extended the double staining method. medium of diameter to 20 nm. Cell Proliferation Quantifying Drug Delivery Efficiency Cell counting method was used to quantify the cell pro- After being incubated with different DNA tetra struc- liferation with time. In details, after being treated with tures for 24 h, the intracellular efficiency of Dox was folic acid-DNA tetra-Dox for a period of time at evaluated with fluorescence imaging, where the intracel- 100 μM, cells were digested by 0.25% trypsin for 30 s, lular red fluorescence is the characteristic fluorescence and then, the equal volume of complete medium was of Dox; hence, cells incubated with DNA tetra did not added to terminate reaction. Supernatant was discarded exhibit any fluorescent signal as shown in Fig. 3a, and after centrifugation in 1000 rpm for 3 min, and cells the red signals of cells incubated with folic acid-DNA were re-suspended with complete medium. The number tetra-Dox and DNA tetra-Dox complexes were obviously of cells in each detection point was recorded under higher than that with Dox, meaning the significantly optical microscope using cell count plates, so as to draw enhanced cellular uptake efficiency of Dox resulting the cellular proliferation curve. from the DNA tetra facilitated penetration through the membrane, as well as the intracellular stability of the Statistics Analysis conjugates between Dox and DNA tetra. In this research, significances of differences were deter- The further quantitative analysis of fluorescence inten- mined using Student’s t test (two-tailed; two-sample sity (Fig. 3b) evidenced the visual findings, and there equal variance). P < 0.05 means significant differences was no significant difference between folic acid-DNA between different groups. tetra-Dox and DNA tetra-Dox complexes (P < 0.05). The methodology of co-incubation of drugs and cells Results obstacles the complete exhibition of targeting ability of Preparation of Folic Acid-DNA tetra-Dox folic acid. The relative high concentration confirmed the In specific synthesis process, Dox was mixed with DNA specific recognition of folate receptors, which is theoret- tetrahedron in different proportions to complete loading ically more obvious in the in vivo applications with the and assembling of Dox (w = 543.52) and folic acid complex circulating system. In brief, the DNA tetra (w = 441.4). As shown in Fig. 2a, due to that Dox and delivery guaranteed the anticancer efficiency of Dox. folic acid are monomers with relative low and similar molecular weight, DNA in a tetrahedron and the conju- gates with one functional molecule had roughly equiva- Cytotoxicity and Anticancer Efficiency lent molecular weights. However, Dox or folic acid First, the viability of HT-29 cells co-incubated with dif- assembling with all the four DNA vertices of the tetrahe- ferent concentrations of DNA tetra were examined using dron made molecular size of DNA tetra increased obvi- MTT assay. There were no obvious cytotoxicity of DNA ously. As shown in Fig. 2b, most of DNA tetrahedral tetra treatment in HT-29 cells at 0–100 μM for 24 and Fig. 2 Characterization of DNA tetra with different constituents. a Left to right: DNA ladder, DNA tetra, folic acid-DNA tetra, folic acid-DNA tetra-Dox, and DNA tetra-Dox imaged in 8% SDS-PAGE. b The size distribution of DNA tetra, folic acid-DNA tetra, folic acid-DNA tetra-Dox, and DNA tetra-Dox detected via dynamic light scattering Zhang et al. Nanoscale Research Letters (2017) 12:495 Page 5 of 7 Fig. 3 The uptake efficiency of HT-29 cells after being incubated with different complexes for 24 h. a Confocal microscopy detection (red is fluorescence excitation light of Dox, scale bar = 10 μm). b Intensity of cellular fluorescence. **P < 0.05 when compared with the group of DNA tetra 48 h (Fig. 4a). Hence, the nano-sized DNA tetrahedron The flow cytometry (Fig. 5b–e) further proved that 6-h provided a bio-safe drug carrier platform. incubation with folic acid-DNA tetra-Dox at 100 μM Secondly, resulting from the difference on Dox deliv- induced 68.7% apoptosis. Meanwhile, the apoptosis ery efficiency, folic acid-DNA tetra-Dox and DNA tetra- levels induced by Dox and DNA tetra-Dox were only Dox exhibited more significant inhibition efficiency 32.8 and 60.5% with the same incubation condition. within 48 h (Fig. 4b). Due to the lack of effective anti- cancer components, DNA tetra-folic acid lead to Inhibition on Cell Proliferation negligible decrease (< 10%) of viability during the 48-h Based on the MTT assays, 100 μM was the effective co-incubation. Due to that Dox cannot objectively pene- concentration for apoptosis induction and was selected trate the membrane, Dox-induced decrease of cellular in the cellular proliferation experiment. As shown by the viability was less than the other groups. Particularly, The cellular proliferation curve (Fig. 6), due to the time- folic acid-DNA tetra-Dox showed a more earlier and consuming process of cellular uptake, the inhibition on significant decrease at 6 h post incubation, proving that cell proliferation was not totally shown during the early the folic acid targeting help the drugs to locate at the stage of co-incubation. A significant inhibitory effect was membrane, and make the Dox take effect more timely. presented after being incubated with folic acid-DNA By contrast, DNA tetra-Dox began to take effect tetra-Dox for more than 6 h, proving the existence of obviously at 12 h post incubation, demonstrating the im- DNA tetra-enhanced endocytosis. Meanwhile, 6 h is a portance of targeting groups. comparative time period with the effective time period Furthermore, HT-29 cells were treated with folic acid- detected in Fig. 4b. DNA tetra-Dox at 0–200 μM to detect the effective concentration (Fig. 4c). Cellular viability of HT-29 cells Discussion declined rapidly with increasing concentration (0– As the common means of DNA modification, click chemis- 100 μM) of the complex, but there were no significant try reaction was of advantages of high reaction efficiency, differences between the concentration of 100 and well-control, and facile operation [21]. Electrophoretogram 200 μM, indicating a dose-dependent manner of folic displayed with a single brand (Fig. 2a), indicating that the acid-DNA tetra-Dox, where 100 μM can be considered products with high purity were obtained by covalent coup- as an effective concentration for antitumor effect. In ling via click chemistry reaction. The fluorescent property addition, cellular viability changed significantly from 24 of Dox make it trackable in vitro and in vivo; meanwhile, to 48 h for the groups of 10, 20, and 50 μM, indicating the fluorescence inside the tumor cells indirectly proved that the time period of incubation was also a factor in the stability of folic acid-DNA tetra-Dox during the process affecting folic acid-DNA tetra-Dox based anticancer of penetration through the cell membrane. Therefore, the efficiency. folic acid-DNA tetra-Dox was capable and stable for bio- medical applications. Apoptosis Induced by Folic Acid-DNA tetra-Dox DNA tetra has the capability to deliver drugs into cells. Accordingly, based on the western blot assays (Fig. 5a), All DNA sequences used in this research were not there were significant increase of the expression of encoded for any genetic information. Thus, no side effects caspase-3 induced by folic acid-DNA tetra-Dox and on both gene expression and cell metabolism were DNA tetra-Dox, proving that apoptosis was the main reported in all examinations. Meanwhile, due to the high way of Dox-induced cell death. expression of folate receptor on surface of tumor cells, Zhang et al. Nanoscale Research Letters (2017) 12:495 Page 6 of 7 Fig. 5 The cellular expression of apoptosis-related caspase-3 after being treated with different complexes (a) and the flow cytometry of HT-29 cells after being incubated with DNA tetra, Dox, DNA tetra-Dox, and folic acid-DNA tetra-Dox (b–e) folic acid is selected as specific targeting molecules of the drug delivery system to enhance the uptake efficiency of DNA tetra complexes. However, with the circumstances of DNA tetra at relatively high concentration, the advan- tage of folate receptor targeting was not fully reflected Fig. 4 The cytotoxicity and antitumor efficiency of Dox complexes and DNA tetra for HT-29 cells. a The viability of HT-29 cells incubated with DNA tetra at different concentrations for 24 or 48 h. b The antitumor efficiency of Dox complexes at 100 μM. c The viability of HT-29 cells incubated with folic acid-DNA tetra-Dox at different concentrations for 24 or 48 h Fig. 6 Inhibition of cell proliferation induced by the incubation with folic acid-DNA tetra-Dox at 100 μM for different time periods Zhang et al. Nanoscale Research Letters (2017) 12:495 Page 7 of 7 in vitro. Accordingly, for in vivo application, the folic 2. Wang Y, Wei X, Zhang C, Zhang F, Liang W (2010) Nanoparticle delivery strategies to target doxorubicin to tumor cells and reduce side effects. 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Fu J, Yan H (2012) Controlled drug release by a nanorobot. Nat Nanotech cer cells, and provides a promising inspiration and idea 30:407–408 for drug design. 16. Jiang Q, Song C, Nangreave J, Liu X, Lin L, Qiu D et al (2012) DNA origami as a carrier for circumvention of drug resistance. J Am Chem Soc 134: Funding 13396–13403 The authors gratefully acknowledge the financial supports provided by Hebei 17. Ouyang X, Li J, Liu H, Zhao B, Yan J, Ma Y et al (2013) Rolling circle Province Science and Technology Support Program (CN) (no. 142777101D). amplification-based DNA origami nanostructrures for intracellular delivery of immunostimulatory drugs. Small 9:3082–3087 Authors’ Contributions 18. Xu X, Zhao Y, Lu H, Fu C, Li X, Jiang L et al (2016) G4-tetra DNA duplex GZ and ZZ designed the research. GZ, ZZ, and JY performed the induce lung cancer cell apoptosis in A549 cells. Nanoscale Res Lett 11:437 experiments and statistics. All the authors read and approved the 19. Kim H, Akagi T, Akashi M (2010) Preparation of CpG ODN-encapsulated anionic manuscript. poly (amino acid) nanoparticles for gene delivery. Chem Lett 39:278–279 20. Zheng B, Yang S, Wang M, Yang X, Teng L, Xie J et al (2015) Non-covalent Competing Interests nanocomplexes of folic acid and reducible polyethylenimine for survivin The authors declare that they have no competing interests. siRNA delivery. Anticancer Res 35:5433–5441 21. AH EI-s, Brown T (2010) Click chemistry with DNA. Chem Soc Rev 39:1388–1405 22. Li J, Fan C, Pei H, Shi J, Huang Q (2013) Smart drug delivery nanocarriers Publisher’sNote with self-assembled DNA nanostructures. Adv Mater 25:4386–4396 Springer Nature remains neutral with regard to jurisdictional claims in 23. Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME et published maps and institutional affiliations. al (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399:271–275 Author details 24. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F et al (2006) A Department of Infectious Diseases, Tangshan Gongren Hospital, Tangshan, microRNA expression signature of human solid tumors defines cancer gene Hebei 063000, China. Department of Pathology, Tangshan Gongren targets. Proc Natl Acad Sci U S A 103:2257–2261 Hospital, Tangshan, Hebei 063000, China. Department of Cardiology, 25. Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H et al (2004) Tangshan Gongren Hospital, Tangshan, Hebei 063000, China. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor Received: 3 May 2017 Accepted: 10 August 2017 progression and angiogenesis. Cancer Res 64:7099–7109 References 1. Litwiniec A, Grzanka A, Helmin-Basa A, Gackowska L, Grzanka D (2010) Features of senescence and cell death induced by doxorubicin in A549 cells: organization and level of selected cytoskeletal proteins. J Cancer Res Clin Oncol 136:717–736 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nanoscale Research Letters Springer Journals

DNA Tetrahedron Delivery Enhances Doxorubicin-Induced Apoptosis of HT-29 Colon Cancer Cells

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Abstract

As a nano-sized drug carrier with the advantage of modifiability and proper biocompatibility, DNA tetrahedron (DNA tetra) delivery is hopeful to enhance the inhibitory efficiency of nontargeted anticancer drugs. In this investigation, doxorubicin (Dox) was assembled to a folic acid-modified DNA tetra via click chemistry to prepare a targeted antitumor agent. Cellular uptake efficiency was measured via fluorescent imaging. Cytotoxicity, inhibition efficiency, and corresponding mechanism on colon cancer cell line HT-29 were evaluated by MTT assay, cell proliferation curve, western blot, and flow cytometry. No cytotoxicity was induced by DNA tetra, but the cellular uptake ratio increased obviously resulting from the DNA tetra-facilitated penetration through cellular membrane. Accordingly, folic acid-DNA tetra-Dox markedly increased the antitumor efficiency with increased apoptosis levels. In details, 100 μM was the effective concentration and a 6-h incubation period was needed for apoptosis induction. In conclusion, nano-sized DNA tetrahedron was a safe and effective delivery system for Dox and correspondingly enhanced the anticancer efficiency. Keywords: DNA tetrahedron, Doxorubicin, Folic acid, Apoptosis, Colon cancer Background make Dox more specific and effective targeting, more Doxorubicin (Dox) is one of the most widely used anti- easily to be encapsulated, and of an excellent intake cap- neoplastic agent, and numerous clinical studies demon- acity and bio-compatibility. strated that Dox can strikingly hinder the growth of Nano-sized drug carriers, such as liposome and inorganic tumor cells in various cellular growth cycles by inhibit- nanoparticles, may facilitate Dox penetrating the tumor cell ing the synthesis of RNA and DNA [1, 2]. Previous stud- membrane, improving the targeting efficiency [8]. Nonethe- ies suggested that the proliferation of tumor cells were less, liposome-based delivery cannot reduce the side effects effectively blocked in G1 phase, and the metastasis was to normal cells because of the relatively poor targeting; also inhibited by Dox at a certain concentration [3]. In meanwhile, inorganic carriers, such as mesoporous silica addition, effective inhibition can be achieved in a rela- nanoparticles, cannot be completely biodegraded in vivo, tively smaller dose when compared with other antican- hampering the process of further drug uptake and bringing cer drugs [4]. However, Dox usually induced side effects potential bio-toxicity. For these functional delivery systems, resulted from the lack of specific targeting for tumor the complexity of preparation, inhomogenization of nano- cells and nonselective inhibition of DNA and RNA, particles structures, and low encapsulation efficiency which seriously limited the clinical applications [5, 6]. obstacle the clinical expansions [9–11]. As a nano-sized Meanwhile, the low cellular intake capacity reduces the drug carrier with the excellent performance on drug deliv- accumulation of Dox in tumor cells [7]. Therefore, an ef- ery, DNA-based structures, such as DNA tetrahedron ficient delivery system for Dox should be developed to (DNA tetra), may penetrate the membrane via avoiding the incompatibility between electro-negative DNA and plasma membrane [12–17]. Drugs and targeting molecules can be * Correspondence: tsyang_jn@sina.com both covalently attached to DNA tetrahedron. Furthermore, Department of Cardiology, Tangshan Gongren Hospital, Tangshan, Hebei 063000, China easy absorption and biodegradation of DNA tetrahedral Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Zhang et al. Nanoscale Research Letters (2017) 12:495 Page 2 of 7 sequences avoid long-time retention. Guanine-rich aptamer in a humidified atmosphere containing 95% air and 5% drugs, such as AS1411, have been successfully delivered carbon dioxide at 37 °C. into A549 tumor cells and performed as targeting agents and inhibitors [18]. Immune regulatory factors, such as Synthesis and Purification of DNA Tetrahedron CpG and siRNA, can also be delivered to tumor cells via In this investigation, synthesis followed the schematic DNA tetra carrier to regulate immune responses [19]. With procedures in Fig. 1, and the single-strand DNA the advantages of low-toxicity, proper biocompatibility, and (ssDNA) sequences of DNA tetrahedron are provided in adjustable targeting, DNA tetra showed bio-safety and po- Fig. 1 as well. In details, each ssDNA was dissolved in tentials for Dox delivery. 0.5× TE buffer, and the corresponding optical density In this study, functional particles of DNA tetrahedron (OD) value of DNA was determined by UV spectropho- assembled with Dox as anticancer drug, and folic acid as tometer at 260 nm. Additional TE buffer was supple- specific recognition molecules [20], were designed, syn- mented to make four chains at the same concentration. thesized, and characterized. The anticancer efficiency The mixing ratio of four ssDNAs was 1:1:1:1 at 1 μMin was evaluated on colon cancer cells considering signifi- 100 μL. The reaction was performed in a polymerase cantly upregulated folate receptors on the surface of cell chain reaction (PCR) machine with the cycling condi- membrane. Specifically, the level of cellular uptake, the tions: 95 °C, 10 min, naturally cooled to 4 °C. All of the degree of Dox-induced apoptosis, and the inhibition on single-strand DNA were purified by HPLC with 260 nm cellular proliferation were measured on HT-29 cell lines. as characteristic absorption peak. In HPLC spectrum, the peak time of DNA tetra was faster than that of single strand, and product was collected at the corresponding Methods time point. Regents and Equipment DNA oligonucleotide chains were purchased from Synthesis and Characterization of Folic Acid-DNA tetra-Dox TAKARA in Dalian, China. Dulbecco’s modified Eagle’s Free hydrogen groups of Dox and folic acid were modi- medium (DMEM) and fetal bovine serum were pur- fied with azide group and then coupled with 3′-OH of chased from Gibco in NY, USA. Penicillin and strepto- ssDNA via click chemistry reaction [21]. When adding a mycin were purchased from Beyotime biotechnology in different amount of functional group tagged ssDNA, the Shanghai, China. Folic acid, doxorubicin, 3-(4,5-di- ratio of functional groups could be stoichiometrically methyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium brom- controlled via specific hybridization of side chains. For ide (MTT), and agarose were purchased from Sigma- synthesis of folic acid-DNA tetra, the molar ratio of folic Aldrich in MO, USA. All the antibodies were purchased acid and DNA tetrahedron was set as 1:1. For synthesis from Abcam Company in Shanghai, China. Other re- of DNA tetra-Dox, the molar ratio of Dox and DNA agents were purchased from Sinopharm Chemical Re- tetrahedron was set as 4:1. For synthesis of folic acid- gent Co., Ltd., in Shanghai, China. DNA tetra-Dox, the molar ratio of folic acid, DNA tetra- UV-Vis spectrophotometer (Thermo Evolution 201) hedron, and Dox was respectively set as 1:1:3. All the and constant temperature incubator were purchased synthesis were carried out at micromolar level at 37 °C from Thermo Fisher in the USA; centrifugal machine and then stored at 4 °C [22]. (GT10-1) was purchased from Beijing Era Beili In this study, the series of DNA tetra complexes was Centrifuge Co., Ltd.; fluorescence spectrophotometer characterized by polyacrylamide gel electrophoresis (UV-1800) was purchased from Shimadzu Corpor- (PAGE) with 8% separation gel (39% Acr-Bis) to check ation. Confocal laser scanning microscopy (Visitech) the purity and relative molecular sizes. The samples con- was purchased from Leica companies; polymerase sisted of the different DNA tetra structures and 6× load- chain reaction instrument (PCR, T100), protein elec- ing buffer with mixing ratio of 2:1. The samples were trophoresis, and nucleic acid electrophoresis apparatus stained and analyzed after gel electrophoresis for 90 min were purchased from Bio-Rad Company; dynamic at 110 V. To figure out the differences on particle sizes, light particle size analyzer was purchased from Beck- DNA tetra samples were also scanned by dynamic light man Company; cell culture plates with 96-well or 24- scattering (DLS) instruments with dynamic light. well were purchased from Dow Corning Corporation. High-performance liquid chromatography (HPLC, DNA tetra-Facilitated Cellular Uptake Agilent 1200) with C18 column was purchased from For the different coupling structures designed in this Agilent Technologies. research, the uptake rates by HT-29 cells were compared The human colon cancer cell line HT-29 was main- to figure out the drug delivery efficiency. Cellular uptake tained in DMEM supplemented with 10% fetal bovine efficiency was evaluated and quantified utilizing the serum, 100 U/mL penicillin, and 100 μg/mL streptomycin characteristic fluorescence spectrum of Dox, i.e., excitation Zhang et al. Nanoscale Research Letters (2017) 12:495 Page 3 of 7 Fig. 1 Schematic diagram of folic acid-DNA tetra-Dox, DNA tetra-Dox, and folic acid-DNA tetra. The single-strand DNA sequences of DNA tetrahedron were provided. The process of targeting for tumor cells, penetrating through the cellular membrane, and inserting the DNAs were depicted light at 470 nm and emission light at 590 nm. The HT-29 bio-safety, and inhibition efficiency; on the other side, cells at 2 × 10 /mL were seeded in 24-well plates and whether the Dox of DNA tetra-Dox has comparable antitu- cultured for 24 h. Cells were incubated with various DNA mor properties as before or not was also explored. tetra structures at 10 μM for another 24 h. The medium Therefore, cytotoxicity and antitumor efficiency of DNA was then discarded, and the cells were rinsed with PBS tetra complexes were evaluated. Complexes respectively at three times. For cell fixation, 4% paraformaldehyde was 100 μM were incubated with HT-29 cells. Cell samples immediately added at room temperature for a 30-min were collected every 6 h and then detected by MTT assay co-incubation, and the cells were rinsed with PBS three to evaluate the effect of incubation period. Particular times again. At last, the 24-well plates were observed by attentions were paid for the difference on anticancer laser confocal microscope to compare the cellular uptake efficiency resulted from the variations of structures. efficiency based on the light intensity of emission light. Furthermore, concentration effects on inhibition of HT-29 after being treated with folic acid-DNA tetra-Dox were Cytotoxicity and Anticancer Efficiency evaluated at 0–200 μM via MTT assays. Cytotoxicity and anticancer efficiency were evaluated using MTT assay, where a redox reaction occurs between the MTT in DMSO and intracellular succinate Western Blot and Flow Cytometry dehydrogenase. HT-29 cells were seeded in 96-well To characterize the cellular apoptosis induced by Dox culture plates and cultured for 24 h. facilitated by DNA tetra delivery, protein samples from For the cytotoxicity of DNA tetra as drug carrier, treated cells were heated and cracked using pyrolysis medium containing DNA tetra structures at the concen- liquid, analyzed by 12% SDS-PAGE under the condition tration of 0–100 μM was added for another 24- or 48-h of 100 V at constant current to separate samples, and incubation. Then, 100 μL of MTT solution (5 mg/mL) then blotted to 0.22-μm-diameter PVDF membranes for was added to each well, and the mixture was incubated 1 h. The samples were blocked with skimmed milk for at 37 °C for 4 h. The liquid was then removed, and the 1 h. After being washed for three times with PBST, cells were lysed and dissolved with 200 μL DMSO. The samples were incubated overnight with rabbit anti- absorbance of supernatant was measured at 570 nm by caspase-3. Then, the samples were probed with a goat- Microreader. The non-treated HT-29 sample was deemed anti-rabbit IgG secondary antibody for 1 h and then as control group. washed with PBST and imaged via Bio-Rad protein For the DNA tetrahedron coupled with folic acid or Dox, imaging system. Further, GAPDH was chosen as internal on the one side, the research focused on the stability, reference protein since its stable expression in cells. Zhang et al. Nanoscale Research Letters (2017) 12:495 Page 4 of 7 Flow cytometry was further utilized to quantify the monomer size was less than 15 nm. With the increase of levels of apoptosis at the selected concentration and coupling groups, the symmetrical structure of DNA tetra time point via MTT assays. HT-29 cells were incubated was destroyed and the medium of particle sizes of with inhibitors for a period of time and then measured compounds increased significantly. In particular, size via quantitative flow cytometry using Annexin V-PI expansion of folic acid-DNA tetra-Dox extended the double staining method. medium of diameter to 20 nm. Cell Proliferation Quantifying Drug Delivery Efficiency Cell counting method was used to quantify the cell pro- After being incubated with different DNA tetra struc- liferation with time. In details, after being treated with tures for 24 h, the intracellular efficiency of Dox was folic acid-DNA tetra-Dox for a period of time at evaluated with fluorescence imaging, where the intracel- 100 μM, cells were digested by 0.25% trypsin for 30 s, lular red fluorescence is the characteristic fluorescence and then, the equal volume of complete medium was of Dox; hence, cells incubated with DNA tetra did not added to terminate reaction. Supernatant was discarded exhibit any fluorescent signal as shown in Fig. 3a, and after centrifugation in 1000 rpm for 3 min, and cells the red signals of cells incubated with folic acid-DNA were re-suspended with complete medium. The number tetra-Dox and DNA tetra-Dox complexes were obviously of cells in each detection point was recorded under higher than that with Dox, meaning the significantly optical microscope using cell count plates, so as to draw enhanced cellular uptake efficiency of Dox resulting the cellular proliferation curve. from the DNA tetra facilitated penetration through the membrane, as well as the intracellular stability of the Statistics Analysis conjugates between Dox and DNA tetra. In this research, significances of differences were deter- The further quantitative analysis of fluorescence inten- mined using Student’s t test (two-tailed; two-sample sity (Fig. 3b) evidenced the visual findings, and there equal variance). P < 0.05 means significant differences was no significant difference between folic acid-DNA between different groups. tetra-Dox and DNA tetra-Dox complexes (P < 0.05). The methodology of co-incubation of drugs and cells Results obstacles the complete exhibition of targeting ability of Preparation of Folic Acid-DNA tetra-Dox folic acid. The relative high concentration confirmed the In specific synthesis process, Dox was mixed with DNA specific recognition of folate receptors, which is theoret- tetrahedron in different proportions to complete loading ically more obvious in the in vivo applications with the and assembling of Dox (w = 543.52) and folic acid complex circulating system. In brief, the DNA tetra (w = 441.4). As shown in Fig. 2a, due to that Dox and delivery guaranteed the anticancer efficiency of Dox. folic acid are monomers with relative low and similar molecular weight, DNA in a tetrahedron and the conju- gates with one functional molecule had roughly equiva- Cytotoxicity and Anticancer Efficiency lent molecular weights. However, Dox or folic acid First, the viability of HT-29 cells co-incubated with dif- assembling with all the four DNA vertices of the tetrahe- ferent concentrations of DNA tetra were examined using dron made molecular size of DNA tetra increased obvi- MTT assay. There were no obvious cytotoxicity of DNA ously. As shown in Fig. 2b, most of DNA tetrahedral tetra treatment in HT-29 cells at 0–100 μM for 24 and Fig. 2 Characterization of DNA tetra with different constituents. a Left to right: DNA ladder, DNA tetra, folic acid-DNA tetra, folic acid-DNA tetra-Dox, and DNA tetra-Dox imaged in 8% SDS-PAGE. b The size distribution of DNA tetra, folic acid-DNA tetra, folic acid-DNA tetra-Dox, and DNA tetra-Dox detected via dynamic light scattering Zhang et al. Nanoscale Research Letters (2017) 12:495 Page 5 of 7 Fig. 3 The uptake efficiency of HT-29 cells after being incubated with different complexes for 24 h. a Confocal microscopy detection (red is fluorescence excitation light of Dox, scale bar = 10 μm). b Intensity of cellular fluorescence. **P < 0.05 when compared with the group of DNA tetra 48 h (Fig. 4a). Hence, the nano-sized DNA tetrahedron The flow cytometry (Fig. 5b–e) further proved that 6-h provided a bio-safe drug carrier platform. incubation with folic acid-DNA tetra-Dox at 100 μM Secondly, resulting from the difference on Dox deliv- induced 68.7% apoptosis. Meanwhile, the apoptosis ery efficiency, folic acid-DNA tetra-Dox and DNA tetra- levels induced by Dox and DNA tetra-Dox were only Dox exhibited more significant inhibition efficiency 32.8 and 60.5% with the same incubation condition. within 48 h (Fig. 4b). Due to the lack of effective anti- cancer components, DNA tetra-folic acid lead to Inhibition on Cell Proliferation negligible decrease (< 10%) of viability during the 48-h Based on the MTT assays, 100 μM was the effective co-incubation. Due to that Dox cannot objectively pene- concentration for apoptosis induction and was selected trate the membrane, Dox-induced decrease of cellular in the cellular proliferation experiment. As shown by the viability was less than the other groups. Particularly, The cellular proliferation curve (Fig. 6), due to the time- folic acid-DNA tetra-Dox showed a more earlier and consuming process of cellular uptake, the inhibition on significant decrease at 6 h post incubation, proving that cell proliferation was not totally shown during the early the folic acid targeting help the drugs to locate at the stage of co-incubation. A significant inhibitory effect was membrane, and make the Dox take effect more timely. presented after being incubated with folic acid-DNA By contrast, DNA tetra-Dox began to take effect tetra-Dox for more than 6 h, proving the existence of obviously at 12 h post incubation, demonstrating the im- DNA tetra-enhanced endocytosis. Meanwhile, 6 h is a portance of targeting groups. comparative time period with the effective time period Furthermore, HT-29 cells were treated with folic acid- detected in Fig. 4b. DNA tetra-Dox at 0–200 μM to detect the effective concentration (Fig. 4c). Cellular viability of HT-29 cells Discussion declined rapidly with increasing concentration (0– As the common means of DNA modification, click chemis- 100 μM) of the complex, but there were no significant try reaction was of advantages of high reaction efficiency, differences between the concentration of 100 and well-control, and facile operation [21]. Electrophoretogram 200 μM, indicating a dose-dependent manner of folic displayed with a single brand (Fig. 2a), indicating that the acid-DNA tetra-Dox, where 100 μM can be considered products with high purity were obtained by covalent coup- as an effective concentration for antitumor effect. In ling via click chemistry reaction. The fluorescent property addition, cellular viability changed significantly from 24 of Dox make it trackable in vitro and in vivo; meanwhile, to 48 h for the groups of 10, 20, and 50 μM, indicating the fluorescence inside the tumor cells indirectly proved that the time period of incubation was also a factor in the stability of folic acid-DNA tetra-Dox during the process affecting folic acid-DNA tetra-Dox based anticancer of penetration through the cell membrane. Therefore, the efficiency. folic acid-DNA tetra-Dox was capable and stable for bio- medical applications. Apoptosis Induced by Folic Acid-DNA tetra-Dox DNA tetra has the capability to deliver drugs into cells. Accordingly, based on the western blot assays (Fig. 5a), All DNA sequences used in this research were not there were significant increase of the expression of encoded for any genetic information. Thus, no side effects caspase-3 induced by folic acid-DNA tetra-Dox and on both gene expression and cell metabolism were DNA tetra-Dox, proving that apoptosis was the main reported in all examinations. Meanwhile, due to the high way of Dox-induced cell death. expression of folate receptor on surface of tumor cells, Zhang et al. Nanoscale Research Letters (2017) 12:495 Page 6 of 7 Fig. 5 The cellular expression of apoptosis-related caspase-3 after being treated with different complexes (a) and the flow cytometry of HT-29 cells after being incubated with DNA tetra, Dox, DNA tetra-Dox, and folic acid-DNA tetra-Dox (b–e) folic acid is selected as specific targeting molecules of the drug delivery system to enhance the uptake efficiency of DNA tetra complexes. However, with the circumstances of DNA tetra at relatively high concentration, the advan- tage of folate receptor targeting was not fully reflected Fig. 4 The cytotoxicity and antitumor efficiency of Dox complexes and DNA tetra for HT-29 cells. a The viability of HT-29 cells incubated with DNA tetra at different concentrations for 24 or 48 h. b The antitumor efficiency of Dox complexes at 100 μM. c The viability of HT-29 cells incubated with folic acid-DNA tetra-Dox at different concentrations for 24 or 48 h Fig. 6 Inhibition of cell proliferation induced by the incubation with folic acid-DNA tetra-Dox at 100 μM for different time periods Zhang et al. Nanoscale Research Letters (2017) 12:495 Page 7 of 7 in vitro. Accordingly, for in vivo application, the folic 2. Wang Y, Wei X, Zhang C, Zhang F, Liang W (2010) Nanoparticle delivery strategies to target doxorubicin to tumor cells and reduce side effects. 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Kim H, Akagi T, Akashi M (2010) Preparation of CpG ODN-encapsulated anionic manuscript. poly (amino acid) nanoparticles for gene delivery. Chem Lett 39:278–279 20. Zheng B, Yang S, Wang M, Yang X, Teng L, Xie J et al (2015) Non-covalent Competing Interests nanocomplexes of folic acid and reducible polyethylenimine for survivin The authors declare that they have no competing interests. siRNA delivery. Anticancer Res 35:5433–5441 21. AH EI-s, Brown T (2010) Click chemistry with DNA. Chem Soc Rev 39:1388–1405 22. Li J, Fan C, Pei H, Shi J, Huang Q (2013) Smart drug delivery nanocarriers Publisher’sNote with self-assembled DNA nanostructures. Adv Mater 25:4386–4396 Springer Nature remains neutral with regard to jurisdictional claims in 23. Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME et published maps and institutional affiliations. al (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399:271–275 Author details 24. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F et al (2006) A Department of Infectious Diseases, Tangshan Gongren Hospital, Tangshan, microRNA expression signature of human solid tumors defines cancer gene Hebei 063000, China. Department of Pathology, Tangshan Gongren targets. Proc Natl Acad Sci U S A 103:2257–2261 Hospital, Tangshan, Hebei 063000, China. Department of Cardiology, 25. Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H et al (2004) Tangshan Gongren Hospital, Tangshan, Hebei 063000, China. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor Received: 3 May 2017 Accepted: 10 August 2017 progression and angiogenesis. Cancer Res 64:7099–7109 References 1. Litwiniec A, Grzanka A, Helmin-Basa A, Gackowska L, Grzanka D (2010) Features of senescence and cell death induced by doxorubicin in A549 cells: organization and level of selected cytoskeletal proteins. J Cancer Res Clin Oncol 136:717–736

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Nanoscale Research LettersSpringer Journals

Published: Aug 15, 2017

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