Functionalized DNA tetrahedron for concomitant delivery of doxorubicin and paclitaxel

Functionalized DNA tetrahedron for concomitant delivery of doxorubicin and paclitaxel | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Functionalized DNA tetrahedron for concomitant delivery of doxorubicin and paclitaxel Denise Eymael, Karina Carneiro, Marco Magalhaes This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2184757/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Breast cancer is a complex disease and the most common cancer among women. Chemotherapy is commonly used in patients with late-stage breast cancer, which can lead to significant adverse effects. The development of new drug delivery systems is fundamental to improving survival and decreasing morbidity for these patients. Previous studies have shown promising results using DNA nanostructures for doxorubicin (DOX) delivery into drug-resistant breast cancer cells. Here, we successfully modify a DNA tetrahedron (TDN) to include alkyl chains attached to the core of the nanostructure for encapsulation of the hydrophobic drug paclitaxel (PTX). The functionalized TDN (fTDN) simultaneously delivered PTX and DOX into MCF7 cells, leading to improved cell death compared to freely diluted drugs. Our fTDN is a stable and effective delivery system that allows dual drug delivery and shows encouraging results for improving breast cancer chemotherapy. Biological sciences/Biotechnology/Nanobiotechnology Biological sciences/Biotechnology/Nanobiotechnology/Nanostructures Health sciences/Oncology Health sciences/Oncology/Cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Invasive breast carcinoma (IBC) affects over 26,800 people per year in Canada, and over 5,000 died in 2019 due to the disease. 1 Early-stage tumors of IBC are primarily treated by surgery, and chemotherapy may be used before or after surgery. This treatment commonly involves chemotherapeutic agents such as anthracyclines (doxorubicin) or taxanes (paclitaxel); these can significantly increase the morbidity of the treatment, and adverse effects can vary from mucositis, organ damage and death. 2 , 3 Clinically, the most successful approach is to use a combination therapy in which drugs that work by different mechanisms are administered simultaneously, thereby decreasing the likelihood of multidrug-resistant cell syndrome. 4 Among breast cancer survivors, side effects directly related to treatment can be devastating, including premature ovarian failure, osteoporosis, lymphedema and long-term cardiac problems. 5 In most cases, these result from chemotherapy-based treatments that are not specific to cancer cells leading to significant cytotoxicity. 6 , 7 The development of new cancer therapies is essential to decrease cancer mortality and morbidity. Various nanocarriers such as self-assembled polymers in the shape of micelles, liposomes and carbon nanoparticles, 8 , 9 have been proposed to facilitate anti-cancer drug delivery. 9 However, the construction of selective, biocompatible and effective drug delivery agents remains a challenge. 10 – 13 Self-assembled DNA nanostructures have been used in therapeutic and diagnostic applications due to effective cellular internalization and high drug loading capability. 14 – 16 Recently, DNA nanostructures with spatially addressable features were used for controlled delivery of DOX to cancer cells. 17 These drug-loaded DNA nanostructures increase the efficacy of chemotherapy, decrease the adverse side effects and show no significant cytotoxicity in vivo and in vitro . 18 , 19 The DNA tetrahedron (TDN) is one of the most promising nanoconstructs due to its straightforward and predictable assembly in a quantitative manner. 19 , 20 Turberfield et al. previously demonstrated the successful cellular uptake of TDN into mammalian cells 21 and Jiang et al. described its’ use as a DOX nanocarrier for targeted delivery into drug-resistant MCF7 cells. 22 The uptake of TDN into mammalian cells is an energy-dependent endocytosis process. The TDN interacts with the plasma membrane non-specifically, followed by rapid cell internalization through the caveolae-mediated pathway. Once inside the cell, it is directed to lysosomes in a microtubule-dependent manner for degradation. 23 , 24 Similar to lipoproteins, 25 lipoprotein-like complexes, 26 and lipid droplet encapsulation, 27 functionalized DNA nanostructures with confined nonpolar pockets have recently been designed to encapsulate hydrophobic cargo. 28 – 31 Typically, DNA strands are functionalized with alkyl chains at a specific location within the nanostructure. Upon assembly, a hydrophobic compartment is generated with the ability to encapsulate nonpolar cargo such as hydrophobic drugs 32 . As described previously, combining chemotherapeutic agents such as taxanes, cisplatin and anthracyclines can significantly increase the morbidity of the treatment, and adverse effects can vary from mucositis, organ damage and death. 2 , 3 Therefore, an optimized system to deliver these agents could significantly decrease treatment complications while improving disease control. Herein we describe the synthesis of a DNA nanocarrier, a functionalized DNA tetrahedron (capable of DOX intercalation) containing hydrophobic anchors (for PTX encapsulation), for the concomitant delivery of two cancer therapeutic drugs. Our results indicate that the functionalized TDN (fTDN) can deliver DOX and PTX concomitantly into cancer cell lines. To our knowledge, this is the first time a single DNA carrier has been used to deliver two drugs that work by different mechanisms simultaneously. This approach can potentially overcome the multidrug-resistant cell syndrome in invasive breast cancer treatment, decrease morbidity, and improve treatment outcomes for patients with breast cancer. 2. Materials And Methods Reagents Tris(hydroxymethyl)aminomethane base (ultra-pure, TRS001), ethylenediaminetetraacetic acid disodium dihydrate (EDTA, reagent grade, EDT001), formamide (biotechnology grade, min. 99.5%, FOR001), tetramethylethylenediamine (TEMED, electrophoresis grade, TEM001), 40% acrylamide/bis-acrylamide solution 19:1, ACR005) and urea (ultra pure, molecular biology grade, URE-001) were purchase from Bioshop Canada. Boric acid (B6768), ammonium persulfate (≥ 98%, A3678), doxorubicin (suitable for fluorescence, 98.0-102.0% (HPLC), 44583), paclitaxel (from semisynthetic, ≥ 97%, T7191), Nile Red (suitable for fluorescence, ≥ 98.0% (HPLC), 19123) were purchase from Sigma-Aldrich. Magnesium chloride hexahydrate (crystalline, USP/FCC, M35-500) was purchased from Fisher Chemical™. Annexin V FITC apoptosis staining / detection kit (ab14085), cleaved caspase-3 staining kit (ab65613), Phalloidin-iFluor 488 (ab176753) and anti-CY5 mouse monoclonal (ab52061) were purchase from Abcam. Acetic acid glacial (lab grade, 00615 − 540) from Anachemia. PAGE GelRed® Nucleic Acid Gel Stain (41008) and GelGreen® Nucleic Acid Gel Stain (41005) were purchased from Biotium. Illustra micro spin G-25 columns (27-5325-01) were purchased from GE Healthcare. AlamarBlue™ Cell Viability Reagent (DAL1025) was purchased from Invitrogen. Alpha Minimum Essential Medium (098150) and fetal bovine serum (FBS, 310-010-CL) were purchased from Multicell. CellLight™ Plasma Membrane-CFP, BacMam 2.0 (C10606) and pHrodo™ Red Dextran, 10,000 MW, for Endocytosis (P10361) were purchased from Invitrogen. Unmodified DNA tetrahedron sequences were obtained from a previous publication. 33 Strands S1-4 were purchased from IDT DNA with polyacrylamide gel electrophoresis purification. The functionalized DNA strands were purchased from GeneLink with PAGE purification. When desired, Cy5 was attached to the 5’-end of strand S1, while an alkyl (C12) was attached to the 3’-end of strands S1-4 on a DNA synthesizer. All DNA sequences used and their modifications are described in Table S1 . DNA tetrahedron assembly DNA tetrahedron was assembled by adding strands S1-4 (20 nM) in 1xTAEMg buffer and performing a 95-4 o C thermal anneal cycle conducted using Flexigene Techne 96 well thermocycler. DNA characterization was done by denaturing PAGE 15% in TB buffer for strand purity and native PAGE 8% gel in TAEMg buffer for proper assembly into a tetrahedron ( Suppl. Figure 1 ). Gel electrophoresis experiments were carried out on an acrylamide 20 x 20 cm vertical Hoefer 600 electrophoresis unit. Gels were stained using GelRed® (Biotium) or GelGreen® (Biotium), and images were collected using a Gene genius bioimaging system (SYNGENE). Cells Human MCF7 (HTB22) cells were purchased from Wisent and maintained in modified Eagle’s Medium (AMEM; Wisent Bioproducts, Quebec, Canada) and supplemented with 10% FBS and 1% pen/strep at 37°C and 5% CO 2 . Cells were passaged with 0.25% trypsin containing one mM EDTA. For TDN efficacy experiments, cells were plated overnight, grown until confluent, then treated with fTDN with or without DOX and PTX. Nile Red encapsulation The hydrophobic fluorescent molecule Nile Red (Sigma-Aldrich) was used to test the hydrophobic anchor functionality. A DNA solution was prepared with a concentration range of 1 µM to 0.75 nM, a final volume of 150 µL, and a concentration of 2.5 µM Nile Red in 1xTAEMg in a 96-well (CORNING) microplate. The samples were incubated at room temperature for 1 hour and an additional 1 hour at 37°C. Cytation 3 Cell Imaging (Bio Tek) was used for fluorescent measurements (Ex 535 nm Em 560 nm and 750 nm). Paclitaxel and doxorubicin loading Paclitaxel was slowly added to the DNA nanostructure (1 µL/s) on a shaker at room temperature. Next, doxorubicin (Sigma-Aldrich, 62.5 µM) was added dropwise to the sample solution and incubated at 37°C for 1 hour for intercalation. To remove unbound drugs, the sample was filtered with an Illustra micro spin G-25 column (GE Healthcare life sciences). Serum stability For degradation studies, a solution of 20 nM of assembled TDN was prepared in 1xTAEMg buffer. The nanostructure was added into 200 µL cell culture medium (AMEM + 10% FBS + 1% penicillin-streptomycin). At t = 0, an aliquot (10 µL) was collected, formamide (5 µL) was added, and the sample was stored at -20°C. The remaining sample was then incubated at 37°C, and aliquots were collected and treated as described above at specific intervals for up to 48hours. Digested products were analyzed by native PAGE (8%, 15 mA, 80V, 16 hrs). MCF7 TDN uptake Fifty thousand cells were cultured in a glass bottom dish (Mattek®) coated with clear gelatin for 24 h, incubation with different TDN solutions was performed for three hours and cells were fixed with 4% paraformaldehyde. After fixation, cells were stained using Phalloidin-iFluor 488 (green), anti-Cy5 mouse monoclonal (white) and DAPI (blue). Images were acquired with confocal Zeiss Axio Observer 7 equipped with LSM 880 scan head in all channels, including DOX autofluorescence (red). Cell apoptosis Twenty thousand cells were cultured in 96-well plates (CORNING) for 24 h. Then cells were incubated with different TDN treatments described in Fig. 4 followed by staining with annexin V-FITC/PI (ABCAM) following the manufacturer’s instructions at different time points (6, 12 and 24 h). Cells were imaged using an inverted epifluorescence microscope (DMIRE2) and analyzed using ImageJ software based on the number of positive cells per field (3 fields per well). Statistical analysis Mean and standard errors were calculated for all continuous variables. Where appropriate, two-sample comparisons were made using Student’s T-test or Mann-Whitney Rank sum test, depending on the nature of the data distribution. A type I error rate of p < 0.05 was set for statistical significance. All experiments were performed at least three times. 3. Results And Discussion Development of a functionalized TDN (fTDN). The DNA tetrahedron (TDN) is composed of four single DNA strands with distinct sequences and has been investigated as a drug carrier for cancer therapeutics. 34 TDN synthesis, self-assembly and safety have been previously described. 35 The functionalized DNA tetrahedron fTDN described here has two main features: 1) hydrophobic anchors facing the core of the nanostructure; 2) the ability to load two drugs simultaneously. Previously, several DNA nanostructures have been investigated for drug delivery, but they were loaded with only one drug. 36 – 40 Loading the TDN with two drugs will enable the delivery of two drugs with different action mechanisms, decreasing the chances of cancer cell survival. To incorporate the hydrophobic anchors, an alkyl tail (C12) was attached to the 3' end of each of the TDN DNA strands through phosphoramidite chemistry on a DNA synthesizer. Our final tetrahedron construct fTDN was functionalized with four C12 chains (Fig. 1 ), one in each vertex of the structure (facing inside, acting as hydrophobic anchors). We added all four modified strands in 1xTAEMg buffer and thermally annealed the solution 95-4 o C over 12 hours to assemble the tetrahedron (Fig. 2 A-B). Native PAGE and circular dichroism (CD) experiments confirmed the proper assembly of the functionalized tetrahedron fTDN with the hydrophobic anchors (Fig. 2 C, Suppl. Figure 2 ). To test the ability of the hydrophobic anchors to encapsulate hydrophobic molecules, we used Nile Red, a molecule used as a lipid stain due to its increased fluorescence in hydrophobic environments. 26 , 28 , 41 Briefly, solutions containing fTDN (with a concentration range of 1 mM to 0.75 nM) and Nile Red (2.5 mM) in 1xTAEMg were prepared with a final volume of 150 µL. The samples were incubated at room temperature for 1 hour and then at 37°C for 1 hour. Fluorescence was measured in a 96-well top‐read microplate at excitation 560 nm and emission at 635 nm. As shown in Fig. 2 C, fluorescent signals from Nile Red were higher in the solutions containing fTDN and Nile Red than in the control solutions containing fTDN, Nile Red, or fTDN + Nile Red. These results indicate that the hydrophobic anchors within fTDN can encapsulate hydrophobic molecules such as Nile Red, presumably at the core of the nanostructure. Our hydrophobic anchors' essential feature is that they have a short size (calculated length of 1.69 nm each) and have a very high critical micelle concentration (CMC). 42 Therefore, it is unlikely that the C12 chains act as hydrophobic anchors at the concentrations used unless assembled into the DNA nanostructure. Loading of doxorubicin (DOX) and paclitaxel (PTX). In the context of IBC, chemotherapy may be used before (neo-adjuvant) or after surgery (adjuvant) and commonly involve anthracyclines (doxorubicin) or taxanes; therefore, we have chosen to test combination doxorubicin (DOX) and paclitaxel (PTX) in our model. 43 To determine the amount of DOX loaded (intercalated) in the fTDN, a standard fluorescence curve with serial dilution DOX solutions was first prepared, as shown in Fig. 3 A (ex 480 nm, em 590 nm). Next, we prepared a solution containing fTDN (1 µM) and DOX (62.5 µM); the solution was filtered to remove unbound DOX molecules, and the sample fluorescence intensity was measured (Fig. 3 A-B). Using regression analysis, the concentration of loaded DOX was determined to be 0.0325 µM. Further, we confirmed the structural integrity of TDN and fTDN after DOX addition by native PAGE and CD (Fig. 3 C). For drug loading into fTDN, first paclitaxel (PTX) was dissolved in DMSO, and the solution was diluted in Tris-Mg buffer to a final concentration of 100 nM. The solution was slowly (0.14 µL/s) added on a shaker to an fTDN solution (0.5 nM) at room temperature to a final volume of 200 µL and incubated at 37°C for 30 minutes. Unbound PTX molecules were removed by filtering through an Illustra microspin G-25 column. Next, for DOX incorporation, 20 µL of 62.5 µM DOX solution was incubated with 160 µL of 1 µM fTDN + PTX solution for 1 hour at 37°C. Unbound DOX molecules were removed by filtering through an Illustra microspin G-25 column. After purification, the calculated concentration of drugs loaded in the functionalized TDN was 32.5 nM DOX and 0.5 nM PTX. fTDN structure was preserved upon loading both drugs, as characterized by gel electrophoresis and CD, as shown in Fig. 3 C-F. The successful loading of two drugs (DOX and PTX) into a single nanostructure provides more effective treatment options for cancer patients. For example, Ahn and co-workers demonstrated that TDN loaded with DOX significantly inhibits the growth of multidrug resistance (MDR) human breast adenocarcinoma cancer cells (MCF7/ADR). 44 With the incorporation of hydrophobic anchors, we have shown that fTDN can be simultaneously loaded with two chemotherapeutic drugs, including hydrophobic drugs that otherwise could not be used due to their toxicity. Determining the stability of loaded fTDN Since the biological media used for cell experiments is known to contain nucleases capable of degrading DNA, we performed a serum stability assay in 10% fetal bovine serum (FBS) and cell culture media. Briefly, fTDN was added into a solution containing TAEMg buffer + 10% FBS and stability was assessed by native PAGE at different time points (Fig. 3 F). Our results show that TDN starts degrading 1 hour after incubation; however, a small number of intact TDN was detected by gel staining after 48 hours. The degree of degradation for each group was quantified by ImageJ and plotted in Fig. 3 G. Importantly, when the fTDN was loaded with PTX and DOX, the degradation rate was significantly slower than all other groups. We hypothesize that DOX intercalation and the hydrophobic interactions stabilize the nanostructure against denaturation and degradation. Other studies showed that TDNs could survive nearly intact for ≥ 48h inside cells, 45 due to their higher DNA density and well-defined oligonucleotide sequences. 46 While these studies were performed in TDN alone; our results indicate that the fTDN has enhanced stability after being loaded with drugs. As described below, our results indicate that fTDN is stable for enough time to reach its target and release the drugs only once inside the cancer cells. We confirmed the proper assembly of the nanostructure upon loading both drugs by native PAGE and CD (Fig. 3 E). CD is a valuable technique for monitoring DNA conformation changes resulting from environmental conditions and the interaction between DNA ligands (including small molecules and proteins). 47 CD spectra of DNA arise from the asymmetric backbone sugars and the helical structures often adopted by nucleic acids. The B-form is the most common structure of DNA. Base pairs are perpendicular to the double-helix axis, which presents only a soft chirality on the molecule so that the peak forces are moderately short. 48 CD characterization of fTDN shows that its structure remains intact upon drug loading. Further, CD melting temperature experiments indicate that the nanostructure resists melting up to 95 o C (Supplemental Fig. 2), corroborating the increased stability aspect of the fTDN. 49 Biological activity of loaded fTDN To determine the biological activity of the drug-loaded fTDN, we evaluated cell apoptosis and necrosis in MCF7 cells incubated with fTDN for up to 24 hours. Cell apoptosis and necrosis were evaluated under confocal microscopy using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide. As seen in Fig. 4 A, there was a significant decrease in the number of attached cells noted in the bright field and a corresponding increase in cell apoptosis highlighted by Annexin V and loss of membrane integrity highlighted by the increase in PI. The overall cell mortality was more prominent in cells treated with the loaded fTDN [ DOX (32.5 nM) + PTX (0.5 µM)] compared to the control or DOX (1 µM) and PTX (100 nM) diluted in the media. We have evaluated cell cytotoxicity and cell mortality based on loss of confluency (Fig. 4 B), development of round morphology (Fig. 4 C) and relative positive area of Annexin V (Fig. 4 D) and PI (Fig. 4 E). For these experiments, we have included free diluted drugs at the same concentration of the loaded TDN labelled low concentration (LC), as well as clinically relevant concentrations labelled high concentration DOX (1 µM) and PTX (100 nM). The loaded fTDN [DOX (32.5 nM) + PTX (0.5 µM)] induced significantly greater loss of confluency and cell apoptosis/necrosis (Fig. 4 D-E) when compared to the drugs at both concentrations, while the unloaded fTDN showed no increase in cell mortality. These results suggest that co-delivery of DOX and PTX using the TDN is significantly more efficient than treating cells with a much higher concentration (30-fold higher for DOX and 5-fold higher for PTX) of free drugs released in the media. Intracellular localization of TDN and drugs After confirming the co-delivery of the hydrophobic PTX and DOX to MCF7 cells, we further examined the intracellular localization of the fTDN and DOX. MCF7 cells were treated with drug-loaded fTDN for 3 hours, fixed and stained with primary antibodies against Cy5 to increase the signal of the labelled TDNCy5. DOX was visualized at the 595 nm range, while phalloidin was used to visualize the actin cytoskeleton. By optimizing the imaging of a confocal laser scanning microscope, we could detect the signal of the fTDN at the membrane and cytoplasm of the cells. Figure 5 A shows the discrete accumulation of Cy5-labelled TDN at the plasma membrane and in variably sized cytoplasmic vesicles, consistent with previous reports that suggested the presence of TDN in endosomes. 22 There was no significant TDN signal at the nucleus suggesting degradation at the cytoplasm. DOX was primarily seen at the nuclei, colocalizing with DAPI (Fig. 5 A high magnification panels ); which is the expected intracellular localization of this drug. The normalized mean fluorescence intensity (MFI) of the TDNCy5 and DOX was significantly increased in the intracellular compartment, confirming successful uptake (Fig. 5 B-D). 4. Conclusions We successfully functionalized a TDN with hydrophobic anchors in its inner vertices to make a fTDN, capable of successfully loading two chemotherapeutic drugs simultaneously (doxorubicin and paclitaxel) ( Fig. 6 ) . This approach for combination therapy using drugs targeting different mechanisms is ideal for treating solid tumours and minimizing the possibility of selecting resistant cancer cells. Our functionalized nanostructure induced significantly higher mortality levels measured by the area of attached cells, Annexin-V and propidium iodide staining than the free diluted drugs at a much lower concentration. This could potentially lead to lower doses needed in clinic and decrease adverse effects and safety for breast cancer treatment. The proposed DNA TDN model can also be easily modified to explore new targeting motifs and drug combinations, such as other hydrophobic chemotherapeutic drugs that are too toxic to be used in the clinic. Based on our results, it is plausible that the efficient delivery of drugs by the fTDN can cause an increase in the intracellular concentration of the drugs. This could lead to higher cellular toxicity despite the lower concentration of drugs used with the fTDN. Preclinical studies are needed to determine the bioavailability, pharmacokinetics and pharmacodynamics of this fTDN. As the results point to very efficient drug delivery mechanisms, targeting specific cells or tissues will be essential to decrease off-target effects. Further preclinical studies are needed to define the off-target effects, pharmacokinetics and dynamics of the fTDN. Finally, the feasibility and scalability of the TDN production have not been assessed, and further analysis is needed to estimate costs to synthesize the DNA to achieve the same drug levels used in chemo will require tens of grams of the loaded construct; rapid and non-specific clearance as after intravenous administration, Declarations Acknowledgements DE is funded by the Oral and Maxillofacial Pathology Graduate Research Fund, Faculty of Dentistry, MM is funded by the Canadian Institutes of Health Research (CIHR) project grant (PJT-17512) (MM). Data availability statement The datasets used during the current study are available from the corresponding author on reasonable request. All DNA sequences generated during this study are included in this published article (Supplemental table 1). References Canadian Cancer Statistics - A 2018 special report on cancer incidence by stage (Canadian Cancer Statistics Advisory Committee) (2018). Ship JA, Fox PC, Baum BJ. How much saliva is enough? 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Drug Delivery . 2017/01/01 2017;24(1):1295-1301. doi:10.1080/10717544.2017.1373166 Zhang G, Zhang Z, Yang J. DNA Tetrahedron Delivery Enhances Doxorubicin-Induced Apoptosis of HT-29 Colon Cancer Cells. Nanoscale Research Letters . 2017/08/15 2017;12(1):495. doi:10.1186/s11671-017-2272-9 Bagalkot V, Farokhzad OC, Langer R, Jon S. An Aptamer–Doxorubicin Physical Conjugate as a Novel Targeted Drug-Delivery Platform. Angewandte Chemie International Edition . 2006;45(48):8149-8152. doi:https://doi.org/10.1002/anie.200602251 Jiang Q, Song C, Nangreave J, et al. DNA Origami as a Carrier for Circumvention of Drug Resistance. Journal of the American Chemical Society . 2012/08/15 2012;134(32):13396-13403. doi:10.1021/ja304263n Zhao Y-X, Shaw A, Zeng X, Benson E, Nyström AM, Högberg B. DNA Origami Delivery System for Cancer Therapy with Tunable Release Properties. ACS Nano . 2012/10/23 2012;6(10):8684-8691. doi:10.1021/nn3022662 Liu M, Ma W, Li Q, et al. Aptamer-targeted DNA nanostructures with doxorubicin to treat protein tyrosine kinase 7-positive tumours. 2019;52(1):e12511. doi:10.1111/cpr.12511 Greenspan P, Mayer EP, Fowler SD. Nile red: a selective fluorescent stain for intracellular lipid droplets. Journal of Cell Biology . 1985;100(3):965-973. doi:10.1083/jcb.100.3.965 Long JA, Rankin BM, Ben-Amotz D. Micelle Structure and Hydrophobic Hydration. Journal of the American Chemical Society . 2015/08/26 2015;137(33):10809-10815. doi:10.1021/jacs.5b06655 Gehl, M. Boesgaard, T. Paaske, Jensen3 BV, Dombernowsky1 P. Combined doxorubicin and paclitaxel in advanced breast cancer: Effective and cardiotoxic. Annals of Oncology . 1996;7:6. doi:10.1093/oxfordjournals.annonc.a010717 Kim K-R, Kim D-R, Lee T, et al. Drug delivery by a self-assembled DNA tetrahedron for overcoming drug resistance in breast cancer cells. Chemical communications . 02/04 2013;49doi:10.1039/c3cc38693g Walsh AS, Yin H, Erben CM, Wood MJA, Turberfield AJ. DNA cage delivery to mammalian cells. ACS Nano . 2011/07/26 2011;5(7):5427-5432. doi:10.1021/nn2005574 Zagorovsky K, Chou LYT, Chan WCW. Controlling DNA–nanoparticle serum interactions. 2016;113(48):13600-13605. doi:10.1073/pnas.1610028113 %J Proceedings of the National Academy of Sciences Garbett NC, Ragazzon PA, Chaires JB. Circular dichroism to determine binding mode and affinity of ligand–DNA interactions. Nature Protocols . 2007/12/01 2007;2(12):3166-3172. doi:10.1038/nprot.2007.475 Kypr J, Kejnovská I, Renčiuk D, Vorlíčková M. Circular dichroism and conformational polymorphism of DNA. Nucleic Acids Research . 2009;37(6):1713-1725. doi:10.1093/nar/gkp026 Pérez-Arnaiz C, Busto N, Leal JM, García B. New insights into the mechanism of the DNA/Doxorubicin interaction. The Journal of Physical Chemistry B . 2014/02/06 2014;118(5):1288-1295. doi:10.1021/jp411429g Additional Declarations No competing interests reported. Supplementary Files SupplementalMaterialSciReports.pdf Gel1TDN1FBSnativegel12Nov2020.tif Gel2C12FBSnativegel12NOV2020.tif Gel3C12DOXFBSnativegel12Nov2020.tif Gel4C12PACFBSnativegel13Nov2020.tif Gel5C12BothFBSNativegel13Nov2020.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2184757","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":147391692,"identity":"47c3af62-7d1b-4f0d-a4f2-a9a0af0ec33f","order_by":0,"name":"Denise Eymael","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Denise","middleName":"","lastName":"Eymael","suffix":""},{"id":147391693,"identity":"7c0e9ba8-8ba0-4e54-ba8c-b1ab998ae933","order_by":1,"name":"Karina Carneiro","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Karina","middleName":"","lastName":"Carneiro","suffix":""},{"id":147391694,"identity":"c2b7262e-aa3f-427d-823b-b1896e17a9ed","order_by":2,"name":"Marco Magalhaes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYHAD5jYQKUeKFkawFmMGNjDPgHgtiQ2EtOi2n3344QPDNnl594NtD35U1KVvuN987MPHHX/kGNgPP8CmxexMurHkDIbbhhvPJLYb9pw5nLvhGFvyzJlnDIwZeNKwWmV2II2NmYfhNuPGhsQ2Cd62A0AtPMbMvG0GiQ0S2F1ndv4ZWIv9xv6HbZJ//9WlGyC0sH/AquUGxJbE+RKJbdK8DcwJSFp4sNty4xmz5AyD28kbJB62ScscO2w481haMuPMNmNjNp6cAuwOS2P88KHitu38/uRjkm9q6uT5Dh8+zPCxTU6On/34BmxaIADoAIMD6IJsuNVDgHwDIRWjYBSMglEwYgEAAalfYBktzfgAAAAASUVORK5CYII=","orcid":"","institution":"University of Toronto","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Magalhaes","suffix":""}],"badges":[],"createdAt":"2022-10-20 02:29:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2184757/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2184757/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":28458326,"identity":"95abdbbd-5d3e-4eb8-8f0a-279290f5d2be","added_by":"auto","created_at":"2022-10-31 14:06:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":346314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSchematic representation of TDN assembly and drug loading (fTDN).(A) DNA C12 strands (please see suppl. Table 1 for sequence), (B) assembled TDN and (C) fTDN after drug loading\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2184757/v1/030c6b4745036c58f37b8119.png"},{"id":28458337,"identity":"724ff5df-3311-4bab-a94b-cb370dc3495a","added_by":"auto","created_at":"2022-10-31 14:06:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":136998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSelf-assembly of fTDN. A) 8% denaturing gel of DNA-C12 strand (lanes 1-4); B) Circular dichroism of fTDN (red) and TDN (black). C) Fluorescence intensity measurements of buffer, Nile Red (1xTAEMg), TDN + PTX, and fTDN + PTX. (n=3, p=0.01).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2184757/v1/f8745089b1c4e77582f52198.png"},{"id":28459018,"identity":"6769e2c7-3e8c-4fcf-8b4a-ad94fb7f3c7e","added_by":"auto","created_at":"2022-10-31 14:11:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":298866,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDoxorubicin (DOX) loading into DNA tetrahedron. A) Standard fluorescence curve with serial dilution DOX (excitation 480 nm, emission 590 nm). B) Fluorescence intensity measurements of DOX intercalated into DNA TDN before and after filtration (n=3, p=0.01). C) Circular dichroism of fTDN (red) and fTDN + DOX (blue). D) Circular dichroism of fTDN (red) and fTDN + PTX (green). E) Circular dichroism of fTDN (pink) and fTDN + PTX + DOX (purple). F) 8% native PAGE of TDN, fTDN\u003c/em\u003e\u003csub\u003e\u003cem\u003e, \u003c/em\u003e\u003c/sub\u003e\u003cem\u003efTDN\u003c/em\u003e\u003csub\u003e\u003cem\u003e \u003c/em\u003e\u003c/sub\u003e\u003cem\u003e+ PTX, fTDN\u003c/em\u003e\u003csub\u003e\u003cem\u003e \u003c/em\u003e\u003c/sub\u003e\u003cem\u003e+ DOX, and fTDN\u003c/em\u003e\u003csub\u003e\u003cem\u003e \u003c/em\u003e\u003c/sub\u003e\u003cem\u003e+ PTX + DOX incubated in 10% FBS at 37\u003c/em\u003e\u003csup\u003e\u003cem\u003eo\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eC for 0, 3, 5, 12, 24 and 48 hours. The images were cropped and converted to a dark background to facilitate viewing. G) Quantification of DNA degradation based on the optical density of native gels\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2184757/v1/c344ec1a4884e4129ba85b31.png"},{"id":28459020,"identity":"416aafec-2c3e-4762-a571-52a7798e8f39","added_by":"auto","created_at":"2022-10-31 14:11:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":823850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMCF7 survival after fTDN treatment. A) Where indicated the cells were treated with media only (control), DOX\u0026nbsp; (1 µM) + PTX 100 nM (High concentration - HC), DOX 32.5 nM + PTX 0.5 µM (low concentration LC), fTDN or fTDN +\u003c/em\u003e\u003csub\u003e\u003cem\u003e \u003c/em\u003e\u003c/sub\u003e\u003cem\u003eDOX 32.5 nM + PTX 0.5 µM and stained with Annexin V and propidium iodide. The images shown are bright-field (top), propidium iodide-TRITC (middle) and annexin V-FITC (bottom images). B) The images were quantified according to the percentage of attached cells coverage per field using ImageJ. C) Cell morphology was calculated manually as the number of round cells compared to the total cells in each field. The Annexin V (D) and PI (E) signals were calculated as the positive area normalized by the total cell area in the field. The results shown are based on six fields per sample in a total of 3 experiments.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2184757/v1/a2826ca065a7b9fba260a0ed.png"},{"id":28458329,"identity":"70e6d8bc-3024-4714-b70b-f1e0f68e3d59","added_by":"auto","created_at":"2022-10-31 14:06:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":475413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eConfocal microscopy of MCF7 cells with TDN. A) Fixed MCF7 cells were incubated with fTDN + DOX + PTX for 3 hours and were fixed and stained with Phalloidin-iFluor 488 (green), anti-Cy5 mouse monoclonal (white), DAPI (blue), DOX autofluorescence (red). The mean fluorescence intensity (MFI) of each channel was normalized to the extracellular signal. The cytoplasm (C) was highlighted by Phalloidin and nuclei (N) by DAPI signal. The MFI of phalloidin (B), fTDN (C) and doxycycline are shown (D). n=3, *p\u0026lt;0.05 ***p\u0026lt;0.0001\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2184757/v1/32159248a22f60b2f9ac3283.png"},{"id":28459021,"identity":"594a7d55-3a12-4632-aff1-2d8c389b2460","added_by":"auto","created_at":"2022-10-31 14:11:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":265845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSchematics of self-assembly of fTDN and proposed mechanism. (A) Assembled DNA tetrahedron. (B) DNA tetrahedron functionalized with four alkyl chains (C12) as anchor strands at each edge, followed by loading of the nanostructure with doxorubicin (DOX) (C) and paclitaxel (PTX) (D). The fTDN enters the cell in endosomes (E) and is released directly in the cytoplasm (F)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-2184757/v1/4755e7259c3dbde362bd7ccd.png"},{"id":30304771,"identity":"100943a3-e232-4808-87c7-454af6335334","added_by":"auto","created_at":"2022-12-14 07:59:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2024045,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2184757/v1/4eb0c4d8-5881-46c8-8896-2f6ab004b220.pdf"},{"id":28460088,"identity":"a36eab31-3145-4bbf-a00b-25b027279136","added_by":"auto","created_at":"2022-10-31 14:16:10","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":251739,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterialSciReports.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2184757/v1/976d6789187bc1aeb7734ca6.pdf"},{"id":28459024,"identity":"403f635e-f0d4-4625-be10-96909b01536f","added_by":"auto","created_at":"2022-10-31 14:11:10","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2204348,"visible":true,"origin":"","legend":"","description":"","filename":"Gel1TDN1FBSnativegel12Nov2020.tif","url":"https://assets-eu.researchsquare.com/files/rs-2184757/v1/65bfb0e046bcc953f1ba9c21.tif"},{"id":28458331,"identity":"2d5f0afd-d04e-4a83-9c6c-89a1bb27d70a","added_by":"auto","created_at":"2022-10-31 14:06:10","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2055716,"visible":true,"origin":"","legend":"","description":"","filename":"Gel2C12FBSnativegel12NOV2020.tif","url":"https://assets-eu.researchsquare.com/files/rs-2184757/v1/1a48c7d250fdd93f892c7c08.tif"},{"id":28460089,"identity":"dcda1cff-d6d7-43d6-ab0b-b0160b0445c8","added_by":"auto","created_at":"2022-10-31 14:16:10","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2096956,"visible":true,"origin":"","legend":"","description":"","filename":"Gel3C12DOXFBSnativegel12Nov2020.tif","url":"https://assets-eu.researchsquare.com/files/rs-2184757/v1/59872309bf4142df58b39aae.tif"},{"id":28460557,"identity":"037b8ccc-7659-4873-8002-d617b5c25469","added_by":"auto","created_at":"2022-10-31 14:26:10","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1816952,"visible":true,"origin":"","legend":"","description":"","filename":"Gel4C12PACFBSnativegel13Nov2020.tif","url":"https://assets-eu.researchsquare.com/files/rs-2184757/v1/7ad75022c184b3ebd5eb53b0.tif"},{"id":28460432,"identity":"3b090717-aff2-410d-9cee-8cf2c1db42e8","added_by":"auto","created_at":"2022-10-31 14:21:10","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1627448,"visible":true,"origin":"","legend":"","description":"","filename":"Gel5C12BothFBSNativegel13Nov2020.tif","url":"https://assets-eu.researchsquare.com/files/rs-2184757/v1/b849af47b731b66e95e3026c.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functionalized DNA tetrahedron for concomitant delivery of doxorubicin and paclitaxel","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eInvasive breast carcinoma (IBC) affects over 26,800 people per year in Canada, and over 5,000 died in 2019 due to the disease.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Early-stage tumors of IBC are primarily treated by surgery, and chemotherapy may be used before or after surgery. This treatment commonly involves chemotherapeutic agents such as anthracyclines (doxorubicin) or taxanes (paclitaxel); these can significantly increase the morbidity of the treatment, and adverse effects can vary from mucositis, organ damage and death.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Clinically, the most successful approach is to use a combination therapy in which drugs that work by different mechanisms are administered simultaneously, thereby decreasing the likelihood of multidrug-resistant cell syndrome.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Among breast cancer survivors, side effects directly related to treatment can be devastating, including premature ovarian failure, osteoporosis, lymphedema and long-term cardiac problems. \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e In most cases, these result from chemotherapy-based treatments that are not specific to cancer cells leading to significant cytotoxicity.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e The development of new cancer therapies is essential to decrease cancer mortality and morbidity. Various nanocarriers such as self-assembled polymers in the shape of micelles, liposomes and carbon nanoparticles,\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e have been proposed to facilitate anti-cancer drug delivery.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e However, the construction of selective, biocompatible and effective drug delivery agents remains a challenge.\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSelf-assembled DNA nanostructures have been used in therapeutic and diagnostic applications due to effective cellular internalization and high drug loading capability.\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Recently, DNA nanostructures with spatially addressable features were used for controlled delivery of DOX to cancer cells.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e These drug-loaded DNA nanostructures increase the efficacy of chemotherapy, decrease the adverse side effects and show no significant cytotoxicity \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e The DNA tetrahedron (TDN) is one of the most promising nanoconstructs due to its straightforward and predictable assembly in a quantitative manner. \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Turberfield \u003cem\u003eet al.\u003c/em\u003e previously demonstrated the successful cellular uptake of TDN into mammalian cells\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and Jiang \u003cem\u003eet al.\u003c/em\u003e described its\u0026rsquo; use as a DOX nanocarrier for targeted delivery into drug-resistant MCF7 cells.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e The uptake of TDN into mammalian cells is an energy-dependent endocytosis process. The TDN interacts with the plasma membrane non-specifically, followed by rapid cell internalization through the caveolae-mediated pathway. Once inside the cell, it is directed to lysosomes in a microtubule-dependent manner for degradation.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Similar to lipoproteins,\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e lipoprotein-like complexes,\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and lipid droplet encapsulation,\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e functionalized DNA nanostructures with confined nonpolar pockets have recently been designed to encapsulate hydrophobic cargo.\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Typically, DNA strands are functionalized with alkyl chains at a specific location within the nanostructure. Upon assembly, a hydrophobic compartment is generated with the ability to encapsulate nonpolar cargo such as hydrophobic drugs\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs described previously, combining chemotherapeutic agents such as taxanes, cisplatin and anthracyclines can significantly increase the morbidity of the treatment, and adverse effects can vary from mucositis, organ damage and death. \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Therefore, an optimized system to deliver these agents could significantly decrease treatment complications while improving disease control. Herein we describe the synthesis of a DNA nanocarrier, a functionalized DNA tetrahedron (capable of DOX intercalation) containing hydrophobic anchors (for PTX encapsulation), for the concomitant delivery of two cancer therapeutic drugs. Our results indicate that the functionalized TDN (fTDN) can deliver DOX and PTX concomitantly into cancer cell lines. To our knowledge, this is the first time a single DNA carrier has been used to deliver two drugs that work by different mechanisms simultaneously. This approach can potentially overcome the multidrug-resistant cell syndrome in invasive breast cancer treatment, decrease morbidity, and improve treatment outcomes for patients with breast cancer.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cp\u003e \u003cb\u003eReagents\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTris(hydroxymethyl)aminomethane base (ultra-pure, TRS001), ethylenediaminetetraacetic acid disodium dihydrate (EDTA, reagent grade, EDT001), formamide (biotechnology grade, min. 99.5%, FOR001), tetramethylethylenediamine (TEMED, electrophoresis grade, TEM001), 40% acrylamide/bis-acrylamide solution 19:1, ACR005) and urea (ultra pure, molecular biology grade, URE-001) were purchase from Bioshop Canada. Boric acid (B6768), ammonium persulfate (\u0026ge;\u0026thinsp;98%, A3678), doxorubicin (suitable for fluorescence, 98.0-102.0% (HPLC), 44583), paclitaxel (from semisynthetic, \u0026ge;\u0026thinsp;97%, T7191), Nile Red (suitable for fluorescence, \u0026ge;\u0026thinsp;98.0% (HPLC), 19123) were purchase from Sigma-Aldrich. Magnesium chloride hexahydrate (crystalline, USP/FCC, M35-500) was purchased from Fisher Chemical\u0026trade;. Annexin V FITC apoptosis staining / detection kit (ab14085), cleaved caspase-3 staining kit (ab65613), Phalloidin-iFluor 488 (ab176753) and anti-CY5 mouse monoclonal (ab52061) were purchase from Abcam. Acetic acid glacial (lab grade, 00615\u0026thinsp;\u0026minus;\u0026thinsp;540) from Anachemia. PAGE GelRed\u0026reg; Nucleic Acid Gel Stain (41008) and GelGreen\u0026reg; Nucleic Acid Gel Stain (41005) were purchased from Biotium. Illustra micro spin G-25 columns (27-5325-01) were purchased from GE Healthcare. AlamarBlue\u0026trade; Cell Viability Reagent (DAL1025) was purchased from Invitrogen. Alpha Minimum Essential Medium (098150) and fetal bovine serum (FBS, 310-010-CL) were purchased from Multicell. CellLight\u0026trade; Plasma Membrane-CFP, BacMam 2.0 (C10606) and pHrodo\u0026trade; Red Dextran, 10,000 MW, for Endocytosis (P10361) were purchased from Invitrogen.\u003c/p\u003e \u003cp\u003eUnmodified DNA tetrahedron sequences were obtained from a previous publication.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Strands S1-4 were purchased from IDT DNA with polyacrylamide gel electrophoresis purification. The functionalized DNA strands were purchased from GeneLink with PAGE purification. When desired, Cy5 was attached to the 5\u0026rsquo;-end of strand S1, while an alkyl (C12) was attached to the 3\u0026rsquo;-end of strands S1-4 on a DNA synthesizer. All DNA sequences used and their modifications are described in \u003cb\u003eTable S1\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDNA tetrahedron assembly\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDNA tetrahedron was assembled by adding strands S1-4 (20 nM) in 1xTAEMg buffer and performing a 95-4\u003csup\u003eo\u003c/sup\u003eC thermal anneal cycle conducted using Flexigene Techne 96 well thermocycler. DNA characterization was done by denaturing PAGE 15% in TB buffer for strand purity and native PAGE 8% gel in TAEMg buffer for proper assembly into a tetrahedron (\u003cb\u003eSuppl. Figure\u0026nbsp;1\u003c/b\u003e). Gel electrophoresis experiments were carried out on an acrylamide 20 x 20 cm vertical Hoefer 600 electrophoresis unit. Gels were stained using GelRed\u0026reg; (Biotium) or GelGreen\u0026reg; (Biotium), and images were collected using a Gene genius bioimaging system (SYNGENE).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHuman MCF7 (HTB22) cells were purchased from Wisent and maintained in modified Eagle\u0026rsquo;s Medium (AMEM; Wisent Bioproducts, Quebec, Canada) and supplemented with 10% FBS and 1% pen/strep at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were passaged with 0.25% trypsin containing one mM EDTA. For TDN efficacy experiments, cells were plated overnight, grown until confluent, then treated with fTDN with or without DOX and PTX.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNile Red encapsulation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe hydrophobic fluorescent molecule Nile Red (Sigma-Aldrich) was used to test the hydrophobic anchor functionality. A DNA solution was prepared with a concentration range of 1 \u0026micro;M to 0.75 nM, a final volume of 150 \u0026micro;L, and a concentration of 2.5 \u0026micro;M Nile Red in 1xTAEMg in a 96-well (CORNING) microplate. The samples were incubated at room temperature for 1 hour and an additional 1 hour at 37\u0026deg;C. Cytation 3 Cell Imaging (Bio Tek) was used for fluorescent measurements (Ex 535 nm Em 560 nm and 750 nm).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePaclitaxel and doxorubicin loading\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePaclitaxel was slowly added to the DNA nanostructure (1 \u0026micro;L/s) on a shaker at room temperature. Next, doxorubicin (Sigma-Aldrich, 62.5 \u0026micro;M) was added dropwise to the sample solution and incubated at 37\u0026deg;C for 1 hour for intercalation. To remove unbound drugs, the sample was filtered with an Illustra micro spin G-25 column (GE Healthcare life sciences).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSerum stability\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor degradation studies, a solution of 20 nM of assembled TDN was prepared in 1xTAEMg buffer. The nanostructure was added into 200 \u0026micro;L cell culture medium (AMEM\u0026thinsp;+\u0026thinsp;10% FBS\u0026thinsp;+\u0026thinsp;1% penicillin-streptomycin). At t\u0026thinsp;=\u0026thinsp;0, an aliquot (10 \u0026micro;L) was collected, formamide (5 \u0026micro;L) was added, and the sample was stored at -20\u0026deg;C. The remaining sample was then incubated at 37\u0026deg;C, and aliquots were collected and treated as described above at specific intervals for up to 48hours. Digested products were analyzed by native PAGE (8%, 15 mA, 80V, 16 hrs).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMCF7 TDN uptake\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFifty thousand cells were cultured in a glass bottom dish (Mattek\u0026reg;) coated with clear gelatin for 24 h, incubation with different TDN solutions was performed for three hours and cells were fixed with 4% paraformaldehyde. After fixation, cells were stained using Phalloidin-iFluor 488 (green), anti-Cy5 mouse monoclonal (white) and DAPI (blue). Images were acquired with confocal Zeiss Axio Observer 7 equipped with LSM 880 scan head in all channels, including DOX autofluorescence (red).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell apoptosis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTwenty thousand cells were cultured in 96-well plates (CORNING) for 24 h. Then cells were incubated with different TDN treatments described in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e followed by staining with annexin V-FITC/PI (ABCAM) following the manufacturer\u0026rsquo;s instructions at different time points (6, 12 and 24 h). Cells were imaged using an inverted epifluorescence microscope (DMIRE2) and analyzed using ImageJ software based on the number of positive cells per field (3 fields per well).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMean and standard errors were calculated for all continuous variables. Where appropriate, two-sample comparisons were made using Student\u0026rsquo;s T-test or Mann-Whitney Rank sum test, depending on the nature of the data distribution. A type I error rate of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was set for statistical significance. All experiments were performed at least three times.\u003c/p\u003e"},{"header":"3. Results And Discussion","content":"\u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eDevelopment of a functionalized TDN (fTDN).\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe DNA tetrahedron (TDN) is composed of four single DNA strands with distinct sequences and has been investigated as a drug carrier for cancer therapeutics.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e TDN synthesis, self-assembly and safety have been previously described.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e The functionalized DNA tetrahedron fTDN described here has two main features: 1) hydrophobic anchors facing the core of the nanostructure; 2) the ability to load two drugs simultaneously. Previously, several DNA nanostructures have been investigated for drug delivery, but they were loaded with only one drug.\u003csup\u003e\u003cspan additionalcitationids=\"CR37 CR38 CR39\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e Loading the TDN with two drugs will enable the delivery of two drugs with different action mechanisms, decreasing the chances of cancer cell survival.\u003c/p\u003e \u003cp\u003eTo incorporate the hydrophobic anchors, an alkyl tail (C12) was attached to the 3' end of each of the TDN DNA strands through phosphoramidite chemistry on a DNA synthesizer. Our final tetrahedron construct fTDN was functionalized with four C12 chains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), one in each vertex of the structure (facing inside, acting as hydrophobic anchors). We added all four modified strands in 1xTAEMg buffer and thermally annealed the solution 95-4\u003csup\u003eo\u003c/sup\u003eC over 12 hours to assemble the tetrahedron (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Native PAGE and circular dichroism (CD) experiments confirmed the proper assembly of the functionalized tetrahedron fTDN with the hydrophobic anchors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cb\u003eSuppl. Figure\u0026nbsp;2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo test the ability of the hydrophobic anchors to encapsulate hydrophobic molecules, we used Nile Red, a molecule used as a lipid stain due to its increased fluorescence in hydrophobic environments.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Briefly, solutions containing fTDN (with a concentration range of 1 mM to 0.75 nM) and Nile Red (2.5 mM) in 1xTAEMg were prepared with a final volume of 150 \u0026micro;L. The samples were incubated at room temperature for 1 hour and then at 37\u0026deg;C for 1 hour. Fluorescence was measured in a 96-well top‐read microplate at excitation 560 nm and emission at 635 nm. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, fluorescent signals from Nile Red were higher in the solutions containing fTDN and Nile Red than in the control solutions containing fTDN, Nile Red, or fTDN\u0026thinsp;+\u0026thinsp;Nile Red. These results indicate that the hydrophobic anchors within fTDN can encapsulate hydrophobic molecules such as Nile Red, presumably at the core of the nanostructure.\u003c/p\u003e \u003cp\u003eOur hydrophobic anchors' essential feature is that they have a short size (calculated length of 1.69 nm each) and have a very high critical micelle concentration (CMC).\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e Therefore, it is unlikely that the C12 chains act as hydrophobic anchors at the concentrations used unless assembled into the DNA nanostructure.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eLoading of doxorubicin (DOX) and paclitaxel (PTX).\u003c/span\u003e \u003c/p\u003e \u003cp\u003eIn the context of IBC, chemotherapy may be used before (neo-adjuvant) or after surgery (adjuvant) and commonly involve anthracyclines (doxorubicin) or taxanes; therefore, we have chosen to test combination doxorubicin (DOX) and paclitaxel (PTX) in our model.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e To determine the amount of DOX loaded (intercalated) in the fTDN, a standard fluorescence curve with serial dilution DOX solutions was first prepared, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA (ex 480 nm, em 590 nm). Next, we prepared a solution containing fTDN (1 \u0026micro;M) and DOX (62.5 \u0026micro;M); the solution was filtered to remove unbound DOX molecules, and the sample fluorescence intensity was measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). Using regression analysis, the concentration of loaded DOX was determined to be 0.0325 \u0026micro;M. Further, we confirmed the structural integrity of TDN and fTDN after DOX addition by native PAGE and CD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor drug loading into fTDN, first paclitaxel (PTX) was dissolved in DMSO, and the solution was diluted in Tris-Mg buffer to a final concentration of 100 nM. The solution was slowly (0.14 \u0026micro;L/s) added on a shaker to an fTDN solution (0.5 nM) at room temperature to a final volume of 200 \u0026micro;L and incubated at 37\u0026deg;C for 30 minutes. Unbound PTX molecules were removed by filtering through an Illustra microspin G-25 column. Next, for DOX incorporation, 20 \u0026micro;L of 62.5 \u0026micro;M DOX solution was incubated with 160 \u0026micro;L of 1 \u0026micro;M fTDN\u0026thinsp;+\u0026thinsp;PTX solution for 1 hour at 37\u0026deg;C. Unbound DOX molecules were removed by filtering through an Illustra microspin G-25 column. After purification, the calculated concentration of drugs loaded in the functionalized TDN was 32.5 nM DOX and 0.5 nM PTX. fTDN structure was preserved upon loading both drugs, as characterized by gel electrophoresis and CD, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F.\u003c/p\u003e \u003cp\u003eThe successful loading of two drugs (DOX and PTX) into a single nanostructure provides more effective treatment options for cancer patients. For example, Ahn and co-workers demonstrated that TDN loaded with DOX significantly inhibits the growth of multidrug resistance (MDR) human breast adenocarcinoma cancer cells (MCF7/ADR).\u003csup\u003e44\u003c/sup\u003e With the incorporation of hydrophobic anchors, we have shown that fTDN can be simultaneously loaded with two chemotherapeutic drugs, including hydrophobic drugs that otherwise could not be used due to their toxicity.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eDetermining the stability of loaded fTDN\u003c/span\u003e \u003c/p\u003e \u003cp\u003eSince the biological media used for cell experiments is known to contain nucleases capable of degrading DNA, we performed a serum stability assay in 10% fetal bovine serum (FBS) and cell culture media. Briefly, fTDN was added into a solution containing TAEMg buffer\u0026thinsp;+\u0026thinsp;10% FBS and stability was assessed by native PAGE at different time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Our results show that TDN starts degrading 1 hour after incubation; however, a small number of intact TDN was detected by gel staining after 48 hours. The degree of degradation for each group was quantified by ImageJ and plotted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG. Importantly, when the fTDN was loaded with PTX and DOX, the degradation rate was significantly slower than all other groups. We hypothesize that DOX intercalation and the hydrophobic interactions stabilize the nanostructure against denaturation and degradation. Other studies showed that TDNs could survive nearly intact for \u0026ge;\u0026thinsp;48h inside cells,\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e due to their higher DNA density and well-defined oligonucleotide sequences.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e While these studies were performed in TDN alone; our results indicate that the fTDN has enhanced stability after being loaded with drugs. As described below, our results indicate that fTDN is stable for enough time to reach its target and release the drugs only once inside the cancer cells.\u003c/p\u003e \u003cp\u003eWe confirmed the proper assembly of the nanostructure upon loading both drugs by native PAGE and CD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). CD is a valuable technique for monitoring DNA conformation changes resulting from environmental conditions and the interaction between DNA ligands (including small molecules and proteins).\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e CD spectra of DNA arise from the asymmetric backbone sugars and the helical structures often adopted by nucleic acids. The B-form is the most common structure of DNA. Base pairs are perpendicular to the double-helix axis, which presents only a soft chirality on the molecule so that the peak forces are moderately short.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e CD characterization of fTDN shows that its structure remains intact upon drug loading. Further, CD melting temperature experiments indicate that the nanostructure resists melting up to 95\u003csup\u003eo\u003c/sup\u003eC (Supplemental Fig.\u0026nbsp;2), corroborating the increased stability aspect of the fTDN.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eBiological activity of loaded fTDN\u003c/span\u003e \u003c/p\u003e \u003cp\u003eTo determine the biological activity of the drug-loaded fTDN, we evaluated cell apoptosis and necrosis in MCF7 cells incubated with fTDN for up to 24 hours. Cell apoptosis and necrosis were evaluated under confocal microscopy using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, there was a significant decrease in the number of attached cells noted in the bright field and a corresponding increase in cell apoptosis highlighted by Annexin V and loss of membrane integrity highlighted by the increase in PI. The overall cell mortality was more prominent in cells treated with the loaded fTDN [ DOX (32.5 nM)\u0026thinsp;+\u0026thinsp;PTX (0.5 \u0026micro;M)] compared to the control or DOX (1 \u0026micro;M) and PTX (100 nM) diluted in the media. We have evaluated cell cytotoxicity and cell mortality based on loss of confluency (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), development of round morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and relative positive area of Annexin V (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and PI (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). For these experiments, we have included free diluted drugs at the same concentration of the loaded TDN labelled low concentration (LC), as well as clinically relevant concentrations labelled high concentration DOX (1 \u0026micro;M) and PTX (100 nM). The loaded fTDN [DOX (32.5 nM)\u0026thinsp;+\u0026thinsp;PTX (0.5 \u0026micro;M)] induced significantly greater loss of confluency and cell apoptosis/necrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E) when compared to the drugs at both concentrations, while the unloaded fTDN showed no increase in cell mortality. These results suggest that co-delivery of DOX and PTX using the TDN is significantly more efficient than treating cells with a much higher concentration (30-fold higher for DOX and 5-fold higher for PTX) of free drugs released in the media.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIntracellular localization of TDN and drugs\u003c/span\u003e \u003c/p\u003e \u003cp\u003eAfter confirming the co-delivery of the hydrophobic PTX and DOX to MCF7 cells, we further examined the intracellular localization of the fTDN and DOX. MCF7 cells were treated with drug-loaded fTDN for 3 hours, fixed and stained with primary antibodies against Cy5 to increase the signal of the labelled TDNCy5. DOX was visualized at the 595 nm range, while phalloidin was used to visualize the actin cytoskeleton. By optimizing the imaging of a confocal laser scanning microscope, we could detect the signal of the fTDN at the membrane and cytoplasm of the cells. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows the discrete accumulation of Cy5-labelled TDN at the plasma membrane and in variably sized cytoplasmic vesicles, consistent with previous reports that suggested the presence of TDN in endosomes.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e There was no significant TDN signal at the nucleus suggesting degradation at the cytoplasm. DOX was primarily seen at the nuclei, colocalizing with DAPI (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u003cb\u003ehigh magnification panels\u003c/b\u003e); which is the expected intracellular localization of this drug. The normalized mean fluorescence intensity (MFI) of the TDNCy5 and DOX was significantly increased in the intracellular compartment, confirming successful uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eWe successfully functionalized a TDN with hydrophobic anchors in its inner vertices to make a fTDN, capable of successfully loading two chemotherapeutic drugs simultaneously (doxorubicin and paclitaxel) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. This approach for combination therapy using drugs targeting different mechanisms is ideal for treating solid tumours and minimizing the possibility of selecting resistant cancer cells.\u003c/p\u003e \u003cp\u003eOur functionalized nanostructure induced significantly higher mortality levels measured by the area of attached cells, Annexin-V and propidium iodide staining than the free diluted drugs at a much lower concentration. This could potentially lead to lower doses needed in clinic and decrease adverse effects and safety for breast cancer treatment. The proposed DNA TDN model can also be easily modified to explore new targeting motifs and drug combinations, such as other hydrophobic chemotherapeutic drugs that are too toxic to be used in the clinic.\u003c/p\u003e \u003cp\u003eBased on our results, it is plausible that the efficient delivery of drugs by the fTDN can cause an increase in the intracellular concentration of the drugs. This could lead to higher cellular toxicity despite the lower concentration of drugs used with the fTDN. Preclinical studies are needed to determine the bioavailability, pharmacokinetics and pharmacodynamics of this fTDN. As the results point to very efficient drug delivery mechanisms, targeting specific cells or tissues will be essential to decrease off-target effects. Further preclinical studies are needed to define the off-target effects, pharmacokinetics and dynamics of the fTDN. Finally, the feasibility and scalability of the TDN production have not been assessed, and further analysis is needed to estimate costs to synthesize the DNA to achieve the same drug levels used in chemo will require tens of grams of the loaded construct; rapid and non-specific clearance as after intravenous administration,\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDE is funded by the Oral and Maxillofacial Pathology Graduate Research Fund, Faculty of Dentistry, MM is funded by the Canadian Institutes of Health Research (CIHR) project grant (PJT-17512) (MM).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used during the current study are available from the corresponding author on reasonable request. All DNA sequences generated during this study are included in this published article (Supplemental table 1).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCanadian Cancer Statistics - A 2018 special report on cancer incidence by stage (Canadian Cancer Statistics Advisory Committee) (2018).\u003c/li\u003e\n\u003cli\u003eShip JA, Fox PC, Baum BJ. How much saliva is enough? \u0026apos;Normal\u0026apos; function defined. \u003cem\u003eThe Journal of the American Dental Association\u003c/em\u003e. Mar 1991;122(3):63-9. doi:https://doi.org/10.14219/jada.archive.1991.0098\u003c/li\u003e\n\u003cli\u003eGarg AK, Malo M. Manifestations and treatment of xerostomia and associated oral effects secondary to head and neck radiation therapy. \u003cem\u003eThe Journal of the American Dental Association\u003c/em\u003e. Aug 1997;128(8):1128-33. doi:https://doi.org/10.14219/jada.archive.1997.0371\u003c/li\u003e\n\u003cli\u003eNooter K, Stoter G. 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Chemotherapy is commonly used in patients with late-stage breast cancer, which can lead to significant adverse effects. The development of new drug delivery systems is fundamental to improving survival and decreasing morbidity for these patients. Previous studies have shown promising results using DNA nanostructures for doxorubicin (DOX) delivery into drug-resistant breast cancer cells. Here, we successfully modify a DNA tetrahedron (TDN) to include alkyl chains attached to the core of the nanostructure for encapsulation of the hydrophobic drug paclitaxel (PTX). The functionalized TDN (fTDN) simultaneously delivered PTX and DOX into MCF7 cells, leading to improved cell death compared to freely diluted drugs. Our fTDN is a stable and effective delivery system that allows dual drug delivery and shows encouraging results for improving breast cancer chemotherapy.\u003c/p\u003e","manuscriptTitle":"Functionalized DNA tetrahedron for concomitant delivery of doxorubicin and paclitaxel","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-31 14:06:08","doi":"10.21203/rs.3.rs-2184757/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"968287bb-bae2-4c32-b6e7-6cb08479dfe0","owner":[],"postedDate":"October 31st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":16531220,"name":"Biological sciences/Biotechnology/Nanobiotechnology"},{"id":16531221,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Nanostructures"},{"id":16531222,"name":"Health sciences/Oncology"},{"id":16531223,"name":"Health sciences/Oncology/Cancer"}],"tags":[],"updatedAt":"2022-12-14T07:59:18+00:00","versionOfRecord":[],"versionCreatedAt":"2022-10-31 14:06:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2184757","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2184757","identity":"rs-2184757","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}