DNA Nanostructure Complex

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Biological and Medical Applications of Materials and Interfaces

Isothermal Self-Assembly of Spermidine/DNA Nanostructure Complex as A Functional Platform for Cancer Therapy Dong Wang, Qian Liu, Di Wu, Binfeng He, Jin Li, Chengde Mao, Guansong Wang, and Hang Qian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03464 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 14, 2018

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ACS Applied Materials & Interfaces

Isothermal Self-Assembly of Spermidine/DNA Nanostructure Complex as A Functional Platform for Cancer Therapy Dong Wang,a Qian Liu,a Di Wu,a Binfeng He,a Jin Li,a Chengde Mao, bGuansong Wang a* and Hang Qian a* a. Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China b. Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA Corresponding Author Prof. Guansong Wang and Dr. Hang Qian. Address: Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China. E-mail: [email protected], [email protected] KEYWORDS: DNA nanostructures, spermidine, magnesium-free, self-assembly, cellular uptake

ABSTRACT: Programmable DNA nanostructure self-assembly offer great potentials in nanomedicine, drug delivery, biosensing and bioimaging. However, the intrinsically negativecharged DNA backbones, the instability of DNA nanostructures in physiological settings pose serious challenges to their practical applications. To overcome this challenge, a strategy that

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combines the magnesium-free DNA self-assembly and functionalization is proposed in this study. We hypothesize that naturally abundant spermidine may not only mediate the self-assembly of DNA nanostructures, but also shield them from harsh physiological environments. As a proof-ofconcept, a DNA nanoprism is designed and synthesized successfully through spermidine. It is found that spermidine can mediate the isothermal self-assembly of DNA nanoprisms. Compared with conventional Mg2+ assembled DNA nanostructures, the spermidine/DNA nanoprism complex shows higher thermal stability and better enzymatic resistance than Mg2+ assembled DNA nanoprisms; and more importantly, it has a much higher cellular uptake efficacy in multiple cancerous cell lines. The internalization mechanism is identified as clathrin-mediated endocytosis. To demonstrate the suitability of this new nanomaterial for biomedical applications, an mTOR siRNA, after being conjugated into the complex, is efficiently delivered into cancer cells and shows excellent gene knockdown efficacy and anticancer capability. These findings indicate that the spermidine/DNA complex nanomaterials might be a promising platform for biomedical applications in the future.

Introduction Programmable self-assembled DNA nanostructures for diagnostic and therapeutic applications have increasingly attracted attention in recent years. They have been explored as drug delivery vehicles1-6, bioimaging agents7-8 and biosensors.9-11 However, their practical applications are still limited by several challenges.12 For example, the negative charges carried by DNA nanostructures greatly impede their cellular uptake due to the negatively charged cell membranes. Although there have been several reports showing that DNA origami objects and other small polyhedron structures can be internalized by certain cancerous cell lines without transfection agents,13-16 their efficiencies are far lower than that in the case with the assistance of transfection


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agents. Another challenge is their stability in physiological settings. In addition to nuclease degradation, the synthesis of DNA nanostructures normally necessitates relatively high ion concentrations compared with physiological settings. The Mg2+ concentration used in DNA selfassembly ranges from ~6 to 20 mM, which is greater than ten times higher than that in physiological fluids (~0.4 mM); thus, the integrity of DNA nanostructures might be compromised in such environments.17-18 Moreover, the excess Mg2+ accompanying DNA nanostructures might be harmful to certain intracellular enzymes.19 Thus, developing new, Mg2+ free and bio-interface friendly DNA self-assembly methods and structures is highly desirable. Much efforts have been devoted to the self-assembly of DNA nanostructures along this direction for potential biomedical applications. There are two main ways to tackle the cellular delivery and stability issues: (1) cover the DNA nanostructures with a protective layer through electrostatic interactions and (2) chemically modify the component DNA strands of DNA nanostructures. A few cationic materials, including polymers,20-21 proteins22-23 and peptides,24-25 have been explored as protective layers of DNA nanostructures. For example, Shih et al. have utilized oligolysine or oligolysine-PEG copolymer as protective layers and found that the coated DNA origami objects show much higher DNase I resistance and survival in low salt environments than their uncoated analogs. Moreover, oligolysine-PEG-stabilized DNA objects showed enhanced pharmacokinetic bioavailability in vivo.25 Kostiainen et al. have employed capsid proteins to encapsulate DNA origami structures and achieved enhanced cellular delivery.23 Another strategy is to chemically modify component DNA strands to enhance the stability of the assembled DNA nanostructures. Manetto et al. have modified selected component DNA strands with alkyne groups and azides at the ends of DNA oligonucleotide, and then interlocked them by click reaction.26 The obtained DNA nanostructure is remarkably stable


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under extreme solvent conditions and at high temperatures up to 95 °C.

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However, the

aforementioned strategies also have their limitations. Chemical modification of DNA oligonucleotides is expensive and the potential immune responses might be a concern for practical applications. The post-treatment of DNA nanostructures with a protective layer is difficult and the systemic toxicity of certain cationic polymers remains unexplored. We reason that spermidine could be used to overcome these limitations. Spermidine is one of the naturally abundant polyamines in ribosomes and living tissues, and has been widely used to investigate DNA condensation in vitro.27 It has three positive charges, which can bring negatively charged DNA together efficiently. Spermidine-DNA complexes are potentially stable because of multivalent DNA-spermidine interaction, and this spermidine-DNA tight interaction may partially inhibit DNase activity towards DNA. Furthermore, we envision that this tight DNA-spermidine interaction can neutralize DNA charges and cover DNA nanostructure as a protective layer in physiological settings. The abundant primary and secondary amino groups existed in the hypothesized spermidine/DNA complex nanostructure represent another advantage for the further functionalization of DNA nanostructures for their applications. Thus, spermidine is highly desirable and promising to enable the DNA self-assembly and functionalization. Recently, Simmel et al. reported that polyamines can facilitate the formation of DNA origami structures through a ramped annealing process.19 The assembled DNA origami were stable under high electric field pulses and were successfully electrotransfected into mammalian cells. Nevertheless, whether spermidine can mediate tile-based DNA self-assembly and their potential performances for biomedical applications remain unexplored. In this study, we demonstrated that spermidine can not only mediate the isothermal self-assembly of tile-based DNA nanostructures such as DNA nanoprisms, but also serve as a coating layer to protect the assembled DNA


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nanostructure. To validate the suitability of spermidine assembled DNA nanoprism for potential biomedical applications, a mTOR siRNA binding site is incorporated into the DNA nanoprism design. The spermidine/DNA nanoprism complex (SNP) exhibits high stability and enhanced cellular uptake in multiple cancerous cell lines without the assistance of transfection agents. The strategy proposed here combines DNA self-assembly and functionalization and might be a promising way to extend DNA nanostructures towards real-world applications. Experimental Section Materials and Reagents: All DNA sequences were designed using the sequence design program SEQUIN28 and then ordered from Sangon Biological Engineering. RNA sequences were designed and purchased from Genepharma. All the DNA/RNA sequences were described in supporting information. The Spermidine trihydrochloride, Acryl/Bis 40% Solution (19:1), Tris base, EDTA-Na, Acetic Acid, Boric Acid, Magnesium acetate and Formamide were acquired from Sangon Biological Engineering (Shanghai, China). The Stains-all was obtained from Sigma Aldrich, USA. The LysoTracker green DND-26 was ordered from Life Technologies Corporation, USA. The Hoechst 33342, N-Acetyl-L-cysteine Chlorpromazine, Chloroquine, Genistein, Nystatin and 100bp DNA Ladder were obtained from Sangon Biological Engineering. Endothelial cell medium (ECM), RPMI-1640 Medium, F-12K (Kaighn's) Medium, DMEM, FBS and trypsin (0.25%) were purchased from Gibco (USA).

MTT Cell Proliferation and

Cytotoxicity Assay Kit was acquired from Beyotime Biotechnology (Shanghai, China). Pierce Protein Concentrator (PES) 30KW were purchased from Thermo Fisher Scientific (USA). XtremeGENE siRNA Transfection Reagent and FastStart Essential DNA Green Mastermix were purchased from Roche (Basel, Switzerland).


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Cell Culture: F-12K (Kaighn's) Medium was used to culture A549 cell; RPMI-1640 Medium was used for the culture of H1299 and PC-9 cells; DMEM was used for Hela cell culture and ECM was used for HUVEC cell and HPMVEC cell culture. These different kinds of medium all contained FBS (10%). All cells were grown at 37 °C in a humidified atmosphere containing 5% CO2. Cells were trypsinized when they grew to 80%-90% confluence. Synthesis and Characterization of DNA Nanoprisms: The DNA strands P1, P2 and P1, P2' were mixed together according to a molar ratio of 1:3 in serial concentrations of spermidine trihydrochloride solution (from 50 µM to 1 mM). For regular annealing: 95 °C for 5 min, 65 °C, 50 °C, 37 °C, and 22 °C for 30 min. After the annealing process, the component DNA strands formed two half-nanoprism (triangular shaped structure), then made these two half-nanoprism mix together at the following conditions: 37 °C /60 min, and 22 °C /60 min and these nanoparticles were analyzed by 6% native polyacrylamide (diluted from 40% 19:1 acrylamide: bisacrylamide solution) gel electrophoresis in an electrophoresis unit (Bio-Rad) at room temperature (constant voltage, 125 V) for 1 h with TAE/Mg2+ buffer. Then 0.01% ‘Stains-All’ solution was employed to stain the gels for 2 h before scanned by a Hewlett-Packard scanner. For isothermal self-assembly, the DNA strands mixtures were subjected to constant temperature annealing 4, 22, 37 and 45 °C for 60 min, separately. The following steps were the same with abovementioned. The spermidine trihydrochloride concentration used in this experiment was 100 µM. For mTOR conjugated DNA nanoprism preparation, single-stranded mTOR siRNA were added into the assembled DNA nanoprism at a molar ratio of 6:1 at 22 °C for another 30 min.


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The spermidine trihydrochloride concentration used in this experiment was 100 µM. DNA nanoprism assembled via Mg2+ was also prepared using the same procedures as control. Hydrochloric acid and NaOH solution were used to adjust spermidine trihydrochloride solution pH values by pH meter. The pH for DNA nanoprism synthesis (100 µM spermidine) was selected at 5.8, 6, 7, 8, 9, and 10. Dynamic Light Scattering of DNA Nanoprisms: The DNA nanoprism concentration for DLS measurement was 100 nM (Malvern Zetasizer, Malvern Instruments Laser Target Designator, UK). The solvents were either spermidine trihydrochloride solution or 1×TAE/Mg2+ buffer. Stability Assay of DNA Nanoprisms: Thermal stability was analyzed by 6% PAGE in an electrophoresis unit (Bio-Rad) at 37 °C (constant voltage, 125 V) with 1×TAE/Mg2+ running buffer for 30 mins. And the stability of DNA was examined by incubating with 10% FBS and PBS, then put the mixture at 37 °C for a serial time points and characterized by 6% PAGE in a Hoefer SE 600 Chroma Vertical Electrophoresis System at room temperature (constant voltage, 250 V) with TAE/Mg2+ buffer for 1 h. 0.01% ‘Stains-All’ solution was employed to stain the gels for 2 h before scanned by a HP scanner. Cellular Uptake Studies: The cellular uptake was observed with CLSM and microplate reader. Fluorophore modified P1 was used to form the DNA nanoprism in TAE/Mg2+ buffer (MNP-Cy3) and in spermidine trihydrochloride solution (100 µM, SNP-Cy3). A549 cells (2×104 cells per cm2) were pre-cultured in a 24-well plate with coverslip over each well for 24 h, then incubated with 150 nM MNP-Cy3 and 150 nM SNP-Cy3 for another 24 h. The volume ration between DNA sample and F12-K medium was kept at 1:9. After these treatments, the cells was stained with Hoechst 33342 dye (Final concentration: 1 µg mL-1). Then they were washed with PBS


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buffer for 5 times for 15 min and fixed with 4% paraformaldehyde for 15 min and observed by Leica DMRA2 laser scanning confocal microscope. Different kinds of cells (2×104 cells per cm2) (A549 cell, Hela cell, PC-9 cell, H1299 cell, HUVEC and HPMVEC) were cultured overnight in a 96-well plate (Costar, black), and then treated with medium containing with 150 nM MNP-Cy3 and 150 nM SNP-Cy3 for 12 hour. Whereafter, the cells were dyed with Hoechst 33342 dye (final concentration: 1 µg/mL) for 40 min and washed with PBS buffer for 5 times for 15 min before measured with microplate reader. The excitation and emission wavelengths used for Cy3 and Hoechst 33342 were 540/566 nm and 346/460 nm, respectively. The measurement was conducted using ThemoFisher microplate reader. Endocytosis Pathway Studies: A549 cells were plated in a 96-well plate (Costar, black) overnight. Later, they were pre-incubated for 2 h with the following inhibitors: chlorpromazine (10 µg mL-1), chloroquine (5 µg mL-1), genistein (50 µg mL-1), nystatin (10 µg mL-1). Afterwards, the cells were treated with F12-K medium containing 150 nM MNP-cy3 and 150 nM SNP-cy3 for 12 h. The following steps were similar to the methods which was used in cellular uptake experiment. The uptake of DNA nanoprism was quantified using ThemoFisher microplate reader. Colocalization Study of DNA Nanoprism and Lysosomes: A549 cells (1×104 cells per cm2) were cultured in 35 mm Glass Bottom Dishes (Nunc) overnight, then incubated with 150 nM MNP-Cy3 and 150 nM SNP-Cy3 for another 24 hours. Afterwards, they were stained with LysoTracker green DND-26 (final concentration: 100 nM) for 2 h and Hoechst 33342 dye (final


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concentration: 1 µg mL-1), then washed with PBS buffer for 5 times for 15 min and observed by Leica DMRA2 laser scanning confocal microscope. Cytotoxicity Analysis of Spermidine: A549 cells were cultured in a 96-well plate for 12 h. The cytotoxicity of various concentration spermidine was evaluated by MTT. To analyze the cytotoxicity of spermidine with N-Ac, the cells were disposed with aforesaid steps, the difference was that the cells treated with F12-K medium containing various concentrations of spermidine and 2 mM N-Acetylcysteine. To filter the excess spermidine in SNP solution, the SNP solution was diluted by deionized water in concentrator (30 KW) and centrifuged at 3000 rpm for 3 mins for twice and we harvest the filtered SNP (FSNP). The MTT assay was used to analyze the cytotoxicity of SNP and MNP, A549 cells (1×105 cells per cm2) were cultured overnight, then the cells were treated with F12-K medium containing MNP (150 nM), SNP (150 nM), FSNP (150 nM). Cell Viability Determination by MTT Assay: The MNP and SNP were mixed with mTOR /NC siRNA at a molar ratio of TNP/SNP: mTOR /NC siRNA =1:6 to form the DNA nanoprisms with mTOR siRNA (MNP/mTOR, SNP/mTOR, SNP/NC) at room temperature for 30 mins. A549 cells were cultured in a 96-well plate for 12 h. Then the cells were incubated with mTOR siRNA with transfection agent (300 nM), MNP/mTOR with transfection agent (50 nM), MNP/mTOR (50 nM), SNP (50 nM), SNP/NC (50 nM), SNP/mTOR (50 nM) separately for another 36 h (The set which used transfection agents was washed by fresh medium after 12 h). RT-qPCR Analysis of Gene Expression: The cells (5×105 cells per cm2) were pre-cultured in 6-well plates overnight, followed by replacing the medium and adding different sets of DNA samples (the same with the aforementioned sets). After another 36 h, the treated cells were


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collected and total RNA was extracted from treated A549 cells. cDNAs were acquired from the total RNA using a RT-PCR kit according to the manual. RT-qPCR reactions were carried out by using FastStart Essential DNA Green Mastermix. To correct the experimental variations between samples, the internal control was β-actin. The primers for qPCR measurement are described in supporting information. The 2-∆∆Ct method was used to calculated the mRNA levels. The results were analyzed by Bio-Rad CFX Manager version 2.00. Results and Discussion Design, preparation and characterization of the spermidine/DNA nanoprism complexes A DNA nanoprism was designed to validate the spermidine-mediated self-assembly strategy. As shown in Figure 1A, one copy of central DNA strand P1 and three short strands P2 or P2' form a triangular-shaped structure (detailed sequences were showed in Figure S1). Both P2 and P2' contain a piece of sequence (15 bases) that can hybridize with single-stranded mTOR siRNA at their 5' ends. There are 21 bases at the 3' ends of P2 and P2' (yellow segment in Figure 1B) that can completely associate with each other. Two DNA triangles assembled from P1 and P2, or P1and P2' further assemble into a prism structure (Figure 1B). The self-assembly of DNA triangles or prisms was either mediated by spermidine trihydrochloride (Spd3+) or performed in conventional TAE/Mg2+ buffer (12.5 mM Mg2+).

DNA strands P1, P2 and P1, P2' were mixed together separately and annealed, and these two triangles were then mixed together for another 2 h at 37 °C to form the DNA nanoprisms. Native polyacrylamide gel electrophoresis (PAGE) was used to analyze the formation of the designed DNA nanoprism. In comparison to single-stranded DNA or a triangular-shaped structure, the DNA nanoprisms moved slower and showed as a dominant band on the gel (Figure 2A).


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Figure 1. Schematic illustration of the designed DNA nanoprism. (A) A DNA triangle consists of one core strand P1 and three copies of side strand P2. One protruding segment of P2 (at the 5' end) hybridizes to mTOR siRNA; the other segment is intended to link to another DNA triangle. (B) Two DNA triangles form a DNA prism by the base pairing between P2 and P2' (Yellow). (C) SNP further hybridizes with mTOR siRNA at a molar ratio of 1:6 to form an mTOR siRNA-loaded DNA nanoprism. Typically, a spermidine concentration of 100 µM at pH 7.0 was applied to facilitate the DNA nanoprism self-assembly.

The whole prism structure was approximately 243 bp, and its mobility was comparable to the DNA ladder on the gel. The DNA nanoprisms prepared in TAE/Mg2+ buffer (12.5 mM Mg2+) (MNP) were also used as a control. Apparently, the SNP and MNP have very similar mobilities, suggesting that spermidine indeed mediated the DNA nanoprism self-assembly. We further characterized the designed DNA nanostructures (Figure 3A) with dynamic light scattering (DLS). The DNA nanoprisms prepared in 1×TAE/Mg2+ buffer were also evaluated for comparison (Figure 3B). The hydrodynamic diameters of the triangular-shaped structure, naked DNA nanoprism and DNA nanoprism prepared via Mg2+ were estimated to be 8.7 nm, 10.2 nm and 13.5 nm, respectively (Figure 3C). These sizes are reasonable and in accordance with their theoretical values. The spermidine assembled DNA nanoprism, naked nanoprism and triangle were 16.3%, 14.7% and 16.1% larger than their canonical versions, suggesting that the spermidine surrounding the DNA nanostructures expanded their hydrodynamic sizes. Moreover, the zeta potential of SNP was found to be slightly more positive than MNP, which probably arises from the fact that each spermidine molecule carries three positive charges, whereas Mg2+ is a divalent cation. Interestingly, although SNP and MNP may have different molecular weights and differently positioned cations, their mobilities on PAGE gel are almost the same.


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Figure 2. PAGE analysis of DNA nanoprisms self-assembled under different synthetic conditions. (A) Different combinations of individual DNA strands were mixed and annealed regularly (see the detailed regular annealing procedure in the experimental section). The spermidine concentration was 100 µM, and the synthesis solution pH was set to 7.0. (B) To evaluate the spermidine concentration needed for successful assembly of a DNA nanoprism,


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DNA nanoprisms were prepared with spermidine concentrations varying from 50 µM to 1 mM and run on native PAGE. All samples were annealed through a regular annealing process. (C) The isothermal self-assembly of DNA nanoprisms at different temperatures. The spermidine concentration was kept at 100 µM and the incubation time was 2 hours. DNA nanoprisms assembled with spermidine through regular annealing were also prepared as controls. (D) Native PAGE showing DNA nanoprisms prepared in 100 µM spermidine solution at different pH. All DNA nanoprism samples were annealed through regular annealing. DNA nanoprisms prepared in 1×TAE/Mg2+ buffer and a 100 bp DNA ladder were always used as controls in the gels.

The spermidine-mediated DNA nanoprism assembly was highly dependent on the spermidine concentration. As shown in Figure 2B, DNA nanoprisms were first subject to a stepwise annealing process at different spermidine concentrations. The highest DNA nanoprism yield was found to be approximately 90% at 100 µM spermidine. Spermidine concentrations greater

than 200 µM resulted in large aggregates in the bottom of the well on the gel. The DNA nanoprism concentration in this experiment was 0.88 µM; thus, the negative charged nucleotide concentration is 425 µM (0.88 µM times 243 bp for each DNA nanoprism). This value matches well with the 75~150 µM trivalent cation spermidine. The optimum spermidine concentration is close to charge neutralization and this fact was also found in another work.19 Rapid and high-yielding isothermal self-assembly of complex DNA nanostructures29-33 is technically interesting and desirable in DNA nanotechnology, as it could potentially expand the scope of the practical applications of such nanomaterials. Yin et al. reported the tile-based DNA isothermal self-assembly under non-canonical conditions with deliberately designed singlestranded DNA tiles.32 Simmel et al. demonstrated that denaturing agents such as formamide or urea could mediate the isothermal self-assembly of DNA origami structures.30 In this study, surprisingly, we found that spermidine can mediate DNA nanoprism self-assembly at constant


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Figure 3. DLS analysis of the DNA triangle, naked DNA prism and DNA prism. (A) Structures of the DNA triangle (half-prism), naked nanoprism and nanoprism. (B) DLS characterization of the three DNA structures prepared in 1×TAE/Mg2+ buffer. (C) DLS characterization of the three DNA structures prepared with 100 µM spermidine at pH 7.0. (D) A summary of the hydrodynamic diameters of all DNA nanostructures prepared under different synthetic conditions and the zeta potentials of the DNA nanoprisms. All DNA samples were diluted to 100 nM (normalized DNA nanoprism) for measurements.

temperatures. Figure 2C shows native PAGE analysis of the isothermally self-assembled SNP from 4 °C to 45 °C at the optimized spermidine concentration (100 µM). The highest yield of DNA nanoprisms was achieved at 37 °C. At 45 °C, the DNA nanoprisms were partially disassociated to DNA triangles, whereas at lower temperatures, the assembled products were either single-stranded component DNA oligonucleotides or condensated irregular DNA oligonucleotide aggregates. Notably, the longest component DNA oligonucleotide P1 is up to 66 bases, yet spermidine can still mediate the correct folding of individual DNA oligonucleotides into well-defined prism structures. Additionally, a DNA “shuriken”,5 a DNA nanotube34-35 and an elongated DNA nanoprism were also successfully assembled via spermidine (Figure S2). We reasoned that through rational structural design and optimization of spermidine concentration and annealing temperatures, the isothermal self-assembly of tile-based DNA nanostructures involving short DNA oligonucleotides can be achieved. We next studied the effect of the protonated state of spermidine on DNA nanaoprism selfassembly. A series of DNA nanoprisms were synthesized with 100 µM spermidine at different pH. Native PAGE gel analysis showed that the DNA nanoprism assembled from pH 6.0 to 10.0 exhibited very similar mobilities on the gel (Figure 2D). There were no significant differences in yield within this pH range. These results suggested that the spermidine-mediated DNA nanoprism self-assembly is robust and applicable to diverse synthetic conditions.


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Figure 4. Stability and cytotoxicity of DNA nanoprisms under physiological conditions. (A) Native PAGE run at 37 °C with different DNA strand combinations, indicating that the SNP is stable at 37 °C. (B) Native PAGE gel showing the DNA nanoprisms incubated with 10% FBS at 37 °C for different times. (C) Native PAGE showing that the SNP was intact after intense washing with ultrapure water, whereas it aggregated heavily after being washed with the same concentration of spermidine. (D) An MTT assay evaluating cytotoxicity showed that the cytotoxicity of spermidine can be eliminated by filtering off the free spermidine in the sample solution. (E) Cytotoxicity of spermidine incubated with A549 cells for 24 hours at different concentrations. (F) Alternatively, the cytotoxicity of spermidine can be neutralized by adding 2 mM N-Ac into culture medium during the incubation with cells. The mean ± S.E., n = 5 Statistical P-values: *P < 0.05, #P < 0.05.

Stability and cytotoxicity of spermidine/DNA nanoprism complex A major issue that hinders the practical applications of self-assembled DNA nanostructures is their instability in physiological settings.36 This instability mainly comes from the enzymatic digestion or low ionic environment in physiological fluids. Thus, the stability issue must be comprehensively evaluated and considered prior to any realistic applications. In our case, we first tested the thermal stability of the SNP by running a PAGE gel at 37 °C. Both the SNP and MNP appeared as a sharp and dominant band on the gel (Figure 4A), indicating that the DNA nanoprism design is robust and that the prism nanoparticles were intact at 37 °C.

The

extracellular stability of the SNP was also examined by incubation with 10% FBS for different times at 37 °C. PAGE analysis showed that SNP stayed intact for at least 12 hours and that approximately 40% of the SNP was intact after 48 hours. However, the MNP was quickly degraded after 3 hours and almost totally digested after 48 hours. These results suggest that spermidine can not only mediate DNA self- assembly but also serve as a protective layer for DNA in the unfriendly extracellular environment. Although spermidine was already demonstrated to be able to promote DNA self-assembly, the binding strength between


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spermidine cations and DNA is still unknown compared with the interaction between Mg2+ and DNA. We utilized a spin-column with a molecular weight cut-off of 30k to remove the free spermidine cations from the reaction solution. Ultrapure water or spermidine solution (at the same concentration as that applied during self-assembly, 100 µM) was used as a washing solvent. After spin-column purification, the SNP or MNP was collected and analyzed by native PAGE. As shown in Figure 4C, the SNP washed with ultrapure water remained intact, suggesting that

Figure 5. Cellular uptake study of DNA nanoprisms by CLSM. DNA nanoprisms tagged with Cy3 were incubated


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with A549 cells for 24 hours before imaging. Cell nuclei were stained with Hoechst. The volume ratio between the DNA nanoprism solutions and medium was kept at 1:9. DNA nanoprisms prepared with Mg2+ were also used for comparison. The Cy3 concentration used in this experiment for both DNA nanoprisms was kept at 300 nM (150 nM DNA nanoprsims). Scale bar: 20 µm

spermidine is tightly bound to DNA and can survive low spermidine level environments. Interestingly, the washing spermidine solution that was meant to keep SNP intact led to large aggregates. This phenomenon probably can be explained by the nature of spermidine as a DNA condensing agent and the centrifugal force during the purification process. To roughly determine the amount of spermidine that bounded to DNA after filtration, a ninhydrin reagent assay was conducted. It was found that approximately half of the spermidine were remained in the SNP (Figure S3). The cytotoxicity of spermidine was studied by MTT assay. The IC50 of pure spermidine was measured to be between 75 and 100 µM. At spermidine concentration of 20 µM, the cell viability was decreased to 83.0% in current experiment setting (Figure 4E). Nevertheless, for the DNA self-assembly procedure developed in this study, the spermidine concentration is required to be greater than 50 µM. From Figure 4D, it was also found that the SNP was more toxic than MNP. However, after removal of the excess spermidine in the sample, the cytotoxicity was negligible compared to the control. Furthermore, N-acetylcysteine (N-Ac) has been reported to able to neutralize the toxicity of spermidine. Thus, we added 2 mM N-Ac into the spermidine to culture cells and found that the cytotoxicity of spermidine was indeed neutralized (Figure 4F). This approach, together with the purification method, successfully solved the cytotoxicity issue of spermidine for potential biological applications in the future. Cellular internalization of spermidine/DNA nanoprism complexes and its underlying mechanism


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Figure 6. Cell line-dependent uptake of DNA nanoprisms and the underlying mechanism. The microplate reader assay quantified the cellular uptake efficiencies by measuring the red fluorescence intensities emitted by the internalized DNA nanoprisms. Multiple-point scanning for each well of the 96-well plate was applied to ensure accuracy. All fluorescence intensity data obtained in this study were normalized to the blue fluorescence emitted by


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the Hoechst-stained cell nucleus to eliminate variations resulting from cell density. For all cells, the Cy3 concentration was kept at 300 nM (150 nM DNA nanoprisms). No transfection agents were used in these experiments. (A) Cellular uptake of the MNP and SNP after incubation with A549 cells for 24 hours. (B) The enhanced cellular uptake capability of the SNP was evaluated in a series of primary (HUVEC, HPMVEC) and cancerous (H1299, PC-9 and Hela) cell lines. The MNP was included for all cell lines for comparison. (C) Endocytosis inhibitors chloropromazine and chloroquine (Clathrin inhibitor), genistein and nystatin (Caveolin inhibitor) were used to determine the endocytic pathways of SNP. All inhibitor concentrations were maintained throughout the experiments to ensure the inhibitory effect; and these concentrations were low enough to keep high cell viability. (D) Endocytic pathways of MNP examined by microplate reader assay. The same protocols as with SNP in (C) were applied. The mean ± S.E., n = 4 Statistical P-values: *P < 0.05, #P < 0.05.

The cellular uptake of conventional DNA nanostructures (both tile-based DNA nanostructures and DNA origami objects) assembled from Mg2+ is highly cell line dependent, and the absolute internalized amounts are at very low level without transfection agents.37 To investigate the interactions between cells and the SNP, we first examined the cellular uptake of the SNP in A549 cell line by confocal laser scanning microscopy (CLSM). Both the SNP and MNP were found to be taken up by A549 cells after 24 hours incubation with cells at 37 °C (Figure 5, red dots). However, according to the confocal micrographs, the internalized amount of the SNP was much more than that of the MNP. To more accurately evaluate the uptake efficiency for SNP and MNP, a microplate reader assay was conducted (Figure 6A). Quantitative analysis indicated that almost 2 times more SNP was internalized than MNP. This result is consistent with the CLSM images in Figure 5 and further confirmed that the SNP is more readily taken up by A549 cells than the MNP. We reasoned that this phenomenon might result from the spermidine surrounding the DNA nanoprisms and thus might exist in other cancerous cell lines. Therefore, we further investigated their cellular uptake without any


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transfection agent in three other cancerous cell lines H1299, PC-9, Hela and two primary cell line HUVEC and HPMVEC. Normally, primary cells are harder to transfect than cancerous cells. It was found that the three cancerous cell lines and two primary cell lines hardly took up any MNP, whereas all three cancerous cell lines and HUVECs took up substantial amounts of SNP (Figure 6B). The internalization efficiency of SNP was also cell line dependent. Moreover, the absolute internalized amount of SNP was comparable to that of MNP in the presence of a commercial transfection agent (Figure S4). These findings demonstrated that the SNP indeed exhibits enhanced cellular internalization in vitro.

Figure 7. Intracellular localization of the SNP. CLSM was employed to examine the intracellular localization. A549 cells were treated with 150 nM (300 nM Cy3) DNA nanoprisms (SNP-Cy3) for 36 hours. Scale bar: 10 µm

To elucidate the underlying cellular internalization mechanisms of the spermidinemediated self-assembled DNA nanostructures, a series of endocytosis inhibitors were employed to identify the endocytic pathways. The MNP was also studied for comparison. Figure 6C shows the cellular uptake efficiencies of the SNP with different endocytosis inhibitors. The cellular


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uptake of the SNP was reduced by 50% in the presence of chlorpromazine and chloroquine, both of which are commonly used to block clathrin-mediated endocytosis. In contrast, for caveolinmediated endocytosis, there were no significant differences in the cellular uptakes at the P = 0.05 level between the blocked cells (genistein or nystatin) and those in the control group. Additionally, the cytotoxicity of the four endocytosis inhibitors was evaluated by methyl thiazolyl tetrazolium assay to ensure appropriate inhibitor doses (Figure S5). These results suggest that the SNP was internalized mainly through clathrin-mediated endocytosis. We also studied the endocytic behaviors of the MNP. In contrast to the SNP, the cells blocked with the caveolin inhibitors nystatin and genistein were found to take up only approximately 30% MNP compared with the cells in the control group (Figure 6D), indicating that the main endocytic pathway is mediated by caveolin. The caveolin-mediated endocytosis of the conventional Mg2+ assembled small DNA nanostructures in current study is in consistent with another report.13 We further located the internalized SNP using CLSM (Figure 7). LysoTracker green was used to stain the lysosomes, and the SNP was tagged with Cy3. After 36 hours of internalization, most of the SNPs were aggregated and colocalized with lysosomes (yellow dots in Figure 7). We quantified the colocalization data to evaluate the endosomal escape efficiency of the spermidine/DNA nanoprism. It was found that about 72% of the DNA nanoprism were trapped in lysosomes. It is well recognized that nanoparticles internalized through clathrin-mediated endocytosis normally end up in lysosomes;12 thus, the colocalization of the SNP and lysosomes further confirmed that these nanoprisms were internalized by A549 cells mainly via the clathrinmediated endocytic pathway. The spermidine/DNA nanoprism complex as a drug delivery system for lung cancer therapy


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Figure 8. Spermidine/DNA nanoprism complex as an siRNA delivery vehicle for gene therapy. (A) PAGE gel analysis of the formation of SNP-mTOR. (B) mTOR mRNA expression analysis by RT-qPCR. Cells were treated with a siRNA concentration of 300 nM (50 nM DNA nanoprism). (C) The anticancer effect of the SNP-mTOR examined by MTT assay. siRNA concentration was kept at 300 nM for all sample groups. The mean ± S.E., n = 4 Statistical P-values: *P < 0.05, #P < 0.05.


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To test the suitability of the SNP for potential biological applications, we conjugated an mTOR siRNA with SNP and validated its capability as a drug carrier for gene therapy (Figure 1A). The mTOR gene is a well-studied oncogene in lung cancer and has been proved to be connected to the cancer cell proliferation.38 The overhangs on the DNA nanoprisms have a sequence that can partially hybridize with mTOR siRNA. The successful synthesis of mTOR siRNA-conjugated SNP (SNP-mTOR) was demonstrated by native PAGE in Figure 8A. After incorporation of mTOR-RNA into the DNA nanoprisms, the resultant SNP-mTOR moved slower than SNP on the gel, indicating that the SNP-mTOR has a higher molecular weight than SNP because of the six conjugated mTOR siRNA strands. RT-qPCR was employed to evaluate the gene knockdown efficiency of SNP-mTOR in the NSCLC cell line A549. mTOR siRNAconjugated DNA nanoprisms assembled with Mg2+ (MNP-mTOR) were also synthesized and used for comparison. The SNP-mTOR was found to have an excellent gene knockdown ability in all groups (Figure 8B). The mTOR mRNA level was reduced to 32% by the SNP-mTOR, whereas the same dosage of MNP-mTOR knocked down the target mRNA only to 61%. DNA nanoprisms carrying negative control mTOR siRNA (SNP-NC) were also designed and prepared as a control. As expected, both the SNP-NC and pure DNA nanoprsim groups had negligible gene knockdown efficacy compared with the control group, indicating that the mTOR mRNA was knocked down by siRNA instead of by an off-target effect or by the cytotoxicity of the nanomaterials. We further conducted an MTT assay to investigate the effect of the SNP-mTOR on inhibiting the proliferation of cancer cell. Figure 8C shows that with knockdown mTOR siRNA, the cell viability of the SNP-mTOR-treated group was greatly suppressed to 67.8% compared with the control. Again, the SNP-mTOR exhibited a better killing effect than the MNP-mTOR. It is rational to infer that the excellent gene knockdown efficiency and anticancer


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capability of the SNP result from its higher stability in physiological settings than DNA nanomaterials prepared in conventional ways. Notably, the cellular uptake of unstructured DNA/siRNA complex was also tested for comparison (Figure S6), it was found that the DNA nanoprism structure itself has an effect in the enhanced cellular uptake and gene knockdown performances. These results demonstrated that the SNP is an excellent drug delivery vehicle and that the strategy of combining DNA self-assembly and functionalization together to meet the requirements of biological applications is promising. Conclusion In summary, we demonstrated that spermidine can not only mediate the isothermal selfassembly of tile-based DNA nanostructures but also serve as a protective layer for DNA. The designed spermidine/DNA nanoprism complex exhibited higher stability in physiological settings and enhanced cellular uptake efficiencies in certain cancerous cell lines than conventional DNA nanostructures assembled with Mg2+. Further studies showed that the spermidine/DNA nanoprism complex could be efficiently internalized by cells via clathrinmediated endocytosis, which is quite different from the uptake mechanism of conventional small DNA nanostructures. As a proof-of-concept, the spermidine/DNA nanoprism complex was also tested as a drug delivery vehicle for gene therapy. It showed excellent gene knockdown efficiency and anticancer activity. Our work in this study demonstrated that the strategy combining DNA self-assembly and functionalization is a promising way to apply DNA nanomaterials to cancer therapy and other biomedical applications in the future.39 Although the current study is preliminary and limited to in vitro experiments, we anticipate that spermidine/DNA complex nanomaterials with abundant amino groups hold great potential for further functionalization and applications.


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ASSOCIATED CONTENT Supporting Information. Additional experimental data Figures S1-5 are available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION *Corresponding Author: H. Qian; E-mail: [email protected]; Phone:+86-13594377088 G.S. Wang; E-mail: [email protected]; Phone:+86-13883193995 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank Professor Jianxiang Zhang for the support of the experimental facility. This work is supported by the National Natural Science Foundation of China (31700863 to HQ; China Joint Research Fund for Overseas Chinese, Hong Kong and Macao Scholars 81429001 to CM and GW); the Chongqing Research Program of Basic Science and Frontier Technology, China (No.cstc2017jcyjA1289); the Chongqing Scientific Research Foundation for the Returned Overseas Chinese Scholars (No.CX2017111) and the Clinical Research Projects of Xinqiao Hospital, Third Military Medical University (No.2016YLC09). REFERENCES


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