Responsive Drug Delivery

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Biological and Medical Applications of Materials and Interfaces

Magnesium Stabilized Multifunctional DNA Nanoparticles for Tumor-Targeted and pH-Responsive Drug Delivery Haoran Zhao, Xuexia Yuan, Jiantao Yu, Yishun Huang, Chen Shao, Fan Xiao, Li Lin, Yan Li, and Leilei Tian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01932 • Publication Date (Web): 20 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

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ACS Applied Materials & Interfaces

Magnesium Stabilized Multifunctional DNA Nanoparticles for Tumor-Targeted and pHResponsive Drug Delivery Haoran Zhaoa, Xuexia Yuana, Jiantao Yua, Yishun Huanga, Chen Shaoa, Fan Xiaoa, Li Lina, Yan Lib and Leilei Tiana* a

Department of Materials Science and Engineering, Southern University of Science and

Technology, 1088 Xueyuan Blvd., Nanshan District, Shenzhen, Guangdong 518055, P. R. China. b

Department of Biology, Southern University of Science and Technology, 1088 Xueyuan Blvd.,

Nanshan District, Shenzhen, Guangdong 518055, P. R. China. KEYWORDS tumor-target chemotherapy, DNA nanostructure, drug delivery, stimuli-responsive release, aptamer

ACS Paragon Plus Environment

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ABSTRACT

Functional nucleic acids, that can target cancer cells and realize stimuli-responsive drug-delivery in tumor microenvironment, have been widely applied for anti-cancer chemotherapy. At present, the high cost, unsatisfactory biostability, and complicated fabrication process are the main limits for the development of DNA-based drug-delivery nanocarriers. Here a doxorubicin (Dox)delivery nanoparticle for tumor-targeting chemotherapy is developed taking advantage of rollingcircle-amplification (RCA) technique, by which a high quantity of functional DNAs can be efficiently collected. Furthermore, Mg2+, a major electrolyte in human body showing superior biocompatibility, can sufficiently condense the very long sequence of a RCA product and better preserve its functions. The resultant DNA nanoparticle exhibits a high biostability, making it a safe and ideal nanomaterial for in vivo application. Through cellular and in vivo experiments, we thoroughly demonstrate that this kind of Mg2+ stabilized multi-functional DNA nanoparticles can successfully realize tumor-targeted Dox delivery. Overall, exploiting RCA technique and Mg2+ condensation, this new strategy can fabricate nanoparticles with a non-toxic composition through a simple fabrication process and provides a good way to preserve and promote DNA functions, which will show a broad application potential in the biomedical field.


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INTRODUCTION Chemotherapy has become the primary method to treat cancer today, however, it has an Achilles’ heel, i.e. the anti-cancer drugs can also attack normal cells that are not cytopathic and result in significant side effects.1, 2 Therefore many research efforts have been paid to develop tumor-targeted chemotherapeutics, among which DNA technique plays an important role.3-9 The synthetic oligonucleotide ligands, which are able to recognize a variety of targets with a high affinity and specificity, are named aptamer; and developed against cancer-related targets, like tumor cell-membrane proteins, such aptamers have become a very powerful tool for tumor tracking and therapy.10-13 What’s more, controllable drug release through DNA conformational switches can be triggered by cancer microenvironments, such as pH,14, 15 temperature,16 redox,17 and some proteins.18 Therefore, a great many of chemotherapeutic systems are integrated with functional nucleic acids, in order to realize targeted drug delivery and release.19, 20 Recently, the pure DNA nanostructures,21 like DNA origami, attracted more and more attentions. Recently, Lin et al. developed a series of therapeutic systems based on DNA tetrahedral structures,22-25 showing considerable effects on improving tumor targeting and suppressing. Jiang et al. have applied triangle DNA origami to load Doxorubicin (Dox), which can inhibit lysosomal acidification and promote cellular redistribution of the drugs.26 Zhao et al. exploited different degrees of twist of a DNA nanotube structure to rationally control Dox release kinetics.27 Meanwhile, pure DNA nanostructures still face many challenges, such as the unsatisfactory biostability for intracellular and in vivo application, the complicated fabrication process, and the high cost on a large number of oligonucleotides. The low-stability in serum-containing medium is the foremost problem, for which the primary strategies including reducing the amount of nick sites, improving compaction density of DNA, and protecting by external encapsulation.28, 29


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Recently, a kind of DNA nanostructures, produced from rolling circle amplification (RCA),3033

have attracted a lot of attentions and have been quickly applied to the biomedical field. The

reason behind is that such RCA nanostructures exhibit a good combination of properties, such as low-cost, high-stability, and facile-functionalization. RCA is a cost-friendly method to produce a high quantity of long single-stranded DNA, whose sequence is well defined by and repeatedly copied from the circular template, as a result, a high density of functional sites is introduced to the RCA product. The enzymatically amplified RCA products with build-in functions are condensed by magnesium pyrophosphate (MgPPi), a byproduct of RCA reaction, to form the final hybrid micro/nanostructures, which are called MgPPi-RCA-NanoClew (MgPPi-RNC) in the following part. As the MgPPi-RNCs are highly condensed, they showed good stability in serum environment, making this material very promising for biomedical applications. For instance, Hu et al. constructed multifunctional RCA nanostructures that were integrated with aptamers, bioimaging agents, and drug loading sites for targeted Dox delivery.34 Herein, we reported a new method to fabricate chemotherapeutic nanostructures from RCA products. Instead of using MgPPi, we found that just a certain concentration of Mg2+ can efficiently condense and protect the RCA products from enzymatic degradation. The RCA nanoparticles condensed by excessive Mg2+ show a more appropriate size of ~100 nm, which are called Mg-RCA-NanoClew (Mg-RNC) in the following part. Although Mg2+ is a relatively weaker condensation agent compared with other high-valence cations, such as Cd3+ and Al3+, it is one of the body’s electrolytes which will show better biocompatibility and lower toxicity. Moreover, after removing the excessive Mg2+ via the desalting treatment, the Mg-RNCs can still keep intact and stable, without interior Mg2+ release during a long incubation time. All of these results demonstrate that Mg-RNC is an ideal nanomaterial for in vivo application. On the other


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side, unlike immobilized by MgPPi, the RCA products display electrostatic interactions with Mg2+; such dynamic electrostatic interaction will better preserve DNA functions and facilitate the drug-release process. Through introducing an aptamer structure for cancer cell targeting and a hairpin structure for Dox loading and pH-responsive release, the resultant Mg-RNC drugcarrier system exhibited high bio-stability, intracellular and in vivo targeted Dox-delivery, and pH-stimulated sustained Dox release (Scheme 1).

Scheme 1. The schematic illustration of the formation of Mg2+ stabilized DNA-based Doxdelivery nanoparticles. EXPERIMENTAL SECTION Reagents and Instruments. DNA sequences listed in Table S1, Doxorubicin (Dox), MgCl2, ZnCl2, and thiazolyl blue tetrazolium bromide (MTT) were purchased from Shanghai Sangon


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Biotech. Phi 29 polymerase was purchased from Thermo Fisher. Deoxyribonucleotide triphosphates (dNTPs)，1 kb DNA ladder, 6× loading buffer and T4 ligase were purchased from Takara. Low molecular DNA ladder was purchased from New England Biolabs. 5Fluorescein- 2´-Deoxyuridine, 5´-Triphosphate (dUTP-FAM) was purchased from GeneCopoeia. Ethylene

Diamine

Tetraacetic

Acid

Disodium

Salt

(EDTA-Na2),

Tris(hydroxymethyl)aminomethane (Tris), hydrochloric acid (HCl), phenol/chloroform /isoamyl alcohol (25:24:1), sodium acetate (NaAc), ethyl alcohol (99.9%), and 5× tris/boric acid/EDTA (TBE) buffer were purchased from Bioson Corporation. GelRed (×10,000) was obtained from Biotium. Phosphotungstic acid (PTA) was purchased from Macklin. Phosphate buffered saline (PBS) was prepared by solving the tablets obtained from Amresco in ultrapure water according to the instruction manual (10 mM PBS, containing 137 mM Na+ and 2 mM K+). Agarose RA was purchased from AMRESCO (Solon, USA). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), and penicillin/streptomycin were obtained from Gibco BRL Co., Ltd.. All the cell lines were purchased from Fu Cheung Biotechnology. Centrifugal desalting column was purchased from G-Biosciences. Athymic male BALB/c nude mice were obtained from Vital River Laboratory Animal Technology. Deionized water was prepared from Synergy UV-R (Merckmillipore, Germany). Fluorescence and absorption spectra, and nucleic acid quantification were measured on BioTek Cytation 3 (BioTek, USA). Circular dichroism spectra (CD) were recorded by Chirascan (Applied Photophysics, UK). RCA reactions proceeded on an Eppendorf ThermoMixer C mixer (Eppendorf, Germany). Laser light scattering and Zeta potential were measured on Brookhaven BI-200SM (Brookhaven, USA). Transmission electron microscope (TEM) was measured on Hitachi HT7700 (Hitachi, Japan). Agarose gel electrophoresis was run on Bio-Rad MINI-


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PROTEAN Tetra system (Bio-Rad, USA) and imaged by Tannon 3500 (Tannon, Shanghai). Cellular fluorescence images were taken by Leica TCS-SP8 laser scanning confocal microscope (LSCM, Leica, Germany). The in vivo fluorescence images were captured by IVIS Spectrum System (PerkinElmer, USA). Inductively coupled plasma mass spectrometry (ICP-MS) was performed on Agilent 7700X (Agilent, USA). Protocols for a RCA reaction and sample preparation. There are three steps for a standard RCA reaction. (1) Annealing: Mixing DNA template (T1 or T2, 0.3 µM) with its primer in T4 ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM Dithiothreitol, 1 mM ATP, pH=7.5). After heating at 90 °C for 10 min, the sample was cooled to room temperature gradually. (2) Ligation: Mixing the annealing products with T4 DNA ligase (10.4 U/µL), cultured at 16 °C for 16 h. Subsequently, the ligation product was heated to 65 °C and kept for 10 min to inactivate ligase. (3) Amplification: The ligation product, dNTPs (2 mM each), RCA reaction buffer (50 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM MgCl2, 4 mM Dithiothreitol) and Phi 29 DNA polymerase (0.2 U/µL) were mixed together, which was cultured at 30 °C for 24 h, and after that, was heated at 90 °C for 10 min to inactivate Phi 29 DNA polymerase. The purification of RCA products: (1) RCA product (50 µL) was heated at 90°C and diluted to 75 µL via deionized water. (2) EDTA (0.5 M, 10 µL) was slowly added to the diluted RCA product at 90°C and mixed well. DNA sample was changed from viscous and turbid to clear solution with a low viscosity. (3) Deionized water (165 µL) was added to the mixture to dilute 5 times. (4) Equal volume (250 µL) of mixed organic solvent (Phenol/Chloroform/Isoamyl alcohol=25:24:1) was fully mixed and vibrate with former mixture. (5) Centrifuge the sample and remove organic phase. (6) Add NaAc (3 M, 50 µL) to the aqueous phase. (7) Another 700 µL ice ethanol was added to separate DNA. After storing at -24 °C for 3 h, DNA precipitation


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was separated from the mixture by centrifugation. (8) The precipitation was re-dissolved by deionized water and mixed well. (9) DNA was quantified through the absorption at 260 nm by using a BioTek microplate reader. Preparation of the Mg-RNCs: Purified RCA product (0.1 mg/mL) and MgCl2 (66.67 mM) was mixed thoroughly in deionized water and cultured at 30 °C for 6 h. According to the different templates (T1 and T2 as shown in Table S1), different RCA products, RCA1 and RCA2 were synthesized, and Mg-RNC1 and Mg-RNC2 represent the nanoparticles fabricated from RCA1 and RCA2, respectively. Analysis of the conformation of DNAs in the presence of Mg2+. Laser light scattering was utilized to detect conformation of RCA with various Mg2+ via a laser scattering spectrometer (BI-200SM), which was equipped with a solid laser (35 mW and 637 nm) and a digital correlator (BI-9000AT). The radius of gyration (Rg) was obtained through over an angular range of 35~155° that based on Berry Plot, and the BI-SLSW software figured out Rg directly. The hydrodynamic radius (Rh) was studied over an angular range of 35~90° and obtained by the BIDLSW software. Zeta potential analysis: Samples were tested in DI water. Each sample run 10 times and the final results was gained by get rid of max and min and taking average of remaining data (Table S3). TEM characterization. PTA (1%) was utilized for staining. First 5 µL samples was dropped on the copper grid and wait for 2 h till the samples were roughly dry. Thereafter, drop 5 µL PTA to the samples, wait for 10 min, and use a filter paper to remove the excess PTA. The sample was completely dried in a vacuum oven prior to the test.


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Gel electrophoresis and CD analysis. 0.5% agarose gels stained by GelRed were fabricated. The samples were treated by EDTA to remove the Mg2+ prior to loading on the gel. A solution of H5 (10 µM) was tested at pH 5.0 and pH 7.4 phosphate buffer by CD spectroscopy to check the i-motif forming abilities. CD spectra were collected from 220 to 320 nm at room temperature and the background from the blank buffer has been subtracted. In vitro investigation of Dox loading and release. First, various DNA samples were mixed with Dox (with a constant concentration of 2 µM) in PBS buffer or non-phenolphthalein serum medium (pH 7.4). The fluorescence intensity of Dox (2 µM) at 590 nm in the absence of DNA is defined as I0; The fluorescence of Dox decreased as the increase of DNA concentration; when the fluorescence decreased to the lowest value, the concentration of DNA at this point was used to calculate the maximum Dox-loading capability, and the minimum fluorescence is defined as If. The Dox loaded DNA samples were kept at pH 7.4 or adjusted to pH 5.0, whose fluorescence changes along incubation time were monitored, the fluorescence intensity at a certain time is defined as I. Finally, the ratio of drug release was calculated according to the following equation: Drug release ratio = (I-If) / (I0-If) ×100% Cell culture. CCRF-CEM (a human T lymphocytic leukemia cell line) and Ramos cells (a human B immuneblastic lymphomas cell line) were cultured in DMEM medium mixed with 1% penicillin/streptomycin and 10% (v/v) FBS at 37 °C in INCO2/153 CO2 incubator (Memmert, Germany; 5% CO2). Cytotoxicity analysis of Mg2+, Zn2+ and Mg-RNC. CCRF-CEM cells were seeded by 8000 cells/well in 96 well plates, to which Mg2+ (38 µM), Zn2+ (38 µM), and Mg-RNC1 of different concentrations were added and incubated for 24 h. Thereafter, MTT assays were conducted


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under the manufacturer’s instruction. The relative cell viability of each tested group was normalized by setting the cell viability of control group (in absence of ions or DNA) as “1”. The in vitro cellular characterization. CCRF-CEM and Ramos cells were seeded by 5000 cells/well in 96-well plates with DMEM medium containing 1% penicillin/streptomycin and 10% (v/v) FBS. Cell uptake characterization: Cells were incubated with dUTP-FAM functionalized Mg-RNC1 or Mg-RNC2 (the samples were quantified according to the concentrations of DNA at 31.24 µg/mL) at 37 °C for 6 h. Thereafter the medium was removed and the cells were washed twice by PBS buffer. The cell images were taken under LSCM with an excitation wavelength at 488 nm and the collected emissions of FAM dye from 500 to 560 nm. Dox intracellular release: CCRF-CEM and Ramos cells were incubated with Mg-RNC1@Dox (Mg-RNC1 with maximally loaded Dox), Mg-RNC2@Dox (Mg-RNC2 with maximally loaded Dox) or free Dox (all the samples were unified by the concentration of Dox at 0.024 µM) at 37 °C. After a 6-h incubation, the medium was removed and the cells were washed twice by PBS buffer. And then, colorless complete mediums were added to cultures, which were incubated at 37 °C for another 42 h. The cell images were taken under LSCM with an excitation wavelength at 488 nm and the collected emissions of Dox from 550 to 610 nm. The cytotoxicity of Mg-RNC1@Dox and free Dox: CCRF-CEM and Ramos cells were incubated with various concentrations of Mg-RNC1@Dox or free Dox (the concentrations of the two samples were unified by the concentrations of Dox) at 37 °C. After a 3-h incubation, the medium was removed and the cells were washed twice by PBS buffer. And then, new mediums were added to the cultures, which were incubated at 37 °C for another 45 h. The cell viabilities were calculated according to MTT assays.


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The in vivo bio-distribution of Dox. All animal operations were on the basis of Institutional Animal Use and Care Regulations, according to protocol No. SUSTC-JY2017078, approved by the laboratory animal ethics committee of the Southern University of Science and Technology. Purchased four-week-old mice were acclimated and tested for infectious diseases for one week before use. Subcutaneous tumor model was established by injecting 100 µL of CCRF-CEM cells (5×107 cells/mL) into the back, above the right leg. After two weeks, tumor bearing mice were administrated with 100 µL of saline suspensions of Dox (1.47 mM), Mg-RNC1@Dox (Dox 1.47 mM and DNA 15.17 mg/mL) and Mg-RNC2@Dox (Dox 1.47 mM and DNA 15.17 mg/mL) by intravenous injection. The same volume of saline through intravenous injection was set as a blank control. Notably, to ensure vital signs of mice, Mg-RNC1@Dox and Mg-RNC2@Dox were desalted and concentrated before intravenous injection. In brief, Mg-RNC@Dox was fabricated under a low concentration (DNA: 0.05 mg/mL), followed by freeze-drying to concentrate the sample; the concentrated samples went through desalting column prior to intravenous injection. The mice were sacrificed at 24 h post-injection to collect the tumor tissues and major organs including heart, liver, spleen, lung, kidney. Fluorescence images of the tissues were captured via PerkinElmer IVIS Spectrum with an exciting wavelength of 465 nm and an emission wavelength of 580 nm. RESULTS AND DISCUSSION A double-insured targeted delivery system for chemotherapeutic application was designed to realize both cancer cell targeting and acid-responsive properties. For this purpose, a hierarchical functional nucleic acids (FNA) has been carefully designed, which is comprised of the aptamer segment for cancer cell targeting (red part) and a hairpin structure for Dox loading (green part)


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and pH-responsive release (blue part) (Figure 1). In normal physiological environment, the hairpin structure is closed with Dox loading at the stem region; while in acidic tumor environment, the i-motif sequences at the loop region will fold, which will drive the hairpin structure open and resulting in subsequent drug release. The sequence and length of the stem region of the hairpin are very crucial, which have to promise a high Dox-loading capability and also a sensitive conformational switch in response to pH changes.35-40 Seven different hairpins (H1-H7), with the same i-motif loop sequence but different stems (their sequences are listed in Table S1), were carefully screened. When Dox intercalated within the stem region of DNA hairpin, the fluorescence of Dox will decrease; on the contrary, the fluorescence will recover when Dox is released. Hence the processes of drug loading and release were investigated by the fluorescence spectroscopy and compared among H1-H7 (Figure S1). H5 was determined to be the optimal sequence, which exhibited a high Dox-loading capability of ~ 2 Dox per hairpin in PBS buffer (Figure S2a) and a quick response to the pH change (Figure S2b and S2c).

Figure 1. Illustration the design of RCA sequences to realize cancer cell targeting and pHresponsive Dox loading and releasing functions.


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To demonstrate that the drug release is related to the conformational switch of H5, further characterization by circular dichroism (CD) was conducted. As shown in Figure S2d, in the CD spectrum of H5, the positive main band at ~278 nm was redshifted to ~290 nm when the pH was switched from 7.4 to 5.0, corresponding to the spectral characteristics of an i-motif folding. Combined with the fact that the fluorescence of Dox gradually recovered to the original intensity during an hour after pH switch, we confirm that the drug release is the consequence of the pHdriving conformational switch of H5. On the other hand, the 41-mer sgc8 sequence was chosen as the tumor-targeting aptamer, which shows specific binding to cell membrane receptor protein tyrosine kinase 7 (PTK7),41 a biomarker for T-cell acute lymphoblastic leukemia (T-ALL). PTK7 is overexpressed on the plasma membrane of CCRF-CEM cells (CEM), a human precursor TALL cell line; and a PTK7 under expressed cell line, Ramos, was used as a control.42 Later, by virtue of the RCA technique, the selected sequence is amplified to reach a satisfactory quantity of functional DNAs for the subsequent chemotherapeutic applications. The resultant template and RCA products are called T1 and RCA1; at the same time, we synthesized another template T2 as a control with the corresponding RCA products named RCA2. Being different from RCA1, RCA2 has no targeting function, in which the sgc8 is replaced by a strand with the same length but a random sequence. The amplified products were characterized by agarose gel electrophoresis, RCA1 and RCA2 exhibited similar bands near the sample-loading zones, which indicates that they show comparable molecular size over 15,000 nucleotides (Figure S3). The functional sites have been copied hundreds of times to form a single RCA chain without any nicks; also, a high quantity of RCA products (~ 150 µg/50 µL reaction) was yielded, which provides a cheap way to fabricate functional DNA materials. It has been reported that RCA products can form micro/nanostructures together with MgPPi, which will be called MgPPi-


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RNCs to ease the following discussion. MgPPi-RNCs can be well functionalized through the design of RCA template, and they show good stability in serum environment as a result of their highly condensed structures. However, the difficulty in getting elegant sizes for cellular and in vivo chemotherapeutic application becomes the major limit for the development of MgPPi-RNC. A recent work inspired us,43 which utilized bivalent metallic ions as transfection agents to deliver plasmid DNA and siRNAs. We attempted to use Mg2+ instead of MgPPi to construct the RCA nanostructures. Mg2+ has been chosen according to the following reasons. At the working concentration, Mg2+ showed a relatively lower cytotoxicity than Zn2+ (Figure S4). Besides, Mg2+ is one of the body’s electrolytes; its concentration (0.4-0.6 mM) in human bloodstream is an order magnitude higher than that of Zn2+ (0.01-0.02 mM). Therefore, using Mg2+ can reduce the risk to bring cytotoxicity; Moreover, such sufficient Mg2+ in bloodstream is also benefit to keeping the nanostructures stable. The raw RCA1 products that fabricated by T1 were first purified by methanol precipitation method to remove MgPPi. The purified RCA products (0.1 mg/mL) were mixed with Mg2+ at various Mg/P ratios (the mole ratios of Mg2+ and the phosphate groups of DNAs); and the mixtures were incubated at 30 °C for 6 h. Laser light scattering (LLS) was used to monitor the condensation process of the RCA product in the presence of Mg2+, which is a classic method to study the conformations of bio-macromolecules. According to the LLS data (Table 1), as the Mg/P ratios increased from 0 to 200, the Rg gradually decreased from 192.6 nm down to 90.1 nm, indicating that the RCA1 products shrank in the presence of excessive Mg2+; thereafter, Rg increased to 104.5 nm when the Mg/P ratios further increased to 400. At the same time, the Rg/Rh ratios changed from 1.48 for a Mg/P ratio of 0 to 0.71 for Mg/P a ratio of 400. Based on the established principle, the date indicated that Rg/Rh ratios around 1.5 indicate a random-coil


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conformation, while Rg/Rh ratios reducing to 0.7 demonstrate a solid-sphere conformation. The results are well coincided with the facts that DNA will adopt a coil conformation due to the strong repulsion between its negatively charged phosphate backbone. We also noted that the values of Rg and Rh increased when the Mg/P ratios surpassed 200. It is assumed that the DNA compaction would be along with the multi-chain aggregation process when more cations are added, resulting in the size increase of condensation structures. Therefore, the optimal Mg/P ratio of 200 was utilized in the remaining experiments. The properties of RCA products, which are long single-stranded DNAs showing high local charge-density, also contribute to the formation of Mg-RNCs. As a straightforward evidence, the repeat segment of RCA1 products, which contains both a drug-loading-releasing hairpin and a sgc8 aptamer, is synthesized and named U1 (Table S1). U1 (0.1 mg/mL) was mixed with excessive amount of Mg2+ to reach a Mg/P ratio of 200, thereafter which was processed to the similar incubation procedure. However, no significant conformational change was observed from the LSS data (Table S2), which indicating that, in addition to producing a desired quantity, the high molecular weight of RCA product is also very critical for the formation of Mg-RNCs.

Table 1. Laser light scattering data of RCA products (0.1 mg/mL) in the presence of different amounts of Mg2+; Mg/P ratio is the ratio between Mg2+ and the negative charges of the DNA


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phosphate backbone; Rg is the radius of gyration and Rh is the hydrodynamic radius. The right is an illustration of the conformational changes of DNA as the increase of the amount of Mg2+. The synthesized Mg-RNC1 (synthesized from RCA1) was carefully characterized to evaluate its capability in further biomedical applications. Analyzed by transmission electron microscope (TEM) as shown in Figure 2a, Mg-RNC1 displayed a “fried egg” like nanostructure with a relatively denser inner and irregular periphery. In addition, the morphology of both Mg-RNCs is a kind of similar to that of MgPPi-RNCs (Figure S5b) but is relatively loosely packed and much smaller. Mg-RNC showed a diameter of ~100 nm, which is well consistent with the LLS data. The drug-loading capability of Mg-RNC1 (Figure 2b) was investigated through monitoring the fluorescence of Dox (2 µM) in the presence of different concentrations of RCA1 (5.16~41.28 µg/mL). The fluorescence of Dox decreased to a minimum when the concentration of RCA1 increased to 20.64 µg/mL, corresponding to ~2.5 Dox per repeat unit of RCA1. The drug-loading capability of Mg-RNC1 is well consistent with that of H5, which demonstrates that Dox drugs have been mainly intercalated in the hairpin structure as we designed; moreover, the Mg2+ induced condensation will not reduce the drug-loading capability of the FNAs. On the contrary, MgPPi-RNC1 was demonstrated a relatively low drug-loading capability of 0.67 Dox per repeat unit of RCA1 (Figure S6), which may be due to the more condensed structure of MgPPi-RNC1 (Figure S5b). The stability of a drug-delivery system in serum is very important in determining its in vivo performances. Mg-RNC1 and RCA1 (the purified RCA product without the condensation by Mg2+) were incubated with fetal bovine serum for different periods of time, and the samples were subsequently analyzed by agarose gel electrophoresis. As displayed in Figure 2c, Mg-RNC1, basically, showed no apparent degradation after cultured in serum medium for a long time of 12 h; while RCA1 without Mg2+ condensation was degraded obviously from the


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beginning. The results indicated that the condensation induced by Mg2+ significantly protected the RCA products from degradation in biological environment. Thereafter the in vitro pHtriggered drug release experiment was performed in non-phenolphthalein serum medium (avoiding the interference to Dox fluorescence) during a 72-h period at two distinct pH values of 7.4 and 5.0 (Figure 2d). At pH 7.4, Mg-RNC1@Dox (@Dox represents the corresponding nanoparticle with maximally loaded Dox) released a small part (< 8 %) during the whole period; for MgPPi-RNC1@Dox, more Dox was further adsorbed into the particles gradually. At pH 5.0, Mg-RNC1@Dox exhibited a sustained release of drug during the whole 72-h period; while a burst drug-release at the very beginning was observed for MgPPi-RNC1@Dox. These drugrelease behaviors imply that Dox might be loaded to the two kinds of particles in different ways. The more densely compacted MgPPi-RNC1@Dox will further adsorbed Dox at pH 7.4 and exhibited a faster release at pH 5.0. As MgPPi-RNC1 has a very rough surface, it is likely that, except a small part would be intercalated into the sequences exposed on the surface, most of Dox was mainly adsorbed on the surface of MgPPi-RNC1 through electrostatic interactions. As a result, at pH 7.4 more Dox was absorbed during a long incubation time due to the large surface area of MgPPi-RNC1, exhibiting a negative drug release rate; on the other side, at pH 5.0 the Dox absorbed on surface showed a fast release due to the weak interactions with DNAs. For MgRNC1, Dox can be efficiently intercalated into DNA, as a result, drug release can be precisely controlled by the conformational switch of DNA when pH is changed.


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Figure 2. (a) TEM image of the as-prepared Mg-RNCs; (b) The fluorescence of Dox (2 µM) mixed with different concentrations of RCA products; the insert gives the relationship between fluorescence changes and DNA concentrations; (c) Agarose electrophoresis analysis of the stability of Mg-RNCs and pure RCA products in serum for a variety of incubation time; (d) The Dox-releases from Mg-RNCs and MgPPi-RNC during a 72-h period at two distinct pH values of 7.4 and 5.0.


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Next, cellular experiments were conducted to demonstrate the cancer cell targeting drugrelease capability of Mg-RNC1. First of all, the cytotoxicity of Mg-RNC1 was investigated by a MTT assay. As shown in Figure S7, Mg-RNC1s (DNA concentrations in the nanostructures were 5.16~25.80 µg/mL) were incubated with CEM cells for 24 h; and negligible decreases to cell viability were observed. In order to evaluate the cell uptake capability and specificity, the RCA products were modified by FAM dyes through the addition of dUTP-FAM (1% of the total dNTPs) during the RCA amplification process. Hence the resultant Mg-RNC1-FAM and MgRNC2-FAM (DNA nanoparticles were synthesized from RCA2) showed bright green fluorescence, which were incubated with the target CEM cell line as well as the non-target Ramos cell line for 6 h to evaluate the cellular uptake capability. As shown in Figure S8a, fluorescence in CEM suggested that Mg-RNC1-FAM entered CEM obviously, while the fluorescence intensity in Ramos was too weak to say the particles entered (Figure S8b). Taking advantage of the RCA technique, the sgc8 sequence have been quantitatively amplified in a single RCA chain, resulting in a very high density of sgc8 functional sites on Mg-RNC1, which can well explain why Mg-RNC1 showed a very high cell uptake efficiency and a superior selectivity for target cells. On the contrary, when incubated Mg-RNC2-FAM, both cells showed weak fluorescence with no difference, which indicated that the DNA nanostructures without aptamer functionalization could not enter both cells very efficiently (Figure S8c and S8d). Therefore, we demonstrated that the introduction of sgc8 functional sites actually can promote intracellular uptake in addition to cancer targeting. Furthermore, Dox was loaded to Mg-RNCs; CEM and Ramos cells were treated with Mg-RNC1@Dox, as well as Mg-RNC2@Dox and free Dox at the equivalent concentration of Dox (0.024 µM) as controls. Cells were incubated with drug-delivery systems for 6 h at 37 °C, washed twice by PBS buffer (pH 7.4), and followed by


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another 42-h incubation in medium containing serum. As shown in Figure 3a, the CEM cells cocultured with Mg-RNC1@Dox emitted strong red fluorescence, indicating that Dox was localized inside the cells obviously. By contrast, Ramos co-cultured with Mg-RNC1@Dox showed weak fluorescence from Dox. For the case of CEM incubated with Mg-RNC2@Dox, very weak fluorescence from Dox was observed. Combined with the fact that when free Dox was co-incubated with CEM and Ramos cells, both cells exhibited obvious fluorescence from Dox (Figure 3a and Figure S9); but less internalized Dox than CEM incubated with Mg-RNC1@Dox. We concluded that Mg-RNC1@Dox showed a specific targeting ability and improved intracellular uptake to CEM cells, meanwhile, Dox were well protected by Mg-RNCs before cell uptake to minimize the side effect. The cytotoxicity of Dox delivery by Mg-RNCs and in free state was evaluated in Figure 3b and 3c. As the concentration of Dox increased up to 2.5 µM, the viability of CEM cells incubated with Mg-RNC1@Dox was decreased down to ~20%; while Mg-RNC1@Dox showed no cytotoxicity to Ramos cells even at a high concentration of Dox. On the other hand, free Dox showed high cytotoxicity to both cells without significant difference. The results further confirm that the drug-delivery system, Mg-RNC1@Dox, not only showed a great chemotherapeutic effect to target cells but also a low cytotoxicity to non-target cells. In addition, the viability of CEM that co-cultured with free Dox at 2.5 µM declined to ~40%, therefore, Mg-RNC1@Dox showed an improved capability to induce CEM apoptosis compared with free Dox. This result is consistent with the previous finding that Mg-RNC1@Dox showed a promoted cell uptake capability due to the presence of a high density of sgc8 functional sites on its surface.


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Figure 3. (a) Fluorescence images of CEM and Ramos cells incubated with Mg-RNC1@Dox, Mg-RNC2@Dox, and free Dox, respectively. (b) The cytotoxicity of Dox delivery by MgRNC1@Dox. (c) The cytotoxicity of free Dox. To further confirm the capability of Mg-RNC1@Dox in tumor-targeted Dox delivery, in vivo experiments were conducted. As a much higher concentration of Mg-RNC@Dox is required for in vivo application than that for a cellular experiment, we concentrated the Mg-RNC@Dox samples (the final concentration with respect to DNA is 15.17 mg/mL and for Dox is 1.47mM) and removed the excessive Mg2+ by utilizing salt-removal columns. Interestingly, MgRNC@Dox went through the salt-removal column thoroughly without any residue of free Dox. By contrast, free Dox alone would be completely trapped in the column (Figure S10a). It


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indicated that Dox was still loaded in the DNA nanostructure even when excessive Mg2+ was removed. In addition, through TEM characterization (Figure S10b), Mg-RNC1@Dox samples before and after the salt removal treatment showed identical morphologies. These results demonstrated that an excessive amount of Mg2+ was required to drive DNA condensation; and once the condensation formed, Mg-RNCs would be stabilized by the very few interior Mg2+ (Table S4), whose structure would not be affected by the removal of exterior ions. Furthermore, we found that, when Mg-RNCs were incubated at pH 7.4 and pH 5.0, respectively, no apparent Mg2+ releases were observed for both situations during a 24-h process (Figure S11). The results indicate that the electrostatic interaction between DNAs and Mg2+ is very strong; on one side, this interaction can stabilize the condensation structures of DNA nanoparticles, and moreover, it can efficiently avoid Mg2+ release from the nanoparticles, ensuring good biocompatibility and lower toxicity of this kind of DNA nanoparticles. The CEM subcutaneous tumor-bearing mice were administered with Mg-RNC1@Dox, Mg-RNC2@Dox, free Dox, and saline (except saline, all the samples had equivalent doses of Dox at 4 mg) via intravenous tail injection. The mice were sacrificed at 24 h post-injection, ex vivo Dox fluorescence images of the organs (heart, lung, liver, spleen, and kidney) and tumors were shown in Figure 4. Compared with free Dox, there were stronger fluorescence in tumor tissues of the two DNA nanostructures, both MgRNC1@Dox and Mg-RNC2@Dox, which is due to the enhanced permeability and retention effects (EPR).44-46 The fluorescence images showed that Mg-RNC2@Dox was distributed not only in tumor but also in liver and kidney, while Mg-RNC1@Dox was mainly accumulated in tumor and slightly in liver. More importantly, Dox delivered by Mg-RNC1@Dox accumulated in tumor was much more in quantity than that by Mg-RNC2@Dox. This result demonstrated that,


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in contrast to the passive accumulation of Mg-RNC2@Dox, Mg-RNC1@Dox functionalized with sgc8 aptamers exhibited active targeting effect.

Figure 4. Ex vivo images of organs and tumors excised from CEM tumor-bearing mice at 24 h post-injection of Mg-RNC1@Dox, Mg-RNC2@Dox, free Dox, and saline. CONCLUSION A tumor-targeting Dox-delivery nanoparticle was synthesized by the condensation of RCA products in the presence of an excessive amount of Mg2+. A cancer cell targeting sgc8 aptamer and a hairpin structure showing pH-responsive Dox loading-releasing capability were integrated and quantitatively amplified into the RCA products through a DNA amplification reaction. Due to the very long sequence of a RCA product taking a high charge density, an excessive amount of Mg2+ was demonstrated to be able to condense and sufficiently stabilize the functionalized


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DNAs. The resultant Mg-RNC structure with a diameter of ~100 nm showed excellent biostability in serum, considerable Dox loading capability, specific cancer-targeting ability, and pHresponsive sustained Dox release. The cellular experiment demonstrated that Mg-RNC1@Dox showed a promoted uptake to target cells due to the presence of a high density of sgc8 functional sites on its surface, which not only exhibited a great chemotherapeutic effect to target cells but also a low cytotoxicity to non-target cells. Furthermore, the in vivo experiment confirmed that Mg-RNC1@Dox is a safe drug-delivery nanoparticle for living mice, more importantly, it exhibited an active targeting effect in addition to the passive EPR effect. Prospectively, this general method utilizing RCA technique and Mg2+ condensation will open a new avenue for the development of functional DNA nanostructures for chemotherapeutic applications.

ASSOCIATED CONTENT Supporting Information The following files are available free of charge. List of DNA sequences; Titration curves for Dox loading and release; CD analysis of the conformational changes of H5 under different pH values; Agarose gel electrophoresis analysis of RCA1 and RCA2; TEM images of Mg-RNC2, the RCA product, and MgPPi-RNC; Cytotoxicity of metal ions and Mg-RNC1; Zeta potential analysis of RCA products in presence of different amounts of Mg2+, Cell-uptake capabilities of Mg-RNC1 and Mg-RN2 toward CEM and Ramos cells; TEM characterization of Mg-RNC1 after the salt removal treatment; Mg2+ content in the nanoparticle and the dynamic release of Mg2+ during incubation. (PDF)


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AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT The work is supported by grants from the National Natural Science Foundation of China (No. 51503096), Shenzhen Fundamental Research Programs (No. JCYJ20150630145302244 and JCYJ20160226193029593), and Guangdong Innovative and Entrepreneurial Research Team Program (No. 2016ZT06G587).

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Responsive Drug Delivery

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