Toroidal Packaging of pDNA into Block Ionomer Micelles Exerting

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Toroidal Packaging of pDNA into Block Ionomer Micelles Exerting Promoted in vivo Gene Expression Yanmin Li†, Kensuke Osada†,∥,*, Qixian Chen‡, Theofilus A. Tockary‡, Anjaneyulu Dirisala‡, Kaori M. Takeda‡, Satoshi Uchida§, Kazuya Nagata§, Keiji Itaka§, Kazunori Kataoka†,‡,§,* †

Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-

1 Hongo, Bunkyo, Tokyo 113-8656, Japan. ‡

Department of Materials Engineering, Graduate School of Engineering, The University of

Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656, Japan. §

Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine,

Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 1130033, Japan. ∥Japan

Science and Technology Agency, PRESTO, Japan.

KEYWORDS. DNA packaging, Polyion complex, Polyplex micelles, Toroid, Gene delivery

ABSTRACT. Selectively spooling single plasmid DNA (pDNA), as a giant polyelectrolyte, into nano-sized toroidal structure or folding it into rod-like structure has been accomplished by polyion complexation with block catiomers to form polymeric micelles in varying NaCl

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concentrations. The interactive potency between the pDNA and block catiomers was determined to play a critical role in defining the ultimate structure of the pDNA; the formation of toroidal or rod-like structures was achieved by complexation in 600 mM or 0 mM NaCl solutions, respectively. Compared to the rod-like structure, the toroidal structure possessed superior biological functions not only capable of elevating in vitro transcription but also of elevating in vivo gene transduction efficiency. This demonstrated the great utility of the toroidal pDNA packaging as a distinct structured gene carrier. Furthermore, the fact that the NaCl concentration at which the toroidal structure was specifically formed corresponds to seawater stimulates interest on this ordered nanostructure as a possible inherent structure for DNA.

INTRODUCTION Controlling biological macromolecules into particular ordered structure is crucial objective for maximizing their functionality in practical applications. Moreover, the knowledge from these studies could provide important information to infer native structures of these biomacromolecules. Plasmid DNA (pDNA), a giant polyelectrolyte with a contour length of micrometers and a characteristic supercoiled closed-circular topology, is an interesting target to challenge this objective with respect of utility as gene delivery system,1-4 because the controlled packaging of pDNA encapsulated in gene carriers is presumed to correlate with its biological performance. Furthermore, studies that reveal relationship between packaging structure of pDNA and its biological activity may further our understanding of chromatin formation in genome packaging.5,6 The packaging of pDNA is achieved by complexation with cationic compounds to neutralize its negative charge, by which process, DNA subsequently experiences a volume transition from the expanded coil to compacted form,7-9 referred to as DNA condensation. Morphological studies have revealed that pDNA is often condensed into characteristic structures


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of a rod or a donut (toroid) shapes.10-12 These structures are often found to coexist among multiple DNA-associated particles,13-15 and the selective packaging of a single molecule of pDNA into each individual structure has not yet been achieved. Therefore, its successful demonstration would provide considerable impact to approach the above-mentioned objectives. To challenge this, two problems must be solved: the occurrence of spontaneously formed secondary association to induce aggregation and the regulation of DNA packaging process in a controllable manner. To address these problems, we focused on 1) block catiomers containing an additional neutral hydrophilic poly(ethylene glycol) (PEG) block segment to the DNA condensing cationic segment, and 2) varying ionic strengths to control the condensation process by regulating electrostatic interactions. As previously demonstrated, block catiomers allowed a single pDNA molecule to be packaged into the core compartment of spontaneously formed polyplex micelles (PMs),16-18 in which the tethering PEG shell compartment inhibits inter-complex secondary associations that produce aggregates. NaCl was used to regulate the kinetics of the DNA packaging process by modulating electrostatic interactions19,20 between the DNA and block catiomers. Based on these methodologies, we aimed to control the formation of pDNA packaging structures selectively into rod-like or toroidal structures and explored the potential biological activity of each structure as a gene delivery system.

MATERIALS AND METHODS Materials. α-Methoxy-ω-amino-PEG (Mw 12K) was obtained from NOF Co., Ltd. (Tokyo, Japan). β-Benzyl-L-aspartate N-carboxyanhydride (BLA-NCA) was obtained from Chuo Kaseihin Co., Inc. (Tokyo, Japan). Diethylenetriamine (DET), N,N-dimethylformamide (DMF), n-butylamine, benzene, dichloromethane, and trifluoroacetic acid were purchased from Wako


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Pure Chemical Industries, Ltd. Alexa Fluor 488 carboxylic acid succinimidyl ester was purchased from Invitrogen (Carlsbad, CA). Plasmid of pBR322 (4,361 bp) was commercially obtained (Takara Bio Inc., Shiga, Japan) and used for packaging structure studies, while luciferase T7 control DNA (4,315 bp) used for in vitro transcription assays and pCAG-Luc2 (6,477 bp) used for in vivo gene transfer studies were obtained by amplification in competent DH5α Escherichia coli, and purified with a QIAGEN HiSpeed Plasmid MaxiKit (Germantown, MD). Ethidium bromide (EtBr) used as an intercalating agent for DNA was purchased from Sigma-Aldrich Japan G.K. BALB/c mice (female, 7 weeks old) were purchased from Charles River Laboratories (Tokyo, Japan). All animal experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals as stated by the University of Tokyo. Synthesis

of

block

catiomer

PEG-b-poly[N'-[N-(2-aminoethyl)-2-

aminoethyl]aspartamide] [PEG-PAsp(DET)]. Block catiomer of PEG-PAsp(DET) was synthesized according to a procedure as previously described.17,21,22 Briefly, the polymerization of monomer BLA-NCA was initiated from the ω-NH2 terminal group of α-methoxy-ω-amino-PEG to produce PEG-PBLA. The yielded block copolymer was determined to have a unimodal and narrow molecular weight distribution (Mw/Mn = 1.05) by gel permeation chromatography (GPC) measurement. The ultimate product of PEG-PAsp(DET) was obtained through aminolysis reaction by introducing diethylenetriamine into the side chain of PBLA. The degree of polymerization of PAsp(DET) segment was determined to be 61 by comparing 1H-NMR integration ratios between methylene protons in PEG chain (-OCH2CH2-) and the methylene groups in the bis-ethylamine of PAsp(DET) [NH2(CH2)2NH(CH2)2NH-] in D2O at 25 °C. Note


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that all the experimental procedures involved with polymer solution were carried out at low temperature, e.g. 4 °C refrigerator or ice bath to avoid polymer degradation.17 Preparation of pDNA/PEG-PAsp(DET) PMs. Synthesized PEG-PAsp(DET) and pDNA were separately dissolved in 10 mM HEPES buffer (pH 7.4) containing varying NaCl concentration ranging from 0 to 2,000 mM. PMs were prepared by simply mixing both solutions under N/P ratio of 3 [amine groups (N) in PAsp(DET) to phosphate groups (P) in pDNA]. Of note, protonation degree of residing two amino groups in the side chain of PAsp(DET) segment was estimated to be 0.53 at physiological pH 7.4,22 thus N/P ratio of 3, which is slightly higher than the charge stoichiometric ratio, was used to ensure complete complexation. The final pDNA concentration of the complexes was adjusted to 33.3 µg/ml for all PM solutions in all the following experiments unless specifically noted. Post-salt adjustment. Post-dialysis of PM solutions were needed for further insight on the thermodynamic behaviors of rod and toroid structures as well as investigation of their biological activity in the physiological condition. In this regard, the solutions of so-prepared PMs were subjected to dialysis for adjustment of salt concentration to a targeted one in a one-step manner or a stepwise manner. One-step adjustment was conducted as follows: rod-shaped PMs (prepared at 0 mM NaCl) solution was dialyzed against deionized water containing 600 or 1,500 mM NaCl overnight at 4 °C, respectively. Likewise, toroid-shaped PMs (prepared at 600 mM NaCl) solution was dialyzed against deionized water containing 0 or 1,500 mM NaCl overnight at 4 °C, respectively. PM solution prepared at 1,500 mM NaCl was dialyzed against deionized water containing 0 or 600 mM NaCl overnight at 4 °C, respectively. As for stepwise adjustment, rodshaped PM solution (prepared at 0 mM NaCl) was subjected to sequential dialysis against deionized water with NaCl concentration in an upward order of 150, 300, 500, 600, 700, 800,


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900, 1,000, 1,100, 1,200, 1,300, 1,400, and 1,500 mM. Similarly, toroid-shaped PMs (prepared at 600 mM NaCl) was also subjected to sequential dialysis against deionized water with NaCl concentration in a downward order of 500, 400, 300, 150, and 0 mM, or an upward order of 700, 800, 900, 1,000, 1,200, 1,300, 1,400, and 1,500 mM. Following similar way, PM solution prepared at 1,500 mM NaCl was sequentially dialyzed against deionized water with NaCl concentration in a downward order of 1,400, 1,300, 1,200, 1,100, 1,000, 900, 800, 700, 600, 500, 400, 300, 150 and 0 mM. Transmission electron microscopy (TEM) measurement. TEM observation for PMs was conducted using an H-7000 electron microscope (Hitachi, Tokyo, Japan) operated at 75 kV acceleration voltages. The sample was stained by mixing it with equal volume of uranyl acetate (UA) solution [2% (w/v)]. Copper TEM grids (400 mesh) with carbon-coated collodion film, which were previously glow-discharged using an Eiko IB-3 ion coater (Eiko Engineering Co. Ltd., Osaka Japan), were dipped into PMs/UA mixture solution, followed by blotting with filter paper and air-dried. Note that the TEM images show the packaged pDNA within PMs whereas PEG shell is invisible due to its low affinity with UA. The obtained TEM images were further analyzed by Image J 1.44 (available online at http://rsb.info.nih.gov/ij/download. html) to quantify the long axis length of rod/string structures and circumference of toroid structures. Approximate 150 individual PMs were measured for length and frequency analysis for each sample, except for the toroid structures at the 0 mM NaCl due to the lack of enough frequency (Figure 1A). The sample of naked DNA was also subjected to a similar procedure for sample preparation, yet a higher resolution TEM, JEM-1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan), was used.


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Electrophoretic mobility evaluation. The electrophoretic mobility of naked pDNA in 10 mM HEPES buffer (pH 7.4) solution with varying NaCl concentrations was determined by the laser-Doppler electrophoresis measurement using the ELSZ-1000 instrument (Otsuka Electronics Co. Ltd, Osaka, Japan) at 25 ± 0.2 °C. Note that this instrument is capable of accurate measurement up to NaCl concentration of 1,000 mM. Electrophoretic mobility was determined by the equation: U = vL/V [v: velocity of particle (cm/s); V: voltage (V); L: the distance of electrode (cm)]. The results were expressed as absolute value of the average three independent measurements. Determination of binding number of PEG-PAsp(DET) to pDNA in the presence of varying NaCl concentrations. The binding behaviors of block catiomers with pDNA were investigated through ultracentrifuge method. As reported previously,23 ultracentrifugation of complex solution allows selective sedimentation of PMs while free polymers unbound to pDNA remained in the supernatant due to significantly different molecular weight. To quantify the binding number with pDNA, Alexa 488-labled PEG-PAsp(DET) was prepared according to protocol provided by the manufacturer. Successful labeling of PEG-PAsp(DET) was confirmed using GPC with UV, IR, and fluorescence detectors, and the conjugation efficiency was determined to be 0.43 Alexa-488 molecules per block catiomer. Ultracentrifugation of PMs, prepared from Alexa 488-labeled PEG-PAsp(DET) and pBR322 pDNA (4,361 bp) in the presence of varying NaCl concentrations, was carried out for 2 h under 38,000 rpm (Optima TLX, Beckman Coulter, Inc., Fullerton, CA). The fluorescence of supernatant (unbound free polymers) was measured at excitation at λ = 495 nm (emission at λ = 519 nm) using a spectrofluorometer (ND-3300, NanoDrop, Wilmington, DE) and converted to polymer concentration according to a calibration curve of Alexa488-labeled polymer solutions. The


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binding fraction of polymer was determined by subtracting the free polymer from the fed polymer prior to ultracentrifugation. The binding quantity in terms of charge ratio was calculated according to the following method: (binding number × 61 × 2 × 0.53) / (4,361 × 2), given that the polymerization degree of PAsp(DET) segment is 61 with two amine groups residing in one PAsp(DET) unit and the degree of protonation is 0.53 at pH 7.4.22 EtBr exclusion assay. The relative condensation degree of DNA by block catiomers in the presence of varying NaCl concentrations was estimated using EtBr fluorescence quench assay. A series of EtBr, pDNA and block catiomer solutions were prepared to have a final concentration of 16.6 µg/ml, 50 µg/ml and 0.217 mg/ml (each also containing NaCl ranging from 0 to 2,000 mM in concentration) with 10 mM HEPES buffer (pH 7.4). DNA was premixed by EtBr (0.332 µg EtBr relative to 1µg of pDNA) and incubated at 4 °C under dark for 2 h followed by complexation with block catiomer solution in the presence of varying NaCl concentrations. The fluorescence intensity of each samples at λem = 590 nm (λex = 510nm) was measured at 25 °C using a spectrofluorometer (FP-6500, JASCO, Tokyo, Japan). The relative fluorescence intensity was calculated as: Fr = (Fsample - F0)/(F100 - F0), where Fsample, F100, and F0 represent the fluorescence intensity of the samples, free pDNA, and background, respectively. All data represent the mean of three independent measurements. In vitro transcription efficiency. Solution containing all required elements for transcription, i.e., 10 mM ribonucleotides mixture (ATP, CTP, GTP, and UTP), 5 mM DTT, RNA polymerase reaction buffer [40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine(HCl)3, and 25 mM NaCl] (Promega Co., Madison, WI), was added to rod- or toroid-shaped PMs solution containing 20 µg/ml of DNA (both were prepared in the optimal NaCl concentration followed by stepwise dialysis to physiological NaCl concentration of 150 mM). T7 RNA


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polymerase (30 units) (Promega Co., Madison, WI) was then added to each sample, followed by transcription reaction for 2 h at 37 °C. The reaction was stopped by addition of excess ethylenediamine tetraacetic acid (EDTA) to chelate Mg2+. Extraction and purification of the transcribed messenger RNA (mRNA) were conducted by using RNeasy mini kit (QIAGEN Co., Japan). Note that the template DNA was removed by the on column DNase digestion with RNase-Free DNase set (QIAGEN Co., Japan). Quantification of the mRNA was determined by ultraviolet light (UV) absorbance measurement at 260 nm using Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE). Note that the absorption spectra showed typical unimodal profile for RNA with peak top at 260 nm. In vivo gene transfer to skeletal muscle. Hydrodynamic gene introduction into skeletal muscle was performed according to a procedure reported previously.24-26 Briefly, a tourniquet was placed on the proximal thigh of mice to transiently restrict blood flow after anesthetizing with 2.5 % isoflurane (Abbott Laboratories, North Chicago, IL). Rod- or toroid-shaped PM solution (300 µL, 30 µg of luciferase gene-coded pDNA, 150 mM NaCl, prepared at the optimal NaCl concentrations, respectively, followed by adjustment by the stepwise dialysis) was injected into a distal site of the great saphenous vein for approximately 5 s. The tourniquet was kept for 5 min after the injection. Ten minutes prior to in vivo imaging with an imaging system (IVIS) (Xenogen, Alameda, CA), the mice were administered with substrate D-luciferin (Biosynth, Itasca, IL) with a concentration of 150 mg/kg in PBS by intraperitoneal injection and then were anesthetized using 1-3% isoflurane (Abbott Laboratories, North Chicago, IL). Subsequently, the mice were immobilized onto warmed stage (37 °C) within the camera box and were exposed to 1-2% isoflurane to maintain sedation during imaging. Luciferase expression was determined 1 day post-injection by measuring bioluminescence and electronically displayed as a pseudo color


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overlay onto a gray scale animal image (n = 6 for each structure). Images were acquired and processed using Living Image software (Xenogen, Alameda, CA). Note that the background was subtracted from relative light unit (RLU).

RESULTS AND DISCUSSION The block catiomer PEG-PAsp(DET), which has been validated as safe and efficient for use as a gene-delivery carrier,17,21,22,27 was complexed with pDNA in solutions containing NaCl in concentrations ranging from 0 mM to 2,000 mM in 10 mM HEPES (pH 7.4) at a fixed N/P ratio of 3. The prepared PMs were observed using TEM to examine the effect of the salt concentration on the packaging structures of pDNA. The TEM images for PMs prepared at 0 mM NaCl revealed uniform short rod-shaped structures, consistent with our previous reports,27-30 with a markedly high selectivity of 95% among all PM formations (Figure 1A and M). As the NaCl concentration increased, toroidal structures appeared and gradually increased in frequency alongside a gradual drop in the frequency of rod structures (Figure 1B-D and M). The frequency of toroidal structures reached a maximum of approximately 90% at 600 mM NaCl (Figure 1E). As the NaCl concentration increased above 600 mM, the frequency of toroidal structures decreased (Figure 1M). Ultimately, neither rod-like nor toroidal structures were observed at exceedingly high NaCl concentrations (e.g., 2,000 mM); instead, seemingly unpackaged DNA structures were observed (Figure 1K). Because this morphology is similar to that of naked pDNA at 2,000 mM NaCl (Figure 1L), we presume that complex formation between pDNA and block catiomers did not proceed at such a high salt concentration. Note that a possibility of structural change during the sample preparation for the TEM observation due to solvent-evaporation may be excluded because complementary investigations using cryogenic-TEM without any solvent-


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evaporation process also showed the formation of identical rod/toroid polyplex micelle structures.28,,29 Meanwhile, the size of complexed structures was markedly affected by NaCl concentration. As shown in Figure 1N, the average rod length increased with increasing salt concentration. Long rod structures, which may be better described as “strings,” were observed at high NaCl concentrations (> 1,000 mM; Figure 1J). However, the size of the toroidal structures did not appear to depend on salt concentration, with the average circumference remaining within the range of 100-200 nm (Figure 1N). These observations demonstrate the crucial role of NaCl in regulating pDNA packaging structure. Specifically, selective packaging into rod-like or toroidal structures was accomplished by complexation in either a 0 mM or 600 mM NaCl environment, respectively. Notably, the complex formed by the homo-catiomer PAsp(DET) with a DP of 55, which is similar to the DP of the cationic segment in the block catiomer, exhibited no such distinctive packaging structures but formed large aggregates when prepared at 0 or 600 mM NaCl. A fraction of samples prepared at the 600 mM NaCl solution exhibited toroidal structures, but these were indistinct among the coagulates (Figure S1). These observations verified the essential role of PEG block catiomers in achieving uniform pDNA packaging.


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Figure 1. Structural characterization of pDNA complexed with block catiomers at varying NaCl concentrations. Representative TEM images of samples prepared in the presence of (A) 0, (B) 150, (C) 300, (D) 500, (E) 600, (F) 700, (G) 800, (H) 900, (I) 1,200, (J) 1,500, or (K) 2,000 mM NaCl. (L) Naked pDNA in the presence of 2,000 mM NaCl. All scale bars represent 200 nm. Inset in (A) is a magnified image. (M) Frequency of rods/strings (open circle), toroids (closed circle), and other structures, including racket-like shapes (indicated by arrows in TEM images, triangle). (N) Average long axis length (open circle) and toroid circumference (closed circle). Scale bars are 200 nm.

Analysis of the rod/string length or toroid circumference could predict the packaging behavior of pDNA within PMs. As described in our previous report,28, 30 the rod structure prepared at 0


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mM NaCl was analyzed. Note that DNA is observed to be 83.7% of its original length in TEM images of the collodion membrane grid by UA staining.31 The rod length (ln) is represented as a multiple of 1/[2(n + 1)] of the pDNA contour length (1474 nm) by n times folding.30 The apparent average rod length of 57.6 nm with a unimodal distribution (Figure S2A) could describe the rod as a bundle consisting of a pDNA folded 10 times involving 22 double-stranded DNA packed in an orthogonal cross section (SI 3, Table S1). The string structures observed at 1,500 mM NaCl (Figure 1J) had discrete string length distributions at 610, 305, and 200 nm (Figure S2C) consistent with quantized folding30 and were determined to be bundles consisting of pDNA that was either unfolded, folded once, or folded twice (SI 3, Table S1). Similarly, the toroidal structure prepared at the 600 mM NaCl was analyzed by assuming that the toroid was formed by circumferentially winding around a circle (SI 4) (i.e., spooling). 32-34 The average circumference of the toroidal structures measured as 175 nm with a unimodal distribution (Figure S2B) could assign a spooling number of 6 (Table S2). Accordingly, this indicates that 7 double-stranded DNAs were packed in the orthogonal cross section of the toroid. Interestingly, 7 is the critical number required to form a hexagonal lattice, as illustrated in Scheme S1, which implies that DNA chains inherently prefer the hexagonal packing as found in genomic DNA packing in bacteriophages.35,36 The relative invariance of toroidal circumference over a range of salt concentrations (Figure 1N) may suggest the energetic favorability of this packing pattern.37 The effect of salt on packaging structure was investigated to understand the principles underlying the formation of the distinctive packaging structures. First, a change in the helical structure of DNA due to NaCl concentration38,39 can be excluded as an explanation of the different packaging structures observed, because the circular dichroism (CD) spectra of naked pDNA exhibited identical profiles over the investigated range of 0 - 2,000 mM NaCl (SI 5,


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Figure S3). Thus, we turned our focus to the interaction between block catiomers and pDNA over a range of salt concentrations. Salt is known to have a marked effect on electrostatic interactions due to charge shielding. To examine this effect, the electrophoretic mobility of naked pDNA as a function of NaCl concentration was measured to estimate the electrostatic “strength” of pDNA. As shown in Figure 2A, the magnitude of the electrophoretic mobility decreased as the NaCl concentration increased from 0 mM to 600 mM, but appeared to reach a plateau when the NaCl concentration exceeded 600 mM. This profile suggests that the potency of the electrostatic interaction is preserved up to 600 mM NaCl, with its contribution decreasing as the NaCl concentration increased before reaching a limit at NaCl concentrations above 600 mM. Complexation behavior was then examined by monitoring the number of block catiomers bound to pDNA using the ultracentrifuge method. At 0 mM NaCl, 144 block catiomers bound to each pDNA, corresponding to a block catiomer to pDNA charge ratio of 1.07, given a protonation degree of the PAsp(DET) side chain of 0.53 at pH 7.4.22 This result indicated that complex formation was driven by charge neutralization, which is supported by evidence of the neutral ζ-potential of this PM (SI 6, Figure S4). The binding number decreased with increasing NaCl concentration and almost no block catiomers were bound to pDNA at 2,000 mM (Figure 2B), which is consistent with previous observations of a non-packaged DNA structure at that salt concentration (Figure 1K). A slight decrease in the binding number was observed at 600-700 mM NaCl, followed by a consistent decrease until 2,000 mM NaCl. The NaCl concentration corresponding to this slight decrease was coincident with the plateau in electrophoretic mobility (Figure 2A). An EtBr exclusion assay, which is commonly used to measure the degree of DNA condensation, also verified a significant change in the degree of condensation within this NaCl concentration range, with a plateau in EtBr fluorescence intensities beginning at 700 mM NaCl


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(Figure 2C). Intensities above 700 mM NaCl were comparable to those of naked DNA, which suggested that the DNA was not fully condensed at this salt concentration, although complex formation was still observed via TEM imaging (Figure 1) and binding number measurement (Figure 2B). Together, these experiments demonstrate that salt concentrations of 600-700 mM may be regarded as critical for complexation. It should be noted that the major driving force for polyion complexation is recognized to be entropy gain due to the liberation of counter-ions to bulk solution.40,41 Accordingly, at low salt concentration (< 600 mM NaCl), this scheme plays a substantial role in block catiomer association with pDNA. However, at higher salt concentration (> 700 mM NaCl), entropy gain due to counter-ion release may not be substantial for the complex formation because of the presence of abundant ions in bulk media. Then, enthalpy favorability between the charge-shielded polyelectrolytes might serve as a primary interaction mode for complexation, even though complexation was not promoted, as the binding number was far below the charge stoichiometry (Figure 2B). A fine balance of interactive potency between pDNA and block catiomers may intricately commit in the complexation mode and play a critical role in determining the pathway to develop the most favored structure at each condition, where an NaCl concentration of 0 or 600 mM could be the most favorable condition for the formation of rod-like or toroidal structures, respectively.


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Figure 2. NaCl concentration affects the structure of naked pDNA and its complexation with block catiomers. (A) The electrophoretic mobility of naked pDNA at various NaCl concentrations. (B) Quantification of the number of block catiomers binding to pDNA. (C) The fluorescence quenching activity of complexed pDNA with block catiomers in the presence of EtBr. Each fluorescence value was normalized to the fluorescence of naked pDNA at the corresponding NaCl concentration.

To promote these distinct structures in a variety of applications, it is crucial to establish a methodology for preserving each structure in different environments. To this end, we adjusted the salt concentration of the complex solution by dialysis treatment of samples prepared at each favored condition. The solution used to selectively prepare rod-like structures at 0 mM NaCl was adjusted to a targeted NaCl concentration of 600 mM by dialysis against 600 mM NaCl, while the solution used to selectively prepare toroidal structures at 600 mM NaCl was adjusted to a targeted NaCl concentration of 0 mM by dialysis against 0 mM NaCl. Following this treatment, the toroidal structures were preserved (Figure S5F, G and I), while the rod-like structures were partially converted to toroidal structures (Figure S5A and B) with the frequency of toroid 72% (Figure S5D). We experimented with another post-salt treatment – stepwise dialysis – expecting a different impact on the formation of structures due to the abrupt change in salinity. In this case, both the toroidal and rod-like structures, prepared at optimized salt concentrations, were preserved (Figure 3); the rod structure was preserved in 0-800 mM NaCl (Figure 3A-C, F, and Figure S6A) and the toroid structure was preserved in 0-1,200 mM NaCl (Figure 3G-J, L and Figure S6B), thus achieving our objective of preserving individual structures over a wide range of NaCl concentrations. All structures became string structures after dialysis to an exceeding


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high NaCl solution (1,500 mM), regardless of the dialysis method used (Figure S5C and H and Figure 3E and K). String structures prepared at 1,500 mM NaCl were preserved at any diluted salt condition (Figure S5K-O and Figure 3M-R, Figure S6C). Notably, a fairly high frequency of toroidal (98%) structures was obtained at 1,200 mM NaCl through stepwise dialysis (Figure 3D and F) of the rod-like structures, although the structures finally became string structures with a further increase in the salt concentration (Figure 3E). As a consequence, by changing the NaCl concentration of the medium containing PM with definite morphology, transitions from rod to toroid and toroid to string occurred, but the toroid to rod or string to any other structures did not occur. This unidirectional morphological change was consistently observed irrespective of the post-salt treatment procedures, whether it was by abrupt (Fig. S5) or gradual (Fig. 3) change. It is thus reasonable to assume that this definite course of direction in the morphological change may reflect the relative thermodynamic stability of each structure as string > toroid > rod. It should be noted that the post-salt treatment procedures also affected the maintenance of the structures. Rod-structures preserved the structures up to 800 mM by conducting the stepwise dialysis (Fig. 3), while the structures partially transformed to toroid structures at 600 mM NaCl by conducting the one-step dialysis (Fig. S5b). Presumably, the abrupt salinity change granted sufficient impact capable for transformation from rod to toroid structure overcoming the necessary energetic barrier, while the gradual change might grant insufficient impact for the transformation at the 600 mM NaCl, although most of the structures finally transformed into toroid structures at 1200 mM NaCl concentration (Fig. 3).


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A

B

C

D

E

G

H

I

J

K

M

N

O

P

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0 mM F

150 mM L

600 mM

1200 mM

1500 mM

R

Figure 3. Representative TEM images of PMs prepared at the indicated NaCl concentrations with arrows followed by a stepwise dialysis treatment in different NaCl concentrations. The corresponding frequency of characteristic structures – rods/strings (open circle), toroids (closed circle), and others (triangle) – are provided. (A)-(E) TEM images of PMs prepared at 0 mM NaCl followed by stepwise dialysis to elevate the NaCl concentrations to 150, 600, 1,200, and 1,500 mM, respectively, and the variation in structural frequency (F). (G)-(K) TEM images of PMs prepared at 600 mM NaCl followed by stepwise dialysis to adjust the NaCl concentration to 0, 150, 1,200, and 1,500 mM, respectively, and the variation in structural frequency (L). (M)-(Q) TEM images of PMs prepared at 1,500 mM NaCl followed by stepwise dialysis to adjust the NaCl concentration to 0, 150, 600, and 1,200 mM, respectively, and the variation in structural


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frequency (R). All scale bars represent 200 nm. Insets in (A)-(C) represent corresponding magnified images.

The aforementioned post-salt treatment enabled the investigation of the relevance of the structures and their functions at identical salt concentrations. To this issue, we evaluated transcription efficacy, as an essential process of DNA function for gene expression, using a cellfree system. In this way, we can directly address how the packaging structure influences transcription efficacy without possible interferences involved in the assay using a model cell line, such as variations in the efficacy of cellular uptake and endosomal escape. The rod and toroid structures were separately prepared at an identical physiological NaCl concentration (150 mM) by stepwise dialysis, and their frequency and size were confirmed as identical to the original condition (i.e., 0 mM for rods and 600 mM for toroids; Figures 3B and 3H). Note that Luciferase T7 control pDNA used for in vitro transcription assay exhibited the change in the packaging structure identical to pBR322 pDNA with the variation in NaCl concentration (SI 9). The evaluation revealed an appreciable effect of structure on efficiency: the toroid structure demonstrated a two-fold increase in transcription activity compared to that of the rod (Figure 4A). Indeed, various differences between the rod and toroid structures were observed, including the binding number of polymers to pDNA (Figure 2B), PEG density, ζ-potential (Figure S4, neutral for rods, and -9.5 mV for toroids), and the extent of DNA condensation (Figure 2C). It is therefore difficult to identify the primary factors contributing to the increased transcription efficiency of the toroid. Nevertheless, it is important to note that the integrity of double-stranded DNA structure within PMs may serve as a potential contributing factor. Previously, we clarified that the double-stranded structure locally dissociates at rod ends to permit folding.30 We therefore


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speculate that the double-stranded structure of the toroid is intact because no sharp folding is required. This difference may influence the ability of the transcription machinery to traverse DNA strands during transcription. To investigate this scenario, the integrity of double-stranded DNA in packaged structures was examined at 150 mM NaCl using the S1 nuclease, an enzyme that specifically cleaves single-stranded DNA.42 Interestingly, clear bands corresponding to intact pDNA were observed for the toroidal structure but not for the rod-like structure (SI 10, Figure S8). This result revealed a difference in the status of double-stranded DNA structure within rodlike and toroidal structures. Further, the morphology may also be a potential factor influencing to the transcription efficiency. Given that transcription machinery slides along DNA in transcriptional process,43 it could be reasonable to assume that the process may continuously proceed in the toroid structure along an infinite loop of DNA. Alternatively, in the rod structure, the transcriptional process could proceed along the rod axis but might be interfered at the rod end because of the impaired integrity of double stranded structure of DNA.26,30 These feasible mechanisms involved in the transcriptional process is consistent with the observation that toroidal structure has higher transcriptional efficacy than rod structure.


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Figure 4. Evaluation of the biological properties of rod-like and toroidal PMs. (A) In vitro transcription efficiency was evaluated in a solution containing all elements needed for transcription (n = 4, mean ± SD). In vivo gene transfer targeted at skeletal muscle following intravenous injection with a tourniquet: (B) IVIS images and (C) quantified results (n = 6, mean ± SD). Two-tailed Student’s t-test: ****p < 0.0001.

To further explore the properties of the toroidal structure, an in vivo gene transfer trial was performed in skeletal muscle cells via intravenous injection with the aid of a transient tourniquet. Note that the method permits transportation of pDNA directly into cytoplasm of skeletal muscle cells,24 skipping the usual intracellular tracking route of particulate systems via endosomal uptake; therefore the effect of the packaging structure on transcription efficacy, as demonstrated in cell-free system, can directly be evaluated in animal model.26 Of note, pCAG-Luc2 used for the in vivo test underwent the same mode of transition in the packaging structure with other pDNAs according to the change in NaCl concentration (SI 9). As observed in IVIS for luciferase expression (Figure 4B and 4C), the toroidal structure exhibited approximately two-fold greater transgene expression than the rod-like structure, thus confirming the superior function of the toroidal structure in vivo. We previously reported the significant anti-tumor effect25 by this administration method to the subcutaneous xenograft of an intractable pancreatic tumor using our intensively studied rod-structured PMs.18,23,26-30 Therefore, the attained further increased efficacy of transgene expression using toroidal structures compared to rod-like structures would address the appreciable potential of this toroid structure and thus it should be explored in future studies as a distinct toroid-structured gene delivery system. Finally, it is noteworthy that 600 mM NaCl, at which the toroidal structure was specifically formed, corresponds to the NaCl concentration of seawater. Given that the sea is the origin of life


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and viruses, as primitive species from ancient period, package their genomic DNA through toroidal manner within their capsid,35,36 this coincidental match stimulates us to deduce various hypothesis of the mysterious DNA packaging with relevance to evolution. These remarks encourage further investigation of the ordered structure of the toroid not only to promote gene therapy mediated by this distinct virus-like gene-delivery system but also to infer the mechanism of genomic packaging in nature.

CONCLUSIONS: Selective packaging of a single pDNA molecule into distinct rod-like or toroidal structures was achieved with the highest frequency reported thus far by use of different NaCl concentrations during polyion complexation. Stepwise dialysis permitted the retention of these two specific structures over a wide range of NaCl concentrations, which enabled a direct comparison of the structures and their biological functions. The toroidal structure, which resembles the viral genomic packaging structure and was formed at a NaCl concentration coincident to that of seawater, was revealed to have markedly higher transcription efficiency and higher in vivo gene transfer efficacy compared to the rod-like structure. These features encourage further investigation of the toroidal structure to promote gene therapy and to infer scheme of genome packaging in nature.

ASSOCIATED CONTENT Supporting Information Available: This information is available free of charge via the Internet at http://pubs.acs.org/.


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Supporting Information: Packaging structure of pDNA with homo-PAsp(DET) catiomer, distributions of long axis lengths for rods and circumference for toroids, quantized folding scheme to form rod/string-structured PMs, spooling scheme to form toroid-structured PMs, CD spectroscopy measurement, ζ-potential of rod- and toroid-structured PMs, effect of salt change on pDNA packaging structure by the one-step dialysis treatment, the average long-axis length of rod structures and circumference of toroid structures prepared at given NaCl concentration followed by the stepwise dialysis, Characterization of complexed pDNA structures prepared from Luciferase T7 control DNA and pCAG-Luc2 DNA at 0 and 600 mM NaCl, and S1 nuclease activity assay. These materials are available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *(K.O.) Email: [email protected]; *(K.K.) Email: [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. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by Precursory Research for Embryonic Science and Technology (PRESTO) and the Center of Innovation (COI) program from the Japan Science and Technology Corporation (JST), KAKENHI Grant-in-Aid for Specially Promoted Research and Core to Core Program for A. Advanced Research Networks from the Japan Society for the Promotion of Science (JSPS), and Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) through “Nanotechnology Platform. Y. Li acknowledges a fellowship from


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the MEXT. The authors are grateful to Dr. S. Fukuda from the University of Tokyo Hospital and Mr. H. Hoshi for their valuable assistance in conducting TEM and to Dr. Y. Mochida from the Graduate School of Medicine at the University of Tokyo for technical support and assistance. REFERENCES (1) Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S. Nat. Rev. Drug Discov. 2005, 4, 581593. (2) Glover, D. J.; Lipps, H. J.; Jans, D. A. Nat. Rev. Genet. 2005, 6, 299-310. (3) Schaffert, D.; Wagner, E. Gene Ther. 2008, 15, 1131-1138. (4) Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. R.; Anderson, D. G. Nat. Rev. Genet. 2014, 15, 541-555. (5) Kornberg, R. D. Science 1974, 184, 868-871. (6) Annunziato, A. T. Nature education 2008, 1, 26. (7) Lifshitz, I. M.; Grosberg, A. Yu.; Khokhlov, A. R. Rev. Mod. Phys. 1978, 50, 683-713. (8) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K.; Khokhlow, A. R.; Doi, M. Biopolymers 1994, 34, 555-558. (9) Kabanov, A. V.; Kabanov, V. A. Bioconjugate Chem. 1995, 6, 7-20. (10) Eickbush, T. H.; Moudrianakis, E. N. Cell 1978, 13, 295-306. (11) Bloomfield, V. A. Biopolymers 1997, 44, 269-282.


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Title: Toroidal Packaging of pDNA into Block Ionomer Micelles Exerting Promoted in vivo Gene Expression Authors: Yanmin Li, Kensuke Osada, Qixian Chen, Theofilus A.

Tockary,

Anjaneyulu

Dirisala, Kaori M. Takeda, Satoshi

Uchida,

Kazuya

Nagata, Keiji Itaka, Kazunori Kataoka


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Toroidal Packaging of pDNA into Block Ionomer Micelles Exerting

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