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Poly(ethylene glycol) crowding as critical factor to determine pDNA packaging scheme into polyplex micelles for enhanced gene expression Kaori M. Takeda, Kensuke Osada, Theofilus Agrios Tockary, Anjaneyulu Dirisala, Qixian Chen, and Kazunori Kataoka Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01247 • Publication Date (Web): 02 Dec 2016 Downloaded from http://pubs.acs.org on December 4, 2016

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Poly(ethylene glycol) crowding as critical factor to determine pDNA packaging scheme into polyplex micelles for enhanced gene expression Kaori M. Takeda†, Kensuke Osada‡,ø,◊,*, Theofilus A. Tockary†,◊, Anjaneyulu Dirisala‡,◊, Qixian Chen†, Kazunori Kataoka†,‡,§,◊,* †

Department of Materials Engineering, The University of Tokyo, ‡Department of Bioengineering,

Graduate School of Engineering, The University of Tokyo, §Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-8656, Japan, øJapan Science and Technology Agency, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan, ◊

Innovation Center of NanoMedicine, Institute of Industrial Promotion - KAWASAKI, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan

KEYWORDS polyion complex micelles, PEG steric repulsion, DNA packaging, non-viral gene delivery

ABSTRACT

ACS Paragon Plus Environment

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A critical role of polyethylene glycol (PEG) crowding in the packaging of plasmid DNA (pDNA) into polyplex micelles (PMs) was investigated using a series of PEG-b-poly(L-lysine) (PEG-PLys) block copolymers with varying molecular weights of both PEG and PLys segments. Rod-shaped PMs preferentially formed when the tethered PEG chains covering pDNA in a precondensed state were dense enough to overlap one another (reduced tethering density (RTD) > 1), whereas globular PMs were obtained when they were not overlapped (RTD < 1). These results submitted a scheme that steric repulsive effect of PEG regulated packaging pathways of pDNA either through folding into rod-shape or collapsing into globular depending on whether the PEG chains are overlapped or not. The rod-shaped PMs gave significantly higher gene expression efficacies in a cell-free system compared to the globular PMs, demonstrating the practical relevance of regulating packaging structure of pDNA for developing efficient gene delivery systems.

INTRODUCTION

Supramolecular assemblies formed from block copolymers have received considerable attention as a means of developing ordered structures with unique morphologies and exotic functions.1–3 In particular, assemblies formed from plasmid DNA (pDNA) and oppositelycharged block copolymers composed of a non-ionic hydrophilic segment, typically polyethylene glycol (PEG), and a cationic segment are appealing due to their fundamental interest in the construction of novel higher-ordered structures from giant DNA molecules,4–7 and also their potency as non-viral gene vectors.8–14 The polyion-mediated assembly process between pDNA and a block copolymer spontaneously undergoes structural ordering to form PMs with a coreshell architecture in an aqueous solution, wherein the ion-complexed core of pDNA with the


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cationic segment of the block copolymer is surrounded by a dense hydrophilic shell of PEG.15 An essential interest in this structural ordering is a scheme of packaging the rigid supercoiled giant pDNA molecule of micrometer length into the sub-100 nanometer-sized core of PM. Our detailed studies to gain insight into this fundamental issue have revealed a unique packaging mechanism of quantized folding as follows:16 pDNA is folded to form a bundled rod structure with regulated rod lengths, which are multiples of 1/2(n + 1) of its contour length upon folding n times. It was also revealed that the rod-length depends on the number of associated PEG chains constituting the shell layer of the PMs.17–19 This can be successfully explained by considering the balance between condensation of the polyplex core to minimize the development of unfavorable interfacial free energy upon charge neutralization (the origin of DNA condensation), and the anticondensation factors derived from the steric repulsive effects of PEG in the shell layer along with the rigidity of DNA packaged as a bundle.18,20–22 This model illustrates the crucial role of PEG steric repulsion in preventing the rod-shape from becoming a globule with minimal surface area. In fact, the rod-length shortens as the number of associated PEG chains in the PMs decreases.17– 19

Moreover, pDNA is condensed into a globular shape when complexed with non-PEG

polycations.17,23 The significance of PEG in the rod-shaped formation is further corroborated by a study that used PEG detachable PMs. This study observed that rod-shaped structures changed to globular shape upon removal of associated PEG chains from PMs.24 These studies have depicted a crucial role of PEG in the rod-shaped packaging of pDNA within PMs. To consolidate the understanding of the scheme of pDNA packaging by block copolymers, the present study further modulated the contribution of PEG over a wide range, using a series of PEG-poly(L-lysine) (PLys) block copolymers, by changing the degree of polymerization (DP) of the PLys segment (20 ~ 145) as well as the molecular weight (Mw) of the


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PEG segment (0 ~ 42 kDa). Note that variation of PLys DP changes the number of block copolymer strands associated with pDNA for charge compensation18 and, thus, changes the number of PEG chains involved in the PEG shell. In this way, the balance between the steric repulsive effect of PEG strands in the shell layer and the condensation of polyplex core to minimize interfacial free energy was systematically evaluated to clarify the scheme for modulating the shape and structure of PMs. The study also focused on integrity of the doublestranded structure in polyplexed pDNA to address the issue of the folding mechanism to package rigid double-stranded pDNA into a core of PM. Finally, gene expression capability of a series of shape-modulated PMs was investigated to ascertain the relevance of these packaging structures in constructing efficient non-viral gene vectors. MATERIALS α-Methoxy-ω-amino-PEG (MeO-PEG) with Mw of 2, 12, 21, 30 and 42 kDa were obtained from NOF Co., Ltd. (Tokyo, Japan). A series of PEG-poly(L-lysine) (PEG-PLys) block copolymers, composed of a PEG segment with varying Mw and a PLys segment with varying DP (referred to as PEG Mw (kDa)-PLys DP), were synthesized via ring-opening polymerization as previously reported.25 The DPs of the PLys segments were determined by comparing the peak intensity of the methylene protons from the PEG chain (CH2CH2O) and the methylene protons from the PLys side chain [(CH2)3CH2NH3] in the 1H-NMR spectra (JEOL EX300 spectrometer, JEOL Ltd., Tokyo, Japan). Gel permeation chromatography (GPC) measurements (HLC-8220, Tosoh Co., Ltd., Tokyo, Japan) were used to determine the molecular weight distribution (Mw/Mn) of the block copolymers. All of the synthesized block copolymers showed a narrow molecular weight distribution below Mw/Mn = 1.1. Alexa-labeled PEG-PLys was prepared by conjugating an Alexa Fluor® 680 carboxylic acid, succinimidyl ester (Life Technologies Inc.,


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Rockville, MD) to the primary amine of the PLys segment in the block copolymer following the manufacturer’s protocol, and was used to determine the binding stoichiometry between the PEGPLys and the pDNA. Unreacted Alexa molecules were removed by column purification.18 EDTA and sodium dextran sulfate were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). S1 nuclease and the pDNA pBR322 were purchased from Takara Bio Inc. (Shiga, Japan). pCAG-Luc1 and pT7-Luc2 were amplified in Escherichia coli and obtained for experimental use by subsequent extraction and purification procedures using a NucleoBond Xtra Maxi Plus Endotoxin Free Kit (Qiagen Science Co., Inc., Hilden, Germany). pBR322 was used for structural investigations (transmission electron microscope (TEM) observations and S1 nuclease reactions), whereas pCAG-Luc1 was used to investigate complex structures at increased concentrations of pDNA by TEM. pT7-Luc2 was used for the cell-free transcription and gene expression experiments. METHODS Preparation of PMs. The PEG-PLys and pDNA (50 µg/mL) were dissolved separately in 10 mM HEPES buffer (pH 7.4). The PEG-PLys solution was added to the pDNA solution while being vortexed to prepare the PMs at a stoichiometric charge ratio, [residual amino groups in the PLys segment of the block copolymers (N)]/[residual phosphate groups in the pDNA (P)] (N/P) = 1, with a final pDNA concentration of 33.3 µg/mL. For complexation at elevated concentrations, both the concentration of PEG-PLys and pDNA solutions were elevated to maintain N/P = 1. The complex solution was then diluted to 33.3 µg/mL in pDNA concentration for the TEM imaging. TEM imaging. TEM imaging was conducted using an H-7000 electron microscope (Hitachi Co., Ltd., Tokyo, Japan) operated at 75 kV of acceleration voltage. Copper TEM grids (Nisshin


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EM Co., Ltd., Tokyo, Japan) with carbon-coated collodion membranes were hydrophilized by an ion coater (Eiko IB-3, Eiko Engineering Co. Ltd., Tokyo, Japan) before sample preparation. The hydrophilized grid was dipped into the PM solution previously mixed with a 2-% (w/v) uranyl acetate (UA) solution for staining. The excess solution on the sample grid was blotted with filter paper, air dried for 10 min. DNA is selectively observed in the TEM images as the UA preferentially stains DNA; the surrounding PEG is not visible. It is reported that the DNA contour length in TEM images obtained with uranyl acetate staining on collodion membrane substrate is observed to be 83.7% of the actual length calculated from 0.338 nm/bp;26 therefore, the rod length measured in the TEM images was corrected accordingly to present the actual length in the histograms. S1 nuclease assay. The integrity of the double-stranded DNA structure was investigated using S1 nuclease, which specifically cleaves single-stranded DNA. S1 nuclease was allowed to react with the DNA at 37 °C in a solution of 30 mM sodium acetate buffer (pH 4.6) containing 1 mM ZnSO4. After 60 min incubation, the solution was placed on ice and an excess of EDTA was added to stop the reaction. Then, a solution of sodium dextran sulfate (Mw 25,000) at 10 equivalents relative to the nucleotides was added to the solution and incubated at 4 °C for 2 h to induce the disassembly of the PMs. The solution was electrophoresed in a buffer (20 mM TrisAcOH, 10 mM NaOAc, 0.5 mM EDTA, and pH 7.8) to visualize the cleavage pattern of the DNA. A 0.9% agarose gel was used for the PMs prepared from the PEG-PLys 12-20 and 12-45, whereas a 2% gel was used for the PMs prepared from 12-103 and 12-145 to allow for detection of shorter fragments. Note that the size of each gel image was adjusted to compare different samples according to the size of the marker (Wide-Range DNA Ladder (50-10,000), Takara Bio Inc., Shiga, Japan).


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Transcription and gene expression efficiencies in the cell-free system. Transcription efficacy was

evaluated

by

measuring

the

mRNA

product

in

the

TnT

Quick

Coupled

Transcription/Translation System (Promega Co., Fitchburg, WI) using real-time polymerase chain reaction (RT-PCR) (Applied Biosystems® 7500 Fast Real-time PCR System, Life Technologies Inc., Rockville, MD). A stock solution containing 200 µL of TnT T7 Quick Master Mix, 5 µL of 1 mM methionine, and 45 µL of 10 mM HEPES was prepared, and a 10 µL aliquot of the stock solution was mixed with 5 µL of the PM solution (containing 0.17 µg of pT7-Luc2). After reacting for 30 min at 37 °C, the transcribed mRNA was extracted using the RNeasy Mini Kit (Qiagen Science Co., Inc., Hilden, Germany) according to the manufacturer’s protocol. The mRNA was converted to cDNA using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd., Osaka, Japan), according to the manufacturer’s protocol. Then, the cDNA was quantified using the Fast Start Universal SYBR Green Master (Roche Ltd., Basel, Switzerland) (n = 3). Cell-free gene expression efficiency of the PMs was also evaluated in the TnT Quick Coupled Transcription/Translation System according to the manufacturer’s protocol. This was conducted under the same conditions as the transcription efficacy evaluation. Luciferase gene expression was evaluated by monitoring luminescence intensity upon addition of the luciferase substrate (Luciferase Assay Reagent, Promega Co., Fitchburg, WI) (n = 3). The GloMax®-96 Microplate Luminometer (Promega Co., Fitchburg, WI) was used to monitor for the experiment with PMs prepared from PEG-PLys with fixed PLys DP of around 70 and various PEG Mw, whereas a Mithras LB 940 luminometer (Berthold Technologies GmbH & Co., Bad Wildbad, Germany) was used for the experiment with PMs prepared from PEG-PLys with a fixed PEG Mw of 12 k and various PLys DPs. Note that background value was subtracted from each datum.


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RESULTS AND DISCUSSION Structure of pDNA packaged by a series of PEG-PLys with varying PEG Mw and PLys DPs. A series of PEG-PLys block copolymers comprising varying PEG Mw (0 ~ 42 kDa) and PLys DP (20 ~ 145) (listed in Table 1) was prepared according to a previously reported procedure.25 These block copolymers were mixed with pDNA at charge stoichiometric ratio of N/P = 1 to prepare PMs. Of note, our previous study using isothermal titration calorimetry (ITC) revealed that, upon the addition of PEG-PLys, pDNA underwent abrupt condensation at N/P = 1 to form PMs.27 Accordingly, in the present study, all the experiments were done for the samples prepared at the charge stoichiometric condition. The long axis lengths of the packaged pDNA within the PMs were then examined using TEM to observe a general trend for the rod-shaped micelle formation (Figure 1a shows the results for the micelles with PEG Mw = 12 k as representative series; Figure S1 shows all the other series). Figure 1b showed that the long axis length of the PMs shows a trend towards becoming shorter by increasing PLys DP, which is consistent with previous results.18,19,25 The same trend was also observed in the other series of PMs with different PEG Mw ranging from 2 ~ 42 k (Figure S2). The dependence of the long axis length as a function of PLys DP can be reasonably explained by considering the balance between the steric repulsive force (or the osmotic pressure) of PEG and the condensation power of the polyplex core due to charge neutralization.18 An increase in PLys DP concomitantly decreases the number of the associated block copolymer strands with pDNA to neutralize the charge, which is evidenced in the quantification using the ultracentrifuge method (Table 1, SI 3),18 and this results in a decrease in the PEG steric repulsive force to facilitate the multiple folding of pDNA into shorter rod structures. Alternatively, by comparing PMs with a similar PLys DP, thus having the


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same number of PEG strands in the outer shell layer, a tendency was observed that a decrease in the PEG Mw resulted in shorter lengths of the long axis length (Figure S3). As the radius of gyration (Rg) correlates with PEG Mw (Table S1),28,29 it is reasonable to assume that the contribution of steric repulsive force from the same number of PEG strands surrounding the core of the PMs may decrease as the Mw decreases. This view again supports PEG steric repulsion as an important factor in regulating the folding number of pDNA packaged within PMs inversely correlated with the long axis length.

Table 1. Characterization of polyplex micelles prepared from a series of PEG-PLys PEG

Codesa 0-20 0-40 0-70 2-40 2-69 12-20 12-38 12-45 12-72 12-103 12-145 21-24 21-42 21-69 21-81 21-110 21-122 30-41 30-77 42-42 42-75 42-132

PLys

Mw (kDa) DP

DP

2 2 12 12 12 12 12 12 21 21 21 21 21 21 30 30 42 42 42

20 40 70 40 69 20 38 45 72 103 145 24 42 69 81 110 122 41 77 42 75 132

45 45 272 272 272 272 272 272 477 477 477 477 477 477 681 681 953 953 953

No. of associated polymer to pDNAb N. A. N. A. N. A. 218e 126 e 431 230 192 121 84.6 56.0 431 206 126 107 79.0 71.4 210 112 191 116 65.4

N/P ratio of associated polymer to pDNAb N. A. N. A. N. A. 1.0 e 1.0 e 1.0 1.0 0.99 1.0 1.0 1.0 1.0 1.0 1.0 0.99 1.0 1.0 1.0 0.99 0.92 0.99 0.99

PEG tethering densityc

σ (chain/nm2) 0 0 0 0.024 e 0.014 e 0.047 0.025 0.021 0.013 0.0086 0.0065 0.047 0.022 0.014 0.012 0.0086 0.0077 0.023 0.012 0.022 0.013 0.0071

RTDd πRg2σ 0 0 0 0.20 e 0.12 e 3.2 1.7 1.4 0.89 0.63 0.45 5.1 2.9 1.8 1.5 1.1 1.0 4.5 2.4 6.1 3.7 2.1

a

Denoted as “PEG Mw (kDa)-PLys DP”. bDetermined by quantifying the amount of free polymer presented in supernatant after application of ultracentrifugation to selectively sediment PMs (SI 3). cDetermined by dividing the number of PEG-PLys molecules associated with coiled pDNA


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strand by the surface area of the naked pDNA. dRg of PEG in different Mw is provided in SI 4. e Number of associated polymers could not be accurately measured due to the formation of inter complex aggregation. Therefore, values were calculated under the assumption of charge stoichiometric complex formation (SI 3).

(a)

(b) 25

(i) Counts

20

Average length 278 nm

15 10 5 0 25

(ii) Counts

20


15 10 5 0 30

(iii) Counts

25 20


15 10 5 0 40

(iv) Counts

30


20 10 0 40

(v) 30 Counts

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20 10 0 0 100 200 300 400 500 600 700 Length [nm]

Figure 1. Packaging structures of pDNA in polyplex micelles (PMs) prepared from PEG-PLys with PEG 12 kDa and various PLys DPs. (a) Representative TEM images and (b) distributions of the long axis lengths measured from the TEM images for PMs of (i) 12-20, (ii) 12-45, (iii) 12-72, (iv) 12-103, and (v) 12-145.


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Notably, in TEM images of the PMs with higher PLys DPs, or lower PEG Mw, the fraction that did not appear to be distinctly rod-shape but, rather, elliptical or globular is observed, as shown in Figure 1a (iv, v) and Figure S1 (indicated by arrows). This observation suggests that the pDNA may be packaged into such globular shape when the PEG steric repulsive force is lowered by decreasing the Mw of PEG as well as increasing PLys DP. The globular shape has apparently a minimized surface area, and indeed agrees with the request of DNA condensation to reduce the interfacial free energy of a charge-neutralized polyplex. The size of the globular structures was measured to be comparable or even shorter than the persistence length of double-stranded DNA (~ 46 nm based on measurements taken in an NaCl concentration of 1000 mM,30 in which the effects of electrostatic repulsion along the DNA may be negligible, thus probably mimicking the persistence length of the charge-neutralized DNA), as seen in the histogram (Figure1b, Figure S2, S3). Apparently, this packaging scheme of pDNA into the size lower than its persistence length is inconsistent with assuming the integrity of the double-stranded DNA structure, suggesting an alternative scheme with a change in the DNA structure.

Integrity of double-stranded DNA within PMs and their classification into rod-shaped and globular structures. The integrity of the double-stranded DNA structure within the PMs was subsequently examined using S1 nuclease, an enzyme that specifically cleaves single-stranded DNA. Our previous study revealed that pDNA in a rod-shape adopts regular sharp folding with localized dissociation into the single-stranded form only at the rod ends, which explains how rigid DNA


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can fold into rod-shaped structures.16 S1 nuclease specifically cleaves DNA at the rod ends, yielding DNA fragments with lengths correlating with the rod lengths, which can be visualized as characteristic band patterns by gel electrophoresis.16,25,31 Consistently, upon S1 nuclease treatment of the PMs prepared from the PEG 12 k series, the expected band patterns were observed for the PMs formed with PLys DP 20 and 45 (Figure 2), which both presented distinct rod-shaped micelles (Figure 1a), and their long axis lengths were found to be mostly above 46 nm (Figure 1b). However, the gel electropherogram did not show such band patterns, and instead showed a faint smear for the PMs formed with PLys DP 103 and 145 (Figure 2), which involves globular micelles (Figure 1a) with long axis lengths of below 46 nm (Figure 1b). Consistent results were also observed in the PMs of the PEG 21 k series (See Section 5 in SI). These smear patterns observed for the PMs containing the fraction with sizes smaller than the persistent length of double-stranded DNA indicate that a substantial number of sites in pDNA are in the singlestranded form, and susceptible to S1 nuclease attack, suggesting that the random dissociation from double-stranded to single-stranded DNA may occur along the pDNA strands in these globular PMs. The flexibility of the single-stranded form of DNA with a persistence length of only a few nanometers32 is consistent with the formation of the globules whose size is below the persistence length of double-stranded DNA.


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PEG-PLys Size

12-20

12-45 12-103 12-145

Figure 2. DNA bands showing S1 nuclease susceptibility of pDNA within PMs prepared from PEG-PLys of the PEG Mw 12 k series.

To comprehensively understand the general trends in the packaging structure of pDNA within the PMs as a function of PLys DP and PEG Mw, we attempted to classify the PMs observed in TEM images into two fractions: rod-shaped or globular according to the length of the long axis with a boundary at 46 nm, corresponding to the persistent length of double-stranded DNA. Here, we assumed that the rod structure with its long axis length equal to the persistent length of double-stranded DNA would be the minimum length of rod that could be formed by the regular rod-folding scheme. With respect to the quantized folding scheme, such a rod structure is assigned as n = 14 with 48.7 nm in length and 18.5 nm in width, and thus 2.6 in aspect ratio (See Section 6 in SI). Accordingly, the long and short axis lengths of each shape were measured from the TEM images, and the aspect ratios were determined. Those structures with aspect ratios above and below 2.6 were classified as rod-shaped or globular, respectively. The plot of the globular fraction against the PLys DP revealed the general trend that the globular PMs only


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formed when the PLys DP was higher than ~ 50 (Figure 3a) except for cases in which the PEG Mw was substantially shorter at 2 k, suggesting that there may be a critical parameter to determine the packaging scheme. A decrease in the globular fraction with an increase in the Mw of PEG is consistent with the assumed contribution of the steric repulsive force of PEG to maintain rod-shaped packaging of pDNA.

(a)

(b)

Figure 3. Variation of the globular fraction as a function of (a) PLys DP, and (b) PEG crowding in RTD for the PMs prepared with various PEG Mw and PLys DP.

Mechanistic analysis of the formation of rod-shaped and globular PMs based on PEG crowding. Mechanistically, DNA condensation is initiated by charge neutralization upon complexation with polycations, and followed by a large conformational transition from a


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hydrated coil to a dehydrated condensed state for minimizing the interfacial free energy of the charge-neutralized polyplex.4 Referring this general description to the block copolymer-mediated condensation, the presence of the PEG chains apparently interferes with the initiation process of DNA condensation as a sterically repulsive barrier. Thus, PEG density at the initiation stage of condensation; i.e., that tethered on the unperturbed pDNA strand, was calculated by dividing the number of PEG-PLys molecules associated with coiled pDNA strand (Table 1) by the surface area of the naked pDNA [for pBR322, 4361 (bp): 2π × 1 (nm) × 0.338 (nm/bp) × 4361 (bp) = 9261.5 (nm2)]. For a more intuitive depiction of PEG crowding, the density (σ = chains/nm2) was converted to reduced tethering density33 (RTD, πRg2σ) (Table 1), which is defined as the number of chains occupying the area of a single unperturbed chain. This quantity illustrates the extent of PEG overlapping (RTD > 1; overlapping). Then, data shown in Figure 3a were replotted in Figure 3b converting the X-axis to log(RTD) to represent the PEG crowding for a series of PMs with different PEG Mw and PLys DP. Notably, all the data appeared to be on the single master curve with a clear reverse trend between the fraction of globular micelles and PEG crowding (Figure 3b). Further notably, the boundary discriminating the preferential shape of the micelles (rod-shaped vs. globular) is located close to log(RTD) = 0. This observation is interesting because an RTD value of 1 corresponds to the state where unperturbed chains contact just each other, as illustrated in Figure 3b inset. From this analysis, it is plausible to interpret that pDNA preferentially undergoes a transition into the globular form, involving substantial and random dissociation of doublestranded DNA, when the neighboring PEG chains associated with the unperturbed doublestranded pDNA do not overlap (RTD < 1, Scheme 1a) at the initiation stage of condensation. As a steric repulsive effect cannot be expected between PEG chains tethered to pDNA without


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overlapping, pDNA neutralized with PLys segments of the block copolymer is thought to undergo prompt condensation into the globular form. This transition accompanies collapsing of the double-stranded structure to give more flexible single-stranded form through the sacrifice of the hydrogen bonds between nucleic acid bases (Scheme 1b). Of note, PLys segments has more freedom in their alignment on flexible single-stranded DNA compared to rigid double-stranded DNA, thereby maximizing the entropy gain. This may also drive the globular transition of pDNA accompanying the dissociation of double-stranded structure. Alternatively, the pDNA preferentially undergoes a transition to the rod-shaped form when the tethered PEG chains are crowded enough to overlap (RTD > 1, Scheme 1c) at the initial stage of condensation. The steric repulsive barrier of tethered PEG chains competes with the condensing force to minimize the interfacial free energy and maximizing the entropy gain upon DNA/PLys complexation, and eventually induces the regular folding of pDNA into rod-shaped structures preserving the hydrogen bonds between nucleic acid bases, except at rod ends (Scheme 1d). This scheme highlights the significance of PEG effect on rod-shaped PM formation by block copolymers. These views are in line with a consideration that rod-shaped and globular PMs are thermodynamically distinguished structures formed via different kinetic pathways with a differential boundary at RTD = 1. This is consistent with the results of our previous study on the PEG-detachable PMs, revealing that transition from the rod-shape to the globular form took place in a discontinuous manner despite the fact that PEG chains were continuously removed from the shell.24

Scheme 1. Schematic illustration of the critical role of PEG crowding in the process of pDNA packaging by the PEG-PLys block copolymers


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Given that these shapes are formed via different kinetic processes, it is presumable that the rate of DNA condensation may be different between the rod and globule transitions. To corroborate this hypothesis, PMs were prepared from pDNA and PEG-PLys of the PEG 12 k series at elevated concentrations. Concentrations of up to 50 µg/mL for pDNA induced no substantial change in the condensation scheme, undergoing single-pDNA packaging into rodshaped or globular micelles as presented in Figure 1a. Alternatively, preparation at concentrations high enough for pDNA strands to overlap each other may lead them to interfere with each other may lead to the formation of secondary aggregates, unless the condensation rate to complete PM formation is enough higher than the rate of their collision due to translational motion. When the initial concentration of pDNA was elevated to 350 µg/mL, the polyplexes from PEG-PLys block copolymers with PLys DP 20 and 45, which form rod-shaped single pDNA micelles in low initial concentrations of pDNA, underwent secondary association to form network-like structures composed of multiple pDNA strands as shown in Figure 4a, b, and Figure S5. In contrast, the polyplexes from PEG-PLys block copolymers with PLys DP 103 and 145, which form globular micelles preferentially in the diluted condition, still kept small globular micelles without any secondary associates (Figure 4c, d, Figure S5), suggesting that the condensation process may be completed rapidly enough to avoid interference between


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surrounding pDNA strands. By further elevating the pDNA concentration up to 2 mg/mL, the network-like structures became obvious even for the polyplexes from PEG-PLys 12-145 (Figure 4e). These observations are consistent with the view that the collapsed globular forms much faster than the regularly folded rod shape in the process of pDNA condensation triggered by the complexation with PEG-PLys block copolymers, and support the hypothesis that there are different kinetic pathways between rod- and globular PMs as illustrated in Scheme 1. It may be natural to assume that steric repulsive forces generated from crowded PEG strands may prevent pDNA from abruptly transitioning to the globular state, and sacrificing the intact double-stranded structure. Eventually, polyplexes surrounded by crowded PEG strands may undergo the relatively slow transition into the rod-shape following the process of quantized folding, managing both steric repulsive force from the crowded PEG layer and the condensing force of the polyplex core to minimize interfacial free energy. The slow kinetic process of the quantized pDNA folding is likely to be interfered with by neighboring pDNA molecules, particularly at the concentrated state, to induce secondary associates as seen in Figure 4a, b, and Figure S5.

(a)

(b)

(c)

(d)

(e)

Figure 4. Representative TEM images of PEG-PLys/pDNA complexes prepared at elevated solute concentrations. Complexation at 350 µg/mL of pDNA solution and PEG-PLys solution of (a) 12-20, (b) 12-45, (c) 12-103 and (d) 12-145. (e) Complexation at 2 mg/mL of pDNA solution and PEG-PLys 12-145 solution.


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Effect of pDNA packaging structures on capability for gene expression. The potential of these PMs to be used for gene vectors was investigated. A cell-free transcription/translation system was used for this evaluation. Firstly, gene expression was evaluated for PMs with fixed PLys DP around 70 and varied PEG Mw (12, 21, 30, and 42 k). Note that the immediate dissociation of the PMs prior to transcription is unlikely to occur because PMs were confirmed to be stable in physiological buffer25 and even within 90 % serum.18 Further, the solution of PMs contained negligible free polymers, as PMs were prepared at the charge stoichiometry, and that was indeed confirmed by the ultracentrifuge analysis of all the examined samples with varying Mw of PEG as well as DP of PLys (PEG Mw: 12k, 21k, 30k, and 42k, PLys DP: 20 ~ 145) (Table 1). Therefore, it is unlikely that free polymers interact with the transcribed mRNA and inhibit translation in this evaluation. As seen in Figure 5a, a significant difference was observed between PMs of PEG 12 k and 21 k and those of PEG 30 k and 42 k, showing lower gene expression capabilities for the latter groups compared with the former group. It is conceivable that several factors participate in this capability for gene expression, which is the result of the foregoing transcription, such as PEG interference, bound PLys segment on DNA, and the form of the packaged pDNA within PMs. Among them, PEG interference may be most likely to influence the result, given that the binding affinity of PLys with DNA was comparable by the similar PLys DP, and the form of the PMs were not significantly different (comparable rod-shapes) though PMs of PEG 12 k included some globular PMs (Figures 3 and S1). Therefore, PEG crowding of PMs was calculated according to the previously described method18 in terms of /2Rg [; averaged closest distance between two neighboring PEG tethering sites located in a square lattice (See details in Section 8 in SI)], as this parameter was used to evaluate permeability of proteins through PEG,28 and thus it may


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be possible to infer potential permeability of relevant proteins for transcription through a PEG shell. The previous investigation presented that the permeability of serum proteins in a PEGmodified surface is reduced with an increase in PEG crowding for overlapping (/2Rg < 1), and ultimately minimized when /2Rg fell below 0.47.28 The PEG crowding analysis indicated that PMs of the former group have a level of crowding sufficient to somewhat reduce protein permeation (/2Rg = 0.68 and 0.47 for PMs of PEG 12k and 21 k, respectively), whereas those of the latter group have a level of crowding sufficient to minimize protein permeation (/2Rg = 0.38 and 0.33 for PMs of PEG 30 and 42 k, respectively). Thus, it could be presumed that the substantially crowded PEG shell in the PMs of the latter group exceeding the critical level of crowding interfered with the transcription machinery subunits encountering pDNA for initiating transcription. The crowded PEG may also interfere with the subsequent sliding process of the formed transcription machinery along DNA for producing mRNA. These presumptions based on the analysis of PEG crowding are consistent with the observed limited gene expression capability in PMs of the latter group, indicating PEG as an interfering factor for gene expression with a critical level of crowding.


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(a) Luminescence intensity [a. u.]

7000

(b) 1.2 1 0.8 0.6 0.4 0.2 0

5000 4000 3000 2000 1000 0

12-72

21-69

30-77

42-75

3000

Luminescence intensity [a. u.]

Relative mRNA content [a. u.]

1.4

**

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100

Transcription Gene expression

2500

80

2000 60 1500 40 1000 20

500 0

0

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100

Globular fraction [%]

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0 150

PLys DP

Figure 5. Gene expression capability of PMs in a cell-free transcription/translation system. (a) Gene expression efficiencies of the PMs prepared from PEG-PLys of PLys DP around 70 and various PEG Mw (**p < 0.01; Student’s t test). (b) Transcription and gene expression efficiencies of the PMs prepared from PEG-PLys of PEG 12 k series, and the globular fraction as a function of PLys DP. Transcription efficiency was normalized to that of the PMs prepared from PLys DP 20.

Effect of PLys DP on gene expression capability was then evaluated for PMs with varied PLys DP and fixed PEG Mw 12 k, noticing its permissibility for gene expression. As found in Figure 5b, significant PLys DP dependence was observed that gene expression efficiency showed a remarkable decrease above the PLys 45. Subsequent investigation to gain further insight into the observed gene expression confirmed that transcription is a major contributing factor in the resulting gene expression, as the profile of the transcription closely followed the profile of the


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gene expression (Figure 5b). Several factors were thought to contribute to this result because PLys DP changes not only PEG crowding but also the affinity of PLys to bind with DNA as well as the form of the packaged pDNA. Firstly, the interference of PEG is unlikely to cause the result in this case because the PEG crowding analysis indicated that it decreased in the order of PLys DP 20 > 38 > 45 > 72 (Table S4), thus the capability for transcription should increase in the order of PLys DP 20 < 38 < 45 < 72, which is opposite to the observed trend. Instead, the affinity of PLys binding to DNA may be a factor because PLys with a higher DP has a stronger binding affinity to DNA than PLys with a lower DP. Therefore, the ability of the transcription machinery to slide along the DNA may be experience greater hindrance in PMs prepared with higher PLys DP. Note that block copolymers bind pDNA via electrostatic interaction, not covalent bonding, so they may permit sliding of the transcription machinery along DNA. The shape may also be an important factor because it is significantly affected by PLys DP. Indeed, this possibility was strongly suggested by the plot of the fraction of globular PMs against PLys DP. Figure 5b clearly showed that the transcription efficiency was a reverse function of the fraction of globular PMs, revealing a strong correlation between transcription efficiency and shape. It appears that finding the promoter region for initiating transcription is more difficult in the globular-collapsed pDNA than the rod-shaped folded pDNA, and the sliding of transcription machinery along DNA seems to occur less in the globular form with impaired double-stranded structure (Figure 2) compared to the rod-shaped form with a regular double-stranded structure. Considering the inferior gene expression capability of the globular form, the apparent discrepancy that PMs of 12-72 showed comparable gene expression efficiency to PMs of 21-69 (Figure 5a), despite the former having less PEG crowding than the latter, could be explained as a result of the undermined efficiency of PMs of 12-72 by the fraction of the involved globular form.


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Although the detailed underlying mechanisms are yet be fully elucidated, the observed different capabilities in transcription and gene expression indicates the importance of controlling various parameters such as PEG Mw, PLys DP, and with a particular emphasis on shapes to obtain efficient function. To this end, the discovery of an essential factor to determine packaging structure of pDNA to be rod-shaped or globular within PMs should provide fundamental insight into the challenge of developing an efficient gene delivery system. CONCLUSION With a view to clarify the packaging scheme of pDNA within PMs, the effect of PEG crowding in the shell layer on the micelles was examined systematically by varying PEG Mw and PLys DP in the PEG-PLys block copolymers. The critical factor determining the packaging schemes for regular rod-folding or random globule-collapsing was found to be the overlapping of tethered PEG strands covering pDNA, quantitatively elucidated by the reduced tethering density (RTD). In the composition with RTD > 1, fast collapsing of pDNA into a globular form is hindered, and eventually quantized folding into a bundled rod structure occurs in a regulated manner within the constraints of the inherent rigidity of the double-stranded DNA. Notably, PMs with regular rod-shaped structure exerted higher transcription and gene expression capability compared to those having randomly collapsing globular form, indicating the proper manipulation of pDNA packaging into the polyplex core provides a convincing basis in the quest for establishing efficient gene delivery systems. ASSOCIATED CONTENT Supporting

Information.

The

following

files

are

available

free

of

charge.

TEM observations of pDNA packaged by PEG-PLys block copolymers with varied PEG Mw and PLys DP, Distributions of the long axis lengths of packaged pDNA within PMs, Quantification of


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number of the associated PEG-PLys block copolymer strands with pDNA, Radius of gyration for PEG in aqueous media, S1 nuclease susceptibility of pDNA within PMs with PEG Mw 21 k, Theoretical dimension of folded rods within PMs in the quantized folding scheme for classification of rod-shaped and globular structures, TEM observations of PEG-PLys/pDNA complexes prepared at elevated solute concentrations, and Calculation of PEG crowding in terms of /2Rg for tethered PEG on packaged pDNA. (PDF) AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *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. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by Center of Innovation (COI) program and “Precursory Research for Embryonic Science and Technology” (PRESTO) in “Molecular Technology and Creation of New Functions” from the Japan Science and Technology Corporation (JST), and the Japan Society for the Promotion of Science (JSPS) through Specially Promoted Research Program, and Core to Core Program for A. Advanced Research Networks.


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