Emergence of DNA-Encapsulating Liposomes from a DNA–Lipid

Article pubs.acs.org/JPCB

Emergence of DNA-Encapsulating Liposomes from a DNA−Lipid Blend Film Shunsuke F. Shimobayashi* and Masatoshi Ichikawa* Department of Physics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan S Supporting Information *

ABSTRACT: Spontaneous generation of DNA-enclosing liposomes from a DNA−lipid blend film is investigated. The special properties of the lipid vesicles, namely, micrometer size, unilamellarity, and dense polymer encapsulation acquired by the dehydration−rehydration process, are physicochemically revealed. We found that the formation of giant unilamellar vesicles encapsulating DNAs are governed by micropatterns of the films, such as dots and network patterns. From the results, we proposed a plausible physical mechanism for the dehydration−rehydration process, making it possible to optimize the encapsulation of any agent.

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film patterns on a substrate to realize efficient encapsulation of DNA molecules inside of cell-sized GUVs.

INTRODUCTION A cell-sized giant unilamellar vesicle (GUV) densely encapsulating biomacromolecules is a physicochemically designed life-mimicking model. During the emergence of life or in the early stage of the cell birth, these biomacromolecules might have become encapsulated in the semipermeable compartment with special properties, namely, micrometer size, unilamellarity, and suitable concentration. It is expected that physicochemical processes on polymer encapsulation within a cell-sized compartment would give insights into the story of the origin of life. Amphiphilic molecules with a dual hydrophilic−hydrophobic character self-assemble into a vesicular-shaped bilayer membrane. The size, lamellarity, and encapsulated solution in vesicles strongly depend on how the vesicles are prepared. Generally, GUVs do not exist in thermodynamic equilibrium but in a kinetically trapped state; therefore, their formation must involve a kinetic procedure. Two methodologies, both based on the kinetic processes, have been investigated to create GUVs. In the first method, known as the gentle swelling or natural swelling, a flat dried lipid film is prepared and hydrated by aqueous solution, containing the solutes to be encapsulated. This is a simple technique but mostly yields giant multilamellar vesicles with lower solute uptake in comparison to outside solution.1 Recently, low uptake efficiency has been improved to concentrations approximating that of the swelling solution by optimizing a solid substrate under the lipid film.4,5 The second method is the dehydration−rehydration technique proposed by Deamer et al., which generates GUVs with higher polymer encapsulation in comparison to outside solution.2,3,6 Therefore, the dehydration−rehydration process satisfies the three requirements, that is, microscale size, unilamellarity, and dense polymer encapsulation, of the precursors of life. However, despite the significance of life’s origin, the intrinsic physicochemical mechanism of drying and hydration in a lipid− biopolymer mixture has not been elucidated. The present study reveals the crucial elements of this process by controlling dried © 2014 American Chemical Society

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METHODS SECTION Materials. Sonicated salmon DNA (300−700 bp, BioDynamics Laboratory Inc.), λ phage DNA (48 kbp, Nippon Gene), and T4 phage DNA (166 kbp, Nippon Gene) were used. Sonicated DNA (152 mM, in base pair unit) dissolved in deionized distilled water, 697 μM λ DNA including 10 mM Tris-HCl (pH 7.9) and 1 mM EDTA, and 682 μM T4 DNA including 10 mM Tris-HCl (pH 7.9) and 1 mM EDTA were diluted by ultrapure water purified by a Milli-Q system (Millipore). DNA was fluorescently stained by YOYO-1 (Abs/Em 491/509 nm, Molecular Probes, U.S.A.). The 1 mM YOYO-1 dissolved in DMSO was diluted by the ultrapure water. The YOYO-1 was added to the DNA solution and incubated for more than 20 h to establish an adsorption equilibrium. Dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), cholesterol, and rhodamine dioleoylphosphatidylethanolamine (rho-PE, Abs/Em 560/583 nm) were dissolved in ultrapure water and ultrasonicated into SUVs for 2 h at 50 °C. The DOPC-rich phase was visualized with rho-PE, and the molar ratio of rho-PE to DOPC was 0.3 mol %. Liposome Preparation. After ultrasonication of 10 mM DOPC and 0.1 g/L rho-PE for 10 min at 25 °C, the DOPC (25 μL), the rho-PE (10 μL), and the 150 μM DNA in base pair units (20 μL) stained by YOYO-1 were gently mixed, in protocol A. The detailed procedure of DNA staining by YOYO1 was previously reported.18 The mixture was pipetted onto a glass base dish with a 35 mm diameter (Iwaki). In protocol B, 150 μM DNA (20 μL) was replaced with 15 μM DNA (20 μL). For liposomes composed of DOPC, DPPC, and cholesterol, Received: June 19, 2014 Revised: August 18, 2014 Published: August 18, 2014 10688

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Figure 1. Dehydration processes. (A−C) Second to last dehydration process. Confocal fluorescence images of DOPC (red) and T4 DNA (green) after (A) 4 h and 25 min, (B) 4 h and 26 min, and (C) 4 h and 27 min of sample preparation. (D−G) Last dehydration process. Confocal images after (D) 2 h and 30 min, (E) 2 h and 35 min, (F) 2 h and 45 min, and (G) 2 h and 50 min of sample preparation. The two last dehydration processes were observed by use of two different samples, resulting in the difference of the acquired times of images. DNA: T4 phage DNA. Protocol: A.

Figure 2. Confocal fluorescence images of DNA/DOPC dried films. (A,B) Films of fragmented salmon DNA (300−700 base pairs, green) and DOPC (red) in protocol A (A) and protocol B (B) on a glass substrate (see the Methods Section). (C,D) Films of λ phage DNA (48 kilo base pairs, green) and DOPC (red) in protocol A (C) and protocol B (D). (E,F) Films of T4 phage DNA (166 kbp, green) and DOPC (red) in protocol A (E) and in protocol B (F). (G) Schematic of protocols A and B before drying. The total amount of DNA in protocol A is a factor of 10 higher than that in protocol B.

150 μM DNA (20 μL), 10 mM DOPC (8 μL), 10 mM DPPC (8 μL), and 10 mM cholesterol (4 μL) were used. The mixtures

on the glass substrates were dried at approximately 20 °C under atmospheric pressure. The dried film was hydrated by pure 10689

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Figure 3. DNA-encapsulated liposomes. Confocal fluorescence images of liposomes encapsulating (A,D) fragmented salmon DNA, (B,E) λ phage DNA, and (C,F) T4 phage DNA growing on the film substrate (A−C) and floating freely in solution (D−F). DNA is stained by YOYO-1 (green), and GUVs are stained by Rho-PE (red).

water (200 μL) for 30 min at room temperature (or approximately 40 °C in the case of DOPC/DPPC/cholesterol liposomes), and the generated DNA-encapsulated liposomes were harvested by gentle pipetting. The obtained solutions were diluted four times (or twice) with pure water for microscopy observation. Confocal Microscopy and Imaging Analysis. Confocal acquisition was performed on an Olympus inverted microscope equipped with a CSU-X1 Yokogawa confocal head and an Andor EMCCD camera. An UplanApo oil ×100/1.35 objective lens or a UPlan SApo ×20/0.75 (Olympus) was used. The system was operated by Andor iQ imaging software (Andor Technology). Fluorescent intensity analysis and measurement of the liposome diameter were performed with Andor iQ imaging software or ImageJ (http://rsb.info.nih.gov/ij). The GUV diameters were first measured from confocal equatorial images captured in rho-PE mode (red). The fluorescence was then switched to YOYO-1 mode (green) to obtain the average fluorescence intensity. In the intensity analysis, we used the band path filter, whose center wavelength and width are 520 and 17.5 nm, respectively, to eliminate the inner filter effect by rho-PE. Negligible crosstalk on the fluorescence path was confirmed by use of laterally phase separated vesicles (Figure S7, Supporting Information). Background fluorescence captured without DNA and YOYO-1 was subtracted from the measured value.

drying, as the effective concentration increased, micrometersized tubular assembled structures emerged (Figure 1). These structures consisted of both DNA and DOPC, and they adhered on the substrate while retaining their tubular shape (Figure 1). Subsequently, the complexes were cooperatively fused into a terrace-like morphology composed of DNA and DOPC on the submillimeter scale. In the final stage of drying, segregation of DNA and lipid proceeded on micrometer to submillimeter scales. Eventually, the morphology was determined by the competitive kinetics between the domain coarsening and freezing by drying. Under a rapid dehydration, microphase separation structures became frozen at the early stage of spinodal decomposition by use of short DNA molecules fragmented by sonication into length approximating the Kuhn length (Figures 2 and S1a-A,a-B, Supporting Information). On the other hand, phase separation in giant genomic DNAs from λ and T4 phages led to network-like patterns, indicating a unique feature of viscoelastic phase separation that occurs in mixtures of components having asymmetrical dynamic properties (Figures 2 and S1b-A,c-A,c-B, Supporting Information).7,8 In this case, viscoelastic phase separation would arise from the large difference in relaxation time between giant DNA and DOPC.7,8 The relaxation time is highly sensitive to polymer length and concentration. In fact, as the DNA concentration decreased, these web structures were minimized or disappeared altogether (Figures 2 and S1b-B,c-B, Supporting Information). Furthermore, for all DNA, the characteristic domain sizes increased with decreasing DNA concentration. On the basis of the features of viscoelastic phase separation, the phase separation morphology in the dried film can be controlled by DNA length and concentration. Welldeveloped structural segmentation in the film provides an indication of efficient preparation of DNA-encapsulated

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RESULTS DNA and small unilamellar vesicles (SUVs) of dioleoyl-L-αphosphatidylcholine (DOPC) separately dissolved were mixed on a glass substrate and dried to form a film. Dramatic changes were observed in the last for about 30 min, where the evaporation was reaching the plateau. In the late process of 10690

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Figure 4. Effect of DNA concentration in liposomes on the vesicle growth rate. (A) Confocal fluorescence imaging of DNA observed after approximately 5 min of natural swelling. Numbered and colored arrows indicate liposomes growing on the substrate film, respectively. (B) Temporal development of each liposome diameter. Shape deformation occurs with liposome growth, and thus, each liposome diameter was estimated by the mean value of the short and long axes (see the Supporting Information movie). Each dashed line represents a linear least-squares fit for each data. (C) Temporal development of the normalized liposome diameter. Each dashed line represents a linear least-squares fit for each data. (D) Dependence of DNA intensity in the liposome, which is assumed to be proportional to the DNA concentration, on the vesicle growth rate. The growth rate and its error, which mean the slope of the fitting line in (B) and its standard deviation, respectively, are plotted as a function of DNA intensity inside of a vesicle. The sizes of liposomes 4 and 5, exhibiting much lower intensity than that of the outside, change little; on the other hand, the sizes of liposomes 1−3, showing much higher intensity, extremely increase. Arrow indicates the mean outside intensity, whose value and its standard deviation are 738 and 26 (arbitrary units), respectively. DNA: T4 phage DNA. Protocol: A.

show that osmotic pressure due to the inner solute, namely, sugar, enhances the unbinding, consistent with our results.10,11 We collected the vesicles encapsulating DNA by brief pipetting (∼10 s) from the substrate and measured the diameters and the average intensities of DNA inside of suspended vesicles from confocal fluorescence images, where approximately 50 vesicles for each sample were selected randomly (Figures 3D,E,F, and 5). The measured intensities were converted into DNA concentrations using DNA-only standard solution, indicating that the maximum DNA concentration is about 2 orders of magnitude less than the average DNA concentration in Escherichia coli cells (∼10 mM) (Figures S4 (Supporting Information) and 5B).14 This implies that DNA uptake, particularly that of fragmented salmon DNA, is enhanced relative to the outside. Most of the vesicles show dense DNA encapsulation on the film substrate, regardless of the DNA length and concentration (Figure 3A,B,C). However, in a moderate number of the recovered vesicles, the encapsulation of giant λ and T4 DNA was comparable to the outside (Figure 5C,D,E,F). By contrast, fragmented salmon DNA was densely encapsulated by nearly all vesicles (Figure 5A,B). The ratios of the average DNA intensity inside of liposomes to that of the outside solution were 17.0 ± 7.5 (fragmented DNA, protocol A), 20.4 ± 1.9 (fragmented DNA,

liposomes, as demonstrated in the following sections. As each film was hydrated, cell-sized unilamellar vesicles with high DNA entrapment were rapidly formed (within 20 min) at large yields (Figures 3A,B,C, and S2, Supporting Information). In contrast, the film composed of DOPC alone yielded multilamellar vesicles even after 6 h of hydration (Figure S3, Supporting Information); the considerable difference can be attributed to interactions between bilayers. Vesicle formation requires the unbinding of lipid bilayers, and in neutral lipid layers, such as DOPC membranes, unbinding occurs by competition between van der Waals attraction, hydration repulsion, and Helfrich repulsion due to undulation of the layers.9 In the present system, the density difference of DNA at layer interfaces in the vicinity of the DNA-rich phase establishes an osmotic pressure difference in these regions. Therefore, unbinding should be enhanced by the additional repulsion force driven by the osmotic pressure. In fact, the vesicle growth rate was strongly correlated with the DNA concentration inside of liposomes (Figure 4 and Supporting Information movie), strongly suggesting that DNA-rich domains in the dried films were the nucleus of GUV formation; this coupling leads to efficient entrapment of solutes. Recently, the hydration process of lipid films doped with sugar was investigated in detail by microscopic observation and small-angle X-ray scattering. The results also 10691

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Figure 5. Efficient uptake. (A,C,E) Average fluorescence intensity histogram of (A) fragmented salmon DNA, (C) λ phage DNA, and (E) T4 phage DNA inside of liposomes determined from confocal fluorescence images, respectively. Each arrow represents the average intensity of the outside solution. The values and the standard deviations in arbitrary units are 55.5 ± 24.6 (A, protocol A), 25.1 ± 2.4 (A, protocol B), 114.0 ± 11.9 (C, protocol A), 12.2 ± 1.8 (C, protocol B), 9.1 ± 2.6 (E, protocol A), and 17.8 ± 1.6 (E, protocol B). Approximately 50 randomly selected freely suspended liposomes were counted. (B,D,F) Average fluorescence intensity of (B) fragmented salmon DNA, (D) λ phage DNA, and (F) T4 phage DNA as a function of liposome diameter, respectively. Filled circles and triangles represent the average intensities of the outside solution.

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protocol B), 3.4 ± 0.4 (λ DNA, protocol A), 13.3 ± 2.0 (λ DNA, protocol B), 2.2 ± 0.6 (T4 DNA, protocol A), and 4.6 ± 0.4 (T4 DNA, protocol B). Furthermore, the proportion of vesicles with dense encapsulation shows the tendency to decrease with increasing DNA length and concentration. Here, we must question the large differences in DNA encapsulation by the recovered vesicles when most of the vesicles formed on the substrate show densely encapsulated DNAs of any length and concentration. Moreover, in the recovered vesicles, encapsulation strikingly depended on DNA length and concentration.

DISCUSSIONS

To elucidate this result, we reconsidered the viscoelastic properties of thin films during swelling, which correlate with the DNA length and concentration. DNAs considerably longer than the Kuhn length, that is, λ and T4 DNA, exhibit viscoelastic properties above a threshold concentration, corresponding to the overlap concentration, because of chain entanglements. In fact, the hydrated films can be regarded as greatly entangled semidilute solutions (see the Supporting Information). The application of stress over a finite time (called the terminal relaxation time τ) gives an indication of the 10692

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Figure 6. Dehydration−rehydration process. Schematic illustration of the dehydration−rehydration process for fragmented DNA and λ/T4 DNA. A mixture of separately dissolved DOPC and DNA forms submillimeter-sized terrace morphologies while drying (yellow), followed by phase separation. Fragmented DNA forms spinodal decomposition patterns, while λ and T4 DNAs form network-like structures. In the case of fragmented DNA, the vesicles formed on the rehydrated substrate are detached by thermal fluctuation. In the case of λ and T4 DNAs, on the other hand, the stress application for a critical time (terminal relaxation time τ) is required to destroy the entanglements of chains.

fluidic devices, the proportion of vesicles showing enhanced entrapment will increase.

destruction of the entanglements.12 Hence, if the stress is applied over the time required to destroy all of the entanglements in the boundary regions between the formed vesicles and the film substrate, the vesicles containing DNA molecules would be detached from the film substrate. In the present experimental system, the recovered liposomes were subjected to shear force imposed by the flow during pipetting. Therefore, the terminal relaxation time τ must be compared with the pipetting time (∼10 s). According to the reptation model by Doi and Edwards,12,13 the terminal relaxation time τ is derived from the time correlation function of the end-to-end vector of the chain with scaling arguments as the following τ≈

6ηa15/2N3C 3/2 πkT

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CONCLUSIONS We have elucidated the physical mechanism underling a series of the dehydration−rehydration processes by controlling two parameters, DNA length and concentration. Our findings indicate that the fundamental physical phenomena, including osmotic pressure, microphase separation, and polymer entanglement, play a critical role in the formation of micrometer-sized unilamellar vesicles with high DNA uptake efficiency, up to genome length. The dehydration−rehydration process is schematized in Figure 6. Regarding the harvest of the vesicles pinned to the film, the present results are consistent with reptation predictions. By tuning of polymer length and concentration, we can regulate domain segmentation and polymer entanglement in blend dried films and thereby efficiently prepare GUVs with dense encapsulation of the polymer. GUVs based on the present study are readily transformed into nanosized liposomes encapsulating polymers by extrusion techniques.17 Therefore, the present study is applicable for making nanosized liposomes. In a broader context, these observations are potentially useful in medical and pharmaceutical applications, such as drug delivery and gene therapy, because the present optimized dehydration−rehydration method is a simple, rapid, noninvasive means of producing large-scale GUVs.

(1)

where η, a, N, C, k, and T represent the solvent viscosity, the segment length relative to the Kuhn length, the total segment number, the segment concentration, the Boltzmann constant, and the temperature, respectively (see the Supporting Information). According to eq 1, τ is proportional to the cube of the polymer length and to the three-halves power of the polymer concentration. These exponents are supported by force Rayleigh scattering and single-molecule observation.15,16 Smith and colleagues measured the diffusion coefficients of λ DNA in entangled solutions by single-molecule observation and observed that the experimentally derived scaling law (τ ≈ N2.8C3/2) was close to reptation predictions.16 On the basis of their results, the relaxation times τ in our experiments, corrected for length and concentration differences, were estimated as τ ≈ 19 min for λ DNA (protocol A), 36 s for λ DNA (protocol B), 6.3 × 102 min for T4 DNA (protocol A), and 20 min for T4 DNA (protocol B). The concentration of each DNA was estimated in the hydrating thin films (see the Supporting Information). According to these presumptions, the relaxation times τ are comparable with the pipetting time for λ DNA (protocols B) and about 1 order of magnitude larger for λ DNA (protocols A) and T4 DNA (protocols B). On the other hand, τ is more than 2 orders of magnitude larger than the time for T4 DNA (protocols A). Therefore, our results are qualitatively consistent with the predictions of the reptation model for detachment and recovery of the vesicles formed on the film substrate. This model also predicts that if the stress duration time is increased using equipment, such as micro-

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

DNA/DOPC dried films, liposome growth on the film substrate, a dried DOPC film and its swelling, the linear relationship between DNA concentration and the measured intensity, derivation of the terminal relaxation time by the reptation model, order estimation of the DNA concentration in the film, and phase-separated GUVs encapsulating DNA. This material is available free of charge via the Internet at http:// pubs.acs.org. Corresponding Authors

*E-mail: [email protected] (S.F.S.). *E-mail: [email protected] (M.I.). Notes

The authors declare no competing financial interest. 10693

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ACKNOWLEDGMENTS We thank Professor K. Yoshikawa for valuable comments. This work was supported by the Grant-in-Aid for JSPS Fellows Grant (No. 25-1270), by JSPS KAKENHI Grant Number 26707020 and by MEXT KAKENHI Grant Numbers 25103012 and 26115709.

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