Shaurya
Sachdev
,
Aswin
Muralidharan
,
Dipendra K.
Choudhary
,
Dayinta L.
Perrier
,
Lea
Rems‡
,
Michiel T.
Kreutzer
and
Pouyan E.
Boukany
*
Department of Chemical Engineering, Delft University of Technology, van der Maasweg 9, 2629 HZ, Delft, The Netherlands. E-mail: p.e.boukany@tudelft.nl
First published on 20th September 2019
Delivery of naked DNA molecules into living cells via physical disruption of the membrane under electric pulses has potential biomedical applications ranging from gene electro-transfer, electro-chemotherapy, to gene therapy, yet the mechanisms involved in DNA transport remain vague. To investigate the mechanism of DNA translocation across the cell membrane, giant unilamellar vesicles (GUVs) were electroporated in the presence of DNA molecules keeping the size of the DNA molecules as a variable parameter. We experimentally determined the translocation efficiency for each size of the DNA molecule, to compare the results with the existing and conflicting theories of the translocation mechanism i.e. stochastic threading and bulk electrophoresis. We observed that the translocation efficiency is independent of DNA size (ranging from 25–20000 bp, bp = base pairs), implying that DNA molecules translocate freely across the electro-pores in the lipid membrane in their native polymer conformation, as opposed to unravelling and threading through the electro-pore. Bulk electrophoretic mobility determines the relationship between translocation efficiency and the size of the DNA molecule. This research provides experimental evidence of the mechanistic understanding of DNA translocation across lipid membranes which is essential for devising efficient and predictable protocols for electric field mediated naked DNA delivery.
Experiments on lipid vesicles have revealed much needed insights into the mechanism of DNA electro-transfer across the cell membrane. For instance, Chernomordik et al. suggested that large native T7 DNA (∼40000 bp) and plasmid DNA (or pDNA ∼ 4700 bp) followed an endocytosis-like mechanism of translocation into large unilamellar vesicles (LUVs, mean diameter ∼ 500 nm) during electroporation.8 On the other hand, Lurquin et al. observed no endocytosis of DNA molecules (∼7600 bp) into giant unilamellar vesicles (GUVs, mean size ∼ 2.5–20 μm) during electroporation, suggesting a mechanism of direct entry (or transport) through the electro-pores formed during electroporation.9 To resolve this discrepancy, recently Portet et al. (2011) conducted experiments using GUVs (mean diameter ∼ 20 μm) and pDNA (∼4700 bp).10 Combining experiments with a proposed theoretical framework, they concluded direct entry of DNA molecules through electro-pores via electrophoresis as the mode of transport instead of electro-induced endocytosis. A similar mechanism of direct translocation of small interfering RNA through electro-pores was also observed during nano-second electroporation of GUVs.11 Investigating electroporation of GUVs in the presence of DNA molecules has thus been conducive in revealing how DNA molecules are transported across the cell membrane during electroporation. This was otherwise often challenging to unveil with experiments on living cells due to complexities associated with the coupling of the cell membrane and cytoskeleton entities.
GUVs provide the opportunity to obtain precise and mechanistic understanding of DNA electro-transfer. For instance, in the experiments and the theoretical framework of Portet et al. (2011) it was assumed that the pores were large enough such that DNA molecules can translocate across the electro-pores in their native polymer conformation.10 In this scenario, the transport of DNA molecules should depend on the bulk electrophoretic mobility of DNA molecules. However, only one size (pDNA ∼ 4700 bp) was tested for the theoretical framework established. If pores are not large enough compared to the size of the DNA molecule (radius of gyration), Yu and Lin12 proposed a different theoretical model for DNA transport by considering that the pore is small enough to allow only a single base pair (bp) to pass through it at a time. In this scenario, the DNA molecules are not transported across the cell membrane in their native polymer conformation but rather translocate through the electro-pore one base pair at a time. This is equivalent to a single-file translocation or stochastic threading of DNA molecules across artificial nano-pores.13 According to this model, the DNA transport or translocation efficiency (TE) scales with the size of the DNA as TE ∼ N−1.5, where N represents the number of base pairs. Thus, by conducting systematic experiments on model cell membranes such as GUVs and varying the size of the DNA molecules, the diverse and conflicting theories can be tested in order to obtain a more accurate understanding of DNA transport across the cell membrane during electroporation.
In this research, GUVs are electroporated in the presence of DNA molecules of different sizes (25 bp, 100 bp, 500 bp, 1000 bp, 10000 bp, 15000 bp and 20000 bp) individually, in order to test the different mechanisms of DNA translocation i.e. the theoretical framework of Portet et al. (2011)10 that claims direct entry of the DNA molecules and the theoretical framework of Yu and Lin12 that claims single-file translocation of DNA molecules across the electro-pores. By comparing the experimental translocation efficiencies with the predictions from the theoretical frameworks, it was inferred that DNA molecules directly enter the GUVs during electroporation in their native configuration as proposed by Portet et al. (2011).10 The results of this study provide a mechanistic understanding of DNA translocation across an electro-pore which is not only necessary for understanding DNA translocation across real cell membranes, but also for predictable loading and dosage control of nucleic acids into vesicles using electroporation. Moreover, with such a diverse range of DNA sizes tested that span three orders of magnitude, these results can also be utilized to optimize loading of vesicles with small nucleic acids (such as siRNA, ∼20 bp) for gene silencing applications, and large nucleic acids (such as pDNA, ∼5000 bp) for gene therapeutic applications, using liposome mediated transfection.
A representative experiment describing the uptake of DNA molecules by the GUV during electroporation, for a DNA size of 100 bp, is shown in Fig. 2. Before the application of electric field pulses, Fig. 2(a), fluorescence from the DNA molecules (shown in green) could be seen to be homogeneously distributed outside the GUV (shown in red). The fluorescence intensity of DNA molecules outside the GUV corresponds to a concentration of 2.5 μg ml−1 and labelled as Iout. The negligible green fluorescence that could be seen inside the GUV was attributed to the sensor noise (Inoise). On the application of electric field pulses at t ∼ 0 s, the DNA molecules could be seen entering the GUV from the cathode facing side of the electrode. A simultaneous decrease in the GUV diameter was also observed. A representative snapshot of this process is shown in Fig. 2(b). After the application of electric field pulses, no uptake of DNA or a decrease in GUV diameter was observed. The final state of the GUV is shown in Fig. 2(c).
Fig. 2 Quantifying the decrease in GUV diameter and the uptake of DNA molecules during electroporation. (a) State of the GUV before the application of electric field pulses (t < 0 s). The GUV is shown in red and the DNA molecules are shown in green. The GUV detected is shown using a white-dotted circle (see Section 2.5 in Materials and methods). The mean fluorescence intensity of DNA molecules outside the GUV is depicted as Iout corresponding to a concentration of 2.5 μg ml−1. The green fluorescence due to sensor noise found inside the GUV is labelled as Inoise. Scale bar = 30 μm. (b) State of the GUV during the application of electric field pulses (t ∼ 15 s). 10 pulses of an electric field amplitude of 0.45 kV cm−1 were applied, each of duration 5 ms and at a frequency of 0.33 Hz. This corresponds to a time frame of t ∼ 0 s to t ∼ 30 s. The mean fluorescence intensity inside the GUV due to uptake of DNA molecules during electroporation is depicted as Iin. (c) State of the GUV after the application of electric field pulses. For visualization purposes, the images shown in (a)–(c) are enhanced using the same adjustments as for Fig. 1. Detected GUV diameter and normalized mean fluorescence intensity I/I0 = (Iin − Inoise)/(Iout − Inoise), as a function of time, are plotted as black dotted lines in (d) and (e), respectively. The beginning and the end of pulses are marked by arrows. The solid red line represents smoothed data using the ‘smooth()’ function in MATLAB®. |
From these experiments, the transient data of the effect of electric field pulses on the GUVs and the simultaneous uptake of DNA molecules during electroporation could be extracted from the sequence of images captured for the process. Fig. 2(d) shows the GUV diameter as a function of time. Before the application of electric field pulses, the diameter was constant at ∼33 μm (labelled as Di). The initial diameter, Di, was calculated by taking the average of the diameters before the application of electric field pulses. During the application of electric field pulses (from t ∼ 0 s till t ∼ 30 s, as marked by arrows), the diameter decreased continuously. The decrease in diameter has been observed previously for fluid phase GUVs and is attributed to lipid loss during electroporation.20,21 After the application of electric field pulses, the diameter attained a steady-state value of ∼25 μm (labelled as Df). The final diameter, Df, was calculated by taking the average of the diameters after the application of pulses.
Similarly, the uptake of DNA by the GUV during electroporation was determined. The mean fluorescence intensity of the DNA molecules inside the GUV (Iin) was determined by calculating the mean fluorescence intensity of the DNA molecules inside a circle having a diameter corresponding to 0.9 times the detected GUV diameter (see Section 2.5 in Materials and methods). This was done to minimize edge effects. The initial mean fluorescence intensity corresponding to sensor noise (Inoise) was subtracted from the mean fluorescence intensity of the DNA molecules detected inside the GUV in each frame as I = Iin − Inoise. The sensor noise was also subtracted from the fluorescence intensity of DNA molecules outside the GUV (Iout) in each frame as I0 = Iout − Inoise. Iout corresponded to the mean fluorescence intensity of the DNA molecules outside a circle having a diameter corresponding to 1.2 times the detected GUV diameter. Normalized mean fluorescence intensity I/I0 was then plotted as a function of time as shown in Fig. 2(e). Before the application of electric field pulses, no DNA uptake could be observed inside the GUV. During application of electric field pulses (from t ∼ 0 s till t ∼ 30 s, as marked by arrows), the mean normalized fluorescence intensity continuously increased linearly. It finally reached a steady state value (I/I0)f = 0.38 after the end of the electric field pulses. This final steady state value (I/I0)f was calculated by taking the average of (I/I0) values after the application of electric field pulses. Other quantities such as DNA uptake time and the slope of normalized intensity (I/I0) vs. time during uptake of DNA by the GUV are shown in Section 1 of the ESI.†
The sequence of images representing the electroporation of GUVs were analysed for a number of experiments corresponding to different DNA sizes. The evolution of the diameter of the GUV and the fluorescence intensity corresponding to the uptake of DNA molecules were extracted as shown previously (see Fig. 2). The diameter ratio (Df/Di) was calculated for each DNA size and is shown in Fig. 3(a). The diameter of the GUVs did not decrease by more than ∼20%, after the application of electric field pulses. Similarly, the uptake of DNA molecules due to electroporation (I/I0)f from Fig. 2(e) was determined for each DNA size and is plotted in Fig. 3(b), as filled squares. Also plotted on the same figure are the theoretical predictions from Yu and Lin12 (solid line) and Portet et al. (2011)10 (dashed lines). For the theoretical prediction of Yu and Lin,12 the final probability of successful translocation (F–PST) was used as a measure for translocation efficiency, (I/I0)f, and for the theoretical prediction of Portet et al. (2011),10 the following equation was used from the proposed theoretical framework:
(1) |
Fig. 3 Size reduction of GUVs and DNA uptake as a function of DNA size. (a) The ratio of the final diameter (Df), after the application of the electric field pulses, to the initial diameter (Di), before the application of electric field pulses. The error bars represent the standard deviation. (b) Filled squares represent the translocation efficiency (I/I0)f as a function of the size of the DNA molecules. The error bars represent standard deviation. Also plotted are the theoretical predictions of the translocation efficiency from Yu and Lin (solid line),12 and Portet et al. (2011) (dashed line).20 (c) Electrophoretic mobility as a function of DNA size calculated using eqn (1) taking (c/c0) as (I/I0)f from (b). Two different values of the flux factor were used; f′(0)θ = 0.26, corresponding to filled black circles and f′(0)θ = 0.15 corresponding to open white circles. Electrophoretic mobilities determined from the literature (see Section 2 of the ESI† for detailed values) are also plotted for TAE buffer22 and low conductivitybuffers,25 as dashed and solid lines, respectively. |
The conductivity of the medium has been shown to influence the electrophoretic mobility of the DNA molecules.22–25 The buffers used in the current experiments consist of 260 mM glucose as the external medium and 240 mM sucrose as the internal medium. This corresponds to very low conductivity or ionic strength compared to TAE buffers. The electrophoretic mobility of DNA molecules in low conductivity buffers can be estimated by systematically reducing the ionic strength, as was done for dsA5 DNA molecules (20 bp).25 In this case, the electrophoretic mobility of dsA5 DNA at zero ionic strength was estimated to be μ = 4.6 × 10−8 m2 S−1 V−1, by the linear extrapolation of electrophoretic mobilities in the range of low ionic strength. A quantitatively similar increase in electrophoretic mobility was observed for dsA5 DNA molecules (20 bp) and pUC19 DNA molecules (2686 bp) with systematic reduction of ionic strength.24 Therefore, a similar quantitative increase in electrophoretic mobilities was considered for the sizes of the DNA molecules considered in this work corresponding to the low conductivity glucose and sucrose buffers. See Section 2 of the ESI† for the exact values of electrophoretic mobilities used, corresponding to TAE (Table ST1, ESI†) and the low conductivity sucrose and glucose buffers (Table ST2, ESI†), for the different DNA sizes in this work. The theoretical prediction according to eqn (1) with an increased electrophoretic mobility corresponding to the low conductivity buffers, and the same flux factor (f′(0)θ = 0.26), is shown in Fig. 3(b) as a dotted line with a legend; f′(0)θ = 0.26, glucose/sucrose buffer. Also shown in the same figure are the predictions from eqn (1) with the same increased electrophoretic mobility, however a low value of flux factor (f′(0)θ = 0.15) as a dashed line (legend; f′(0)θ = 0.15, glucose/sucrose buffer) which shows better agreement with the experimental values.
Based on the theoretical framework of Portet et al. (2011),10 the electrophoretic mobility can be back-calculated using eqn (1). Taking (c/c0) as (I/I0)f and using the electric field parameters from the experiments; E0 = 0.45 kV cm−1, N = 10, tp = 5 ms and R = 15 μm the electrophoretic mobility can be estimated for each size of the DNA molecule. These values are plotted in Fig. 3(c), as filled black and open white circles for f′(0)θ = 0.26 and f′(0)θ = 0.15, respectively. On the same figure, electrophoretic mobilities determined from the literature (see Section 2 of the ESI†) are also plotted for TAE buffer and low conductivity buffers, as dashed and solid lines, respectively. The close match between the experimentally determined electrophoretic mobilities and the values determined from the literature further validates the applicability of the theoretical framework of Portet et al. (2011)10 as the dominant mode of DNA translocation during electroporation of GUVs.
The model of Portet et al. (2011)10 suggests that the DNA molecules can cross the electro-pores in their native polymer conformation, or that the electro-pores are large enough to allow the DNA molecules to cross freely. The bulk electrophoretic mobility governs the transport across the pores during electroporation. According to the theoretical framework adapted by Portet et al. (2011),10 the normalized increase in the concentration of DNA molecules inside the GUVs after the application of electric field pulses is given by eqn (1). The prediction is plotted in Fig. 3(b) for different values of electrophoretic mobility and flux factors f′(0)θ, as dashed lines. This prediction matches with the experimental values of normalized mean intensity of DNA molecules inside the GUVs, (I/I0)f, for values of electrophoretic mobilities of DNA molecules in glucose/sucrose buffer, and for a flux factor of f′(0)θ = 0.15. However, these values are slightly different from the values used in the theoretical framework of Portet et al. (2011).10
The electrophoretic mobility and the flux factor used in the theoretical framework of Portet et al. (2011) were μ = 3.75 × 10−8 m2 S−1 V−1 and f′(0)θ = 0.26.10 This value of electrophoretic mobility was measured for DNA molecules with size greater than 400 bp and for a TAE buffer (40 mM Tris, 1 mM EDTA, pH 8.0).22 The conductivity of TAE buffer is found to be in the range of 0.38–3.11 mS cm−1 depending upon the concentration of Tris (10–80 mM).23 The conductivity of the external buffer used by Portet et al. (2011) (260 mM glucose, 1 mM KH2PO4/K2HPO4, 1 mM NaCl, pH 7) was measured to be 0.45 mS cm−1.10 This lies within the range of conductivities of the TAE buffers used to measure the electrophoretic mobilities of DNA molecules. Addition of 2–50 mM NaCl to TAE buffers has shown to influence the value of the electrophoretic mobility, albeit only slightly for 2 mM NaCl.24 Therefore, ignoring the effects of addition of 1 mM NaCl to the KH2PO4/K2HPO4 buffer, a value of electrophoretic mobility of μ = 3.75 × 10−8 m2 S−1 V−1 seems to be an appropriate choice for a buffer adopted by Portet et al. (2011).10 The buffers used in this work correspond to 260 mM glucose as the external solution and 240 mM sucrose as the internal solution. The conductivities of 200 mM glucose and 200 mM sucrose solution were measured to be 0.0045 and 0.006 mS cm−1,26 respectively. The conductivity of 240 mM sucrose solutions was measured to be 0.015 mS cm−1.10 Thus, it can be assumed that the conductivities of the glucose and sucrose solutions used in this work are O(10−3–10−2) mS cm−1. For such low conductivity solutions, the electrophoretic mobilities are expected to be higher.22–25 Using values of electrophoretic mobilities corresponding to the low conductivity sucrose/glucose buffers (see Section 2 of the ESI† for precise values used) and a flux factor f′(0)θ = 0.26, the translocation efficiency or the normalized concentration according to eqn (1) plotted in Fig. 3(b) captures the qualitative trend, however it over-predicts the experimental translocation efficiency.
The value of the flux factor in the theoretical framework by Portet et al. (2011) was f′(0)θ = 0.26.10 The authors determined this value by comparing their experimental results with the theory. According to the theoretical framework, for conductivity ratios (external solution conductivity/internal solution conductivity) between 1 and 10, the experimentally determined flux factor f′(0)θ should lie between 0.1–0.5.10 The authors obtained a distribution of experimentally determined f′(0)θ, with a mean value of the distribution as 〈f′(0)θ〉 = 0.26, thus validating the theoretical framework for a DNA size of 4700 bp. According to Fig. 3(b), the theoretical prediction (eqn (1)) matches with the experimental translocation efficiencies in this work, for electrophoretic mobilities corresponding to low conductivity sucrose/glucose buffers, and for a flux factor value of f′(0)θ = 0.15. This value of flux factor f′(0)θ also lies between 0.1 and 0.5, suggesting the validity of the theoretical framework by Portet et al. (2011)10 for DNA sizes ranging from 25–20000 bp.
A lower value of flux factor, f′(0)θ = 0.15 in this work, as opposed to f′(0)θ = 0.26 in Portet et al. (2011),10 could be due to multiple reasons. f′(0) depends on the conductivity ratio, and θ represents the angle that the permeabilized area, accessible to DNA translocation, subtends at the center of the GUV.10 A constant value of f′(0) = 2.34 rad−1 corresponding to a conductivity ratio of 1 was considered.10 A similar value of f′(0) = 3 rad−1 was obtained for the buffers, the electric field pulsing conditions and the GUV diameters corresponding to this work (see Section 3 of the ESI†). The distribution, as reported by Portet et al. (2011),10 thus arises due to different θ values corresponding to different permeabilized areas.10 The authors attributed this to different electric field intensities, pulse durations and GUV diameters considered which could lead to different permeabilized areas accessible to DNA translocation.10 Thus, f′(0)θ = 0.15 for the constant electric field pulsing conditions, the GUV diameters and the buffers used in this work could correspond to a value lying in the lower spectrum of the distribution reported by Portet et al. (2011).10
Considering f′(0) = 3 rad−1, a permeabilized area subtending an angle θ ∼ 2.9° (for f′(0)θ = 0.15) is obtained. Using Dperm = Rθc, where Dperm is the diameter of the permeabilized area (assuming a circular area), R is the radius of the vesicle and θc is the permeabilized angle θ in radians, a permeabilzed area with a diameter Dperm ∼ 0.75 μm is obtained for θ ∼ 2.9°. This permeabilized area could be considered as a single macropore. Although macropores are observed during the electroporation of GUVs,20,27 they are only observed during the last few μs of a ms pulse, and remain open for tens of milliseconds after the pulse ends.27 Since diffusion of DNA molecules is negligible compared to electrophoresis,10 majority of the DNA transport thus occurs during, and not after the pulse. However, the experiments in this work suggest a mode of transport where the pores are large enough during the electric field pulse to allow DNA molecules to translocate freely in their native polymer conformation. This implies that the pores formed are comparable to the coil size (or radius of gyration Rg) of the DNA molecules. The largest DNA molecule used in this work (20000 bp) has an Rg ∼ 1 μm (see Section 4 of the ESI†), similar in size to the permeabilized area (Dperm ∼ 0.75 μm). Thus, a permeabilized area with diameter Dperm ∼ 0.75 μm can be considered as a macropore. When the size of electro-pores is smaller than Rg of the DNA molecules, DNA translocation across the membrane follows more complex mechanisms. For instance, DNA molecules form a DNA–membrane complex (for DNA sizes ≥ 25 bp) at the cell membrane of living cells under electropermeabilization conditions, which can influence the DNA translocation efficiency during electric pulses.3,28,29 These results, apart from providing a mechanistic understanding of DNA translocation, also provide information on the pore size during electroporation of GUVs.
Footnotes |
† Electronic supplementary information (ESI) available: Fig. S1–S5 and Tables ST1–ST4. See DOI: 10.1039/c9sm01274e |
‡ Present address: Science for Life Laboratory, Department of Applied Physics, KTH Royal Institute of Technology, Tomtebodavägen 23, SE-171 65 Solna, Sweden. |
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