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Effects of pulse strength and pulse duration on in vitro DNA electromobility
Abstract
Interstitial transport of DNA is a rate-limiting step in electric field-mediated gene delivery in vivo. Interstitial transport of macromolecules, such as plasmid DNA, over a distance of several cell layers, is inefficient due to small diffusion coefficient and inadequate convection. Therefore, we explored electric field as a novel driving force for interstitial transport of plasmid DNA. In this study, agarose gels were used to mimic the interstitium in tissues as they had been well characterized and could be prepared reproducibly. We measured the electrophoretic movements of fluorescently labeled plasmid DNA in agarose gels with three different concentrations (1.0%, 2.0% and 3.0%) subjected to electric pulses at three different field strengths (100, 200 and 400 V/cm) and four different pulse durations (10, 50, 75, 99 ms). We observed that: (1) shorter pulses (10 ms) were not as efficient as longer pulses in facilitating plasmid transport through agarose gels; (2) plasmid electromobility reached a plateau at longer pulse durations; and (3) plasmid electromobility increased with applied electric energy, up to a threshold, in all three gels. These data suggested that both pulse strength and duration needed to be adequately high for efficient plasmid transport through extracellular matrix. We also found that electric field was better than concentration gradient of DNA as a driving force for interstitial transport of plasmid DNA.
... The mobility of charged molecules such as RNA are described by Nernst-Planck equation, which incorporates both diffusion and electrophoresis [5,[69][70][71]. Before pulse application diffusion dominates, while during pulse application strong electric field results in an electrophoretic force that drags siRNA in the opposite direction of the applied electric field. ...
... During the pulse application the electrophoretic force F e drags siRNA in the opposite direction of the field. The electrophoretic displacement (L E ) during pulse application can be calculated [5,64,69,73]: ...
... We performed our analysis on an in vitro system, but the main conclusions are valid also for tissues. Since siRNA is relatively small molecule and its radius gyration (R g ~ 1.8 nm) is smaller than pores sizes in the extracellular matrix in tissue (20-200 nm), the electrophoresis and diffusion of siRNA will be hindered much less compared to larger plasmid DNA molecules (Zimm model) [69,70,72,77]. Therefore, we expect that in tissues the electroporation parameters that enable delivery of small molecules will enable also efficient delivery of siRNA, however, the silencing efficiency could be affected due to poor RNA stability. ...
Background
Electrotransfection is based on application of high-voltage pulses that transiently increase membrane permeability, which enables delivery of DNA and RNA in vitro and in vivo. Its advantage in applications such as gene therapy and vaccination is that it does not use viral vectors. Skeletal muscles are among the most commonly used target tissues. While siRNA delivery into undifferentiated myoblasts is very efficient, electrotransfection of siRNA into differentiated myotubes presents a challenge. Our aim was to develop efficient protocol for electroporation-based siRNA delivery in cultured primary human myotubes and to identify crucial mechanisms and parameters that would enable faster optimization of electrotransfection in various cell lines.
Results
We established optimal electroporation parameters for efficient siRNA delivery in cultured myotubes and achieved efficient knock-down of HIF-1α while preserving cells viability. The results show that electropermeabilization is a crucial step for siRNA electrotransfection in myotubes. Decrease in viability was observed for higher electric energy of the pulses, conversely lower pulse energy enabled higher electrotransfection silencing yield. Experimental data together with the theoretical analysis demonstrate that siRNA electrotransfer is a complex process where electropermeabilization, electrophoresis, siRNA translocation, and viability are all functions of pulsing parameters. However, despite this complexity, we demonstrated that pulse parameters for efficient delivery of small molecule such as PI, can be used as a starting point for optimization of electroporation parameters for siRNA delivery into cells in vitro if viability is preserved.
Conclusions
The optimized experimental protocol provides the basis for application of electrotransfer for silencing of various target genes in cultured human myotubes and more broadly for electrotransfection of various primary cell and cell lines. Together with the theoretical analysis our data offer new insights into mechanisms that underlie electroporation-based delivery of short RNA molecules, which can aid to faster optimisation of the pulse parameters in vitro and in vivo.
... One of main causes of low in vivo electrotransfer efficiency is relatively low mobility of DNA in tissue compared to mobility in in vitro conditions. In different tissues, extensive network of extracellular matrix hinders DNA mobility to migrate towards the cell by reducing especially its diffusion and its electrophoretic mobility during electric pulse application [35][36][37][38][39]. ...
... where c (z, t) describes a time-dependent spatial concentration distribution of pDNA and z is the distance from the top of the gel to the given point inside the gel (see Fig. 5A). For estimation of the diffusion constant D, we have used measured diffusion coefficients from Zaharoff and Yuan 2004 [36] for 0.5-3% agarose gel. Since our 3D collagen gel with [36] to lower gel percentages (0.35% w/w collagen) and obtained D = 3 × 10 −8 cm 2 /s, which we have used in our theoretical analysis of pDNA diffusion. ...
... For estimation of the diffusion constant D, we have used measured diffusion coefficients from Zaharoff and Yuan 2004 [36] for 0.5-3% agarose gel. Since our 3D collagen gel with [36] to lower gel percentages (0.35% w/w collagen) and obtained D = 3 × 10 −8 cm 2 /s, which we have used in our theoretical analysis of pDNA diffusion. In a more dense 3% gel D is reduced to ~ 0.01 × 10 −8 cm 2 /s, while in water media (culture media) D ~ 5 × 10 −8 cm 2 /s. ...
Background
Gene electrotransfer is an established method that enables transfer of DNA into cells with electric pulses. Several studies analyzed and optimized different parameters of gene electrotransfer, however, one of main obstacles toward efficient electrotransfection in vivo is relatively poor DNA mobility in tissues. Our aim was to analyze the effect of impaired mobility on gene electrotransfer efficiency experimentally and theoretically. We applied electric pulses with different durations on plated cells, cells grown on collagen layer and cells embedded in collagen gel (3D model) and analyzed gene electrotransfer efficiency. In order to analyze the effect of impaired mobility on gene electrotransfer efficiency, we applied electric pulses with different durations on plated cells, cells grown on collagen layer and cells embedded in collagen gel (3D model) and analyzed gene electrotransfer efficiency.
Results
We obtained the highest transfection in plated cells, while transfection efficiency of embedded cells in 3D model was lowest, similarly as in in vivo. To further analyze DNA diffusion in 3D model, we applied DNA on top or injected it into 3D model and showed, that for the former gene electrotransfer efficiency was similarly as in in vivo. The experimental results are explained with theoretical analysis of DNA diffusion and electromobility.
Conclusion
We show, empirically and theoretically that DNA has impaired electromobility and especially diffusion in collagen environment, where the latter crucially limits electrotransfection. Our model enables optimization of gene electrotransfer in in vitro conditions.
... One of main causes of low in vivo electrotransfer efficiency was found to be relatively low mobility of DNA in tissue compared to mobility in in vitro conditions. In different tissues, extensive network of extracellular matrix hinders DNA mobility to migrate towards the cell by reducing its diffusion and especially its electrophoretic mobility during electric pulse application [35][36][37][38][39]. ...
... Electrophoresis is another mechanism which was shown to be important for the delivery of DNA molecules into cells by electric pulses [32,[34][35][36]. During pulse application, the electrophoretic driving force acts (F) on the negatively charged DNA molecule and drags it toward the cathodic side of the cell membrane. ...
... where the effective charge depends on the ionic strength of the solution and length of the pDNA (eeff = 0.066 per base pair × 4.7 k bp for our 4.7 kbp pDNA). DNA molecule moving in an aqueous solution under external electric field E reaches the steady state velocities v practically immediately -in approximately 3 × 10 -11 s [36], therefore during pulse application steady-state conditions can be assumed. Under steady-state condition frictional force equals electrophoretic force, therefore electrophoretic mobility μ is: ...
Background: Gene electrotransfer is an established method that enables transfer of DNA into cells with electric pulses. Several studies analyzed different parameters; however the question of the mechanisms involved in gene electrotransfer remains open. One of main obstacles toward efficient gene electrotransfer in vivo is relatively poor DNA mobility in tissues.
Objective and method: In order to analyze the effect of impaired mobility on gene electrotransfer efficiency, we applied electric pulses with different durations on plated cells, cells grown on collagen layer and cells embedded in collagen gel (3D model) and compared gene electrotransfer efficiency and viability of cells.
Results: We obtained the highest transfection of plated cells, while transfection efficiency of embedded cells in 3D model was lowest and similar as in in vivo. To further analyze poor DNA mobility in 3D model, we applied DNA in top of or injected it into 3D model and showed that former way increases gene electrotransfer efficiency as was shown in in vivo studies.
Conclusion: We reported empirically and theoretically evidence that DNA has impaired mobility and diffusion in collagen environment. In addition our method provides resembling in vivo situation, where gene electrotransfer mechanisms can be studied.
... The complex presents binding to the membrane or partial insertion of DNA in the permeabilized cell membrane 15,16,19,29,30 . The first visualization performed by labeling DNA with fluorescent dye TOTO-1 41 showed complex formation only on the cathodic side of the cell. In addition, within tissues the extracellular matrix represents another barrier which reduces the amount of DNA interacting with the target cells, by hindering the homogeneous distribution of the injected DNA and by decreased DNA mobility during electric pulses [40][41][42] . ...
... The first visualization performed by labeling DNA with fluorescent dye TOTO-1 41 showed complex formation only on the cathodic side of the cell. In addition, within tissues the extracellular matrix represents another barrier which reduces the amount of DNA interacting with the target cells, by hindering the homogeneous distribution of the injected DNA and by decreased DNA mobility during electric pulses [40][41][42] . The free diffusion of DNA is almost negligible compared to electrophoretic drag, therefore, the selection of sufficiently high and long pulses is important 12,16,17,21,40,43 . ...
... The process of DNA transfer across the cell membrane has not been directly visualized yet. The DNA enters the cytoplasm several minutes after pulse application 41,48 ; first the contact of the DNA with the permeabilized cell membrane is formed 15,19,34,44 , then the DNA is either translocated across the cell membrane by an unidentified mechanism 20,23 , or alternatively, the DNA enters the cells by electric-field-stimulated endocytosis 32,45,46 . ...
Gene electrotransfer is a promising non-viral method of gene delivery. In our in vitro study we addressed open questions about this multistep process: how electropermeabilization is related to electrotransfer efficiency; the role of DNA electrophoresis for contact and transfer across the membrane; visualization and theoretical analysis of DNA-membrane interaction and its relation to final transfection efficiency; and the differences between plated and suspended cells. Combinations of high-voltage and low-voltage pulses were used. We obtained that electrophoresis is required for the insertion of DNA into the permeabilized membrane. The inserted DNA is slowly transferred into the cytosol, and nuclear entry is a limiting factor for optimal transfection. The quantification and theoretical analysis of the crucial parameters reveals that DNA-membrane interaction (NDNA) increases with higher DNA concentration or with the addition of electrophoretic LV pulses while transfection efficiency reaches saturation. We explain the differences between the transfection of cell suspensions and plated cells due to the more homogeneous size, shape and movement of suspended cells. Our results suggest that DNA is either translocated through the stable electropores or enters by electo-stimulated endocytosis, possibly dependent on pulse parameters. Understanding of the mechanisms enables the selection of optimal electric protocols for specific applications.
... Currently both theoretical models [42] and experimental quantification of DNA electrophoresis were studied on model gel systems [43] and ex-vivo on tumors [44,45], however full quantitative description of this process in different tissues is very complex and is still lacking [46]. ...
... This is in agreement with studies by Bureau et al. and Satkauskas et al. [33][34][35] where LV pulses markedly increased transfection in vivo conditions. In vivo we can expect that local plasmid concentrations are sub-optimal due to limited plasmid mobility in the tissue and availability of plasmid DNA at the cell membrane level [43][44][45][46]49]. ...
... Therefore in in vivo conditions LV pulses are crucial for efficient transfection as they provide electrophoretic force needed to drag negatively charged DNA molecules toward the cell membrane. Our explanation is also in agreement with conclusions of Golzio et al. [20,50] that crucial step in gene electrotransfer is the interaction of the DNA molecule with the cell membrane, as well as with the experimental and theoretical studies of Zaharoff et al. that long pulses are important for electrophoretic movement of DNA in gels [43] and for improving interstitial transport of DNA during gene delivery in tissues [43,44,46]. ...
Gene electrotransfer is an established method for gene delivery which uses high-voltage pulses to increase permeability of cell membrane and thus enables transfer of genes. Currently, majority of research is focused on improving in vivo transfection efficiency, while mechanisms involved in gene electrotransfer are not completely understood. In this paper we analyze the mechanisms of gene electrotransfer by using combinations of high-voltage (HV) and low-voltage pulses (LV) in vitro. We applied different combinations of HV and LV pulses to CHO cells and determined the transfection efficiency. We obtained that short HV pulses alone were sufficient to deliver DNA into cells for optimal plasmid concentrations and that LV pulse did not increase transfection efficiency, in contrast to reported studies in vivo. However, for sub-optimal plasmid concentrations combining HV and LV pulses increased transfection rate. Our results suggest that low-voltage pulses increase transfection in conditions where plasmid concentration is low, typically in vivo where mobility of DNA is limited by the extracellular matrix. LV pulses provide additional electrophoretic force which drags DNA toward the cell membrane and consequently increase transfection efficiency, while for sufficiently high concentrations of the plasmid (usually used in vitro) electrophoretic LV pulses do not have an important role.
... However, the interplay between HV and LV pulses, and how the parameters of HV and LV pulses influenced DNA electrotransfer, were still unclear. In elegant in vitro experiments using 1, 2, and 3% agarose gels and pulses similar to our LV pulses, Zaharoff and Yuan (2004) analyzed DNA electromobility: they showed that, using pulses of 10 to 99 msec at 100 to 400 V/cm (comparable to our LV pulses), plasmids were transported over distances longer, by two to three orders of magnitude, than those achieved with pulses of 99 sec at 2.0 kV/cm (comparable to our HV pulses). We discuss here both old and new data on the influence of HV and LV pulses on DNA electrotransfer efficacy in light of these in vitro data. ...
... Each column represents results from 10 muscles treated in one experiment. be shown by taking into account the in vitro results of Zaharoff and Yuan (2004) on the mobility in agarose gels of DNA molecules exposed to pulse durations similar to those of the LV pulses described here. The agarose gels used by these authors are supposed to mimic interstitial barriers in biological tissues. ...
... They found that the dependence of plasmid electromobility on pulse duration was not linear and displayed a sigmoid shape: at shorter durations electromobility is low, whereas with longer durations it increases, reaching a plateau at 50 msec in 1% agarose gels, and at higher pulse duration (80-100 msec) in more concentrated gels (2-3% agarose). Indeed, for a plasmid to move through the narrow passages in agarose gels, random coiled DNA should elongate in the direction of motion and shrunk in the perpendicular direction ( Zaharoff and Yuan, 2004). Then, if a long LV pulse is substituted by multiple shorter LV pulses, separated by 1 sec, the elongated plasmid relaxes between the pulses. ...
Efficient DNA electrotransfer can be achieved with combinations of short high-voltage (HV) and long low voltage (LV) pulses that cover two effects of the pulses, namely, target cell electropermeabilization and DNA electrophoresis within the tissue. Because HV and LV can be delivered with a lag up to 3000 sec between them, we considered that it was possible to analyze separately the respective importance of the two types of effects of the electric fields on DNA electrotransfer efficiency. The tibialis cranialis muscles of C57BL/6 mice were injected with plasmid DNA encoding luciferase or green fluorescent protein and then exposed to various combinations of HV and LV pulses. DNA electrotransfer efficacy was determined by measuring luciferase activity in the treated muscles. We found that for effective DNA electrotransfer into skeletal muscles the HV pulse is prerequisite; however, its number and duration do not significantly affect electrotransfer efficacy. DNA electrotransfer efficacy is dependent mainly on the parameters of the LV pulse(s). We report that different LV number, LV individual duration, and LV strength can be used, provided the total duration and field strength result in convenient electrophoretic transport of DNA toward and/or across a permeabilized membrane.
... Despite its potential significance, the in vivo electrophoresis component of electric fieldmediated gene delivery is only the focus of a few studies in the literature [33][34][35][36][37]. The significance of the electrophoretic component has been demonstrated in vivo in muscle by studying the transfection levels obtained using a combination of low voltage, non-porating pulses with long duration (LV), and high voltage, porating pulses with short duration (HV). ...
... In this study, we investigated the ability of an applied pulsed electric field to overcome the interstitial barrier by quantifying the magnitude of in vivo electric field-induced transport of pDNA in tumor interstitium. Zaharoff et al. have previously reported pDNA electromobility ex vivo in tumor interstitium [36], as well as pDNA electromobility in agarose tissue phantoms [37]. Here, we used a similar technique to quantify interstitial transport of pDNA in vivo in two types of tumors (4T1 and B16.F10) grown in mouse dorsal skin-fold chambers (DSCs), and correlated the observed electrophoretic movement with the tissue collagen content as has been observed in previous studies [36,38]. ...
... The distance of pDNA movement was statistically greater than zero only if a pulsed field was applied to tumors. This observation confirmed a notion that DNA electrophoresis was several orders of magnitude faster than passive diffusion [36,37]. As a result, the interstitial electrophoresis could effectively push more pDNA molecules towards the transient pores in the plasma membrane of cells. ...
Local pulsed electric field application is a method for improving non-viral gene delivery. Mechanisms of the improvement include electroporation and electrophoresis. To understand how electrophoresis affects pDNA delivery in vivo, we quantified the magnitude of electric field-induced interstitial transport of pDNA in 4T1 and B16.F10 tumors implanted in mouse dorsal skin-fold chambers. Four different electric pulse sequences were used in this study, each consisted of 10 identical pulses that were 100 or 400 V/cm in strength and 20 or 50 ms in duration. The interval between consecutive pulses was 1 s. The largest distance of transport was obtained with the 400 V/cm and 50 ms pulse, and was 0.23 and 0.22 microm/pulse in 4T1 and B16.F10 tumors, respectively. There were no significant differences in transport distances between 4T1 and B16.F10 tumors. Results from in vivo mapping and numerical simulations revealed an approximately uniform intratumoral electric field that was predominantly in the direction of the applied field. The data in the study suggested that interstitial transport of pDNA induced by a sequence of ten electric pulses was ineffective for macroscopic delivery of genes in tumors. However, the induced transport was more efficient than passive diffusion.
... These barriers can be studied separately to improve the transfection efficiency since transport processes through the barriers occur in a sequential manner. Electric field-mediated pDNA transport through the interstitium has been quantified in previous studies (4)(5)(6)(7). The data suggest that interstitial transport of pDNA is largely ineffective for macroscopic delivery of genes but is several orders of magnitude faster than passive diffusion. ...
... where C 0 is the concentration in the extracellular medium and P is the permeability coefficient of FD across the membrane. Equations 1 through 4 can be solved analytically and the solution is (5) where erfc is the complementary error function and h is the ratio of P and D (46). The average intracellular concentration (C mean ) of FD was defined as the total amount of FD in a cell per unit volume. ...
... The average intracellular concentration (C mean ) of FD was defined as the total amount of FD in a cell per unit volume. It was calculated by integrating Equation 5. The result is, (6) For 1-D diffusion in a membrane with homogeneous structures, P can be determined by, (7) where α is the fractional area of pores in a permeabilized cap and δ is the membrane thickness. ...
Pulsed electric field has been widely used as a nonviral gene delivery platform. The delivery efficiency can be improved through quantitative analysis of pore dynamics and intracellular transport of plasmid DNA. To this end, we investigated mechanisms of cellular uptake of macromolecules during electroporation. In the study, fluorescein isothiocyanate-labeled dextran (FD) with molecular weight of 4,000 (FD-4) or 2,000,000 (FD-2000) was added into suspensions of a murine mammary carcinoma cell (4T1) either before or at different time points (ie, 1, 2, or 10 sec) after the application of different pulsed electric fields (in high-voltage mode: 1.2-2.0 kV in amplitude, 99 microsec in duration, and 1-5 pulses; in low-voltage mode: 100-300 V in amplitude, 5-20 msec in duration, and 1-5 pulses). The intracellular concentrations of FD were quantified using a confocal microscopy technique. To understand transport mechanisms, a mathematical model was developed for numerical simulation of cellular uptake. We observed that the maximum intracellular concentration of FD-2000 was less than 3% of that in the pulsing medium. The intracellular concentrations increased linearly with pulse number and amplitude. In addition, the intracellular concentration of FD-2000 was approximately 40% lower than that of FD-4 under identical pulsing conditions. The numerical simulations predicted that the pores larger than FD-4 lasted <10 msec after the application of pulsed fields if the simulated concentrations were on the same order of magnitude as the experimental data. In addition, the simulation results indicated that diffusion was negligible for cellular uptake of FD molecules. Taken together, the data suggested that large pores induced in the membrane by pulsed electric fields disappeared rapidly after pulse application and convection was likely to be the dominant mode of transport for cellular uptake of uncharged macromolecules.
... Each column represents results from 10 muscles treated in one experiment. be shown by taking into account the in vitro results ofZaharoff and Yuan (2004)on the mobility in agarose gels of DNA molecules exposed to pulse durations similar to those of the LV pulses described here. The agarose gels used by these authors are supposed to mimic interstitial barriers in biological tissues. ...
... They found that the dependence of plasmid electromobility on pulse duration was not linear and displayed a sigmoid shape: at shorter durations electromobility is low, whereas with longer durations it increases, reaching a plateau at 50 msec in 1% agarose gels, and at higher pulse duration (80–100 msec) in more concentrated gels (2–3% agarose). Indeed, for a plasmid to move through the narrow passages in agarose gels, random coiled DNA should elongate in the direction of motion and shrunk in the perpendicular direction (Zaharoff and Yuan, 2004). Then, if a long LV pulse is substituted by multiple shorter LV pulses, separated by 1 sec, the elongated plasmid relaxes between the pulses. ...
... Another possible role of electrophoresis (specific to in vivo) could be to transport DNA in the interstitial space through the dense network of the Extra-Cellular Matrix (ECM) fibres. For instance, diffusion is negligible compared to electrophoresis in the ECM, and the DNA molecules primarily rely on electrophoresis as the dominant mode of transport [59][60][61]. Thus, another role of electrophoresis could be to overcome the interstitial barriers by transporting DNA in the tissue and improving the interstitial distribution of DNA molecules. ...
Gene therapies are revolutionizing medicine by providing a way to cure hitherto incurable diseases. The scientific and technological advances have enabled the first gene therapies to become clinically approved. In addition, with the ongoing COVID-19 pandemic, we are witnessing record speeds in the development and distribution of gene-based vaccines. For gene therapy to take effect, the therapeutic nucleic acids (RNA or DNA) need to overcome several barriers before they can execute their function of producing a protein or silencing a defective or overexpressing gene. This includes the barriers of the interstitium, the cell membrane, the cytoplasmic barriers and (in case of DNA) the nuclear envelope. Gene electrotransfer (GET), i.e., transfection by means of pulsed electric fields, is a non-viral technique that can overcome these barriers in a safe and effective manner. GET has reached the clinical stage of investigations where it is currently being evaluated for its therapeutic benefits across a wide variety of indications. In this review, we formalize our current understanding of GET from a biophysical perspective and critically discuss the mechanisms by which electric field can aid in overcoming the barriers. We also identify the gaps in knowledge that are hindering optimization of GET in vivo.
... High-voltage pulse causes electroporation of cell membrane, while the low-voltage pulse helps highly charged DNA entrance into the cell interior. A low-voltage pulse thus provides electrophoretic movement of DNA into the cell in in vitro conditions, or it can be a powerful driving force for improving interstitial transport of DNA during gene delivery in vivo (Klenchin et al. 1991, Sukharev et al. 1992Zaharoff et al. 2002;Zaharoff and Yuan 2004). The effect of electrophoretic pulses was successfully used and demonstrated in in vivo experiments in mammalian tissues (Bureau et al. 2000;Somiari et al. 2000;Šatkauskas et al. 2002;Šatkauskas et al. 2005;Andre and Mir 2004;Zampaglione et al. 2005;Pavšelj and Preat 2005a). ...
... Taking this into account, it becomes more understandable why a short-duration protocol, where a relatively small interaction is observed, can lead to a high percentage of absolute transfection compared to longerduration electric pulse protocols. On the other hand, longer pulses are preferable if only high 'relative' percentage transfection is needed as is the case, for example, for certain biotechnological applications [15], or in vivo [64] and under similar conditions [65,66] where it was shown that longer pulses are more efficient. Namely, in vivo, lextracellular matrix hinders the transport of DNA in the proximity of cells, consequently leading to relatively low transfection; therefore, the electrophoretic force of longer pulses enables the efficient contact of the DNA molecule with the cell membrane [66][67][68]. ...
Background:
Gene electrotransfer is a nonviral method used for DNA delivery into cells. Several steps are involved. One of them is the interaction of DNA with the cell membrane, which is crucial before DNA can enter the cell. We analysed the level of DNA-membrane interaction in relation to electrotransfer efficiency and the importance of the electrophoretic accumulation of DNA at the cell membrane. Systematic comparison of long-duration, short-duration and combinations of electropermeabilizing short (high-voltage; HV) and electrophoretic long (low-voltage; LV) pulses were performed. The effect of Mg(2+) ion concentrations on electrotransfer and their effect on DNase activity were explored.
Methods:
To visualize the DNA-membrane interaction, TOTO-1 labeled DNA was used. Transfection efficiency was assessed with plasmid DNA coding for green fluorescent protein.
Results:
Higher relative electrotransfer efficiency was obtained by using longer pulses, whereas shorter pulses preserved cell viability. Short-duration pulses enabled higher (24%) overall transfection yield compared to long-duration pulses (12%), although a higher DNA-membrane interaction was observed. No significant difference in transfection was obtained between different HV-LV pulsing protocols, although the highest DNA-membrane interaction was observed with HV + LV pulses. The formation of the DNA-membrane complex depended on the Mg(2+) concentration, whereas DNase inhibitor did not affect gene expression.
Conclusions:
Gene electrotransfer is a complex phenomenon, where many factors mutually affect the process and the DNA-membrane interaction only comprises the first step. We showed that longer electric pulses are optimal for higher transfection efficiency but reduce viability, whereas shorter pulses enable moderate transfection efficiency and preserve viability. Thus, each application needs a careful choice of pulsing protocol.
... High-voltage pulse causes electroporation of cell membrane, while the low-voltage pulse helps highly charged DNA entrance into the cell interior. A low-voltage pulse thus provides electrophoretic movement of DNA into the cell in in vitro conditions, or it can be a powerful driving force for improving interstitial transport of DNA during gene delivery in vivo (Klenchin et al. 1991, Sukharev et al. 1992 Zaharoff et al. 2002; Zaharoff and Yuan 2004). The effect of electrophoretic pulses was successfully used and demonstrated in in vivo experiments in mammalian tissues (Bureau et al. 2000; Somiari et al. 2000; ˇ Satkauskas et al. 2002; ˇ Satkauskas et al. 2005; Andre and Mir 2004; Zampaglione et al. 2005; Pavšelj and Preat 2005a). ...
In this chapter, basics and mechanisms of electroporation are presented. Most important electric pulse parameters for electroporation
efficiency for different applications that involve introduction of small molecules and macromolecules into the cell or cell
membrane electrofusion are described. In all these applications, cell viability has to be preserved. However, in some biotechnological
applications, such as liquid food sterilization or water treatment, electroporation is used as a method for efficient cell
killing. For all the applications mentioned above, besides electric pulse parameters, other factors, such as electroporation
medium composition and osmotic pressure, play significant roles in electroporation effectiveness. For controlled use of the
method in all applications, the basic mechanisms of electroporation need to be known. The phenomenon was studied from the
single-cell level and dense cell suspension that represents a simplified homogenous tissue model, to complex biological tissues.
In the latter, different cell types and electric conductivity that change during the course of electric pulse application
can significantly affect the effectiveness of the treatment. For such a complex situation, the design and use of suitable
electrodes and theoretical modeling of electric field distribution within the tissue are essential. Electroporation as a universal
method applicable to different cell types is used for different purposes. In medicine it is used for electrochemotherapy and
genetherapy. In biotechnology it is used for water and liquid food sterilization and for transfection of bacteria, yeast,
plant protoplast, and intact plant tissue. Understanding the phenomenon of electroporation, its mechanisms and optimization
of all the parameters that affect electroporation is a prerequisite for successful treatment. In addition to the parameters
mentioned above, different biological characteristics of treated cell affect the outcome of the treatment. Electroporation,
gene electrotransfer and electrofusion are affected by cell membrane fluidity, cytoskeleton, and the presence of the cell
wall in bacteria yeast and plant cells. Thus, electroporation parameters need to be specifically optimized for different cell
types.
... electric field intensity was well above the threshold to permeabilize cells [33] and LV (80 V/cm, 500 msec.) was at a level and duration sufficient to induce DNA electrophoresis [34]. Transfection vs permeabilization relationship transfection (data not shown), as observed by other authors with COS7 cells [18]. ...
Electrotransfer can be obtained by the successive delivery of a high voltage short duration pulse (HV) inducing membrane destabilization and then a low voltage long duration pulse (LV), allowing DNA electrophoresis (HVLV mode). Pluronic® L64 (L64) (Fluka, Sigma-Aldrich, L'Isle-d'Abeau Chesnes, Saint-Quentin Fallavier, France) has permeabilizing properties and amplifies the expression of DNA. We aimed to determine whether L64 could have an adjuvant effect on transfection by electrotransfer and whether the sequence L64 injection and then application of a LV pulse could induce transfection comparable to that observed with the HVLV mode.
In vitro, we used fluorescence-activated cell sorting to evaluate Chinese hamster ovary (CHO) cell transfection by a plasmid coding green fluorescent protein, and permeabilization to propidium iodide. In vivo, the transfection efficiency of mice tibial cranial muscle was evaluated by optical imaging using a plasmid DNA encoding luciferase. For the same animals, permeabilization indices were evaluated by magnetic resonance imaging from the uptake of a T(1) contrast agent.
Using the HVLV mode, transfection efficiency was low in vitro on CHO cells but high for muscles in vivo. Pre-treatment by L64 increased the transfection efficiency of electrotransfer for CHO cells but not for muscle. In mice muscles, the L64 amplified the expression of DNA. Nevertheless, neither transgene expression, nor permeability indices were further amplified by subsequent delivery of one LV pulse.
A major finding of the present study is that the nature of the membrane modification induced by electric pulses is not comparable to that mediated by L64. The electrophoretic LV pulse does not induce additive effects to that of L64 for transfection improvement.
... The study revealed that the mannitol solution could significantly increase the extent of tissue penetration of the dextran molecule with molecular weight of 2,000,000 (2M), and that the penetration depth was correlated to the change in the available volume fraction (KAV) of dextran 2M. The present study was designed to investigate whether the pretreatment of 4T1 and B16.F10 tumors with the hyperosmotic solution of mannitol would similarly increase the tumor interstitial space, and therefore the extent of pDNA transport in vivo since previous studies have shown that the rate of interstitial transport depends strongly on the pore sizes in the extracellular matrix 29, 30. In the study, a series of in vitro and ex vivo experiments were first performed to determine the kinetics of tumor cell or tissue volume reduction following the mannitol treatment. ...
Pulsed electric fields can enhance interstitial transport of plasmid DNA (pDNA) in solid tumors. However, the extent of enhancement is still limited. To this end, the effects of cellular resistance to electric field-mediated gene delivery were investigated. The investigation used two tumor cell lines (4T1 (a murine mammary carcinoma) and B16.F10 (a metastatic subline of B16 murine melanoma)) either in suspensions or implanted in two in vivo models (dorsal skin-fold chamber (DSC) and hind leg). The volume fraction of cells was altered by pretreatment with a hyperosmotic mannitol solution (1 M). It was observed that the pretreatment reduced the volumes of 4T1 and B16.F10 cells, suspended in an agarose gel, by 50 and 46%, respectively, over a 20-min period, but did not cause significant changes ex vivo in volumes of hind-leg tumor tissues grown from the same cells in mice. The mannitol pretreatment in vivo improved electric field-mediated gene delivery in the hind-leg tumor models, in terms of reporter gene expression, but resulted in minimal enhancement in pDNA electrophoresis over a few microns distance in the DSC tumor models. These data demonstrated that hyperosmotic mannitol solution could effectively improve electric field-mediated gene delivery around individual cells in vivo by increasing the extracellular space.
Electrotransfection (ET) is a non-viral method for delivery of various types of molecules into cells both in vitro and in vivo. Close to 90 clinical trials that involve the use of ET have been performed, and approximately half of them are related to cancer treatment. Particularly, ET is an attractive technique for cancer immunogene therapy because treatment of cells with electric pulses alone can induce immune responses to solid tumors, and the responses can be further enhanced by ET of plasmid DNA (pDNA) encoding therapeutic genes. Compared to other gene delivery methods, ET has several unique advantages. It is relative inexpensive, flexible, and safe in clinical applications, and introduces only naked pDNA into cells without the use of additional chemicals or viruses. However, the efficiency of ET is still low, partly because biological mechanisms of ET in cells remain elusive. In previous studies, it was believed that pDNA entered the cells through transient pores created by electric pulses. As a result, the technique is commonly referred to as electroporation. However, recent discoveries have suggested that endocytosis plays an important role in cellular uptake and intracellular transport of electrotransfected pDNA. This review will discuss current progresses in the study of biological mechanisms underlying ET, and future directions of research in this area. Understanding the mechanisms of pDNA transport in cells is critical for development of new strategies for improving the efficiency of gene delivery in tumors.
Gene electrotransfer is one of the promising nonviral methods for introducing genes into the cell by using high-voltage electric pulses which transiently permeabilize cell membrane. Gene delivery, which uses electric pulses, is a complex phenomenon consisting of different steps: cell membrane electropermeabilization, formation of the contact between DNA and cell membrane, translocation across the membrane and transfer to the cell nucleus, resulting in final gene expression. In this chapter, we discuss different aspects of electric pulse-mediated plasmid DNA (pDNA) delivery. We focus on the importance of electric pulse parameters for efficient gene electrotransfer, analyze the relation between cell membrane electropermeabilization and gene electrotransfer. We present state of the art of different steps involved in electric pulse-mediated pDNA delivery. Furthermore, we focus on theoretical analysis of gene electrotransfer, calculation of pDNA mobility during electric pulse application with special emphasis on electrophoresis of highly charged pDNA. We discuss the importance of DNA availability at cell membrane level and estimate the number of DNA molecules in contact with the membrane for different concentrations of pDNA used in experiments. A novel study of DNA mobility and gene electrotransfer efficiency in cells embedded in 3D collagens will be presented. Finally, from all the data obtained in studies of mechanisms of electric pulse-mediated pDNA delivery we present also optimization of parameters for gene electrotransfer in muscle tissue using 3D numerical modeling.
The application of voltage has been used to control the movement of charged molecule such as DNA, some proteins and phospholipid in recent year. In this study, we first applied voltage to amyloidogenic protein, α-synuclein, which is related to Parkinson's disease. To offer the new approach using electric field and new insight into aggregation and fibrillation mechanism of α-synuclein, we tried to control the aggregation of α-synuclein, which has a negative charge, by applying voltage to it. The aggregation of α-synuclein without conformational change occurred rapidly when a voltage of 1 V was applied. The protein did not form amyloid-like fibrils, but it did form small aggregates. These results demonstrate that this technique might be useful not only to efficiently control aggregation of α-syn but also to understand the mechanism of aggregation and fibrillation of α-syn.
Abstract This study concerns the modeling and treatment of mass transport associated with electroporation of the skin. Electroporation involves the exposure of relatively impermeable phospholipid membranes to an intense electric field, resulting in order of magnitude increases in permeability to mass transport and increased electrical conductivity. Electroporation is currently conducted in clinical settings to increase the permeability of cells, as well as in the treatment of the skin’s barrier layer. Skin electroporation is used to enhance the success of localized transdermal transport and is the focus of this chapter. This chapter reviews the current understanding of skin electroporation and the methods that have been developed to model the physical process of the alteration of the skin’s barrier structure under the influence of an intense electric field.
The subject of this study concerns modeling and treatment of mass transport associated with electroporation of the skin. A model based on observed phenomena documented in experimental skin electroporation studies is developed in which the evolution of the Local Transport Region is described in terms of the thermal behavior of the Stratum Corneum lipids. This study conducts a parametric investigation of the transport of large charged solute through the skin during a 300 ms 300V electroporation pulse. It is determined that the total solute transported into the skin during an electroporation pulse is more sensitive to factors influencing electrical conductivity than to factors influencing the molecular permeability of the skin.
Electroporation is an approach used to enhance transdermal transport of large molecules in which the skin is exposed to a series of electric pulses. Electroporation temporarily destabilizes the structure of the outer skin layer, the stratum corneum, by creating microscopic pores through which agents, which ordinarily are unable to pass into the skin, are able to pass through this outer barrier. Long duration electroporation pulses can cause localized temperature rises which result in thermotropic phase transitions within the lipid bilayer matrix of the stratum corneum. Chemical agents applied to the skin can reduce the lipid phase transition temperatures. This paper studies the benefits of the combination of the chemical enhancer, terpene d-limonene with low voltage electroporation pulses in order to further aid in electroporation pore development resulting from fluidization of the lipid structures within the stratum corneum. A transient finite volume model is developed in which the thermal and electrical behavior associated with electroporation of in vivo human skin is analyzed and lipid phase transition is represented by a melting process. The Nernst–Planck model is used to represent the electrophoretic-assisted transport of large charged molecules through the skin. The results show that the lower lipid phase transition temperatures associated with the topical application of chemical enhancers to the skin allow for increased solute delivery and solute penetration of the skin reaching radial locations much further than in the untreated case. Solute transport solutions of both cases exhibit local accumulation of concentrations below the stratum corneum – epidermis interface which exceed concentration values initially contained within the applicator gel.
Gene electrotransfer is a promising nonviral method that enables transfer of plasmid DNA into cells with electric pulses. Although many in vitro and in vivo studies have been performed, the question of the implied gene electrotransfer mechanisms is largely open. The main obstacle toward efficient gene electrotransfer in vivo is relatively poor mobility of DNA in tissues. Since cells are mechanically coupled to their extracellular environment and act differently compared to standard in vitro conditions, we developed a three-dimensional (3-D) in vitro model of CHO cells embedded in collagen gel as an ex vivo model of tissue to study electropermeabilization and different parameters of gene electrotransfer. For this purpose, we first used propidium iodide to detect electropermeabilization of CHO cells embedded in collagen gel. Then, we analyzed the influence of different concentrations of plasmid DNA and pulse duration on gene electrotransfer efficiency. Our results revealed that even if cells in collagen gel can be efficiently electropermeabilized, gene expression is significantly lower. Gene electrotransfer efficiency in our 3-D in vitro model had similar dependence on concentration of plasmid DNA and pulse duration comparable to in vivo studies, where longer (millisecond) pulses were shown to be more optimal compared to shorter (microsecond) pulses. The presented results demonstrate that our 3-D in vitro model resembles the in vivo situation more closely than conventional 2-D cell cultures and, thus, provides an environment closer to in vivo conditions to study mechanisms of gene electrotransfer.
The interstitial space is a rate limiting physiological barrier to non-viral gene delivery. External pulsed electric fields have been proposed to increase DNA transport in the interstitium, thereby improving non-viral gene delivery. In order to characterize and improve the interstitial transport, we developed a reproducible single molecule detection method to observe the electromobility of DNA in a range of pulsed, high field strength electric fields typically used during electric field-mediated gene delivery. Using agarose gel as an interstitium phantom, we investigated the dependence of DNA electromobility on field magnitude, pulse duration, pulse interval, and pore size in the interstitial space. We observed that the characteristic electromobility behavior, exhibited under most pulsing conditions, consisted of three distinct phases: stretching, reptation, and relaxation. Electromobility depended strongly on the field magnitude, pulse duration, and pulse interval of the applied pulse sequences, as well as the pore size of the fibrous matrix through which the DNA migrated. Our data also suggest the existence of a minimum pulse amplitude required to initiate electrophoretic transport. These results are useful for understanding the mechanisms of DNA electromobility and improving interstitial transport of genes during electric field-mediated gene delivery.
A hypothesis is presented that a transduction mechanism for low frequency electric fields of physiological strength ( approximately 1 V/cm) is the same as that for sinusoidal fluid shear stresses, the force exerted on an integrin. Simple calculations show that the forces exerted on a model integrin by transverse electric fields and fluid shears that produce cellular effects are comparable in magnitude, about 1 fN. The electric force is provided by the interaction of the surface charges on the integrin with the tangential component of the applied field. The mechanical shear force is the transverse fluid drag force exerted on the cylindrical surface of the integrin. Either force is coupled mechanically to the actin cortex within the cell. The mechanical network which exists within a cell and connects a cell to its surroundings would then be directly coupled to an applied electric field. The fundamental transduction mechanism for some electric field effects may then be ultimately mechanical in nature.
The intratumoral field, which determines the efficiency of electric field-mediated drug and gene delivery, can differ significantly from the applied field. Therefore, we investigated the distribution of the electric field in mouse tumors and tissue phantoms exposed to a large range of electric stimuli, and quantified the resistances of tumor, skin, and electrode-tissue interface. The samples used in the study included 4T1 and B16.F10 tumors, mouse skin, and tissue phantoms constructed with 1% agarose gel with or without 4T1 cells. When pulsed electric fields were applied to samples using a pair of parallel-plate electrodes, we determined the electric field and resistances in each sample as well as the resistance at the electrode-tissue interface. The electric fields in the center region of tissue phantoms and tumor slices ex vivo were macroscopically uniform and unidirectional between two parallel-plate electrodes. The field strengths in tumor tissues were significantly lower than the applied field under both ex vivo and in vivo conditions. During in vivo stimulation, the ratio of intratumoral versus applied fields was approximately either 20% or 55%, depending on the applied field. Meanwhile, the total resistance of skin and electrode-tissue interface was decreased by approximately 70% and the electric resistance at the center of both tumor models was minimally changed when the applied field was increased from 50 to 400 V/cm. These results may be useful for improving electric field-mediated drug and gene delivery in solid tumors.
One of the key issues in electric field-mediated molecular delivery into cells is how the intracellular field is altered by electroporation. Therefore, we simulated the electric field in both the extracellular and intracellular domains of spherical cells during electroporation. The electroporated membrane was modeled macroscopically by assuming that its electric resistivity was smaller than that of the intact membrane. The size of the electroporated region on the membrane varied from zero to the entire surface of the cell. We observed that for a range of values of model constants, the intracellular current could vary several orders of magnitude whereas the maximum variations in the extracellular and total currents were less than 8% and 4%, respectively. A similar difference in the variations was observed when comparing the electric fields near the center of the cell and across the permeabilized membrane, respectively. Electroporation also caused redirection of the extracellular field that was significant only within a small volume in the vicinity of the permeabilized regions, suggesting that the electric field can only facilitate passive cellular uptake of charged molecules near the pores. Within the cell, the field was directed radially from the permeabilized regions, which may be important for improving intracellular distribution of charged molecules.
Gene therapy has a great potential in cancer treatment. However, the efficacy of cancer gene therapy is currently limited by the lack of a safe and efficient means to deliver therapeutic genes into the nucleus of tumor cells. One method under investigation for improving local gene delivery is based on the use of pulsed electric field. Despite repeated demonstration of its effectiveness in vivo, the underlying mechanisms behind electric field-mediated gene delivery remain largely unknown. Without a thorough understanding of these mechanisms, it will be difficult to further advance the gene delivery. In this review, the electric field-mediated gene delivery in solid tumors will be examined by following individual transport processes that must occur in vivo for a successful gene transfer. The topics of examination include: (i) major barriers for gene delivery in the body, (ii) distribution of electric fields at both cell and tissue levels during the application of external fields, and (iii) electric field-induced transport of genes across each of the barriers. Through this approach, the review summarizes what is known about the mechanisms behind electric field-mediated gene delivery and what require further investigations in future studies.
Externally applied electric fields play an important role in many therapeutic modalities, but the fields they produce inside cells remain largely unknown. This study makes use of a three-dimensional model to determine the electric field that exists in the intracellular domain of a 10-μm spherical cell exposed to an applied field of 100 V/cm. The transmembrane potential resulting from the applied field was also determined and its change was compared to those of the intracellular field. The intracellular field increased as the membrane resistance decreased over a wide range of values. The results showed that the intracellular electric field was about 1.1 mV/cm for R<sub>m</sub> of 10 000 Ω·cm<sup>2</sup>, increasing to about 111 mV/cm as R<sub>m</sub> decreased to 100 Ω·cm<sup>2</sup>. Over this range of R<sub>m</sub> the transmembrane potential was nearly constant. The transmembrane potential declined only as R<sub>m</sub> decreased below 1 Ω·cm<sup>2</sup>. The simulation results suggest that intracellular electric field depends on R<sub>m</sub> in its physiologic range, and may not be negligible in understanding some mechanisms of electric field-mediated therapies.
Recent reports have shown the successful delivery of plasmid DNA to skeletal muscle by in vivo electroporation. However, most of these reports have used what has been characterized as low-voltage, millisecond pulses. One of the purposes of this study was to examine a wide range of parameters to establish a better understanding of the effects of field strength and pulse length. Two electroporation conditions, one utilizing low-voltage millisecond pulses and the other high-voltage microsecond pulses, were compared for their ability to deliver a molecule that can be measured systemically, IL-12. Not only was this molecule efficiently delivered, but one of the molecules it induces (IFN-γ) was also measured systemically, with expression peaking at greater than 200 pg/ml serum levels on day 7 and still as high as 40 pg/ml on day 21 when using electrical parameters of 100 V/cm, 20-ms pulses. Of the two types of parameters tested, low-voltage, millisecond pulses resulted in higher, prolonged expression of plasmid DNA than high-voltage, microsecond pulses. In summary, this report provides evidence that in vivo electroporation is an efficient method for the delivery of plasmid DNA, and its use in immunotherapy protocols should be furthered examined.
We present an experimental study of single-stranded DNA electrophoresis in polyacrylamide gels. We demonstrate the existence of an entropic trapping regime, situated between the Ogston and reptation regimes, in which the mobility scales as 1/M1+gamma, where M is the DNA molecular size. The exponent gamma>0 increases for denser gels but decreases for higher fields. Entropic trapping disappears for electric fields E exceeding a critical size-dependent value E*\(M\). We also present various estimates of the gel's mean pore size. Finally, we propose a phase diagram describing the observed DNA migration regimes.
Overview summary The present study evaluates the pharmacokinetic half-life and tissue distribution of plasmid DNA following intravenous injection in mice. This study extends the time frame of previous in vivo analyses to 6 months following i.v. injection. Injected mice exhibit no expression of the encoded gene as assayed by immunofluorescence. This represents the first systematic in vivo pharmacokinetic study of intravenously injected DNA complexed with cationic lipids, and is relevant to many gene therapy protocols utilizing direct injection of plasmid DNA plus lipids. The results provide a preliminary basis for the safe initiation of cancer immunotherapy clinical trials in which plasmid DNA is directly injected into tumors. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/63149/1/hum.1995.6.5-553.pdf
An extensive series of experiments has been performed to study the mobility of DNA fragments ranging in size from 2.0 to 48.5 kilobose pairs. By varying the agarose concentration in the gels and the electric field strength, three DNA electrophoresis regimes were clearly identified: the Ogston regime (small DNA fragments in large pores of agarose), the reptation regime without DNA chain stretching (small pores of agarose and weak electric fields), and the reptation regime with DNA chain stretching (small pores of agarose, strong electric fields, and large DNA fragments). Here we report on the experimental identification of these regimes and on the conditions governing the transition between each of them. The onset of reptation and of stretching of DNA chains in gel electrophoresis are described quantitatively for the first time, and a phase diagram for the dynamics of DNA during electrophoresis is presented.
The transient orientation of lambda DNA and lambda-DNA oligomers has been measured during pulsed field gel electrophoresis.
The DNA becomes substantially aligned parallel to the electric field E. In response to a single rectangular pulse, orientation
shows an overshoot with a peak at 1 second, then a small undershoot, and finally a plateau. When the field is turned off,
the orientation dissipates in two distinct exponential phases. Field inversion leads to periods of orientation with intervening
periods of reduced orientation as the chains reverse direction. Field inversion pulses applied to linear oligomers of lambda-DNA
show that orientation responses slow down but increase in amplitude as molecular weight increases, for a given field. Because
DNA stretching and alignment parallel to E are expected to correlate with DNA velocity, the velocity in response to a pulsed
field is also expected to exhibit an overshoot.
The fate of plasmid DNA complexed with cationic lipids delivered intravenously in mice was evaluated at selected timepoints up to 6 months postinjection. Blood half-life and tissue distribution of plasmid DNA and potential expression in tissues were examined. Southern blot analyses of blood indicated that intact plasmid DNA was rapidly degraded, with a half-life of less than 5 min for intact plasmid, and was no longer detectable at 1 hr postinjection. Southern analyses of tissue demonstrated that intact DNA was differentially retained in the lung, spleen, liver, heart, kidney, marrow, and muscle up to 24 hr postinjection. After 7 days, no intact plasmid DNA was detectable by Southern blot analysis; however, the plasmid was detectable by the polymerase chain reaction (PCR) in all tissues examined at 7 and 28 days postinjection. At 6 months postinjection, femtogram levels of plasmid were detected only in muscle. Immunohistochemical analyses did not detect encoded protein in the tissues harboring residual plasmid at 1 or 7 days postinjection.
Partition of sized FITC-dextrans in polyacrylamide gel showed a relationship between Kav and solute radius as predicted by the theory of Ogston, which is based solely on geometry of the spaces. Permeability data for the same dextrans were fit to several theories, including those based on geometry and those based on hydrodynamic interactions, and the gel structure predicted by the partition and permeability data were compared. The Brinkman effective-medium model (based on hydrodynamic interactions and requiring a measure of the hydraulic conductivity of the matrix) gave the best fit of permeability data with the values for fiber radius (rf) and void volume of the gel (epsilon) that were obtained from the partition data. The models based on geometry and the hydrodynamic screening model of Cukier, using the rf and epsilon from partition data, all predicted higher rates of permeation than observed experimentally, while the effective-medium model with added term for steric interaction predicted lower permeation than that observed. The size of cylindrical pores appropriate for the partition data predicted higher rates of permeation than observed. These relative results were unaffected by the method of estimating void volume of the gel. In sum, it appears that one can use data on partition of solute, combined with measurement of hydraulic conductivity, to predict solute permeation in polyacrylamide gel.
The diffusion coefficients (D) of different types of macromolecules (proteins, dextrans, polymer beads, and DNA) were measured by fluorescence recovery after photobleaching (FRAP) both in solution and in 2% agarose gels to compare transport properties of these macromolecules. Diffusion measurements were conducted with concentrations low enough to avoid macromolecular interactions. For gel measurements, diffusion data were fitted according to different theories: polymer chains and spherical macromolecules were analyzed separately. As chain length increases, diffusion coefficients of DNA show a clear shift from a Rouse-like behavior (DG congruent with N0-0.5) to a reptational behavior (DG congruent with N0-2.0). The pore size, a, of a 2% agarose gel cast in a 0.1 M PBS solution was estimated. Diffusion coefficients of the proteins and the polymer beads were analyzed with the Ogston model and the effective medium model permitting the estimation of an agarose gel fiber radius and hydraulic permeability of the gels. Not only did flexible macromolecules exhibit greater mobility in the gel than did comparable-size rigid spherical particles, they also proved to be a more useful probe of available space between fibers.
Gene transfer into muscle by electroporation with low-voltage and long-pulse (LV/LP, 100 V/50 msec) currents was shown to be more efficient than simple intramuscular DNA injection. Nevertheless, transgene expression declined from day 7 and only reached 10% of the maximum 3 weeks after electroporation. We have optimized electroporation conditions including voltage, pulse number, and the amount of injected luciferase-encoding plasmid DNA in the tibialis anterior muscle. Using high-voltage and short-pulse (HV/SP, 900 V/100 microsec) currents, we observed an average 500-fold increase in luciferase expression, in comparison with nonelectroporated muscle. Moreover, sustained and long-lasting gene expression was observed for at least 6 months. When we compared HV/SP currents with LV/LP currents, luciferase expression was similar 24 hr after electroporation. One month later, whereas luciferase expression was stable in muscle electroporated with HV/SP currents, it decreased 600-fold in muscle electroporated with LV/LP currents. In conclusion, electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression in muscle.
Gene therapy by direct delivery of plasmid DNA has several advantages over viral gene transfer, but plasmid delivery is less efficient. In vivo electroporation has been used to enhance delivery of chemotherapeutic agents to tumors in both animal and human studies. Recently, this delivery technique has been extended to large molecules such as plasmid DNA. Here, the successful delivery of plasmids encoding reporter genes to rat hepatocellular carcinomas by in vivo electroporation is demonstrated.
Efficient cell electrotransfection can be achieved using combinations of high-voltage (HV; 800 V/cm, 100 micros) and low-voltage (LV; 80 V/cm, 100 ms) pulses. We have developed equipment allowing the generation of various HV and LV combinations with precise control of the lag between the HV and LV pulses. We injected luciferase-encoding DNA in skeletal muscle, before or after pulse delivery, and measured luciferase expression after various pulse combinations. In parallel, we determined permeabilization levels using uptake of (51)Cr-labeled EDTA. High voltage alone resulted in a high level of muscle permeabilization for 300 seconds, but very low DNA transfer. Combinations of one HV pulse followed by one or four LV pulses did not prolong the high permeabilization level, but resulted in a large increase in DNA transfer for lags up to 100 seconds in the case of one HV + one LV and up to 3000 seconds in the case of one HV + four LV. DNA expression also reached similar levels when we injected the DNA between the HV and LV pulses. We conclude that the role of the HV pulse is limited to muscle cell permeabilization and that the LV pulses have a direct effect on DNA. In vivo DNA electrotransfer is thus a multistep process that includes DNA distribution, muscle permeabilization, and DNA electrophoresis.
Interstitial transport is a crucial step in plasmid DNA-based gene therapy. However, interstitial diffusion of large nucleic acids is prohibitively slow. Therefore, we proposed to facilitate interstitial transport of DNA via pulsed electric fields. To test the feasibility of this approach to gene delivery, we developed an ex vivo technique to quantify the magnitude of DNA movement due to pulsed electric fields in two tumor tissues: B16.F10 (a mouse melanoma) and 4T1 (a mouse mammary carcinoma). When the pulse duration and strength were 50 ms and 233 V/cm, respectively, we found that the average plasmid DNA movements per 10 pulses were 1.47 microm and 0.35 microm in B16.F10 and 4T1 tumors, respectively. The average plasmid DNA movements could be approximately tripled, ie to reach 3.69 microm and 1.01 microm, respectively, when the pulse strength was increased to 465 V/cm. The plasmid DNA mobility was correlated with the tumor collagen content, which was approximately eight times greater in 4T1 than in B16.F10 tumors. These data suggest that electric field can be a powerful driving force for improving interstitial transport of DNA during gene delivery.
The large size of many novel therapeutics impairs their transport through the tumor extracellular matrix and thus limits their therapeutic effectiveness. We propose that extracellular matrix composition, structure, and distribution determine the transport properties in tumors. Furthermore, because the characteristics of the extracellular matrix largely depend on the tumor-host interactions, we postulate that diffusion of macromolecules will vary with tumor type as well as anatomical location. Diffusion coefficients of macromolecules and liposomes in tumors growing in cranial windows (CWs) and dorsal chambers (DCs) were measured by fluorescence recovery after photobleaching. For the same tumor types, diffusion of large molecules was significantly faster in CW than in DC tumors. The greater diffusional hindrance in DC tumors was correlated with higher levels of collagen type I and its organization into fibrils. For molecules with diameters comparable to the interfibrillar space the diffusion was 5- to 10-fold slower in DC than in CW tumors. The slower diffusion in DC tumors was associated with a higher density of host stromal cells that synthesize and organize collagen type I. Our results point to the necessity of developing site-specific drug carriers to improve the delivery of molecular medicine to solid tumors.
To reach cancer cells in a tumor, a blood-borne therapeutic molecule or cell must make its way into the blood vessels of the tumor and across the vessel wall into the interstitium, and finally migrate through the interstitium. Unfortunately, tumors often develop in ways that hinder each of these steps. Our research goals are to analyze each of these steps experimentally and theoretically, and then integrate the resulting information in a unified theoretical framework. This paradigm of analysis and synthesis has allowed us to obtain a better understanding of physiological barriers in solid tumors, and to develop novel strategies to exploit and/or to overcome these barriers for improved cancer detection and treatment.
Microhydrodynamics of Macromolecules and ParticlesBrownian MotionViscosity of Dilute SuspensionsSedimentation under GravitySedimentation in a Centrifugal FieldUltrafiltrationHydrodynamic Chromatography
Hindered transport coefficients for a spherical macromolecule in a spatially periodic fibrous medium were calculated using two different methods. The first method is an effective medium approach based on Brinkman's equation and can be readily applied to disordered fibrous media. The second and more rigorous set of calculations makes use of generalized Taylor dispersion theory. Results from these two approaches are compared for two different spatially periodic lattices of bead-and-string fibers, which were chosen to illustrate the effects of inhomogeneity in the distribution of fibers. In addition, ratios of solute radius to fiber radius ranging from 0.5 to 5.0 were considered. Qualitative agreement between the two methods was obtained for each case studied, and quantitative agreement was obtained for volume fractions at which the hindering effects of the fibers were not too severe.
The swelling behavior of gelatin gels containing proteoglycans (sulphated proteoglycans from bovine intervertebral discs and a hyaluronate proteoglycan from bovine synovial fluid) when immersed in osmotically active solutions of dextran have been measured. The presence of the proteoglycans markedly affects the internal osmotic contribution to the swelling pressure of the gel. These internal osmotic pressures are considerably in excess of the sum of the osmotic activities of the individual components. This behavior is understood in terms of an entropic interaction between the gelatin and the proteoglycan molecules. By use of the “dilute solution” treatment of Flory, the osmotic pressure excesses are related to the volumes and hence dimensions of the interact acting species. A comparison of these values with those calculated by other means shows good agreement. The osmotic behavior of the complex gels can be understood on a mechanistic basis, if we regard the gelatin and sulphated proteoglycans as spheres and the hyaluronate proteoglycan as a rod.
Purpose. We examined the stability and disposition characteristics of a naked plasmid DNA pCAT as a model gene after intravenous injection in mice to construct the strategy of in vivo gene delivery systems.
Methods. After the injection of pCAT to the mice, stability, tissue distribution, hepatic cellular localization, and effect of some polyanions on the hepatic uptake were studied.
Results. The in vitro study demonstrated that the pCAT was rapidly degraded in mouse whole blood with a half-life of approximately 10 min at a concentration of 100 g/ml. After intravenous injection, pCAT was degraded at a significantly faster rate than that observed in the whole blood, suggesting that pCAT in vivo was also degraded in other compartments. Following intravenous injection of [32P] pCAT, radioactivity was rapidly eliminated from the plasma due to extensive uptake by the liver. Hepatic accumulation occurred preferentially in the non-parenchymal cells. The hepatic uptake of radioactivity derived from [32P] pCAT was inhibited by preceding administration of polyanions such as polyinosinic acid, dextran sulfate, maleylated and succinylated bovine serum albumin but not by polycytidylic acid. These findings indicate that pCAT is taken up by the liver via scavenger receptors on the non-parenchymal cells. Pharmacokinetic analysis revealed that the apparent hepatic uptake clearance was fairly close to the liver plasma flow.
Conclusions. These findings provide useful information for the development of delivery systems for in vivo gene therapy.
In vivo targeted gene transfer by non-viral vectors is subjected to anatomical constraints depending on the route of administration. Transfection efficiency and gene expression in vivo using non-viral vectors is also relatively low. We report that in vivo electropermeabilization of the liver tissue of rats in the presence of genes encoding luciferase or β-galactosidase resulted in the strong expression of these genetic markers in rat liver cells. About 30–40% of the rat liver cells electroporated expressed the β-galactosidase genetic marker 48 h after electroporation. The marker expression was also detected at least 21 days after transfection at about 5% of the level 48 h after electroporation. The results indicate that gene transfer by electroporation in vivo may avoid anatomical constraints and low transfection efficiency.
A new electrooptical apparatus has been used to characterize the dichroism decay time constants for a collection of nine blunt-ended DNA restriction fragments in the range of chain lengths from 41 to 256 base-pairs at physiological salt concentrations. The experimental data show an increase of rotational diffusion coefficients, when the monovalent salt concentration is increased from a few mM, used previously for standard electrooptical experiments, to the range of salt concentrations around 100 mM. The presence or absence of 10 mM Mg2+ in a buffer with 100 mM NaCl does not induce any large change of the rotational diffusion. Bending of double helices is reflected by a fast component in the dichroism decay for fragments greater than or equal to 90 bp; the time constant of the first bending mode is 7-9% relative to the time constant of overall rotational diffusion for fragments with 90 to 179 bp at the temperatures 2, 10 and 20 degrees C. Interpretation of the overall rotational diffusion time constants by different models on the hydrodynamics of flexible polymer chains leads to diverging values of the persistence length. The most accurate description is expected from a combination of the rotational diffusion coefficient for rigid rods given by Tirado and Garcia de la Torre (J. Chem. Phys. 73 (1980) 1986) with correction factors derived from Monte Carlo simulations (P.J. Hagerman and B.H. Zimm, Biopolymers 20 (1981) 1481). This model leads to 'average' values of the persistence length of 440, 400 and 380 A at the temperatures 20, 10 and 2 degrees C, respectively (in 110 mM Na+ and 10 mM Mg2+, pH 7.0); the hydrodynamic radius of the helix is approx. 12.5 A. The persistence lengths measured at various monovalent salt concentrations can be represented as a linear function of the reciprocal square root of the ionic strength. The rotational time constants measured for individual fragments at physiological salt show clearly larger deviations from the model average than corresponding time constants measured previously at low salt; 'apparent' persistence lengths of individual fragments as well as their temperature dependence show strong variations. Thus, it is hardly possible to define a 'standard' persistence length for mixed sequences--even though the sequences used in the present investigation do not show clear deviations from standard gel mobilities. These data indicate that formation of individual, sequence-directed structures of DNA fragments is favoured under physiological salt conditions.
Individual ethidium-stained DNA molecules, embedded in an agarose gel made with electrophoresis buffer (0.05 molar salt), are observed using a fluorescence microscope. In the first experiment, open circular 66 kilobase pair (kbp) plasmids, immobilized by agarose fibers threaded through their centers, display entropic "rubber" elasticity. The charged molecules extend in an electric field of several volts per centimeter and contract to a compact random coil when the field is removed. The extension of the plasmids as a function of field strength is consistent with the freely jointed chain model when the effective electrophoretic charge density is set at 15 e-per persistence length. In a second experiment, stained linear 48.5 kbp DNA molecules are observed as random coils immobilized in agarose. A measure of their size, here named the "maximal-X-extent," is taken for 100 molecules and found to average 1.47 mu. A Monte Carlo computer simulation of random coils (freely jointed chain model) gives the same maximal-X-extent value when the persistence length is set at 0.08 mu.
The understanding, on a molecular level, of the mechanisms responsible for the improved separation in DNA gel electrophoresis when using modulated electric fields requires detailed information about conformational distribution and dynamics in the DNA/gel system. The orientational order due to electrophoretic migration (“electrophoretic orientation”) is an interesting piece of information in this context that can be obtained through linear dichroism spectroscopy [M. Jonsson, B. Åkerman, and B. Nordén, (1988) Biopolymers 27 , 381–414]. The technique permits measurement of the orientation factor S of DNA ( S = 1 corresponds to perfect orientation) within an electrophoretic zone in the gel during the electrophoresis. It is reported that the degree of orientation of T2 DNA [170 kilo base pairs (kpb)] is considerable ( S = 0.17 in 1% agarose at 10 V/cm) compared to relatively modest orientations of short fragments found earlier (for 23‐kbp DNA, S = 0.03 in 1% agarose at 10 V/cm), showing that large DNA coils are substantially deformed during the migration. Growth and relaxation dynamics of the orientational order of the T2 DNA are also reported, as functions of gel concentration (0.3–2%), electric field strength (0–40 V/cm), and pulse characteristics.
The rise profile of the DNA orientation, when applying a constant field, is a nonmonotonic function that displays a pronounced overshoot, followed by a minor undershoot, before it reaches steady‐state orientation (after 12 s in 1% agarose, 9 V/cm). The orientational relaxation in absence of field shows a multiexponential decay in a time region of some 10 s, when most of the DNA anisotropy has disappeared. A surprising phenomenon is a memory over minutes of the DNA/gel system to previous pulses: with two consecutive rectangular pulses (of the same polarity), the orientational overshoot and undershoot as a response to the second pulse are significantly reduced compared to the first pulse. The time required to recover 90% of their amplitudes is typically 1200 s (1% agarose, 9 V/cm), which may be compared to the time required to relax 90% of the DNA orientation, which is only 6 s. The major part of the over‐ and undershoot recovery is thus a reorganization of a system in which DNA is already randomly oriented.
The different response amplitudes and relaxation times, including the amplitude and recovery time of the overshoot, of the orientational order of DNA in the electrophoretic gel have been studied as functions of gel concentration and field strength. The results are discussed against relevant theories of polymer dynamics.
Transient electric birefringence has been used as an analytical tool to study the orientation of DNA in agarose gels, and to study the orientation of the matrix alone. The sign of the birefringence of DNA oriented in an agarose gel is negative, as observed in free solution, indicating that the DNA molecules orient parallel to the direction of the electric field. If the median pore diameter of the gel is larger than the contour length of the DNA molecule, the DNA effectively does not see the matrix and the birefringence relaxation time is the same as observed in free solution. However, if the median pore diameter of the gel is smaller than the contour length of the DNA, the DNA molecule becomes stretched as well as oriented. For DNA molecules of moderate size (⩽ 4 kb), stretching in the gel causes the birefringence relaxation times to increase to the values expected for fully stretched molecules. Complete stretching is not observed for larger DNA molecules. The orientation and stretching of DNA molecules in the gel matrix indicates that end‐on migration, or reptation, is a likely mechanism for DNA electrophoresis in agarose gels.
When the electric field is rapidly reversed in polarity, very little change in the orientation of the DNA is observed if the DNA molecules were completely stretched and had reached their equilibrium orientation before the field was reversed in direction. Hence completely stretched, oriented DNA molecules are able to reverse their direction of migration in the electric field with little or no loss of orientation. However, if the DNA molecules were not completely stretched or if the equilibrium orientation had not been reached, substantial disorientation of the DNA molecules is observed at field reversal. The forced rate of disorientation in the reversing field is faster than the field‐free rate of disorientation. Complicated patterns of reorientation can be observed after field reversal, depending on the degree of orientation in the original field direction.
The effect of pulsed electric fields on the orientation of the agarose gel matrix itself was also investigated. If very short pulses of high amplitude ( e.g. 1–10 kV/cm in amplitude and 10–1000 μs in duration) were applied to the gel, the sign of the birefringence was small and positive, the Kerr law was obeyed, and the birefringence decayed to zero after the removal of the electric field with a relaxation time varying from 10–220 μs, depending on the length of the orienting pulse. These results indicate that individual agarose chains or bundles of chains, or dangling ends of the matrix, could be oriented by very short pulses of high amplitude. However, when smaller electric fields were used ( e.g. , 10–100 V/cm applied to the gel for 0.5–2 s), the amplitude of the birefringence of the gel matrix increased markedly and the signal passed through an extremum several seconds after removal of the electric field before decaying slowly to zero. These slow time‐dependent effects indicate that domains in the agarose matrix were being oriented by the longer pulses used at the lower electric field strengths. The sign of the birefringence of the agarose gel could be reversed from positive to negative, or vice versa , by reversing the direction of the applied electric field, indicating that the domains apparently change their direction of orientation from parallel to perpendicular (or vice versa ) after field reversal. Orientation and reorientation of microdomains of the matrix in alternating pulsed electric fields would increase the fluidity of the matrix, making it easier for very large DNA molecules to migrate through the gel during electrophoresis.
A method for in situ study of orientation of DNA during gel electrophoresis has been developed. Linear dichroism spectra measured by this phase-modulation technique can sensitively and selectively detect orientation of DNA during electrophoretic migration in gel. [Measurement of “electrophoretic orientation” was first reported in 1985 by B. Åkerman, M. Jonsson, and B. Nordén (1985) (J. Chem. Soc. Chem. Commun. 422–423)]. Restriction fragments of duplex DNA of lengths in the ranges of 300–2319 base pairs (bp) and 4361–23130 bp have been studied in 5% polyacrylamide and 1% agarose gels, respectively. The fragments become preferentially oriented with the DNA helix axis parallel to the migration direction. In agarose the orientation is found to increase sigmoidally, and in polyacrylamide, linearly, with the electric field strength, within the field ranges accessible to measurement (0–40 and 5–40 V/cm, respectively). In both types of gels a considerable increase in orientation with length of DNA was observed. Compared to dipole orientation in electric fields, the electrophoretic orientation is high: orientation factor S = 0.027 in agarose for 23130 bp at 10 V/cm and S = 0.004 in polyacrylamide for 2319 bp at 10 V/cm. In addition to orientation of DNA, the electrophoresis also leads to orientation effects in the gel structure owing to Joule heating. In agarose there is also an effect that is associated with the migrating DNA zones and that produces different orientations of the gel at the front and rear parts of a zone. Evidence is presented that this effect is due to a DNA-induced electroosmotic flow causing a contraction of the gel in the front of the zone and an expansion in the rear. The experimental results on DNA orientation are compared with the reptation theories for gel electrophoresis. The theory of Lumpkin et al. [O. J. Lumpkin, P. Dejardin, and B. H. Zimm (1985) Biopolymers24, 1573–1593] predicts no orientation length dependence, but it does predict a shape of the field dependence that resembles the shape observed in agarose. The theory of Slater and Noolandi [G. W. Slater and J. Noolandi (1986) Biopolymers25, 431–454] predicts an orientational length dependence that is an order of magnitude less than the experimental one, and a field dependence that agrees neither with the sigmoidal shape observed in agarose nor with the linear dependence in polyacrylamide.
Intensities of polarized fluorescence from ethidium bound to phage λ DNA undergoing agarose gel electrophoresis were measured. The intensities were strongly field dependent at voltage gradients of 8 V/cm, consistent with a partial orientation of DNA helices in the direction of electrophoresis about 500 times larger than seen in the same field in solution. Such an orientation was predicted by a reptation model of gel electrophoresis advanced by Lumpkin et al. [(1985) Biopolymers, 24, 1573–1593]. The present results can be fit successfully to this theory with a single adjustable parameter, the gel–DNA contact distance. Also, λ DNA electrophoretic mobilities in the same concentration gel were determined using the same buffer system. Both orientation and mobility measurements can be fit to the reptation theory within a factor of two using the same values of two parameters, the gel–DNA contact distance and the ratio of DNA charge to frictional coefficient.
The transport of fluid and solute molecules in the interstitium is governed by the biological and physicochemical properties of the interstitial compartment as well as the physicochemical properties of the test molecule. The composition of the interstitial compartment of neoplastic tissues is significantly different from that of most normal tissues. In general the tumor interstitial compartment is characterized by large interstitial space, high collagen concentration, low proteoglycan and hyaluronate concentrations, high interstitial fluid pressure and flow, absence of anatomically well-defined functioning lymphatic network, high effective interstitial diffusion coefficient of macromolecules, as well as large hydraulic conductivity and interstitial convection compared to most normal tissues. While these factors favor movement of macromolecules in the tumor interstitium, high interstitial pressure and low microvascular pressure may retard extravasation of molecules and cells, especially in large tumors. These differences in transport parameters have major implications in tumor growth and metastases, as well as in tumor detection and treatment.
Agarose gel electrophoresis of spheres (radius = R) has been used to determine the effective radius (PE) of the pores of an agarose gel (percentage of agarose in a gel = A). The value of PE at a given A was taken to be the R of the largest sphere that enters the gel. When log PE is plotted as a function of log A, the results can be represented by: PE = 118A-0.74 for 0.2 less than or equal to A less than or equal to 4.0 (PE in nm). However, the data suggest significant nonlinearity in this plot, the magnitude of the exponent of the PE vs A relationship increasing by about 20% as A increases from 0.2 to 4.0. From these data, PE's as big as 1500 nm and as small as 36 nm can be achieved with agarose gels formed with unmodified, unadulterated agarose and usable for electrophoresis.
We studied reporter gene expression in synovial tissue after intra-articular administration of an expression plasmid into the knees of rabbits and rats. In both species, administration of a plasmid encoding beta-galactosidase led to gene expression in the synovial cells lining the joint. Expression correlated with the presence of plasmid DNA in synovial tissue extracts. Studies with a plasmid encoding chloramphenicol acetyltransferase demonstrated that gene expression persists for 2-5 days after administration. Southern blotting demonstrated that the administered plasmid was taken up rapidly by synovial tissue and degraded. By 24 hr after administration, no intact plasmid could be detected by Southern blotting, although small amounts of plasmid could be amplified by PCR up to 7 days. Administration of a plasmid encoding human growth hormone demonstrated that this product could be expressed from synovial cells and secreted into the synovial fluid. The histological distribution of gene expression in synovium resembles the known distribution of particulate materials injected into the joint and suggests that plasmid DNA is taken up by nonspecific endocytosis like other particulate materials during the remodeling of synovial fluid.
Intratracheal administration of plasmid DNA resulted in gene expression in mouse airways in the absence of any enhancing agent. Administration of plasmid DNA encoding the chloramphenicol acetyltransferase gene (CAT) in sterile water lead to CAT transgene expression that peaked between 1 and 3 days and was detected up to 28 days after DNA administration. Transgene expression was independent of mouse gender, age and strain. Levels of expression from DNA in various isotonic solutions did not differ from levels obtained with DNA administered in water, suggesting that transfection is not dependent on damage to airway cells caused by a hypo-osmotic delivery vehicle. Pharmacokinetic studies using radiolabeled plasmid DNA showed that DNA was rapidly degraded, while higher levels of radioactivity were retained for longer duration following administration of cationic liposome-DNA complexes in the airway. Southern blot and PCR analysis confirmed that DNA complexed with DOTMA-DOPE was retained in the airways for a longer period. However, cationic liposomes DOTMA-DOPE (1:1) or DOTAP complexed with DNA, did not enhance expression over DNA alone. These results suggest that 'naked' plasmid DNA should be included as a control in all studies on intratracheal gene delivery using nonviral systems.
Direct injection of DNA expression vectors into muscle leads to expression of encoded recombinant gene products in mature muscle cells. This phenomenon is not shared by most other organs. We have surveyed various organs in the rabbit to identify other cell types that would express DNA vectors after direct injection. We observed that thyroid follicular cells were capable of acquiring plasmid DNA and expressing recombinant gene products after direct interstitial injection of plasmid vectors into the thyroid gland. The level of expression of a chloramphenicol acetyltransferase (CAT) reporter gene in thyroid tissue was similar to that seen in muscle tissue three days after injection in controlled experiments. Using a beta-galactosidase reporter gene, expression was localized to thyroid follicular cells. CAT activity decreased with first-order kinetics and a half-life t1/2 of 40 hr. DNA was identified in thyroid tissue by polymerase chain reaction (PCR) analysis and displayed first-order elimination kinetics with a half-life t1/2 of 10 hr. The persistence of the gene and gene product in the thyroid was significantly different from that observed after injection of DNA vectors into muscle or delivery of DNA vectors to the liver using asialoglycoprotein/polylysine/DNA complexes, suggesting that there are significant differences in the process of DNA uptake or compartmentalization in these experimental systems. These results introduce the possibility of developing the thyroid as a novel target for treating certain thyroid or systemic diseases using DNA vectors.
The orientation of DNA induced by electrophoretic transport in agarose gel has been studied by optical birefringence. From its field-free decay, it is clearly demonstrated that the degree of orientation results from two processes: alignment along the electric field (stretching of the end-to-end vector) and elongation of the primitive path in the gel (overstretching). Separation of the two contributions allows the experimental determination of the effective charge per base pair, the gel pore size seen by the reptating molecule, the reptation time, the degree of overstretching and the mean relaxation time of the overstretching. Their field and DNA length dependences compare well with theoretical predictions. Similarly, the time at which overstretching presents an overshoot in the rise of the orientation follows closely the predictions of a model based on the evolution of J-shaped conformations. The recovery of such conformations is studied by a sequence of two pulses with variable delay time. The use of directly measured or extrapolated characteristic times and fields in the design of efficient pulse schemes for pulsed-field gel electrophoresis is emphasized.
The equilibrium partition coefficient (K) and diffusion coefficient (Dgel) of two proteins and two linear polymers were measured as a function of polymer content of a 2.7% cross-linked polyacrylamide (PA) gel. The gel concentration, expressed as a volume percentage of PA in the gel (phi), varied between 0 and 14%. The measurements were made by fluorescence spectroscopy; fluorescent dyes were covalently attached to the macromolecules. The dependence of K on phi for the proteins agrees with a model of the gel network as randomly placed, impenetrable rods. The diffusion data are interpreted in terms of an effective medium theory for the mobility of a sphere in a Brinkman fluid. Using values of the Brinkman parameter in the literature, the effective medium model with no adjustable parameters fits the diffusion data for the proteins very well but underpredicts Dgel for the linear polymers. The gel effect on partitioning is significantly greater than that on diffusion. The permeability (KDgel) of bovine serum albumin decreased by 10(3) over the range phi = 0 --> 8%, and the ratio of permeabilities for ribonuclease compared to BSA increased from 2 to 30.
The pharmaceutical approach to somatic gene therapy is based on consideration of a gene as a chemical entity with specific physical, chemical and colloidal properties. The genes that are required for gene therapy are large molecules (> 1 x 10(6) Daltons, > 100 nm diameter) with a net negative charge that prevents diffusion through biological barriers such as an intact endothelium, the plasma membrane or the nuclear membrane. New methods for gene therapy are based on increasing knowledge of the pathways by which DNA may be internalized into cells and traffic to the nucleus, pharmaceutical experience with particulate drug delivery systems, and the ability to control gene expression with recombined genetic elements. This article reviews two themes in the development of gene therapies: first, the current approaches involving the administration of cells, viruses and plasmid DNA; second, the emerging pharmaceutical approach to gene therapy based on the pharmaceutical characteristics of DNA itself and methods for advanced drug delivery.
Despite exciting progress in the biology underlying a variety of proposed molecular medicines, an unmet challenge Remainsdelivery. This problem, how to better target the new generation of therapeutics, cuts across all diseases. The solution offers unprecedented opportunities for multidisciplinary teams of bioengineers to work with biological and medical scientists to realize the fruits of our nation's investment in molecular and cellular medicine.
Electrophoretic velocity and orientation have been used to study the electric-field-induced trapping of supercoiled and relaxed circular DNA (2926 and 5386 bp) in polyacrylamide gels (5% T, 3.3% C) at 7.5-22.5 V/cm, using as controls linear molecules of either the same contour length or the same radius of gyration. The circle-specific trapping is reversible. From the duration of the reverse pulse needed to detrap the molecules, the average trap depth is estimated to be 90 A, which is consistent with the molecular charge and the field strengths needed to keep molecules trapped. Trapped circles exhibit a strong field alignment compared to the linear form, and there is a good correlation between the enhanced field alignment for the circles and the onset of trapping in both constant and pulsed fields. The circles do not exhibit the orientation overshoot response to a field pulse seen with linear DNA, and the rate of orientation growth scales as E(-2+/-0.1) with the field, as opposed to E(-1.1+/-0.1) for the linear form. These results show that the linear form migrates by cyclic reptation, whereas the circles most likely are trapped by impalement on gel fibers. This proposal is supported by very similar velocity and orientation behavior of circular DNA in agarose gels, where impalement has been deemed more likely because of stiffer gel fibers. The trapping efficiency is sensitive to DNA topology, as expected for impalement. In polyacrylamide the supercoiled form (superhelical density sigma = -0.05) has a two- to fourfold lower probability of trapping than the corresponding relaxed species, whereas in agarose gels the supercoiled form is not trapped at all. These results are consistent with existing data on the average holes in the plectonemic supercoiled structures and the fiber thicknesses in the two gel types. On the basis of the topology effect, it is argued that impalement during pulsed-field electrophoresis in polyacrylamide gels may be useful for the separation of more intricate DNA structures such as knots. The results also indicate that linear dichroism on field-aligned molecules can be used to measure the supercoiling angle, if relaxed DNA circles are used as controls for the global degree of orientation.
DNA electrophoresis is now a fairly mature technology. Nevertheless, as we approach the 21st century, new ideas are frequently suggested that could lead to a revolution for DNA sequencing and mapping. Here, we review some of the novel concepts that have been studied since ca. 1990. Our review focuses on new separation mechanisms, new sieving matrices and recent conceptual advances.
Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. However, present DNA delivery technologies have to be improved with regard to both the level of expression and interindividual variability. We report very efficient plasmid DNA transfer in muscle fibers by using square-wave electric pulses of low field strength (less than 300 V/cm) and of long duration (more than 1 ms). Contrary to the electropermeabilization-induced uptake of small molecules into muscle fibers, plasmid DNA has to be present in the tissue during the electric pulses, suggesting a direct effect of the electric field on DNA during electrotransfer. This i.m. electrotransfer method increases reporter and therapeutic gene expression by several orders of magnitude in various muscles in mouse, rat, rabbit, and monkey. Moreover, i.m. electrotransfer strongly decreases variability. Stability of expression was observed for at least 9 months. With a pCMV-FGF1 plasmid coding for fibroblast growth factor 1, this protein was immunodetected in the majority of muscle fibers subjected to the electric pulses. DNA electrotransfer in muscle may have broad applications in gene therapy and in physiological, pharmacological, and developmental studies.
In vivo electroporation is increasingly being used to deliver small molecules as well as DNA to tissues. The aim of this study was to quantitatively investigate in vivo electroporation of skeletal muscle, and to determine the threshold for permeabilization. We designed a quantitative method to study in vivo electroporation, by measuring uptake of (51)Cr-EDTA. As electrode configuration influences electric field (E-field) distribution, we developed a method to calculate this. Electroporation of mouse muscle tissue was investigated using either external plate electrodes or internal needle electrodes placed 4 mm apart, and eight pulses of 99 micros duration at a frequency of 1 Hz. The applied voltage to electrode distance ratio was varied from 0 to 2.0 kV/cm. We found that: (1) the threshold for permeabilization of skeletal muscle tissue using short duration pulses was at an applied voltage to electrode distance ratio of 0.53 kV/cm (+/-0.03 kV/cm), corresponding to an E-field of 0.45 kV/cm; (2) there were two phases in the uptake of (51)Cr-EDTA, the first indicating increasing permeabilization and the second indicating beginning irreversible membrane damage; and (3) the calculated E-field distribution was more homogeneous for plate than for needle electrodes, which was reflected in the experimental results.
DNA degradation is a fundamental problem for any gene therapy or genetic immunization approach, since destruction of incoming genes translates into loss of gene expression. To characterize the biology of DNA degradation after naked DNA injection, the location and levels of tissue nucleases were assessed. Extracts from the serum, kidney, and liver of mice had high levels of calcium-dependent endonuclease activity. High levels of acidic endonuclease activity were identified in the spleen, liver, kidney, and skin with little activity in skeletal or cardiac muscle. Relatively little exonuclease activity was observed in any tissue. The presence of endonucleases in the skin and muscle mediated degradation of 99% of naked DNA within 90 min of injection. This degradation most likely occurred in the extracellular space upstream of other cellular events. Despite this massive destruction, gross tissue nuclease levels did not determine skin-to-muscle transfection efficiency, or site-to-site transfection efficiency in the skin. While gross tissue nuclease levels do not appear to determine differences in transfection efficiency, the presence of robust tissue nuclease activity still necessitates that massive amounts of DNA be used to overcome the loss of 99% of expressible DNA. In addition to destroying genes, the nucleases may play a second role in genetic immunization by converting large plasmids into small oligonucleotides that can be taken up more easily by immune cells to stimulate CpG-dependent Th1 immune responses. For genetic immunization, vaccine outcome may depend on striking the right balance of nuclease effects to allow survival of sufficient DNA to express the antigen, while concomitantly generating sufficient amounts of immunostimulatory DNA fragments to drive Th1 booster effects. For gene therapy, all nuclease effects would appear to be negative, since these enzymes destroy gene expression while also stimulating cellular immune responses against transgene-modified host cells.
To reach cancer cells in a tumor, a blood-borne therapeutic molecule or cell must make its way into the blood vessels of the tumor and across the vessel wall into the interstitium, and finally migrate through the interstitium. Unfortunately, tumors often develop in ways that hinder each of these steps. Our research goals are to analyze each of these steps experimentally and theoretically, and then integrate the resulting information in a unified theoretical framework. This paradigm of analysis and synthesis has allowed us to obtain a better understanding of physiological barriers in solid tumors, and to develop novel strategies to exploit and/or to overcome these barriers for improved cancer detection and treatment.
Intratracheal gene delivery to the mouse airway: characterization of plasmid DNA expression and pharmacokinetics
- Meyer
Role of tumor-host interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors
- Pluen
Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electropermeabilization and DNA electrophoresis
- Satkauskas