Gene Therapy (1997) 4, 710–715
 1997 Stockton Press All rights reserved 0969-7128/97 $12.00
Shock wave permeabilization as a new gene transfer
method
U Lauer1,2, E Bürgelt1, Z Squire2, K Messmer 3, PH Hofschneider1, M Gregor2 and M Delius3
1
Max-Planck-Institut für Biochemie, Martinsried; 3Institut für Chirurgische Forschung, LMU, München; and 2Medizinische Klinik
und Poliklinik, Universität Tübingen, Germany
Uptake of naked functional DNA into mammalian cells can
be achieved by a number of physical methods. However,
for most of these techniques possibilities for therapeutic in
vivo applications – especially to solid organs – are often
limited. In this report, we describe shock wave permeabilization as a new physical gene transfer method, which can
be easily applied, provides great flexibility in the size and
sequence of the DNA molecules to be delivered, and which
should exhibit an advantageous security profile in vivo.
Upon exposure to lithotripter-generated shock waves
eukaryotic cells display a temporary increase in membrane
permeability. This effect was shown to be caused by cavitation resulting in the transient generation of cell pores
which allows the direct transfer of naked plasmid DNA.
Shockwave transfection of a variety of cell lines was demonstrated. Since shock waves can be well focused within
particular body regions, future applications of extracorporally generated shock waves to tissues simultaneously perfused with DNA solutions might open up the possibility of
achieving a regionally enhanced in vivo gene transfer.
Keywords: nonviral gene delivery; physical DNA transfer; lithotripter shock waves; cavitation
Introduction
A major challenge in human gene therapy approaches
remains the site-specific nonviral delivery of genes,
especially to solid organs. A number of physical methods
for the uptake of plasmid DNA into mammalian cells
have been developed in the past, including coprecipitation with calcium phosphate,1 use of polycations or lipids to form complexes with DNA,2 encapsidation of plasmid DNA into liposomes or erythrocyte ghosts,3–5
exposure of cells to rapid pulses of high-voltage current,
ie electroporation,6,7 receptor-mediated endocytosis of
DNA–ligand complexes8 and introduction of DNA into
cells by direct microinjection9 or on high-velocity tungsten microprojectiles.10 However, in vivo therapeutic
applications of these techniques are often impossible (eg
in the case of calcium phosphate coprecipitation and
electroporation) or limited by the availability of suitable
tissue specifically expressed receptors (receptor-mediated
endocytosis) or restricted to distinct tissues like muscle
(microinjection) and skin (particle bombardment). Therefore, alternative physical gene transfer approaches supporting a transient increase in cell membrane permeability have to be investigated. Lithotripter shock
waves are single pressure pulses of microsecond duration
with peak pressures of 35–120 MPa followed by a tensile
wave of 5–10 MPa and are widely employed for the treatment of kidney stones and gallstones. 11 They are generated in water outside the target body by the focusing of
pulses, propagated in tissues and focused on the acoustic
Correspondence: Dr U Lauer, Medizinische Klinik und Poliklinik, Universität Tübingen, Otfried-Müller-Str 10, D-72076 Tübingen, Germany
Received 31 January 1997; accepted 11 April 1997
target area by means of an ultrasound or radiograph
localizing device. Lithotripter shock waves are responsible for the physical phenomenon of cavitation, which is
defined as the movement of newly formed and pre-existing gas bubbles containing gas or vapor in a fluid.12 Cavitation is well known as a powerful mechanism of tissue
damage induction based on severe ultrastructural alterations at the cellular and subcellular level.13 Whereas a
defined portion of shock wave-exposed cells is completely destroyed and reduced to cell debris (dependent
upon the applied number of discharges and their pulse
energy), the majority of cells surviving the shock waveexposure procedure has been shown to continue to proliferate at a near normal rate.14 Interestingly, different cell
lines are known to differ in their sensitivity to shock
waves only by a factor of two15 and to react similarly
during different phases of the cell cycle. 16
Based on these observations, we have investigated
whether shock waves are able to induce a transient state
of cell membrane permeability in a defined proportion of
shock wave-exposed target cells allowing the uptake of
nucleic acid molecules. We report for the first time the
application of lithotripter shock waves as a new physical
gene transfer method, which functions in a receptor-independent way and exhibits the potential of an in vivo plasmid DNA delivery to almost any solid organ of interest.
Results
Starting from the observation that cells upon exposure to
shock waves display a temporary increase in cell
membrane permeability,17 we investigated whether this
phenomenon might support the transfer of plasmid DNA
into eukaryotic cells. For this purpose, HeLa cell suspensions (107 cells per ml) in a total volume of 2.5 ml were
Shock wave permeabilization as gene transfer method
U Lauer et al
mixed with 30 mg of reporter plasmid pRSVb-gal encoding the cytoplasmic enzyme b-galactosidase or with 30
mg of negative control reporter plasmid pSV-MHBs encoding the middle surface protein (MHBs) of hepatitis B
virus (HBV). Polypropylene vials were filled with the
DNA/cell suspensions which were then exposed to 250
shock wave discharges generated at 25 kV with an
experimental XL1 lithotripter (Figure 1). Shock wavetreated cells were subsequently seeded into Petri dishes.
When cell attachment had taken place 4 h later nonviable,
shock wave-disrupted cells (approximately 30%) were
eliminated by washing with PBS followed by a subsequent medium replacement. Cells were analyzed histochemically 48 h later for expression of reporter protein
b-galactosidase. Between 0.1% and 0.5% of HeLa cells
shock wave-permeabilized in suspension with b-galactosidase reporter plasmid pRSVb-gal stained blue, whereas
untreated HeLa cells (data not shown) or cells shock
wave-permeabilized in suspension with reporter plasmid
pSV-MHBs – used as a negative control – did not. This
finding demonstrates that eukaryotic cells upon exposure
to shock waves can take up naked plasmid DNA.
A possible mechanism responsible for this phenomenon is the process of acoustic cavitation, ie shock wavetriggered generation and subsequent collapse of bubbles
containing gas or vapor,18 which results in the formation
of high velocity water jetstreams moving in the direction
of the pulse.19 When eukaryotic cell membranes are hit
by these jetstreams their integrity is supposed to be disturbed leading to a shock wave-induced transient permeabilization.20 Acoustic cavitation is known to be
completely abolished under hyperbaric pressure.14 To
investigate whether shock wave-mediated transfection of
eukaryotic cells depends on the phenomenon of acoustic
cavitation, 107 HeLa cells mixed with 30 mg of reporter
plasmid pSV-MHBs were exposed to 1000 shock waves
in a pressure chamber. At ambient pressure, a constant
increase in MHBs reporter protein accumulation in the
cell culture supernatant was observed (Figure 2, 0 MPa
curve), indicating that shock wave permeabilization was
not affected by the pressure chamber setting. However,
no MHBs reporter protein secretion was detected at 10
MPa hyperbaric pressure, indicating a complete inhibition of shock wave-mediated gene transfer under these
conditions (Figure 2, 10 MPa curve). Concomitantly, cell
disruption was found to be entirely absent at hyperbaric
pressure in accordance with the prevention of cell membrane damage under these conditions (data not shown).
These results strongly suggest that the mechanism of
shock wave transfection depends on acoustic cavitation.
Next, we further characterized the parameters
determining the efficiency of shock wave transfection.
For this purpose, HeLa cell suspensions containing
reporter plasmids pSV-MHBs or pRSVb-gal at a concentration of 30 mg/ml were exposed to variable numbers of
shock waves (125, 250, 500), seeded into Petri dishes and
analyzed 24–72 h later for reporter protein expression.
Increased numbers of applied shock waves resulted in a
linear increase in both MHBs reporter protein expression
(Figure 3a) as well as in the number of blue-staining cells
due to b-galactosidase activity (Figure 3b). Concomitantly, a linear increase in the cell disruption rate was
found, resulting in a linear decrease of viability rates of
80, 65 and 50% at 125, 250 and 500 discharges, respectively, as estimated by trypan blue exclusion staining.
Integrity of plasmid DNA was not disturbed when
Figure 1 Shock wave permeabilization equipment. Shock waves were generated in a 140 l water tank with an experimental XL1 lithotripter by
underwater spark discharge between the two tips of an electrode which
were located in the first focus of a hemi-ellipsoid (15.6 cm ellipsoid
diameter). The propagation wave was focused to the second focus of the
ellipsoid (indicated by the intersection of a positioning laser beam split
into two partial beams meeting at the second focus) where a polypropylene
vial containing the respective suspended cells was positioned and held by
a sample holder. Water in the lithotripter tank was degassed (O2 content
0.5–1 mg/l) using a vacuum pump and maintained at 35–37°C.
Figure 2 Complete inhibition of shock wave-mediated gene transfer under
static excess pressure conditions. One thousand shock waves were applied
to 1 × 107 HeLa cells mixed with reporter plasmid pSV-MHBs at a DNA
concentration of 30 mg/ml at normal atmospheric pressure (0 MPa) or at
static excess pressure (10 MPa), respectively. Afterwards, shock waveexposed cells were plated in 60-mm Petri dishes at a density of 1 × 106
cells per 60-mm diameter plate. MHBs reporter protein accumulation
within cell culture supernatants (5 ml total volume) was determined at
24, 48 and 72 h by testing with a commercial HBsAg-ELISA kit (Auszyme
Monoclonal). The cut-off of the assay is defined as the mean extinction at
492 nm of control values +0.050. Extinction values of the samples corrected for the mean value of negative controls (calculated from three independent experiments) are indicated.
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Shock wave permeabilization as gene transfer method
U Lauer et al
712
Figure 3 Expression of reporter genes after shock wave-mediated gene transfer: dependence on discharge numbers and DNA concentration. Values
indicate the average MHBs reporter protein expression (a, c) or absolute numbers of transfected cells staining blue after histochemical treatment (b, d)
(mean values from five independent shock wave transfection experiments each are given). 125, 250, 500 shock waves were applied to 3 × 106 HeLa cells
mixed with reporter plasmids pSV-MHBs (a) or pRSVb-gal (b) at a DNA concentration of 30 mg/ml, respectively. 3 × 106 HeLa cells mixed with 3,
10, 30, 90 mg/ml of reporter plasmids pSV-MHBs (c) or pRSVb-gal (d) were transfected via application of 250 shock waves, respectively. Shock wavetreated HeLa/pSV-MHBs suspensions (a, c) accumulated increasing amounts of MHBs reporter protein over time (values are given at 24, 48 and 72
h) within cell culture supernatants (5 ml total volume) as determined by the respective extinctions at 492 nm in the Auszyme-ELISA kit. No significant
extinction was detected in control experiments with shock wave treatment of HeLa/pRSVb-gal suspensions or without shock wave treatment of HeLa/pSVMHBs suspensions, respectively. b-Galactosidase activity of shock wave-treated HeLa/pRSVb-gal suspensions (b, d), plated in 60-mm diameter Petri
dishes at a density of 1 × 106 cells per plate, was documented by histochemical staining 48 h after shock wave exposure. Numbers of positive-staining,
transfected cells were determined under the light microscope as the mean value of five representative fields. No positively staining cells were detected
in control experiments with shock wave treatment of HeLa/pSV-MHBs suspensions or without shock wave treatment of HeLa/pRSVb-gal
suspensions, respectively.
exposed to up to 1000 discharges, as determined by agarose gel electrophoresis followed by ethidium bromide
staining (data not shown).
Furthermore, HeLa cell suspensions were mixed with
variable amounts of reporter plasmids pSV-MHBs or
pRSVb-gal (3, 10, 30, 90 mg/ml) and exposed to 250 shock
wave discharges each. Shock wave-treated cells were
analyzed 24–72 h later for MHBs reporter protein
accumulation in the supernatant (Figure 3c) or for the
number of blue-staining cells due to b-galactosidase
activity (Figure 3d), respectively. The efficiency of shock
wave-mediated transfection was found to be directly proportional to the applied DNA concentrations over a wide
range (3, 10, 30, 90 mg/ml) irrespective of the reporter
plasmid used (Figure 3c and d).
In addition, shock wave treatment using different cell
numbers (105 up to 3 × 107 HeLa cells) demonstrated a
constant percentage of transfected cells of about 0.5%
under a standard setting of 250 discharges and at reporter
plasmid concentrations of 30 mg/ml (either pSV-MHBs
or pRSVb-gal) (data not shown). This finding shows that
variations in cell concentration are of no importance for
the overall efficiency of shock wave transfection.
The general applicability of this new method for the
transfer of plasmid DNA into eukaryotic cells originating
from different species and tissues was then tested. Chang
liver cells, HepG2 hepatoma cells and HeLa cells (all of
human origin), mouse L-M (TK−) fibroblast cells, CV-1
monkey kidney cells as well as L1210 mouse lymphocytic
leukemia cells were mixed with reporter plasmid pSV-
Shock wave permeabilization as gene transfer method
U Lauer et al
MHBs at a DNA concentration of 30 mg/ml and exposed
to 250 shock wave discharges. MHBs reporter protein
accumulation within cell culture supernatants was
determined 48 h later. Interestingly, MHBs expression
levels displayed large differences dependent on the transfected cell type ranging from 0.24 ng/ml for L1210 cells
(corresponding to an extinction value about two times
over the assay’s cut-off) to up to 18.0 ng/ml for CV-1
cells (Figure 4). Taken together, these results indicate a
general applicability of shock wave permeabilization for
the transfer of plasmid DNA into eukaryotic cells. The
observed cell type-specific differences in the efficiency of
shock wave-mediated reporter protein expression can
only be partially related to cell type-dependent transcriptional differences of the SV40 expression cassette and
might be primarily explained by a different behavior
towards shock wave-induced share forces, potentially
determined by the cellular shape and volume as well as
by the individual membrane composition.
Discussion
Taken together, these results demonstrate that naked
plasmid DNA can easily be delivered to eukaryotic cells
of different origin upon exposure to lithotriptergenerated shock waves. Since the first clinical applications of extracorporal shock wave lithotripsy,21 several
million patients have been treated world-wide, mainly
for kidney stone and also gall bladder fragmentation and
stones at other locations (bile duct, pancreas, salivary
gland). Shock waves are further used for the treatment
of nonunion fractures. Adverse clinical effects of this
nowadays standard technique are quite rare.11 Most
importantly, shock waves can be focused deep within the
body, thereby making nearly every organ or tissue of
interest accessible. Depending on the chosen lithotripter
setting, focal volumes (defined as the volume in which
the pressure of the shock waves exceeds 50% of the peak
focal pressure) can be varied over a wide range (eg the
experimental XL1 lithotripter used in our experiments
has a focal volume of 600 mm3 whereas the first renal
stone lithotripter22 has one of nearly 20 000 mm3 ). This
opens up the possibility of achieving a regionally
enhanced nonviral in vivo gene transfer even throughout
a whole organ due to perfusion with plasmid DNA solutions while simultaneously applying extracorporally
generated shock waves.
In earlier experiments we were already able to demonstrate that shock wave permeabilization is effective over
a wide range of molecular weights (molecules up to 2
MDa have been successfully transferred as demonstrated
for fluorescence-labeled dextrans).20 Interestingly, this
also applies to naked nuclear acid molecules, since various plasmid DNA molecules up to 10 kb have been successfully shock wave-transferred into recipient cells (our
unpublished data). All in all, these features make shock
wave transfection a gene delivery system which can be
easily applied, which provides great flexibility in the size
and sequence of the DNA molecules to be delivered, and
which should exhibit an advantageous security profile in
vivo. Importantly, the wave energy applied in our in vitro
experiments can also be achieved in vivo. A recent comparison has demonstrated that – under the same setting –
in vivo pressures are only 15–25% lower than the ones
measured in vitro.23 This loss does not increase even over
long shock wave propagating paths in vivo and can easily
be compensated by increasing the output of the shock
wave device. Furthermore, it has been demonstrated in
vivo that shock waves – applied under conditions comparable to our in vitro setting – do induce much much less
cellular disruption and tissue damage in normal as well
as in tumor tissue (,1%) than upon in vitro exposure of
Figure 4 Shock wave transfection of cell lines originating from different tissues and species. Chang liver cells, HepG2 cells, HeLa cells, mouse L-M
(TK−) cells, CV-1 monkey kidney cells as well as L1210 mouse lymphocytic leukemia cells were mixed with reporter plasmid pSV-MHBs at a DNA
concentration of 30 mg/ml, respectively. Subsequently, 250 shock waves were applied. Shock wave-transfected cells were plated in 60-mm diameter Petri
dishes or cultivated in suspension (L1210) at a density of 1 × 106 cells per plate or 1 × 106 cells per 5 ml Dulbecco’s minimal essential medium
supplemented with 10% fetal calf serum (L1210). MHBs reporter protein expression within cell culture supernatants (5 ml total volume) was determined
at 48 h by testing in Auszyme-ELISA. Extinction values (E492 nm) represent the result of a representative experiment and were corrected for the mean
value of the negative controls. The cut-off of the assay, defined as the mean extinction at 492 nm of control values +0.050, is indicated by a horizontal
line next to the base line of the chart.
713
Shock wave permeabilization as gene transfer method
U Lauer et al
714
cells kept in suspension. 24,25 This phenomenon might be
related to a reduced in vivo level of cavitation forces,
which however is sufficient to promote a transient
increase in membrane permeability enabling an intracellular accumulation of therapeutic molecules (eg
cisplatin) under in vivo conditions.24,26
Improvements in the achievable overall efficiency of
shock wave transfection and expression seem to be possible: (1) The minimal immunogenicity of purified, double-stranded plasmid DNA should allow sequential
rounds of shock wave transfection in vivo. (2) A masking
of negative charges of the DNA molecules as well as of
cell membrane molecules by mixing with polycations
could possibly reduce electrostatic repulsion forces. (3)
Confocal laser scanning of shock wave-exposed cells
mixed with fluorescein-labeled dextran molecules has
demonstrated a diffuse intracellular staining pattern indicating direct access of transferred molecules to the cytoplasm.20 Thus, the disadvantageous usage of the endosomal pathway, which is employed in receptor-mediated
gene transfer, is omitted.8 (4) The ineffectiveness of plasmid DNA transfer across the nuclear membrane – limiting expression efficiency of successfully transferred, cytoplasmically localized nucleic acids – can be overcome
potentially by utilization of nucleus-independent
transcription/translation systems, eg by usage of T7 RNA
polymerase autogene vectors.27
Of further interest will be the tissue distribution/
specificity of the shock wave permeabilization procedure.
Under our in vitro conditions, we never observed any
transfection event with eukaryotic cells mixed with
naked plasmid DNA in the absence of shock wave application. In a mouse model, two patterns of transgene
expression after intravenous injection of plasmid DNA
complexed with liposomes have been reported: 28 (1) generalized expression throughout the tissue (as seen in the
lung); and (2) expression largely confined to the vascular
endothelial compartment (as seen for example in the
heart and kidney). In this context, shock wave-induced
damage of small blood vessels which is known to be
restricted to the high pressure area along the central axis
of the shock wave field 11 could potentially enhance
uptake of naked plasmid DNA by parenchymal cells in
a strictly localized manner.
Currently, the transfer of naked plasmid DNA does not
permit continuous expression of genes as indicated by a
transient reporter gene expression in the lung after intravenous injection of naked plasmid DNA29 and a somehow prolonged expression of episomal transgenes in
nondividing cell types after intramuscular injection of
naked plasmid DNA into mice.30 Nevertheless, nonpermanent expression of genes may prove to have clinical
applications, particularly if the in vivo gene transfer can
be performed repeatedly without immunological sideeffects in a setting where only naked DNA will be used.
Therefore, it is suggested that in vivo studies involving
animal models are carried out in order to clarify further
the therapeutic prospects of the shock wave permeabilization procedure.
Materials and methods
Cell lines and cell culture
Chang liver cells (ATCC CCL13; American Type Culture
Collection, Rockville, MD, USA), HepG2 hepatoma cells
and HeLa cells (all of human origin), mouse L-M (TK−)
fibroblast cells, CV-1 monkey kidney cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) in a 5% CO2
atmosphere at 37°C. L1210 mouse lymphocytic leukemia
cells were kindly supplied by Dr Kraemer
(Behringwerke, Marburg, Germany) and grown as suspension culture in RPMI 1640 medium supplemented
with 15% heat inactivated FCS and 2% sodium pyruvate.
Shock wave generation and shock wave
permeabilization procedure
Shock waves were generated with an experimental XL1
lithotripter (Dornier Medical Systems, Germering,
Germany) by underwater spark discharge between the
two tips of an electrode which were located in the first
focus of a hemi-ellipsoid (Figure 1). An 80 nF capacitance
was loaded with 25 kV and subsequently unloaded. The
propagation wave was focused to the second focus of the
ellipsoid where a 2.5 ml silicone-stopper sealed polypropylene vial (Nunc, Wiesbaden, Germany) containing
the respective suspended cells was positioned without
any residual air in the vial. The position of the second
focus was indicated by the intersection of two positioning
laser beams (Figure 1). The center of the vial was positioned within the second focus 10 mm above the vial
bottom. Shock wave duration is less than 2 ms. The peak
positive focal pressure in the vial exceeded 80 MPa. The
pressure fell off rapidly comprising only 50% of the focal
value at a distance of 2.55 mm lateral (x-axis) and 11 mm
longitudinal (z-axis). The discharge rate was set to 100
per min. Water in the lithotripter tank was degassed (O2
content 0.5–1 mg/l) using a vacuum pump and maintained at 35–37°C. For the investigation of the shock wave
exposure effects at static excess pressure target cell suspensions were mixed with the respective reporter plasmid and exposed to 1000 shock waves in a pressure
chamber at ambient pressure (0 MPa) and at hyperbaric
pressure (10 MPa). Viability of shock wave treated cells
was determined by the trypan blue dye exclusion
method.
Reporter plasmids
Reporter plasmid pRSVb-gal contains the E. coli lacZ
gene, encoding the cytoplasmic enzyme b-galactosidase,
under control of the RSV LTR.31 Reporter plasmid pSVMHBs encodes the secretable middle surface protein
(MHBs) of hepatitis B virus (HBV) under control of the
SV40 enhancer/early promoter regulatory cassette.32
b-Galactosidase reporter protein detection assay
For detection of lacZ-encoded b-gal activity, cells were
washed twice with PBS, fixed 48 h after shock wave permeabilization in ice-cold 2% paraformaldehyde/0.2%
glutaraldehyde solution, washed again twice with PBS
(containing 2 mm MgCl2) and incubated at 37°C with the
histochemical reaction mixture containing X-gal chromagen (1 mg/ml; Boehringer, Mannheim, Germany) until
the blue colour developed.26
MHBs reporter protein detection assay
MHBs reporter protein accumulation within cell culture
supernatants (5 ml total volume) was determined at 24,
48 and 72 h by testing with a commercial HBsAg-ELISA
kit (Auszyme Monoclonal) according to the manu-
Shock wave permeabilization as gene transfer method
U Lauer et al
facturer’s recommendations (Abbott Laboratories, North
Chicago, IL, USA). In brief, 200 ml samples were harvested and dispensed into the respective wells of the test
plate. Subsequently, test pellets covered with mouse antiHBsAg monoclonal antibodies were added and incubated together with 50 ml of peroxidase-coupled mouse
anti-HBsAg monoclonal antibodies at room temperature
for 16 h. Next, incubated pellets were washed five times
with 5 ml of distilled water. Finally, 300 ml of the o-phenylendiamine solution was added to the washed pellets
and incubated for 30 min followed by a final addition of
1 ml 1 N sulphuric acid. The respective extinction values
were measured at 492 nm. The cut-off of the Auszyme
Monoclonal assay is defined as the mean extinction at 492
nm of control values + 0.050.
Acknowledgements
We would like to thank DA Brenner for providing us
with reporter plasmid pRSVb-gal, G Adams for his excellent technical assistance, S Werner for her critical reading
of the manuscript, T Coutts for her linguistic reading of
the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (De 531/1–1, De 531/1–2,
La 649/12–1).
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