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Shock Wave Based Biolistic Device for DNA and Drug Delivery
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2008 Jpn. J. Appl. Phys. 47 1522
(http://iopscience.iop.org/1347-4065/47/3R/1522)
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Shock Wave Based Biolistic Device for DNA and Drug Delivery
Mutsumi NAKADA, Viren MENEZES1, Akira KANNO, S. Hamid R.
HOSSEINI2, and Kazuyoshi TAKAYAMA3
Graduate School of Life Sciences, Tohoku University, Sendai
980-8577, Japan1Department of Aerospace Engineering, Indian
Institute of Technology Bombay, Powai, Mumbai 400-076,
India2Department of Bioengineering, University of Washington, 1705
N.E. Pacic St., Box 355061, Seattle, WA 98195, U.S.A.3Biomedical
Engineering Research Organization (TUBERO), Tohoku University,
Sendai 980-0872, Japan
(Received September 18, 2007; accepted December 3, 2007;
published online March 14, 2008)
A shock wave assisted biolistic (biological ballistic) device
has been developed to deliver DNA/drug-coated micro-projectilesinto
soft living targets. The device consists of an Nd:YAG laser, an
optical setup to focus the laser beam and, a thin aluminum(Al) foil
(typically 100 mm thick) which is a launch pad for the
micro-projectiles. The DNA/drug-coated micro-particles to
bedelivered are deposited on the anterior surface of the foil and
the posterior surface of the foil is ablated using the laser
beamwith an energy density of about 32 109W/cm2. The ablation
launches a shock wave through the foil that imparts an impulseto
the foil surface, due to which the deposited particles accelerate
and acquire sucient momentum to penetrate soft targets.The device
has been tested for particle delivery by delivering 1 mm size
tungsten particles into liver tissues of experimentalrats and in
vitro test models made of gelatin. The penetration depths of about
90 and 800 mm have been observed in the liverand gelatin targets,
respectively. The device has been tested for in vivo DNA [encoding
-glucuronidase (GUS) gene] transferby delivering plasmid
DNA-coated, 1-mm size gold (Au) particles into onion scale, tobacco
leaf and soybean seed cells. TheGUS activity was detected in the
onion, tobacco and soybean cells after the DNA delivery. The
present device is totally non-intrusive in nature and has a
potential to get miniaturized to suit the existing medical
procedures for DNA and/or drugdelivery. [DOI:
10.1143/JJAP.47.1522]
KEYWORDS: shock wave, laser ablation, biolistic, DNA/drug
delivery, gene expression
1. Introduction
The biolistic approach has been proved to be quiteecacious in
transferring DNA into plant cells for geneticmodications.14) The
DNA-coated particles could be deliv-ered into intact plant cells
and tissues without enzymaticremoval of cell walls using the
biolistic process. Severaldevices were developed and tested for
accelerating micro-projectiles to high velocities to accomplish the
task ofbiolistic drug delivery.2,57) Among these devices, the
shocktube based particle delivery device6,7) was the rst one to
beused on a human organ for a pharmacological eect. Thedevice was
reported to be successful in delivering powderedvaccines into human
epidermis for immunotherapies.Gene therapy, which alters the
genetic information
contained in specic cells, can be useful for the treatmentof
several inherited and acquired human diseases.810) By farthe most
ecient DNA administration could be achieved bya localized biolistic
delivery of DNA coated micro-particlesinto intact epidermal
cells.6,11) The treatment sites in suchtherapies could also be
internal body organs8,9) and thetreatment modality may have to be
non-invasive. In suchcases, a totally non-intrusive drug delivery
device that has agood controllability and a potential to get
miniaturized tosuit the existing non-invasive surgical devices,
would hold agreat promise to clinicians.Here we describe a new
biolistic device that uses a laser
ablation generated shock wave to deliver powdered vaccinesand/or
DNA-coated particles into living cells and tissues.12)
The bench-top prototype of the device, as shown inFig. 1(a), has
a 1064 nm wavelength Nd:YAG laser thatgenerates pulses of 5.5 ns
duration and 1.4 J energy. Asuitable optical set up is used to
collimate and focus the laserbeam on to a 100 mm thick aluminum
foil, the anterior sideof which contains the drug in
particle/powder form. A 5-
mm-thick BK7 glass cover has been used on the posteriorside of
the foil to conne the laser ablation.Unlike other biolistic
devices, this device does not use any
additional substance, such as a gas, to carry the particlesonto
the target, and hence can be used to deliver drugs intointernal
body organs in medical procedures. The deviceis laser driven and
has an advantage over the explosivedriven devices as far as the
controllability is concerned.Moreover, it is possible to
miniaturize this device such thatit can be integrated with the
existing, non-invasive surgicalprocedures.
(a)
(b)
Fig. 1. (Color online) (a) Schematic of the bench-top prototype
of the
device. (b) The device physics; 1: Lens. 2: Laser beam. 3: Glass
overlay.
4: Foil. 5: Target. 6: Particles. 7: Shock wave. 8: Conned
ablation.
9: Expansion wave. 10: Micro-crater due to ablation.
E-mail address: [email protected]
Japanese Journal of Applied Physics
Vol. 47, No. 3, 2008, pp. 15221526
#2008 The Japan Society of Applied Physics
1522
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The device has been tested for particle delivery bydelivering 1
mm size tungsten projectiles into soft targetssuch as liver tissues
of experimental rats and in vitro testmodels made of gelatin. The
device has been tested for DNAdelivery by delivering plasmid
DNA-coated, 1-mm size goldprojectiles into onion scale, tobacco
leaf and soybean seedcells. The expression of an introduced gene
was detected inthe onion, tobacco and soybean cells.
2. Materials and Methods
2.1 Optics and launch padThe Q-switched, pulsed neodymium-doped
yttrium
aluminum garnet (Nd:YAG) laser (Thales Laser) wasoperated with
its basic wavelength of 1064 nm to derivethe maximum energy of 1.5
J/pulse. Installation of anoptical isolator in the laser head
caused an energy loss of0.1 J/pulse and the laser energy available
for the applica-tion was 1.4 J/pulse. The optical isolator was an
additionalaccessory installed to prevent the possible damage to
thelaser due to the reection of the beam from the metallictarget.
The pulse duration of the laser was 5.5 ns, whichwas aptly adequate
for the application, as larger pulseduration would hold a risk of
melting away the foil anddamaging the drug.The laser beam that was
initially 9mm in diameter was
expanded and collimated using a combination of concaveand convex
lenses and focused on the foil through the BK7glass using a
focusing lens. The diameter of the focal spot onthe foil was about
4mm. As can be seen in Fig. 1(a), thelaser beam had to be taken
through several mirrors beforepassing through the lenses and in a
real model, these mirrorscan be replaced by a miniature optical arm
or an opticalber, making the device exible and user friendly.
Thelenses, foil holder, BK7 glass and the foil, in their
miniatureform, can be attached to the end of the optical arm or
theconduit of the optical ber.The aluminum foil (99.2% purity;
Nilaco), used as a
particle launch pad in the operation, was chosen for its
highacoustic speed. The objective was to maximize the speed ofthe
loaded shock wave and the unloading expansion wave,thereby
maximizing the velocity of the foil, as the wavespeeds are directly
proportional to the speed of sound in themedium of propagation.
2.2 Micro-particlesThe micro-particles of 1 mm size (Bio-Rad)
chosen in
the present study were apt for gene therapy. A
particlesuspension was prepared using 70% ethanol and a
smallportion (typically 5 ml) of this suspension was deposited
onthe metal foil. The alcohol evaporated leaving behind a thintrace
of the particles. While testing the device for particledelivery,
tungsten particles of 1 mm size were used, and forin vivo DNA
delivery we used pure gold particles of thesame size. Since the
density of gold is almost equal to thedensity of tungsten, use of
tungsten at the testing stage couldminimize the consumable
expenses, while the particledynamics remained the same. The
deposited layer ofmicro-particles on the launch pad often had
clusters thatranged from 2 to 10 mm in size, but these were
disintegratedinto almost individual, 1 mm size particles on shock
waveloading into the launch pad.
2.3 Plant materialThree plant materials were used. These were,
the scales
of onion (Allium cepa) that were cut into 1 1 cm2, theleaf-discs
of tobacco (Nicotiana tabacum) with a diameterof about 1 cm and,
the seeds of soybeans with cotyledoncells (Glycine max). The onion
was purchased locally; thetobacco plant and the soybeans were grown
in the greenhouse and the experimental elds, respectively, at
TohokuUniversity, Japan.
2.4 Plasmid DNA and particle coatingThe plasmid DNA, pIG121Hm,
which contained the nptII
(neomycin phosphotransferase II) gene under the control ofthe
nos promoter, the hpt (hygromycin phosphotransferase)gene under the
control of the CaMV (cauliower mosaicvirus) 35S promoter, and the
-glucuronidase (GUS) genewith an intron (GUSintron) under the
control of the CaMV35S promoter,13) was used. The closed circular
form of theplasmid DNA was puried, and coated onto gold particles(1
mm in size) by co-precipitation in ethanol at a DNAconcentration of
15 mg of DNA/mg of particles.
2.5 Histochemical GUS assayAfter particle bombardment, the
samples were transferred
onto MS medium,14) containing sucrose (30 g/l) and gellangum (2
g/l), and kept for 48 h in dark. Onion cells wereincubated at 25 C,
and tobacco leaves and soybean seedswere incubated at 28 C. Each
sample was put into thebuer, which contained 1.5ml of
lter-sterilized GUSsubstrate mixture. The substrate mixture
consisted of50mM sodium hydrogenphosphate, 50mM disodium
di-hydrogenphosphate, 1.9mM 5-bromo-4-chloro-3-indolylglucuronide
(X-gluc: the substrate of GUS), and 0.1% (v/v)Triton X-100.
Further, the tissues were incubated for 24 hat 37 C, and then 5ml
of 70% (v/v) ethanol was added tothe cellGUS substrate mixture in
order to stop the reactionand to keep aseptic conditions.
GUS-expressing cells weredetected as blue-colored spots.
3. Results and Discussion
The physical process of device operation is depicted inFig.
1(b). Laser focusing launches a shock wave through thefoil, which
propagates longitudinally, and reects back as anexpansion wave on
reaching the foilair boundary. At thisinstant of time, the foil
gets unloaded or decompressed andacquires a high velocity in the
direction of the initial motionof the shock. The drug particles,
deposited on the anteriorsurface of the foil also move along with
the foil surfaceand get ejected out of the foil surface due to
inertia. Themomentum acquired by the powdered drug is high enough
topenetrate soft targets.The acceleration of the micro-particles
from the surface
of a 100-mm-thick aluminum foil on laser ablation wasanalyzed
through photography using a high-speed videocamera (Shimadzu
HyperVision HPV 1) in a standardshadowgraph system. The photography
was carried out at asampling rate of 1 Mega frames per second with
a spatialresolution of 312 260 pixels per frame. Figure 2 shows500
mg of 1 mm size tungsten particles getting ejected out ofthe foil
surface, and the velocity of these particles, analyzedbased on the
visualized pictures is plotted in Fig. 3. The
Jpn. J. Appl. Phys., Vol. 47, No. 3 (2008) M. NAKADA et al.
1523
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particles have a high velocity initially, but soon
aredecelerated due to the resistance oered by the
surroundingatmospheric air. The visualized pictures also show
theincident shock wave getting transmitted into the atmosphericair
from the foil. The transmitted shock wave has been foundahead of
the particles and the steep deceleration of theparticles observed
at the initial stage can be attributed tothis shock wave, as the
pressure and density of the air itpropagates through are increased.
Further, at a later stage ofthe particle ight, the mass motion
behind the transmittedshock wave aides the particle motion, which
is indicated byan almost uniform velocity of the particles at a
later stage oftheir ight, as shown in Fig. 3.In vitro targets such
as gelatin test-beds were initially
used to test the device for particle delivery. Tungstenparticles
of 1 mm size were delivered into 3% gelatin test-beds (2025 bloom,
cooled at 10 C for 1 h.) that modelhuman blood clots. Figure 4(a)
shows the delivered tungstenparticles in a 3% gelatin model.
Tungsten particles of 1 mm
size penetrated through about 800 mm in 3% gelatin. Softbody
tissues were also used as targets to test the devicefor particle
delivery. Tungsten particles of 1 mm size weredelivered into liver
tissues of Sprague Dawley male(experimental) rats. Figure 4(b)
shows hematoxylineosinstained micrographs of the sections (30 mm
thick) of theliver tissues. The tungsten particles were found
penetratedthrough about 90 mm in the rat liver.
Experimentallyobserved depths of particle penetration in liver and
gelatinare plotted in Fig. 4(c). All the animal
experimentsconducted were within the animal welfare regulations
andguidelines in Japan.Figures 5(a)5(c) show the in vivo results of
DNA trans-
fer in onion scale, tobacco leaf and soybean seed
cells,respectively. The blue spots in the plant targets indicate
theGUS activity in the transformed cells. No blue spots
weredetected on bombarding the targets with uncoated goldparticles,
and likewise, un-bombarded samples had no bluespots (data not
shown). The blue spots in tobacco leaf and
(a)
(b)
(c)
Fig. 2. (Color online) (a) Acceleration of micro-particles from
the launch pad on shock wave loading, visualized using a
high-speed
video camera, at an interframe (time dierence between two
frames) of 1ms. The frame-sequence is from left to right. Laser
peakpower was 0.25GW. 100-mm-thick Al foil was used as the launch
pad for 1mm size tungsten particles that were about 500 mg
inquantity, which is a higher than the usual quantity of particles
that was used to facilitate the process of visualization. Ablation
spot
diameter on the foil was 4mm. Legend: S, Transmitted shock wave;
P, Particle cloud. Scale bar (horizontal line in the top-leftcorner
of rst frame): 5mm. (b) Enlarged view of frame No. 5, showing
transmitted shock waves. (c) Schematic describing the
photographs of the particle launch.
Jpn. J. Appl. Phys., Vol. 47, No. 3 (2008) M. NAKADA et al.
1524
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soybean seed samples were smaller in size and weaker incolor
when compared to those of onion, and this observationcan be
attributed to the smaller size of the tobacco leaf andsoybean seed
cells. Several tests have been performed on theplant cells to
ascertain the repeatability of the device and theprocess of DNA
transfer. The transfection eciency of thepresent biolistic process,
in terms of number of transformedcells per mm2 area, is depicted in
Fig. 6(a). The gure alsogives the eciency of another biolistic
device, Bio-RadPDS-1000/He,15) for a very similar experiment. This
analy-sis could be carried out only for onion cells, as these
cellswere larger in size and more robust in structure. Figure
6(b)shows the sections of onion scales, indicating the depth ofgene
expression in the cells. The blue spots extended up to adepth of
106 to 139 mm in onion scales as exhibited in thegure.GUS gene of
Escherichia coli is the most widely used
reporter gene, which has been developed as a gene fusionmarker
for animals and plants. GUS is a hydrolase thatcatalyses the
degradation of a wide variety of -glucur-onides. The hydrolysis of
the substrate 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc)
results in an indigo blueprecipitate. The blue spots indicate that
the exogenous GUS
genes are delivered to the nucleus and expressed in
thetransformed cells. Most of the plants do not have endoge-nous
GUS genes or functionally similar genes, and thereforethe
GUSreporter system is very useful to analyze thetransgene
activity.The plant targets used in the present study were of
assorted types. The tobacco leaf-discs are very fragile innature
and the device could successfully transfer the DNA-coated particles
into the leaf cells without causing anynoticeable damage. DNA
transfer into soybean seed cellsprovides evidence for the
controllability of the device asthese cells are quite minute for a
biolistic process. Moreover,delivery of the vaccines onto a specic
spot on the target,without much of a diversion in the ight path of
the particlesis the specialty of this device.
Fig. 3. (Color online) The velocity of the ejected
micro-particles with
respect to distance from the launch pad and time, deduced from
the high-
speed photography that was carried out at 1ms interframe.
Reference line(0mm) indicated in the plot is the lower edge of the
foil holder. Foil
location is 0.5mm upwards from the reference line.
(a)
(b)
(c)
Fig. 4. (Color online) (a) A 3% gelatin test model with
penetrated 1mmsize tungsten particles. (b) Micro-sections of the
rat liver tissues with
penetrated 1 mm size tungsten particles. Scale bar: 50mm. (c)
Exper-imentally observed particle penetration depths in 3% gelatin
and Rat
liver.
Jpn. J. Appl. Phys., Vol. 47, No. 3 (2008) M. NAKADA et al.
1525
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In summary, we describe a shock wave based biolisticdevice that
can be used to deliver powdered vaccines/DNAinto intact living
cells. The device employs an Nd:YAG laserto drive a shock wave
through a thin aluminum foil that
functions as a launch pad for the powdered drug. The devicehas
been tested for in vivo DNA delivery into living plantcells. The
biolistic device being proposed is non-intrusiveand can be
miniaturized to integrate with non-invasivesurgical devices to have
potential applications in medicaltherapies.
Acknowledgement
The authors thank Mr. Taichi Kamimura for the technicalsupport
during the experiments. This work was supported inpart by a
Grant-in-Aid for Scientic Research from theMinistry of Education,
Culture, Sports, Science and Tech-nology, Japan.
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(a) (b) (c)
Fig. 5. (Color online) Bombarded plant cells showing GUS
expression. (a) The onion block. Scale bar: 500 mm. (b) The tobacco
leafdisc. Scale bar: 100mm. (c) The soybean seed. Arrows indicate
transformed cells. Scale bar: 100 mm.
(b)
(a)
Fig. 6. (Color online) (a) Transfection eciency of the device.
The plot
is the average of 4 onion scale samples. 4.5 mg of DNA was used
for eachshot. Transformed cells were counted per square millimeter
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Jpn. J. Appl. Phys., Vol. 47, No. 3 (2008) M. NAKADA et al.
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