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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
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Japanese Journal of Applied Physics

Vol. 47, No. 3, 2008, pp. 15221526
#2008 The Japan Society of Applied Physics

Shock Wave Based Biolistic Device for DNA and Drug Delivery
Mutsumi N AKADA, Viren M ENEZES1 , Akira KANNO, S. Hamid R. H OSSEINI2 , and Kazuyoshi TAKAYAMA3
Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan
1
Department of Aerospace Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400-076, India
2
Department of Bioengineering, University of Washington, 1705 N.E. Pacic St., Box 355061, Seattle, WA 98195, U.S.A.
3
Biomedical 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-projectiles
into 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 be
delivered are deposited on the anterior surface of the foil and the posterior surface of the foil is ablated using the laser beam
with an energy density of about 32  109 W/cm2 . The ablation launches a shock wave through the foil that imparts an impulse
to 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 experimental
rats and in vitro test models made of gelatin. The penetration depths of about 90 and 800 mm have been observed in the liver
and gelatin targets, respectively. The device has been tested for in vivo DNA [encoding -glucuronidase (GUS) gene] transfer
by delivering plasmid DNA-coated, 1-mm size gold (Au) particles into onion scale, tobacco leaf and soybean seed cells. The
GUS activity was detected in the onion, tobacco and soybean cells after the DNA delivery. The present device is totally nonintrusive in nature and has a potential to get miniaturized to suit the existing medical procedures for DNA and/or drug
delivery. [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 quite

ecacious in transferring DNA into plant cells for genetic
modications.14) The DNA-coated particles could be delivered into intact plant cells and tissues without enzymatic
removal of cell walls using the biolistic process. Several
devices were developed and tested for accelerating microprojectiles to high velocities to accomplish the task of
biolistic drug delivery.2,57) Among these devices, the shock
tube based particle delivery device6,7) was the rst one to be
used on a human organ for a pharmacological eect. The
device was reported to be successful in delivering powdered
vaccines into human epidermis for immunotherapies.
Gene therapy, which alters the genetic information
contained in specic cells, can be useful for the treatment
of several inherited and acquired human diseases.810) By far
the most ecient DNA administration could be achieved by
a localized biolistic delivery of DNA coated micro-particles
into intact epidermal cells.6,11) The treatment sites in such
therapies could also be internal body organs8,9) and the
treatment modality may have to be non-invasive. In such
cases, a totally non-intrusive drug delivery device that has a
good controllability and a potential to get miniaturized to
suit the existing non-invasive surgical devices, would hold a
great promise to clinicians.
Here we describe a new biolistic device that uses a laser
ablation generated shock wave to deliver powdered vaccines
and/or DNA-coated particles into living cells and tissues.12)
The bench-top prototype of the device, as shown in
Fig. 1(a), has a 1064 nm wavelength Nd:YAG laser that
generates pulses of 5.5 ns duration and 1.4 J energy. A
suitable optical set up is used to collimate and focus the laser
beam on to a 100 mm thick aluminum foil, the anterior side
of which contains the drug in particle/powder form. A 5

E-mail address: viren@aero.iitb.ac.in

(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.

mm-thick BK7 glass cover has been used on the posterior

side 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 particles
onto the target, and hence can be used to deliver drugs into
internal body organs in medical procedures. The device
is laser driven and has an advantage over the explosive
driven devices as far as the controllability is concerned.
Moreover, it is possible to miniaturize this device such that
it can be integrated with the existing, non-invasive surgical
procedures.

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M. NAKADA et al.

The device has been tested for particle delivery by

delivering 1 mm size tungsten projectiles into soft targets
such as liver tissues of experimental rats and in vitro test
models made of gelatin. The device has been tested for DNA
delivery by delivering plasmid DNA-coated, 1-mm size gold
projectiles into onion scale, tobacco leaf and soybean seed
cells. The expression of an introduced gene was detected in
the onion, tobacco and soybean cells.
2.

Materials and Methods

2.1 Optics and launch pad

The Q-switched, pulsed neodymium-doped yttrium
aluminum garnet (Nd:YAG) laser (Thales Laser) was
operated with its basic wavelength of 1064 nm to derive
the maximum energy of 1.5 J/pulse. Installation of an
optical isolator in the laser head caused an energy loss of
0.1 J/pulse and the laser energy available for the application was 1.4 J/pulse. The optical isolator was an additional
accessory installed to prevent the possible damage to the
laser due to the reection of the beam from the metallic
target. The pulse duration of the laser was 5.5 ns, which
was aptly adequate for the application, as larger pulse
duration would hold a risk of melting away the foil and
damaging the drug.
The laser beam that was initially 9 mm in diameter was
expanded and collimated using a combination of concave
and convex lenses and focused on the foil through the BK7
glass using a focusing lens. The diameter of the focal spot on
the foil was about 4 mm. As can be seen in Fig. 1(a), the
laser beam had to be taken through several mirrors before
passing through the lenses and in a real model, these mirrors
can be replaced by a miniature optical arm or an optical
ber, making the device exible and user friendly. The
lenses, foil holder, BK7 glass and the foil, in their miniature
form, can be attached to the end of the optical arm or the
conduit of the optical ber.
The aluminum foil (99.2% purity; Nilaco), used as a
particle launch pad in the operation, was chosen for its high
acoustic speed. The objective was to maximize the speed of
the loaded shock wave and the unloading expansion wave,
thereby maximizing the velocity of the foil, as the wave
speeds are directly proportional to the speed of sound in the
medium of propagation.
2.2 Micro-particles
The micro-particles of 1 mm size (Bio-Rad) chosen in
the present study were apt for gene therapy. A particle
suspension was prepared using 70% ethanol and a small
portion (typically 5 ml) of this suspension was deposited on
the metal foil. The alcohol evaporated leaving behind a thin
trace of the particles. While testing the device for particle
delivery, tungsten particles of 1 mm size were used, and for
in vivo DNA delivery we used pure gold particles of the
same size. Since the density of gold is almost equal to the
density of tungsten, use of tungsten at the testing stage could
minimize the consumable expenses, while the particle
dynamics remained the same. The deposited layer of
micro-particles on the launch pad often had clusters that
ranged from 2 to 10 mm in size, but these were disintegrated
into almost individual, 1 mm size particles on shock wave
loading into the launch pad.

2.3 Plant material

Three plant materials were used. These were, the scales
of onion (Allium cepa) that were cut into 1  1 cm2 , the
leaf-discs of tobacco (Nicotiana tabacum) with a diameter
of about 1 cm and, the seeds of soybeans with cotyledon
cells (Glycine max). The onion was purchased locally; the
tobacco plant and the soybeans were grown in the green
house and the experimental elds, respectively, at Tohoku
University, Japan.
2.4 Plasmid DNA and particle coating
The plasmid DNA, pIG121Hm, which contained the nptII
(neomycin phosphotransferase II) gene under the control of
the nos promoter, the hpt (hygromycin phosphotransferase)
gene under the control of the CaMV (cauliower mosaic
virus) 35S promoter, and the -glucuronidase (GUS) gene
with an intron (GUSintron) under the control of the CaMV
35S promoter,13) was used. The closed circular form of the
plasmid DNA was puried, and coated onto gold particles
(1 mm in size) by co-precipitation in ethanol at a DNA
concentration of 15 mg of DNA/mg of particles.
2.5 Histochemical GUS assay
After particle bombardment, the samples were transferred
onto MS medium,14) containing sucrose (30 g/l) and gellan
gum (2 g/l), and kept for 48 h in dark. Onion cells were
incubated at 25  C, and tobacco leaves and soybean seeds
were incubated at 28  C. Each sample was put into the
buer, which contained 1.5 ml of lter-sterilized GUS
substrate mixture. The substrate mixture consisted of
50 mM sodium hydrogenphosphate, 50 mM disodium dihydrogenphosphate, 1.9 mM 5-bromo-4-chloro-3-indolyl
glucuronide (X-gluc: the substrate of GUS), and 0.1% (v/v)
Triton X-100. Further, the tissues were incubated for 24 h
at 37  C, and then 5 ml of 70% (v/v) ethanol was added to
the cellGUS substrate mixture in order to stop the reaction
and to keep aseptic conditions. GUS-expressing cells were
detected as blue-colored spots.
3.

Results and Discussion

The physical process of device operation is depicted in

Fig. 1(b). Laser focusing launches a shock wave through the
foil, which propagates longitudinally, and reects back as an
expansion wave on reaching the foilair boundary. At this
instant of time, the foil gets unloaded or decompressed and
acquires a high velocity in the direction of the initial motion
of the shock. The drug particles, deposited on the anterior
surface of the foil also move along with the foil surface
and get ejected out of the foil surface due to inertia. The
momentum acquired by the powdered drug is high enough to
penetrate soft targets.
The acceleration of the micro-particles from the surface
of a 100-mm-thick aluminum foil on laser ablation was
analyzed through photography using a high-speed video
camera (Shimadzu HyperVision HPV 1) in a standard
shadowgraph system. The photography was carried out at a
sampling rate of 1 Mega frames per second with a spatial
resolution of 312  260 pixels per frame. Figure 2 shows
500 mg of 1 mm size tungsten particles getting ejected out of
the foil surface, and the velocity of these particles, analyzed
based on the visualized pictures is plotted in Fig. 3. The

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M. NAKADA et al.

(b)

(c)

(a)
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 1 ms. The frame-sequence is from left to right. Laser peak
power was 0.25 GW. 100-mm-thick Al foil was used as the launch pad for 1 mm size tungsten particles that were about 500 mg in
quantity, 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 4 mm. Legend: S, Transmitted shock wave; P, Particle cloud. Scale bar (horizontal line in the top-left
corner of rst frame): 5 mm. (b) Enlarged view of frame No. 5, showing transmitted shock waves. (c) Schematic describing the
photographs of the particle launch.

particles have a high velocity initially, but soon are

decelerated due to the resistance oered by the surrounding
atmospheric air. The visualized pictures also show the
incident shock wave getting transmitted into the atmospheric
air from the foil. The transmitted shock wave has been found
ahead of the particles and the steep deceleration of the
particles observed at the initial stage can be attributed to
this shock wave, as the pressure and density of the air it
propagates through are increased. Further, at a later stage of
the particle ight, the mass motion behind the transmitted
shock wave aides the particle motion, which is indicated by
an almost uniform velocity of the particles at a later stage of
their ight, as shown in Fig. 3.
In vitro targets such as gelatin test-beds were initially
used to test the device for particle delivery. Tungsten
particles of 1 mm size were delivered into 3% gelatin testbeds (20 25 bloom, cooled at 10  C for 1 h.) that model
human blood clots. Figure 4(a) shows the delivered tungsten
particles in a 3% gelatin model. Tungsten particles of 1 mm

size penetrated through about 800 mm in 3% gelatin. Soft

body tissues were also used as targets to test the device
for particle delivery. Tungsten particles of 1 mm size were
delivered into liver tissues of Sprague Dawley male
(experimental) rats. Figure 4(b) shows hematoxylineosin
stained micrographs of the sections (30 mm thick) of the
liver tissues. The tungsten particles were found penetrated
through about 90 mm in the rat liver. Experimentally
observed depths of particle penetration in liver and gelatin
are plotted in Fig. 4(c). All the animal experiments
conducted were within the animal welfare regulations and
guidelines in Japan.
Figures 5(a)5(c) show the in vivo results of DNA transfer in onion scale, tobacco leaf and soybean seed cells,
respectively. The blue spots in the plant targets indicate the
GUS activity in the transformed cells. No blue spots were
detected on bombarding the targets with uncoated gold
particles, and likewise, un-bombarded samples had no blue
spots (data not shown). The blue spots in tobacco leaf and

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M. NAKADA et al.

(a)

(b)

Fig. 3. (Color online) The velocity of the ejected micro-particles with

respect to distance from the launch pad and time, deduced from the highspeed photography that was carried out at 1 ms interframe. Reference line
(0 mm) indicated in the plot is the lower edge of the foil holder. Foil
location is 0.5 mm upwards from the reference line.

soybean seed samples were smaller in size and weaker in

color when compared to those of onion, and this observation
can be attributed to the smaller size of the tobacco leaf and
soybean seed cells. Several tests have been performed on the
plant cells to ascertain the repeatability of the device and the
process of DNA transfer. The transfection eciency of the
present biolistic process, in terms of number of transformed
cells per mm2 area, is depicted in Fig. 6(a). The gure also
gives the eciency of another biolistic device, Bio-Rad
PDS-1000/He,15) for a very similar experiment. This analysis could be carried out only for onion cells, as these cells
were larger in size and more robust in structure. Figure 6(b)
shows the sections of onion scales, indicating the depth of
gene expression in the cells. The blue spots extended up to a
depth of 106 to 139 mm in onion scales as exhibited in the
gure.
GUS gene of Escherichia coli is the most widely used
reporter gene, which has been developed as a gene fusion
marker for animals and plants. GUS is a hydrolase that
catalyses the degradation of a wide variety of -glucuronides. The hydrolysis of the substrate 5-bromo-4-chloro-3indolyl glucuronide (X-Gluc) results in an indigo blue
precipitate. The blue spots indicate that the exogenous GUS

(c)
Fig. 4. (Color online) (a) A 3% gelatin test model with penetrated 1 mm
size tungsten particles. (b) Micro-sections of the rat liver tissues with
penetrated 1 mm size tungsten particles. Scale bar: 50 mm. (c) Experimentally observed particle penetration depths in 3% gelatin and Rat
liver.

genes are delivered to the nucleus and expressed in the

transformed cells. Most of the plants do not have endogenous GUS genes or functionally similar genes, and therefore
the GUSreporter system is very useful to analyze the
transgene activity.
The plant targets used in the present study were of
assorted types. The tobacco leaf-discs are very fragile in
nature and the device could successfully transfer the DNAcoated particles into the leaf cells without causing any
noticeable damage. DNA transfer into soybean seed cells
provides evidence for the controllability of the device as
these 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 particles
is the specialty of this device.

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M. NAKADA et al.

(b)

(a)

(c)

Fig. 5. (Color online) Bombarded plant cells showing GUS expression. (a) The onion block. Scale bar: 500 mm. (b) The tobacco leaf
disc. Scale bar: 100 mm. (c) The soybean seed. Arrows indicate transformed cells. Scale bar: 100 mm.

functions as a launch pad for the powdered drug. The device

has been tested for in vivo DNA delivery into living plant
cells. The biolistic device being proposed is non-intrusive
and can be miniaturized to integrate with non-invasive
surgical devices to have potential applications in medical
therapies.
Acknowledgement
The authors thank Mr. Taichi Kamimura for the technical
support during the experiments. This work was supported in
part by a Grant-in-Aid for Scientic Research from the
Ministry of Education, Culture, Sports, Science and Technology, Japan.
(a)

(b)
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 each
shot. Transformed cells were counted per square millimeter area on the
target. (b) Sections of onion scales indicating the depth of gene
expression in the target. The horizontal lines are the scale bars and are
1 mm in length. The vertical lines indicate the depth of blue spots from
the edge of the sections.

In summary, we describe a shock wave based biolistic

device that can be used to deliver powdered vaccines/DNA
into intact living cells. The device employs an Nd:YAG laser
to drive a shock wave through a thin aluminum foil that

1) J. C. Sanford: Trends Biotechnol. 6 (1988) 299.

2) T. M. Klein, E. D. Wolf, R. Wu, and J. C. Sanford: Nature (London)
327 (1987) 70.
3) T. M. Klein, M. Fromm, A. Weissinger, D. Tomes, S. Schaaf, M.
Sletten, and J. C. Sanford: Proc. Natl. Acad. Sci. U.S.A. 85 (1988)
4305.
4) H. Daniell, J. Vivekananda, B. L. Nielsen, G. N. Ye, K. K. Tewari, and
J. C. Sanford: Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 88.
5) J. C. Sanford, T. M. Klein, E. D. Wolf, and N. Allen: Part. Sci.
Technol. 5 (1987) 27.
6) D. Chen, R. L. Endres, C. A. Erickson, K. F. Weis, M. W. McGregor,
Y. Kawaoka, and L. G. Payne: Nat. Med. 6 (2000) 1187.
7) N. J. Quinlan, M. A. F. Kendall, B. J. Bellhouse, and R. W. Ainsworth:
Shock Waves 10 (2001) 395.
8) J. L. Swain: Circulation 80 (1989) 1495.
9) H. Lin, M. S. Parmacek, G. Morle, S. Bolling, and J. M. Leiden:
Circulation 82 (1990) 2217.
10) E. G. Nabel, G. Plautz, and G. J. Nabel: Science 249 (1990) 1285.
11) E. F. Fynan, R. G. Webster, D. H. Fuller, J. R. Haynes, J. C. Santoro,
and H. L. Robinson: Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 11478.
12) V. Menezes, K. Takayama, T. Ohki, and J. Gopalan: Appl. Phys. Lett.
87 (2005) 163504.
13) S. Ohta, S. Mita, T. Hattori, and K. Nakamura: Plant Cell Physiol. 31
(1990) 805.
14) T. Murashige and F. Skoog: Physiol. Plant. 15 (1962) 473.
15) J. R. Kikkert: Plant Cell Tissue Organ Cult. 33 (1993) 221.

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