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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: viren@aero.iitb.ac.in

Japanese Journal of Applied Physics

Vol. 47, No. 3, 2008, pp. 15221526

#2008 The Japan Society of Applied Physics

1522

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

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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.

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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.

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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 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

1mm in length. The vertical lines indicate the depth of blue spots from

the edge of the sections.

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