HUMAN GENE THERAPY 18:1233–1243 (December 2007)© Mary Ann Liebert, Inc.DOI: 10.1089/hum.2007.063

Hydrodynamics- and Ultrasound-Based Transfection of Heart with Naked Plasmid DNA

LEONARDO PINTO DE CARVALHO,1,2 DANIELA TAKESHITA,1 BRUNO A. CARILLO,3

BIANCA CRISTINA GARCIA LISBOA,1 GUSTAVO MOLINA,1 ABRAM BEUTEL,3

EDUARDO GALLATTI YASUMURA,1 CHRISTINA MAEDA TAKIYA,4 VALDEREZ BASTOS VALERO,1

RUY RIBEIRO DE CAMPOS, JR.,3 HANS FERNANDO DOHMANN,2 and SANG WON HAN1,5

ABSTRACT

A novel, efficient transfection method, based on ultrasound and hydrodynamics, has been developed to trans-fect heart tissue with plasmid DNA. An ultrasound probe was aimed at the heart of anesthetized rats for 30sec, at an intensity of 1 MHz and 2 W/cm2. The aorta was clamped and a phosphate-buffered saline (PBS)solution containing pSV-LacZ was quickly injected into the left ventricle. Each animal was maintained in thiscondition for 20 sec, and then the clamp was opened and the needle was removed. Electrocardiography, per-formed after 4 weeks, showed mild or no sign of ischemia in all groups. Visual evaluation of heart tissue sam-ples from rats that received 100 �g of pSV-LacZ in 100 �l had only a few blue cells, indicating transfection,and those that received only PBS had no blue cells. However, all heart tissue samples from rats transfectedwith 100 to 500 �g of pSV-LacZ in 200 �l, or with 200 to 500 �g of pSV-LacZ in 100 �l, had many blue cells.The base and epicardium of the heart tissue samples had many more blue cells than did the rest of the sam-ples. Histological results, based on staining with hematoxylin and eosin, showed similar results between con-trol and transfected groups. Therefore, we concluded that gene delivery by plasmid vector in association withultrasound and hydrodynamics was highly effective in transfecting rat heart.

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INTRODUCTION

CARDIOVASCULAR DISEASES have high socioeconomic impactworldwide and affect 16.7 million people each year (World

Health Organization, 2003). Cardiovascular surgeries and newdrugs are available, but there are limitations to their use re-sulting from individual variation in effectiveness and side ef-fects, and their high cost. Therefore, alternative therapies arenecessary to deal with untreatable diseases and to improve theefficacy of current therapies.

Use of genes for the treatment of cardiovascular diseases hashigh therapeutic potential, and in some cases, such as cardio-vascular congenital abnormalities, gene therapy might be theonly option. Successful gene therapy depends mainly on the ef-fectiveness of gene transfer to heart cells, and adenoviral andplasmid vectors have typically been used for this purpose

(Muller et al., 2007). Adenoviral vectors transduce heart cellsin vivo efficiently and they can be introduced directly into themyocardium, or by intracoronary injection (Hammond andMcKirnan, 2001). However, these vectors are highly immuno-genic and the process of manipulation and production is morecomplex and expensive than with plasmid vectors. In the caseof a virus leak from the local injection, cells of other tissuescan easily be infected, provoking undesired effects or evendeath (Somia and Verma, 2000; Hammond and McKirnan,2001). Naked plasmid DNA is the safest vector because it issimply a neutral and nontoxic molecule and much less im-munogenic than the adenoviral vector (Bessis et al., 2004).Therefore, naked plasmid DNA can be administered repeatedlyif the therapeutic gene is not immunogenic. In addition, nakedDNA is easier to handle, produce, and purify than is adenovi-ral vector. Cells transfected with plasmid express the exoge-

1Interdisciplinary Center for Gene Therapy (CINTERGEN), Federal University of São Paulo, São Paulo-SP, 04044-010, Brazil.2PROCEP, ProCardiaco Hospital, Rio de Janeiro-RJ, 22280-000, Brazil.3Department of Physiology, Federal University of São Paulo, São Paulo-SP, 04023-062, Brazil.4Department of Histopathology, Federal University of Rio de Janeiro, Rio de Janeiro-RJ, 21949-900, Brazil.5Department of Biophysics, Federal University of São Paulo, São Paulo-SP, 04023-062, Brazil.

nous gene transiently, which could be another advantage whenexpressing vascular growth factor genes to avoid the growth oftumor cells.

In vivo transfection with naked plasmid DNA was firstly de-scribed by Wolff et al. (1990). Since that time a large numberof studies have been carried out with plasmid DNA to transfectdifferent types of cells in vivo. Basically, skeletal and cardiacmuscle cells and hepatocytes are well transfected with plasmidby local injection (Herweijer and Wolff, 2003). Use of electricshock (electroporation) and ultrasound (sonoporation) has im-proved significantly the rate of transfection of those cells withplasmid. Intravascular delivery of naked DNA using increasedpressure has also demonstrated to improve significantly the rateof transfection in vivo (Zhang et al., 2004).

Although there are many reports of the use of naked DNA,alone or associated with the above-mentioned physical meth-ods or with chemical compounds, as a vector for transfectionof many cell types in vivo, heart transfection has been performedin animals and in humans exclusively by local injection of DNA(Losordo et al., 1998; Symes et al., 1999; Tio et al., 1999; Aokiet al., 2000; Lee et al., 2000; Schwarz et al., 2000; Vale et al.,2000), because the administration of plasmid by intracoronaryinjection leads to a low rate of transfection of heart cells (Symes,2001; Isner, 2002). Local injection of DNA solution limits thetransfection to only a limited number of local cells near the in-jected area and, consequently, the therapeutic effect is limitedto certain local cells or to their proximity. To gain the maximaltherapeutic effect of gene therapy in cardiovascular disease, itis necessary to transfer genes to a large number of heart cellsefficiently.

With the intent to efficiently transfect heart cells with nakedplasmid DNA in vivo, we developed a new method based on acombination of hydrodynamics (Hagstrom et al., 2004) and ul-trasound (Newman and Bettinger, 2007) methods to deliver vec-tor to cells in the whole heart; that method is described and dis-cussed here.

MATERIALS AND METHODS

Vector preparation

Vector pSV-LacZ expresses the bacterial �-galactosidasegene under the control of the simian virus 40 (SV40) promoter.Large-scale preparation of this plasmid was carried out with aplasmid giga kit (Qiagen, São Paulo, Brazil), in accordance withthe manufacturer’s instructions.

Transfection of heart in vivo

All rats used for experiments were from the animal facilityof the Federal University of São Paulo (São Paulo, Brazil). Allexperiments were carried out with due protocol, in accordancewith recommendations for the proper care and use of labora-tory animals promulgated by the Ethics Committee of the Fed-eral University of São Paulo.

Male 8-week-old Wistar rats were anesthetized with keta-mine plus xylazine (40 and 20 mg/kg, respectively) and main-tained with a breathing apparatus (Ugo Basile, Comerio, Italy).In the left parasternal area an ultrasound (US) probe (Sonacel

III; Bioset, São Paulo, Brazil) was positioned toward the heartfor 30 sec with an intensity of 1 MHz and power of 2 W/cm2.Soon after, an incision was made in the fourth intercostal spaceto expose the heart. The aorta was clamped and a phosphate-buffered saline (PBS) solution containing pSV-LacZ was in-jected into the left ventricle over 3 sec with a 1-ml insulin sy-ringe. Each animal was maintained in this condition for 20 sec,after which the clamp was opened, the needle was removed,and finally the incision was closed. After 48 hr, one group ofanimals was killed and the hearts were removed and sliced intofour parts for staining with 5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside (X-Gal) and histological analysis. Anothergroup was used for evaluation of heart function by electrocar-diography.

Electrocardiogram

Peritoneal anesthesia was performed with ketamine plus xy-lazine (40 and 20 mg/kg, respectively). The animals were thenarranged in the dorsal decubitus position and probes were im-planted in each limb. Electrocardiography was performed(model EKG-5; FUNBEC, São Paulo, Brazil) before and 1 weekand 1 month after transfection, using all bipolar leads and twounipolar leads (V1 and V6).

Creatine phosphokinase fraction MB determination

Blood samples were collected from the tail vein of rats be-fore and 24 hr after transfection to determine the MB fractionof creatine phosphokinase (CPK-MB), using the automated Vit-ros 5,1 FS system (Johnson & Johnson, New Brunswick, NJ).

X-Gal staining

Forty-eight hours after transfection, the animals werekilled and the hearts were removed. Each heart was slicedinto four pieces and immediately fixed with 4% formalde-hyde (Formol) for 1 hr at 4°C. The sliced hearts were washedwith rinse buffer (100 mM NaH2PO4 [pH 7.3], 2 mM MgCl2,0.01% sodium deoxycholate, 0.02% Nonidet P-40 [NP-40])three times for 20 min and were incubated in X-Gal solution[5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, X-Gal(1 mg/ml) in dimethyl sulfoxide (DMSO)] at 37°C. After 12hr, the slices were washed with PBS, which was also used topreserve the stained slices.

Blood pressure

The rats were initially anesthetized with ketamine and xy-lazine (40 and 20 mg/kg respectively); aortic pressure and leftventricular pressure (LVP) were then measured with a PE-50catheter inserted via the right carotid artery and connected to apressure transducer and analog–digital converter system (Pow-erLab; ADInstruments, Bella Vista, Australia) (Tanaka et al.,2005).

For the measurement of intraventricular pressure, the heartwas exteriorized as described above and a 27-gauge needlelinked to the pressure transducer by a three-way valve was in-serted through the myocardium in the left ventricle cavity. In-jection of DNA (100 and 200 �l) was performed through aninsulin syringe connected to the three-way valve.

CARVALHO ET AL.1234

Histopathology

Rat hearts were sliced into four pieces, washed with salinesolution to remove blood, fixed in 37% Formol, dehydrated,and embedded in paraffin. Sections (thickness, 6 �m) were ob-tained and stained with hematoxylin and eosin (H&E) for anal-ysis.

RESULTS AND DISCUSSION

The strategy of our heart transfection method with nakedDNA is based on the simultaneous use of two principles: hy-drodynamics and sonoporation. Transfection consisted of USinsonation, exteriorization of the heart, clamping of the aorta,injection of DNA into the left ventricle, opening of the clamp,internalization of the heart, and suturing of the incision (Fig.1). The whole procedure is relatively simple and can be carriedout within 2 min, but some steps are critical: exteriorization andinternalization of the heart, and conditions for US insonation.While handling the heart to pass it through the intercostalspaces, lesions can be caused in the epicardium that conse-quently can affect the results, or even kill the animal. Goodtraining is necessary to minimize the danger of epicardial le-sions. The determination of US insonation conditions, such aspower, frequency, and time of insonation, is also important. Weused 2 W/cm2 of 1-MHz radiation for 30 sec and no animalsdied, but if the insonation time was doubled, the postirradiation

mortality rate among the rats increased (data not shown). Ahigher intensity of US (higher power and extended exposure)is dangerous because it increases local temperature significantlyand can provoke tissue damage. In addition, it also leads to lesseffective transfection (Chen et al., 2003).

To determine the effect of US and hydrodynamics on trans-fection, 100 to 200 �l of DNA solution was injected, soon af-ter insonation, into the exposed heart and maintained for 20 sec.The aorta was kept clamped during this step to keep the in-jected DNA in the heart and also to increase local pressure. Un-der these conditions, the difference between the pSV-LacZ-transfected and control groups is clear: the hearts from allpSV-LacZ-transfected groups were stained blue, but controlgroup hearts had no blue cells (Fig. 2). Three control groupswere included in this experiment: in two groups saline or pSV(without the lacZ gene) was injected and treated with US, andin the other control group pSV-LacZ was injected without US.These data apparently indicate that the transfection of cardiaccells occurred mainly because of US insonation. On comparingthe groups transfected with pSV-LacZ and treated with US,there is a clear difference between the groups transfected with100 and 200 �l: when doubling the volume of DNA from 100to 200 �l the area of transfected cells increased significantly.These results indicate that preparation of the heart for trans-fection with US is the first essential step. Once the heart is prepared, a large injection volume appears to improve the trans-fection rate, as happened with hydrodynamics-based transfec-tion, because it caused an increase in heart pressure (Fig. 3).

HEART TRANSFECTION WITH NAKED DNA 1235

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FIG. 1. (A) Overview of heart transfection method. U.S., ultrasound. For steps I–IV, see (B), which provides pictures of eachstep. (I) US insonation; (II) clamping of the aorta after exteriorization of the heart; (III) injection of DNA into the left ventricle;and (IV) suturing of the incision.

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The possible effects of US and mechanisms of transfection arediscussed below.

In the next step, the influence of DNA mass and volume wereevaluated. Concentrations of DNA equal to or higher than 200�g in 200 �l yielded similar results (Fig. 2A). One hundred mi-crograms in 200 �l still worked well, but when the volume wasreduced to 100 �l the rate of transfection decreased drastically.This result is important, as it indicates that the minimal volumenecessary for transfection is 100 �l. However, in this case the

transfection rate could be increased if a higher quantity of DNAwas used. The maximal concentration of DNA tested was 500�g in 200 �l and it produced a slightly higher number of trans-fected cells compared with 200 �g of DNA in 200 �l. There-fore, under our experimental conditions we believe that 200 �gof DNA in 200 �l produces close to the maximal transfectionefficiency.

Distribution of transfected cells in the heart was similar inthe various groups, but the base and epicardium of the hearts

CARVALHO ET AL.1236

FIG. 2A.

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were more extensively transfected than were the endocardiumand myocardium (Fig. 2A). Initial intraventricular systolic pres-sures were 110 and 70 mmHg for 200- and 100-�l DNA in-jections, respectively, but these values dropped quickly by about50% within 20 sec. Meanwhile, during all procedures diastolicpressures were maintained between 10 and 20 mmHg in bothcases (Fig. 3F). This means that soon after DNA injection, thesolution circulated quickly and was distributed to the main ar-teries around the heart. However, we interpret the rapid decreasein pressure as indicating that after initial good perfusion the rateof blood flow dropped rapidly and was not enough to reach all

arterioles and capillaries, which are located mainly in the my-ocardium and endocardium. In addition to this, with the dias-tolic pressure increased from 0 to 10–20 mmHg, it is under-standable that the tension of the myocardium may be elevatedand, consequently, small vessels may be compressed. There-fore, it is reasonable to think that the reduced flow and increaseddiastolic pressure could be responsible for the low rate of trans-fection in the myocardium and endocardium.

A segment of septal and lateral wall showed an increase inthe number of transfected cells in some animals (Fig. 2A). Con-sidering that the rat heart rate is about 80 beats/min during trans-

HEART TRANSFECTION WITH NAKED DNA 1237

FIG. 2. Staining of hearts with X-Gal 48 hr after transfection: (A) pSV-LacZ group; (B) negative controlgroups. Hearts were sliced into fourpieces and positioned as indicated. n,number of animals.

B

fection and the diameter of the rat heart is 1 cm, the injectionof DNA into the left ventricle cavity is not an easy task. Tomake sure that the needle was in the left ventricle, we pulledout the syringe piston and tested for blood before DNA injec-tion. Even so, the needle position can change during the 3 secof injection as a result of heart movement while beating, andconsequently injection into a wall or septum was possible.

It is important to note that in our transfection procedure theUS was applied to animals before the injection of DNA. Thisfact apparently contradicts the principle of sonoporation, whichis based on the entrance of DNA into cells during US insonationby the temporary opening of membrane pores; for this reason,DNA is typically added to the medium before sonoporation

(Brayman et al., 1999; Taniyama et al., 2002; Duvshani-Eshetet al., 2006). In our experiments, there was a time lapse of 50to 60 sec between US insonation and DNA injection. This is along time for the effect of sonoporation to be maintained. Inaddition, if the pores were kept open for longer than a few sec-onds, those cells should experience osmotic shock. It is knownthat insonation by US produces free radicals such as nitric ox-ide, which is a potent vasodilator and its action is very fast(Feril and Kondo, 2006; Milowska and Gabryelak, 2007).

Nitric oxide is also quickly produced under hypoxic condi-tions, which was the situation in our transfection experiment.Transfection by hydrodynamics in vessels occurs due to vesseldilation, which is provoked by high volume pressure. Under our

CARVALHO ET AL.1238

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FIG. 3. Variation of heart pressure over time. (A) Aortic pressure before transfection. (B) Intraventricular pressure before trans-fection. (C) Intraventricular pressure 1 month after transfection. (D and E) Intraventricular pressure recorded during various partsof the procedure [(D) DNA volume injected, 100 �l; (E) DNA volume injected, 200 �l]: (I) interference during exposure ofheart; (II) intraventricular pressure during heart manipulation for clamping; (III) intraventricular pressure during clamping of ex-posed heart; (IV) interference during injection; (V) intraventricular pressure of exteriorized heart after injection. (F) Maximal in-traventricular pressure of exteriorized heart after injection. Values represent means � SD (n � 3). Each horizontal bar in (A–C)represents 0.5 sec.

conditions, injection of 200 �l of DNA led to systolic and di-astolic pressures of 110 � 14 and 12 � 1 mmHg (Fig. 3F), re-spectively, and therefore there was no augmentation of bloodpressure. However, as the vessels were already dilated by USand hypoxia, we believe that transfection was facilitated tem-porarily. Favorable conditions created for transfection by USinsonation and ischemia lasted for several minutes, becausewhen injecting DNA even 5 min after US insonation a signif-icant number of blue-stained cells could still be observed (datanot shown). Duvshani-Eshet and collaborators (2006) showedthat alterations of the surface of the cell membrane occurred af-ter US insonation, and they also reported that this effect lastedfor several hours. If plasmid DNA was added to the mediumduring this period, a good level of transfection was observed,as we have also observed in our experiments with rats. To un-derstand this result, the authors suggested that US should in-duce local shear forces and/or acoustic microstreaming ratherthan cavitation.

Cardiac function of animals subjected to transfection wasmonitored by electrocardiography and CPK-MB concentration.CPK-MB is one of the main indicators of infarction, and it isreleased into the circulation soon after such an event. Twenty-four hours after infarction, the CPK-MB concentration in theblood reaches a maximum and this value extends through 48hr and then returns to its basal value (Wagner et al., 1973).Therefore, the concentration of CPK-MB is an excellent pa-rameter by which to monitor acute heart lesion. In the group ofanimals subjected to transfection, the CPK-MB value rose sig-nificantly after 24 hr and lasted for 4 days, in comparison withthe control group; however, these values returned to basal levelin 10 days (Fig. 4). These results indicate only a transient le-sion of the heart in some animals. Acute heart lesion after trans-fection was expected as a result of heart manipulation for injection of plasmid DNA. This was confirmed by electrocar-diography of animals 1 week and 1 month after transfection,

with results showing no sign of significant heart ischemia; inaddition, the heart axes did not undergo significant alteration(Fig. 5). Furthermore, histological analysis of hearts by H&Estaining 1 month after transfection also showed no visible al-teration (Fig. 6). Variation of heart rate had been observedamong different groups and also in the same animals duringdifferent steps of the procedure. It is likely that xylazine (brady-cardia agent) and ketamine, which were used to induce anal-gesia and anesthesia of animals during handling, were respon-sible for these alterations in heart rate (Hsu et al., 1986, 1987).For this reason, we did not consider heart rate a parameter forevaluation.

Exposure of the heart and temporal blockage of heart bloodcirculation for transfection appears to be an aggressive and in-vasive procedure, especially when considering humans as thetarget of gene therapy. However, this practice is common incardiovascular surgeries, during which the heart is exposed formanipulation and the heart blood circulation can be stoppedtemporally.

In most cases of sonoporation, microbubbles such as Op-tison (GE Healthcare, Piscataway, NJ), PESDA (perfluoro-carbon-exposed sonicated dextrose albumin), or BR14(Bracco, Geneva, Switzerland) are used to increase the trans-fection effect (Pislaru et al., 2003). Ultrasound provokes anexplosion of microbubbles, which forces transference ofDNA into the cells (Ay et al., 2001; Rahim et al., 2006). Thistype of transfection, used to bring about a higher level oftransfection than with naked DNA, causes rupture of mi-crovessels and, in some cases, embolisms in vital organs(Skyba et al., 1998; Miller and Quddus, 2000). In the case ofheart transfection with microbubbles mixed with DNA, whichwere treated by US after injection of the mixture into theheart, apparently only a small number of cells were trans-fected (Kondo et al., 2004). However, as the experimentalconditions were different, we cannot compare those results

HEART TRANSFECTION WITH NAKED DNA 1239

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FIG. 4. Concentration of CPK-MB was determined in rats under the following conditions: (A) nontreated; (B) exteriorized butnot transfected; (C) 48 hr after transfection; (D) 4 days after transfection; (E) 10 days after transfection. Five rats were used ineach group. Dashed line, normal mean CPK-MB value in rats was 1020 U/L.

directly with ours, but apparently our method produced moretransfected cells without microbubbles.

In summary, gene delivery by plasmid vector in associa-tion with ultrasound and hydrodynamics was highly effectivein transfecting rat heart cells, without affecting the heart func-tions. The concept of sonoporation should be reanalyzed, be-cause US insonation can be done well before the injection ofnaked DNA.

ACKNOWLEDGMENTS

The authors acknowledge Bioset Company for providingultrasound equipment. The authors thank Sophie Thatcherfor assistance with English. This work was supported byFAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo: Processo 06/59630-0) and UNIFESP-PROCEPprogram.

CARVALHO ET AL.1240

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FIG. 5. (A and B) Variation of electrical axes was determined by electrocardiography before and 1 week and 1 month aftertransfection in (A) five rats subjected to transfection and in (B) five negative control group rats. (C and D) ECG of a represen-tative rat before (C) and 1 month after (D) gene transfer.

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HEART TRANSFECTION WITH NAKED DNA 1241

FIG. 6. Microscopic analysis of rats subjected to transfection (A) and sham operated (B). Anterior wall (AW), posterior wall(PW), and septal (SEP) and lateral (LAT) segments were stained with H&E. There were no visible alterations between the twogroups. Scale bars: 100 �m.

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AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

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Address reprint requests to:Dr. Sang Won Han

CINTERGEN - UNIFESPRua Mirassol 207

São Paulo-SP, Brazil 04044-010

E-mail: sang@biofis.epm.br

Received for publication May 31, 2007; accepted after revisionOctober 8, 2007.

Published online: November 20, 2007.

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