1.

Introduction

Regeneration in the peripheral nerve is poor after severe or chronic denervation. Promoting facial nerve regeneration is an important area of study because facial paralysis markedly decreases the quality of life. Previous reports demonstrated that topically applied growth factors, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and basic fibroblast growth factor (bFGF), enhance facial nerve regeneration.^{1, 2, 3, 4} Secretion of such proteins, upregulated by gene transfection, may be a promising treatment.

Viral and nonviral vectors are two major categories of gene delivery vehicles that are currently being applied for gene therapy. The viral delivery system has been shown to be efficient for gene transduction in muscle. Adeno-associated viral vectors, for example, can transduce muscle with high efficiency. However, therapeutic application in human gene therapy would require the resolution of serious problems such as mutagenesis and unexpected immune responses.⁵

For nonviral gene delivery, physical methods, such as electroporation^{6, 7} and ultrasound,⁸ have been investigated. They are relatively safe and easy to apply, but have low transfer efficiencies and can cause tissue damage. Optical transfection, especially laser-mediated gene transfection has received much attention as a new method for target gene transfection because of the high spatial controllability of laser energy. Most reports on laser-mediated gene transfer have mainly dealt with single-cell-basis transfection by direct irradiation of the cell membrane with a focused laser beam; this approach cannot be applied to deep tissue because of limited optical penetration. However, we recently demonstrated that genes can be transferred both in vitro and in vivo by the use of laser-induced stress waves (LISWs) or photomechanical waves.^{8, 9, 10, 11, 12}

Delivery of drug molecules by LISWs was developed by Lee and Doukas¹³ and Doukas and Flotte.¹⁴ We have extended the application of LISW to the delivery of genes. Using this method, a large number of cells can be transfected simultaneously. It also enables the treatment of deeper tissue than the methods based on direct laser irradiation, because stress waves can be propagated much more efficiently in tissue than can laser light. Using LISWs, we have previously transfected various types of cell lines with plasmid deoxyribose nucleic acid (DNA) for the expression vector of enhanced green fluorescent protein (EGFP).¹¹ We have also delivered luciferase, EGFP, and Lac-Z genes to rat skin,¹² and EGFP genes to the mouse central nerve system in vivo.¹⁰

Polyethylenimene (PEI) is a synthetic cationic vector and has been shown to be an efficient and versatile gene transfer tool. The plasmid DNA complexes formulated with 22 kDa PEI in 5% glucose are sufficiently small and stable in physiological fluid to provide high diffusibility and enhance gene transfection.^{15, 16}

In this study, we investigated the feasibility of gene transfer using LISWs and the DNA/PEI complex. We demonstrated that efficient, site-specific, and minimally invasive gene transfer can be achieved by applying this novel technique based on LISWs.

2.

Materials and Methods

2.2.

Laser-Induced Stress Waves

Figure 1a shows the experimental arrangement. A black natural rubber disk with a thickness of 0.5 mm and a diameter of 12 mm was used as the laser target. A 15-mm square transparent polyethylene terephthalate sheet with a thickness of 1 mm was bonded to the rubber disk surface to confine the laser-induced plasma. Plasma confinement increases the peak pressure and pulse width of the generated stress waves.^{9, 11, 12} LISWs were generated by irradiating a target with 6-ns pulsed laser light from the second harmonics (532 nm) of a Q-switched Nd:YAG laser (Brilliant b; Japan Laser, Tokyo, Japan); the laser beam was focused with an uncoated planoconvex quartz lens (f = 200 mm). The spot diameter on the target can be changed but was kept constant at 6 mm in the present study [Fig. 1b].

Fig. 1

Depictions of gene transfer using LISWs.

3.

Results

Figure 2 shows the sections of the whole facial tissue seven days after gene transfection using LISWs. Lac-Z expression was observed only in the tissue exposed to LISWs. The muscles existing several millimeters deep in the skin were especially well stained (arrowheads). Figure 3a shows the hematoxylin-eosin-stained tissue section taken from the tissue exposed to LISWs without DNA injection. Almost no detectable damage and inflammatory infiltrating cells were observed in the muscles seven days after application of LISWs. Figure 3b shows the facial muscles seven days after DNA transfection by LISWs. Intensive Lac-Z expression was observed in myofibers (arrowheads). No obvious damage was observed, but some myofibers seemed to be slightly atrophic.

Fig. 2

Whole facial tissue obtained at seven days after gene transfer with LISWs. Scale bar: 2 mm.

Fig. 3

(a) Hematoxylin-eosin staining of muscle fibers at seven days after exposure of LISWs without injection of DNA. Scale bar: 10 μm. (b) X-gal staining of muscle fibers at seven days after gene transfection using LISWs. Scale bar: 10 μm.

Figure 4 shows the difference in intensity of Lac-Z expression at seven days after gene transfer under four experimental conditions. Detectable expression of Lac-Z was observed neither in the muscle layer of control tissue (group a) nor in the muscle layer only with pDNA injection (group b). On the other hand, apparent gene expression was observed in both cases of LISW application (groups d and e). In high-power fields, strong Lac-Z expression was observed in the deep muscle layer exposed to LISWs. When the DNA-PEI complex was used (if both LISWs and DNA-PEI complex were used), gene expression seemed to be slightly stronger than that with pDNA alone. Figure 5 shows the count of Lac-Z-positive cells in the tissue of groups b–e at seven days after gene transfection. A highly significant increase in the number of Lac-Z-positive cells was observed in both cases of LISW application (groups d and e) (both p < 0.01). A significant increase was also observed in case of DNA-PEI complex alone (group c) (P < 0.05). There was significant difference between group c and groups d and e (p < 0.05, P < 0.01, respectively), but there was no significant difference between groups d and e, regardless of use of Jet-PEI.

Fig. 4

Comparison of the intensities of Lac-Z expression under the four conditions at seven days after gene transfer. Scale bar: 200 μm.

Fig. 5

Comparison of the transfection efficiencies evaluated by counting of Lac-Z-positive cells at seven days after gene transfer. Statistical analysis was performed using the Mann-Whitney U-test. The error bars represent standard error (*p < 0.05, **p < 0.01).

Figure 6 shows the time course of expression in cases of using LISWs (groups d and e). The peak expression was observed at seven days after gene transfer, and thereafter, expression tended to decrease. However, gene expression was sustained for at least 14 days after gene transfer.

Fig. 6

Time course of the Lac-Z-positive cells in two conditions, using LISWs and a combination of LISWs and the DNA/PEI complex. Statistical analysis was performed using the Mann-Whitney U-test. The error bars represent standard error. (n.s. p > 0.05).

4.

Discussion

We previously showed that plasmid DNA could be transfected into skin and central nervous system (CNS) cells in vivo by use of LISWs.^{10, 12} Moreover, we delivered a therapeutic vector construct carrying the hHGF gene into a rat skin graft for autografting and observed enhanced angiogenesis.⁸ We believe this to be the first experimental demonstration of LISW-mediated gene transfection showing therapeutic efficacy.

In the present study, we demonstrated the applicability of LISW-mediated gene transfer into rat facial muscles as a basic experiment to test facial nerve regeneration. Muscle is an ideal target for gene therapy because it is easily accessible and able to induce the local and systemic secretion of therapeutic proteins.^{5, 6, 7} We proved that LISWs enabled efficient DNA delivery into facial muscles existing several millimeters deep from the face surface under the laser spot on the target. No skin incision or exposure of muscles was required. Moreover, only limited damage and inflammatory change were caused by LISW application in the target muscle and its surrounding tissue. To be precise, some muscle fibers showed slight atrophic change. Several studies have suggested that toxicity could be caused by the intracellular presence and expression of DNA themselves.^{6, 17, 18} Thus, some tissue alteration might be inevitable in gene transfer using plasmid DNA.

In the condition using LISWs with the DNA-PEI complex, the intensity of Lac-Z expression was slightly stronger than in experiments using only LISWs. The results showed some transduction efficiency of PEI, but a significantly small number of βGal positive cells are observed compared to that of LISWs. PEI could possibly have an additional effect on gene transfer using LISWs, but sufficient gene expression can be achieved by using LISWs alone. The most obvious Lac-Z expression was observed at seven days after gene transfection, and the expression tended to decrease with elapsed time. This time-dependent decrease in gene expression indicates that the transgene was hardly inserted into chromosomal DNA, suggesting a lower risk of adverse effects such as tumorigenesis due to insertion mutagenesis or chromosomal aberrations compared to the viral methods. These results suggest that this method might be suitable to prevent muscle atrophy and to promote nerve regeneration during at least the initial two weeks.

According to a previous report on gene transfer by electroporation, transient gene expression is observed mainly by transfection of mature muscle fibers. When satellite cells that are involved in the growth and regeneration of muscle are activated, and DNA is taken into these cells, stable gene expression was shown to last for several months up to a year.⁸ If we apply LISWs to a degeneration muscle model, DNA will also be transfected into proliferated satellite cells and longer-term gene expression will be observed in regenerated myofibers.

Topically applied growth factors are known to enhance peripheral nerve regeneration. Watabe demonstrated that adenoviral gene transfer of GDNF, BDNF, TGFβ2, and HGF into the facial canal prevented death of injured motoneurons after facial nerve avulsion.² Kohmura showed that locally applied BDNF aterocollagen minipellets accelerated facial nerve regeneration in the rat model of facial nerve transection and immediate repair with suture.¹ Araki proved that in a rat crush model for the recurrent nerve, a cranial motor-nerve-like facial nerve, treatment with topical injection of an adenovirus vector of GDNF led to significantly improved motor nerve conduction velocity and vocal fold movement at two and fout weeks after treatment and a significantly larger axonal diameter and improved remyelination compared to controls.¹⁹ By use of LISWs, therapeutic genes can be more safely transfected into muscles and can impart neurotrophic effects on cases of acute nerve degeneration such as those that occur in facial injury and paralysis.

The mechanism of gene transfer using LISWs is not clear, but there are two possibilities: acceleration of exogenous genes and/or transient disruption of the plasma membrane by sheer force. We consider that exogenous genes are accelerated by large impulse pressure and enter the cells through lipid bilayers. Sheer force induced by LISWs may also cause reversible disruption of the cell membrane, through which exogenous genes are diffused into the cells due to the concentration gradient.^{9, 11} The mechanisms of LISWs appear to be different from those of conventional ultrasound; waveforms of LISWs were dominated by positive pressure components, unlike those of ultrasound. Increased permeabilization of the plasma membrane due to interaction with ultrasound is mediated mostly by cavitation. A contrast agent and a large number of shots are required to achieve efficient gene transfection based on the cavitation effect.^{18, 20} With the present method, on the other hand, efficient gene transfer can be achieved with only a few pulses, without the need for any contrast medium or long time of operation.

The most notable advantage of our method compared to other physical methods is its high spatial controllability. Genes can be delivered to a pinpointed target tissue by applying finely focused laser light, while large-area treatment is also possible by using expanded or scanned laser light. Additionally, as laser light can be delivered through fiber optics, catheter-based gene transfer into deep and inaccessible target organs may be possible. With electroporation, it would be difficult to realize such highly controllable targeted gene delivery because it is difficult to control the path of electric current in tissue. Furthermore, electroporation easily causes tissue damage, making it unsuitable for large-area treatment.

Another advantage of LISW-based gene transfer is its minimal invasiveness and easy applicability. Q-switched Nd:YAG lasers are widely used in head and neck surgery and proven to be safe. They are commercially available, cost effective, and very easy to operate and maintain. Recently, gene transfer by the use of a titunium-sapphire femtosecond laser has been reported.²¹ DNA-injected muscle in mice was directly irradiated with focused laser pulses in vivo, and efficient gene expression was observed in the irradiated tissue. However, Q-switched Nd:YAG lasers are cheaper, more compact, and easier to control when compared to femtosecond lasers. Therefore, this LISW-mediated method seems to have fewer problems that need to be solved before clinical use than other nonviral gene delivery method.

5.

Conclusions

In this study, we successfully demonstrated gene transfer into the facial muscle by applying LISWs. This technique enables site-specific, safe, and convenient gene transfer. We plan to study the therapeutic effect of facial nerve regeneration based on this technique.