BACKGROUND OF THE INVENTION Administration of substances such as drugs, nutrients, vaccines and metabolites into tissues or via membranes, which may be biological or artificial (such as in implants), is often faced with difficulties due to the barrier healthy tissues and biological or artificial membranes present against undesired penetration of foreign components.

Various techniques have been developed for the facilitation of transport of substances across tissues. Examples of such techniques are: building up concentration gradients of the compounds to be administered; iontophoresis utilizing an electromagnetic field carried out both to increase the driving force of the administered substance and to cause formation of pores in the tissue or membranes; and utilization of ultrasound.

Ultrasound is defined as a sound having a frequency greater than 20 kHz and is used in a plurality of medical and diagnostic procedures such as imaging of internal organs, sterilization, degassation, superficial eye-lens-epithelium surgery, bile-stone perforation and anti-cancer treatment.

Ultrasound is also used for facilitation of transport of various compounds across tissues, typically skin (Mitragotri, M., et al., Science, 269:850-853 (1995)).

U.S. Patent 5,076,208 discloses a method for the delivery of molecules, the example being gonadotropin-releasing hormone analogue (GnRHa), to aquatic animals in an aquatic medium. The molecule to be administered is added to the medium and ultrasound is then applied to enhance the effect of the uptake of the compound by the animal. The ultrasound is usually applied at a single frequency for a relatively long period of time, typically 10-15 minutes at an intensity of 1.7 W/cm2.

The ultrasonic delivery was improved by using ultrasound in conjunction with chemical permeation enhancer and/or iontophoresis (U.S. Patent 5,231,975). Other methods use ultrasonic waves to exite the local nerves in the way that trauma does, and the nerve excitation opens the epidermal/dermal junction membrane and the capillary endothelial cell joints, which enables the transfer of drugs through the skin and into the blood stream (U.S. Patent No. 5,421,816).

Prior art administration methods utilizing ultrasound are suitable for the administration of substances such as proteins, nucleic acids and drugs having a small size, which are typically dissolved in a liquid medium, i.e. soluble substances.

At times, it is desirable to administer through tissues or biological membranes large complex particles having a size in the range of 1 nm to tens or hundreds of microns, these complex particles are essentially inert in the liquid medium in which they are carried. Examples of such complex particles are dead or attenuated virions, bacteria, fungi or parasites or their fragments administered for the purpose of immunization; plasmids administered to tissues or to cultured cells for the purpose of genetic manipulation nuclei of gametes administered to oocytes for the purpose of

fertilization; particles impregnated with medicaments and capable of releasing them at a slow rate to the surrounding tissue administered for the purpose of therapeutic treatment; particles containing compounds that were coated with a protective coating, for example, in order to change the compounds to prevent oxidation, to prevent a hygroscopic effect, to increase resistance to heat or to protect the contents of the particle from biological effects (such as degradation); and administration, topical or systemic administration of particles, for example, magnetic beads or dye particles for local or systemic therapeutic, cosmetic or research purposes.

State-of-the-art ultrasound- facilitated administration methods are unsuitable to administration of such large complex particles, since applica- tion of ultrasound pulses, sufficient to drive a small-sized molecule through a tissue is insufficient to drive those large complex particles through tissues or biological or artificial membranes. Increase of the duration, frequency or intensity of the ultrasound pulses to levels which are presumably sufficient to drive the large complex particles through the tissue or cell membrane, has not been reported probably since it results in irreversible damage to the tissue and in massive cell-death. Similarly, irreversible damage occurs in non-biological membranes of e.g., polyethylene or elastomer (for example those used in implants), when increased intensities or durations of ultrasound irradiation have been used.

It would have been highly desirable to provide a method for administering substances to tissues, cells or membranes (both natural and artificial) utilizing ultrasound, while minimizing the damage to the tissue or cells. It would have further been desirable to provide an ultrasound facilitated method for administration of complex particles having a relatively large size.

SUMMARY OF THE INVENTION The present invention provides a novel method allowing the introduction of substances into or through cells, tissues or membranes. This, in accordance with the invention, is achieved by utilizing a complex ultrasound stimulus, consisting of a first irritant stimulus and a second driving stimulus. By utilizing these two stimuli, it was discovered that it was possible to introduce to the tissues or cells large complex particles without causing irreversible damage. The method for the administration of substances to cells, tissues or membranes, in accordance with the invention, comprises the following steps: (a) exposing the cells, tissues or membranes to a first irritant ultrasound stimulus, being such as to cause transient formation of openings in said cells, said tissues or said membranes without causing any irreversible damage to the bulk of said cells or to most of the cells of said tissues or without causing irreversible damage to the membranes, the openings being of a size allowing entry therethrough of said substances; and (b) within a time period in which at least a portion of said openings remains open, exposing the cells, tissues or membranes to a second driving ultrasound stimulus, said exposure being carried out in the presence of said substances in a medium which is in contact with said cells, tissues or membranes; said second ultrasound stimulus being effective in driving at least part of said substances through said openings without causing any irreversible damage to the bulk of said cells or most of the cells of said tissues or to the membranes.

The method of the present invention may be used for therapeutic and cosmetic purposes, as well as for diagnostic and experimental purposes, according to the type of substance to be administered.

The substances to be administered may be soluble substances such as various medicaments for therapeutic treatment, macromolecules such

as DNA molecules for the purpose of genetic manipulation, various dyes for the purpose of diagnosis inside cells, or within a tissue and the like.

By a preferred embodiment, the substances to be administered are complex particles. The term "complex particle" refers generally to a particle having the size of at least 1 nm ranging to tens or hundreds of microns which is usually composed of several types of molecules, although at times it may be composed of a single type of molecule. The complex particles are essentially insoluble in the medium in which they are carried. Examples of complex particles are attenuated disease-causing agents or parts thereof such as bacteria, virions, fungi, protozoa or parasites administered for the purpose of vaccination; plasmids containing DNA to be inserted into tissues or cultured cells for the purpose of genetic manipulations; nuclei of gametes administered into oocytes for the purpose of fertilization; particles impreg- nated with medicaments capable of releasing them at a slow rate to the surrounding tissue for the purpose of therapy; particles containing compounds that were coated with a protective coating, for example, in order to form particles having different solubility, to prevent oxidation, to prevent a hygroscopic effect, to increase resistance to heat or to protect the contents of the particle from biological effects (such as degradation); particles comprising a biologically compatible dye for the purpose of tattooing, as for example, in the case of permanent makeup; particles comprising a detectable marker for the purpose of diagnosis, and the like.

The cells to which the substances are administered, can be any type of eukaryotic or prokaryotic cells, typically cells cultured in a medium.

The cells may be obtained from a single-cell organism or cells obtained from multicellular organisms.

The tissue to which the substances are administered, are typically epithelial tissues which may be an artificially moistened keratinized epithelial tissues such as skin, or moist-non-keratinized epithelial tissues, for

example, the epithelium lining the eyes, digestive, respiratory, or reproductive systems. The tissue may also be the moist epithelial tissue covering aquatic animals such as fish, crustaceans or molluscs at different stages of rearing.

The membranes may be either natural membranes or artificial membranes such as polyethylene or elastomer membranes which form a part of an implant.

The term "openings" when used herein in connection with cells refers to pores formed in the membrane of the cells. The term "openings" when used herein in connection with tissue refers to pores formed in the membranes of the cells forming the tissue, pores formed in the basal lamina lining the tissue, or opening of the intercellular junctions of the tissue and/or increase of the intercellular space which may be due to elimination of some cells from the tissue, or vibration at or close to the tissue resonance frequency.

The term "opening" used in connection with membranes (both natural and artificial) refers to gaps i.e. incontinuous spaces in the natural or artificial membranes.

The two stimuli of the ultrasound are applied when the cells, the tissue or the membrane are inside or in contact, as the case may be, with a liquid medium. When the tissue is keratinized skin the liquid medium may be a gel adapted for ultrasound at exposure. The liquid medium may also be water or water with additives needed for the maintenance of particular cells, tissue or membrane.

The substance to be administered, may be present in the liquid medium a priori, i.e. also during the first irritant stimulus, or alternatively may be present only during the time the second stimulus is applied, either by adding the substance to the liquid medium immediately prior to the application of the second driving stimulus, or by transferring the tissue or cells into a medium containing the substance after the first stimulus has been applied. When the two stimuli are applied simultaneously the substances

should be present, a priori in the medium. The substances may be added manually to the medium. Alternatively, it is possible to construct a venturi tube and use the pumping effect created by the ultrasound wave itself, to deliver the substances present in specially constructed reservoirs to the medium, at rates according to the specific requirements.

The specific parameters of the first irritant stimulus, capable of causing a formation of transient openings in the cells or cells in the tissue, and the second driving stimulus capable of driving the administered substances through said openings to the cells or the tissue, should be determined empirically, depending on the exact nature of the cells or tissue and the nature of the administered substance.

In order to determine the parameters of the first irritant stimulus, a first set of samples of the tissue, cells or membranes to be administered should be exposed to a variety of ultrasound pulses varying in intensities, frequencies, pulse modes and durations, and concomitantly or immediately after the application of the stimulus, fixated and examined under electron-microscope. Preferably a corresponding set of stimuli should be given to a second set of samples which are fixated for electron-microscope after several hours from the end of the stimulus in order to determine the level of recovery. Parameters which should be chosen are those which are able to cause formation of openings of a desired size, as determined in the first set of samples, preferably without causing irreversible damage to the bulk of the cells or tissue, as determined by the second set of samples.

The second driving stimulus should be applied while the openings are still open, which is usually within the range of several seconds to several minutes from administration of the first irritant stimulus. The exact time window in which the openings caused by the first stimulus are still open may also be determined empirically by monitoring the number and size of

openings at various time periods after application of the first stimulus.

Alternatively, the two stimuli may be applied simultaneously.

The exact parameters of the second driving stimulus, should also be determined empirically, for example, by using electron dense particles (i.e. tracer) having about the same size as the size of the substance to be administered, and determining which are the exact parameters of the ultrasound required to successfully drive the tracer through the openings formed by the first irritant stimulus, without causing substantial damage to the bulk of cells or tissue. With respect to the fish skin, the total effect which should be considered as not constituting an irreversible damage is an effect which should never exceed necrosis followed by loss of the superficial pavement cells at the irradiated zone, i.e. a skin peeling of about the one-twelfth of skin layers at ultrasound irradiated zones. Acceptable alterations which can be observed one layer deeper are formation of pores in the distal (towards the external part) part of cell membranes. All deeper cells should remain naive, i.e., should show essentially no ultrastructural or physiological alterations. A two-fold enlargement of the intercellular spaces in the 3-4 outermost epithelial layers is also acceptable. Substantial recovery of these phenomena, i.e., that re-epithelization occurred, should be observed within about ten minutes.

Generally speaking, the first irritant stimulus has the following parameters: Frequency: 20 kIIz to 3 Mllz, preferably 100 kHz to 1.5 MHz, most preferably, 1 MHz or less.

Duration: 0.01 sec. to 20 mins. preferably 0.1 sec. to 30 secs. most preferably less than but close to 1 sec.

Intensity: 0.1 - 500 W/cm2, preferably 0.1 - 100 W/cm2, most preferably 3-50 W/cm2.

The parameters of the second driving stimuli are: Frequency: 20 kHz to 50 MHz, preferably 2 MHz to 15 MHz, most preferably, 3-5 MHz.

Duration: 0.01 to 20 mins. preferably 0.1 to 5 mins., most preferably 1-10 secs.

Intensity: 0.1 - 50 W/cm2, preferably 0.1 to 10 W/cm2 most preferably 0.5 to 5 W/cm2.

It should be appreciated that there exists a reversal proportion between the intensity and the duration. Where the intensity is increased, for example, due to use of focusing system such as acoustic lenses, the duration should be decreased. Furthermore, cells present in vitro should be treated with lower intensities and shorter durations than cells present in vivo.

Occasionally, cells under in vitro conditions might have to be adhered, prior to ultrasound application, to a particular sheet to enable controlled irradiation in order to reduce streaming effects on particular cells.

Preferably, the duration and frequency of the second driving stimulus should be longer and higher, respectively, than those of the first irritant stimulus, while the intensity should be lower.

In accordance with a second aspect of the invention, there is provided a method of destroying cells or tissues by irradiation of irradia- tion-activated compounds, which may be activated by light, by ultrasound or by other energy sources. These compounds are administered according to the administration method of the invention. The irradiation-activated compounds are several substances known to be activated by irradiation and to release free radicals. They are therefore used in medicine to cause damage to the surrounding tissues. Several substances are activated by light, in photodynamic therapy, in order to selectively destroy target tissue, typically neoplasmic tissue (Orenstein et al., Br. j Cancer, 73:937-944, (1996)). There have been reports (Miyoshi et al., Radiat. Res., 143:194-202 (1995)) of cancer

treatment based on the combined effect of a photosensitizer and ultrasound which apparently is capable of activating some compounds previously reported as being activated by light.

Prior art methods do not teach, however, the manner in which the irradiation-activated compounds, which may be soluble or particulate reach their target zone by topical application. This can be achieved by the method of the present invention. Once they reach their desired zone, for example, a certain epithelial-layer several layers deep, the activating radiation, which may be light, ultrasound or another source of energy is applied. By this mode, only cells or tissue in the region which the administered compound reached are selectively destroyed, while cells and tissue present outward of this region can remain intact.

By a second mode, the activating irradiation, which again may be light, ultrasound or another source of energy, may be applied simultaneously with the second driving stimulus. This caused the irradiation-activated substances to cause their tissue-destroying affect already during their penetration route so that essentially all the region from the site of administration (the outer layer of the skin) to the region where the substances reached is essentially destroyed.

Examples of irradiation-activated substances which are activated by light are photofrin, pheophorbide, porphyrin, boronated porphyrin, phtalocyanine, hematoporphyrin and chlorin.

Examples of irradiation-activated substances which are activated by ultrasound are dimethylformamide, N-methylformamide, dimethylsulfoxide and gallium porphyrin.

The present invention also concerns a system for use in the above method.

The system generally comprises a multi-frequency signal generator, a signal amplifier, a matching unit and at least one transducer

which may be attached to a focusing system (for example focusing lens) in order to increase the intensity at a desired site. The system may be also attached to any ultrasonic pumping unit attached to the first or second transducer.

In the following the invention will be further illustrated with reference to some non-limiting drawings and examples.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 1 MHz, 1.5 w/cm2 for 5 min.

(x 3,900); Fig. 2 shows electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 1 MHz, 1.5 w/cm2 for 50 sec.

(x 7,500); Fig. 3 shows electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 1 MHz, 3 w/cm2 for 1 sec.

(x 12,450); Figs. 4,5 show electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 3 MHz, 1.7 w/cm2 for 5 min. (Fig. 4 - x 4,800; Fig. 5 - x 3,900); Fig. 6 shows electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 3 MHz, 1.7 w/cm2 for 50 sec.

(x 8,700); Figs. 7,8 show electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 3 MHz, 1.7 w/cm2 for 10 sec. (Fig. 7 - x 18,000; Fig. 8 - x 19,400); Figs. 9,10 shows electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 3 MFIz in the presence of

the tracer lanthanum (arrow) 1.7 w/cm2 for 5 mins. (Fig. 9 - x 19,500, Fig. 10 - x 9,900); Figs. 11,12 and 13 shows electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 1 Nm and the presence of the tracer lanthanum (arrow) in the cells and intercellular spaces 1.7 w/cm2 for 5 mins. (Fig. 11 - x 12,000;, Fig. 12 - x 15,000; Fig. 13 - x 3,000); Fig. 14 shows a higher magnification of part of Fig. 13 (x 19,000); Fig. 15 shows electron microscopy of the epidermis after irradiation of 1 MMz at 2 w/cm2 for 10 sec., followed by irradiation at 3 Nm for 50 sec. in the presence of the tracer (arrows) in the tissue and sampled immediately after irradiation (x 9,000); Figs. 16-18 show the ultrastructure of fish skin treated as indicated in Fig. 15 above, 10 min. after irradiation. Tracer is present in naive cells (Fig. 16 - x 24,000; Fig. 17 - x 24,000; Fig. 18 - x 27,000); Fig. 19 shows a schematic representation of the ultrasound system of the invention; Fig. 20 shows a schematic drawing of acoustic lens used in the system of the invention; and Fig. 21 shows a schematic representation of an ultrasonic pump.

DETAILED DESCRIPTION OF THE INVENTION I. EXPERIMENTAL PROCEDURES A. Ultrasound instruments The therapeutic ultrasound used was a Sonicator 720 (Mettler Electronics, California, USA), with a probe surface area of 5 cm2 and 10 cm2 for two possible frequencies of 1.0 and 3.0 Nm (i 5%), respectively. Power output up to 2.2 w/cm2, pulse mode 20% duty cycle or continuous wave.

B. Application of ultrasound to fish Over 120 specimens of fish (tilapia and carp species) weighing 4-100 gr. were treated with ultrasound in sea water. Ultrasonic irradiation was performed on 4-6 fish concomitantly, when the fish were held in the same container (of about 3,000 ml) and each of the fish was stabilized to his location using a special holder. Ultrasound irradiation was performed from above as demonstrated in the schematic drawing of Fig. 17. Except for one exposure, in which the fish were transferred to another container comprising the tracer, in all other experiments the fish remained in the same container during the two stimuli. The lanthanum tracer was added either immediately before the second irradiation or was present in the medium from the beginning of the first irradiation. The time of introducing the tracer did not significantly affect the results. The experiment was later carried on again.

This time the lanthanum tracer was replaced by bacterins (streptococcus) that was introduced to the water during the second irradiation.

C. Histological preparation for electron microscopy analysis Skin biopsies (3x3 mm and thickness of 0.5 mm) were taken at different times after the ultrasound irradiation. The biopsies were taken from the dorsal part of the head of fish that were lightly anesthetized (MS222) and the biopsies were taken without killing the fish. The tissues were fixed in 3% glutaraldehyde in sodiumcacodylate buffer (0.09M, pH 7.3), washed in the same buffer and post fixed in osmium-tetroxide (1%) in the same buffer.

Ethanol dehydrated tissues were embedded in Agar 100 resin. Thin sections were collected in 300 mesh copper grids. Samples of fish treated without tracer were further contrasted with uranyl acetate and lead citrate. Samples of fish treated in the presence of the tracer were not contrasted so that visualization of the tracer was enhanced. All samples were examined in a

Jeol 100 CX transmission electron microscope (Iger Y. and Wendelaar Bonga, S.E., Cell Tissue Res., 275:481-492 (1994)).

D. Ultrasound system The system 1 shown in Fig. 19, is a conceptual model which was used to perform the following experiments is composed of multi frequency signal generator and signal amplifier and matching unit all designated as 2 and a transducer 3. Irradiation is always carried out via aquatic medium 4, i.e. gel, or water as shown in the figure inside container 5.

The active side of the transducers 6 is placed in aquatic medium 4. The transducer for the irritant stimuli may be attached to focusing lens (not shown; to increase its intensity) via regular inertic sonic-coupling gel. The lens is made of condensed material, preferably transparent plexiglass.

In practice, an irritant stimulus is applied and later, or concomi- tantly, the tissue or cells are exposed to the second driving stimuli. The substances to be delivered are then introduced to the medium 4 either, a priori, before or simultaneously with the exposure to the second stimulus.

The second transducer (not shown) might be also attached to lens in order to further increase the intensity. The fish 7 are held in place by a particular stand 8 acting as a clasp to immobilize them during the irradiation. A rubber lamina 9 is placed in the far part of the irradiated tissue, to absorb the energy and prevent formulation of standing wave resulting in unneeded elevation of the ultrasonic intensity.

E. Acoustic lenses Plexiglass lenses were made from plexiglass cylinder. The curvature of the etched lens was 28 mm, i.e. the physical focus was at r- 28 mm. The F, (acoustic focus) was F-40 mm. A schematic drawing of the lens is shown in Fig. 20, wherein F represents the acoustic focus and r the

curvature of lens. Region 10 is made of plexiglass and region 11 is made of water. Calculation of lens' parameter was carried out in accordance with Gordon S.K., Acoustic waves, devices, imaging and analog signal processing, Prentice Hall Inc., Englewood Cliffs, New Jersey, p. 652.

The calculation of F is as follows: <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> F= 1<BR> <BR> <BR> 1 n wherein r = curvature of lens; <BR> <BR> <BR> <BR> <BR> C <BR> <BR> n= P 2 <BR> <BR> <BR> <BR> Cw Cp = the speed of sound in matrix (in plexiglass 2.7/km/hr); Cw = the speed of sound in water at 200C (1.48 km/hr).

F can be determined also experimentally. Since the intensity is the highest and the affected area is the smallest at the exact focal point, it should cause a smallest mark to appear in the least amount of time on exposed materials. For calibration, a moveable thin disc of plastic was used. The distance where ultrasound caused the desired smallest "scar" on the plastic was then chosen.

F. Ultrasound pump The ultrasonic force may be used in order to cause flow of the medium and thus bring the substances to be delivered to the site of administration.

The medium flow causes suction activity at its surroundings, based on the effect known as "venturi tube", and this suction activity can be used to introduce the desired elements into the medium. This will occur if the wave is enclosed in a hollowed shape, for example, a cylinder.

Fig. 21 shows an administration system 20. The system contains a hollow cylinder 21 closed at one end by a flat disc 22 and closed at

the other end by acoustic lens 23 attached to an ultrasound transducer 24.

Cylinder 21 has about in its middle zone three openings 25 (only one shown in the figure) allowing passage of medium 29 from the tank 30 in which the cylinder is held into the cylinder 21 in the direction of the curved arrows.

Disk 22 has a hole 26 which allows passage of liquid from the cylinder outward in the direction of the straight arrow. On top of the disc 22 are present two open-ended tubes 27 each attached at one end to reservoir 28 containing the substance to be administered and having the other open-ended tube present adjacent to opening 26 or disc 22.

Upon ultrasound application the medium 29 present in tank 30 will flow into cylinder 21 through openings 25 and then out of the cylinder through hole 26 in the direction ofthe arrows. While passing through hole 26, the suction activity initiated will pump out substances present in reservoir 28 through tubes 27. The substances will then be released into medium 29. The rate of substance release, from the reservoir via the tube, is proportional, among other parameters, to the relation between areas of openings 25 and the diameter of the cylinder 21. The intensity of suction and release can be justified according to the needs.

Example 1 Morphological effects of ultrasound application Over 120 specimens of fish (tilapia and carp species), weighing 4-100 gr, were treated with ultrasound. All fish survived the irradiation and the periods after that for all the time they were still monitored (30 to 50 days).

Macroscopically, fish (either naive or anesthetized) exposed to 1 Nm ultrasound were covered with cavitation bubbles, whereas fish (if under 10 gr) exposed to 3 MHz, were slightly forced downwards by the acoustic pressure.

Using different ultrasound parameters, the rupture of cellular junctions was initiated in the epidermis, which was (macroscopically)

followed by skin sloughing. Vasodilation and skin hemorrhages was also initiated when high intensities were used for prolonged periods.

Example 2 Ultrastructural effects of ultrasound application Effect of ultrasound phases having various parameters and the results are shown in Figs. 1 to 8.

Application of 1 Nm at an intensity of 1.5 w/cm2 for 5 min.

(Fig. 1) resulted in a local degeneration effect. Outmost cells shown at the top of the figure featured severe damage and numerous membrane holes. Cells positioned in deeper levels were less affected and inner cell layers (bottom of Fig.) appeared normal. Application of 1.5 w/cm2 for 50 sec. (Fig. 2) resulted in necrotic superficial cells. Remnants of outer cells are visible while cells positioned at a deeper level appeared normal. Application of 30 w/cm2, carried out by applying an acoustic lens for a period of 1 sec. (Fig. 3) caused degeneration of cells which feature agglutination of nuclear hetrochronation and rupture of membranes.

Application of 3 Nm at 1.7 w/cm2 for 5 min. (Figs. 4 and 5) caused rupture and fusion of cells located at the 4-5 epidermal layers from the surface while outer cells remain in tact. Five to fifteen minutes after application of the ultrasound skin peeling of outer layers occurred.

Application of 3 Nm at 1.7 w/cm2 for 50 sec. (Fig. 6) caused detachment of cells at the site of ultrasound administration. Cells located outer to the place of detachment were sloughed from the epidermis as compact layers. Remnants of ruptured membranes can be observed.

Application of 3 Nm 1.7 w/cm2 for 10 sec. (Figs. 7 and 8) caused formation of vesicles unbound by membranes, which are probably cavitation bubbles in superficial cells.

It was found that application of 1 Nm of ultrasound could break and cause sloughing of the previous unstirred microclimate of mucus,

surrounding the epithelium. Formation of holes in cell membranes and membranes rupturing, up to necrosis, of the 2-3 outermost layers of the epidermis (the outermost 20-30 performed for longer than 2 minutes, sloughing of the affected epithelial tissues followed the application. Application of such an intensity for periods below 2 minutes allowed many of the cells to recover and remain an integral part of the epidermal tissue. Deeper cells were, at least apparently, unaffected. The intercellular spaces of the external epithelial layers were slightly increased.

Using special focusing lenses described in Experimental Procedure D, time of exposure of the fish was dropped to 1-2 seconds, yet having effects similar to those mentioned above.

Example 3 Sonophoresis Lanthanum hydroxide (LH) having a size of about 60-90 nm was used as a colloidal tracer. Fish were immersed in a bath containing 0.1% w/w of LH. LH was added to the water immediately before the second ultrasonic stimuli. In biopsies of control fish exposed to LH without ultrasound, the tracer was only rarely observed on the skin surface. In fish irradiated with 3 Nm in the presence of LH, and sampled immediately afterwards, LH had adhered heavily to the epidermal surface, (Figs. 9-10), but the tracer did not penetrate the cells. In fish irradiated with 1 Nm and sampled immediately afterwards, the tracer penetrated well into the cells with ruptured membranes and also into the intercellular spaces of deeper and naive cells located one or two levels deeper (Figs. 11-14).

Fish were irradiated with 1 Nm followed by irradiation with 3 MHz, in the presence of a tracer. In fish sampled immediately afterwards (Fig. 15) the tracer penetrated deeper, up to 5-6 layers from the surface and at a higher quantity into the epidermal intercellular spaces than was possible

only by a single application of 1 MHz. In fish sampled 10 minutes after the irradiation (Figs. 16-18), particles of tracer were found not only in the intercellular spaces, but also in the cytoplasm of naive cells, located at deeper epidermal zones- close to the basal lamina. The uptake of such particles into the cells was probably carried out by phagocytosis since they were mostly engulfed into phagosomes. After 10 minutes particles were found also in the dermal zone.

Similar results were obtained when LH was replaced by bacterins (which are formalin inactivated streptococcus) that was introduced to the water just before the second irradiation to create concentrations of 5x107 bacterins/ml. Bacterin particles were not observed in tissue immersed in bacterin-containing water, without ultrasound irradiation. After irradiation with 1 Nm followed by 3 MHz, bacterin particles penetrated the skin. They were found in the intercellular spaces of the epidermis and in the cytoplasm of superficial cells, as well as in the cytoplasm of deeper and naive cells.

These results demonstrate that it is possible to bypass the nonspecific barriers at the fish skin surface, at the cellular and intercellular levels, to force delivery of particles into epithelial cells and deeper dermal zones, when a two phase delivery of the invention is used.