Abstract:
An apparatus for sonoporation for transdermal delivery of a microparticles suspension containing microencapsulated drugs includes a container containing said microparticles suspension and an ultrasound horn having a tip submerged in said microparticles suspension containing microencapsulated drug or the like. The ultrasound radiation is applied to generate cavitation bubbles, thus causing pores to be formed in the skin of a patient. The ultrasound radiation intensity and distance from the skin are also effective in generating ultrasonic jets driving the microparticles through the formed pores into the skin. The ultrasound radiation is desirably applied at a frequency other than a resonant frequency of the microparticles to avoid rupturing them.

Description:
RELATED APPLICATION 
   This is a continuation-in-part of U.S. patent application Ser. No. 10/058,627, which claims benefit under 35 U.S.C. 119(e) of U.S. provisional patent application 60/264,803, both incorporated by reference herein. 

   FIELD OF THE INVENTION 
   This invention relates to a method and apparatus for in vivo intradermal incorporation of microparticles containing an encapsulated drug or other beneficial substance such as a therapeutic agent or cosmetic for topical or subcutaneous application using low-frequency ultrasound. 
   BACKGROUND OF THE INVENTION 
   Intradermal delivery of drugs offers several advantages over conventional delivery methods including oral and injection methods. It delivers a predetermined drug dose to a localized area with a controlled steady rate and uniform distribution, is non-invasive, convenient and painless. 
   U.S. Pat. No. 6,487,447, commonly assigned and incorporated by reference herein, describes a method and apparatus for sonoporation of biological barriers such as Stratum Corneum (SC), most commonly referred to as the outermost layer of human skin. Sonoporation is the noninvasive transdermal delivery of pharmaceutical drug molecules through the SC and into the cardiovascular system a human body via ultrasound radiation. Significant improvement made to existing sonoporation and sonophoresis methods of transdermal drug delivery may be leveraged to increase market share. Drug encapsulation is a known practice in therapeutic application of the potent but very unstable drugs. Using sonoporation and sonophoresis in the area of delivery of encapsulated drugs would provide many additional pharmaceutical benefits compared to the benefits of a present ways of sonoporetic or sonophoresic drug delivery. Some of these benefits would include delivery of peptide-based drugs that range from bed-wetting to gastric bleeding to cancer and immune disorders such as HIV. 
   SUMMARY OF THE INVENTION 
   In one aspect, the invention provides for an apparatus for sonoporation for intradermal delivery of a microparticles suspension containing one or more microencapsulated drugs or other beneficial substances including a container having an end covered with a porous membrane and containing the microparticles suspension; an ultrasound horn having a tip submerged in the microparticles suspension and applying ultrasound radiation to the microparticles suspension wherein the ultrasound radiation is applied at a frequency, an intensity, for a period of time, and at a distance from the skin, effective to generate cavitation bubbles, wherein the cavitation bubbles collapse and transfer their energy into the skin area thus causing the formation of pores in the skin area; and wherein the ultrasound radiation intensity and distance from the skin area are also effective in generating ultrasonic jets, the ultrasonic jets driving the microparticles suspension through an optional porous membrane and the formed pores into the skin area. 
   Implementations of the invention may include one or more of the following features. The porous membranes desirably have pores with a diameter of 100 microns (μm) or greater. Larger pores make it easier for the microparticles suspension to penetrate the membrane. Alternatively, the porous membrane may be microporous; that is, it won&#39;t allow liquid to run through without pressure, and typically has pores in the range of about 1 μm to 100 μm. The optional membrane may be hydrophobic. 
   The ultrasound radiation has a frequency in the range of 1 kHz and 1 MHz. Preferably the frequency is between 5 kHz and 30 kHz. Most preferably it is between 10 kHz and 20 kHz. In a preferred embodiment it is about 20 kHz. 
   The tip may be removably connected to the ultrasound horn and it may have a distal end surface, which may be flat, concave or it may be open. The distal end surface may have a plurality of depressions. The tip may also have a body having markings indicating the amount of microparticles suspension containing microencapsulated drugs contained in the container or intended to be introduced therein. A removable protective film may cover the membrane, if any; otherwise it directly covers the end of the tip. 
   The container may include an outer wall, and optionally an inner wall and an absorbent wick placed between the inner and outer wall. The wick absorbs any excess microparticles suspension containing microencapsulated drugs that is not driven into the skin area through the formed pores and it may be made of highly absorbent and hydrophilic material such as PVA sponge CLINICEL™ from M-Pact Company, HYDROFERA PVA sponge from Hydrofera LLC, Sodium CMC and any other similar spongy material. The container inner wall may have first and second grooves and the tip may have a body having first and second grooves. The tip is inserted into the container and placed so that the first and second grooves of the tip body are opposite the first and second grooves of the container inner wall. This arrangement defines the first and second spaces for accommodating the first and second o-rings, respectively. The container may also have an inlet septum for filling it with the solution. The container may be a cylinder made of a transparent material and/or a plastic material. 
   Optionally at the end of the container, a ring of skin-removable resilient medical adhesive material is provided, which functions as a temporary barrier seal around the area where the microparticles suspension is being applied to the patient&#39;s skin while the method of the present invention is being performed. (The term “patient” refers to a human or animal receiving treatment according to the present invention.) 
   The ultrasound frequency may be for example 20 kHz, and the ultrasound intensity may be in the range of 5 W/cm 2  and 55 W/cm 2 . The tip may have a distal end located at a distance from the membrane in the range of 1 millimeter (mm) to 10 millimeters. The ultrasound radiation may be continuous or pulsed and it may be applied for a period of time in the range of 30 seconds to 5 minutes, preferably 1 minute for continuous exposure or about 10 to 20 minutes for pulsed exposure with a 5% duty cycle, respectively. The formed pores may have a diameter in the range of 1 micrometer (μm) to 100 micrometers. 
   The microparticles may have an average diameter from 0.1 μm to about 50 μm. For many applications they desirably have an average diameter from 1 μm to 5 μm. 
   In another aspect, the invention features a method for sonoporation for intradermal delivery of a microparticles suspension containing microencapsulated drug or other beneficial substance such as one or more therapeutic agents or cosmetics. The method includes providing a container containing a predetermined amount of said microparticles suspension, and having a first end and a second end, said second end being covered with a porous membrane. Next the tip of an ultrasound horn is submerged in the microparticles suspension through the first end of the container and then the porous membrane is placed in contact with the patient&#39;s skin area. The ultrasound radiation is applied at a frequency, and intensity, for a period of time, and at a distance from the skin, effective to generate cavitation bubbles. The cavitation bubbles collapse and transfer their energy into the skin area thus causing the formation of pores in the skin area. The ultrasound radiation intensity and distance from the skin area are also effective in generating ultrasonic jets, the ultrasonic jets driving the microparticles suspension through a porous membrane and the formed pores into the skin area. 
   In a further aspect, the ultrasound radiation can then applied at a frequency in the range of 1 kHz and 1 MHz selected to avoid a resonant frequency of the particles. If the ultrasound were at a resonant frequency, it would tend to rupture the particles. A feature of the present invention is that at least a majority of the particles are delivered into the skin area without rupturing them. Preferably substantially all of the particles are delivered intact. For each formulation of particles and application, the preferable frequency may desirably be determined empirically by applying various frequencies to the microparticles suspension and determining at which range of frequencies the particles remain un-ruptured. In general, it has been found that a frequency between 1 kHz and 30 kHz is typically applicable. Most preferably it is between 10 kHz and 20 kHz. In a preferred embodiment it is about 20 kHz. 
   One advantage of the present invention is to protect against any unknown effects of ultrasound and cavitation on drugs, or therapeutic agents or cosmetics. 
   A second advantage of the present invention is the controlled release of drugs, or other beneficial substances over time into the stratum corneum and subsequently into the human vascular system. This invention can be used to provide slow and constant intradermal release of drugs, or therapeutic agents or cosmetics, because most or desirably all of the particles delivered into the skin are substantially intact. 
   A third advantage of the present invention is to reduce the need for repeated dosage of drugs, or therapeutic agents since time-released beneficial substances can be administered once and not require repeating for longer periods of time than dosages required using conventional methods. 
   A fourth advantage of the present invention is the ability to apply sensitive, non-soluble or unstable beneficial substances. Drugs, or therapeutic agents or cosmetics can be specially engineered to retain full potency in a stable environment within the microparticle until it is delivered using sonoporation. The encapsulation prevents the premature breakdown of drugs or active agents or cosmetic before they can be effectively delivered into or through the skin. 
   A fifth advantage of the present invention is that the delivery of the drugs and therapeutic agents is painless compared to the side effects or the discomfort and pain associated with injection. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  is a cross sectional side view of an apparatus for intradermal delivery of microparticles using ultrasound. 
       FIG. 1A  is a cross sectional side view of an alternative embodiment of the tip, i.e. the UTDDA, shown in  FIG. 1 . 
       FIG. 2  is a cross sectional side view of an ultrasonic transdermal drug delivery applicator (UTDDA) using microparticles containing encapsulated drugs and therapeutic agents and cosmetics. 
       FIG. 3  is a graphic transcription of the confocal microscopy image of the cross-section of the upper part of the skin after 15 seconds exposure to 20 kHz ultrasound at intensity of 19 W/cm 2  in aqueous suspension of 1 μm particles. 
       FIG. 4  is a cross sectional of a microparticle containing encapsulated drugs, or therapeutic agents or cosmetics. 
       FIG. 5  is a cross sectional side view of a Franz Cell apparatus for determining infusion of microparticles into the human cadaver epidermis. 
       FIG. 6  is a flow chart of a method for transdermal incorporation of microparticles containing encapsulated substance using 20 kHz ultrasound. 
       FIG. 7  is a view similar to  FIG. 2  of an alternative embodiment of a UTDDA having an adhesive ring thereon. 
       FIG. 8  is a view similar to  FIG. 2  of another alternative embodiment of a UTDDA having an open end and adhesive ring thereon. 
       FIG. 9  is a view similar to  FIG. 2  of yet another alternative embodiment of a UTDDA having a ring-shaped reservoir containing the microparticles suspension. 
   

   DETAILED DESCRIPTION 
   The present invention provides an apparatus and method for intradermal incorporation of microparticles containing encapsulated drugs, or therapeutic agents or cosmetics using sonoporation. The apparatus is designed to use ultrasound to deliver a suspension of microparticles into the epidermal layer of the skin. The microparticles contain medication or cosmetic that is encapsulated to provide protection. The encapsulation also provides means of controlled released of the drug, or therapeutic agents or cosmetic into the skin. 
   With reference to the drawings,  FIG. 1  shows a sonoporation device  6  used for in vitro sonoporation of skin that includes an ultrasound equipment assembly  20 , electrically and mechanically connected to an ultrasonic transdermal drug delivery applicator (UTDDA)  100  (described in detail below) via an ultrasonic horn  80 . The ultrasound equipment assembly  20  includes an ultrasound transducer  50 , which in turn is electrically connected to power supply  30  via connecting cable  40 . 
   As shown in  FIG. 1 , the distal end surface of the UTDDA is flat. As shown in  FIG. 1A , the distal end surface of an alternative embodiment of the UTDDA is concave. 
   In operation, the UTDDA  100  is removably connected to the affected skin surface via pressure resistive adhesive. Ultrasonic horn  80  is placed inside the UTDDA  100 . Power supply  30  is activated and provides power to ultrasonic transducer  50 . The ultrasonic transducer  50  converts electrical energy into acoustic pressure waves that are coupled through the ultrasonic horn  80  and into the UTDDA  100 . Sonoporation of the affected skin occurs for a pre-determined interval, which depending upon skin resistivity and can range from 5 to 60 seconds. 
     FIG. 2  is a schematic of one example of an ultrasonic transdermal drug delivery applicator (UTDDA)  100  in accordance with the invention that includes an inner applicator wall  201 , an outer applicator wall  202 , a first O-ring  220 , a second O-ring  230 , a wick  250 , a microparticles suspension inlet septum  240 , a solution level marking  260 , a removable protective film  265 , a porous membrane  280 , a microparticles suspension  290  that includes microparticles containing encapsulated drugs.  FIG. 2  also shows parts of the ultrasonic horn (element  80  of  FIG. 1 ), including a tip of ultrasonic horn  210 , and a bottom of ultrasonic horn tip  270 . 
   The UTDDA  100  is a hollow cylindrically shaped object, preferably made of a transparent hard plastic material and is discarded after use. Between the outer applicator wall  202  and the inner applicator wall  201  is a wick  250 , made of material such as high-absorbency polypropylene. Both the inner and outer applicator walls  201  and  202  are basically cylindrical and axially aligned, with the exception of two locations on the inner applicator wall  201  where two grooves are cut on the inside surface of the inner applicator wall  201  for the two O-rings  220  and  230  to fit into the assembly. The location and orientation of the grooves, and the two O-rings  220  and  230  are shown in  FIG. 2 . As shown in  FIG. 2 , grooves cut into the outer diameter of the ultrasonic horn are matched to the placement of the O-rings  220  and  230  to facilitate a secure fit between the UTDDA  100  and the ultrasonic horn  80 . 
   Again in reference to  FIG. 2 , the drug inlet septum  240  is located between the outer applicator wall  202  and the inner applicator wall  201  approximately halfway between the top and bottom of the UTDDA  100 . The septum is constructed of a silicon rubber material, designed to be impervious to liquids yet allow injection of the microparticles suspension into the UTDDA  100  using a hypodermic needle. The solution marking level  260  is pre-marked on the side of the UTDDA  100  to indicate the proper volumetric measure of microparticles suspension to be administered. 
   At the base of the UTDDA  100 , the porous membrane  280  is fixedly attached to the inner applicator wall  201 . The membrane  280 , constructed of a non-woven polypropylene or other similar hydrophobic material, resists the passage of the aqueous liquid due to its non-wettable surface and small diameter of the pores in range of 1-100 microns. A removable protective film  265 , which is preferably a thin plastic sheet, is removably connected to the porous membrane  280  using silicone or other medical adhesive. 
   In operation, the sterilized UTDDA  100  is placed over the tip of ultrasonic horn  210  with the two O-rings  220  and  230  in place as shown in  FIG. 2 . The microparticles suspension  290  with suspended microparticles containing encapsulated drugs is introduced into the reservoir of the UTDDA  100  through the inlet septum  240  using a hypodermic needle (not shown) to inject a pre-measured amount of microparticles suspension  290 . The protective film  265  is peeled off to expose the porous membrane  280 . When the UTDDA  100  is properly filled, the tip of the ultrasonic horn  210  is partially immersed in the microparticles suspension  290 . Visual inspection of the solution level marking  260  indicates whether the applicator is properly filled, and whether the UTDDA  100  is leaking or defective. 
   Once the UTDDA  100  is filled and determined to be ready for use, the apparatus is placed on the skin, oriented such that the porous membrane  280  is flush with the location where the drugs are to be administered and such that the bottom of the horn tip  270  is immersed in microparticles suspension  290 . A timer (not shown), which is contained in power supply  30 , is set to a predetermined length of time for sonoporation. The power supply is switched on, and the ultrasound sonoporates the skin for an allotted amount of time. The porous membrane  280  is designed to prevent the microparticles suspension from leaking prior to transdermal infusion process, yet simultaneously allow ultrasound waves to freely pass through the membrane  280  and sonoporate the skin surface. Any excess liquid that is transferred to the skin during the ultrasound exposure is absorbed by the wick  250 . After use, the UTDDA  100  is removed form the ultrasound tip and discarded. 
     FIG. 3  shows a skin system  300  that represents a cross section of human skin after exposure to 20 kHz ultrasound in presence of the suspension of the 1 μm particles in aqueous saline solution. The figure shows the environment where the drug, therapeutic agent, or cosmetic, which is encapsulated in the microparticle is delivered into the skin by action of the 20 kHz ultrasound. The cross-section of the skin includes a stratum corneum (the top part of the skin)  320 , an underlying layer called the viable epidermis  330 , and a dermis  340 . Between the epidermal and dermal layers reside the endings of a capillary vascular system  350 . Pores  310  in the stratum corneum  320  and the transient micropores  315  in the viable epidermis  330  are created when the skin is exposed to ultrasound. The size of the pores in stratum comeum is in the range of 1 to 100 micrometers in diameter. The size of the micropores generated in viable epidermis is in range up to 35 micrometers in diameter. The ultrasound intensity is in the range of 11 W/cm to 79 W/cm The figure shows the microparticles  370  migrating through the pores  310  in the stratum corneum  320  then through the transient micropores  315  in the viable epidermis  330  to part of the dermis with the capillary vascular system  350 . 
   In operation, the microparticles suspension  290  containing the microparticles  370  is delivered to the skin using the sonoporation apparatus. The ultrasound assists in propelling the microparticles  370  through the pores  310  and transient micropores  315 . Once the microparticles are lodged in the epidermis, the drug, or therapeutic agent, or cosmetic is released from the microparticles at a controlled rate determined by the microparticle chemical composition. Subsequently the drug, or therapeutic agent released from the microparticles is ultimately absorbed into the capillary vascular system  350 . 
     FIG. 4  shows a schematic of a cross-section of a microparticle  400  containing a drug, or therapeutic agent, or cosmetic  410 , a first protective sheath  420 , and a second protective sheath  430 . Microencapsulation is a known process in the pharmaceutical field and is not described here. Examples of other patents that address the method of creating and using microparticles include U.S. Pat. No. 4,983,401, U.S. Pat. No. 5,792,477, U.S. Pat. No. 5,723,269, U.S. Pat. No. 6,048,550, and U.S. Pat. No. 5,651,990. The microparticle  400  is generally spherical and includes one or more protective sheaths, arranged in concentric and incrementally smaller hollow spheres, with a center core sphere of encapsulated drug, or therapeutic agent, or cosmetic in a solid, liquid or solvated states. The content of encapsulated drugs, or therapeutic agent, or cosmetic  410  is shown in  FIG. 4  at the center of the microparticle  400 . 
   In one example embodiment, drug or a therapeutic agent  410  includes anti-fungal agents, hormones, vitamins, peptides, enzymes, anti-allergic agents, anti-coagulation agents, antituberculars, antivirals, antibiotics, antibacterials, antiinflammatory agents, antiprotozoans, local anesthetics, growth factors, cardiovascular agents, diuretics, and radioactive compounds; scopolamine, nicotine, methyinicotinate, mechlorisone dibutyrate, naloxone, methanol, caffeine, salicylic acid, and 4-cyanophenol; anti-fungal agents selected from the group consisting of ketoconazole, nystatin, griseofulvin, flucytosine, miconazole, and amphotericin B; hormones selected from the group consisting of growth hormone, melanocyte stimulating hormone, estradiol, progesterone, testosterone, bcclomethasone dipropionate, betamethasone, betamethasone acetate and betamethasone sodium phosphate, vetamethasone disodium phosphate, vetamethasone sodium phosphate, cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, flunisolide, hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, triamcinolone hexacetonide and fludrocortisone acetate; vitamins selected from the group consisting of cyanocobalamin neinoic acid, retinoids, retinol palmitate, ascorbic acid, and alpha-tocopherol, B-12 and other vitamins; peptides and enzymes selected from the group consisting of manganese super oxide dismutase and alkaline phosphatase; the anti-allergic agent is amelexanox; the anti-coagulation agents selected from the group consisting of phenprocoumon and heparin; the antituberculars selected from the group consisting of paraminosalicylic acid, isoniazid, capreomycin sulfate cycloserine, ethambutol hydrochloride ethionamnide, pyrazinamide, rifampin, and streptomycin sulfate; the antivirals selected from the group consisting of acyclovir, amantadine azidothymidine, ribavirin and vidarabine monohydrate; the antibiotics selected from the group consisting of dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin, cephradine erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin rifampin and tetracycline; the antiinflammatories selected from the group consisting of diflunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone, phenylbutazone, piroxicam, diclofenac, sulindac, tolmetin, aspirin and salicylates; the antiprotozoans selected from the group consisting of chloroquine, hydroxychloroquine, metronidazole, quinine and meglumine antimonate; the local anesthetics selected from the group consisting of bupivacaine hydrochloride, chloroprocaine hydrochloride, etidocaine hydrochloride, lidocaine hydrochloride, mepivacaine hydrochloride, procaine hydrochloride and tetracaine hydrochloride; the growth factors selected from the group consisting of Epidermal Growth Factor, acidic Fibroblast Growth Factor, Basic Fibroblast Growth Factor, Insulin-Like Growth Factors, Nerve Growth Factor, Platelet-Derived Growth Factor, Stem Cell Factor, Transforming Growth Factor of the alpha family and Transforming Growth Factor of the beta family; the cardiovascular agents are selected from the group consisting of clonidine, propranolol, lidocaine, nicardipine and nitroglycerin; the diuretics are selected from the group consisting of mannitol and urea; and wherein the radioactive particles are selected from the group consisting of strontium, iodine, rhenium and yttrium. 
   In another example embodiment, therapeutic agent  410  includes the following:
         (1) peptides selected from the group consisting of melanin concentrating hormone, melanin stimulating hormone, trypsin inhibitor, Bowman Burk inhibitor, luteinizing hormone releasing hormone, bombesin, cholecystokinin, insulin, gastrin, endorphins, enkephalins, growth hormone, prolactin, oxytocin, follicle stimulating hormone, human chorionic gonadotropin, corticotropin, .beta.-lipotropin, .gamma.-lipotropin, calcitonin, glucagon, thyrotropin, elastin, cyclosporin, and collagen;   (2) monoclonal antibodies;   (3) factors selected from the group consisting of hyaluronic acid, heparin, mad heparin sulfate;   (4) anti-sense peptides and anti-sense oligonucleotides selected from the group consisting of an antisense oligonucleotide capable of binding the DNA encoding at least a portion of Ras, an antisense oligonucleotide capable of binding the DNA encoding at least a portion of basic fibroblast growth factor, and the antisense ras/p53 peptide;   (5) immunosuppressants and anti-inflammatory agents;   (6) chelants and chelating agents selected from the group consisting of penicillamine, citrate, ascorbate, diethylenetriaminepentaa-cetic acid, dihydroxypropylethylenediamine, cyclohexanediaminetetraacetic acid, ethylenediaminetetraacetic acid, ethylene glycol-bis (.beta.-aminoethyl ether)N,N,N′,N′,-tetraacetic acid, etidronic acid, dimethylsulfoxide, dipyridoxylethylenediaminediacetate-bisphosphate, N,N′-(1,2-ethanediylbis(oxy-2,1-phenylene))bis(N-(carboxymethyl), aminophenoltriacetic acid, tetrakis(2-pyridylmethyl) ethylenediamine, cyanins, and salts thereof; and   (7) DNA encoding at least a portion of the following genes: HLA, dystrophin, CFTR, interleukin-2, tumor necrosis factor, adenosine deaminase, HDL receptor, thymidine kinase, HLA-B7, interleukin-4, melanocyte stimulating hormone gene, and melanin concentrating hormone gene.       

   In yet another example embodiment, the cosmetic  410  includes Vitamin A, Vitamin C, Vitamin D, Vitamin E, Vitamin K, beta carotene, collagen, elastin, retinoic acid, aloe vera, lanolin, hyaluronic acid, and nucleosides; a sunscreen agent, said sunscreen agent selected from the group consisting of 5% isobutyl-p-aminobenzoate, 5% diallyl trioleate, 2.5% monoglyceryl p-aminobenzoate, 4% propylene glycol p-aminobenzoate, and a composition comprising 2% benzyl salicylate and 2% benzyl cinnamate; a cosmetic cream, ointment, lotion, skin softener, gel, blush, eye-liner, mascara, acne-medication, cold cream, cleansing cream, or oleaginous foam. 
   In another example embodiment, the composition  410  comprises one or more compounds selected from the following:
         (1) bacteriostatic agents selected from the group consisting of benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, butylparaben, cetylpyridinium chloride, chlorobutanol, chlorocresol, methylparaben, phenol, potassium benzoate, potassium sorbate, sodium benzoate and sorbic acid;   (2) antioxidants selected from the group consisting of tocopherol, ascorbic acid and ascorbyl palmitate;   (3) preservatives selected from the group consisting of parabens, quaternary ammonium compounds, alcohols, phenols, and essential oils;   (4) buffers and neutralizers;   (5) moisture content control agents and humectants;   (6) ointment bases selected from the group consisting of lanolin, lanolin anhydrous, hydrophilic ointment, white ointment, yellow ointment, polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, white petrolatum, rose water ointment, and squalene;   (7) suspending and viscosity-increasing agents selected from the group consisting of acacia, agar, alginic acid, aluminum monostearate, bentonite, purified bentonite, magma bentonite, carbomer 934P, carboxymethylcellulose calcium, carboxymethylcellulose sodium 12, carboxymethylcellulose sodium, carrageenan, microcrystalline cellulose, dextrin, gelatin, guar gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, magnesium aluminum silicate, methylcellulose, pectin, polyethylene oxide, polyvinyl alcohol, povidone, propylene glycol alginate, silicon dioxide, silicon dioxide, zinc oxide, sodium alginate tragacanth, and xanthan gum;   (8) skin absorption enhancing agents selected from the group consisting of pyrrolidones, fatty acids, sulfoxides, amines, terpenes, terpenoids, surfactants, alcohols, urea, glycols, azone, n-alkanols, n-alkanes, orgelase, and alphaderm cream;   (9) bases selected from the group consisting of glycerol, propylene glycol, isopropyl myristate, urea in propylene glycol, ethanol and water, and polyethylene glycol;   (10) other agents selected from the group consisting of glycerin, hexylene glycol, sorbitol, propylene glycol, and calcium silicate;   (11) oleaginous vehicles;   (12) coloring agents; and   (13) foaming agents.       

   In another example embodiment, the composition  410  comprises a gas in range of 0-50% in the interior of the microparticle and an effective amount of drug, or therapeutic agent, or cosmetic. 
   A structural composition of the microparticle  370  or  400  can vary depending on the content of the microparticle and the method of the release of that content once the microparticles are embedded in the skin. 
   In one example embodiment microparticle  370  or  400  includes liposomes, microspheres, nano-spheres or nano-particles. In another example embodiment microparticle  370  or  400  consists of first protective layer  420  as the next largest hollow sphere, and second protective layer  430  as outermost hollow sphere. This design may consist of one, two, or as many protective layers as required for a given microparticle structural composition. 
   The layers of the microparticle&#39;s structure includes but is not limited to lipid conglomerates or polymers preferably biodegradable. 
   In another example embodiment microparticle  370  or  400  is prepared from at least one biocompatible lipid. In another example embodiment microparticle  370  or  400  is prepared from at least one biocompatible polymer selected from the group consisting of polysaccharides, semi-synthetic polymers and synthetic polymers. In another example embodiment microparticle  370  or  400  is prepared from the following:
         (1) from a composition comprising dipalmitoylphosphatidylcholine, glycerol and propylene glycol.   (2) from a composition comprising dipalmitoylphosphatidylethanolamine and phosphatidic acid in an amount of from 0.5 to 30 mole percent.   (3) from a composition comprising dipalmitoylphosphatidylcholine and distearoylphosphatidyl-choline in an amount of from 70 to 100 mole percent.   (4) from a composition comprising: (I) a neutral lipid, (ii) a negatively charged lipid, and (iii) a lipid bearing a hydrophilic polymer; wherein the amount of said negatively charged lipid is greater than 1 mole percent of total lipid present, and the amount of lipid bearing a hydrophilic polymer is greater than 1 mole percent of total lipid present.       

   In another example embodiment microparticle  370  or  400  comprises the following:
         (1) a mono-layer.   (2) a polymer.   (3) a polysaccharide.   (4) a micelle system.   (5) a surfactant.       

   In operation, the microparticles  400  are forced into the ultrasonically disrupted skin as shown in  FIG. 3 . After the particle is embedded in the dermis, the protective layer or layers surrounding the drugs, or therapeutic agents, or cosmetic are released into the skin in one or more of the following manners:
         (1) the microparticle dissolves or biodegrades in the skin with predetermined rate and the drugs, or therapeutic agents, or cosmetic are released The rate of dissolution will depend on the design of the protective sheath and the number of sheaths surrounding the center core of the microparticle.   (2) The microparticles containing gas in their interior are burst open by application of an additional external ultrasound of an resonance frequency that matches the size of the microparticles containing gas along with a drug, or therapeutic agent, or cosmetic. For example, when diameter of the gas containing microparticle is 3 micrometers, which is convenient size for drug, or therapeutic, or cosmetic carrier, the resonance frequency is 2.2 MHz. By the large-amplitude vibration and the rise of the temperature caused by the resonance, the drug, or therapeutic agent, or cosmetic is released in to the skin.       

     FIG. 5  shows Franz Cell apparatus  100  for determining infusion of microparticles into the human cadaver epidermis  140  during exposure of the skin to ultrasound of 20 kHz and intensity of 19 W cm.sup.-2. The apparatus  100  includes the ultrasound transducer  110  with horn  120  and Franz Cell assembly  105 . The ultrasound horn  120  is electrically and mechanically connected to an ultrasound transducer  110 . Ultrasound transducer  110  is electrically connected to a power supply (not shown). The ultrasound horn  120  is submerged in the microparticles suspension  170  placed in the donor compartment  130 . The exposure of the skin to the ultrasound causes formation of pores in the stratum corneum and transient micro-pores in the epidermis of the skin  140 , which allow for transdermal flux of the microparticles from the donor compartment  130  to the saline solution  180  in the receiver compartment  150 . 
     FIG. 6  is a method  500  for transdermal incorporation of microparticles containing encapsulated drugs using sonoporation that includes the following steps: 
   Step  510 : Sterilizing the Ultrasonic Horn Tip 
   The ultrasonic horn tip  270  may be sterilized using an ethylene oxide gas or by exposing the horn tip  270  to elevated heat/steam. The horn tip may also be pre-sterilized and sealed in a protective package. The method then proceeds to Step  520 . 
   Step  520 : Assembling the Tip of Ultrasonic Horn with Ultrasound Horn 
   The sterilized ultrasonic horn tip  270  is attached to the ultrasound horn  80  by screwing the threaded tip into the ultrasound horn. The method then proceeds to Step  525 . 
   Step  525 : Attaching the Ultrasonic Transdermal Drug Delivery Applicator (UTDDA) to the Tip of Ultrasonic Horn 
   The tip of the ultrasonic horn  270  is inserted into the UTDDA  100  so that the O-rings  220  and  230  hold the assemblies together securely. The method then proceeds to Step  530 . 
   Step  530 : Injecting the Microparticles Suspension Containing Microparticles into the UTDDA 
   The microparticles suspension  290  is introduced into the UTDDA  100  via the septum  240  using a hypodermic needle to inject the solution. The method then proceeds to Step  540 . 
   Decision Step  540 : Is Level of the Microparticles Suspension in the UTDDA Adequate 
   By visual inspection, the injected liquid level is compared to the level marking  260 . If the microparticles suspension level is aligned with the marked level  260 , the method proceeds to Step  550 . If the levels are not aligned, the method proceeds to Step  560  to check for the source of inadequate solution level. 
   Step  550 : Placing UTDDA on Skin 
   The removable protective film  265  is removed from the UTDDA  100 . Then the apparatus is oriented such that the porous membrane is fully flush with the skin surface (it must be basically perpendicular to the plane of the skin surface and arranged so that the tip of the horn  270  is immersed in the microparticles suspension  290 ). The method then proceeds to Step  570 . 
   Decision Step  560 : Checking for Leaks or Defects 
   If the microparticles suspension level is inadequate, the apparatus may be leaking or is defective. The apparatus is visually inspected to look for leaks or visible defects. If there are no other sources of error, the amount of solution may be inadequate to fill the reservoir, and the method proceeds to Step  580  to correct this problem. If the UTDDA  100  is leaking or is otherwise defective, the method proceeds to Step  590 . 
   Step  570 : Exposing Skin to Ultrasound and Administering Drugs 
   The power supply  30  is turned on and a timer, which is contained in power supply  30 , is set to a predetermined length of ultrasound exposure (5-60 seconds). The ultrasound is turned on for a predetermined period of time that causes formation of the micropores  310  in the skin and subsequent transfer of the drug from the reservoir of the UTDDA  100  into the micropores formed. The method then proceeds to Step  590 . 
   Step  580 : Adding More Microparticles Suspension 
   If an inadequate volume of solution was initially added to the reservoir, more microparticles suspension  290  is added via the septum. The method then proceeds to Step  540 . 
   Step  590 : Discarding UTDDA 
   If the UTDDA  100  is defective, the applicator must be discarded and a new one used to administer the microparticles suspension  290 . After the UTDDA  100  has been used once, it must be discarded. Prior to discarding the UTDDA  100 , the power supply is set to a stand-by condition. The method ends after the UTDDA  100  has been discarded. 
   If the microparticles embedded in the skin are to be burst open in order to release the content into the skin, the external ultrasound of the resonance frequency is applied to skin. 
     FIG. 7  is a schematic of another example of an ultrasonic transdermal drug delivery applicator (UTDDA)  100 B in accordance with the invention. It includes an inner applicator wall  701 , an outer applicator wall  702 , a first O-ring  720 , a second O-ring  730 , a wick  750 , a microparticles suspension inlet septum  740 , a solution level marking  760 , a removable protective film  765 , here shown partially removed, a porous membrane  780 , and a microparticles suspension  790  that includes microparticles containing encapsulated drugs. The figure also shows parts of the ultrasonic horn (element  80  of  FIG. 1 ), including a tip of ultrasonic horn  710 , and a bottom of ultrasonic horn tip  770 . 
   The UTDDA  100 B is a hollow cylindrically shaped object, of construction similar to that described above with respect to  FIG. 2 . Between the outer applicator wall  702  and the inner applicator wall  201  is a wick  750 , made of material such as high-absorbency polypropylene. Both the inner and outer applicator walls  701  and  702  are basically cylindrical and axially aligned, with the exception of two locations on the inner applicator wall  701  where two grooves are cut on the inside surface of the inner applicator wall  701  for the two O-rings  720  and  730  to fit into the assembly. The location and orientation of the grooves, and the two O-rings  770  and  730  are shown in  FIG. 7 . As shown in  FIG. 7 , grooves cut into the outer diameter of the ultrasonic horn are matched to the placement of the O-rings  720  and  730  to facilitate a secure fit between the UTDDA  100 B and the ultrasonic horn  80 . 
   Again in reference to  FIG. 7 , the drug inlet septum  740  is located between the outer applicator wall  702  and the inner applicator wall  701  approximately halfway between the top and bottom of the UTDDA  100 B. The septum is constructed of a silicone rubber material, designed to be impervious to liquids yet allow injection of the microparticles suspension into the UTDDA  100 B using a hypodermic needle. The solution marking level  760  is pre-marked on the side of the UTDDA  100 B to indicate the proper volumetric measure of microparticles suspension to be administered. 
   At the base of the UTDDA  100 B, the porous membrane  780  is fixedly attached to the inner applicator wall  701 . The membrane  780 , constructed of a non-woven polypropylene or other similar hydrophobic material, resists the passage of the aqueous liquid due to its non-wettable surface and small diameter of the pores in range of 1-100 microns (μm). A removable protective film  765 , which is preferably a thin plastic sheet, is removably connected to the porous membrane  780  by a ring  785  of medical adhesive which has two purposes. 
   The first purpose of the adhesive ring  785  is to adhere the protective film  765  to the base of the UTDDA  100 B. The second purpose is to form a seal when the UTDDA  100 B is applied to the patient&#39;s skin, preventing leakage of fluid into the surroundings. 
   The adhesive ring  785  may be composed, for example, of an acrylic adhesive such as DuraTak 87-2516, Duratack 87-2353 or Duro-Tak 87-4098 (National Starch) or Gelva 737, Gelva 1151, or Gelva 1430 (Solutia). Alternatively the adhesive ring may be composed, for example, of a rubber-type adhesive such as Duro-Tak 87-6430 or Duro-Tak 87-6173 (National Starch). Alternatively the adhesive ring may be composed of a silicone adhesive such as MED-1356, MED 6345 or MED6340 (NuSil) or BIO-PSA 4301 or BIO-PSA 4601. 
   In operation, the sterilized UTDDA  100 B operated substantially as described above with respect to UTDDA  100 . However, after the protective film  765  is peeled away as shown, the adhesive ring  785  is exposed at the bottom of UTDDA  100 B so as to be placed in contact with the patient&#39;s skin and form a temporary seal thereto. Any fluid which nevertheless spills out from the region of the seal is absorbed by the wick  750 . After use, the UTDDA  100 B is removed form the ultrasound tip and discarded. 
     FIG. 8  is a schematic of another example of an ultrasonic transdermal drug delivery applicator (UTDDA)  100 B in accordance with the invention. It includes an inner applicator wall  801 , an outer applicator wall  802 , a first O-ring  820 , a second O-ring  830 , a wick  850 , a microparticles suspension inlet septum  840 , a solution level marking  860 , a removable protective film  865 , here shown partially removed, a porous membrane  880 , and a microparticles suspension  890  that includes microparticles containing encapsulated drugs. The figure also shows parts of the ultrasonic horn (element  80  of  FIG. 1 ), including a tip of ultrasonic horn  810 , and a bottom of ultrasonic horn tip  870 . 
   The UTDDA  100 C is a hollow cylindrically shaped object, of construction similar to that described above with respect to  FIG. 2 . Between the outer applicator wall  802  and the inner applicator wall  201  is a wick  850 , made of material such as high-absorbency polypropylene. Both the inner and outer applicator walls  801  and  802  are basically cylindrical and axially aligned, with the exception of two locations on the inner applicator wall  801  where two grooves are cut on the inside surface of the inner applicator wall  801  for the two O-rings  820  and  830  to fit into the assembly. The location and orientation of the grooves, and the two O-rings  870  and  830  are shown in  FIG. 8 . As shown in  FIG. 8 , grooves cut into the outer diameter of the ultrasonic horn are matched to the placement of the O-rings  820  and  830  to facilitate a secure fit between the UTDDA  100 B and the ultrasonic horn. 
   Again in reference to  FIG. 8 , the drug inlet septum  840  is located between the outer applicator wall  802  and the inner applicator wall  801  approximately halfway between the top and bottom of the UTDDA  100 B. The septum is constructed of a silicone rubber material, designed to be impervious to liquids yet allow injection of the microparticles suspension into the UTDDA  100 B using a hypodermic needle. The solution marking level  860  is pre-marked on the side of the UTDDA  100 B to indicate the proper volumetric measure of microparticles suspension to be administered. 
   At the base of the UTDDA  100 B, the porous membrane  880  is fixedly attached to the inner applicator wall  801 . The membrane  880 , constructed of a non-woven polypropylene or other similar hydrophobic material, resists the passage of the aqueous liquid due to its non-wettable surface and small diameter of the pores in range of 1-100 microns (μm). A removable protective film  865 , which is preferably a thin plastic sheet, is removably connected to the porous membrane  880  by a ring  885  of medical adhesive which has two purposes. 
   The first purpose of the adhesive ring  885  is to adhere the protective film  865  to the base of the UTDDA  100 B. The second purpose is to form a seal when the UTDDA  100 B is applied to the patient&#39;s skin, preventing leakage of fluid into the surroundings. 
   The adhesive ring  885  may be composed, for example, of an acrylic adhesive, a rubber-type adhesive, or a silicone adhesive as described above with respect to adhesive ring  785 . 
   In operation, the sterilized UTDDA  100 C operated substantially as described above with respect to UTDDA  100 B, except that there is no porous membrane (element  780  in  FIG. 7 ) to contain the suspension. Instead the microparticles suspension is introduced therein by injection through septum  840  from a syringe through a cannula (not shown). 
   After the protective film  865  is peeled away as shown, the adhesive ring  885  is exposed at the bottom of UTDDA  100 C so as to be placed in contact with the patient&#39;s skin and form a temporary seal thereto. Fluid which nevertheless spills out from the region of the seal is generally absorbed by the wick  850 . After use, the UTDDA  100 C is removed form the ultrasound tip and discarded. 
   It is to be understood that an alternative embodiment of the UTDDA  100 C (or an other version, as shown in  FIGS. 7 and 9  for example) may omit the wick  850  and outer wall  802 , especially if it is determined empirically that the adhesive ring  885  generally forms such a good seal in use that leakage onto surrounding skin is minimal. 
     FIG. 9  is a schematic of another example of an ultrasonic transdermal drug delivery applicator (UTDDA)  100 D in accordance with the invention. It includes an inner applicator wall  901 , an outer applicator wall  902 , a first O-ring  920 , a second O-ring  930 , a wick  950 , a microparticles suspension inlet septum  940 , a solution level marking  960 , a removable protective film  965 , here shown in place, and a ring-shaped reservoir  945  containing a microparticles suspension that includes microparticles containing encapsulated drugs. The figure also shows parts of the ultrasonic horn (element  80  of  FIG. 1 ), including a tip of ultrasonic horn  910 , and a bottom of ultrasonic horn tip  970 . 
   The UTDDA  100 D is a hollow cylindrically shaped object, of construction similar to that described above with respect to  FIG. 2 . Between the outer applicator wall  902  and the inner applicator wall  201  is a wick  950 . Both the inner and outer applicator walls  901  and  902  are basically cylindrical and axially aligned, with the exception of two locations on the inner applicator wall  901  where grooves are cut on the inside surface of the inner applicator wall  901  for the two O-rings  920  and  930  to fit into the assembly. The location and orientation of the grooves, and the two O-rings  920  and  930  are shown in  FIG. 9 . 
   Again in reference to  FIG. 9 , the drug inlet septum  940  is located between the outer applicator wall  902  and the inner applicator wall  901  approximately halfway between the top and bottom of the UTDDA  100 D. The septum may be constructed of a silicone rubber material, designed to form a seal around a pipe extending from the reservoir  945  into the cavity  990  of UTDDA  100 D. The solution marking level  960  is pre-marked on the side of the UTDDA  100 D to indicate the proper volumetric measure of microparticles suspension to be administered. 
   At the base of the UTDDA  100 D, a protective film  965 , is removably connected to the UTDDA by a ring  985  of medical adhesive, which functions similarly to the adhesive ring  885  described above. 
   In operation, the sterilized UTDDA  100 D operated substantially as described above with respect to UTDDA  100 C, except that the microparticles suspension is provided from the reservoir  945  through a pipe extending through septum  940  rather than by a syringe and needle. After the protective film  965  is peeled away, the adhesive ring  985  is exposed at the bottom of UTDDA  100 D so as to be placed in contact with the patient&#39;s skin and form a temporary seal thereto. Any fluid which nevertheless spills out from the region of the seal is absorbed by the wick  950 . After use, the UTDDA  100 D is removed form the ultrasound tip and discarded. 
   EXAMPLE 
   In the Franz Cell apparatus shown in  FIG. 5 , a 2% microparticles suspension in saline was placed in the donor compartment over the heat-split human cadaver epidermis. The receiver compartment was filled with the saline solution. Ultrasound horn was submerged in microparticles suspension at 6 mm height above the skin surface. Ultrasound of 20 kHz and intensity of 19 W cm.sup.-2 was turned on for periods of 15 sec and off for 59.9 sec. The total exposure time to ultrasound was 90 sec. The AC current of 1 V and 10 Hz was measured during the ultrasound exposure time (not shown) to determine skin permeability in presence of different size microparticles. The following microparticle sizes were investigated: 1.5 μm, 5.2 μm, 11.9 μm, 25 μm, 40 μm and 173 μm. The 2% suspensions in saline solution with microparticles of the following sizes 1.5 μm, 5.2 μm, 11.9 μm, and 173 μm. were obtained from Seradyn Mitsubishi Kasei Corp., 1200 Madison Ave., Indianapolis, Ind. 46225. Particles of other two sizes 25 μm and 40 μm were obtained from Aldrich). The effect of the microparticle size on the transdermal flux and skin conductivity is shown in Table 1. 
   
     
       
             
             
           
             
             
             
             
             
             
             
             
           
             
             
             
             
             
             
             
             
           
         
             
                 
               TABLE 1 
             
           
           
             
                 
                 
             
             
                 
               CONDUCTANCE ACROSS THE SKIN (10 −6 /Ω) 
             
           
        
         
             
               ULTRASOUND 
               WITHOUT 
                 
                 
                 
                 
                 
                 
             
             
               EXPOSURE 
               MICRO- 
             
             
               TIME (sec) 
               PARTICLES 
               1.51 μm 
               5.2 μm 
               11.9 μm 
               25 μm 
               40 μm 
               173 μm 
             
             
                 
             
           
        
         
             
               0 
               36 
               23 
               37 
               34 
               Not measured 
               24 
               40 
             
             
               15 
               53 
               40 
               45 
               50 
               Not measured 
               28 
               52 
             
             
               30 
               55 
               50 
               50 
               60 
               Not measured 
               35 
               60 
             
             
               45 
               67 
               90 
               70 
               83 
               Not measured 
               36 
               61 
             
             
               60 
               76 
               128 
               85 
               120 
               Not measured 
               46 
               61 
             
             
               75 
               124 
               140 
               110 
               225 
               Not measured 
               50 
               62 
             
             
               90 
               151 
               165 
               180 
               290 
               Not measured 
               51 
               65 
             
             
               TOTAL 
               N/A 
               YES 
               YES 
               YES 
               YES 
               NO 
               NO 
             
             
               PENETRATION 
             
             
               OF SKIN BY 
             
             
               MICRO- 
             
             
               PARTICLES 
             
             
                 
             
           
        
       
     
   
   It is apparent from Table 1 that microparticles of up to 25 μm penetrated into the skin under the conditions tested, whereas particles larger than about 40 μm did not penetrate. In general, suspensions consisting essentially of microparticles of diameters less than 40 μm are preferred for use with the present invention, although if the conditions are varied to produce relatively large micropores in the skin, somewhat larger microparticles would also be effective and could be used if desired. Desirably the average diameter of microparticles useful in the invention are from 1 μm to 35 μm in diameter, and commonly could be from 5 μm to 25 μm. 
   The many features and advantages of the present invention are apparent from the detailed specification, and thus, it is intended by the appended claims to cover all such features and advantages of the described method and apparatus that follow the true spirit and scope of the invention. Furthermore, since numerous modifications and changes will readily occur to those of skill in the art, it is not desired to limit the invention to the exact construction and operation described herein. Moreover, the method and apparatus of the present invention, like related apparatus and methods used in medical applications tend to be complex in nature and are often best practiced by empirically determining the appropriate values of the operating parameters or by conducting computer simulations to arrive at a best design for a given application. Accordingly, other embodiments are within the scope of the following claims.