2013在经皮给药载体的超音波

Sonophoresis in transdermal drugdeliverysDonghee Park a ,Hyunjin Park b ,Jongbum Seo a ,⇑,Seunghun Lee caDepartment of Biomedical Engineering,Yonsei University,Wonju 220-710,Republic of KoreabSchool of Electronic and Electrical Engineering,Sungkyunkwan University,Suwon 440-746,Republic of Korea cDepartment of Dermatology,YongdongSeverance Hospital,Yonsei University College of Medicine,Seoul 135-720,Republic of Koreaa r t i c l e i n f o Article history:Received 23February 2013Received in revised form 1June 2013Accepted 2July 2013Available online 16July 2013Keywords:Sonophoresis Inertia Cavitation Microstreaminga b s t r a c tTransdermal drug delivery (TDD)has several significant advantages compared to oral drug delivery,including elimination of pain and sustained drug release.However,the use of TDD is limited by low skin permeability due to the stratum corneum (SC),the outermost layer of the skin.Sonophoresis is a tech-nique that temporarily increases skin permeability such that various medications can be delivered non-invasively.For the past several decades,various studies of sonophoresis in TDD have been performed focusing on parameter optimization,delivery mechanism,transport pathway,or delivery of several drug categories including hydrophilic and high molecular weight compounds.Based on these various studies,several possible mechanisms of sonophoresis have been suggested.For example,cavitation is believed to be the predominant mechanism responsible for drug delivery in sonophoresis.This review presents details of various studies on sonophoresis including the latest trends,delivery of various therapeutic drugs,sonophoresis pathways and mechanisms,and outlook of future studies.Ó2013Elsevier B.V.All rights reserved.Contents 1.Introduction ..........................................................................................................561.1.The use of ultrasound in transdermal drug delivery.....................................................................562.Mechanisms of sonophoresis.............................................................................................572.1.Thermal effects ..................................................................................................572.2.Cavitation.......................................................................................................572.2.1.Stable cavitation..........................................................................................572.2.2.Inertial cavitation .........................................................................................583.Transport pathway in the skin ...........................................................................................593.Transport pathway in the skin ...........................................................................................604.Synergistic effects ofsonophoresis.........................................................................................605.Applications of sonophoresis.............................................................................................616.New trends in sonophoresis .............................................................................................627.Conclusion ...........................................................................................................63Acknowledgements ....................................................................................................63References ...........................................................................................................631.Introduction1.1.The use of ultrasound in transdermal drug deliveryTransdermal drug delivery (TDD)is a non-invasive,topical administration method for therapeutic agents.TDD represents a considerable technological advance compared to oral delivery be-cause gastrointestinal degradation and first-pass metabolism through the liver are avoided [1].Despite these advantages,the0041-624X/$-see front matter Ó2013Elsevier B.V.All rights reserved./10.1016/j.ultras.2013.07.007⇑Corresponding author.Address:Department of Biomedical Engineering,Yonsei University,304Medical Industry Techno Tower,Wonju,Gangwon 220-710,Republic of Korea.Tel.:+82(0)337602961;fax:+82(0)337655483.E-mail address:jongbums@yonsei.ac.kr (J.Seo).use of TDD has been limited by innate barrier functions of the skin [2].The skin,which consists of several layers including the stratum corneum(SC),epidermis,and dermis,is the primary defense sys-tem of the body[3,4](Fig.1).The functions of skin include main-tenance of body temperature,blockage of ultra-violet radiation, and providing a defense against harmful external pathogens such as bacteria and toxins.The most important layer of the skin that provides substantial protection from pathogens is the SC[5].The SC is composed of corneocytes interspersed in a laminate of com-pressed keratin and intercorneocyte lipid lamellae,functions to conserve water and electrolytes,and is selectively permeable to certainsubstances such as drugs(see Fig.1).Naturally,the SC has a very low permeability to foreign molecules and is thus the main obstacle of TDD[6].Because of this barrier,available transdermal medicines are small molecules(<500Da)that are active at low blood concentrations of a few ng/ml or less[7,8].A number of methods have been proposed to overcome the natural barrier func-tion of the skin,such as skin patches[9,10],iontophoresis[11–13], use of chemical enhancers[14,15],and ultrasound methods [16–18].The traditional TDD systemincludes an impermeable patch and allows for controlled drug release over time.Occlusion of the skin traps the natural epidermal moisture and causes an increase in the water content and skin membrane swelling,which result in a compromise of the skin barrier function[19].Iontopho-resis,which utilizes small electric currents,can enhance transport across the skin by a number of possible mechanisms such as elec-trophoretic and electro-osmotic driving forces[20,21].Chemical enhancers such as Azone(1-dodecylazacycloheptan-2-one or laurocapram),DMSO(dimethyl sulphoxide),and surfactants in-crease transdermal drug transport via several mechanisms such as increased drug solubility in the donor formulation and drug par-titioning into the SC because of the solvent properties of these compounds[22–24].Specifically,DMSO,a commonly used topical analgesic,anti-inflammatory,and antioxidant,has been used in studies of skeletal muscle as a selective antioxidant or as a solvent for numerous drugs[25].Lastly,sonophoresis,which uses ultra-sound as a physical enhancer for systemic drug delivery,has also been shown to effectively deliver various types of drugs regardless of their electrical characteristics and can easily be coupled with other TDD methods to enhance drug delivery rates[26–28].Histor-ically,sonophoresis wasfirst reported in the1950s along with other therapeutic ultrasound applications such as noninvasiveIn this review,we focused on sonophoresis among TDD meth-ods.The remainder of this article is divided into six sections.The mechanism of sonophoresis is explained in section two,and sec-tion three covers the transport pathway of sonophoresis.In section four,a number of applications using various drugs are introduced. Sectionfive introduces new approaches to enhance sonophoresis. Thefinal section summarizes sonophoresis and provides an out-look of future studies.2.Mechanisms of sonophoresisAlthough sonophoresis is known to increase skin permeability, the fundamental mechanism is still not clearly understood or char-acterized.Several proposed mechanisms of sonophoresis include thermal effects by absorption of ultrasound energy and cavitation effects caused by collapse and oscillation of cavitation bubbles in the ultrasoundfield.Between these two effects,cavitation is be-lieved to be the predominant mechanism responsible for sonopho-resis[32–34].2.1.Thermal effectsWhen ultrasound passes through a medium,energy is partially absorbed[35].In the human body,ultrasound energy absorbed by tissue causes a local temperature increase that is dependent upon ultrasound frequency,intensity,area of the ultrasound beam,dura-tion of exposure,and the rate of heat removal by bloodflow or con-duction[36].The resultant temperature increase of the skin may enhance permeability due to an increase in diffusivity of the skin. Merino et al.reported enhanced transdermal permeability caused by this temperature increase[37].The skin temperature was in-creased by20°C with low frequency ultrasound(20kHz),and the delivery of mannitol was enhanced35-fold.However,the delivery of mannitol was only25%when the skin was heated to a similar level without ultrasound.These contradictory results sug-gest that thermal energy alone may not play a significant role in TDD,even though the temperature increase may affect the skin permeability.2.2.CavitationFig.1.Cross section of human skin(left).Barrier function of the stratum corneum(right).Thisfigure is reproduced from Ref.[3].D.Park et al./Ultrasonics54(2014)56–6557inertial cavitation,according to the activity of gaseous bubbles in relation to the acousticfield[16].2.2.1.Stable cavitationStable cavitation corresponds to a continuous oscillation of bub-bles around the equilibrium radius in response to relatively lower acoustic pressures in an acousticfield[39].Bubble oscillation around asymmetric boundary conditions by stable cavitation leads to a phenomenon called microstreaming,which can generate high velocity gradients and hydrodynamic shear stresses[40].[44,45].Second-order microstreamingflow may generateflow fields that develop shear stresses over a cell membrane,resulting in tension and stretching on membrane walls that cause channel activation,allowing delivery of compounds such as DNA for thera-peutic purposes[46,47].Fig.3shows possible sites in which the cavitation effect may occur.Cavitation nuclei and air pockets in TDD can be formed in intracellular and/or intercellular structures. In addition,bubbles can be formed on the skin and coupling med-ium at the skin surface.However,small gaseous nuclei are more resistant to oscillation within densely packed tissue.This becomes especially apparent during high frequency(P1MHz)sonophoresis when skin density variations occur rapidly due to relatively short wavelengths in comparison to that at low frequency.Therefore, cavitation may preferentially occur within the coupling medium between the ultrasound transducer and skin surface and may locally increase skin permeability[40,43,48,49].2.2.2.Inertial cavitationInertial cavitation corresponds to the violent growth and col-lapse of bubbles that can occur within a period of a single cycle or a several cycles and is dependent on acoustic pressure as well as frequency and the size of bubble[40,50,51].Apfel and Holland derived a mechanical index(MI)to represent the likelihood of iner-tial cavitation[52].Organizations such as the AIUM,NEMA,andFig.2.Illustration of the differences between uniform shear and surface diver-gence.(a)Uniform,high shear stress,no divergence and thus no stretch of cellsurface,(b)high rate of positive divergence(as well as shear stress),cell/cellmembrane in stretch-activated state,(c)high rate of negative divergence(as wellshear stress),and cell/cell membrane compressed together.Thisfigure is repro-duced from Ref.[43].Schematic sketching of cavitation occurring in a variety of sites such as coupling medium,skin surface,and skin tissue.Cavitation occurs preferentially keratinocytes and lipid bilayers.54(2014)56–65spherical bubble collapse yields high-pressure cores that emit shock waves with amplitudes exceeding 10kbar [54].The disrup-tion of a target exposed to such a pressure wave may occur through relative particle displacement,compressive failure,tensile stress,or shear strain [55].When a bubble collapses asymmetrically near boundary,it generally produces a well–defined,high velocity mi-cro-jet [56,57].Micro-jet distortion due to bubble collapse depends on the surface encountered by the bubble.If the surface is larger than the resonant size of the bubble (radius of 1–150l m at approximately 5MHz–20kHz),the resulting collapse will be in the form of a micro-jet (Fig.4)[33,52].As shown in Fig.3,inertial cavitation has the potential to take place in keratinocytes,lipid bi-layer regions,and crevices of hair follicles filled with coupling medium [47].Shock waves generated by inertial cavitation can cause structural alterations in the surrounding keratinocyte-lipid Fig.4.Three possible modes through which inertial cavitation may enhance SC permeability.(a)Spherical collapse near the SC surface emits shock waves,(b)impact of an acoustic micro-jet on the SC surface,and (c)micro-jets physically penetrating into the SC.The figure is reproduced from Ref.[32].Ultra-structural changes of the SC after treatment with low frequency sonophoresis.(a)After 5min of treatment with low frequency sonophoresis,m-sized pores (b,arrowheads)were observed on the SC surfaces that were observed on the normal SC surface.(b)Scanning electron microscopy findings surface of mouse skin treated with low frequency sonophoresis.l m.The figure is reproduced from Ref.[59].Fig.6.Schematic,cross-sectional drawing of skin.Skin is composed of a dermis and epidermis.Cells proliferate in the basal layer of the epidermis.Upon leaving the basal layer,cells begin to differentiate and migrate toward the skin surface.At the stratum granulosum and stratum corneum interface,final differentiation occurs,during which viable cells transform into dead cells filled with keratin (corneocytes).The corneocytes are surrounded by a cell envelope composed of cross-linked proteins and a covalently bound lipid envelope (see arrow).The corneocytes are embedded in lipid lamellar regions,which are orientated parallel to the corneocyte surface.Substances diffuse across the skin following the tortuous pathway in the intercellular lamellar regions (a)or other sites of structural disorganization caused enhancer compounds (b).C =corneocyte filled with keratin.Bar =100nm.This figure is reproduced from Ref.[63].nuclei on the skin plays a major role in enhancing cavitation effects.However,resonant frequencies of cavitation nuclei on the skin cannot be predicted.Therefore,adopting low frequency(e.g., 100kHz)ultrasound is considered suitable to increase cavitation effects in TDD[61].3.Transport pathway in the skin tional to the lipophilicity of drugs but is rather dependent on the creation of aqueous pathways across the SC(the trans-keratinocyte route).This thought is supported by recent research using nano-particles to show the importance of the penetration pathway in TDD.Lee et al.studied the ultra-structural changes on the SC sur-face and the penetration pathway of lanthanum nitrate(LaNO3) tracer in viable epidermis after combined treatment with low fre-quency sonophoresis and tape stripping(Fig.7)[46].After the application of sonophoresis,LaNO3was distributed within the cor-pathways of lanthanum nitrates after treatment with low frequency sonophoresis,and TEM images of SC after treatment ruthenium tetroxidefixation.(a)TEMfindings in the SC after application of LaNO3.In the absence of low frequency sonophoresis, spaces of the upper SC.(b)In contrast,after5min of treatment with low frequency sonophoresis,LaNO3wascorneocytes in the upper SC.(c,arrow)After5min of treatment of low frequency sonophoresis,2-to4-l m expansionsspaces of the SC.(d,arrow)In some sections,rolled-up lipids were also observed in the dilated lacunae.Scale bar=260 D.Park et al./Ultrasonics54(2014)56–65calcein,they observed that skin permeability was enhanced up to5 times by microneedles in comparison with passive diffusion,7 times for sonophoresis,and9times by a combination low fre-quency ultrasound with microneedles.Similar results were achieved with bovine serum albumin.Increment ratios of TDD were increased7times by microneedles,8.5times by sonophore-sis,and12times by a combination of low frequency ultrasoundand microneedles.These results showed the combination of sonopho-resis and microneedles greatly enhanced TDD rates compared to passive diffusion and either microneedles or ultrasound alone. Sonophoresis also exhibited a synergistic effect with iontophoresis [66].Le et al.investigated the synergistic effects of sonophoresis and iontophoresis using heparin as a model drug in the SC of pig skin.The penetrationflux of heparin applied with sonophoresis (20kHz)was13times higher than passiveflux over24h.Ionto-phoresis increased theflux of heparin by10times compared to theflux of the passive control.Transdermal haparinflux during iontophoresis across skin pretreated with ultrasound is approxi-mately2-fold higher compared to either ultrasound or iontophore-sis alone.In addition,a synergistic effect of sonophoresis and chemical treatment has been shown to enhance transdermal trans-port.Meidan et al.reported a synergistic effect with a combination technique of sonophoresis(1.1MHz and3.3MHz)and Azone as chemical enhancer[67].Synergistic effects between phonophore-sis and Azone treatment was observed for enhancement of percu-taneous hydrocortisone transport.Pretreatment using Azone enhancer increased the hydrocortisone permeability almost6-fold. Additionally,sonication of Azone-pretreated skin enhanced hydro-cortisone permeation by a further2.5-fold compared to Azone pre-treatment alone.Synergistic effects of sonophoresis and various enhancers of TDD have been confirmed through several studies. Combining certain technologies and techniques with sonophoresis may offer an advantageous method for TDD.The practicality and usefulness of combinations of these techniques will require further research.5.Applications of sonophoresisNumerous studies have been performed to confirm an enhance-ment of permeability by sonophoresis.Various applications of sonophoresis in TDD are summarized in Table1,including param-eter optimization,mechanisms,and delivery of numerous drugs such as hydrophilic and large molecules.Since the initial treatment of polyarthritis with hydrocortisone ointment by Fellingher and Schmidt in the1950s,the transdermal delivery of therapeutic drugs such as fentanyl,caffeine,heparin,ketoprofen and insulin has become a major concern for clinical medicine.Borcaud et al. evaluated effects of sonophoresis at20kHz with2.5W/cm2on transdermal transport of fentanyl and caffeine across both hairless rat and human skin[72].Fentanyl is generally used to relieve pain in either surgery or cancer patients,while caffeine is the most com-mon stimulant used for treatment of lipodystrophy[100,101].The results showed that sonophoresis enhanced TDD of fentanyl(about 35-fold greater than control)and caffeine(about4-fold greater than control)across human and hairless rat skin.In general,hepa-rin,which is a commonly used anticoagulant,is administered by intravenous or subcutaneous injections for the treatment and pre-vention of venous thromboembolism.Mitragotri et al.performed in vitro experiments with sonophoresis of20kHz with7W/cm2 (I SAPA)to deliver heparin or low-molecular weight heparin across the skin[79].Biologic activity of transdermally delivered heparin was measured using activated clotting time assays and by measur-ing anti-Xa(aXa)activity.Transdermally delivered low molecular weight heparin resulted in raised aXa levels in the blood.The transdermalflux of heparin was found to be approximately21-fold higher through sonicated skin compared to non-sonicated skin. This result was in strong contrast to subcutaneous or intravenous injections of LMWH,which resulted in only temporary elevations of aXa levels.Mitragotri et al.proposed that patients might use a product based on this technology throughout the day to provide sustained heparin concentration in the blood.The dose of heparin may be controlled through skin permeability,treatment area,or LMWH concentration in the reservoir of the patch system under the effects of sonophoresis.Ketoprofen is a non-steroidal anti-inflammatory drug predominantly used for treatment of rheuma-toid arthritis and osteoarthritis[88]as well as to relieve minor aches and menstrual pain[102].Herwadkar et al.tested the effects of sonophoresis at20kHz with6.9W/cm2for delivery of ketopro-fen across the skin[89].In vitro experiments were performed on excised hairless rat skin over a period of24h using Franz diffusion cells.Sonophoresis significantly enhanced the permeation of keto-profen from74.87±5.27l g/cm2with passive delivery to 491.37±48.78l g/cm2with sonophoresis.Furthermore,drug level in skin layers increased from34.69±7.25l g following passive per-meation to212.62±45.69l g following sonophoresis.These re-sults show that sonophoresis at a frequency of20kHz is an effective enhancement technique to improve transdermal and top-ical delivery of ketoprofen.Among the therapeutic drugs used in TDD,noninvasive trans-dermal delivery of insulin has received great attention because of the increasing incidence of diabetes,one of the most costly dis-eases in all patient populations and age groups[103].Management of diabetes often requires painful,repetitive insulin injections up to three or four times daily[86].Non-invasive insulin delivery through the skin may be a preferable technique for diabetic pa-tients over traditional invasive and painful subcutaneous insulin injections.Insulin,a hormone produced by the pancreas,plays a central role in the regulation of carbohydrate and fat metabolism in the body.Numerous studies of transdermal insulin delivery have been performed by many researchers[3,16,83,84,86,87].The feasi-bility of sonophoresis for insulin delivery has been evaluated by Smith et al.[84]in an effort to enhance the in vitro transport of insulin across human skin.Specifically,experiments were carried out with two types of lightweight cymbal transducer arrays,a stack array with an intensity(I SPTP)of15.4±0.6mW/cm2and a standard array with an intensity(I SPTP)of173.7±1.2mW/cm2at pared with passive transmission(4.1±0.5U)over an exposure period of1h,the standard array facilitated a greater than 7-fold increase in the non-invasive transdermal transport of HumulinÒR insulin(45.9±12.9U).The results using HumalogÒinsulin with the standard array showed a4-fold increase in the sonophoresis-facilitated transmission over that seen in the control group.Additionally,Mitragotri et al.tested the recovery of the skin barrier properties after short(1h)as well as long(5h)ultrasound exposures(20KHz,125mW/cm2,100ms pulses applied every sec-ond).They measured the electrical resistance and transdermalflux of water for up to12h after ultrasound exposure.After a1h expo-sure,the epidermal permeability to water measured within2h postexposure was comparable to the passive epidermal permeabil-ity to water.Within2h of ultrasound treatment,resistance in-creased by a factor of1.2compared to the value immediately after ultrasound treatment.In the case of a5h exposure,the permeability was approximately6times higher than the passive permeability to water.This value continued to decrease to a factor of2times the passive permeability of water at12h post-exposure. In this case,2h after discontinuation of the ultrasound,the resis-tance increased by approximately2-fold compared to that at the end of ultrasound exposure.This result suggests that the sonopho-reticflux of permeants exhibited significant recovery after ultra-D.Park et al./Ultrasonics54(2014)56–6561sound was discontinued,and sonophoresis does not cause long-term changes in the epidermal barrier properties of skin[40].Maruani et al.surveyed adverse effects that were systematically recorded by the research engineer and a dermatologist[80].The most frequent adverse effects reported during or after sonophore-sis included skin erythema,pain,and tinnitus.These events in-creased with increasing ultrasound intensity,which generates relatively high thermal and cavitation effects on the skin.However, erythema and purpura quickly regressed after sonophoresis was stopped.In addition,tinnitus,reported in23.5%of cases,also re-solved after ultrasound was terminated.The pain associated with ultrasound was not constant and showed large variations among subjects.The pain disappeared with the use of a discontinuous ultrasound mode.These results suggest that optimization of ultra-sound parameters is essential to reduce adverse effects of sonophoresis.6.New trends in sonophoresisUnder the assumption that cavitation is the principal mecha-nism in sonophoresis due to the formation of intercellular lipid channels and disordering of lipid bilayers,enhancement of sono-phoresis cavitation activity has been recently attempted.MI,which was developed as a method to express the likelihood of cavitation, increases with decreasing center frequency and increasing peak rarefaction pressure[53].Adopting low frequency sonophoresis is one way to increase existing cavitation bubbles[18,31,32].When the frequency is low enough,a quasi-staticfield of sufficiently small bubbles can be assumed.Given that small bubbles rapidly grow and collapse with exposure to quasi-static rarefaction pres-sure,this observed activity may be related to inertial cavitation or asymmetric bubble collapse[104,105].Low-frequency sonopho-resis has thus been the topic of extensive research over the last 20years.The effects of low frequency sonophoresis have beenTable1Research on sonophoresis in TDD.Compound M.W(Dalton)Skin tissue Ultrasound parameter Ref No.Frequency Intensity Sonication time DutyAldosterone360Human(in vitro)20kHz125mW/cm210%[40] Arnica Montana Rat(in vitro)1MHz0.5W/cm23min33%[68] Ascorbic acid176Pig ear(in vitro)1MHz 2.3,3.2W/cm21,10,20min CW mode[69] Butanol74Human(in vitro)20kHz125mW/cm210%[40] Bovine serum albumin66000Rat(in vivo)(in vitro)20kHz30%amplitude2min50%[70] Caffeine194Pig(in vitro)3MHz0.2W/cm2240min CW mode[71] Caffeine194Human(in vitro),Rat(in vitro)20kHz 2.5W/cm210min,1h CW mode10%[72] Caffeine194Pig(in vitro)20kHz0.37–3.7W/cm25–1200s10,33,100%[73] Calcein623Pig(in vitro)20,40,60kHz7.5W/cm220min50%[74] Calcium40Rat(in vivo)20kHz1W/cm2Less than5min50%[31] Corticosterone346Human(in vitro)20kHz125mW/cm210%[40] Dexamethasone392Human(in vivo)1,3MHz1W/cm210min CW mode[75] Diclofenac296Human(in vivo)1MHz0.5W/cm25min[76] Estradiol272Human(in vitro)20kHz125mW/cm210%[40] Fentanyl336Human(in vitro),Rat(in vitro)20kHz 2.5W/cm210min,1h CW mode10%[72] FITC-dextran4000,20,000,150,000Rat(in vivo) 1.12,2.47MHz330mW/cm230min1%[77] Glycerol92Pig(in vitro) 1.1MHz600kPa60min10%[78] Heparin Average MW of18,000Pig(in vitro)20kHz7W/cm210min50%[79] Histamine184Human(in vivo)36kHz 2.7,3.50W/cm25min28.6,37.5%[80] Hyaluronan1000Rabbit(in vivo)1MHz400mW/cm210min CW mode[81] Hydrocortisone362Human(in vitro)20kHz 3.7W/cm230,45s10%[82] Insulin5807Rabbit(in vivo)105kHz5kPa90min50%[83] Insulin5807Human(in vitro)20kHz12.5–225mW/cm24h10%[16] Insulin5807Human(in vitro)20kHz173.7±1.2mW/cm21h20%[84]Insulin5807Rabbit(in vivo)20kHz100mW/cm220%[85] Insulin5807Pig(in vivo)20kHz100mW/cm260min20%[86] Insulin5807Rabbit(in vivo)225.8–1016kHz10min[87] Ketoprofen254Human(in vivo)100Hz 1.5W/cm25min CW mode20%[88]1MHzKetoprofen254Rat(in vitro)20kHz 6.9W/cm20.5–2min50,100%[89] Ketorolac-tromethamine376Rat(in vitro)1MHz1–3W/cm230min CW mode[90]Lanthanum nitrate433Mouse(in vivo)25kHz800m W/cm25min CW mode[59] Mannitol183Pig(in vitro)20kHz 1.6–14W/cm2 1.5h10,50%[31] Mannitol183Rat(in vivo)1MHz 1.5.3W/cm23–5min CW mode[69] Morphine285Mouse(in vitro)40kHz0.13–0.44W/cm212–18min CW mode[91] Oligonucleotides Second generationchemistriesPig(in vitro)20kHz 2.4W/cm210min50%[92]Peptide dendrimer Human(in vitro)20kHz7–8W/cm230min50%[93] Quantum dot20nm diameter Pig(in vitro)20kHz 2.4W/cm250%[94] Salicylic acid138Rat(in vivo)20kHz125mW/cm210%[40] Salicylic acid138Guinea pig(in vivo)2,10,16MHz0.2W/cm25–20min[17] Sodium lauryl sulfate288Pig(in vitro)20,40,60kHz7.5W/cm220min50%[95] Sucrose342Human(in vitro)20kHz125mW/cm210%[40] Sucrose342Pig(in vitro)20kHz7.5mW/cm22min50%[96] Tetanus toxoid150,000Mouse(in vivo)20kHz 2.4W/cm280s50%[97]Triamcinolone-acetonide 434Mouse(in vitro)1,3MHz 1.0,2.5mW/cm210h CW mode[98]Pulse modeUrea60Human(in vitro)20kHz7.2mW/cm27–8min50%[99] Water18Human(in vitro)20kHz125mW/cm210%[40] 62 D.Park et al./Ultrasonics54(2014)56–65。