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Ultrastructural Evidence of Stratum Corneum Permeabilization Induced by Photomechanical Waves

2003; Elsevier BV; Volume: 121; Issue: 1 Linguagem: Inglês

10.1046/j.1523-1747.2003.12302.x

1523-1747

Gopinathan K. Menon, Nikiforos Kollias, Apostolos G. Doukas,

Essential Oils and Antimicrobial Activity

Photomechanical waves (high amplitude pressure transients generated by lasers) have been shown to permeabilize the stratum corneum in vivo and facilitate the transport of macromolecules into the viable epidermis. The permeabilization of the stratum corneum is transient and its barrier function recovers. Sites on the volar forearm of humans were exposed to photomechanical waves and biopsies were obtained immediately after the exposure and processed for electron microscopy. Electron microscopy showed an expansion of the lacunar spaces within the stratum corneum lipid bilayers but no changes in the organization of the secreted lamellar bodies at the stratum corneum–stratum granulosum boundary. The combination of photomechanical waves and sodium lauryl sulfate enhances the efficiency of transdermal delivery and delays the recovery of the barrier function of the stratum corneum. Electron microscopy from sites exposed to photomechanical waves and sodium lauryl sulfate showed that the lacunar spaces expanded significantly more and the secreted lamellar bodies also appeared to be altered. In either case, there were no changes in the papillary dermis. These observations support the hypothesis that the photomechanical waves induce the expansion of the lacunar spaces within the stratum corneum leading to the formation of transient channels. Photomechanical waves (high amplitude pressure transients generated by lasers) have been shown to permeabilize the stratum corneum in vivo and facilitate the transport of macromolecules into the viable epidermis. The permeabilization of the stratum corneum is transient and its barrier function recovers. Sites on the volar forearm of humans were exposed to photomechanical waves and biopsies were obtained immediately after the exposure and processed for electron microscopy. Electron microscopy showed an expansion of the lacunar spaces within the stratum corneum lipid bilayers but no changes in the organization of the secreted lamellar bodies at the stratum corneum–stratum granulosum boundary. The combination of photomechanical waves and sodium lauryl sulfate enhances the efficiency of transdermal delivery and delays the recovery of the barrier function of the stratum corneum. Electron microscopy from sites exposed to photomechanical waves and sodium lauryl sulfate showed that the lacunar spaces expanded significantly more and the secreted lamellar bodies also appeared to be altered. In either case, there were no changes in the papillary dermis. These observations support the hypothesis that the photomechanical waves induce the expansion of the lacunar spaces within the stratum corneum leading to the formation of transient channels. lamellar bodies photomechanical waves stratum corneum sodium lauryl sulfate Transdermal drug delivery has been the subject of extensive research during the past two decades. The main challenge to this mode of delivery is the permeability barrier of skin, located in the stratum corneum (SC). Several physical and chemical methods to surmount the barrier have been devised, with varying degrees of effectiveness (Menon and Elias, 2001Menon G.K. Elias P.M. The epidermal barrier and strategies for surmounting it: An overview.in: Hengge U.R. Volc-Platzer B. The Skin and Gene Therapy. Blackwell Publishing, Berlin2001: 3-26Crossref Google Scholar). Physical methods have the advantage of decreased skin irritant/allergic responses, as well as no interaction with the drugs being delivered. Some of the physical techniques under development or investigation include iontophoresis (Singh and Singh, 1993Singh S. Singh J. Transdermal drug delivery by passive diffusion and iontophoresis: A review.Med Phys Rev. 1993; 13: 569-621Google Scholar;Jadoul et al., 1999Jadoul A. Bouwastra J. Peat V. Effects of iontophoresis and electroporation on the stratum corneum—Review of biophysical studies.Adv Drug Deliv Rev. 1999; 35: 89-105Crossref PubMed Scopus (125) Google Scholar), electroporation (Prausnitz et al., 1993Prausnitz M.P. Bose V.G. Langer R. Weaver J.C. Electroporation of mammalian skin: A mechanism to enhance transdermal drug delivery.Proc Natl Acad Sci USA. 1993; 90: 10504-10508Crossref PubMed Scopus (555) Google Scholar;Jadoul et al., 1999Jadoul A. Bouwastra J. Peat V. Effects of iontophoresis and electroporation on the stratum corneum—Review of biophysical studies.Adv Drug Deliv Rev. 1999; 35: 89-105Crossref PubMed Scopus (125) Google Scholar), and phonophoresis (Bommannan et al., 1992Bommannan D. Okuyama H. Stauffer P. Guy R.H. Sonophoresis 1. The use of high-frequency ultrasound to enhance transdermal delivery.Pharm Res. 1992; 9: 559-564Crossref PubMed Scopus (141) Google Scholar;Mitragotri et al., 1995aMitragotri S. Edwards D.A. Blankschtein D. Langer R. Mechanistic study of ultrasonically-enhanced transdermal drug-delivery.J Pharm Sci. 1995; 84: 697-706Crossref PubMed Scopus (301) Google Scholar,Mitragotri et al., 1995bMitragotri S. Blankschtein D. Langer R. Ultrasound-mediated transdermal protein delivery.Science. 1995; 269: 850-853Crossref PubMed Scopus (694) Google Scholar). It has been recently shown that high amplitude pressure transients generated by lasers photomechanical waves (PW) are effective for transdermal delivery (Lee et al., 1998Lee S. McAuliffe D.J. Flotte T.J. Kollias N. Doukas A.G. Photomechanical transcutaneous delivery of macromolecules.J Invest Dermatol. 1998; 111: 925-929Crossref PubMed Scopus (96) Google Scholar,Lee et al., 1999Lee S. Kollias N. McAuliffe D.J. Flotte T.J. Doukas A.G. Topical delivery in humans with a single photomechanical wave.Pharm Res. 1999; 16: 1717-1721Crossref PubMed Scopus (76) Google ScholarLee et al., 2001aLee S. McAuliffe D.J. Mulholland S.E. Doukas A.G. Photomechanical transdermal delivery of insulin in vivo.Lasers Surg Med. 2001; 28: 282-285Crossref PubMed Scopus (44) Google Scholar,Lee et al., 2001cLee S. McAuliffe D.J. Flotte T.J. Kollias N. Doukas A.G. Photomechanical transdermal delivery. The effect of laser confinement.Lasers Surg Med. 2001; 28: 344-347Crossref PubMed Scopus (59) Google Scholar,Lee et al., 2002Lee S. McAuliffe D.J. Kollias N. Flotte T.J. Doukas A.G. Photomechanical delivery of 100-nm microspheres through the stratum corneum: Implications for transdermal drug delivery.Lasers Surg Med. 2002; 31: 207-211Crossref PubMed Scopus (41) Google Scholar;Gonzàlez et al., 2001Gonzàlez S. Lee S. Gonzàlez E. Doukas A.G. Rapid antigen delivery with photomechanical waves for inducing allergic skin reaction in the DNCB-sensitized hairless guinea pig animal model.Am J Contact Dermat. 2001; 12: 162-165Crossref PubMed Google Scholar;Ogura et al., 2002Ogura M. Sato S. Kuroki M. et al.Transdermal delivery of photosensitizer by the laser-induced stress waves in combination with skin heating.Jpn J Appl Phys. 2002; 41: L814-L816Crossref Google Scholar).Lee et al., 1998Lee S. McAuliffe D.J. Flotte T.J. Kollias N. Doukas A.G. Photomechanical transcutaneous delivery of macromolecules.J Invest Dermatol. 1998; 111: 925-929Crossref PubMed Scopus (96) Google Scholar have shown that 40 kDa dextran could be delivered ≈400 μm deep into the skin in vivo in an animal model. Furthermore, PW facilitated transdermal delivery of insulin in a diabetic rat model in sufficient amount to reduce the concentration of blood glucose (Lee et al., 2001aLee S. McAuliffe D.J. Mulholland S.E. Doukas A.G. Photomechanical transdermal delivery of insulin in vivo.Lasers Surg Med. 2001; 28: 282-285Crossref PubMed Scopus (44) Google Scholar). PW, laser-generated shock waves, and laser-generated stress waves are terms that have been used to describe high amplitude pressure pulses generated during ablation by high-power lasers (Doukas and Flotte, 1996Doukas A.G. Flotte T.J. Physical characteristics and biological effects of laser-induced stress waves.Ultrasound Med Biol. 1996; 22: 152-164Abstract Full Text PDF Scopus (156) Google Scholar;Lee and Doukas, 1999Lee S. Doukas A.G. Laser generated stress waves and their effects on the cell membrane.IEEE J Select Topics Quant Electr. 1999; 5: 997-1003Crossref Scopus (47) Google Scholar;Doukas and Lee, 2000Doukas A.G. Lee S. Photomechanical drug delivery.Biomedical Optoacoustics. 2000; 3916 (Proc SPIE): 188-197Crossref Google ScholarandDoukas and Lee, 2000Doukas A.G. Lee S. Photomechanical drug delivery.Biomedical Optoacoustics. 2000; 3916 (Proc SPIE): 188-197Crossref Google Scholar). In ablation, the laser radiation produces plasma on the surface of the target, which moves away from the surface at supersonic speed (Srinivasan, 1986Srinivasan R. Ablation of polymers and biological tissue by ultraviolet light.Science. 1986; 234: 559-565Crossref PubMed Scopus (497) Google Scholar). A high-pressure pulse (PW) is generated inside the target by the imparted recoil momentum (Perri, 1973Perri A.N. Theory of momentum transfer to a surface with a high-power laser.Phys Fluids. 1973; 16: 1435-1440Crossref Scopus (144) Google Scholar). The PW propagates through the target and impinges on to the skin. The PW generated by ablation are unipolar compression waves that do not show a measurable tensile component, and thus exclude biologic effects induced by cavitation. PW have also been shown to induce transient permeabilization of the cell membrane in vitro (Lee et al., 1996Lee S. Anderson T. Zhang H. Flotte T.J. Doukas A.G. Alteration of the plasma membrane by stress transients in vitro.Ultrasound Med Biol. 1996; 22: 1285-1293Abstract Full Text PDF PubMed Scopus (100) Google Scholar;McAuliffe et al., 1997McAuliffe D.J. Lee S. Flotte T.J. Doukas A.G. Stress-wave-assisted transport through the plasma membrane in vitro.Laser Surg Med. 1997; 20: 216-222Crossref PubMed Scopus (47) Google Scholar;Mulholland et al., 1999Mulholland S.E. Lee S. McAuliffe D.J. Doukas A.G. Cell loading with laser-generated stress waves. The role of stress gradient.Pharm Res. 1999; 16: 514-518Crossref PubMed Scopus (62) Google Scholar;Soughayer et al., 2000Soughayer J.S. Krasieva T. Jacobson S.C. Ramsey J.M. Tromberg B.J. Allbritton N.L. Characterization of cellular optoporation with distance.Anal Chem. 2000; 72: 1342-1347Crossref PubMed Scopus (101) Google Scholar), as well as facilitate the delivery of molecules into microbial biofilms (Soukos et al., 2000Soukos N.S. Socransky S.S. Mulholland S.E. Lee S. Doukas A.G. Photomechanical drug delivery into bacterial biofilms.Pharm Res. 2000; 17: 405-409Crossref PubMed Scopus (58) Google Scholar). The mechanism of the SC permeabilization by PW is not known. Molecular delivery can occur, however, whether the molecules are present during the application of the PW or introduced after the PW (Lee and Doukas, 1999Lee S. Doukas A.G. Laser generated stress waves and their effects on the cell membrane.IEEE J Select Topics Quant Electr. 1999; 5: 997-1003Crossref Scopus (47) Google Scholar,Lee et al., 2001bLee S. McAuliffe D.J. Kollias N. Flotte T.J. Doukas A.G. Permeabilization and recovery of the stratum corneum in vivo: The synergy of photomechanical waves and sodium lauryl sulfate.Lasers Surg Med. 2001; 29: 145-150Crossref PubMed Scopus (31) Google Scholar). Given the short duration of the PW (≈300 ns), this observation suggests that the action of the PW is probably limited to the permeabilization of the SC. The molecular transport through the SC is carried out by diffusion of the molecules through the channels induced by the PW. Presently, we have investigated the ultrastructural changes in human skin in vivo and ex vivo following PW exposure in order to elucidate the mechanism of SC permeabilization under these conditions. The research on human subjects followed the tenets of the declaration of the Helsinki Convention. The protocol was approved by the Massachusetts General Hospital Subcommittee on Human Studies and informed consent from the subjects was obtained. This investigation formed part of an ongoing study on PW in transdermal delivery. Seven panelists were involved in these studies, and a total of eight biopsies were taken (four each from two panelists) for electron microscopy. For the ex vivo measurements, full thickness human skin was obtained from NDRI (Presbyterian Hospital, Philadelphia, PA) and stored in the freezer until ready to use. The skin was thawed to room temperature prior to the experiments. It was blotted dry and set on the bottom of a Petri dish, supported on the bench, and exposed to PW as was done for the in vivo experiments. Figure 1(a–c) show the procedure of applying the PW on the volar forearm of a panelist. The schematic of the experimental set-up used is shown in Figure 1(d). A flexible washer, ≈1.5 mm thick and ≈7 mm inside diameter, was used as the reservoir for the acoustic coupling medium. The washer was attached to the skin of a panelist with grease (Figure 1a). The grease was applied only around the edge of the washer as a sealant to prevent the solution from leaking out. The reservoir was filled with either sterilized water or an aqueous solution of sodium lauryl sulfate (SLS) 2% w/v (Sigma, St Louis, Missouri) (Figure 1a). The water or the SLS solution in the reservoir acted as a coupling medium, which allowed the PW to propagate from the target to the surface of the skin. In addition, the water or the SLS solution could diffuse into the SC and modify the SC architecture. The rationale for using SLS was to investigate the effects of anionic surfactants on the SC. The combination of SLS and PW has been shown to enhance the efficiency of transdermal delivery and delay the recovery of the barrier function of the SC (Lee et al., 2001bLee S. McAuliffe D.J. Kollias N. Flotte T.J. Doukas A.G. Permeabilization and recovery of the stratum corneum in vivo: The synergy of photomechanical waves and sodium lauryl sulfate.Lasers Surg Med. 2001; 29: 145-150Crossref PubMed Scopus (31) Google Scholar). PW were generated by ablation of a target by a laser pulse from a Q-switched ruby laser. The ruby laser produced pulses of 23 ns duration at 694.3 nm and 2 J pulse energy. The laser was operating at 1 Hz repetition rate. The target was taken from the sides of black polystyrene tissue culture plates (Dynatech Laboratories, Chantily, Virginia). The target was ≈1 mm thick and was placed on top of the washer in contact with the solution Figure 1(b). An articulated arm was used to deliver the laser on to the target Figure 1(c). The beam size at the target was ≈6 mm in diameter to achieve fluence of ≈7 J per cm2. The laser pulse was completely absorbed by the target so that only the PW were applied on to the skin with no discomfort or pain to the panelists. Under the experimental conditions, the PW characteristics were as follows: Peak pressure ≈500 bar (1 bar=0.987 atmosphere), rise time ≈30 ns and duration ≈300 ns. Sites on the inner volar forearm were exposed to a single PW with water or SLS solution as the acoustic coupling medium. In addition, one of the sites was exposed to 10 PW with water as the coupling medium. The water or the SLS solution was removed immediately after the application of the PW, the skin was wiped clean with lab towels (Kimwipes, Kimberly Clark) and shave biopsies from the subjects were obtained. The same procedure was followed for the control sites except that no PW was applied. Experiments were also performed on human cadaver skin ex vivo on full thickness epidermis. Sites on the cadaver skin were exposed to a single PW with water acting as the coupling medium. The biopsies as well as the samples from the cadaver skin were obtained immediately after the experiments and fixed in Karnovsky's fixative for 1 h at room temperature and then overnight at 4°C in the same fixative for electron microscopy. The samples then were washed in cacodylate buffer and postfixed either in 1% OsO4 or 0.5% RuO4 (Madison et al., 1987Madison K.C. Swartzendruber D.C. Wertz P.W. Downing D.T. Presence of intact intercellular lipid lamellae in the upper layers of the stratum corneum.J Invest Dermatol. 1987; 88: 714-718Abstract Full Text PDF PubMed Google Scholar). Subsequently, the samples were dehydrated in graded series of ethanol, embedded in a low viscosity epon-epoxy mixture and sectioned (McNutt and Crain, 1981McNutt N.S. Crain W.L. Quantitative electron microscope comparison of lymphatic nuclear contours in mycosis fungoides and in benign infiltrates of the skin.Cancer. 1981; 47: 163-166Crossref Scopus (75) Google Scholar). Thin sections were double stained with uranyl acetate and lead citrate and examined under an electron microscope operating at 80 kV. Routine electron microscopy did not reveal any noticeable differences between the control sites and PW exposed sites when water was used as the coupling medium (micrographs not shown). The nucleated epidermis as well as the dermis maintained their typical ultrastructural features with no indication of damage either in the extracellular matrix or the cellular components. Comparison of the SC exposed to one PW with that exposed to 10 PW with water as the coupling medium showed expansion of the extracellular areas in the lowermost SC layers (Figure 2a, b). On the other hand, one PW treatment with SLS as the coupling medium resulted in large-scale dilation in the SC extracellular space even in the stratum compactum (Figure 2c). The SC–stratum granulosum boundary showed normal features of the lamellar body's secretion indicating an unaltered barrier function. When the SLS solution was used as the coupling medium, however, the secreted lamellar bodies' contents appeared to have been disrupted both at the stratum granulosum–SC boundary as well as between the outermost and the underlying stratum granulosum cells (Figure 3a, b).Figure 3(a) Ultrastructure of epidermis shows no noticeable structural alterations following a single PW with water as the coupling medium. (b) When sodium lauryl sulfate solution was used instead of water, morphologic signs of lipid extraction at SC–stratum granulosum interface as well as between the two layers of stratum granulosum (arrows).View Large Image Figure ViewerDownload (PPT) Electron microscopy of RuO4 postfixed samples of control SC showed the basic extracellular lamellar unit, i.e., the minimal repeating unit, comprising of a series of electron-dense and electron-lucent lamellae (Figure 4). Following exposure to PW, either one or 10 pulses with water as the coupling medium, the SC extracellular domains showed many highly expanded near continuous lacunar domains (Figure 5); however, they were not present at every level and almost normal bilayers could be seen in the extracellular spaces of corneocytes that were immediately below. When SLS was used as the coupling medium, the lacunar domains were much more prominent with clumping of electron-dense precipitates either lining the expanded lacunar domains (Figure 6a) or occupying much of the lacunar domains (Figure 6b). No ultrastructural changes were seen in the morphology of individual corneocytes. The dermis showed no structural changes following PW treatment, whether water or SLS was used as the coupling medium (micrographs not shown).Figure 5High magnification electron micrograph of the SC exposed to a single PW (water as coupling medium) shows expanded lacunar domains within the intercellular lamellae.View Large Image Figure ViewerDownload (PPT)Figure 6(a) Exposure of the SC to a single PW with water as the acoustic coupling medium. (b) Exposure of the SC to a single PW with sodium lauryl sulfate as the acoustic coupling medium resulted in large-scale expansions of the lacular domains, which often showed precipitates or flocculant materials representing the surfactant.View Large Image Figure ViewerDownload (PPT) When cadaver skin was used, the microscopic features were inconsistent. The lacunar domains were found to be expanded in both of the controls as well as the PW exposed samples. Additionally, in both of the samples, cytosol of the granulocytes showed some structural disruption, and the secreted lamellar bodies' contents at the SC–stratum granulosum interface also appeared disordered (Figure 7a, b). Previous studies have shown that a single PW can permeabilize the SC and facilitate the delivery of macromolecules into the epidermis and dermis (Lee et al., 1998Lee S. McAuliffe D.J. Flotte T.J. Kollias N. Doukas A.G. Photomechanical transcutaneous delivery of macromolecules.J Invest Dermatol. 1998; 111: 925-929Crossref PubMed Scopus (96) Google Scholar,Lee et al., 1999Lee S. Kollias N. McAuliffe D.J. Flotte T.J. Doukas A.G. Topical delivery in humans with a single photomechanical wave.Pharm Res. 1999; 16: 1717-1721Crossref PubMed Scopus (76) Google ScholarLee et al., 2001aLee S. McAuliffe D.J. Mulholland S.E. Doukas A.G. Photomechanical transdermal delivery of insulin in vivo.Lasers Surg Med. 2001; 28: 282-285Crossref PubMed Scopus (44) Google Scholar,Lee et al., 2002Lee S. McAuliffe D.J. Kollias N. Flotte T.J. Doukas A.G. Photomechanical delivery of 100-nm microspheres through the stratum corneum: Implications for transdermal drug delivery.Lasers Surg Med. 2002; 31: 207-211Crossref PubMed Scopus (41) Google Scholar;Gonzàlez et al., 2001Gonzàlez S. Lee S. Gonzàlez E. Doukas A.G. Rapid antigen delivery with photomechanical waves for inducing allergic skin reaction in the DNCB-sensitized hairless guinea pig animal model.Am J Contact Dermat. 2001; 12: 162-165Crossref PubMed Google Scholar;Ogura et al., 2002Ogura M. Sato S. Kuroki M. et al.Transdermal delivery of photosensitizer by the laser-induced stress waves in combination with skin heating.Jpn J Appl Phys. 2002; 41: L814-L816Crossref Google Scholar). For example, 40 kDa dextran was delivered into the dermis to a depth of ≈400 μm in a rat animal model (Lee et al., 1998Lee S. McAuliffe D.J. Flotte T.J. Kollias N. Doukas A.G. Photomechanical transcutaneous delivery of macromolecules.J Invest Dermatol. 1998; 111: 925-929Crossref PubMed Scopus (96) Google Scholar). The maximum size that has been presently delivered through the SC is 100 nm latex microspheres (Lee et al., 2002Lee S. McAuliffe D.J. Kollias N. Flotte T.J. Doukas A.G. Photomechanical delivery of 100-nm microspheres through the stratum corneum: Implications for transdermal drug delivery.Lasers Surg Med. 2002; 31: 207-211Crossref PubMed Scopus (41) Google Scholar). The permeabilization of the SC is transient and the barrier function recovers (Lee et al., 1998Lee S. McAuliffe D.J. Flotte T.J. Kollias N. Doukas A.G. Photomechanical transcutaneous delivery of macromolecules.J Invest Dermatol. 1998; 111: 925-929Crossref PubMed Scopus (96) Google Scholar,Lee et al., 1999Lee S. Kollias N. McAuliffe D.J. Flotte T.J. Doukas A.G. Topical delivery in humans with a single photomechanical wave.Pharm Res. 1999; 16: 1717-1721Crossref PubMed Scopus (76) Google ScholarLee et al., 2001bLee S. McAuliffe D.J. Kollias N. Flotte T.J. Doukas A.G. Permeabilization and recovery of the stratum corneum in vivo: The synergy of photomechanical waves and sodium lauryl sulfate.Lasers Surg Med. 2001; 29: 145-150Crossref PubMed Scopus (31) Google Scholar). The application of PW for transdermal delivery has a number of appealing advantages, such as the substantial penetration depth as well as the large size of molecules that can be delivered. PW can be a useful method for delivering proteins and genes. Furthermore, PW can also permeabilize the cell membrane thus facilitating drug delivery into the cytoplasm. The PW do not appear to cause injury to the viable skin and do not cause pain or discomfort (Lee et al., 1999Lee S. Kollias N. McAuliffe D.J. Flotte T.J. Doukas A.G. Topical delivery in humans with a single photomechanical wave.Pharm Res. 1999; 16: 1717-1721Crossref PubMed Scopus (76) Google Scholar). Sites exposed to one and 10 PW with water as the coupling medium, when postfixed with RuO4, showed many highly expanded SC extracellular domains near continuous lacunar domains. The lacunae have been defined as electron-luscent areas embedded within the lipid bilayers spanning the SC extracellular domains, and considered as the putative pores (Menon and Elias, 1997Menon G.K. Elias P.M. Morphologic basis of a pore-pathway in mammalian stratum corneum.Skin Pharmcol. 1997; 10: 235-246Crossref PubMed Scopus (121) Google Scholar). The expansion of the lacunar domains could possibly create transient pores that enable drug delivery through the SC and into the epidermal and dermal compartments. These pores were not present at every level, i.e., between every corneocyte, as normal bilayers could be seen in the extracellular spaces of corneocytes that were immediately below. It should be kept in mind, however, that the lacunae seen in electron micrographs are in essence cross-sections of the three-dimensional trabecular network that may form a continuous, permeable lacunar system or a "pore-pathway". The use of SLS solution as the coupling medium showed a further expansion of the lacunae domains, resulting in larger pores. These observations are consistent with previous studies in which low concentrations of SLS under a patch for 24 h (Fartasch, 1997Fartasch M. Ultrastructure of the epidermal barrier after irritation.Microsc Res Tech. 1997; 37: 193-199Crossref PubMed Scopus (105) Google Scholar) or on skin ex vivo (Hafteck et al, 1998) where lipid bilayers often showed fissuring and splitting along the horizontal plane of the corneocyte spaces.Menon et al., 1998Menon G.K. Lee S.H. Roberts M.S. Ultrastructural effects of some solvents and vehicles on the stratum corneum and other skin components: Evidence for an extended mosaic-partitioning model of the skin barrier.in: Roberts M.S. Walters K.A. Dermal Absorption and Toxicity Assessment. Blackwell Publishing, New York1998: 727-751Google Scholar found that 1 h exposure of human skin ex vivo to 10% and 5% SLS solutions created large-scale phase separations in the SC lipids and created continuities between lacunar domains. Presently, we used a 2% SLS solution with a short exposure time (≈1 min). The effective surfactant delivery with PW into the lacunar system, however, can be expected to delay the repair of the barrier. In fact, experiments with 40 kDa dextran have shown that whereas the recovery of the barrier function of the SC was 30 min (Lee et al., 2001bLee S. McAuliffe D.J. Kollias N. Flotte T.J. Doukas A.G. Permeabilization and recovery of the stratum corneum in vivo: The synergy of photomechanical waves and sodium lauryl sulfate.Lasers Surg Med. 2001; 29: 145-150Crossref PubMed Scopus (31) Google Scholar). There are differing views on the effects of anionic surfactants on SC, such as: (1) selective lipid removal (Proksch et al., 1991Proksch E. Feingold K.R. Man M.Q. Elias P.M. Barrier function regulates epidermal DNA synthesis.J Clin Invest. 1991; 87: 1668-1673Crossref PubMed Scopus (233) Google Scholar), and (2) disruption of the processing of lamellar bodies derived sheets but otherwise no effects on the outer SC bilayers (Fartasch, 1997Fartasch M. Ultrastructure of the epidermal barrier after irritation.Microsc Res Tech. 1997; 37: 193-199Crossref PubMed Scopus (105) Google Scholar). These differences may be due to the mode and duration of surfactant application in our study. As the lacunar dilatations imply, delivery of SLS into putative pores could result in selective removal of lipid species in these domains. Electron microscopy did not reveal any noticeable differences between the control sites and PW exposed sites when water was used as the coupling medium. The epidermis and dermis maintained their typical structural features with no indication of damage either in the extracellular matrix or the cellular components. When 2% SLS solution was used as the coupling medium, however, the secreted lamellar bodies appeared to have been disrupted both at the SC–stratum granulosum interface as well as between the outermost and the underlying stratum granulosum cells, further pointers to selective lipid extraction. The dermis showed no structural changes following PW treatment whether water or SLS solution was used as the coupling medium. In a previous study (Lee and Doukas, 1999Lee S. Doukas A.G. Laser generated stress waves and their effects on the cell membrane.IEEE J Select Topics Quant Electr. 1999; 5: 997-1003Crossref Scopus (47) Google Scholar), transmission electron microscopy of human biopsies taken 24 h following the exposure of a PW with water as the coupling medium showed no damage in the subcellular organelles. The cadaver skin showed features in the PW treated SC that did not differ from that of control samples. Surprisingly, we have been unable to permeabilize the SC ex vivo, although the same PW would permeabilize the SC in vivo (Lee and Doukas, unpublished observations). In view of these findings, it could be surmised that the freezing and subsequent thawing of the cadaver skin resulted in alterations of the physical properties of SC, most probably brought about by ice crystal damage. Such changes may modify the responses of a "smart polymer" such as SC to physical factors relevant in penetration enhancement (Menon and Elias, 2001Menon G.K. Elias P.M. The epidermal barrier and strategies for surmounting it: An overview.in: Hengge U.R. Volc-Platzer B. The Skin and Gene Therapy. Blackwell Publishing, Berlin2001: 3-26Crossref Google Scholar). It should be pointed out, that low-frequency ultrasound has been applied successfully to cadaver skin for transdermal delivery (Mitragotri et al., 1995aMitragotri S. Edwards D.A. Blankschtein D. Langer R. Mechanistic study of ultrasonically-enhanced transdermal drug-delivery.J Pharm Sci. 1995; 84: 697-706Crossref PubMed Scopus (301) Google Scholar); however, ultrasound may interact with the SC differently than the PW. Low-frequency ultrasound works predominantly through cavitation induced by the tensile component. Confocal micrographs show significant bleaching of fluorescein-loaded SC after ultrasound exposure (Mitragotri et al., 1995aMitragotri S. Edwards D.A. Blankschtein D. Langer R. Mechanistic study of ultrasonically-enhanced transdermal drug-delivery.J Pharm Sci. 1995; 84: 697-706Crossref PubMed Scopus (301) Google Scholar). The bleaching was attributed to the oxidation of fluorescein by cavitation-generated free radicals. On the other hand, the PW used in our experiments show no measurable tensile component. The characteristics of the PW are very different from those of ultrasound. The peak pressure of the PW is in the range of ≈500 bar, whereas the peak pressure of ultrasound used in transdermal delivery usually ranges from 0.1 to 5 bar. On the other hand, the duration of a PW is only a few hundred nanoseconds, whereas the application of ultrasound can last several minutes. What is more important is the rate at which this pressure is applied on to the skin. The rise time of the PW in the present experiments was ≈30 ns (30×10-9 s), which produced a rate of pressure increase of ≈15×109 bar per s, a rate increase of billion of atmospheres per second. It is reasonable to assume that the application of this kind of physical forces on to the skin can produce novel effects. Our hypothesis is that the free water in the lacunar system is incompressible in this time scale and is forced through the constricted spaces expanding them, thus forming transient pores. The observation Figure 2(a, b), that the lacunae deep inside the SC, where SC is more hydrated, are dilated more than the surface ones is consistent with this hypothesis. The electron micrographs indicate that PW cause similar structural changes in the SC extracellular lipids as has been reported for high-frequency sonophoresis (Menon et al., 1994Menon G.K. Bommannan D.B. Elias P.M. High-frequency phonophoresis. Permeation pathways and structural basis for enhanced permeability.Skin Pharmcol. 1994; 7: 130-139Crossref PubMed Scopus (56) Google Scholar), i.e., pore formation via the lacunar system. The mode of operation of ultrasound, however, is probably different than that of the PW. In the former, the cavitation generated by the ultrasound modifies the lacunae structure, whereas in the latter, it is the strong mechanical forces that induce lacunae dilations. The fact that the effect of PW is different in ex vivo and in vivo experiments suggests that cadaver skin may not always be an appropriate model for use in transdermal delivery experiments. We propose that the uniqueness of structural organization of the SC may include diversity of structural responses. As it evolved, the mammalian SC is designed to function as a barrier in diverse and specialized environments (low and high humidity, extremes of temperature, ultraviolet radiation, and other physical factors), while functioning as a sensory transducer as well. As a composite biopolymer of proteins and lipids, arranged in a "brick and mortar" organization, the corneocytes provide the scaffolding for the waterproofing lipid bilayers. Physical forces, such as PW, ultrasound, and electric fields, target the weaker lipid domains rather than the tough corneocytes protected by the cornified envelope and the covalently bound lipid envelope. Within the lipid domains, it is the hydrophilic regions such as the lacunae that contain water that are susceptible to most physical and chemical agents used for permeability enhancement. The recent observation byOgura et al., 2002Ogura M. Sato S. Kuroki M. et al.Transdermal delivery of photosensitizer by the laser-induced stress waves in combination with skin heating.Jpn J Appl Phys. 2002; 41: L814-L816Crossref Google Scholar that the combination of heating the skin and PW enhances transdermal delivery is consistent with this view. Finally, it appears to be a contradiction that the PW used in the present experiments can induce such extensive changes in the SC but not damage the viable epidermis and dermis. This is probably due to the differences in the structures of the SC and the rest of the viable epidermis. The study of the biologic effects of PW over the last decade has changed our view of the PW interactions with cells and tissue. Traditionally, it was thought that the interactions of the PW with tissue were nonspecific. We have found that those interactions are specific and depend on the PW parameters, such as peak pressure, rise time, or duration (Yashima et al., 1991Yashima Y. McAuliffe D.J. Jacques S.L. Flotte T.J. Laser-induced photoacoustic injury of skin: Effect of inertial confinement.Lasers Surg Med. 1991; 11: 62-68Crossref PubMed Scopus (43) Google Scholar;Doukas et al., 1993Doukas A.G. McAuliffe D.J. Flotte T.J. Biological effects of laser-induced shock waves: Structural and functional cell damage in vitro.Ultrasound Med Biol. 1993; 19: 137-146Abstract Full Text PDF PubMed Scopus (96) Google Scholar,Doukas et al., 1995Doukas A.G. McAuliffe D.J. Lee S. Venugopalan V. Flotte T.J. Physical factors involved in stress-wave-induced cell injury. The effect of stress gradient.Ultrasound Med Biol. 1995; 21: 961-967Abstract Full Text PDF PubMed Scopus (73) Google Scholar;Lee et al., 1996Lee S. Anderson T. 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In fact, it is possible to control these effects and even target specific structures by generating PW with the appropriate parameters. Our conjecture is that PW interact the strongest with structures that spatial dimensions are of the same magnitude as the spatial length of the PW. For example, PW of short rise time act on the cell membrane (Mulholland et al., 1999Mulholland S.E. Lee S. McAuliffe D.J. Doukas A.G. Cell loading with laser-generated stress waves. The role of stress gradient.Pharm Res. 1999; 16: 514-518Crossref PubMed Scopus (62) Google Scholar), whereas PW of long duration act on larger structures, e.g., the SC (Lee et al., 2001cLee S. McAuliffe D.J. Flotte T.J. Kollias N. Doukas A.G. Photomechanical transdermal delivery. The effect of laser confinement.Lasers Surg Med. 2001; 28: 344-347Crossref PubMed Scopus (59) Google Scholar) and cells (Kodama et al., 2000Kodama T. Hamblin M.R. Doukas A.G. Cytoplasmic molecular delivery with shock waves Importance of impulse.Biophys J. 2000; 79: 1821-1832Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). We thank Shun Lee, Ph.D. and Daniel McAuliffe for assistance with the experiments, and Sheri Lenc, Avon Products, Inc. for her invaluable help with the manuscript. We also thank the Department of Biology, William Paterson University, NJ for the Electron Microscopy facilities. This work was supported at the Wellman Laboratories of Photomedicine by the DoD Medical Free Electron Program under contracts N00014-94-1-0927 and F49620-01-1-0014.