European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 301–306
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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb
Research paper
Influence of massage and occlusion on the ex vivo skin penetration of rigid liposomes and invasomes Sindy Trauer a,b, Heike Richter a, Judith Kuntsche c,d, Rolf Büttemeyer e, Manfred Liebsch b, Michael Linscheid f, Alfred Fahr c, Monika Schäfer-Korting g, Jürgen Lademann a,⇑, Alexa Patzelt a a
Center of Experimental and Applied Cutaneous Physiology, Department of Dermatology, Charité – Universitätsmedizin Berlin, Germany Zebet at Federal Institute of Risk Assessment (BfR), Berlin, Germany Department of Pharmaceutical Technology, Friedrich-Schiller-University Jena, Jena, Germany d Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark e Department of Surgery, Charité – Universitätsmedizin Berlin, Germany f Department of Chemistry, Humboldt-Universität zu Berlin, Berlin, Germany g Institute of Pharmacy (Pharmacology and Toxicology), Freie Universität Berlin, Germany b c
a r t i c l e
i n f o
Article history: Received 5 June 2013 Accepted in revised form 7 November 2013 Available online 16 November 2013 Keywords: Massage technique Franz diffusion cell Follicular penetration Liposomes Invasomes Drug delivery
a b s t r a c t Liposomes are frequently described as drug delivery systems for dermal and transdermal applications. Recently, it has been shown that particulate substances penetrate effectively into hair follicles and that the follicular penetration depth can be increased by massaging the skin, which simulates the in vivo movement of hairs in the hair follicles. In the present study, massage was applied to skin mounted to Franz diffusion cells. By means of confocal laser scanning microscopy, the influence of massage and occlusion on the follicular penetration depths of rigid and flexible liposomes loaded with a hydrophilic and lipophilic dye was investigated. The application of massage increased follicular penetration significantly. Occlusion resulted in an increased follicular penetration depth only for rigid liposomes, whereas invasomes did not penetrate more effectively if occlusion was applied. The results confirm that massage is an important tool for increasing follicular penetration in ex vivo studies using Franz diffusion cells. Occlusion may reduce the efficacy of follicular penetration depending on the specific liposomal preparation. Rigidity in particular appears to be a relevant parameter. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Liposomes have been adopted as drug delivery systems for systemic and topical use. Hydrophilic agents can be enclosed within the inner aqueous sphere, and lipophilic agents can be intercalated into the lipid bilayer [1–3]. Liposomes can effectively deliver drugs to and through the skin [4–6]. Whereas previous studies were aimed elucidating the mechanisms of liposome penetration and subsequent distribution of the active compounds being transported [7–9], optimization of topical drug delivery by penetration of intact liposomes, the adsorption effect and penetration through the transappendageal route are currently the subject of intense research [10]. Other studies investigated the relevance of liposome composition on penetration efficacy [11]. Classical liposomes do not penetrate deeply into the skin, but rather remain confined to the upper layers of the stratum corneum [10], as was evidenced
⇑ Corresponding author. Department of Dermatology, Venerology and Allergology, Charité – Universitätsmedizin Berlin Charitéplatz 1, 10117 Berlin, Germany. Tel.: +49 30 450 518 235; fax: +49 30 450 518 918. E-mail address: [email protected] (J. Lademann). 0939-6411/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2013.11.004
by a study in which intact rigid liposomes were not detected in the granular layers of the epidermis by confocal microscopy [12]. A new class of highly deformable liposomes has recently been developed, i.e., deformable liposomes which can increase dermal and transdermal delivery. In some cases, the efficiency described was compared with subcutaneous administration [13]. Elsayed et al. [10] reviewed the mode of action of deformable liposomes and proposed that both intact vesicular permeation into the stratum corneum and a penetration-enhancing effect contribute to the enhanced topical delivery of drugs, whereby one of the two mechanisms may predominate, depending on the physico-chemical properties of the drug. A comparison between rigid and flexible liposomes can be assumed as penetration-enhancing properties of flexible liposomes do not become relevant during follicular penetration, since penetration is stimulated mechanically by massage. A liposome size of approximately 130 nm (Table 1) was specifically chosen because it is in a range in which the structure of the cuticula of the moving hairs stimulates the transport of the liposomes into the hair follicles by a mechanism that is similar to a geared pump. The comparison serves to assess the use of massage and occlusion to allow
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conclusions to be drawn regarding the penetration behavior of rigid and flexible liposomes, respectively. Thus far, penetration studies investigating the dermal or transdermal delivery of drugs via liposomes were mainly performed ex vivo in Franz diffusion cell experiments [14–16]. It is widely recognized that ex vivo penetration studies can demonstrate whether such delivery systems can enhance penetration into human skin when performed according to the standard guidelines [17]. Ex vivo tests are required as in vivo testing in humans or animals is limited due to ethical reasons. In the past, the penetration of liposomes through the stratum corneum has been studied extensively, whereas the follicular pathway has rarely been regarded as relevant for liposomal transdermal delivery [18,19]. Recently, this opinion has drastically changed. The hair follicle infundibulum represents an invagination of the epidermis that physiologically interrupts the skin barrier, thus providing an additional surface area that is available for penetration of topically applied substances [20–22]. A clear advantage of follicular uptake is that there is no need for manipulation of the physiological function, such as by the use of penetration enhancers, once the agent gains access to the hair follicle infundibulum in sufficient amounts. Nanoparticles and microparticles in particular preferentially penetrate into the hair follicles [23]. It has been hypothesized that movement of hairs on the skin promotes the transportation of particles deeply into the hair follicles where the skin barrier is less efficient and dispersion of the active agents into the viable tissue can occur. In vivo, the movement of the hair is a physiological process, whereas it must be simulated ex vivo [24] such as by massage application, which leads to a significantly deeper penetration of particulate substances into the hair follicle [25]. However, the effect of massage on the penetration depth of rigid and flexible liposomes into skin mounted to Franz cells, the standard method for ex vivo testing [17], is unknown to date, and was therefore investigated in the present study. Additionally, the influence of environmental humidity (e.g., occlusion) on the penetration of rigid and flexible liposomes was examined. While occlusion is known to enhance the delivery of conventional liposomes [26], occlusion may diminish the penetration efficacy of flexible liposomes [27,28]. 2. Material and methods 2.1. Handling of the skin Human full thickness breast or abdominal skin (1700 lm thick on average) was obtained during plastic surgery from 6 different subjects (female, aged 30–67 years). Approval for these experiments had been obtained from the Ethics Committee of the Charité – Universitätsmedizin Berlin. After surgery, the subcutaneous fat was carefully removed and the remaining sample was wrapped in aluminum foil and stored at 20 °C (min 24 h, max 6 months). 2.2. Liposome formulations Flexible and rigid liposomes sized approximately 130 nm (Table 1) were prepared and labeled with fluorescent dyes. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B), ammonium salt (Rh-DOPE, Avanti Polar Lipids, Alabaster, USA) Table 1 Characterization of liposomes. Liposomes
Flexible Rigid
Before dialysis
After dialysis
is a lipophilic fluorescent dye located in the liposomal membrane. The excitation wavelength of rhodamine is 540 nm and the detection wavelength of the red fluorescence is 570 nm. 5,6-Carboxyfluorescein, disodium salt (CF, Sigma Aldrich, Taufkirchen, Germany) is a hydrophilic fluorescent dye, which is enclosed into the aqueous core of the liposomes. The excitation wavelength of carboxyfluorescein is 490 nm and the detection wavelength is 520 nm. Green fluorescence is detectable after degradation of the liposomes and subsequent release of the dye. 2.3. Preparation of rigid liposomes For the preparation of the liposomes with a rigid bilayer, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt (DPPG), were purchased from Genzyme Pharmaceuticals (Liestal, Switzerland). 90 mg DPPC, 10 mg DPPG and 0.5 mg Rh-DOPE were dissolved in approximately 1.5 ml chloroform (Merck, Darmstadt, Germany). After evaporation of the chloroform at 50 °C under a vacuum (RotaVapor, Büchi Labortechnik, Konstanz, Germany), the obtained thin lipid film was hydrated with 1 ml 10 mM Tris buffer pH 7.4 (Sigma Aldrich, Taufkirchen, Germany) containing 20 mM carboxyfluorescein, and shaken for approximately 30 min at 50 °C. After equilibration at room temperature overnight under a nitrogen atmosphere, small unilamellar liposomes were prepared by extrusion (LiposoFast basic, Avestin, Ottawa, Canada) with 21 repetitions through a polycarbonate membrane with a pore size of 100 nm (Armatis, Mannheim, Germany) at 55 °C. 2.4. Preparation of flexible liposomes (invasomes) For the preparation of the flexible liposomes, soybean lecithin in ethanol 75:20 w/w (NAT 8539) was purchased from Phospholipid GmbH (Cologne, Germany). 0.5 mg Rh-DOPE was dissolved in 134 mg of NAT 8539 corresponding to 100 mg soybean lecithin. The mixture was vortexed at room temperature until a clear solution was obtained. 20 ll of a mixture of limonene, citral and cineol (10:45:45 by volume, Sigma Aldrich, Taufkirchen, Germany) was added. Subsequently, 900 ll 10 mM Tris buffer pH 7.4 containing 20 mM carboxyfluorescein was added under vortexing. The mixture was sonicated (sonication bath USR 54 h, Merck EuroLab NV, VWR, Germany) for approximately 15 min at room temperature and small unilamellar liposomes were prepared by successive extrusion (LiposoFast basic) at room temperature twice each through the polycarbonate membranes with pore sizes of 400 nm, 200 nm, 100 nm and 50 nm, respectively. Non-entrapped carboxyfluorescein was removed by dialysis of the liposome formulations (MWCO 12–14 kDa, Visking, Medical International, Great Britain) at room temperature against 10 mM Tris buffer pH 7.4 (rigid liposomes) or against 10 mM Tris buffer pH 7.4 containing 3% ethanol (v/v) and a few droplets of the terpene mixture (flexible liposomes). Removal of the non-entrapped carboxyfluorescein was completed after 30 h for both liposome dispersions with the dialysis fluid being replaced four times during this process. As a consequence, the dispersion was diluted until approximately one third of the dialyzed fluid remained. The loss of entrapped carboxyfluorescein can be excluded as the size of the liposomes increased as a result of the supplied fluid (Table 1). During the entire preparation process and dialysis, the formulations were stored at 4 °C under the exclusion of light.
Z – average (nm)
PDI
Z – average (nm)
PDI
2.5. Reference substance caffeine
139 ± 1.5 115 ± 1.4
0.1 ± 0.005 0.07 ± 0.009
146 ± 0.6 117 ± 1.5
0.11 ± 0.005 0.08 ± 0.003
Caffeine (Sigma Aldrich, Steinhagen, Germany) was used as a control substance. 2.5 g Caffeine was dissolved in 1000 ml
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Dulbecco’s phosphate-buffered saline (DPBS) from PANBiotec GmbH (Aidenbach, Germany). Subsequently, 10 ll/cm2 of the reference solution was applied at a concentration of 2.5 mg/ml DPBS. 2.5.1. Franz diffusion cell experiments Thawed human full thickness skin was mounted to a static Franz diffusion cell (FD-C) (surface area 1.76 cm2; Gauer–Glas, Püttlingen, Germany) [29] with the stratum corneum facing upwards and the dermis positioned to contact the receptor medium DPBS (Dulbecco’s Phosphate-Buffered Saline, PAA, Pasching, Austria), which was maintained at 32 °C ± 1 °C and continuously stirred by a magnetic bar. According to the OECD guideline 428, caffeine was used as reference probe to control the quality of the experimental series [17]. All test formulations (10 lg/cm2, finite dose) were applied for 24 h, during which aliquots of the receptor solution were collected (0, 2, 4, 6 and 24 h after dosing; under replacement) for fluorescence analysis. The lack of a fluorescence signal in the removed receptor medium probe proved a valid test procedure. Parallel tests were performed, i.e., all test formulations and reference substances were treated equally. As caffeine penetrates into the receptor medium and the amount must be analyzed to validate the quality of the tests, it is indispensable that the receptor medium is also removed from the Franz diffusion cells with a test formulation and replaced by fresh medium. Even if the liposomes do not penetrate into the receptor solution, this must be verified by fluorescence measurements. As expected, fluorescence was never detected in the receptor medium. It could be demonstrated that no fluorescent dye reached the receptor reservoir as the follicular pathway was used for penetration. Because vellus hairs do not extend into the subcutaneous fat tissue, the samples did not exhibit any holes as it is usual for split skin, indicating that the full thickness skin was not damaged. Also, the skin sample was not shifted during massage application. As the data established for caffeine are comparable to those already published, the experiment was correctly performed. Each experiment was conducted with at least 6 FD-C (originating from 6 donor subjects) and repeated three times so that the data were acquired over the course of 4 days. The effects of massage (study design 1) and occlusion (study design 2) were derived from experiments on skin samples of the same donor skin, in order to exclude donor-related skin properties. The structural integrity of the liposomes was not affected by sink conditioning as the liposomes penetrated exclusively via the follicular shunt and did not reach the receptor reservoir. The liposomes did not break when subjected to massage or occlusion. This was shown in study design 1, in which occlusion was applied and the effects of massage were investigated. If the liposomes had been broken, the skin measurements would have shown strong carboxyfluorescein coloration. However, the CSLM images disclosed strong rhodamine fluorescence, but only weak carboxyfluorescein coloration. In deeper dermal layers, weaker rhodamine fluorescence and increased carboxyfluorescence intensity was detectable. Intact liposomes are represented by ample red and only minute green colorations in the images. If the liposomes break, carboxyfluorescein solution is released from the core, and the fragments migrate into deeper regions in the hair follicle. 2.5.2. Study design 1 – Application of massage using a device The massage device used was an electrical toothbrush (Braun, Oral B, Germany), in which the brush was replaced with a metallic ball that was covered with a cut-off finger of a latex glove. The vibration of the toothbrush led to an oscillation of the metallic ball and thereby enabled massaging of the topically applied test formulations into the skin.
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After mounting the human full thickness skin on the FD-C and homogeneous application of the liposome formulations, massage was applied for 3 min on the test samples, whereas no massage was applied on the control samples. Tests were regarded valid if a test probe removed from the receptor solution yielded no fluorescent signal. The structural integrity of the liposomes was not affected by sink conditioning as the liposomes penetrated exclusively via the follicular shunt and did not reach the receptor reservoir. The donor compartments of test and control samples were then covered with parafilm and aluminum foil to ensure occlusion (and exclusion of light) during the 24 h exposure. At the end of the experiments, any donor formulation found on the application site was removed using cotton swabs, and 8 mm punch biopsies were taken from the skin samples and shock frozen in liquid nitrogen for preparation of 10 lm cyro-sections. The sections were then analyzed using confocal laser scanning microscopy (CLSM). Anticipating that the application of massage led to significantly increased penetration depths of both liposome preparations, the massage device was also applied for the study of the influence of occlusion on the follicular penetration depth of both liposome preparations. 2.5.3. Study design 2 – Investigation of the influence of occlusion The influence of occlusion on the formulations was investigated in study design 2. Both the test formulations and the reference substances were investigated with and without occlusion, respectively, at 70% environmental humidity, corresponding to OECD guidelines. Thus, the conditions for the occluded skin samples were not significantly different from the non-occluded samples, such that the behavior of the liposome types permits conclusions as to an influence of occlusion only if the humidity of the ambient air dropped significantly. The results shown in Fig. 3 suggest that flexible liposomes are influenced much less by the environmental humidity than rigid liposomes. Investigations were performed in FD-C, whereas the liposome application and massage were performed as described for study design 1. During the experiments, part of the FD-C was occluded by covering the donor chamber with parafilm and aluminum foil to create a microclimate and to exclude light above the epidermis, while the remaining FD-C was non-occluded, i.e., the donor chamber remained open. The humidity of the environment was 70% in the experiments with non-occluded skin as recommended [17]. At the end of the experiment, skin samples were prepared for CLSM as described in the study design 1. 2.5.4. Confocal laser scanning microscopy (CLSM) All cryo-sections of the skin containing hair follicles were analyzed with a CLSM 410 (Zeiss, Jena, Germany) to determine the follicular penetration depths of the liposomes. The field of view magnification of the CLSM was 250 250 lm. As the liposomes were double-labeled with two fluorescent dyes, the penetration of the constituents of the outer (rhodamine) and the inner sphere (carboxyfluorescein) of the liposomes could be detected separately at 570 nm and 520 nm, respectively. The penetration depths of the fluorescent dyes were determined in lm. In order to exclude the influence of autofluorescence of the skin, a corresponding filter was utilized to cut off the fluorescence below 510 nm, which corresponds to the autofluorescence of the skin. 2.5.5. Statistics Statistical analysis was performed with the software program IBM SPSS Statistics 19Ò. Explorative data analysis and the nonparametric Wilcoxon test were used to compare the penetration
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depths of the fluorescence probes of test and control samples. p 6 0.05 was considered as significant.
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It could be demonstrated that liposomes can penetrate effectively into the hair follicles via the follicular penetration pathway. Fig. 2 shows that the hair follicle comprises both fluorescence dyes. Although the hydrophilic carboxyfluorescein dye was also detectable in the tissue adjacent to the follicle, it did not penetrate into the receptor fluid. The carboxyfluorescein was not detectable in the receptor fluid within 24 h as the hair follicle serves as a reservoir. As a result, the fluorescent dye rhodamine, which binds to the lipids of the liposomes, was not detected outside the follicle.
Penetration depth [µm]
3. Results
After massage application, the follicular penetration depths of the fluorescent dyes were determined. Rhodamine covalently binds to phospholipids of the liposome membrane, and thus is a marker for the lipophilic agents forming the outer sphere of the liposomes, while carboxyfluorescein embedded into the inner sphere of the liposomes is a marker of hydrophilic agents dissolved in the aqueous phase. For all four tested variations (rigid and flexible liposomes with and without massage), it can be concluded that carboxyfluorescein penetrated significantly deeper than the rhodamine-conjugate, the marker for the liposome bilayer (p 6 0.05; Fig. 1). Massage application favored penetration into the depth of the hair follicle infundibula of both fluorescent markers in the cases of rigid as well as flexible liposomes (p 6 0.05). Independent of the massage application, the flexible liposomes penetrated significantly deeper than the rigid liposomes (p 6 0.05). The liposomes investigated in the present study penetrated to a depth of 93.2 ± 11.7 lm (rigid liposomes) and 137.3 ± 26.5 lm (flexible liposomes) when applied without massage, meaning that neither the sebaceous gland nor the bulge region was reached. The application of massage led to a penetration depth of 477.2 ± 61 lm and 698.8 ± 90.7 lm, respectively, which enabled the target sites to be reached. Fig. 1 shows the preparation of fresh liposomes. The influence of massage on the penetration depth was investigated using occluded samples. Most liposomes remained undamaged, penetrating to a depth of 500 lm (rigid liposomes) and 700 lm (flexible liposomes), respectively, within 24 h. The released carboxyfluorescein reached penetration depths of approximately 1200 lm, independent of the type of liposomes implemented. The liposomes applied without massage showed penetration depths of less than 200 lm for rhodamine and of less than 300 lm for carboxyfluorescein, independent of the liposome types. In Fig. 2, the penetration of both kinds of liposomes is depicted, demonstrating that the penetration of carboxyfluorescein (Fig. 2b and d) was more efficient than the penetration of rhodamine (Fig. 2a and c). In the case of massage application (Fig. 2c and d), the fluorescence signal could be detected in deeper regions of the hair follicles. 3.2. Effect of occlusion on the follicular penetration depth of rigid and flexible liposomes The microclimate generated by occlusion leads to a stronger hydration of the skin, which in turn promotes the penetration of hydrophilic substances. Once released from the core of the liposomes, carboxyfluorescein, e.g., can automatically penetrate into deeper skin layers because the cells are hydrated and the dye can disperse. Likewise, the effect of occlusion on the follicular penetra-
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3.1. Effect of massage on the follicular penetration of rigid and flexible liposomes
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Rigid-Rh Rigid-CF Rigid-Rh Rigid-CF Flex-Rh Flex-CF Flex-Rh Flex-CF
-M
+M
-M
+M
Fig. 1. Massage effect follicular penetration depths of rigid (Rigid) and flexible (Flex) liposomes with (+M) and without ( M) massage appliance. Rh-DOPE (Rh) was used as a label for the liposome bilayer and CF for the inner, aqueous liposome core. (n = 6; p < 0.05).
Fig. 2. Massage effect laser scanning microscope images of the penetration of flexible liposomes: (a) penetration of rhodamine without massage, (b) penetration of carboxyfluorescein without massage, (c) penetration of rhodamine with massage, and (d) penetration of carboxyfluorescein with massage.
tion depth of rigid and flexible liposomes was investigated. The results are summarized in Fig. 3. Again, for all four tested variations (rigid and flexible liposomes with and without occlusion), hydrophilic carboxyfluorescein penetrated significantly deeper than the rhodamine-conjugate (p 6 0.05). For the rigid liposomes, occlusion had a positive effect on the penetration depth (p 6 0.05), whereas for the flexible liposomes, occlusion did not increase the penetration depth of rhodamine (p > 0.05) and even appeared to have inhibited the penetration of carboxyfluorescein. In the case of
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Penetration depth [µm]
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600 400 *
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0 Rigid-Rh Rigid-CF Rigid-Rh Rigid-CF Flex-Rh Flex-CF Flex-Rh Flex-CF
-O
+O
-O
+O
Fig. 3. Occlusion effect penetration depths of rigid (Rigid) and flexible (Flex) liposomes with (+O) and without ( O) occlusion after massage application. The outer sphere of the liposomes was labeled with Rh-DOPE (Rh), the inner sphere was labeled with CF. (n = 6; p < 0.05).
non-occlusion, the flexible liposomes penetrated significantly deeper into the hair follicles (p 6 0.05). For the investigations on the influence of occlusion, the same batch of liposomes was used as for the investigations on the influence of massage. The investigations were not conducted before the massage experiments were completed and evaluated. Fig. 3 illustrates the penetration of intact and broken liposomes. The broken liposomes migrate to deeper skin layers than intact liposomes, which explains why rhodamine is detectable in liposome fragments up to a depth of approximately 1200 lm. In non-occluded samples, the rigid liposomes exhibit similar penetration depths as shown in Fig. 1 for the occluded samples. This is attributed to the high environmental humidity, which under occlusion and through the development of a microclimate above the dermis can exceed 70%. The results show that the rigid liposomes desiccate more rapidly if a microclimate cannot develop above the skin. In this case, penetration ceases earlier and at lower depths. Once a microclimate has formed above the epidermis, the rigid liposomes exhibit penetration depths comparable to those of the flexible liposomes. The flexible liposomes are less influenced by occlusion, i.e., they penetrate to the same depths, whether or not they are occluded. 4. Discussion The movement of hair in hair follicles has been described as crucial to stimulating follicular penetration. Recent investigations have shown that the pumping effect of the moving hairs is most effective when the particle size of the topically applied substance is in the range of the cuticula thickness of the corresponding hair [24]. Larger and smaller particles did not penetrate as efficiently into the hair follicles [23]. On the contrary, the penetration of liposomes through the stratum corneum decreases as their diameter increases [30]. While the movement of the hair occurs physiologically in vivo and is additionally supported by the user when products are topically applied by rubbing, this phenomenon must be induced in ex vivo studies by the application of massage. The present investigation demonstrated that the application of massage had a significant positive effect on the follicular penetration depth for both liposome preparations. The penetration depth was increased by up to a factor of 5 when massage was applied. Lademann et al. observed a comparable increase in the follicular
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penetration depth of 320 nm-sized particles into ex vivo skin subsequent to massage application [25]. Moreover, the results indicate liposomal degradation as the green fluorescence of carboxyfluorescein, which is the label of the inner aqueous liposome core, was detected independent of rhodamine-related fluorescence of the phospholipid. It can be assumed that after the degradation at a significant depth within the hair follicle, both marker dyes penetrate independently. The results show that liposomes can be utilized to deliver hydrophilic substances such as carboxyfluorescein deeply into the hair follicles. Within the hair follicle, several target sites of interest are located, such as the sebaceous gland, the bulge region, localization of the stem cells, or the infundibulum, which represents an interrupted barrier with increased permeability in the lower part and is surrounded by a high density of immune cells and an extensive capillary network. This capillary network offers new therapeutic options concerning the therapy of sebaceous gland-associated diseases such as acne vulgaris, but also concerning topical vaccinations or regenerative medicine [23]. It has been shown previously that liposomal preparations can induce better results in the treatment of acne vulgaris than conventional treatments [19]. Recently, Vogt et al. [31] investigated the morphometry of vellus and terminal hair follicles. For vellus hair follicles, present mainly in breast and abdominal skin, the average length of the infundibulum, e.g., at the end of which the sebaceous gland is located, is 225 ± 34 lm. Additionally, the influence of environmental humidity on the penetration of rigid and flexible liposomes was analyzed. Percutaneous absorption is generally increased when the site of application is occluded [26]. However, in regard to the implementation of flexible liposomes, occlusion has been described to be counterproductive [28,32]. The results of the present study revealed that occlusion had a significant positive effect on the follicular penetration depth for rigid liposomes, whereas for the flexible liposomes, no significant difference could be detected for the lipids of the outer sphere (rhodamine). This is presumably also the case for other lipophilic agents embedded in the liposomal shell. However, occlusion led to a slightly but significantly decreased penetration depth of the hydrophilic marker carboxyfluorescein. This should also remain true for other hydrophilic agents dissolved in the aqueous core. It has been suggested [28,32] that in the case of non-occlusive application, the increased drug transport can be caused by a more profound interaction between the liposomal constituents and the skin and/or the presence of a hydration gradient in the skin [7]. According to Cevc and Blume [32], the water gradient is an important driving force for drug diffusion. During the occlusive application, the water gradient is expected to be absent [28]. The same explanation might be valid for follicular penetration, but this must be investigated further. The results of the present study allow the conclusion that massage application is an important element when investigating follicular penetration of liposomes and other particulates ex vivo. Massage application simulates the physiological movement of hair, which is important for an effective follicular delivery, and additionally imitates the application behavior of the end user. Although valid for several other dermatological topical preparations, occlusion did not increase the follicular penetration of the flexible liposomes. Thus, a potential beneficial effect of occlusion must to be evaluated for new formulations. Acknowledgment We would like to thank the Foundation ‘‘Skin Physiology’’ of the Donor Association for German Science and Humanities for financial support.
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absorption test for the investigation of the follicular penetration pathway of caffeine, Skin Pharmacol. Physiol. 23 (2010) 320–327. R. Bussiahn, R. Brandenburg, T. Gerling, E. Kindel, H. Lange, N. Lembke, K.D. Weltmann, T. von Woedtke, T. Kocher, The hairline plasma: an intermittent negative dc-corona discharge at atmospheric pressure for plasma medical applications, Appl. Phys. Lett. 96 (2010). G.M. El Maghraby, A.C. Williams, B.W. Barry, Skin hydration and possible shunt route penetration in controlled estradiol delivery from ultradeformable and standard liposomes, J. Pharm. Pharmacol. 53 (2001) 1311–1322. G.M. El Maghraby, A.C. Williams, B.W. Barry, Can drug-bearing liposomes penetrate intact skin?, J Pharm. Pharmacol. 58 (2006) 415–429. N. Otberg, H. Richter, H. Schaefer, U. Blume-Peytavi, W. Sterry, J. Lademann, Variations of hair follicle size and distribution in different body sites, J. Invest. Dermatol. 122 (2004) 14–19. B. Lange-Asschenfeldt, D. Marenbach, C. Lang, A. Patzelt, M. Ulrich, A. Maltusch, D. Terhorst, E. Stockfleth, W. Sterry, J. Lademann, Distribution of bacteria in the epidermal layers and hair follicles of the human skin, Skin Pharmacol. Physiol. 24 (2011) 305–311. N. Luther, M.E. Darvin, W. Sterry, J. Lademann, A. Patzelt, Ethnic differences in skin physiology, hair follicle morphology and follicular penetration, Skin Pharmacol. Physiol. 25 (2012) 182–191. A. Patzelt, H. Richter, F. Knorr, U. Schafer, C.M. Lehr, L. Dahne, W. Sterry, J. Lademann, Selective follicular targeting by modification of the particle sizes, J. Control. Release 150 (2011) 45–48. J. Lademann, A. Patzelt, H. Richter, C. Antoniou, W. Sterry, F. Knorr, Determination of the cuticula thickness of human and porcine hairs and their potential influence on the penetration of nanoparticles into the hair follicles, J. Biomed. Opt. 14 (2009) 021014. J. Lademann, H. Richter, A. Teichmann, N. Otberg, U. Blume-Peytavi, J. Luengo, B. Weiss, U.F. Schaefer, C.M. Lehr, R. Wepf, W. Sterry, Nanoparticles – an efficient carrier for drug delivery into the hair follicles, Eur. J. Pharm. Biopharm. 66 (2007) 159–164. P. Treffel, P. Muret, P. Muret-D’Aniello, S. Coumes-Marquet, P. Agache, Effect of occlusion on in vitro percutaneous absorption of two compounds with different physicochemical properties, Skin Pharmacol. 5 (1992) 108–113. Y. Shimoyama, M. Fujii, Y. Kanda, A. Mizoguchi, H. Oda, N. Koizumi, Y. Watanabe, Effects of application method on skin penetration of carboxyfluorescein incorporated in liposomes, Chem. Pharm. Bull. (Tokyo) 58 (2010) 429–431. M.E.M.J. van Kuijk-Meuwissen, H.E. Junginger, J.A. Bouwstra, Interactions between liposomes and human skin in vitro, a confocal laser scanning microscopy study, Bba-Biomembranes 1371 (1998) 31–39. T.J. Franz, Percutaneous absorption on the relevance of in vitro data, J. Invest. Dermatol. 64 (1975) 190–195. J. de Leeuw, H.C. de Vijlder, P. Bjerring, H.A. Neumann, Liposomes in dermatology today, J. Eur. Acad. Dermatol. Venereol. 23 (2009) 505–516. A. Vogt, S. Hadam, M. Heiderhoff, H. Audring, J. Lademann, W. Sterry, U. BlumePeytavi, Morphometry of human terminal and vellus hair follicles, Exp. Dermatol. 16 (2007) 946–950. G. Cevc, G. Blume, Lipid vesicles penetrate into intact skin owing to the transdermal osmotic gradients and hydration force, Biochim. Biophys. Acta 1104 (1992) 226–232.
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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb
Research paper
Influence of massage and occlusion on the ex vivo skin penetration of rigid liposomes and invasomes Sindy Trauer a,b, Heike Richter a, Judith Kuntsche c,d, Rolf Büttemeyer e, Manfred Liebsch b, Michael Linscheid f, Alfred Fahr c, Monika Schäfer-Korting g, Jürgen Lademann a,⇑, Alexa Patzelt a a
Center of Experimental and Applied Cutaneous Physiology, Department of Dermatology, Charité – Universitätsmedizin Berlin, Germany Zebet at Federal Institute of Risk Assessment (BfR), Berlin, Germany Department of Pharmaceutical Technology, Friedrich-Schiller-University Jena, Jena, Germany d Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark e Department of Surgery, Charité – Universitätsmedizin Berlin, Germany f Department of Chemistry, Humboldt-Universität zu Berlin, Berlin, Germany g Institute of Pharmacy (Pharmacology and Toxicology), Freie Universität Berlin, Germany b c
a r t i c l e
i n f o
Article history: Received 5 June 2013 Accepted in revised form 7 November 2013 Available online 16 November 2013 Keywords: Massage technique Franz diffusion cell Follicular penetration Liposomes Invasomes Drug delivery
a b s t r a c t Liposomes are frequently described as drug delivery systems for dermal and transdermal applications. Recently, it has been shown that particulate substances penetrate effectively into hair follicles and that the follicular penetration depth can be increased by massaging the skin, which simulates the in vivo movement of hairs in the hair follicles. In the present study, massage was applied to skin mounted to Franz diffusion cells. By means of confocal laser scanning microscopy, the influence of massage and occlusion on the follicular penetration depths of rigid and flexible liposomes loaded with a hydrophilic and lipophilic dye was investigated. The application of massage increased follicular penetration significantly. Occlusion resulted in an increased follicular penetration depth only for rigid liposomes, whereas invasomes did not penetrate more effectively if occlusion was applied. The results confirm that massage is an important tool for increasing follicular penetration in ex vivo studies using Franz diffusion cells. Occlusion may reduce the efficacy of follicular penetration depending on the specific liposomal preparation. Rigidity in particular appears to be a relevant parameter. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Liposomes have been adopted as drug delivery systems for systemic and topical use. Hydrophilic agents can be enclosed within the inner aqueous sphere, and lipophilic agents can be intercalated into the lipid bilayer [1–3]. Liposomes can effectively deliver drugs to and through the skin [4–6]. Whereas previous studies were aimed elucidating the mechanisms of liposome penetration and subsequent distribution of the active compounds being transported [7–9], optimization of topical drug delivery by penetration of intact liposomes, the adsorption effect and penetration through the transappendageal route are currently the subject of intense research [10]. Other studies investigated the relevance of liposome composition on penetration efficacy [11]. Classical liposomes do not penetrate deeply into the skin, but rather remain confined to the upper layers of the stratum corneum [10], as was evidenced
⇑ Corresponding author. Department of Dermatology, Venerology and Allergology, Charité – Universitätsmedizin Berlin Charitéplatz 1, 10117 Berlin, Germany. Tel.: +49 30 450 518 235; fax: +49 30 450 518 918. E-mail address: [email protected] (J. Lademann). 0939-6411/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2013.11.004
by a study in which intact rigid liposomes were not detected in the granular layers of the epidermis by confocal microscopy [12]. A new class of highly deformable liposomes has recently been developed, i.e., deformable liposomes which can increase dermal and transdermal delivery. In some cases, the efficiency described was compared with subcutaneous administration [13]. Elsayed et al. [10] reviewed the mode of action of deformable liposomes and proposed that both intact vesicular permeation into the stratum corneum and a penetration-enhancing effect contribute to the enhanced topical delivery of drugs, whereby one of the two mechanisms may predominate, depending on the physico-chemical properties of the drug. A comparison between rigid and flexible liposomes can be assumed as penetration-enhancing properties of flexible liposomes do not become relevant during follicular penetration, since penetration is stimulated mechanically by massage. A liposome size of approximately 130 nm (Table 1) was specifically chosen because it is in a range in which the structure of the cuticula of the moving hairs stimulates the transport of the liposomes into the hair follicles by a mechanism that is similar to a geared pump. The comparison serves to assess the use of massage and occlusion to allow
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conclusions to be drawn regarding the penetration behavior of rigid and flexible liposomes, respectively. Thus far, penetration studies investigating the dermal or transdermal delivery of drugs via liposomes were mainly performed ex vivo in Franz diffusion cell experiments [14–16]. It is widely recognized that ex vivo penetration studies can demonstrate whether such delivery systems can enhance penetration into human skin when performed according to the standard guidelines [17]. Ex vivo tests are required as in vivo testing in humans or animals is limited due to ethical reasons. In the past, the penetration of liposomes through the stratum corneum has been studied extensively, whereas the follicular pathway has rarely been regarded as relevant for liposomal transdermal delivery [18,19]. Recently, this opinion has drastically changed. The hair follicle infundibulum represents an invagination of the epidermis that physiologically interrupts the skin barrier, thus providing an additional surface area that is available for penetration of topically applied substances [20–22]. A clear advantage of follicular uptake is that there is no need for manipulation of the physiological function, such as by the use of penetration enhancers, once the agent gains access to the hair follicle infundibulum in sufficient amounts. Nanoparticles and microparticles in particular preferentially penetrate into the hair follicles [23]. It has been hypothesized that movement of hairs on the skin promotes the transportation of particles deeply into the hair follicles where the skin barrier is less efficient and dispersion of the active agents into the viable tissue can occur. In vivo, the movement of the hair is a physiological process, whereas it must be simulated ex vivo [24] such as by massage application, which leads to a significantly deeper penetration of particulate substances into the hair follicle [25]. However, the effect of massage on the penetration depth of rigid and flexible liposomes into skin mounted to Franz cells, the standard method for ex vivo testing [17], is unknown to date, and was therefore investigated in the present study. Additionally, the influence of environmental humidity (e.g., occlusion) on the penetration of rigid and flexible liposomes was examined. While occlusion is known to enhance the delivery of conventional liposomes [26], occlusion may diminish the penetration efficacy of flexible liposomes [27,28]. 2. Material and methods 2.1. Handling of the skin Human full thickness breast or abdominal skin (1700 lm thick on average) was obtained during plastic surgery from 6 different subjects (female, aged 30–67 years). Approval for these experiments had been obtained from the Ethics Committee of the Charité – Universitätsmedizin Berlin. After surgery, the subcutaneous fat was carefully removed and the remaining sample was wrapped in aluminum foil and stored at 20 °C (min 24 h, max 6 months). 2.2. Liposome formulations Flexible and rigid liposomes sized approximately 130 nm (Table 1) were prepared and labeled with fluorescent dyes. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B), ammonium salt (Rh-DOPE, Avanti Polar Lipids, Alabaster, USA) Table 1 Characterization of liposomes. Liposomes
Flexible Rigid
Before dialysis
After dialysis
is a lipophilic fluorescent dye located in the liposomal membrane. The excitation wavelength of rhodamine is 540 nm and the detection wavelength of the red fluorescence is 570 nm. 5,6-Carboxyfluorescein, disodium salt (CF, Sigma Aldrich, Taufkirchen, Germany) is a hydrophilic fluorescent dye, which is enclosed into the aqueous core of the liposomes. The excitation wavelength of carboxyfluorescein is 490 nm and the detection wavelength is 520 nm. Green fluorescence is detectable after degradation of the liposomes and subsequent release of the dye. 2.3. Preparation of rigid liposomes For the preparation of the liposomes with a rigid bilayer, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt (DPPG), were purchased from Genzyme Pharmaceuticals (Liestal, Switzerland). 90 mg DPPC, 10 mg DPPG and 0.5 mg Rh-DOPE were dissolved in approximately 1.5 ml chloroform (Merck, Darmstadt, Germany). After evaporation of the chloroform at 50 °C under a vacuum (RotaVapor, Büchi Labortechnik, Konstanz, Germany), the obtained thin lipid film was hydrated with 1 ml 10 mM Tris buffer pH 7.4 (Sigma Aldrich, Taufkirchen, Germany) containing 20 mM carboxyfluorescein, and shaken for approximately 30 min at 50 °C. After equilibration at room temperature overnight under a nitrogen atmosphere, small unilamellar liposomes were prepared by extrusion (LiposoFast basic, Avestin, Ottawa, Canada) with 21 repetitions through a polycarbonate membrane with a pore size of 100 nm (Armatis, Mannheim, Germany) at 55 °C. 2.4. Preparation of flexible liposomes (invasomes) For the preparation of the flexible liposomes, soybean lecithin in ethanol 75:20 w/w (NAT 8539) was purchased from Phospholipid GmbH (Cologne, Germany). 0.5 mg Rh-DOPE was dissolved in 134 mg of NAT 8539 corresponding to 100 mg soybean lecithin. The mixture was vortexed at room temperature until a clear solution was obtained. 20 ll of a mixture of limonene, citral and cineol (10:45:45 by volume, Sigma Aldrich, Taufkirchen, Germany) was added. Subsequently, 900 ll 10 mM Tris buffer pH 7.4 containing 20 mM carboxyfluorescein was added under vortexing. The mixture was sonicated (sonication bath USR 54 h, Merck EuroLab NV, VWR, Germany) for approximately 15 min at room temperature and small unilamellar liposomes were prepared by successive extrusion (LiposoFast basic) at room temperature twice each through the polycarbonate membranes with pore sizes of 400 nm, 200 nm, 100 nm and 50 nm, respectively. Non-entrapped carboxyfluorescein was removed by dialysis of the liposome formulations (MWCO 12–14 kDa, Visking, Medical International, Great Britain) at room temperature against 10 mM Tris buffer pH 7.4 (rigid liposomes) or against 10 mM Tris buffer pH 7.4 containing 3% ethanol (v/v) and a few droplets of the terpene mixture (flexible liposomes). Removal of the non-entrapped carboxyfluorescein was completed after 30 h for both liposome dispersions with the dialysis fluid being replaced four times during this process. As a consequence, the dispersion was diluted until approximately one third of the dialyzed fluid remained. The loss of entrapped carboxyfluorescein can be excluded as the size of the liposomes increased as a result of the supplied fluid (Table 1). During the entire preparation process and dialysis, the formulations were stored at 4 °C under the exclusion of light.
Z – average (nm)
PDI
Z – average (nm)
PDI
2.5. Reference substance caffeine
139 ± 1.5 115 ± 1.4
0.1 ± 0.005 0.07 ± 0.009
146 ± 0.6 117 ± 1.5
0.11 ± 0.005 0.08 ± 0.003
Caffeine (Sigma Aldrich, Steinhagen, Germany) was used as a control substance. 2.5 g Caffeine was dissolved in 1000 ml
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Dulbecco’s phosphate-buffered saline (DPBS) from PANBiotec GmbH (Aidenbach, Germany). Subsequently, 10 ll/cm2 of the reference solution was applied at a concentration of 2.5 mg/ml DPBS. 2.5.1. Franz diffusion cell experiments Thawed human full thickness skin was mounted to a static Franz diffusion cell (FD-C) (surface area 1.76 cm2; Gauer–Glas, Püttlingen, Germany) [29] with the stratum corneum facing upwards and the dermis positioned to contact the receptor medium DPBS (Dulbecco’s Phosphate-Buffered Saline, PAA, Pasching, Austria), which was maintained at 32 °C ± 1 °C and continuously stirred by a magnetic bar. According to the OECD guideline 428, caffeine was used as reference probe to control the quality of the experimental series [17]. All test formulations (10 lg/cm2, finite dose) were applied for 24 h, during which aliquots of the receptor solution were collected (0, 2, 4, 6 and 24 h after dosing; under replacement) for fluorescence analysis. The lack of a fluorescence signal in the removed receptor medium probe proved a valid test procedure. Parallel tests were performed, i.e., all test formulations and reference substances were treated equally. As caffeine penetrates into the receptor medium and the amount must be analyzed to validate the quality of the tests, it is indispensable that the receptor medium is also removed from the Franz diffusion cells with a test formulation and replaced by fresh medium. Even if the liposomes do not penetrate into the receptor solution, this must be verified by fluorescence measurements. As expected, fluorescence was never detected in the receptor medium. It could be demonstrated that no fluorescent dye reached the receptor reservoir as the follicular pathway was used for penetration. Because vellus hairs do not extend into the subcutaneous fat tissue, the samples did not exhibit any holes as it is usual for split skin, indicating that the full thickness skin was not damaged. Also, the skin sample was not shifted during massage application. As the data established for caffeine are comparable to those already published, the experiment was correctly performed. Each experiment was conducted with at least 6 FD-C (originating from 6 donor subjects) and repeated three times so that the data were acquired over the course of 4 days. The effects of massage (study design 1) and occlusion (study design 2) were derived from experiments on skin samples of the same donor skin, in order to exclude donor-related skin properties. The structural integrity of the liposomes was not affected by sink conditioning as the liposomes penetrated exclusively via the follicular shunt and did not reach the receptor reservoir. The liposomes did not break when subjected to massage or occlusion. This was shown in study design 1, in which occlusion was applied and the effects of massage were investigated. If the liposomes had been broken, the skin measurements would have shown strong carboxyfluorescein coloration. However, the CSLM images disclosed strong rhodamine fluorescence, but only weak carboxyfluorescein coloration. In deeper dermal layers, weaker rhodamine fluorescence and increased carboxyfluorescence intensity was detectable. Intact liposomes are represented by ample red and only minute green colorations in the images. If the liposomes break, carboxyfluorescein solution is released from the core, and the fragments migrate into deeper regions in the hair follicle. 2.5.2. Study design 1 – Application of massage using a device The massage device used was an electrical toothbrush (Braun, Oral B, Germany), in which the brush was replaced with a metallic ball that was covered with a cut-off finger of a latex glove. The vibration of the toothbrush led to an oscillation of the metallic ball and thereby enabled massaging of the topically applied test formulations into the skin.
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After mounting the human full thickness skin on the FD-C and homogeneous application of the liposome formulations, massage was applied for 3 min on the test samples, whereas no massage was applied on the control samples. Tests were regarded valid if a test probe removed from the receptor solution yielded no fluorescent signal. The structural integrity of the liposomes was not affected by sink conditioning as the liposomes penetrated exclusively via the follicular shunt and did not reach the receptor reservoir. The donor compartments of test and control samples were then covered with parafilm and aluminum foil to ensure occlusion (and exclusion of light) during the 24 h exposure. At the end of the experiments, any donor formulation found on the application site was removed using cotton swabs, and 8 mm punch biopsies were taken from the skin samples and shock frozen in liquid nitrogen for preparation of 10 lm cyro-sections. The sections were then analyzed using confocal laser scanning microscopy (CLSM). Anticipating that the application of massage led to significantly increased penetration depths of both liposome preparations, the massage device was also applied for the study of the influence of occlusion on the follicular penetration depth of both liposome preparations. 2.5.3. Study design 2 – Investigation of the influence of occlusion The influence of occlusion on the formulations was investigated in study design 2. Both the test formulations and the reference substances were investigated with and without occlusion, respectively, at 70% environmental humidity, corresponding to OECD guidelines. Thus, the conditions for the occluded skin samples were not significantly different from the non-occluded samples, such that the behavior of the liposome types permits conclusions as to an influence of occlusion only if the humidity of the ambient air dropped significantly. The results shown in Fig. 3 suggest that flexible liposomes are influenced much less by the environmental humidity than rigid liposomes. Investigations were performed in FD-C, whereas the liposome application and massage were performed as described for study design 1. During the experiments, part of the FD-C was occluded by covering the donor chamber with parafilm and aluminum foil to create a microclimate and to exclude light above the epidermis, while the remaining FD-C was non-occluded, i.e., the donor chamber remained open. The humidity of the environment was 70% in the experiments with non-occluded skin as recommended [17]. At the end of the experiment, skin samples were prepared for CLSM as described in the study design 1. 2.5.4. Confocal laser scanning microscopy (CLSM) All cryo-sections of the skin containing hair follicles were analyzed with a CLSM 410 (Zeiss, Jena, Germany) to determine the follicular penetration depths of the liposomes. The field of view magnification of the CLSM was 250 250 lm. As the liposomes were double-labeled with two fluorescent dyes, the penetration of the constituents of the outer (rhodamine) and the inner sphere (carboxyfluorescein) of the liposomes could be detected separately at 570 nm and 520 nm, respectively. The penetration depths of the fluorescent dyes were determined in lm. In order to exclude the influence of autofluorescence of the skin, a corresponding filter was utilized to cut off the fluorescence below 510 nm, which corresponds to the autofluorescence of the skin. 2.5.5. Statistics Statistical analysis was performed with the software program IBM SPSS Statistics 19Ò. Explorative data analysis and the nonparametric Wilcoxon test were used to compare the penetration
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depths of the fluorescence probes of test and control samples. p 6 0.05 was considered as significant.
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It could be demonstrated that liposomes can penetrate effectively into the hair follicles via the follicular penetration pathway. Fig. 2 shows that the hair follicle comprises both fluorescence dyes. Although the hydrophilic carboxyfluorescein dye was also detectable in the tissue adjacent to the follicle, it did not penetrate into the receptor fluid. The carboxyfluorescein was not detectable in the receptor fluid within 24 h as the hair follicle serves as a reservoir. As a result, the fluorescent dye rhodamine, which binds to the lipids of the liposomes, was not detected outside the follicle.
Penetration depth [µm]
3. Results
After massage application, the follicular penetration depths of the fluorescent dyes were determined. Rhodamine covalently binds to phospholipids of the liposome membrane, and thus is a marker for the lipophilic agents forming the outer sphere of the liposomes, while carboxyfluorescein embedded into the inner sphere of the liposomes is a marker of hydrophilic agents dissolved in the aqueous phase. For all four tested variations (rigid and flexible liposomes with and without massage), it can be concluded that carboxyfluorescein penetrated significantly deeper than the rhodamine-conjugate, the marker for the liposome bilayer (p 6 0.05; Fig. 1). Massage application favored penetration into the depth of the hair follicle infundibula of both fluorescent markers in the cases of rigid as well as flexible liposomes (p 6 0.05). Independent of the massage application, the flexible liposomes penetrated significantly deeper than the rigid liposomes (p 6 0.05). The liposomes investigated in the present study penetrated to a depth of 93.2 ± 11.7 lm (rigid liposomes) and 137.3 ± 26.5 lm (flexible liposomes) when applied without massage, meaning that neither the sebaceous gland nor the bulge region was reached. The application of massage led to a penetration depth of 477.2 ± 61 lm and 698.8 ± 90.7 lm, respectively, which enabled the target sites to be reached. Fig. 1 shows the preparation of fresh liposomes. The influence of massage on the penetration depth was investigated using occluded samples. Most liposomes remained undamaged, penetrating to a depth of 500 lm (rigid liposomes) and 700 lm (flexible liposomes), respectively, within 24 h. The released carboxyfluorescein reached penetration depths of approximately 1200 lm, independent of the type of liposomes implemented. The liposomes applied without massage showed penetration depths of less than 200 lm for rhodamine and of less than 300 lm for carboxyfluorescein, independent of the liposome types. In Fig. 2, the penetration of both kinds of liposomes is depicted, demonstrating that the penetration of carboxyfluorescein (Fig. 2b and d) was more efficient than the penetration of rhodamine (Fig. 2a and c). In the case of massage application (Fig. 2c and d), the fluorescence signal could be detected in deeper regions of the hair follicles. 3.2. Effect of occlusion on the follicular penetration depth of rigid and flexible liposomes The microclimate generated by occlusion leads to a stronger hydration of the skin, which in turn promotes the penetration of hydrophilic substances. Once released from the core of the liposomes, carboxyfluorescein, e.g., can automatically penetrate into deeper skin layers because the cells are hydrated and the dye can disperse. Likewise, the effect of occlusion on the follicular penetra-
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3.1. Effect of massage on the follicular penetration of rigid and flexible liposomes
*
*
Rigid-Rh Rigid-CF Rigid-Rh Rigid-CF Flex-Rh Flex-CF Flex-Rh Flex-CF
-M
+M
-M
+M
Fig. 1. Massage effect follicular penetration depths of rigid (Rigid) and flexible (Flex) liposomes with (+M) and without ( M) massage appliance. Rh-DOPE (Rh) was used as a label for the liposome bilayer and CF for the inner, aqueous liposome core. (n = 6; p < 0.05).
Fig. 2. Massage effect laser scanning microscope images of the penetration of flexible liposomes: (a) penetration of rhodamine without massage, (b) penetration of carboxyfluorescein without massage, (c) penetration of rhodamine with massage, and (d) penetration of carboxyfluorescein with massage.
tion depth of rigid and flexible liposomes was investigated. The results are summarized in Fig. 3. Again, for all four tested variations (rigid and flexible liposomes with and without occlusion), hydrophilic carboxyfluorescein penetrated significantly deeper than the rhodamine-conjugate (p 6 0.05). For the rigid liposomes, occlusion had a positive effect on the penetration depth (p 6 0.05), whereas for the flexible liposomes, occlusion did not increase the penetration depth of rhodamine (p > 0.05) and even appeared to have inhibited the penetration of carboxyfluorescein. In the case of
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0 Rigid-Rh Rigid-CF Rigid-Rh Rigid-CF Flex-Rh Flex-CF Flex-Rh Flex-CF
-O
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-O
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Fig. 3. Occlusion effect penetration depths of rigid (Rigid) and flexible (Flex) liposomes with (+O) and without ( O) occlusion after massage application. The outer sphere of the liposomes was labeled with Rh-DOPE (Rh), the inner sphere was labeled with CF. (n = 6; p < 0.05).
non-occlusion, the flexible liposomes penetrated significantly deeper into the hair follicles (p 6 0.05). For the investigations on the influence of occlusion, the same batch of liposomes was used as for the investigations on the influence of massage. The investigations were not conducted before the massage experiments were completed and evaluated. Fig. 3 illustrates the penetration of intact and broken liposomes. The broken liposomes migrate to deeper skin layers than intact liposomes, which explains why rhodamine is detectable in liposome fragments up to a depth of approximately 1200 lm. In non-occluded samples, the rigid liposomes exhibit similar penetration depths as shown in Fig. 1 for the occluded samples. This is attributed to the high environmental humidity, which under occlusion and through the development of a microclimate above the dermis can exceed 70%. The results show that the rigid liposomes desiccate more rapidly if a microclimate cannot develop above the skin. In this case, penetration ceases earlier and at lower depths. Once a microclimate has formed above the epidermis, the rigid liposomes exhibit penetration depths comparable to those of the flexible liposomes. The flexible liposomes are less influenced by occlusion, i.e., they penetrate to the same depths, whether or not they are occluded. 4. Discussion The movement of hair in hair follicles has been described as crucial to stimulating follicular penetration. Recent investigations have shown that the pumping effect of the moving hairs is most effective when the particle size of the topically applied substance is in the range of the cuticula thickness of the corresponding hair [24]. Larger and smaller particles did not penetrate as efficiently into the hair follicles [23]. On the contrary, the penetration of liposomes through the stratum corneum decreases as their diameter increases [30]. While the movement of the hair occurs physiologically in vivo and is additionally supported by the user when products are topically applied by rubbing, this phenomenon must be induced in ex vivo studies by the application of massage. The present investigation demonstrated that the application of massage had a significant positive effect on the follicular penetration depth for both liposome preparations. The penetration depth was increased by up to a factor of 5 when massage was applied. Lademann et al. observed a comparable increase in the follicular
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penetration depth of 320 nm-sized particles into ex vivo skin subsequent to massage application [25]. Moreover, the results indicate liposomal degradation as the green fluorescence of carboxyfluorescein, which is the label of the inner aqueous liposome core, was detected independent of rhodamine-related fluorescence of the phospholipid. It can be assumed that after the degradation at a significant depth within the hair follicle, both marker dyes penetrate independently. The results show that liposomes can be utilized to deliver hydrophilic substances such as carboxyfluorescein deeply into the hair follicles. Within the hair follicle, several target sites of interest are located, such as the sebaceous gland, the bulge region, localization of the stem cells, or the infundibulum, which represents an interrupted barrier with increased permeability in the lower part and is surrounded by a high density of immune cells and an extensive capillary network. This capillary network offers new therapeutic options concerning the therapy of sebaceous gland-associated diseases such as acne vulgaris, but also concerning topical vaccinations or regenerative medicine [23]. It has been shown previously that liposomal preparations can induce better results in the treatment of acne vulgaris than conventional treatments [19]. Recently, Vogt et al. [31] investigated the morphometry of vellus and terminal hair follicles. For vellus hair follicles, present mainly in breast and abdominal skin, the average length of the infundibulum, e.g., at the end of which the sebaceous gland is located, is 225 ± 34 lm. Additionally, the influence of environmental humidity on the penetration of rigid and flexible liposomes was analyzed. Percutaneous absorption is generally increased when the site of application is occluded [26]. However, in regard to the implementation of flexible liposomes, occlusion has been described to be counterproductive [28,32]. The results of the present study revealed that occlusion had a significant positive effect on the follicular penetration depth for rigid liposomes, whereas for the flexible liposomes, no significant difference could be detected for the lipids of the outer sphere (rhodamine). This is presumably also the case for other lipophilic agents embedded in the liposomal shell. However, occlusion led to a slightly but significantly decreased penetration depth of the hydrophilic marker carboxyfluorescein. This should also remain true for other hydrophilic agents dissolved in the aqueous core. It has been suggested [28,32] that in the case of non-occlusive application, the increased drug transport can be caused by a more profound interaction between the liposomal constituents and the skin and/or the presence of a hydration gradient in the skin [7]. According to Cevc and Blume [32], the water gradient is an important driving force for drug diffusion. During the occlusive application, the water gradient is expected to be absent [28]. The same explanation might be valid for follicular penetration, but this must be investigated further. The results of the present study allow the conclusion that massage application is an important element when investigating follicular penetration of liposomes and other particulates ex vivo. Massage application simulates the physiological movement of hair, which is important for an effective follicular delivery, and additionally imitates the application behavior of the end user. Although valid for several other dermatological topical preparations, occlusion did not increase the follicular penetration of the flexible liposomes. Thus, a potential beneficial effect of occlusion must to be evaluated for new formulations. Acknowledgment We would like to thank the Foundation ‘‘Skin Physiology’’ of the Donor Association for German Science and Humanities for financial support.
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