International Journal of Pharmaceutics 487 (2015) 135–141
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Propranolol hydrochloride-loaded liposomal gel for transdermal delivery: Characterization and in vivo evaluation Yuanyuan Guan, Tiantian Zuo, Minglu Chang, Fang Zhang, Ting Wei, Wei Shao, Guimei Lin * School of Pharmaceutical Science, Shandong University, Jinan 250012, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 January 2015 Accepted 10 April 2015 Available online 13 April 2015
This study was to develop propranolol hydrochloride (PRO)-loaded liposomal gel as a topical drug delivery system. Scanning electron microscope (SEM) revealed that the liposomes were spherical and scattered in the surface of the gel. Pseudoplastic flows of PRO liposomal gel were showed after stored at three different temperatures. Besides, PRO liposomal gel showed non-irritating to the skin of rabbit. Skin deposition studies in vivo demonstrated that PRO liposomal gel can apparently increase drug content in skin compared with PRO gel. Histopathology examination showed that PRO liposomal gel could obviously weaken the barrier function of stratum corneum (SC) by comparison with PRO gel. What is more, the plasma pharmacokinetic showed the maximum concentration in plasma was 0.41 mg/mL and 1.17 mg/mL after topical and oral administration respectively. However, tissue distribution study showed PRO liposomal gel obviously changed drug distribution in tissues, significantly increased drug concentration in skin with about 74 folds compared with PRO gel. In conclusion, liposomes-based gel could be a promising vehicle as a transdermal delivery system of PRO. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Propranolol hydrochloride Liposomal gel Transdermal delivery Pharmacokinetic Tissue distribution
1. Introduction Propranolol hydrochloride (PRO) is one of b-blockers which can antagonize the b-adrenergic pathway, blocking receptors such as in heart, pancreas, liver, and in peripheral blood vessels and bronchi (Sánchez-Carpintero et al., 2011). Since Léauté-Labrèze et al. (2008) first described its occasional antiproliferative effect on severe infantile hemangioma (IH), PRO has gradually become the first-line therapy for IH. IH is one of the most common tumors in infants, which can lead to deformities when they are located in the facial areas of the lip, nasal tip or the ear (Matuszczak et al., 2013). Therefore, it is necessary to treat IH timely. However, PRO given orally shows significant first pass metabolism, some side effects on heart and poor patient compliance (Lawley et al., 2009). As a result, we need to seek for another administration route to solve these problems. Transdermal drug delivery offers potential to overcome disadvantages caused by oral administration. Unfortunately, the stratum corneum (SC) which is recognized as the primary barrier
* Corresponding author at: School of Pharmaceutical Science, Shandong University, 44 Wenhuaxi Rd. Jinan, Shandong 250012, China. Tel.: +86 53188382007; fax: +86 53188382548. E-mail address: [email protected] (G. Lin). http://dx.doi.org/10.1016/j.ijpharm.2015.04.023 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
for transdermal drug delivery makes it difficult for most drugs to enter into skin by this route. In recent years liposomes have been intensively studied as drug carrier systems for topical delivery. Several in vivo and in vitro transport studies reported that liposomes can improve skin permeability (Verma et al., 2003; Dragicevic-Curic et al., 2008; Vyas et al., 2013) and enhanced the skin deposition with reduction in systemic absorption (Patel et al., 2000), which suggested that liposomes were useful for topical drug delivery. As liposomes and biomembranes of skin have lipid bilayer membrane structure in common, liposomes have a good biocompatibility with skin. The major disadvantage of using liposomes topically lies in the liquid nature of the preparation. To achieve the viscosity desirable for topical application, liposomes should be incorporated into a suitable vehicle. It has been well established that liposomes are fairly compatible with carbopol (Elnaggar et al., 2014), which has a good bioadhesive properties and the ability to prolong the retention of the formulation on the mucosal surface. The purpose of this study was to develop a topical delivery carrier for the use of PRO. As a result, scanning electron microscope (SEM), rheological study, skin irritation test, in vivo skin deposition studies, histopathology examination, plasma pharmacokinetic properties and biodistribution characteristics were carried out to evaluated PRO liposomal gel.
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Fig. 1. The SEM photomicrographs of liposomal gel. A (55); B (20,000).
2. Materials and methods 2.1. Materials PRO was purchased from Wuhan Ding Lixin Chemical Co. Ltd., China (99.90%). Phosphatidyl ethanolamine (PE) was obtained from Jianglaibio, Shanghai, China. Cholesterol (Chol) was supplied by Aobox Biotechnology, Beijing, China. Carbopol 934 was obtained from Jingxifang Technology Co. Ltd., Beijing, China. Methanol used was of chromatographic grade and others were of analytical grade.
2.2.3. Rheological measurements Rheological tests of liposomal gel were performed by a rotational viscosimeter (RheoStress 6000, Thermo Scientific, Germany). The flow properties of the developed gel were conducted at 25 1 C (room temperature) by using a cone/ plate-measuring device (cone with D = 35 mm, 1 ). The measurements were carried out by increasing the shear rate from 0.1 s 1 to 100 s 1. In order to explore the effect of storage conditions on the gel properties, rheological measurements were performed after production and 1 week of storage at three different temperatures (4 C, 25 C and 40 C).
2.2. Methods 2.2.1. Preparation of PRO liposomes and liposomal gel PRO liposomes and PRO liposomal gel were prepared as detailed in our previous work (Guan et al., 2014). 2.2.2. SEM of PRO liposomal gel PRO liposomal gel was prefreezed in the ultra-low-temperatue refrigerator of 80 C (DW-86L, Haier, China) for 24 h before they were freeze-dried by the use of freeze drying equipment for 24 h. The final products were fixed on a platform and sprayed with gold and observed by the JSM-6700F scanning electron microscope.
2.2.4. Primary skin irritation test To evaluate and compare skin irritation of PRO liposomal gel and blank liposomal gel, the study was carried out on rabbits. New Zealand rabbits weighing 2.5–3.0 kg were fed with food and water for 1 week to adapt to the environment before the study. The hairs of the dorsal portion were removed with the help of shaver at 24 h prior to application of the formulations. The rabbits were divided into three groups of 3 rabbits each as follows: Group 1: no application (control). Group 2: blank liposomal gel. Group 3: PRO liposomal gel. PRO liposomal gel (1.0 g) and blank liposomal gel (1.0 g) were applied on the hairless skin of the rabbits by uniform spreading within the area of 2 cm 2 cm, respectively. The preparations were removed after 24 h and each of the area was observed for any changes such as erythema or edema. To study the cumulative effect of repeated applications, the respective preparations were applied once daily for next 7 days on the same area of hairless skin. Draize scale was applied to evaluate the skin irritation (Draize et al., 1944). The irritation scores between 0 and 4 were used to grade stimulus intensity which range from no response to a severe response.
Table 1 The irritation scores of gels to the rabbit skin after administration of 7 days. Formulation
Fig. 2. Shear stress–shear rate curves of PRO liposomal gel after production and 1 week of storage at three different temperatures.
Control (Group 1) Blank liposomal gel (Group 2) PRO liposomal gel (Group 3) Stimulus intensity
Irritation index 24 h
Day 7
0 0 0 Nonirritant
0 0 0 Nonirritant
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Fig. 3. The irritation photos after administration of preparations to skin. A – control (24 h); B – control (Day 7). C – PRO liposomal gel (24 h); D – PRO liposomal gel (Day 7). E – blank liposomal gel (24 h); F – blank liposomal gel (Day 7).
2.2.5. In vivo skin deposition studies 2.2.5.1. Animals. Studies were performed on Kunming strain mice (25 2) g, which were provided by the Experimental Animal Center of Shandong University (Jinan, China). All animals were acclimatized for 1 week before use. Prior to formal experiments, mice were kept under food fasting but free access to water overnight. All studies were performed in accordance with the ethics and regulations of animal experiments of pharmaceutical sciences, Shandong University, China. 2.2.5.2. Skin sample processing. The Kunming strain mice were randomly divided into two treatment groups of 4 mice each. PRO liposomal gel and PRO gel were administered transdermally at a dose of 30 mg/kg, respectively. Then animals were sacrificed by
cervical dislocation at the established post-administration time point (0.5, 1, 2, 4, 6, 12, 24 h). Skin samples were collected from the back of the mice and then washed in normal saline and dried using filter paper. All the samples were stored at 20 C until further analysis.
2.2.6. Histopathology examination by light microscopy PRO liposomal gel was given to Kunming strain mice (25 2) g for 24 h. Afterwards, the skins were immediately collected and fixed in 10% formaldehyde solution for 24 h. Then samples were dehydrated using ethanol, embedded in paraffin wax and followed by stained with hematoxylin and eosin (H&E). At last, skins were cut by a cryostat into 3–4 mm and evaluated under light microscopy.
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Fig. 4. Mean concentration–time curves of PRO after topical administration of PRO liposomal gel and PRO gel to mice at 30.0 mg/kg (mean SD; n = 4).
2.2.7. Pharmacokinetic and tissue distribution study The principle of treating with Kunming strain mice was the same as described above except that PRO liposomal gel and PRO suspension were administered transdermally and orally at a dose of 75 mg/kg, respectively. Besides, blood samples were collected from the ocular vein into heparin-containing eppendorf tubes at 0.5, 1, 2, 4, 6, 8 h and then immediately centrifuged at 4000 rpm for 20 min to obtain supernate. The dorsal skins beneath the drug application site and the organs (heart, liver, spleen, lung and kidney) were separated, washed in normal saline and dried with filter paper. Plasma and tissue samples were stored at 20 C until further analysis. The concentrations of PRO at different time points were expressed as mean values and standard deviations, and the mean concentration–time curves were plotted. Pharmacokinetic parameters were calculated using the Drug and Statistics software (DAS, version 2.0). 2.2.8. High performance liquid chromatography (HPLC) analysis The quantification of PRO was performed on an Agilent HPLC system (Agilent, USA). The system was equipped with a variable wavelength UV detector with being set at 289 nm in this study and a reversed-phase C18 column (5 mm, 4.6 mm 250 mm, Welch Materials, Inc.). The mobile phase was consisted of methanol and 0.01 mol/L dipotassium phosphate solution (50:50, v/v) and was used at a flow rate of 1.0 mL/min for chromatographic separation and analysis. The column temperature was set at 25 C and samples were injected into the column at a constant volume of 20 mL. 3. Results and discussion 3.1. SEM of liposomal gel SEM was used to observed liposomes surface morphology. Fig. 1A showed that liposomes were scattered in the surface of the gel. It can clearly show that liposomes were spherical and about 260 nm in size when observed under a greater magnification (Fig. 1B). In other words, liposomes had no obvious change in morphology and sizes after they were incorporated into the gel matrix compared with our previous date monitored by transmission electron microscope (TEM, Hitachi, Japan) and DelsaTM Nano C Particle Analyzer (Beckman Counter Ltd., USA) respectively.
Fig. 5. Photomicrographs of the skin structure of using different preparations (400). A – skin of no preparations; B – skin treated with PRO liposomal gel; C – skin treated with PRO gel.
3.2. Rheological measurements The rheological properties play an important role for topical gel formulations in delivering the molecules onto or across the skin because they can greatly affect spreadibility, adhesiveness, drug release from semisolid formulations, and subsequent penetration
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Table 3 The targeting disposition of PRO after topical administration of PRO liposomal gel and oral administration of PRO suspension (n = 4). AUC (mg h/g)
Tissue
PRO liposomal gel Heart Liver Spleen Lung Kidney r = (AUC
51.31 20.84 87.38 141.80 52.11
0.26 1.35 7.01 6.15 6.37
r PRO suspension 68.99 78.06 92.19 242.08 92.29
7.98 9.82 8.73 22.52 18.86
0.74 0.27 0.95 0.59 0.56
topica/AUCoral).
3.3. Skin irritation test One of the major disadvantages associated with transdermal drug delivery system is skin irritation of preparations. To study security of products, rabbits are often used for the prediction of skin irritation effects in humans because of their high sensitivity to irritations (Ishii et al., 2013). The results of the skin irritation studies were listed in Table 1. It indicated that PRO liposomal gel and blank liposomal gel resulted in no skin irritation compared with control group after 24 h of application and 7 days of repeated applications. Besides, the irritation photos of rabbits (Fig. 3) clearly
Fig. 6. Mean plasma (A) and skin (B) concentration–time curves of PRO after topical and oral administration to mice at 75.0 mg/kg (mean SD; n = 4).
into the skin (Elnaggar et al., 2014). Fig. 2 clearly showed that the slope of rheogram (namely the viscosity of the gel) was decreased with the increase of shear rate, which indicated that the developed gel showed pseudoplastic flow. In fact, with the shear rate increasing the structure of the gel began to disrupt and the particles started to align until it started flowing. On the other hand, the viscosity of the developed gel was high (255.2 Pas at shear rate of 0.1 s 1, date was not shown) to adhere to the skin, which was suitable for the transdermal administration. Besides, no obvious change of the viscosity was observed post 7 days of storage at three different temperatures compared with that of gel on the 0 day seen from Fig. 2. All this indicated that preparations were suitable for the transdermal administration and stable when stored at 40 C.
Table 2 The pharmacokinetic parameters of PRO in mice after topical and oral application. Cmax mg/mL(or mg/g) Tmax (h) AUC0–t mg h/mL Plasma Skin
Topical Oral Topical Oral
0.41 0.04 1.17 0.13 265.10 23.34 13.37 0.93
6.5 1.25 4.0 0.5
1.72 0.17 2.67 0.23 1618.50 109.07 21.84 1.31
Fig. 7. The concentration level of PRO in tissues of mice over 0.5–8 h after topical (A) and oral (B) administration PRO.
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showed no oedema and erythema after administration of 24 h and7 days. Thus, PRO liposomal gel demonstrated evident advantage of non-irritating to skin, which indicated their potential in improving patient acceptance and topical delivery. 3.4. In vivo skin deposition studies The cumulative PRO concentration in skin was showed in Fig. 4. PRO liposomal gel increased rapidly from 0 to 4 h compared with PRO gel group. The max drug concentration was 123.34 mg/g for PRO liposomal gel, obviously remarkable than that of PRO gel (75.65 mg/g). As Fig. 4 showed that PRO liposomal gel was more able to increase drug content in skin, as a result it can provide effective drug concentration for a long time in comparison with PRO gel. The differences can be attributed that the presence of the liposomes. Yokomizo and Sagitani (1996) found that phospholipids had an effect on the permeability barrier of SC. In fact when liposomes act on the skin they can successively fuse with the SC by mixing with the intercellular polar lipids and loosening the bilayered structure of the intercellular mortar of the SC (Sinico and Fadda, 2009). 3.5. Histopathology examination by light microscopy Skin is the outermost and largest organ in the body, which from outside to inside is consisted of epidermis, dermis and subcutis (Lai-Cheong and McGrath, 2009). As the outmost layer of epidermis to protect skin from invasion, SC is composed of about 10 layers of flattened corneocytes. They have a thick insoluble cell envelope, which is consisted of loricrin and involucrin as well as intercellular lipids (Alexander et al., 2012). As a result, SC becomes the major challenge for the penetration of drugs across the skin. As was shown in Fig. 5A, the skin of control group displayed a clear delineation among epidermis, dermis and subcutis. The SC cells were arranged neatly with no inflammation being observed. After the administration of PRO liposomal gel, the intercellular gaps of SC were increased and the SC structure was loose (Fig. 5B), while PRO gel slightly changed the tight junction of the SC (Fig. 5C). Besides, there being no obvious change in dermis and subcutis of all samples demonstrated that the preparation did not cause irritation and was safe to patients. In a conclusion, PRO liposomal gel can significantly weaken the barrier function of SC and promote drug permeation, which explain the phenomenon that PRO liposomal gel had a higher drug deposition in skin than PRO gel. The change was attributed to that liposomes produced an enhancing effect because their lipid components may penetrate deep into the SC or militate with skin lipids to loosen their structure (El Maghraby et al., 2008). 3.6. Plasma and tissue concentration–time curves As is shown in Fig. 6A, plasma concentration–time curves of PRO liposomal gel and PRO suspension were significantly different. The maximum concentration in plasma appeared at a 6.5 h (Cmax = 0.41 mg/mL) and 1.25 h (Cmax = 1.17 mg/mL) after topical and oral administration respectively (Table 2), which indicated that the extension of time for PRO absorbed in the systemic circulation. The maximum concentration in plasma post oral administration was 2.85 folds of that topical administration, while AUC0–8h of PRO after oral administration was 1.55 folds compared with topical administration, both of which indicated that lower lever of PRO was absorbed in the systemic circulation after topical administration compared with oral treatment. Besides, after 4 h drug concentration post topical administration was higher than that of oral administration. The reason can be attributed to the drug repository effect of skin.
As was shown that maximum drug concentration in skin (265.00 mg/g) after local application was significantly higher than oral application (13.37 mg/g, Table 2). The AUC0–8h value post local application was 74-fold higher than oral application, which demonstrated that the topical application was more effective than oral application at the aspect of increasing drug concentration in skin. Besides, PRO can maintain an effective drug concentration for a long time after topical application (Fig. 6B). As a result PRO liposomal gel can prolong time of acting on body and improve patient compliance. The targeting disposition of PRO after topical administration of PRO liposomal gel and oral administration of PRO suspension were shown in Table 3. The re value of heart, liver, spleen, lung, kidney all less than 1, which indicated a lower bioavailability after topical administration compared with oral treatment. What's more, lower drug disposition in skin would reduce the side effects on the organs especially heart mainly reflecting in such as bradycardia, hypotension, and hypoglycemia. In order to more visually show the biodistribution characteristics of PRO after administration, concentration distribution histograms over the time course of 0.5–8 h were plotted. Fig. 7 showed that PRO liposomal gel obviously changed drug distribution in tissues, relatively increased drug concentration in skin, and decreased drug concentration in other tissues. 4. Conclusion The present work demonstrated that PRO liposomal gel as a transdermal preparation was prepared successfully. It could obviously increase drug content in skin to reduce dosing frequency compared with PRO suspension. Besides, it decreased drug content in the systemic circulation. As a result, side effects of PRO on organs especially the heart can be reduced. Thus, the liposomal gel could be a promising vehicle for topical delivery of PRO. Acknowledgements This work was supported by the National Natural Science Foundation of China(Grant No.: 21203112) and the Natural Science Foundation of Shandong Province (Grant No.: ZR2012BQ002).
References Alexander, A., Dwivedi, S., Giri, T.K., Saraf, S., Saraf, S., Tripathi, D.K., 2012. Approaches for breaking the barriers of drug permeation through transdermal drug delivery. J. Control. Release 164, 26–40. Draize, J.H., Woodard, G., Calvery, H.O., 1944. Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharmacol. Exp. Ther. 82, 377–390. Dragicevic-Curic, N., Scheglmann, D., Albrecht, V., Fahr, A., 2008. Temoporfin-loaded invasomes: development, characterization andin vitroskin penetration studies. J. Control. Release 127, 59–69. El Maghraby, G.M., Barry, B.W., Williams, A.C., 2008. Liposomes and skin: from drug delivery to model membranes. Eur. J. Pharm. Sci. 34, 203–222. Elnaggar, Y.S., El-Refaie, W.M., El-Massik, M.A., Abdallah, O.Y., 2014. Lecithin-based nanostructured gels for skin delivery: an update on state of art and recent applications. J. Control. Release 180, 10–24. Guan, Y., Lin, G., Shao, W., 2014. Preparation and evaluation of propranolol hydrochloride liposomal gel. Chin. J. Pharm. Biotechnol. 21, 525–529. Ishii, S., Ishii, K., Nakadate, M., Yamasaki, K., 2013. Correlation study in skin and eye irritation between rabbits and humans based on published literatures. Food Chem. Toxicol. 55, 596–601. Léauté-Labrèze, C., de la Roque, E.D., Hubiche, T., Boralevi, F., Thambo, J.-B., Taïeb, A., 2008. Propranolol for severe hemangiomas of infancy. N. Engl. J. Med. 358, 2649–2651. Lawley, L.P., Siegfried, E., Todd, J.L., 2009. Propranolol treatment for hemangioma of infancy: risks and recommendations. Pediatr. Dermatol. 26, 610–614. Lai-Cheong, J.E., McGrath, J.A., 2009. Structure and function of skin, hair and nails. Medicine 37, 223–226 (Baltimore). Matuszczak, E., Oksiuta, M., De˛bek, W., Dzienis-Koronkiewicz, E., Hermanowicz, A., 2013. Topical timolol gel for the treatment of residual facial hemangioma previously treated with propranolol. Pediatr. Pol. 88, 116–119.
Y. Guan et al. / International Journal of Pharmaceutics 487 (2015) 135–141 Patel, V.B., Misra, A., Marfatia, Y.S., 2000. Topical liposomal gel of tretinoin for the treatment of acne: research and clinical implications. Pharm. Dev. Technol. 5, 455–464. Sinico, C., Fadda, A.M., 2009. Liposomes and niosomes as carriers for dermal drug delivery. Expert Opin. Drug Deliv. 6, 813–825. Sánchez-Carpintero, I., Ruiz-Rodriguez, R., López-Gutiérrez, J., 2011. Propranolol in the treatment of infantile hemangioma: clinical effectiveness, risks, and recommendations. Actas Dermosifiliogr. 102, 766–779. Verma, D.D., Verma, S., Blume, G., Fahr, A., 2003. Liposomes increase skin penetration of entrapped and non-entrapped hydrophilic substances into
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human skin: a skin penetration and confocal laser scanning microscopy study. Eur. J. Pharm. Biopharm. 55, 271–277. Vyas, L.K., Tapar, K.K., Nema, R.K., Parashar, A.K., 2013. Development and characterization of topical liposomal gel formulation for anti-cellulite activity. Int. J. Pharm. Pharm. Sci. 5, 512–516. Yokomizo, Y., Sagitani, H., 1996. The effects of phospholipids on the percutaneous penetration of indomethacin through the dorsal skin of guinea pig in vitro. 2. The effects of the hydrophobic group in phospholipids and a comparison with general enhancers. J. Control. Release 42, 37–46.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Propranolol hydrochloride-loaded liposomal gel for transdermal delivery: Characterization and in vivo evaluation Yuanyuan Guan, Tiantian Zuo, Minglu Chang, Fang Zhang, Ting Wei, Wei Shao, Guimei Lin * School of Pharmaceutical Science, Shandong University, Jinan 250012, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 January 2015 Accepted 10 April 2015 Available online 13 April 2015
This study was to develop propranolol hydrochloride (PRO)-loaded liposomal gel as a topical drug delivery system. Scanning electron microscope (SEM) revealed that the liposomes were spherical and scattered in the surface of the gel. Pseudoplastic flows of PRO liposomal gel were showed after stored at three different temperatures. Besides, PRO liposomal gel showed non-irritating to the skin of rabbit. Skin deposition studies in vivo demonstrated that PRO liposomal gel can apparently increase drug content in skin compared with PRO gel. Histopathology examination showed that PRO liposomal gel could obviously weaken the barrier function of stratum corneum (SC) by comparison with PRO gel. What is more, the plasma pharmacokinetic showed the maximum concentration in plasma was 0.41 mg/mL and 1.17 mg/mL after topical and oral administration respectively. However, tissue distribution study showed PRO liposomal gel obviously changed drug distribution in tissues, significantly increased drug concentration in skin with about 74 folds compared with PRO gel. In conclusion, liposomes-based gel could be a promising vehicle as a transdermal delivery system of PRO. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Propranolol hydrochloride Liposomal gel Transdermal delivery Pharmacokinetic Tissue distribution
1. Introduction Propranolol hydrochloride (PRO) is one of b-blockers which can antagonize the b-adrenergic pathway, blocking receptors such as in heart, pancreas, liver, and in peripheral blood vessels and bronchi (Sánchez-Carpintero et al., 2011). Since Léauté-Labrèze et al. (2008) first described its occasional antiproliferative effect on severe infantile hemangioma (IH), PRO has gradually become the first-line therapy for IH. IH is one of the most common tumors in infants, which can lead to deformities when they are located in the facial areas of the lip, nasal tip or the ear (Matuszczak et al., 2013). Therefore, it is necessary to treat IH timely. However, PRO given orally shows significant first pass metabolism, some side effects on heart and poor patient compliance (Lawley et al., 2009). As a result, we need to seek for another administration route to solve these problems. Transdermal drug delivery offers potential to overcome disadvantages caused by oral administration. Unfortunately, the stratum corneum (SC) which is recognized as the primary barrier
* Corresponding author at: School of Pharmaceutical Science, Shandong University, 44 Wenhuaxi Rd. Jinan, Shandong 250012, China. Tel.: +86 53188382007; fax: +86 53188382548. E-mail address: [email protected] (G. Lin). http://dx.doi.org/10.1016/j.ijpharm.2015.04.023 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
for transdermal drug delivery makes it difficult for most drugs to enter into skin by this route. In recent years liposomes have been intensively studied as drug carrier systems for topical delivery. Several in vivo and in vitro transport studies reported that liposomes can improve skin permeability (Verma et al., 2003; Dragicevic-Curic et al., 2008; Vyas et al., 2013) and enhanced the skin deposition with reduction in systemic absorption (Patel et al., 2000), which suggested that liposomes were useful for topical drug delivery. As liposomes and biomembranes of skin have lipid bilayer membrane structure in common, liposomes have a good biocompatibility with skin. The major disadvantage of using liposomes topically lies in the liquid nature of the preparation. To achieve the viscosity desirable for topical application, liposomes should be incorporated into a suitable vehicle. It has been well established that liposomes are fairly compatible with carbopol (Elnaggar et al., 2014), which has a good bioadhesive properties and the ability to prolong the retention of the formulation on the mucosal surface. The purpose of this study was to develop a topical delivery carrier for the use of PRO. As a result, scanning electron microscope (SEM), rheological study, skin irritation test, in vivo skin deposition studies, histopathology examination, plasma pharmacokinetic properties and biodistribution characteristics were carried out to evaluated PRO liposomal gel.
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Fig. 1. The SEM photomicrographs of liposomal gel. A (55); B (20,000).
2. Materials and methods 2.1. Materials PRO was purchased from Wuhan Ding Lixin Chemical Co. Ltd., China (99.90%). Phosphatidyl ethanolamine (PE) was obtained from Jianglaibio, Shanghai, China. Cholesterol (Chol) was supplied by Aobox Biotechnology, Beijing, China. Carbopol 934 was obtained from Jingxifang Technology Co. Ltd., Beijing, China. Methanol used was of chromatographic grade and others were of analytical grade.
2.2.3. Rheological measurements Rheological tests of liposomal gel were performed by a rotational viscosimeter (RheoStress 6000, Thermo Scientific, Germany). The flow properties of the developed gel were conducted at 25 1 C (room temperature) by using a cone/ plate-measuring device (cone with D = 35 mm, 1 ). The measurements were carried out by increasing the shear rate from 0.1 s 1 to 100 s 1. In order to explore the effect of storage conditions on the gel properties, rheological measurements were performed after production and 1 week of storage at three different temperatures (4 C, 25 C and 40 C).
2.2. Methods 2.2.1. Preparation of PRO liposomes and liposomal gel PRO liposomes and PRO liposomal gel were prepared as detailed in our previous work (Guan et al., 2014). 2.2.2. SEM of PRO liposomal gel PRO liposomal gel was prefreezed in the ultra-low-temperatue refrigerator of 80 C (DW-86L, Haier, China) for 24 h before they were freeze-dried by the use of freeze drying equipment for 24 h. The final products were fixed on a platform and sprayed with gold and observed by the JSM-6700F scanning electron microscope.
2.2.4. Primary skin irritation test To evaluate and compare skin irritation of PRO liposomal gel and blank liposomal gel, the study was carried out on rabbits. New Zealand rabbits weighing 2.5–3.0 kg were fed with food and water for 1 week to adapt to the environment before the study. The hairs of the dorsal portion were removed with the help of shaver at 24 h prior to application of the formulations. The rabbits were divided into three groups of 3 rabbits each as follows: Group 1: no application (control). Group 2: blank liposomal gel. Group 3: PRO liposomal gel. PRO liposomal gel (1.0 g) and blank liposomal gel (1.0 g) were applied on the hairless skin of the rabbits by uniform spreading within the area of 2 cm 2 cm, respectively. The preparations were removed after 24 h and each of the area was observed for any changes such as erythema or edema. To study the cumulative effect of repeated applications, the respective preparations were applied once daily for next 7 days on the same area of hairless skin. Draize scale was applied to evaluate the skin irritation (Draize et al., 1944). The irritation scores between 0 and 4 were used to grade stimulus intensity which range from no response to a severe response.
Table 1 The irritation scores of gels to the rabbit skin after administration of 7 days. Formulation
Fig. 2. Shear stress–shear rate curves of PRO liposomal gel after production and 1 week of storage at three different temperatures.
Control (Group 1) Blank liposomal gel (Group 2) PRO liposomal gel (Group 3) Stimulus intensity
Irritation index 24 h
Day 7
0 0 0 Nonirritant
0 0 0 Nonirritant
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Fig. 3. The irritation photos after administration of preparations to skin. A – control (24 h); B – control (Day 7). C – PRO liposomal gel (24 h); D – PRO liposomal gel (Day 7). E – blank liposomal gel (24 h); F – blank liposomal gel (Day 7).
2.2.5. In vivo skin deposition studies 2.2.5.1. Animals. Studies were performed on Kunming strain mice (25 2) g, which were provided by the Experimental Animal Center of Shandong University (Jinan, China). All animals were acclimatized for 1 week before use. Prior to formal experiments, mice were kept under food fasting but free access to water overnight. All studies were performed in accordance with the ethics and regulations of animal experiments of pharmaceutical sciences, Shandong University, China. 2.2.5.2. Skin sample processing. The Kunming strain mice were randomly divided into two treatment groups of 4 mice each. PRO liposomal gel and PRO gel were administered transdermally at a dose of 30 mg/kg, respectively. Then animals were sacrificed by
cervical dislocation at the established post-administration time point (0.5, 1, 2, 4, 6, 12, 24 h). Skin samples were collected from the back of the mice and then washed in normal saline and dried using filter paper. All the samples were stored at 20 C until further analysis.
2.2.6. Histopathology examination by light microscopy PRO liposomal gel was given to Kunming strain mice (25 2) g for 24 h. Afterwards, the skins were immediately collected and fixed in 10% formaldehyde solution for 24 h. Then samples were dehydrated using ethanol, embedded in paraffin wax and followed by stained with hematoxylin and eosin (H&E). At last, skins were cut by a cryostat into 3–4 mm and evaluated under light microscopy.
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Fig. 4. Mean concentration–time curves of PRO after topical administration of PRO liposomal gel and PRO gel to mice at 30.0 mg/kg (mean SD; n = 4).
2.2.7. Pharmacokinetic and tissue distribution study The principle of treating with Kunming strain mice was the same as described above except that PRO liposomal gel and PRO suspension were administered transdermally and orally at a dose of 75 mg/kg, respectively. Besides, blood samples were collected from the ocular vein into heparin-containing eppendorf tubes at 0.5, 1, 2, 4, 6, 8 h and then immediately centrifuged at 4000 rpm for 20 min to obtain supernate. The dorsal skins beneath the drug application site and the organs (heart, liver, spleen, lung and kidney) were separated, washed in normal saline and dried with filter paper. Plasma and tissue samples were stored at 20 C until further analysis. The concentrations of PRO at different time points were expressed as mean values and standard deviations, and the mean concentration–time curves were plotted. Pharmacokinetic parameters were calculated using the Drug and Statistics software (DAS, version 2.0). 2.2.8. High performance liquid chromatography (HPLC) analysis The quantification of PRO was performed on an Agilent HPLC system (Agilent, USA). The system was equipped with a variable wavelength UV detector with being set at 289 nm in this study and a reversed-phase C18 column (5 mm, 4.6 mm 250 mm, Welch Materials, Inc.). The mobile phase was consisted of methanol and 0.01 mol/L dipotassium phosphate solution (50:50, v/v) and was used at a flow rate of 1.0 mL/min for chromatographic separation and analysis. The column temperature was set at 25 C and samples were injected into the column at a constant volume of 20 mL. 3. Results and discussion 3.1. SEM of liposomal gel SEM was used to observed liposomes surface morphology. Fig. 1A showed that liposomes were scattered in the surface of the gel. It can clearly show that liposomes were spherical and about 260 nm in size when observed under a greater magnification (Fig. 1B). In other words, liposomes had no obvious change in morphology and sizes after they were incorporated into the gel matrix compared with our previous date monitored by transmission electron microscope (TEM, Hitachi, Japan) and DelsaTM Nano C Particle Analyzer (Beckman Counter Ltd., USA) respectively.
Fig. 5. Photomicrographs of the skin structure of using different preparations (400). A – skin of no preparations; B – skin treated with PRO liposomal gel; C – skin treated with PRO gel.
3.2. Rheological measurements The rheological properties play an important role for topical gel formulations in delivering the molecules onto or across the skin because they can greatly affect spreadibility, adhesiveness, drug release from semisolid formulations, and subsequent penetration
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Table 3 The targeting disposition of PRO after topical administration of PRO liposomal gel and oral administration of PRO suspension (n = 4). AUC (mg h/g)
Tissue
PRO liposomal gel Heart Liver Spleen Lung Kidney r = (AUC
51.31 20.84 87.38 141.80 52.11
0.26 1.35 7.01 6.15 6.37
r PRO suspension 68.99 78.06 92.19 242.08 92.29
7.98 9.82 8.73 22.52 18.86
0.74 0.27 0.95 0.59 0.56
topica/AUCoral).
3.3. Skin irritation test One of the major disadvantages associated with transdermal drug delivery system is skin irritation of preparations. To study security of products, rabbits are often used for the prediction of skin irritation effects in humans because of their high sensitivity to irritations (Ishii et al., 2013). The results of the skin irritation studies were listed in Table 1. It indicated that PRO liposomal gel and blank liposomal gel resulted in no skin irritation compared with control group after 24 h of application and 7 days of repeated applications. Besides, the irritation photos of rabbits (Fig. 3) clearly
Fig. 6. Mean plasma (A) and skin (B) concentration–time curves of PRO after topical and oral administration to mice at 75.0 mg/kg (mean SD; n = 4).
into the skin (Elnaggar et al., 2014). Fig. 2 clearly showed that the slope of rheogram (namely the viscosity of the gel) was decreased with the increase of shear rate, which indicated that the developed gel showed pseudoplastic flow. In fact, with the shear rate increasing the structure of the gel began to disrupt and the particles started to align until it started flowing. On the other hand, the viscosity of the developed gel was high (255.2 Pas at shear rate of 0.1 s 1, date was not shown) to adhere to the skin, which was suitable for the transdermal administration. Besides, no obvious change of the viscosity was observed post 7 days of storage at three different temperatures compared with that of gel on the 0 day seen from Fig. 2. All this indicated that preparations were suitable for the transdermal administration and stable when stored at 40 C.
Table 2 The pharmacokinetic parameters of PRO in mice after topical and oral application. Cmax mg/mL(or mg/g) Tmax (h) AUC0–t mg h/mL Plasma Skin
Topical Oral Topical Oral
0.41 0.04 1.17 0.13 265.10 23.34 13.37 0.93
6.5 1.25 4.0 0.5
1.72 0.17 2.67 0.23 1618.50 109.07 21.84 1.31
Fig. 7. The concentration level of PRO in tissues of mice over 0.5–8 h after topical (A) and oral (B) administration PRO.
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showed no oedema and erythema after administration of 24 h and7 days. Thus, PRO liposomal gel demonstrated evident advantage of non-irritating to skin, which indicated their potential in improving patient acceptance and topical delivery. 3.4. In vivo skin deposition studies The cumulative PRO concentration in skin was showed in Fig. 4. PRO liposomal gel increased rapidly from 0 to 4 h compared with PRO gel group. The max drug concentration was 123.34 mg/g for PRO liposomal gel, obviously remarkable than that of PRO gel (75.65 mg/g). As Fig. 4 showed that PRO liposomal gel was more able to increase drug content in skin, as a result it can provide effective drug concentration for a long time in comparison with PRO gel. The differences can be attributed that the presence of the liposomes. Yokomizo and Sagitani (1996) found that phospholipids had an effect on the permeability barrier of SC. In fact when liposomes act on the skin they can successively fuse with the SC by mixing with the intercellular polar lipids and loosening the bilayered structure of the intercellular mortar of the SC (Sinico and Fadda, 2009). 3.5. Histopathology examination by light microscopy Skin is the outermost and largest organ in the body, which from outside to inside is consisted of epidermis, dermis and subcutis (Lai-Cheong and McGrath, 2009). As the outmost layer of epidermis to protect skin from invasion, SC is composed of about 10 layers of flattened corneocytes. They have a thick insoluble cell envelope, which is consisted of loricrin and involucrin as well as intercellular lipids (Alexander et al., 2012). As a result, SC becomes the major challenge for the penetration of drugs across the skin. As was shown in Fig. 5A, the skin of control group displayed a clear delineation among epidermis, dermis and subcutis. The SC cells were arranged neatly with no inflammation being observed. After the administration of PRO liposomal gel, the intercellular gaps of SC were increased and the SC structure was loose (Fig. 5B), while PRO gel slightly changed the tight junction of the SC (Fig. 5C). Besides, there being no obvious change in dermis and subcutis of all samples demonstrated that the preparation did not cause irritation and was safe to patients. In a conclusion, PRO liposomal gel can significantly weaken the barrier function of SC and promote drug permeation, which explain the phenomenon that PRO liposomal gel had a higher drug deposition in skin than PRO gel. The change was attributed to that liposomes produced an enhancing effect because their lipid components may penetrate deep into the SC or militate with skin lipids to loosen their structure (El Maghraby et al., 2008). 3.6. Plasma and tissue concentration–time curves As is shown in Fig. 6A, plasma concentration–time curves of PRO liposomal gel and PRO suspension were significantly different. The maximum concentration in plasma appeared at a 6.5 h (Cmax = 0.41 mg/mL) and 1.25 h (Cmax = 1.17 mg/mL) after topical and oral administration respectively (Table 2), which indicated that the extension of time for PRO absorbed in the systemic circulation. The maximum concentration in plasma post oral administration was 2.85 folds of that topical administration, while AUC0–8h of PRO after oral administration was 1.55 folds compared with topical administration, both of which indicated that lower lever of PRO was absorbed in the systemic circulation after topical administration compared with oral treatment. Besides, after 4 h drug concentration post topical administration was higher than that of oral administration. The reason can be attributed to the drug repository effect of skin.
As was shown that maximum drug concentration in skin (265.00 mg/g) after local application was significantly higher than oral application (13.37 mg/g, Table 2). The AUC0–8h value post local application was 74-fold higher than oral application, which demonstrated that the topical application was more effective than oral application at the aspect of increasing drug concentration in skin. Besides, PRO can maintain an effective drug concentration for a long time after topical application (Fig. 6B). As a result PRO liposomal gel can prolong time of acting on body and improve patient compliance. The targeting disposition of PRO after topical administration of PRO liposomal gel and oral administration of PRO suspension were shown in Table 3. The re value of heart, liver, spleen, lung, kidney all less than 1, which indicated a lower bioavailability after topical administration compared with oral treatment. What's more, lower drug disposition in skin would reduce the side effects on the organs especially heart mainly reflecting in such as bradycardia, hypotension, and hypoglycemia. In order to more visually show the biodistribution characteristics of PRO after administration, concentration distribution histograms over the time course of 0.5–8 h were plotted. Fig. 7 showed that PRO liposomal gel obviously changed drug distribution in tissues, relatively increased drug concentration in skin, and decreased drug concentration in other tissues. 4. Conclusion The present work demonstrated that PRO liposomal gel as a transdermal preparation was prepared successfully. It could obviously increase drug content in skin to reduce dosing frequency compared with PRO suspension. Besides, it decreased drug content in the systemic circulation. As a result, side effects of PRO on organs especially the heart can be reduced. Thus, the liposomal gel could be a promising vehicle for topical delivery of PRO. Acknowledgements This work was supported by the National Natural Science Foundation of China(Grant No.: 21203112) and the Natural Science Foundation of Shandong Province (Grant No.: ZR2012BQ002).
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