Noninvasive and persistent transfollicular drug delivery system using a combination of liposomes and iontophoresis

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Abstract

Iontophoresis is a promising technique for enhancing transdermal administration of charged drugs. However, conventional iontophoresis is not sufficient for effective delivery of large, hydrophilic, or electrically neutral molecules. In this study, we utilized charged liposomes as carriers, focused on a transfollicular route for delivery of the liposomes, and optimized iontophoretic conditions and lipid composition for this method in both in vitro and in vivo conditions. As a result, we identified the optimum condition (lipid composition: DOTAP/EPC/Chol = 2:2:1, current supply: 0.45 mA/cm2, duration: 1 h) for effective iontophoretic delivery of aqueous solution, which cannot be transferred into the skin without charged liposomes. We also examined the pharmacological effects of iontophoresis of liposomes encapsulating insulin (INS-lipo) using a rat model of type I diabetes. Interestingly, iontophoresis of INS-lipo onto a diabetes rat skin resulted in a gradual decrease in blood glucose levels, with levels reaching 20% of initial values at 18 h after administration. These lower blood glucose levels were maintained for up to 24 h. Significant amount of insulin were also detected in plasma 18 h after iontophoresis of INS-lipo. We succeeded in developing a non-invasive and persistent transfollicular drug delivery system that used a combination of liposomes and iontophoresis.

Introduction

Transdermal administration of drug molecules is considered to have numerous clinical benefits, such as avoiding the liver first-pass effect, improvement in patient compliance, and a reduction in adverse effects (Prausnitz and Langer, 2008). However, as the skin functions as a barrier in many cases, an innovative method for enhancing absorption of drug molecules across this barrier is required.

Iontophoresis utilizes an electric field to enhance the delivery of charged compounds across the skin and is recognized as a promising method for transdermal administration of drugs (Tyle, 1986, Varghese and Khar, 1996). Iontophoresis primarily provides an electrical driving force for transport of compounds across the stratum corneum, in addition to increasing skin permeability to drug molecules. As iontophoresis does not change the skin barrier directly, it is particularly applicable for transdermal administration of small, hydrophobic and charged molecules (Prausnitz and Langer, 2008).

In order to deliver drug molecules that are poorly delivered by iontophoresis, such as large, hydrophilic, or electrically neutral molecules, we investigated using charged liposomes as carriers for iontophoretic transdermal delivery. However, it was readily apparent these liposomes, generally having a diameter >100 nm, would not be able to penetrate the horny cell layer of the skin as they were larger than the intercellular spaces (Lasch et al., 1992). To overcome this problem, we considered the hair follicle may provide a suitable pathway for transdermal delivery of charged liposomes using iontophoresis.

There are some reports for follicular drug delivery using the nano- or microparticulate systems (several hundreds nm–several μm) (Alvarez-Roman et al., 2004, Mordon et al., 2003, Rolland et al., 1993, Toll et al., 2004). While the follicular opening constitute only about 0.1% of the total skin area (Meidan et al., 2005), the hair follicle represents an invagination of the epidermis extending deep into the dermis and thus provides a greater actual area for potential absorption (Agarwal et al., 2000, Singh et al., 2000). Furthermore, there is no mature stratum corneum below the ostia of the sebaceuous glands, although the surfaces of the follicular openings are initially keratinised. In addition, there is an extensive capillary network associated with the upper dermal vasculature (Meidan et al., 2005). According to these features, the hair follicle might have great potential for drug delivery into the viable skin layers or the systemic circulation.

In this study, we combined liposome and iontophoresis technology to develop in vitro and in vivo non-invasive transdermal delivery systems. We also examined the in vivo effects of transfollicular administration of insulin encapsulated in liposomes in streptozotocin-induced diabetic model rats, by examining the prolonged action of insulin on blood glucose levels in these animals.

Section snippets

Materials

Sulforhodamine B was purchased from Molecular Probes (Carlsbad, CA, USA) and 4-nitrobenzo-2-oxa-1,3-diazolyl-(NBD-), rhodamine- or non-labeled 1,2-dioleoylphosphatidylethanolamine (DOPE), 1,2-dioleoyl-3(trimethylammonium)propane (DOTAP) and cholesterol (Chol) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Egg phosphatidylcholine (EPC) was purchased from NOF Corp. (Tokyo, Japan), and cholesteryl hemisuccinate (CHEMS) and insulin (porcine) from Sigma (St. Louis, MO, USA). The

Optimization of conditions for in vitro transfollicular delivery of liposomes via iontophoresis

At first, we determined whether it was possible to deliver liposomes into hair follicles using iontophoresis. Confocal laser scanning microscopy was used to observe cross-sections of the skin after in vitro iontophoresis of liposomes in which the lipid membrane was labeled with rhodamine. Cationic liposomes, composed of DOTAP/Chol (7:3), and several iontophoretic conditions of current density and duration were attempted to use for delivery of the liposomes into the hair follicles. As shown in

Discussion

In vitro anodal iontophoresis of cationic liposomes (DOTAP/Chol = 7:3) achieved maximum delivery distance into hair follicles at conditions of 0.45 mA/cm2 for 1 h (Fig. 2). In the condition of 0.45 mA/cm2 for 2 h, delivery distance into hair follicles of liposomes was shorter than that of 0.45 mA/cm2 for 1 h (Fig. 2). Probably, some parts of the liposomes delivered into hair follicles during first 1 h treatment were excluded from the hair follicles by impairments, such as inflammation, induced by longer

Acknowledgment

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

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