Lipid-mediated gene delivery to the skin

European Journal of Pharmaceutical Sciences 43 (2011) 199–211

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Review

Lipid-mediated gene delivery to the skin Barbara Geusens a,⇑,1, Tine Strobbe a,1, Stefanie Bracke a, Peter Dynoodt a, Niek Sanders b, Mireille Van Gele a, Jo Lambert a a b

Department of Dermatology, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium

a r t i c l e

i n f o

Article history: Received 25 April 2010 Received in revised form 16 November 2010 Accepted 9 April 2011 Available online 16 April 2011 Keywords: Lipid-based vesicles Gene delivery Skin Topical immunization

a b s t r a c t Cutaneous gene delivery methods have been developed over the past decades as therapeutic strategies for the treatment of a variety of skin disorders. Both viral and non-viral techniques have been frequently described. Mainly due to safety concerns, the application of viral methods is being questioned and nonviral alternatives are gaining major interest. Lipid-based vesicles for the delivery of plasmid DNA by topical application onto the skin hold great potential and have been investigated thoroughly. Here, we give an overview of the different lipid vesicles that have been described in literature. Next to the conventional phospholipid liposomes, new generation liposomes like niosomes and TransfersomesÒ have been developed for enhanced (trans)dermal delivery. In addition, we draw attention to other lipid-based delivery systems, that could not be classified into one of these categories. Clearly, lipid-based delivery vehicles demonstrate very promising results for DNA delivery into and through the skin, especially for cutaneous vaccination purposes. Apart from simple topical application onto the skin, liposomes have also been described in combination with delivery enhancing techniques. Here we describe this combined approach for some specific skin disorders. Ó 2011 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4.

5. 6.

7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The skin as a target organ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Barriers for topical gene application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liposomes for topically applied cutaneous gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Classical liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Niosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. TransfersomesÒ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Other lipid-containing formulations for topical DNA delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disadvantages and limitations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Combination with physical enhancing techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Immunization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Wound healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Vitiligo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Melanoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Atopic dermatitis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Psoriasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⇑ Corresponding author. Tel.: +32 9332 65 42. 1

E-mail address: [email protected] (B. Geusens). These authors contributed equally to this manuscript.

0928-0987/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2011.04.003

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1. Introduction In general, gene therapy is defined as the introduction of a therapeutic gene into a cell, whose expression can lead to a cure of a disease or offer a transient advantage for e.g. tissue growth, vaccination or anticancer treatment (Morgan and Anderson, 1993). To achieve this goal, a therapeutic gene must first be efficiently delivered to the specific target cell. Secondly, its expression must reach and sustain a certain level to achieve its therapeutic purpose. Thirdly, to optimize the therapy, a pharmacological agent should reversibly control the level and timing of the gene’s expression within the target cells. In this manner, the therapeutic dose can be fine-tuned to the requirements of the treatment, and stopped when it is no longer needed. Two basic approaches towards the therapeutic gene delivery into the skin involve ex vivo and in vivo gene delivery. Ex vivo delivery involves a skin biopsy to harvest cells for growth and gene insertion in culture followed by re-grafting to the patient. In contrast to the ex vivo approach, in vivo gene transfer delivers genetic material directly to the patient’s intact skin tissue and is therefore generally more simple and direct. Many in vivo gene transfer systems have been developed for cutaneous delivery, using both viral and non-viral vectors. These systems include topical gene application, direct intradermal injection, electroporation, iontophoresis, sonophoresis and gene guns (Carretero et al., 2006; Khavari et al., 2002; Kikuchi et al., 2008). Some viral vectors (lentiviral, retroviral) allow prolonged gene expression in vivo because they are capable of integrating into the genome of various skin cells and even epidermal stem cells (Baek et al., 2001; Ghazizadeh et al., 1999). Intradermal or subcutaneous injection is the preferred method of choice for their introduction. For diseases involving recessive loss-offunction mutations, such as those involved in many genodermatoses, simple re-introduction of the wild-type gene via genomic insertion may be sufficient for prolonged correction of the phenotype (Kikuchi et al., 2008). Although recombinant viruses are the most effective vehicles to deliver therapeutic nucleic acids into the cells,

the genetic material that they can carry is limited and they can potentially induce insertional oncogenesis and severe immunogenic responses. As a consequence, several non-viral gene delivery systems have been developed for safe delivery of therapeutic nucleic acids into the skin. Topical application of plasmid DNA is an attractive alternative approach for gene delivery. Although only transient expression can be obtained, it can be useful for various cutaneous gene therapy applications, if successfully developed. Lipid-based delivery vehicles for topical DNA application onto the skin offer advantages over other transfer systems. It is a painless, cheap and easily applicable method for large surface areas on the body. In addition, it is patient compliant, safe and can be used for home-based settings. However, they are considered to be inefficient as they tend to accumulate on the skin surface. Efficient gene therapy depends in the first place on efficient delivery into the target cells. The skin encompasses several extracellular and intracellular barriers that need to be overcome by specialized delivery techniques. The present review summarizes the different vesicular lipid-based systems for topical delivery of DNA to and through the skin, mentioning their limitations and particular applications. 2. The skin as a target organ Human skin is the largest organ of the human body and is an interesting candidate for gene therapy because of its easy accessibility. The skin structure and cell types are well understood, which makes the skin relatively easy to manipulate. Additional advantages include the ability to visually monitor the genetically modified region and the possibility of surgical removal of aberrant tissue if unwanted side-effects occur (Greenhalgh et al., 1994). Human skin consists of three layers, beginning from the surface; the epidermis, the dermis and subcutaneous tissue or subcutis, each containing their own specific cells and respective functions (Kanitakis, 2002) (Fig. 1). The majority of the epidermis is comprised of keratinocytes. The high turnover and self-renewing capacity of the epidermis

Fig. 1. Schematic cross-sectional representation of the skin. Arrows indicate the possible routes of particle penetration through skin; transappendageal, transfollicular, transcellular and intercellular pathways. Several factors determine particle penetration. Depending on the size of the particle, different penetration routes can be followed. The size of the sweat pores and the follicular openings of the skin range between 10 and 210 lm. The lipids of the intercellular route are assembled in parallel head–head tail– tail repeating bilayers, with the tail–tail region known as the intercellular lipidic route of skin absorption and the head–head regions constituting the hydrophilic SC aqueous pores. Particle dimensions below 6–7 or 36 nm might be able to be concurrently and respectively absorbed through these pathways. It is questionable whether the transcellular pathway forms an important route for cutaneous penetration.

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B. Geusens et al. / European Journal of Pharmaceutical Sciences 43 (2011) 199–211 Table 1 Clinical trials for cutaneous gene therapy. Indication

*

Phase

Start

Gene

Vector

In/ex vivo

Application

Author

Ref.

Intra-ulcer Intra-ulcer Intra-ulcer Skin patch administration Intramuscular

D.J. Margolis D.J. Margolis D. Steed A. Kimball

*

Mavilio et al. (2006)

R.J. Powell

Powell et al. (2008)

Skin patch administration Intramuscular

A.T. Lane

*

I. Baumgartner

Baumgartner et al. (2009)

R.P. Hickerson S.A. Leachman S. Nikol

Hickerson et al. (2008) Leachman et al. (2008) Nikol et al. (2008)

M.J. Schurr

*

Foot ulcer Venous leg ulcer Diabetic leg ulcer JEB

I I I I/II

1999 1999 2000 2000

PDGF PDGF PDGF Laminin 5-Beta3

Adenovirus Adenovirus Adenovirus Retrovirus

In vivo In vivo In vivo Ex vivo

Periferic ischemic ulcers RDEB

II

2005–6

HGF

Naked plasmid DNA

In vivo

I/II

2007

III

2007

Retrovirus (keratinocytes) Naked plasmid DNA

In vitro

Critical ischemia with skin lesions Pachyonychia congenita Critical limb ischemia with skin lesions Diaberic ulcers

human collagen type 7 A1 FGF

I

2008

siRNA

siRNA

In vivo

III

2008

FGF

Naked plasmid DNA

In vivo

Intralesional Intradermal Intramuscular

I

2009

Cathelicidin (hCAP18/LL-37)

Naked plasmid DNA

In vitro

Skin graft tissue

In vivo

Margolis et al. (2004) *

The Journal of Gene Medicine Website: www.wiley.co.uk/genmed/clinical.

makes long-term gene expression in keratinocytes very difficult. The genetic material is lost during cell division (‘dilution’) unless stably inserted into the genome. On the contrary, longer expression can be obtained for transiently inserted genes in cells with lower turnover, such as epidermal melanocytes or dermal fibroblasts. Of particular interest would be targeting of the epidermal stem cells that are located in the basal layer of the epidermis and at the base of hair follicles. If the stem cells can be transduced with the gene of interest then expression of that gene should continue throughout adult life. The epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin and form a protective layer. The follicular stem cells are able to give rise to both the hair follicle and to the epidermis. Due to their quiescent and slow-dividing nature under steady-state conditions, so far only viral vectors (retroand lentiviral) have been found to efficiently integrate into cultured stem cells and result in long-term gene expression in vivo after injection (Baek et al., 2001; Ghazizadeh et al., 1999). From a genetic viewpoint, there is an ever-expanding list of skin-related mutations that could potentially be corrected by addition of a normal gene copy (recessive monogenic disorders) (Carretero et al., 2006) or by arresting the expression of a mutant gene (dominant disorders) (Geusens et al., 2009; Wraight and White, 2001). With the increasing knowledge on skin pathobiology to and the genetic basis of many inheritable skin diseases, localized gene therapy could offer an effective treatment. Delivery of therapeutic genes to viable epidermal tissue provides potential for local immune-modulatory DNA-based cure of dermatological pathologies. Further more, given the antigen presenting characteristics of skin tissue, the epidermis could serve as an antigen bioreactor for polynucleotide vaccine delivery. The elucidation of the molecular events of alopecia (Ahmed et al., 2010) also highlights the potential cosmetic interventions for gene therapy targeted to the hair follicle. In some cases, skin disorders do not require the permanent or continuous activity of an introduced gene. Transient expression may often be sufficient or desirable to correct acute cutaneous conditions such as wound repair, infection or other inflammatory processes. Despite the unbalanced ratio of expectation and delivered achievements of gene therapy during the past 20 years, a number of gene therapy studies have led to clinical trials for treatment of acquired and inherited skin disorders (Table 1). 3. Barriers for topical gene application The outermost layer of the epidermis, the stratum corneum (SC), acts as the major skin barrier and limits ingress of foreign

agents into the skin. This statement is however an oversimplification. Actually, barrier action is the resultant of positive cooperation and interactions between SC macro- and microstructure, bi- and 3D supramolecular organization of SC lipidic matrix, and SC whole composition (Baroli, 2010). SC macrostructure generally refers to corneocyte cross-sectional organization that is conventionally described as the ‘‘brick and mortar’’ model, where ‘‘bricks’’ are alternate staggered corneocytes, and ‘‘mortar’’ a lipidic matrix surrounding them (Nemes and Steinert, 1999). The microstructure of the latter refers to supramolecular organization of intercorneocyte lipids that are assembled in parallel head–head tail–tail repeating bilayers, with the tail–tail region known as the intercellular lipidic route of skin absorption (Fig. 1) and the head–head regions constituting the hydrophilic SC aqueous pores (Bouwstra and Ponec, 2006; Plasencia et al., 2007). The chemical composition of the SC determines a nonhomogeneously distributed change in hydrophobicity progressing from the SC to the stratum granulosum and is responsible for the nonlinear pH gradient existing between upper and lower SC. The low pH (acid mantle), the presence of enzymes on the skin and the transcutaneous gradient form additional defensive features of the SC, negatively influencing cutaneous penetration. Briefly, it can be concluded that the ingress of foreign agents into the SC, as well as their further progression towards the viable epidermis, is limited by SC nanoporosity and gradients. Composition and physicochemical properties (e.g. dimensions, o/w partitioning coefficient, solubility, pKa, diffusivity coefficient) of nanometric agents may improve or limit the ingress and diffusion into or through the skin (Baroli, 2010). When penetrating agents are molecules, it is generally believed that small (<500 Da) lipophilic and uncharged compounds are the best candidates for successful percutaneous penetration (Barry, 2001; Brown et al., 2006). As a consequence, it is expected that high molecular weight molecules that are hydrophilic – such as plasmid DNA – will not penetrate spontaneously into the skin through the transepidermal pathway. Despite the compact defensive structure of the SC, both sweat glands and pilosebaceous units open on the skin surface (Fig. 1). It is believed that these appendages in the skin are the main migration pathways through which molecules will diffuse (El Maghraby and Williams, 2009). The size of the sweat pores and the follicular openings of the skin range between 10 and 210 lm (Otberg et al., 2004), thus it is reasonable to expect penetration through skin of DNA or small oligonucleotides such as siRNAs. It should be noted however that the significance of the transappendageal route has been approached with some ambiguity for a long time because

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these skin openings have a low density [sweat glands: 0.01% (Scheuplein, 1967); hair follicles: 0.1% (Szabo, 1962) on average]. In addition, it was commonly argued that sweat and sebum outward excretion could hinder the inward movement of a penetrating agent. Subsequent to overcoming the stratum corneum, migration and transport of nucleic acids through the extracellular matrix of the deeper skin layers encounter resistance due to the dense network of intercellular lipids, polysaccharides and fibrous proteins. Its high molecular weight and negative charge hamper the molecule to diffuse freely into the different skin layers. Once the target cell membrane is reached, cellular uptake might be prevented by electrostatic repulsion considering the cell surface as well as the nucleic acids are both negatively charged. Intracellular uptake generally proceeds via endocytosis. Therefore, the plasmid needs to escape from the endosomes in order to reach the cytoplasm. If DNA is unable to exit the endosome, the decreasing pH and the activation of the acidic nucleases will have disastrous consequences for the stability and integrity of the DNA. Lastly, the plasmid is challenged by cytosolic migration before reaching the nucleus as final destination. Overcoming the nuclear envelope is the final step in the cascade of hurdles that greatly influences the transfection efficiency of nucleic acids in vivo. Keeping this cascade of challenging events in mind, it is very surprising that studies have been describing the successful application of naked plasmids into or onto the skin. In 1995, Hengge et al. first reported the direct injection of DNA coding for interleukin-8 genes in vivo and subsequent significant recruitment of dermal neutrophils (Hengge et al., 1995). By injecting naked DNA into the skin, the stratum corneum is overcome, but in vivo delivery of naked DNA to target cells is subjected to additional challenges (Paroo and Corey, 2004). Naked DNA is susceptible to degradation by endogenous enzymes present in the interstitial space and because of their negative charge and size, nucleic acids do not readily cross the cell membrane (Vogel, 2000). Despite some studies describing the uptake of naked, unmodified DNA by keratinocytes after intradermal injection in animal models (Marchetto et al., 2004; Meng et al., 1998; Sawamura et al., 2002; Woodley et al., 2004), it is still unknown why intradermally injected plasmid DNA is taken up and expressed by keratinocytes. Except for the few studies describing injection of naked plasmids, delivery

enhancing techniques such as electroporation or gene gun were created to increase the efficiency of intracellular uptake. Unfortunately, none of these techniques are applicable for fast and easy transfection into larger areas of the skin. In 1999, Fan et al. attempted topical application of naked plasmid DNA onto the skin. The pDNA induced an antigen-specific immune response against hepatitis B and the transfer was dependent on the presence of normal hair follicles. Hair follicle structure and hair cycle stage seemed to act as important parameters influencing transfection of human hair follicles (Hoffman et al., 1996; Wu et al., 2001). It is clear that, because of size restriction, the follicular route is the preferred pathway of entrance into the skin. The minute amount of protein that is generated is apparently enough to elicit an immune response. Although this approach looked very promising, there is relatively little follow-up to this method. Penetration enhancing techniques to improve dermal or transdermal delivery of nucleic acids in the skin have been described numerously (Hengge and Mirmohammadsadegh, 2000; Ishimoto et al., 2008; Khavari et al., 2002; Mitragotri et al., 2000; Prud’homme et al., 2006; Raghavachari and Fahl, 2002; Vogel, 1999; Wraight and White, 2001). Among these skin delivery techniques, ‘active’ procedures such as tape-stripping (Nakamura et al., 2002), gene gun (Nanney et al., 2000; Oshikawa et al., 2001), intradermal injection with or without electroporation (Baek et al., 2001; Choate and Khavari, 1997; Prud’homme et al., 2006) and depilatory technology are commonly applied (Domashenko et al., 2000; Peachman et al., 2003). Even though successful studies have been performed using these techniques, they all encounter limitations for translation to the clinic, with regard to patient compliance and costeffectiveness. 4. Liposomes for topically applied cutaneous gene transfer Liposomes are vesicles formed by one or multiple lipid bilayers that enclose an aqueous environment (Fig. 2). Their major components are usually phospholipids that contain a hydrophilic head group and lipophilic tail. In an aqueous dispersion medium these phospholipids spontaneously orientate their head groups towards the water unlike the lipidic tails, resulting in a liposome bilayer. The theoretical advantages of liposomes as drug carrier systems have been assigned to their ability to encapsulate water-soluble

Fig. 2. Schematic presentation of a vesicular delivery system. Amphiphilic molecules (e.g. phospholipids), containing a hydrophilic head group and lipophilic tail domain, spontaneously form bilayer vesicles upon contact with an aqueous solution. Surfactants can be included into the liposomes and are often characterized by a single chain hydrophobic tail. Depending on the ratio of phosholipid to surfactant, classical liposomes, niosomes or transfersomes are synthesized.

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and lipophilic substances in their different phases. Moreover, they are similar to the epidermis with respect to their lipid composition which enables them to penetrate the epidermal barrier to a greater extent compared to other dosage forms. Most liposomes are biodegradable and non-toxic which is important in order to avoid sideeffects (Epbaria and Weiner, 1990). Of particular interest are cationic liposomes for ‘passive’ cutaneous delivery of nucleic acids, since their opposite charges spontaneously result in complexation due to electrostatic interactions. Cationic liposomes protect DNA from degradation and they allow introduction of large DNA fragments into the target cells (Fresta and Puglisi, 1996). Topical application of liposome/DNA complexes (lipoplexes) on the skin of living mice was performed for the first time by Li and Hoffman, and resulted in the transfection of mainly hair follicle cells (Li and Hoffman, 1997; Li et al., 1993). As from then, numerous attempts have been made using cationic liposomes for topical plasmid delivery. It was soon concluded that, in general, these non-viral systems exhibit a low delivery efficiency due to their limited ability to cross the stratum corneum barrier before reaching their intracellular target site.

New classes of lipid vesicles were introduced by different researchers to ameliorate some of the features of the previously described ‘classical’ liposomes and to procure an enhanced skin permeation of entrapped molecules. By introducing permeation enhancers into the vesicle composition (e.g. ethanol or surfactants that act as edge activators), vesicle elasticity and deformability were increased, enhancing their skin penetration. Alternative terminology has been adopted to describe different vesicular systems with their different functions and/or compositions, such as Transfersomes, niosomes, ethosomes or vesosomes. For a complete overview of the vesicular systems utilized to deliver small and larger molecules to and through the skin, we refer to other excellent reviews (Cosco et al., 2008; El Maghraby and Williams, 2009; Elsayed et al., 2007; Honeywell-Nguyen and Bouwstra, 2005; Sinico and Fadda, 2009). Thus far, only ‘classical’ liposomes, niosomes and TransfersomesÒ have been used for topical gene delivery. Ethosomes are generally negatively charged (Touitou et al., 2000) and therefore not suitable for complexation with DNA. Vesosomes, on the other hand, have been solely described for the delivery of tetanus toxoid

Table 2 Schematic overview of lipid-mediated gene delivery systems after topical application onto the skin. Delivery system

Composition

DNA/plasmid encoding

Tested on

Effect

Ref.

Liposomes

Egg PC

Histocultured mouse skin

Labeled hair and follicle cells

Li et al. (1993)

Liposomes

PC:Chol:PE

Random mouse DNA fragment labeled with [35S]dATP LacZ reporter

LacZ expression in hair follicles

Liposomes

DOTAP

b-galactosidase reporter

Li and Hoffman (1995) Birchall et al. (2000)

Niosomes

GDL:Chol:POE-10

b-galactosidase reporter

Histocultured mouse skin In vitro mouse skin Rat skin

Niosomes

GDL:Chol:POE-10

Niosomes

Tween61:Chol: DDAB

Human interleukin-1 receptor antagonist (IL-1ra) Luciferase reporter

Niosomes

Span85:Chol

Hepatitis B surface antigen (HBsAg)

Niosomes (spray formulation)

Soybean lecithin

Cy5-labeled pDNA

Mouse skin

GFP reporter

Hamster ears

Reporter expression in viable epidermal tissue Intense staining of follicular and epidermal cells IL-1ra expression in hair follicles

In vitro rat skin Mouse skin

High cumulative amounts in skin and high transdermal fluxes High serum anti-HBsAg titer

Transfersomes

DOTAP:NaC

GFP reporter

In vitro human skin Mouse skin

Transfersomes

DOTMA:NaDC Octadecylamine:Chol GDL:Chol:POE-10:DOTAP

HBsAg

Mouse skin

IL-1ra

Hamster skin

Chloramphenical acetyltransferase/human interferon-a2

Mouse skin

Hybrid non-ioniccationic liposomes Nanoemulsions

Tween80:Span80:olive oil

Solid lipid nanoparticles

Non-ionic emulsifying wax:CTAB:DOPE

b-galactosidase model antigen

Mouse skin

Gemini nanoparticles

DOPE:DPPC:Gemini surfactant 16-316:di-ethylene glycol monoethyl ether SoyaPC:Chol:DC-Chol SoyaPC:Chol:DMPC

IFN-c

Mouse skin

Glycoprotein D (gD)

Mouse skin

Biphasix vesicles

Elevated IL-2 and IFN-c levels Intrafollicular and follicular uptake Cy5-DNA Generation of GFP-specific antibodies GFP expression in liver and lungs Specific anti-HBsAg titers in serum Elevated IFN-c levels Transgene expression in perifollicular cells Gene expression in follicular keratinocytes Expression higher in normal structured follicles compared to abnormal Enhancement of antigen-specific IgG titers and splenocyte proliferation High IFN-c levels in skin and lymph nodes Elevated anti-gD IgG gD-specific cellular response (IL-4) in spleen cells

Raghavachari and Fahl (2002) (Ciotti and Weiner (2002) Manosroi et al. (2009) Vyas et al. (2005)

Meykadeh et al. (2005)

Kim et al. (2004a), Lee et al. (2005) Mahor et al. (2007) Wang et al. (2007) (Niemiec et al., 1997) Wu et al. (2001)

Cui et al. (2003), Cui and Mumper (2002) Badea et al. (2005)

Babiuk et al. (2002)

SoyaPC:Chol:DOPE PC, phosphatidylcholine; PE, phosphatidylethanolamine; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; Chol, cholesterol; GDL, glyceryl dilaurate; POE-10, polyoxyethylene-10 stearyl ether; DDAB, dimethyl dioctadecyl ammonium bromide; Tween61, polyoxyethylene sorbitan monostearate; NaC, sodium cholate; NaDC, sodium deoxycholate; DOTMA, N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium; CTAB, hexadecyltrimethyl-ammonium bromide; DOPE, dioleoyl phosphatidylethanolamine; DC-Chol, 1-cholesteryl 3-N-(dimethylaminoethyl)carbamate; DMPC, dimyristoylphosphatidylcholine.

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(Mishra et al., 2006). It should be noted that other lipid-based systems have also been developed for cutaneous gene delivery. These are not categorized into one of the earlier described subsets but belong to the ‘super family’ of lipidic carrier systems. They include hybrid liposomes, microemulsions, solid lipid nanoparticles and biphasic lipid vesicles and will be discussed in a separate section. Table 2 gives an overview of all the types of lipid-based carrier systems that have been described for topically applied cutaneous pDNA delivery without the aid of chemical or physical enhancing techniques.

veillance mechanism within epidermis and subsequently resulted in an immune response against the antigen encoded by the vector. Another ‘barrier disruption technique’ was described by Watabe et al. (2001) who used adhesive glue stripping prior to topical application of pDNA encoding the M gene of influenza virus. DNA vaccine plus cationic liposome induced higher levels of antibody production than DNA vaccine alone. This immune response was even increased when the liposomes were coated with mannan (the cholesterol component in the liposomes was conjugated to aminoethylcarbamylmethyl mannan).

4.1. Classical liposomes

4.2. Niosomes

The term ‘classical’ liposomes is generally used to distinguish conventional phospholipid-based vesicles from the ‘new’ vesicle formulations. They are composed of double chain phospholipids, with or without cholesterol and are commonly used for drug delivery in various disciplines of pharmaceutical science. When topically applied, the transappendageal follicular pathway (Fig. 1) is believed to be the route of preference for gene delivery with conventional liposomes. Li et al. (Li and Hoffman, 1995; Li et al., 1993) found that liposomal entrapped DNA resulted in specific delivery into the hair follicles of histocultured mouse skin, while aqueous control solution (i.e. naked DNA in water) of these molecules showed no follicular localization. Hair follicle structure and hair cycle stage seemed to act as important parameters influencing transfection of human hair follicles. Because the follicular openings only constitute for 0.1% of the skin surface (Szabo, 1962), penetration through these routes is limited. The majority of topically applied liposomes onto the skin will accumulate in the upper layers of the SC and function as a ‘reservoir’ providing a localized action without effects in deeper layers, as confirmed by several authors (Choi and Maibach, 2005; Cosco et al., 2008; Elsayed et al., 2007; Honeywell-Nguyen et al., 2005). Conflicting results arose when Birchall et al. demonstrated in 2000 that topical application of cationic lipid:pDNA complexes can mediate uptake and expression of reporter pDNA in viable ex vivo mouse epidermal tissue, probably using transepidermal diffusion pathways (Fig. 1). To date, it is generally believed that topical plasmid transfection using classical liposomes can only be achieved in combination with other delivery enhancing techniques, such as ‘disrupting’ the stratum corneum barrier functions prior to lipoplex application onto the skin. Alexander and Akhurst demonstrated in 1995 that chemical depilation of 4-week-old mice followed by topical application of cationic liposomal constructs containing the Lac Z gene, encoding the protein b-galactosidase (b-gal), resulted in an efficient transfection as shown by immunohistochemistry data. Expression was observed in the hair follicle, epidermis and also in the deeper layers of the dermis. In addition, the investigators reported that the topical application of a cationic liposome–pDNA vector to in vivo mouse skin in the anagen or active phase of the hair growth cycle resulted in more efficient reporter gene expression than when applied in the telogen phase of the hair growth cycle. Shi et al. (1999) investigated whether the amount of antigen produced in the skin from a topically applied vector was sufficient for eliciting an immune response against the antigen by complexing human growth hormone (hGH) encoding pDNA (pCMV-hGH) with liposomes made of 1,2-dioleoyl-3-trimethylammoniumpropane (chloride salt)/1,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine (DOTAP/DOPE) and applying the complexes on mouse skin treated with depilatory agent. Results demonstrated that DNA/liposome complexes were less potent than intramuscular injection of naked pCMV-hGH DNA for eliciting an immune response against hGH in mice. Direct comparison with topically applied naked DNA was not made. The expression of antigens in small number of cells within the outer layer of skin was apparently enough to activate the sur-

Evolving from liposomes, niosomes are non-ionic surfactant vesicles made up from single chain surfactant molecules such as polyoxyethylene alkyl ethers, polyoxyethylene alkyl esters, diacyl glycerides or saccharose diesters, often in combination with cholesterol. They alleviate the disadvantages associated with liposomes, like chemical instability and variable purity of phospholipids (Vora et al., 1998). Niosomes have been prepared and studied for drug delivery over the past decades. The mechanism of interaction with the skin has been extensively examined by several authors (Abraham and Downing, 1990; Bouwstra et al., 2003; Vrhovnik et al., 1998) and generally resembles the same characteristics as that of liposomes. Hofland et al. (1995) found that niosomes are thought to improve the horny layer properties, both by reducing transepidermal water loss and by increasing smoothness through replenishment of lost skin lipids. This fusion to corneocytes and formation of lipid stocks was detected by electron microscopy. When side-by-side comparative studies were carried out, using liposomes and niosomes, the latter were found to posses better permeation efficiencies for DNA delivery. Raghavachari and Fahl (2002) conducted a study in which they compared traditional phospholipid-based liposome carriers and non-ionic liposomes for their delivery efficiency and expression of b-galactosidase or luciferase DNAs in rat skin cells. Among the formulations tested, the non-ionic liposome formulation was found to be superior in delivering the reporter gene plasmid DNA. Similarly, Ciotti and Weiner (2002) reported the expression level of human interleukin-1 receptor antagonist (IL-1ra) pDNA by testing different formulations on hamster ears. Delivery into the hair follicle and an enhanced localized absorption and expression of pDNA were significantly greater for the niosomes compared to the PC liposomes and control formulation tested. Cotsarelis confirmed these findings and stated that – as far as liposome composition was concerned – non-ionic liposomes were more efficient than the traditional PCbased liposomes (Cotsarelis, 2002; Domashenko et al., 2000). One possible reason why niosomes enhance permeability is their ability to modify stratum corneum structure; the intercellular lipid barrier in the SC may become looser and more permeable by niosome treatment (Fang et al., 2001). Adsorption and fusion of niosomes with the skins surface may lead to a high thermodynamic activity gradient at the interface. In addition, Jayaraman et al. (1996) ascribed the high efficiency of non-ionic liposomes to the unique lipid composition. Niosomes enhance transdermal adsorption by changing the structure of the SC resulting from the solubilization of their surfactant properties. Manosroi et al. (2009) confirmed this by demonstrating that niosomes exhibit better transcutaneous permeation of a luciferase plasmid when compared to liposomes. This effect was even more pronounced when they increased elasticity of both vesicles by introducing ethanol into the formulations. This gave way to better physicochemical and biological properties in relation to their non-ethanolic counterparts and in the same way, ethanol-containing niosomes were superior to the ethanol-containing liposomes.

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Niosomes composed of Span 85 and cholesterol as constitutive lipids have been investigated for the topical delivery of plasmid DNA encoding hepatitis B surface antigen (HBsAg). This study signifies the potential of niosomes as DNA vaccine carriers for effective topical immunization (Vyas et al., 2005). The immunestimulating activity of these niosome-based systems was investigated in terms of the serum anti-HBsAg titre and also cytokine levels (IL-2 and IFN-c) were registered following the topical application of niosomes to Balb/c mice. After 6 weeks, the titer values were comparable to that elicited by intramuscular injection of pure HBsAg. In addition, a significantly higher level of both IL-2 and IFN-c was observed in mice immunized with vesicular DNA vaccine in contrast with intramuscular injection of recombinant protein. Topical immunization using lipid vesicles has received a great deal of interest since it is painless and easy to administer. Moreover, the skin is rich in potent antigen-presenting dendritic cells such as Langerhans cells in the epidermis (Banchereau and Steinman, 1998). Meykadeh et al. (2005) analyzed the expression of plasmid DNA in vivo and in vitro following topical application of plasmid DNA containing an enhanced green fluorescent protein in non-ionic liposome spray formulations onto mouse or human skin. The results revealed that EGFP mRNA and protein were detectable by RT-PCR and Western blot, and that application of pEGFP-DNA led to the generation of GFP-specific antibodies. 4.3. TransfersomesÒ TransfersomesÒ were described as the first generation of highly elastic or deformable vesicles and were introduced by Cevc and Blume (1992). They are a novel type of liquid-state vesicles that consist of phospholipids and an edge activator (Fig. 2). An edge activator is often a single chain surfactant that destabilizes the lipid bilayers of the vesicles and increases their deformability by lowering the interfacial tension. According to the authors, this feature enables TransfersomesÒ to squeeze themselves through intercellular regions of the stratum corneum under the influence of the transdermal water gradient (Cevc et al., 1998) (Fig. 1, Fig. 3). This proposed mechanism of action is a subject of debate and should be regarded with a certain skepticism. TransfersomesÒ have been reported to penetrate intact skin in vivo, carrying therapeutic concentration of drugs (including macromolecules), with an efficiency similar to subcutaneous administration, provided that the elastic vesicles are topically applied in non-occlusive conditions (Cevc and Blume, 2001; Cevc et al., 1998). These in vivo successes with TransfersomesÒ guided to their introduction as possible carriers for transcutaneous immu-

Fig. 3. Diagram of the brick and mortar model of the stratum corneum. Two possible drug permeation pathways through intact stratum corneum are shown; the transcellular and the tortuous intercellular pathways. Transfersomes, who are able to squeeze themselves through intercellular regions of the stratum corneum, are believed to prefer the latter route of penetration.

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nization and non-invasive gene delivery. Kim et al. (2004a) investigated the effect of cationic (DOTAP)-based TransfersomesÒ on the transdermal application of GFP-expressing DNA in intact mice skin. GFP expression was observed in a few organs such as liver and lungs when GFP was complexed with TransfersomesÒ, whereas GFP dissolved in PBS did not produce any GFP expression. Recently, Lee et al. (2005) reported in vivo transdermal absorption of DNA delivered by egg phosphatidylcholine (PC)-based TransfersomesÒ. The extents of transdermal absorption varied with the type of edge activators used in TransfersomesÒ. When PBS was used as a gene delivery vehicle of naked DNA, it was difficult to detect the absorption of DNA. Among various edge activators, sodium cholate and sodium deoxycholate-based TransfersomesÒ displayed the highest levels of DNA in the liver. In contrast, Tween 80 did not render transdermal absorption of DNA in any organs of the mice. Therefore, the use of cholate seemed to be better than other edge activators in the case of topical DNA delivery. The group of Vyas, who previously reported the use of niosomes for delivery of pHBsAg (Vyas et al., 2005), recently developed cationic TransfersomesÒ for the topical vaccination against hepatitis B, using the same plasmid (Mahor et al., 2007). Similar results were obtained and indicated that a higher immune response was provoked in Balb/c mice when pDNA was used in combination with the TransfersomesÒ. Naked DNA alone is minimally taken up by the antigen presenting cells (APCs) and is susceptible to degradation by DNase attack. In addition, the immunity induced by topical immunization using pDNA appears to be long lasting and the serum antibody titer and endogenous cytokine levels were similar to those produced after intramuscular recombinant HBsAg administration. Wang et al. (2007) performed analogous studies, however using a different TransfersomeÒ composition, and found that an increased production of specific anti-HBsAg antibodies and IFN-c induction upon topical immunization of Balb/c mice. 4.4. Other lipid-containing formulations for topical DNA delivery Hybrid non-ionic–cationic lipid containing liposomes were synthesized by Niemiec et al. (1997) for complexation and topical application of pDNA coding for IL-1ra. They observed consistent levels of transgenic expression of human interleukin-1 receptor antagonist protein in hamster skin following topical application. Especially, transgene expression localized to perifollicular cells was detected. These observations are in concordance with Ciotti and Weiner (2002) and suggested that liposome-mediated delivery of expression plasmid to follicular cells for transient transfection was not only possible but might be optimized by systematic manipulation of the liposomal components (Niemiec et al., 1997). Unfortunate attempts by the group of Roessler to improve the efficiency of transfection using hybrid non-ionic–cationic lipid containing liposomes have contributed to the hypothesis that the limitations of topical transfection using liposomes are fundamentally related to the physical conformation of the complexes that repidly take place after topical application (Wu et al., 2001). Therefore, water-in-oil (W/O) nanoemulsions have been developed. Nanoemulsions are thermodynamically stable liquid isotropic dispersions composed of water, oil and surfactants. They have presented promising potential in enhancing absorption and delivery of water-soluble macromolecules, such as peptides and pDNA (Osborne et al., 1991). Since high shear energies are not required during the preparation of nanoemulsions the likelihood of physical damage to the plasmid DNA degradation is virtually eliminated. Wu et al. (2001) used plasmids encoding chloramphenicol acetyltransferase (CAT) or human interferon-2 and showed that W/O microemulsions prepared from Tween 80, Span 80, and olive oil can be used to facilitate transfection of murine follicular keratinocytes by about 20-fold. The level of gene expression was detected

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to be higher in normal structured follicles compared to abnormal ones. One year later, Cui and Mumper (2002) reported the genetic immunization using solid lipid nanoparticles (SLNP) engineered from microemulsion precursor techniques. Expressed b-galactosidase was used as a model antigen. Plasmid DNA was coated on the surface of preformed cationic nanoparticles engineered directly from warm oil-in-water (O/W) microemulsion precursors comprised of non-ionic emulsifying wax - as the core material of the nanoparticles – and hexadecyltrimethyl-ammonium bromide (CTAB) as the cationic surfactant. Mannan, a dendritic cell ligand, was coated on the nanoparticles with and without entrapped DOPE and cholesterol. The humoral and proliferative immune responses were assessed after topical application of these nanoengineered systems to the skin of shaved Balb/c mice. All pDNA-coated nanoparticles, particularly the mannan-coated pDNA nanoparticles with DOPE resulted in significant enhancement in both antigen-specific IgG titers (16-fold) and splenocyte proliferation over naked DNA alone. Incorporating pDNA into another, novel ethanol-in-fluorocarbon (E/F) microemulsion system, with FSN-100 as the fluorosurfactant, and applying topically to shaved mouse skin also led to enhanced specific total IgG titer to an expressed model antigen, b-galactosidase, in serum by 45-fold compared to naked pDNA alone (Cui et al., 2003). Cutaneous gene therapy for scleroderma was also used by Badea and coworkers (2005) to administer IFN-c gene by means of ‘gemini nanoparticles’ in IFN-c-deficient mice. The particles (NP16) were made up of the gemini cationic surfactants N,N0 bis(dimethylhexadecyl)-1,3-propanediammonium dibromide (163-16), 1,2-dioleyl-sn-glycero-3-phosphatidylethanolamine, 1,2dipalmitoyl-sn-glycero-3 phosphatidyl-choline and diethylene glycol monoethyl ether. IFN- c -deficient mice (0.067 ng/cm2 of protein expressed) treated with plasmid-NP16 nanoparticles showed that gemini nanoparticles improved the transgenetic expression by threefold with respect to a plasmid DNA solution (0.480 versus 0.167 ng/cm2, respectively). Lastly, biphasic vesicles (Biphasix) were developed as an alternative type of liposomal vesicle. They are multi-compartmental lipid vesicles consisting of multiple, concentric mixed-lipid bilayers entrapping lipophilic, micellar and aqueous subunit compartments. Babiuk et al. (2002) demonstrated that topical delivery of plasmid formulated in biphasic vesicles resulted in gene expression in the draining lymph node and in the induction of cellular and humoral immune responses. These vesicles were later evaluated for immunization in pigs and mice using CpG oligonucleotides for subcutaneous, intranasal and mucosal administration (Alcon et al., 2005a,b, 2006, 2003).

5. Disadvantages and limitations It is clear that most studies that use topical application of DNA loaded vesicles were conducted for immunization purposes or were developed in order to evaluate the transdermal adsorption properties of these formulations by using reporter constructs. The major reason is probably that these delivery systems encounter limited penetration and hence reduced efficiency for appropriate expression of therapeutic amounts. However, minute amounts of intraepidermally produced protein, processed and presented by epidermal Langerhans cells, may be sufficient to elicit an immune response (Meykadeh et al., 2005). For therapeutic purposes in order to treat skin diseases, higher transfection efficiency would be necessary (Fan et al., 1999; Khavari, 2000). Therefore, delivery enhancing techniques are often employed in combination with liposomes to increase the transfection efficiency. In the following section, we will describe the use of lipid-mediated gene delivery in

combination with delivery enhancing techniques for various cutaneous purposes. Due to the transient nature of gene expression inherent to this type of delivery, applications for DNA vaccination, wound healing, melanoma or other disorders that require temporary and reversible treatment are of most benefit. For genodermatoses, where stable expression of correction of the mutated gene is required, lipid-mediated gene delivery has not yet offered a solution and probably will never do.

6. Combination with physical enhancing techniques 6.1. Immunization Without any doubt because of its natural barrier function, the skin is perceived as a site that is well-equipped for the induction of adaptive immune responses and the high density of antigenpresenting cells in skin provides indirect support for this notion. Dermal DNA vaccines can be applied by various methods, including topical lipid-mediated application (as described previously), intradermal injection, gene gun and DNA tattoo (Bins et al., 2005). In the next paragraphs, we describe the combination of liposomecomplexed pDNA together with physical enhancing techniques for improved (trans)cutaneous delivery. Heller et al. (2008) applied electroporation (EP)-assisted delivery of intradermally injected DOTAP:DNA formulations into Balb/c mice and compared the expression of the luciferase reporter protein to that obtained by EP of naked DNA and topical DOTAP:DNA application. At 48 h, the topically applied DOTAP:DNA formulation tested tended to increase reporter expression. EP of naked DNA increased expression significantly, nearly 20-fold higher than the liposome formulation. When EP and liposome delivery were combined, expression was not significantly higher compared to expression levels after injection of naked DNA alone. Changing EP parameters has shown to influence levels and duration of expression. Although a reporter construct was used here, the authors believe that this approach holds potential for vaccination purposes. In 2009, Cheng et al. developed liposome/DNA complex-based patches as means of topical DNA vaccine carriers for transcutaneous immunization (Cheng et al., 2009). The plasmid encodes Japanese encephalitis virus (JEV) envelope proteins and liposomes were prepared containing DOTAP or 3b-[N-(N,N-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol)/DOPE. These cationic liposomes were complexed with DNA, applied topically with clinical gauze of non-woven fabrics on chemically depilated abdominal skin of C3H/HeN mice and covered with transparent dressing film. These patches were perceived to effectively induce protective antibodies against the infection with 50 times the 50% lethal dose (LD50) of JEV. The level of immunogenicity and the levels of the correspondingly provoked antibodies were found to be correlated. Recently, van den Berg et al. (2010b) investigated the transfection rate and immune response of cationic liposome–DNA complexes (lipoplexes) when they were intradermally applied by means of DNA tattooing into an ex vivo human skin model. The lipoplexes were formulated with a GFP encoding plasmid that also encodes the influenza A NP366–374 epitope as a genetic fusion construct. It was found that cationic lipoplexes increased transfection efficiency in epidermal cell suspensions but decreased antigen expression in ex vivo human skin and mice. The presence of the extracellular matrix (ECM) in skin tissue reduced free diffusion of particles in intact skin. In addition, several ECM components carry a net negative charge and are likely to interact with the positively charged nanoparticles, consequently becoming immobilized in the ECM. Van den Berg et al. encountered that shielding the cationic surface charge by means of PEG moieties restored the transfection

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efficiency and vaccination-induced antigen expression in ex vivo human skin and murine skin, subsequently leading to a full restoration of vaccine immunogenicity. For a complete overview of liposome-based DNA vaccines we refer to the recently published reviews of Schwendener et al. (2010) and van den Berg et al. (2010a). 6.2. Wound healing For wound healing purposes, it is most likely that expression of a specific transgene product is required only for a limited period, sufficient to adequately stimulate cells toward synthesis of repair. In contrast to genetic diseases, the use of non-viral vectors such as liposomes may be sufficient. Because the skin barrier properties are compromised and therefore the reduced penetration capacity of (conventional) liposomes is of lesser importance in this type of condition, topical application has high expectations. However, the majority of the studies carried out used subcutaneous injection of DNA containing liposomes to improve its efficacy. Several studies have been performed to determine liposomal gene transfer for growth factors (Eming et al., 1999; Jeschke et al., 1999b; Sun et al., 1997). Sun et al. (1997) administered fibroblast growth factor-1 (FGF-1) cDNA using cationic liposomes for topical application and subcutaneous injection to the injured skin of diabetic mice. Transfection with FGF-1 was found to increase tensile strength and improved wound quality when compared to controls. The effect of insulin-like growth factor I (IGF-1) cDNA liposomal constructs subcutaneously applied to thermally injured rat skin has been investigated by Jeschke et al. (2000). The burn wound has been shown to be the major source of pro-inflammatory cytokines and is characterized by a hypermetabolic response (Rodriguez et al., 1993). Thermally injured rats treated with liposomal IGF-1 cDNA showed attenuated pro-inflammatory hypermetabolism and post-traumatic acute phase response (Jeschke et al., 1999a). The rats significantly improved in body weight and showed increased muscle proteins when compared to burn controls. This is indicative for the fact that skin may be used as a delivery system for metabolic disorders (‘metabolic sink’ approach) because gene products have been shown to reach the systemic circulation (Alexander and Akhurst, 1995; Gao and Huang, 1995). An accelerated rate of re-epithelialization of nearly 15% was observed when cationic liposomal complexes were compared to naked IGF-1 protein and IGF-1 protein encapsulated in liposomes. In a preliminary biodistribution study, using thermally injured rat skin, the same authors detected a transfection rate of 70–90% in myofibroblasts, endothelial cells and macrophages, including multinucleated giant cells (Jeschke et al., 2004). The same group recently investigated the feasibility of administration of multiple cDNA constructs, using multiple genes (cDNA for keratinocyte growth factor (KGF) and insulin-like growth factor) and compared this to the administration of the same growth factors individually. Accelerated re-epithelialization, increased proliferation, and decreased skin cell apoptosis was noted compared to KGF cDNA, IGF-1 cDNA and control groups. The re-epithelialization in the burn model reached 225% of the untreated control and significantly improved cell survival (Jeschke and Klein, 2004; Pereira et al., 2007). In 2007, Pereira et al. even managed to transfect epidermal stem cells at the edge of a wound, both on the outer root sheath and the matrix of hair follicles, using cationic liposomes carrying genes encoding for b-galactosidase. 6.3. Vitiligo Vitiligo is a chronic condition in which melanocytes, in the basal layer of the epidermis, are destroyed. To obtain long-term effects, targeted delivery and repeated application of liposome complexes

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are needed since non-viral treatment is only transient. The major hurdle will be penetration and migration throughout the entire viable epidermis for the effective and specific delivery of the gene of interest into the melanocytes. We previously mentioned the group of Manosroi who recently developed ethanol-containing niosomes for transdermal enhancement of plasmid DNA (Manosroi et al., 2009). In a recent study, they investigated the potential application of these elastic cationic niosomes as a topical delivery system for the tyrosinase gene in vitiligo therapy (Manosroi et al., 2010). A plasmid encoding human tyrosinase, pMEL34, was loaded in elastic niosomes and transdermal absorption was evaluated using rat skin by means of Franz-diffusion cells. The phenotypic effect (repigmentation) was not evaluated in vivo. In cultured melanoma cells transfection of pMEL34 niosome complexes showed enhanced tyrosinase activity, about four times higher than the free plasmid. 6.4. Melanoma Due to its unique set of characteristics, melanoma represents a suitable target for the clinical translation of different gene transfer approaches. The goal of gene therapy targeted to melanoma cells is to introduce ‘‘suicide’’ genes, to transfer tumor suppressor genes, to inactivate aberrant oncogene expression, or to introduce genes encoding immunologically relevant molecules. Gene therapy targeted to the host’s immune cells has been developed as an additional strategy to redirect immune responses against melanoma (Sotomayor et al., 2002). A potential advantage of using cationic liposomes for cancer treatment is that they are preferentially taken up by the leaky tumor vasculature, therefore increasing local gene delivery in melanoma tumors. Additionally, immune cells such as macrophages, dendritic cells and M cells, show preferential uptake of charged liposomes, which can trigger additional immune responses that can be critical to tumor rejection. Therefore, liposomes can potentially function as an adjuvant for immunotherapy (Tabata and Ikada, 1988). In many cases, local and targeted delivery is mediated by local injection to increase its efficacy. Liposomes fabricated to target antigen presenting cells (APCs), particularly dendric cells (DCs) and macrophages, for melanoma vaccination have been explored by various researchers. Lu and co-workers (2007) have focused on the mannose receptor on the surface of APCs and developed cholesten-5-yloxy-N-(4-((1-imino-c-b-D-thiomannosyl-ethyl)amino)butyl) formamide (Man-C4-Chol), which exhibits bifunctional properties: an amino group for binding pDNA by electrostatic interaction and mannose residues for binding the mannose receptor on the surface of APCs (Kawakami et al., 2000). A gp100, TRP-2 and ubiquitin co-expressing plasmid (pUb-M) was used as the delivery target. Different routes of administration were investigated and they found that the intraperitoneal route resulted in significantly higher cytotoxic lymphocyte activity compared to intramuscular, intradermal or subcutaneous administration. Inoculation with the mannosylated liposomes produced significantly higher IFN-c, B16BL6-specific lymphocyte proliferation, blocked melanoma propagation and prolonged the survival of immunized mice. Another example of immunotherapy by means of lipid-mediated gene vaccination was described by the group of Gonzalez (Gonzalez et al., 2006; Stopeck et al., 2001). In a phase II trial for metastatic melanoma, melanoma patients were injected intralesionally with a low dose of a HLA-B7/beta2 microglobulin plasmid formulated with cationic lipids consisting of 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium biomide (DMRIE)/DOPE. This formulation is called Allovectin-7 and is a registered trademark of Vical Incorporated (San Diego, California, USA). HLA-B7 antigen is infrequently expressed in the United States population

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(about 20% of the United States Caucasian population) and thus allows for a potential allogeneic immune response in most patients. Loss of b2-microglobulin or mutations in b2-microglobulin have also been reported as possible mechanisms for deficient MHC class I expression in tumor cells. Thus, the b2-microglobulin gene was also included in Allovectin-7 to allow for expression of the complete MHC class I complex on the tumor cell surface. Minimal adverse events were associated with the Allovectin-7 injections. Seven patients (9.1%) had complete or partial response with 4.8 months median duration of response. Allovectin-7 was shown to be safe and exhibits biological activity at this dose. Its safety profile may enable Allovectin-7 to be used at higher doses, which may provide greater clinical activity. Recently, Speroni et al. (2009) investigated the antitumoral effect of IL-12 on the progression of melanoma tumors that were generated by B16F0 cells in C57BL/6 mice. Plasmids carrying the IL-12 gene were associated with liposomes containing the cationic lipid stearylamine (SA). This type of melanoma vaccination results from locally secreted IL-12 that stimulates the proliferation and cytotoxicity of natural killer cells and T lymphocytes, and promotes the generation of Th1 effector cells. Due to chemokine induction, IL-12 also exerts anti-angiogenic effects by decreasing blood supply to tumors. As a result, IL-12 expression had a marked effect on in vivo growth of B16 melanoma tumors, significantly retarding their growth and prolonging host survival. The role of angiogenesis in tumor growth and progression is firmly established and considerable efforts have focused on antiangiogenic therapy as a new form of cancer treatment (Tandle et al., 2004). Next to anti-angiogenic protein therapy that is considered as an effective approach, anti-angiogenic gene therapy has become one of the most attractive concepts to the treatment of cancer. Kim et al. (2004b) have systematically investigated whether co-administration of the mouse angiostatin kringle 1-3 gene (pFLAG-AngioK1/3) and the endostatin gene (pFLAG-Endo) complexed with cationic DOTAP/cholesterol liposomes exhibits enhanced therapeutic efficacy. In vivo, subcutaneous co-administration of pFLAG-AngioK1/3 and pFLAG-Endo lipoplexes inhibited 81% of tumor growth and 90% of formation of pulmonary metastases of B16BL6 melanoma cells in mice. Co-administration resulted in a better outcome than treatment with pFLAG-AngioK1/3 lipoplexes or pFLAG-Endo lipoplexes individually. Similarly, the same group found that subcutaneous administration of the salmosin gene with cationic liposomes resulted in systemic expression of the gene product and concomitant inhibition of the growth of B16BL6 melanoma cells (Kim et al., 2003). The effect of local and systemic delivery of angiostatin genes on human melanoma growth was studied in nude mice by Rodolfo et al. (2001). Liposome-coated plasmids carrying the cDNA coding for murine and human angiostatin (CMVang and BSHang) were injected weekly, locally or systemically, in mice transplanted with melanoma cells. The treatment reduced melanoma growth by 50–90% compared to that occurring in control animals treated with liposome-coated plasmid carrying the lacZ gene or in untreated controls. The growth of both locally injected and contralateral uninjected tumors in mice bearing two melanoma grafts was significantly suppressed after intratumoral treatment. The present report demonstrates that gene therapy with murine and human angiostatin gene by liposome delivery inhibits local and distant human melanoma growth in nude mice, after intratumoral or intraperitoneal administration. Kinet et al. (2009) described the use of plasmid DNA encoding the human 16 kDa fragment of prolactin (16K hPRL) as a potent inhibitor of angiogenesis both in vitro and in vivo. The plasmids were complexed with cationic DOTAP/cholesterol liposomes and were found to prevent tumor growth in xenograft B16F10 mouse models after subcutaneous administration. A reduction of tumor

vessel length and width led to a 57% decrease in average vessel size, reflecting its anti-angiogenetic activities.

6.5. Atopic dermatitis Atopic dermatitis is a chronic inflammatory disease in which a complex network of cytokines and chemokines contributes to establishing a local milieu that favors the permanence of inflammation in the skin. Immune cells as well as keratinocytes are activated and they both are potential target cells for genetic treatment. Of particular interest is anti-sense therapy, since overexpression or overactivation of the NF-jB pathway is believed to be a major underlying cause (Nakamura et al., 2002). However, anti-sense therapy will not be discussed in this review. A pilot study performed by Mueller et al. (2005) evaluated the effects of immunostimulatory liposome–plasmid-DNA complexes combined with specific allergens for immunotherapy of refractory canine atopic dermatitis. Seven dogs with previously diagnosed atopic dermatitis and unsatisfactory response to at least 12 months of conventional allergen-specific immunotherapy underwent a series of six intradermal injections (weeks 0, 2, 4, 6, 10 and 14), with patient-specific allergen extracts contained in cationic liposome-DNA complexes. Degree of pruritus was assessed on a visual analog scale. Lesion scores were determined using the Canine Atopic Dermatitis Extent and Severity Index (CADESI) and medication usage was recorded at weeks 0 and 14. Canine cytokine mRNA expression in peripheral blood mononuclear cells collected prior to treatment and at the completion of the study was determined for IFN-gamma, IL-4, TNF and IL-10 genes using quantitative reverse transcription competitive polymerase chain reaction. Repeated intradermal injections of specific allergens incorporated into liposome–nucleic acid complexes were well tolerated in all seven dogs. There was a significant improvement in pruritus scores (P = 0.0277) and concurrent significant decrease in IL-4 production (P = 0.0428) at the completion of the trial compared to pretreatment values. Medication scores, CADESI and production of other cytokines did not change significantly with treatment. These early results suggest that antigen-specific immunotherapy using a novel liposome– nucleic acid complex vaccine may be beneficial for treatment of established atopic dermatitis in dogs using lower antigen doses.

6.6. Psoriasis Psoriasis is one of the most common human inflammatory dermatologic diseases that result from a combination of genetic and environmental factors. Similar to atopic dermatitis, both skin cells and immune cells are involved in sustaining the pathology (Krueger and Ellis, 2005). The cytokine IL-4 has a potent inhibitory effect on the autoimmune process. A clinical trial of IL-4 in psoriasis has demonstrated that such a pleiotropic cytokine has shown success in inhibiting human psoriasis (Ghoreschi et al., 2003). In a very recent study, Li et al. (2010) used a K14-VEGF transgenic mouse model as a psoriasis model to evaluate the topical transdermal delivery of mIL-4 plasmid using ultradeformable cationic (DOTAP/DOPE) liposomes and its anti-psoriatic effect. Daily application of 15 lg of plasmid DNA was used to cure psoriasis. After 31 days of treatment, the mice in the treatment group appeared to display a milder psoriatic phenotype compared to those in control groups. The data obtained in the present study indicate that gene therapy based on our transdermal IL-4 delivery system is effective in anti-psoriasis in the K14-VEGF transgenic mouse model.

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7. Conclusion and discussion Human skin is a complex organ and many features determine its barrier. In a recent review, Baroli nicely describes the factors that affect the skin absorption of topically applied agents (Baroli, 2010). These factors appear to be primarily size-related; only those nanoparticles and/or nanomaterials whose dimensions are below 6–7 or 36 nm might be able to be concurrently and respectively absorbed through the lipidic transepidermal routes or aqueous pores. In contrast, lager agents (10–210 nm) may preferentially penetrate through the transfollicular route. Additional properties that control skin absorption are described to be size deformability (shape), superficial charges, and composition and properties of penetrating agent dispersing medium. Liposomes are popular nano-sized drug carriers that offer potential value in dermal and transdermal delivery. Although their usefulness for delivery of high molecular weight compounds, such as nucleic acids is often a subject of debate, numerous studies have shown promising results. Through the years, advances and modifications have generated improved therapeutic potential. Alterations in their composition and structure resulted in vesicles with flexible and ultradeformable properties. Such liposomes, especially TransfersomesÒ, claim enhanced transdermal gene delivery with efficiencies comparable to subcutaneous administration. Noninvasive transcutaneous vaccination using these structures has been shown effective. The immunological competence of the skin, the rapid turn-over of skin cells and the various reports that animals can be immunized by DNA-based antigen expressing non-invasive vaccine delivery, may allow for the development of a unique method for vaccination that is simple, safe and painless. It should be kept in mind that we are often confronted with the usual unavailability of data showing clear evidence of intact particle penetration. The different experimental parameters such as formulations, experimental setting, specie, gender, and age of skin donors, often make it challenging to compare similar studies. In addition, most literature reports have generated data from animal models and it is well known that animal skin permeability differs from that in humans. Mouse skin is much thinner and contains more hair follicles, increasing the odds for transdermal delivery. As a consequence, these preclinical studies give an optimistic view and should therefore be evaluated with a certain critical view. Nevertheless, preclinical studies are imperative steps in the evaluation of gene therapy strategies and confirmatory investigations on human skin are still required. Different target applications require different delivery needs. They determine the individualistic approach for each purpose. Current clinical studies for cutaneous disorders suggest that there is still a long way to go before liposomes become an effective topical gene delivery method. Their reduced penetration and concurrent ineffectiveness make them unusable for the therapeutic treatment of the majority of genetic skin disorders. A combination with active delivery systems will probably offer a good alternative. Although FDA has approved injection procedures and other active systems for transdermal delivery in general, they are withdrawn from the market because of safety concerns and the expensiveness of these technologies (Watkinson, 2010). Therefore, it is still a question mark whether liposomes designed for cutaneous gene delivery purposes –with or without penetration enhancing techniques – will ever make it to the market and stay there.

Acknowledgements Stefanie Bracke is a doctoral fellow of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). Peter Dynoodt received financial support from

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