Review
Therapeutic Delivery
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Delivery and targeting of nanoparticles into hair follicles
It has been demonstrated that nanoparticles used for follicular delivery provide some advantages over conventional pathways, including improved skin bioavailability, enhanced penetration depth, prolonged residence duration, fast transport into the skin and tissue targeting. This review describes recent developments using nanotechnology approaches for drug delivery into the follicles. Different types of nanosystems may be employed for management of follicular permeation, such as polymeric nanoparticles, metallic nanocrystals, liposomes, and lipid nanoparticles. This review systematically introduces the mechanisms of follicles for nanoparticulate penetration, highlighting the therapeutic potential of drug-loaded nanoparticles for treating skin diseases. Special attention is paid to the use of nanoparticles in treating appendage-related disorders, in particular, nanomedical strategies for treating alopecia, acne, and transcutaneous immunization.
Background The skin is the largest organ in humans. Topical/transdermal drug delivery via the skin provides an efficient way to prevent or treat diseases, including dermatological and systemic diseases. The stratum corneum (SC) is the outermost layer of the skin tissue, which provides the human organism with a barrier to the external environment. The structure of the SC consists of cornified corneocytes surrounded by lipids. The SC is the predominant barrier for permeation of topically applied medicines and cosmetics [1] . For successful permeation into the skin, a permeant should enter into the SC and then penetrate across it. Percutaneous absorption can occur through three pathways: intercellular, transcellular and transappendageal routes. In the past, the intercellular pathway was regarded as a main route for the diffusion of most drugs or active agents [2] . In the last decades, many investigations have reported that the appendageal pathway should be taken into consideration as an efficient route for drug permeation [3] . The appendageal route can be defined as the transport path via the hair follicles, the sebaceous glands, and the sweat
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ducts. As compared with the reservoir of the SC with a thickness of 10–20 μm, the reservoir of the hair follicles extends deep into the skin tissues up to 2000 μm [4] . The infundibulum of the follicles increases the surface area and disrupts the epidermal barrier toward the lower parts of the follicles, thus serving as an efficient reservoir for drug permeation. Interest in appendages is directed toward their employment as reservoirs for localized delivery to the skin and also as a transport track for systemic drug delivery. The sebaceous glands open into the lower infundibulum of the hair follicles. Each follicle is associated with a sebaceous gland that releases sebum, creating a lipid-enriched environment. Efficient drug transport into the hair follicles depends upon the interaction between the drug and the sebum, as well as the choice of drug vehicles [5] . A number of reports assess the follicular deposition of drugs from different vehicles, especially the formulations of nanoparticles [6] . Nanotechnology is at the leading edge of rapidly developing new therapeutic and diagnostic concepts in all areas of medicine [7] . Nanoparticles are increasingly used in different applications,
Ther. Deliv. (2014) 5(9), 991–1006
Chia-Lang Fang1,2, Ibrahim A Aljuffali3, Yi-Ching Li4,5 & Jia-You Fang*,4,6,7 Department of Pathology, College of Medicine, Taipei Medical University, Taipei, Taiwan 2 Department Pathology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan 3 Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia 4 Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan 5 Chronic Diseases & Health Promotion Research Center, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan 6 Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwan 7 Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan *Author for correspondence: Tel.: +886 883 211 8800 Fax: +886 883 211 8236 [email protected] 1
part of
ISSN 2041-5990
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Review Fang, Aljuffali, Li & Fang including bioimaging, diagnostic technology, and drug/gene delivery [8] . Nanoparticulate systems not only enhance skin absorption but can allow for drug targeting to the skin and/or its substructures [9] . The bioavailability of drugs permeating into the skin can be enhanced by using nanocarriers because the small particulate size ensures close contact to the SC [10] . Nanosized particles can make close contact with superficial junctions of the SC and the furrows between the corneocyte islands, allowing superficial spreading of the active agents. Following the evaporation of water from the nanosystems applied to the skin surface, particles form an adhesive layer occluding the skin. Hydration of the SC thus increases to reduce corneocyte packing and widen the inter-corneocyte gaps, subsequently enhancing drug transport. Figure 1 depicts the possible mechanisms involved in skin permeation enhancement by nanostructured lipid carriers (NLCs). One of the characteristics making nanoparticles interesting for topical application is their tendency to diffuse and accumulate in the hair follicles. Nanoparticles possess the potential for delivering drugs via the follicles [11,12] . The particles can aggregate in the follicular opening and penetrate along the follicular duct when administered onto the skin surface. It is beneficial to treat some dermatological diseases involved in the appendages. Moreover, the nanocarriers can deliver the active
ingredients deep into the skin and into the systemic circulation for therapeutic aims. In this review article, we would comprehensively introduce the nanoparticles that demonstrate the capability of follicular targeting for assisting drug permeation via the skin in the past ten years. This approach is successfully used in a variety of nanoparticulate formulations, such as polymeric, metallic and lipid nanoparticles. The mechanisms of follicular targeting by nanoparticles are also elucidated in this review. The structure & physiology of hair follicles The follicular orifices occupy about 0.1% of the total skin surface area [13] . For the regions in the scalp and the face, the combined areas of the follicular openings can be as much as 10% of the total area [14] . According to the report by Otberg [15] , the mean density is highest on the forehead (292 follicles/cm2), with this density being significantly higher compared with the back (29), thorax (22), forearm (18) and thigh (17). The follicles exhibit great variation in diameter in different body areas. The smallest diameter was found in the forehead (~66 μm) and forearm (~78 μm). The calf shows the largest follicular opening. Hair follicles are the pockets of the epidermis that extend through most or all of the skin depth and enclose a small papilla of dermis at their base (Figure 2) . The hair bulbs lie at the
Water evaporation Nanoparticles
Hair shaft
Nanoparticles
Film occlusion Nanoparticles
Drug Water SC
SC
SC
Sebaceous gland Viable skin
Viable skin
Close contact to skin
Skin hydration by particle occlusion
Viable skin
Hair follicle
Entrance into follicles and sebaceous glands
Figure 1. Possible mechanisms for skin permeation enhancement of drugs or active ingredients from nanoparticles. SC: Stratum corneum.
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Follicular delivery & targeting Nanoparticles can be a permeant for active targeting of hair follicles. The follicles are surrounded by networks of blood vessels, dendritic cells, and stem cells. They are all ideal targets for drug delivery [21] . The follicular route is available for systemic absorption via the blood capillaries near the bulge region. It has been shown that caffeine and minoxidil can be rapidly detected in the plasma after topical administration through the follicular pathway [22,23] . Other cell populations or structures such as Langerhans cells, matrix cells, and melanocytes demonstrate possible targets for drugs and nanocarriers. The follicles can be categorized into four areas for drug and nanoparticulate delivery, including the sebaceous duct, the bulge region, the hair matrix, and the follicular infundibulum [14] . Among these, the epithelium of the uppermost parts displays no difference from the interfollicular epidermis. On the other hand, the epithelium in the vellus and infundibulum is thinner than the interfollicular epidermis. The corneocytes in this region are smaller and not completely differentiated [24] . This part of the follicular structure with reduced barrier function provides a fast and facile approach for topically applied substances (Figure 3) . In addition, follicles can be an efficient reservoir for long-
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Dermis Subcutaneous fat
base of the follicles, which are structures of actively growing cells that eventually produce the long, fine cylinder of a hair. One or more sebaceous glands attach to the follicles. The hair shafts are enveloped in an inner root sheath that consists of keratin-rich cells. The outer root sheath that surrounds the follicles is a stratified epithelium continuous with the epidermis [16] . The superficial area of follicular infundibulum is lined by the epidermis such as a well-developed SC and stratum granulosum. The follicular duct consists of sebum, a complex lipid mixture produced in the sebaceous glands. Sebum is highly complex and comprises triacylglycerols, diacylglycerols and free fatty acids. These components account for 50% –60% of sebum [17] . Wax ester (25%), squalene (13%), and cholesterol (2%) are other principal components of sebum [18] . Hair follicles undergo a growth cycle comprising three stages: anagen (growth phase), catagen (involution) and telogen (resting phase before hair shedding). Follicular stem cells and the strict interaction between epithelial and mesenchymal cells are essential to maintaining and regulating the hair cycle [19] . About 85% of the follicles in the scalp are associated with the anagen stage. The rate of scalp hair shaft elongation is typically 0.3–0.4 mm per day. The anagen phase of the scalp can last 2–6 years as the follicles go through extensive alterations in immune and gene expression status and vascular supply [20] .
Epidermis
Delivery & targeting of nanoparticles into hair follicles
Review
Sebaceous gland Bulge region Outer root sheath Inner root sheath Hair fiber Dermal papilla
Figure 2. The histological structure of hair follicles.
term storage in comparison to the SC, creating a possibility of sustained drug delivery. Based on the investigation by Schaefer–Korting et al. [25] , the nanoparticles can persist in the reservoir of the hair follicles ten times longer than the reservoir of the SC. Another advantage of follicular delivery is the capability of penetrating into deeper skin strata since the follicular infundibulum is located in the dermal layer [26] . Follicles are generally active in sebum secretion and hair growth. Some 30% of follicles are still inactive and closed due to dry sebum [5] . The permeant usually diffuses into active follicles but not inactive follicles due to the blockage of the transport pathway by dry sebum and cell debris [27] . Some nanoparticles can open up the closed follicles for efficient follicular transport (Figure 3) . It has been shown that nanoparticles preferentially permeate into the follicles but not the SC, enabling high accumulation within the follicular reservoir. Once deposited in the follicles, the drug may release from the nanoparticles and exert its effects on the target cells or structures [28] . Additional force such as massage can increase follicular penetration of nanoparticles [29] . Massage induces hair shaft movement, which acts as a geared pump pushing the particles deep into the follicles (Figure 2) . An opposite mechanism for follicular delivery is the hair growth and sebum flow, which may slow down the passage of nanoparticles because of the upward flow compared with the downward delivery of follicular permeation.
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Review Fang, Aljuffali, Li & Fang
Massage increases particle entry
Nanoparticles open inactive follicles (crump site)
Drug release from nanopartciles
Hair growth/sebum flow retards particle penetration
Figure 3. Follicular delivery and the related mechanisms of nanoparticles.
Dermatological diseases treated via follicular delivery Topical delivery of drugs or nanoparticles that selectively target hair follicles are of interest in the treatment of some skin disorders, especially the diseases related to the appendages. Possible application includes therapy for hair growth disorders and inflammation. Hair loss is one of the disorders that can be treated by follicular targeting of drugs and nanomedicines. Alopecia is known as hair loss on the scalp due to an androgenetic process (androgenetic alopecia) or an inflammatory process (alopecia areata). The prevalence of androgenetic alopecia in Caucasian men is 96% [30] while alopecia areata affects 2.1% of the population [31] . Clearly, hair loss affects a large number of the human population. Although the condition is never mortal, it causes deep social implications for the affected individual because of the significant change to the patient’s appearance. Androgenetic alopecia occurs due to a shortening of the anagen phase with the consequence of increased hair loss and the transformation of terminal to vellus follicles. Contrary to this phenomenon, a prolonged anagen stage with conversion of vellus follicles into terminal follicles can be observed during hirsutism and hypertrichosis [32] . The follicular absorption strategy may also be beneficial to treat these
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abnormalities. Follicular targeting can deliver the topically applied substances to the sebaceous glands, providing a promising approach for topical therapy of sebaceous gland dysfunctions such as acne, seborrhoeic eczema, and rosacea. Among these, acne is the most popular disease model for development of nanoparticulate formulations containing active ingredients such as retinoic acid and its derivatives [33–35] . Almost >80% of people suffer at some point during their lives from acne vulgaris, the most frequent disorder of the human skin [36] . Acne is a multifactorial disease with various symptoms such as comedones, nodules, papules, cysts, and pilosebaceous inflammation. Recent studies have shown that a variety of nanoparticulate types can target the hair follicles for successful acne therapy [37] . These include polymeric nanoparticles, solid lipid nanoparticles (SLN), and liposomes. Follicles are also a location of Staphylococcus aureus colonization. Diseases related to S. aureus skin infection include impetigo, furuncles, folliculitis, sycosis, and subcutaneous abscesses via the production of exfoliative toxins [38] . It may be possible to treat the infections of S. aureus by follicular delivery of nanoparticles although related investigation is currently lacking. Nanoparticulate delivery into hair follicles Polymeric nanoparticles
Different types of nanoparticles show a potential application in follicular absorption. Among these, the polymeric nanoparticles have gained the most attention for follicular delivery. The polymer materials such as polystyrene, polyvinylalcohol, polylactic acid (PLA), polyglycolic acid, poly(lactic-co-glycolic) acid (PLGA), polyethyleneimine, and cellulose are utilized for the preparation of polymeric nanoparticles with the aim of delivery into the follicles. Polystyrene nanosystems are the first nanoparticles introduced for follicular targeting. Alvarez–Román et al. [39] had used carboxylate-modified polystyrene nanoparticles with an average diameter of 20 and 200 nm for examining skin penetration and distribution. In this study, confocal laser scanning microscopy (CLSM) was employed to observe the distribution of fluorescein isothiocyanate (FITC)-loaded polymeric nanoparticles in porcine ear skin. CLSM is a noninvasive technique commonly used to visualize skin samples at multiple depths parallel to the sample surface (horizontal sections) [40] . The image is achieved at a high resolution with depth selectivity compared with conventional microscopy. The imaging quality is significantly enhanced due to the unique sectioning method of the laser. In addition, the information from the out-of-focus field is not superimposed on the image in focus. The results of CLSM showed that polystyrene nanoparticles preferentially
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Delivery & targeting of nanoparticles into hair follicles
accumulated in the openings of the follicles. The FITC signal was also observed in the vicinity of the follicles and hair shafts. Wu et al. [41,42] have developed polystyrene nanoparticles with Nile red as the dye for observing in vitro skin uptake by confocal microscopy. Poly-2-hydroxyl methacrylate (HEMA) was associated with polystyrene to modulate the hydrophobicity degree of the nanoparticles. The addition of HEMA decreased the particulate hydrophobicity. The nanoparticulate diameter was below 100 nm (37–74 nm), with the nanoparticles containing a higher percentage of HEMA showing the smaller size. The uptake of nanoparticles in full-thickness porcine skin was assessed after a 6 h application. At the skin surface, Nile red remained partly associated with the nanoparticles, but also escaped from the nanoparticles and permeated into deeper SC layers. Nile red uptake in the skin declined as the percentage of HEMA increased. The CLSM image showed that the nanoparticles could not diffuse beyond the SC, but did reveal an affinity for follicular openings. Fluoresbrite® is a fluorescent polystyrene latex sphere with a particulate size of 50 nm. This nanosystem was utilized to examine the possibility of skin absorption [43] . Both hairless rat skin and porcine ear skin were used as the skin barrier models. In this case, the polymeric nanoparticles did not permeate into intact skin. The nanoparticles only permeated through the skin after needle puncture (i.e., through damaged skin). The fluorescence of Fluoresbrite® was shown around the micropores produced by the needles. The penetration and storage of 5-fluoresceinamincontaining polymer nanoparticles into the hair follicles was analyzed by CLSM [44] . The polymeric nanocarriers were prepared by polyvinylalcohol and Resomer®, a biodegradable polymer composed of PLA and polyglycolic acid. The mean diameter of this nanosystem was 320 nm. In the in vitro porcine skin experiment, nanoparticles transported much deeper into the follicles compared with the free dye solution when a massage was applied. It is assumed that the hair movement may act as a pumping force, pushing the nanoparticles into the follicles. The storage of polymeric nanoparticles in human skin in vivo was also monitored by a differential stripping technique. It was found that the nanoparticles were stored in the follicles up to 10 days, whereas the free dye could be detected for only 4 days. This suggests a superior accumulation of nanoparticles in the follicles for long-term use. Rancan et al. [45] investigated the feasibility of biodegradable PLA nanoparticles as carriers for topical delivery. Two formulations, each with a different particulate size (228 and 365 nm), were prepared for human skin uptake examination by fluorescence microscopy, CLSM, and flow cytometry.
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Review
PLA nanoparticles were absorbed by 50% of the vellus hair follicles, reaching a maximal depth corresponding to the entry of the sebaceous glands in 12% –15% of all observed follicles. There was no significant difference of follicular uptake between the nanodispersions with different diameters. PLA nanoparticles could rapidly release the dye when coming in contact with a lipophilic environment. The release process was triggered by nanoparticulate destabilization. The retention of nanoparticles in the sebaceous glands could last up to 24 h. The size of the nanoparticles may play an important role in the penetration depth of the follicles. Patzelt et al. [46] explored the effect of the particulate size of PLGA nanoparticles on the follicular permeation depth of porcine ear skin in vitro. PLGA nanosystems were prepared in a hydrogel form with the different sizes of 122, 230, 300, 470, 643, and 860 nm. The results of CLSM demonstrated that the increased particulate size led to a significantly deeper penetration into the follicles. The deepest transport was obtained by the nanoparticles with a diameter of 643 nm. The penetration depth was significantly decreased for larger particles (860 nm). This trend can be explained by the thickness of keratinized cell layers. The surface structure of the hair shafts and follicles, determined by the thickness of the keratin cells (~530 nm for human hair), may act as a pumping system that pushes the nanoparticles into the follicles when the hairs are moving [47] . This movement can be achieved by massage. The particles in the range of the thickness of the keratin cells would penetrate most deeply. Morgen et al. [48] assessed the follicular targeting of polymeric nanoparticles loaded with the lipophilic drug UK-157,147. This compound is a potassium channel opener, developed by Pfizer for treating alopecia. The predominant material of the polymeric nanosystems was ethyl cellulose. The average size of the nanoparticles was 96 nm with a polydispersity index of 0.18. As the images of fluorescence microscopy showed, the distribution of the nanoparticles was limited to rabbit ear skin surface and hair follicles during a 2 h application. The effect of UK-157,147-loaded nanoparticles on hair growth of the C3H mouse was tested. Fifty percent of the mice treated with polymeric nanocarriers with 1% drug displayed visible hair growth on the dorsal region at day 25. At the end of the experiment (day 35), hair growth was observed in 70% of the mice treated with nanosystems. No observable skin irritation was detected during the experiment. Retinyl acetate (RA) has been recognized as a potential drug for acne therapy and dermatological inflammation [49] . RA was loaded into polymeric nanoparticles synthesized by ethyl cellulose and poly(ethylene
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Review Fang, Aljuffali, Li & Fang glycol) - 4-methox ycinnamoylp htha loylchitosan for testing in vitro skin absorption [50] . For the RA control solution, no drug was found on the mouse skin surface after washing at 2 h post-application. For RA-loaded nanoparticles, 77.7% of the applied RA dose was detected on the skin surface. Confocal microscopy had shown the entry and accumulation of nanoparticles and RA in the hair follicles. RA release from nanoparticles was confirmed through detection of an increasingly higher RA/nanoparticles fluorescence intensity ratio deeper into the dermis and away from the follicles. Abdel–Mottaleb et al. [51] reported the behavior of ethyl cellulose nanoparticles for selective betamethasone delivery to the inflammatory skin sites. Nanoparticles with different diameters of 50, 100, 500, and 1000 nm were applied to a mouse ear with dithranol-induced dermatitis. According to CLSM, by using Nile red as the fluorescent marker, the particles could transport deeply and selectively into the follicles, around the hair shafts, and deposit in the associated sebaceous glands. The smaller nanoparticles (50 and 100 nm) showed a threefold stronger and deeper permeation into the hair follicles. The measurement of the inflammatory biomarker, myeloperoxidase activity, in mouse skin had confirmed the greater deposition of smaller nanoparticles in the skin. The myeloperoxidase activity of the nontreated control, 1000-nm particles, and 100-nm particles was 1.2, 1.0, and 0.5 U/mg, respectively. Not only nano-sized particles can pass into hair follicles, but micrometer-sized particles (>1 μm) can also achieve this goal. Sumian et al. [52] had used porous nylon microspheres with a diameter of 5 μm to examine the possible passage into the follicular duct. The microspheres were dispersed in two different silicones, fluid silicone and volatile silicone, to avoid alteration of nylon and dye extraction from the particles. This study was conducted in vivo in hairless rats. The penetration depth of rhodamine-6G in the microspheres was monitored by fluorescence microscopy. After administration with massage for 1 minute, dye diffusion was detected by applying ethanol on the skin to extract rhidamine-6G from the microspheres. The results demonstrated the occurrence of follicular targeting. Both the dye diffusion out of the microspheres and dye penetration depth into the follicles were considerably increased using the fluid silicone vehicle. Toll et al. [53] evaluated the influence of polystyrene microsphere size (0.75 to 6 μm) on the efficiency of follicular targeting. Full-thickness skin samples were obtained from the scalp, axillar, or pubic regions of the volunteers after surgical excision. A selective permeation of the microspheres into the follicles was shown. The optimal size for follicular delivery proved to be 1.5 μm, with a 55%
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rate of all follicles and a maximal penetration depth of >2300 μm. Needle-free vaccination is desirable for ease of application, improved patient compliance, and less risk of infection. Transcutaneous immunization provides a noninvasive and sustained release approach for vaccination. It is difficult for vaccines with a large molecular size to penetrate across the SC. Nanocarriers can be designed in a way to be taken up by hair follicles for successful vaccination [54] . Nanoparticles can be an ideal carrier for vaccines to penetrate deeper into the follicles than solution, increase the stability of antigens, and modulate immune responses [55] . Mittal et al. [56] investigated the capability of transfollicular delivery of polymeric nanoparticles using ovalbumin as a model antigen. The nanocarriers were produced by PLGA or chitosan-coated PLGA using polyvinylalcohol as a stabilizer. The average size of PLGA and chitosan-coated PLGA nanoparticles was 170 and 180 nm, respectively, with a polydispersity index of
Therapeutic Delivery
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Delivery and targeting of nanoparticles into hair follicles
It has been demonstrated that nanoparticles used for follicular delivery provide some advantages over conventional pathways, including improved skin bioavailability, enhanced penetration depth, prolonged residence duration, fast transport into the skin and tissue targeting. This review describes recent developments using nanotechnology approaches for drug delivery into the follicles. Different types of nanosystems may be employed for management of follicular permeation, such as polymeric nanoparticles, metallic nanocrystals, liposomes, and lipid nanoparticles. This review systematically introduces the mechanisms of follicles for nanoparticulate penetration, highlighting the therapeutic potential of drug-loaded nanoparticles for treating skin diseases. Special attention is paid to the use of nanoparticles in treating appendage-related disorders, in particular, nanomedical strategies for treating alopecia, acne, and transcutaneous immunization.
Background The skin is the largest organ in humans. Topical/transdermal drug delivery via the skin provides an efficient way to prevent or treat diseases, including dermatological and systemic diseases. The stratum corneum (SC) is the outermost layer of the skin tissue, which provides the human organism with a barrier to the external environment. The structure of the SC consists of cornified corneocytes surrounded by lipids. The SC is the predominant barrier for permeation of topically applied medicines and cosmetics [1] . For successful permeation into the skin, a permeant should enter into the SC and then penetrate across it. Percutaneous absorption can occur through three pathways: intercellular, transcellular and transappendageal routes. In the past, the intercellular pathway was regarded as a main route for the diffusion of most drugs or active agents [2] . In the last decades, many investigations have reported that the appendageal pathway should be taken into consideration as an efficient route for drug permeation [3] . The appendageal route can be defined as the transport path via the hair follicles, the sebaceous glands, and the sweat
10.4155/TDE.14.61 © 2014 Future Science Ltd
ducts. As compared with the reservoir of the SC with a thickness of 10–20 μm, the reservoir of the hair follicles extends deep into the skin tissues up to 2000 μm [4] . The infundibulum of the follicles increases the surface area and disrupts the epidermal barrier toward the lower parts of the follicles, thus serving as an efficient reservoir for drug permeation. Interest in appendages is directed toward their employment as reservoirs for localized delivery to the skin and also as a transport track for systemic drug delivery. The sebaceous glands open into the lower infundibulum of the hair follicles. Each follicle is associated with a sebaceous gland that releases sebum, creating a lipid-enriched environment. Efficient drug transport into the hair follicles depends upon the interaction between the drug and the sebum, as well as the choice of drug vehicles [5] . A number of reports assess the follicular deposition of drugs from different vehicles, especially the formulations of nanoparticles [6] . Nanotechnology is at the leading edge of rapidly developing new therapeutic and diagnostic concepts in all areas of medicine [7] . Nanoparticles are increasingly used in different applications,
Ther. Deliv. (2014) 5(9), 991–1006
Chia-Lang Fang1,2, Ibrahim A Aljuffali3, Yi-Ching Li4,5 & Jia-You Fang*,4,6,7 Department of Pathology, College of Medicine, Taipei Medical University, Taipei, Taiwan 2 Department Pathology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan 3 Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia 4 Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan 5 Chronic Diseases & Health Promotion Research Center, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan 6 Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwan 7 Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan *Author for correspondence: Tel.: +886 883 211 8800 Fax: +886 883 211 8236 [email protected] 1
part of
ISSN 2041-5990
991
Review Fang, Aljuffali, Li & Fang including bioimaging, diagnostic technology, and drug/gene delivery [8] . Nanoparticulate systems not only enhance skin absorption but can allow for drug targeting to the skin and/or its substructures [9] . The bioavailability of drugs permeating into the skin can be enhanced by using nanocarriers because the small particulate size ensures close contact to the SC [10] . Nanosized particles can make close contact with superficial junctions of the SC and the furrows between the corneocyte islands, allowing superficial spreading of the active agents. Following the evaporation of water from the nanosystems applied to the skin surface, particles form an adhesive layer occluding the skin. Hydration of the SC thus increases to reduce corneocyte packing and widen the inter-corneocyte gaps, subsequently enhancing drug transport. Figure 1 depicts the possible mechanisms involved in skin permeation enhancement by nanostructured lipid carriers (NLCs). One of the characteristics making nanoparticles interesting for topical application is their tendency to diffuse and accumulate in the hair follicles. Nanoparticles possess the potential for delivering drugs via the follicles [11,12] . The particles can aggregate in the follicular opening and penetrate along the follicular duct when administered onto the skin surface. It is beneficial to treat some dermatological diseases involved in the appendages. Moreover, the nanocarriers can deliver the active
ingredients deep into the skin and into the systemic circulation for therapeutic aims. In this review article, we would comprehensively introduce the nanoparticles that demonstrate the capability of follicular targeting for assisting drug permeation via the skin in the past ten years. This approach is successfully used in a variety of nanoparticulate formulations, such as polymeric, metallic and lipid nanoparticles. The mechanisms of follicular targeting by nanoparticles are also elucidated in this review. The structure & physiology of hair follicles The follicular orifices occupy about 0.1% of the total skin surface area [13] . For the regions in the scalp and the face, the combined areas of the follicular openings can be as much as 10% of the total area [14] . According to the report by Otberg [15] , the mean density is highest on the forehead (292 follicles/cm2), with this density being significantly higher compared with the back (29), thorax (22), forearm (18) and thigh (17). The follicles exhibit great variation in diameter in different body areas. The smallest diameter was found in the forehead (~66 μm) and forearm (~78 μm). The calf shows the largest follicular opening. Hair follicles are the pockets of the epidermis that extend through most or all of the skin depth and enclose a small papilla of dermis at their base (Figure 2) . The hair bulbs lie at the
Water evaporation Nanoparticles
Hair shaft
Nanoparticles
Film occlusion Nanoparticles
Drug Water SC
SC
SC
Sebaceous gland Viable skin
Viable skin
Close contact to skin
Skin hydration by particle occlusion
Viable skin
Hair follicle
Entrance into follicles and sebaceous glands
Figure 1. Possible mechanisms for skin permeation enhancement of drugs or active ingredients from nanoparticles. SC: Stratum corneum.
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Follicular delivery & targeting Nanoparticles can be a permeant for active targeting of hair follicles. The follicles are surrounded by networks of blood vessels, dendritic cells, and stem cells. They are all ideal targets for drug delivery [21] . The follicular route is available for systemic absorption via the blood capillaries near the bulge region. It has been shown that caffeine and minoxidil can be rapidly detected in the plasma after topical administration through the follicular pathway [22,23] . Other cell populations or structures such as Langerhans cells, matrix cells, and melanocytes demonstrate possible targets for drugs and nanocarriers. The follicles can be categorized into four areas for drug and nanoparticulate delivery, including the sebaceous duct, the bulge region, the hair matrix, and the follicular infundibulum [14] . Among these, the epithelium of the uppermost parts displays no difference from the interfollicular epidermis. On the other hand, the epithelium in the vellus and infundibulum is thinner than the interfollicular epidermis. The corneocytes in this region are smaller and not completely differentiated [24] . This part of the follicular structure with reduced barrier function provides a fast and facile approach for topically applied substances (Figure 3) . In addition, follicles can be an efficient reservoir for long-
future science group
Dermis Subcutaneous fat
base of the follicles, which are structures of actively growing cells that eventually produce the long, fine cylinder of a hair. One or more sebaceous glands attach to the follicles. The hair shafts are enveloped in an inner root sheath that consists of keratin-rich cells. The outer root sheath that surrounds the follicles is a stratified epithelium continuous with the epidermis [16] . The superficial area of follicular infundibulum is lined by the epidermis such as a well-developed SC and stratum granulosum. The follicular duct consists of sebum, a complex lipid mixture produced in the sebaceous glands. Sebum is highly complex and comprises triacylglycerols, diacylglycerols and free fatty acids. These components account for 50% –60% of sebum [17] . Wax ester (25%), squalene (13%), and cholesterol (2%) are other principal components of sebum [18] . Hair follicles undergo a growth cycle comprising three stages: anagen (growth phase), catagen (involution) and telogen (resting phase before hair shedding). Follicular stem cells and the strict interaction between epithelial and mesenchymal cells are essential to maintaining and regulating the hair cycle [19] . About 85% of the follicles in the scalp are associated with the anagen stage. The rate of scalp hair shaft elongation is typically 0.3–0.4 mm per day. The anagen phase of the scalp can last 2–6 years as the follicles go through extensive alterations in immune and gene expression status and vascular supply [20] .
Epidermis
Delivery & targeting of nanoparticles into hair follicles
Review
Sebaceous gland Bulge region Outer root sheath Inner root sheath Hair fiber Dermal papilla
Figure 2. The histological structure of hair follicles.
term storage in comparison to the SC, creating a possibility of sustained drug delivery. Based on the investigation by Schaefer–Korting et al. [25] , the nanoparticles can persist in the reservoir of the hair follicles ten times longer than the reservoir of the SC. Another advantage of follicular delivery is the capability of penetrating into deeper skin strata since the follicular infundibulum is located in the dermal layer [26] . Follicles are generally active in sebum secretion and hair growth. Some 30% of follicles are still inactive and closed due to dry sebum [5] . The permeant usually diffuses into active follicles but not inactive follicles due to the blockage of the transport pathway by dry sebum and cell debris [27] . Some nanoparticles can open up the closed follicles for efficient follicular transport (Figure 3) . It has been shown that nanoparticles preferentially permeate into the follicles but not the SC, enabling high accumulation within the follicular reservoir. Once deposited in the follicles, the drug may release from the nanoparticles and exert its effects on the target cells or structures [28] . Additional force such as massage can increase follicular penetration of nanoparticles [29] . Massage induces hair shaft movement, which acts as a geared pump pushing the particles deep into the follicles (Figure 2) . An opposite mechanism for follicular delivery is the hair growth and sebum flow, which may slow down the passage of nanoparticles because of the upward flow compared with the downward delivery of follicular permeation.
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Massage increases particle entry
Nanoparticles open inactive follicles (crump site)
Drug release from nanopartciles
Hair growth/sebum flow retards particle penetration
Figure 3. Follicular delivery and the related mechanisms of nanoparticles.
Dermatological diseases treated via follicular delivery Topical delivery of drugs or nanoparticles that selectively target hair follicles are of interest in the treatment of some skin disorders, especially the diseases related to the appendages. Possible application includes therapy for hair growth disorders and inflammation. Hair loss is one of the disorders that can be treated by follicular targeting of drugs and nanomedicines. Alopecia is known as hair loss on the scalp due to an androgenetic process (androgenetic alopecia) or an inflammatory process (alopecia areata). The prevalence of androgenetic alopecia in Caucasian men is 96% [30] while alopecia areata affects 2.1% of the population [31] . Clearly, hair loss affects a large number of the human population. Although the condition is never mortal, it causes deep social implications for the affected individual because of the significant change to the patient’s appearance. Androgenetic alopecia occurs due to a shortening of the anagen phase with the consequence of increased hair loss and the transformation of terminal to vellus follicles. Contrary to this phenomenon, a prolonged anagen stage with conversion of vellus follicles into terminal follicles can be observed during hirsutism and hypertrichosis [32] . The follicular absorption strategy may also be beneficial to treat these
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abnormalities. Follicular targeting can deliver the topically applied substances to the sebaceous glands, providing a promising approach for topical therapy of sebaceous gland dysfunctions such as acne, seborrhoeic eczema, and rosacea. Among these, acne is the most popular disease model for development of nanoparticulate formulations containing active ingredients such as retinoic acid and its derivatives [33–35] . Almost >80% of people suffer at some point during their lives from acne vulgaris, the most frequent disorder of the human skin [36] . Acne is a multifactorial disease with various symptoms such as comedones, nodules, papules, cysts, and pilosebaceous inflammation. Recent studies have shown that a variety of nanoparticulate types can target the hair follicles for successful acne therapy [37] . These include polymeric nanoparticles, solid lipid nanoparticles (SLN), and liposomes. Follicles are also a location of Staphylococcus aureus colonization. Diseases related to S. aureus skin infection include impetigo, furuncles, folliculitis, sycosis, and subcutaneous abscesses via the production of exfoliative toxins [38] . It may be possible to treat the infections of S. aureus by follicular delivery of nanoparticles although related investigation is currently lacking. Nanoparticulate delivery into hair follicles Polymeric nanoparticles
Different types of nanoparticles show a potential application in follicular absorption. Among these, the polymeric nanoparticles have gained the most attention for follicular delivery. The polymer materials such as polystyrene, polyvinylalcohol, polylactic acid (PLA), polyglycolic acid, poly(lactic-co-glycolic) acid (PLGA), polyethyleneimine, and cellulose are utilized for the preparation of polymeric nanoparticles with the aim of delivery into the follicles. Polystyrene nanosystems are the first nanoparticles introduced for follicular targeting. Alvarez–Román et al. [39] had used carboxylate-modified polystyrene nanoparticles with an average diameter of 20 and 200 nm for examining skin penetration and distribution. In this study, confocal laser scanning microscopy (CLSM) was employed to observe the distribution of fluorescein isothiocyanate (FITC)-loaded polymeric nanoparticles in porcine ear skin. CLSM is a noninvasive technique commonly used to visualize skin samples at multiple depths parallel to the sample surface (horizontal sections) [40] . The image is achieved at a high resolution with depth selectivity compared with conventional microscopy. The imaging quality is significantly enhanced due to the unique sectioning method of the laser. In addition, the information from the out-of-focus field is not superimposed on the image in focus. The results of CLSM showed that polystyrene nanoparticles preferentially
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Delivery & targeting of nanoparticles into hair follicles
accumulated in the openings of the follicles. The FITC signal was also observed in the vicinity of the follicles and hair shafts. Wu et al. [41,42] have developed polystyrene nanoparticles with Nile red as the dye for observing in vitro skin uptake by confocal microscopy. Poly-2-hydroxyl methacrylate (HEMA) was associated with polystyrene to modulate the hydrophobicity degree of the nanoparticles. The addition of HEMA decreased the particulate hydrophobicity. The nanoparticulate diameter was below 100 nm (37–74 nm), with the nanoparticles containing a higher percentage of HEMA showing the smaller size. The uptake of nanoparticles in full-thickness porcine skin was assessed after a 6 h application. At the skin surface, Nile red remained partly associated with the nanoparticles, but also escaped from the nanoparticles and permeated into deeper SC layers. Nile red uptake in the skin declined as the percentage of HEMA increased. The CLSM image showed that the nanoparticles could not diffuse beyond the SC, but did reveal an affinity for follicular openings. Fluoresbrite® is a fluorescent polystyrene latex sphere with a particulate size of 50 nm. This nanosystem was utilized to examine the possibility of skin absorption [43] . Both hairless rat skin and porcine ear skin were used as the skin barrier models. In this case, the polymeric nanoparticles did not permeate into intact skin. The nanoparticles only permeated through the skin after needle puncture (i.e., through damaged skin). The fluorescence of Fluoresbrite® was shown around the micropores produced by the needles. The penetration and storage of 5-fluoresceinamincontaining polymer nanoparticles into the hair follicles was analyzed by CLSM [44] . The polymeric nanocarriers were prepared by polyvinylalcohol and Resomer®, a biodegradable polymer composed of PLA and polyglycolic acid. The mean diameter of this nanosystem was 320 nm. In the in vitro porcine skin experiment, nanoparticles transported much deeper into the follicles compared with the free dye solution when a massage was applied. It is assumed that the hair movement may act as a pumping force, pushing the nanoparticles into the follicles. The storage of polymeric nanoparticles in human skin in vivo was also monitored by a differential stripping technique. It was found that the nanoparticles were stored in the follicles up to 10 days, whereas the free dye could be detected for only 4 days. This suggests a superior accumulation of nanoparticles in the follicles for long-term use. Rancan et al. [45] investigated the feasibility of biodegradable PLA nanoparticles as carriers for topical delivery. Two formulations, each with a different particulate size (228 and 365 nm), were prepared for human skin uptake examination by fluorescence microscopy, CLSM, and flow cytometry.
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PLA nanoparticles were absorbed by 50% of the vellus hair follicles, reaching a maximal depth corresponding to the entry of the sebaceous glands in 12% –15% of all observed follicles. There was no significant difference of follicular uptake between the nanodispersions with different diameters. PLA nanoparticles could rapidly release the dye when coming in contact with a lipophilic environment. The release process was triggered by nanoparticulate destabilization. The retention of nanoparticles in the sebaceous glands could last up to 24 h. The size of the nanoparticles may play an important role in the penetration depth of the follicles. Patzelt et al. [46] explored the effect of the particulate size of PLGA nanoparticles on the follicular permeation depth of porcine ear skin in vitro. PLGA nanosystems were prepared in a hydrogel form with the different sizes of 122, 230, 300, 470, 643, and 860 nm. The results of CLSM demonstrated that the increased particulate size led to a significantly deeper penetration into the follicles. The deepest transport was obtained by the nanoparticles with a diameter of 643 nm. The penetration depth was significantly decreased for larger particles (860 nm). This trend can be explained by the thickness of keratinized cell layers. The surface structure of the hair shafts and follicles, determined by the thickness of the keratin cells (~530 nm for human hair), may act as a pumping system that pushes the nanoparticles into the follicles when the hairs are moving [47] . This movement can be achieved by massage. The particles in the range of the thickness of the keratin cells would penetrate most deeply. Morgen et al. [48] assessed the follicular targeting of polymeric nanoparticles loaded with the lipophilic drug UK-157,147. This compound is a potassium channel opener, developed by Pfizer for treating alopecia. The predominant material of the polymeric nanosystems was ethyl cellulose. The average size of the nanoparticles was 96 nm with a polydispersity index of 0.18. As the images of fluorescence microscopy showed, the distribution of the nanoparticles was limited to rabbit ear skin surface and hair follicles during a 2 h application. The effect of UK-157,147-loaded nanoparticles on hair growth of the C3H mouse was tested. Fifty percent of the mice treated with polymeric nanocarriers with 1% drug displayed visible hair growth on the dorsal region at day 25. At the end of the experiment (day 35), hair growth was observed in 70% of the mice treated with nanosystems. No observable skin irritation was detected during the experiment. Retinyl acetate (RA) has been recognized as a potential drug for acne therapy and dermatological inflammation [49] . RA was loaded into polymeric nanoparticles synthesized by ethyl cellulose and poly(ethylene
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Review Fang, Aljuffali, Li & Fang glycol) - 4-methox ycinnamoylp htha loylchitosan for testing in vitro skin absorption [50] . For the RA control solution, no drug was found on the mouse skin surface after washing at 2 h post-application. For RA-loaded nanoparticles, 77.7% of the applied RA dose was detected on the skin surface. Confocal microscopy had shown the entry and accumulation of nanoparticles and RA in the hair follicles. RA release from nanoparticles was confirmed through detection of an increasingly higher RA/nanoparticles fluorescence intensity ratio deeper into the dermis and away from the follicles. Abdel–Mottaleb et al. [51] reported the behavior of ethyl cellulose nanoparticles for selective betamethasone delivery to the inflammatory skin sites. Nanoparticles with different diameters of 50, 100, 500, and 1000 nm were applied to a mouse ear with dithranol-induced dermatitis. According to CLSM, by using Nile red as the fluorescent marker, the particles could transport deeply and selectively into the follicles, around the hair shafts, and deposit in the associated sebaceous glands. The smaller nanoparticles (50 and 100 nm) showed a threefold stronger and deeper permeation into the hair follicles. The measurement of the inflammatory biomarker, myeloperoxidase activity, in mouse skin had confirmed the greater deposition of smaller nanoparticles in the skin. The myeloperoxidase activity of the nontreated control, 1000-nm particles, and 100-nm particles was 1.2, 1.0, and 0.5 U/mg, respectively. Not only nano-sized particles can pass into hair follicles, but micrometer-sized particles (>1 μm) can also achieve this goal. Sumian et al. [52] had used porous nylon microspheres with a diameter of 5 μm to examine the possible passage into the follicular duct. The microspheres were dispersed in two different silicones, fluid silicone and volatile silicone, to avoid alteration of nylon and dye extraction from the particles. This study was conducted in vivo in hairless rats. The penetration depth of rhodamine-6G in the microspheres was monitored by fluorescence microscopy. After administration with massage for 1 minute, dye diffusion was detected by applying ethanol on the skin to extract rhidamine-6G from the microspheres. The results demonstrated the occurrence of follicular targeting. Both the dye diffusion out of the microspheres and dye penetration depth into the follicles were considerably increased using the fluid silicone vehicle. Toll et al. [53] evaluated the influence of polystyrene microsphere size (0.75 to 6 μm) on the efficiency of follicular targeting. Full-thickness skin samples were obtained from the scalp, axillar, or pubic regions of the volunteers after surgical excision. A selective permeation of the microspheres into the follicles was shown. The optimal size for follicular delivery proved to be 1.5 μm, with a 55%
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rate of all follicles and a maximal penetration depth of >2300 μm. Needle-free vaccination is desirable for ease of application, improved patient compliance, and less risk of infection. Transcutaneous immunization provides a noninvasive and sustained release approach for vaccination. It is difficult for vaccines with a large molecular size to penetrate across the SC. Nanocarriers can be designed in a way to be taken up by hair follicles for successful vaccination [54] . Nanoparticles can be an ideal carrier for vaccines to penetrate deeper into the follicles than solution, increase the stability of antigens, and modulate immune responses [55] . Mittal et al. [56] investigated the capability of transfollicular delivery of polymeric nanoparticles using ovalbumin as a model antigen. The nanocarriers were produced by PLGA or chitosan-coated PLGA using polyvinylalcohol as a stabilizer. The average size of PLGA and chitosan-coated PLGA nanoparticles was 170 and 180 nm, respectively, with a polydispersity index of
