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Topical and cutaneous delivery using nanosystems

MS Roberts, Y Mohammed, M Pastore, S Namjoshi, S Yousef, A Ali-naghi, IN Haridass, E Abd, VR Leite-Silva, HAE Benson, JE Grice

PII: S0168-3659(16)30614-9DOI: doi: 10.1016/j.jconrel.2016.12.022Reference: COREL 8578

To appear in: Journal of Controlled Release

Received date: 27 August 2016Accepted date: 20 December 2016

Please cite this article as: M.S. Roberts, Y. Mohammed, M. Pastore, S. Namjoshi, S.Yousef, A. Alinaghi, I.N. Haridass, E. Abd, V.R. Leite-Silva, H.A.E. Benson, J.E. Grice,Topical and cutaneous delivery using nanosystems, Journal of Controlled Release (2016),doi: 10.1016/j.jconrel.2016.12.022

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Topical and cutaneous delivery using nanosystems

MS Robertsa,b*

, Y Mohammeda, M Pastore

b, S Namjoshi

a, S Yousef

a,1, A Alinaghi

b, IN

Haridassa,c

, E Abda, VR Leite-Silva

a,2, HAE Benson

c, JE Grice

a.

aTherapeutics Research Centre, School of Medicine, The University of Queensland,

Translational Research Institute, QLD, Australia 4102

bSchool of Pharmacy and Medical Sciences, University of South Australia, Adelaide, Australia

cSchool of Pharmacy, Curtin Health Innovation Research Institute, Curtin University, GPO Box U1987,

Perth, WA, Australia

Present addresses: 1School of Pharmacy, Helwan University, Helwan, Egypt; 2Instituto de Ciências

Ambientais Químicas e Farmacêuticas, Universidade Federal de São Paulo, Diadema SP, Brazil

*Corresponding author:

Prof Michael S. Roberts,

Therapeutics Research Centre,

The University of Queensland School of Medicine – Translational Research Institute,

37 Kent St, Woolloongabba QLD 4102 Australia

Telephone: 61-7-34438031 Fax: 61-7-34437779

Email: m.roberts@uq.edu.au

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Abstract

The goal of topical and cutaneous delivery is to deliver therapeutic and other substances to a

desired target site in the skin at appropriate doses to achieve a safe and efficacious outcome.

Normally, however, when the stratum corneum is intact and the skin barrier is

uncompromised, this is limited to molecules that are relatively lipophilic, small and

uncharged, thereby excluding many potentially useful therapeutic peptides, proteins,

vaccines, gene fragments or drug-carrying particles. In this review we will describe how

nanosystems are being increasingly exploited for topical and cutaneous delivery, particularly

for these previously difficult substances. This is also being driven by the development of

novel technologies, which include minimally invasive delivery systems and more precise

fabrication techniques. While there is a vast array of nanosystems under development and

many undergoing advanced clinical trials, relatively few have achieved full translation to

clinical practice. This slow uptake may be due, in part, to the need for a rigorous

demonstration of safety in these new nanotechnologies. Some of the safety aspects

associated with nanosystems will be considered in this review.

Keywords: topical delivery, cutaneous delivery, nanosystems, nanoparticles, colloidal

nanocarrier systems

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1. Introduction

There has been great interest in nanosystem facilitated delivery of therapeutic, diagnostic,

prophylactic or cosmetic substances into the skin, in which the goal is to overcome the skin

barrier in a controlled manner to reach targets in various skin layers, including the stratum

corneum, as well as deeper tissues, or systemically. On the other hand, nanomaterials are

also applied directly to the skin, for example, when titanium or zinc oxide are used as

sunscreens. Here, the intention is not to penetrate the stratum corneum, but to deliver the

nanomaterials so that they remain on the skin surface. In this review, we will examine some

of the various nanosystems currently in use, with particular reference to their ability to

penetrate the skin barrier. Table 1 summarises the scope of the field of topical and cutaneous

delivery using nanosystems and Table 2 gives a more detailed summary of the properties of

some of the nanodelivery systems to be discussed later in this review. The range of

particulate nanosystems under investigation is illustrated in Figure 1. Some of the

applications of topically applied nanodelivery systems for treatment of skin disorders are

listed in Table 3.

2. The stratum corneum barrier problem

The skin plays a major role in protecting the body from the surrounding environment by

providing a highly efficient barrier to the penetration of exogenous molecules and micro-

organisms, and maintaining homeostasis by preventing excessive loss of water [38]. A

simplified representation of human skin containing its major structures and cell types is

illustrated in Figure 2. Although the location of the barrier within the skin was long debated,

Scheuplein showed conclusively in 1966 that it largely resided in the stratum corneum [39],

due to its unique structure, most simply described as a series of layers of flattened

corneocytes surrounded by a lipid envelope. The most likely route taken by a substance in

penetrating the stratum corneum is via a tortuous pathway through the lipids surrounding the

corneocytes (intercellular route) [40], although the transcellular route though the corneocytes

may be viable under certain circumstances [41]. The stratum corneum is also traversed by

pilosebaceous units (hair follicles and associated sebaceous glands) and sweat glands. While

the average follicular orifice area on the human skin surface is only about 0.1% of the total

surface area [42], under some circumstances these appendages may be potential routes of

entry into the skin [43], as we shall discuss later.

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The fundamental question facing those who seek to develop formulations for topical or

transdermal delivery is; how can a particular active be made to overcome the formidable

stratum corneum barrier,

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so that it can be delivered to the site of action in sufficient quantities to achieve a desired

therapeutic effect, with the least possible adverse effects? The range of substances that are

suitable for diffusion through the stratum corneum is generally limited to those that are

relatively small (< ~500 Da), lipophilic (log 1 – 3) and water soluble [44]. Given the

generally low permeation rates for most molecules, they must also be highly potent [45].

Clearly, there is a vast array of attractive prospects for topical application such as peptides,

proteins, vaccines, nucleotide fragments or powerful cancer therapeutics that do not fit these

criteria. This dilemma has been a major factor in the interest in nanodelivery systems that

could expand the range of active molecules that are available for therapeutic or other

purposes.

3. Solid nanoparticles

In the early 1900s, Ehrlich introduced the concept of drug targeting using a delivery system

he called „Zauberkugeln‟ (Magic Bullets) [46]. This was put into practice in the 1950s and

1960s with the development of miniaturised delivery systems, focusing initially on

polyacrylic beads for oral delivery, with the first nanoparticles for vaccination purposes

appearing in the late 1960s [47]. By 1978, the field was sufficiently advanced for the first

review article on nanoparticles as new drug delivery systems to be published [48]. In 1994,

nanoparticles were defined for pharmaceutical purposes in the Encyclopaedia of

Pharmaceutical Technology as “solid colloidal particles ranging in size from 1 to 1000 nm.

They consist of macromolecular materials and can be used therapeutically as drug carriers, in

which the active principle (drug or biologically active material) is dissolved, entrapped, or

encapsulated, or to which the active principle is adsorbed or attached” [49]. Later

developments saw the use of nanoparticles for intravenous cancer therapy, with Brasseur et

al. reporting an enhanced efficacy of actinomycin D absorbed on polymethylcyanoacrylate

nanoparticles against an experimental subcutaneous sarcoma [50], taking advantage of their

enhanced permeability and retention effects [51]. The first commercial nanoparticle product

containing a drug appeared on the US market in 2005. This contained an injectable

suspension of human serum albumin nanoparticles containing paclitaxel (Abraxane®, Abraxis

BioScience, now Celgene Corporation, Los Angeles, CA, USA) and was indicated for the

treatment of metastatic breast cancer [52].

The first reported topical applications of nanoparticles were to the eye, when Li et al. treated

albino rabbits with polymeric nanoparticles encapsulating progesterone in 1986 [53].

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However, it was not until the end of the 20th

century that reports on the application of

nanoparticles to skin for vaccination [54] and vitamin A delivery [55] appeared in the

literature. In the short time since, the topical application of a range of nanomaterials has been

investigated. Of interest in this review are such materials as quantum dots, metal particles

(gold, silver and beryllium), metal oxides (TiO2, ZnO, carbon nanotubes and organic

fullerene derivatives [1, 56-64]. These are relevant because, as we have already seen,

nanosystems are not only potential drug delivery vectors, but some have also been suspected

of causing harmful effects on the skin. For instance, it has been proposed that surface-coated

quantum dots could be used to carry drugs into the skin [65]. Similarly, drug-coated particles

could be targeted to appendages such as hair follicles, where their payloads could be released

slowly into local sites or into the systemic circulation [66]. Conversely, the unwanted

penetration of nanoparticles through occupational or environmental exposure [64], or through

absorption of nano sunscreen substances could have toxic consequences [67].

3.1. Factors affecting skin penetration of solid nanoparticles

The properties of nanoparticles that could influence penetration of nanoparticles through the

stratum corneum, as well as potential deposition in furrows, appendages and deeper skin

layers include size (both in the native state and in conjunction with associated water or other

solvent molecules), shape, charge and surface properties such as coatings or functional

groups, as well as their aggregation state [68]. In addition, the vehicle in which the particles

are suspended can have an influence by altering the properties of the nanoparticles

themselves [69] or by affecting the permeability of the stratum corneum [58]. Furthermore,

the condition of the skin, including the presence of an altered or impaired barrier due to

specific skin diseases may affect the degree and depth of nanoparticle penetration [70, 71]. A

summary of the factors influencing the skin penetration of nanoparticles, as well as the

potential routes of delivery is illustrated in Figure 3.

In evaluating the large volume of literature on nanoparticle skin penetration, it is important to

be aware of the experimental conditions under which the studies were carried out. This is

highlighted in Figure 4 which shows the wide variation in penetration of various gold based

nanoparticles through excised skin from rats [72], pigs [73] and humans [58, 74, 75]. Figure

4 shows that rat and pig skin was highly permeable to even large (~200 nm) particles,

whereas the 161 nm gold/silica particles applied to human skin remained on the surface or

penetrated into follicles [74]. Moreover, in the studies by Labouta et al, [58, 75], human skin

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penetration was size and vehicle-dependent. The 6 nm particles penetrated readily from

aqueous buffer or toluene, as expected for such a small diameter [68, 70]. The 15 nm

particles applied in aqueous buffer, accumulated in the skin furrows, but in toluene, which

could potentially damage the skin barrier, they were detected in the lower epidermis.

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3.1.1. Quantum dots (QDs)

These are small spherical or ellipsoidal particles, generally less than 20 nm on the major axis,

with a metal core, an outer shell and various coatings or functional groups attached to the

surface. Penetration of QDs has been studied in ex vivo rat, weanling pig and human skin. In

contrast to the previous suggestions [65, 76], little compelling evidence of quantum dot

penetration in intact skin has been found to date. In 2006, Ryman-Rasmussen [63] used a

range of QDs with different shapes and surface charges, applied to dermatomed weanling pig

skin in buffers at pH 8.3 or 9.0. The QDs were found to be localized within the viable

epidermis by 8 or 24 hrs, but none penetrated into the receptor of the flow-through cells used.

The authors concluded that significant penetration through the stratum corneum and into the

viable tissue was possible with these small particles. Later, Prow et al [61] repeated some of

Ryman-Rasmussen‟s key experiments using excised human skin in order to examine the

possibility that the penetration seen previously [63] may have been influenced by an

immature barrier in weanling pig skin. The results, shown in Figure 5, indicate that at neutral

pH none of the QDs penetrated beyond the stratum corneum and remained accumulated in the

skin furrows. On the other hand, they did observe some penetration of the neutral PEG-

coated particles to the viable epidermis at pH 8.3 [61] and suggested that the elevated pH

may indeed cause a weakening of the stratum corneum barrier, in support of Ryman-

Rasmussen‟s findings [63] and others [77]. When 30 tape strips were applied to intact skin to

physically disrupt the barrier, all QDs penetrated as far as the upper dermis by 24 hrs [61].

Zhang et al. [78] performed similar studies in rat skin using the same anionic QDs used by

Ryman-Rasmussen, as well as with different QDs (nail shaped, PEG coated) in dermatomed

weanling pig skin [79]. Interestingly, they saw no penetration of these QDs in intact rat or pig

skin, nor did they detect penetration in flexed or tape stripped rat skin. There was, however,

minimal QD penetration into abraded rat skin at 8 or 24 hrs. Taken together, these findings

support the notion that while QDs may be able to penetrate a weakened or disrupted stratum

corneum barrier to some extent, they are unlikely to penetrate intact human skin under

normal conditions.

3.1.2. Effects of a stressed or compromised skin barrier

We have already seen that even under quite severe treatment such as tape stripping, the skin

barrier is able to limit the penetration of small nanoparticles like QDs. In everyday life

however, human skin is subject to a range of insults and stresses. For example, Tinkle et al

[64] have argued that if skin is exposed to particulate material during repetitive movement or

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mechanical stress, such as might occur in an industrial situation, or regularly walking

barefoot, penetration of those particles may be enhanced. This led them to suggest that the

skin was a route of exposure in industrial chronic beryllium disease, while further support

came from observations of elephantiasis in individuals in certain regions in Africa, associated

with penetration of clay particles through bare skin of their feet [80]. To test this, they

investigated the effect of mechanical flexing on particle penetration in excised human skin

and showed that 30 – 60 minutes of mechanical flexing led to the penetration of 0.5 and 1.0

m FITC-dextran particles as far as the viable epidermis and occasionally to the dermis, as

shown in Figure 6.

While these fluorospheres are far larger than the nanoparticles discussed above, Rouse et al

found that very small fullerene-substituted nanoparticles (about 3.5 nm) also penetrated into

the dermis of dermatomed pig skin by 8 hrs after 60 min mechanical flexing [62]. This

contrasts with the lack of penetration of the somewhat larger QDs through flexed rat skin

noted above [78].

3.1.3. Zinc oxide (ZnO) skin penetration

Nano-sized zinc oxide particles are commonly used in sunscreens to protect against the

effects of UV radiation and are also subject to the same mechanical and repetitive stresses

that are imposed upon skin during normal activities, as discussed above. This prompted

concern about whether the nanoparticles could penetrate the skin barrier and access the viable

epidermal cells [81-83], particularly in view of in vitro evidence suggesting that ZnO

nanoparticles are internalized by cultured human keratinocytes and cause cytotoxic and

genotoxic responses [84].

In an early in vitro study by Cross et al. in 2007 [56], sunscreen formulations containing

micronized ZnO nanoparticles (15-40 nm) were applied to human epidermal membranes in

Franz cells for 24 hours. Electron microscopy showed that the nanoparticles were confined to

the surface and loose outer layers of the stratum corneum, with none detected in the lower

stratum corneum or viable epidermis. Surprisingly however, they found evidence to suggest

Zn penetration though the membrane, with a trend towards increased Zn levels in the receptor

medium, detected by ICP-MS, although the differences were not significant. In an analogous

in vivo study, Gulson et al. [57] also reported penetration of Zn through human skin.

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Sunscreens containing ZnO with >99% of the stable isotope 68

Zn, to distinguish from

naturally occurring Zn, were applied to human volunteers under in use beach conditions over

five consecutive days and small but significant amounts of 68

Zn were detected in blood and

urine of volunteers by ICP-MS. Neither of these studies therefore provided direct evidence

of intact ZnO penetration beyond the outer skin layers and it is likely that the Zn detected by

ICP-MS was ionic Zn2+

ions that penetrated following hydrolysis of ZnO on the skin surface.

In support of the hydrolysis mechanism, the skin surface is known to be relatively acidic,

particularly with moderate sweating as might be experienced in association with sunscreen

use outdoors [85] and significant amounts of Zn2+

have been shown to be released from 4 nm

ZnO nanoparticles at pH 1, 3 and 6 [86]. More direct evidence of increased labile Zn2+

penetration was recently reported by Holmes et al [87], after application of ZnO nanoparticles

suspended in oil (capric caprylic triglycerides, or CCT) or water (pH 6 and 9). Zn2+

was

detected in the stratum corneum and viable epidermis by imaging the fluorescence due to the

complex formed between Zn2+

and the labile zinc selective probe Zinpyr-1, with the highest

levels occurring with the pH 6 application.

Notwithstanding the in vivo findings above [57], it is generally accepted that passive

penetration of ZnO nanoparticles into intact human skin is limited to the surface and upper

layers of the stratum corneum [56, 88]. However, the question remains whether penetration

can occur when the barrier is compromised, or when then skin is stressed or manipulated in

normal activities. We have reported findings with in vivo ZnO nanoparticle applications in

massage and flexing [59], occlusion of the skin surface [1], barrier disruption [1, 59, 89], and

skin conditions such as psoriasis and atopic dermatitis LL [89], where both nanoparticle

penetration and metabolic changes to endogenous fluorophores in skin (representing toxicity)

were assessed by multiphoton tomography with fluorescence lifetime imaging (MPT-FLIM).

The particles used in this work were characterised to be less than 200 nm in size and either

bare, or with a siloxane coating. In none of these studies was there any significant evidence

of penetration of nanoparticles into the viable epidermis or deeper. Unlike the evidence of

microparticle penetration in excised human skin shown by Tinkle et al. [64] seen in Figure 6,

MPT images of flexed in vivo human skin following application of bare (uncoated) and

siloxane coated ZnO nanoparticles [59] show that all ZnO was confined to the skin surface,

furrows and the upper layers of the stratum corneum (Figure 7).

Similar results were obtained with massage [59] (Figure 7) and occlusion of the skin [1]

(Figure 8) No evidence of epidermal cytotoxicity due to effects of ZnO on NAD(P)H and

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FAD fluorescence lifetimes was seen with any of these treatments [1, 59]. Similarly, Figure 9

shows that ZnO nanoparticles applied to psoriatic lesions in human patients also fail to

penetrate into the viable epidermis and remain largely accumulated in the furrows.

In other work examining nanoparticle penetration with a compromised skin barrier,

Monteiro-Riviere et al. applied ZnO and titanium dioxide (TiO2) products to the skin of UVB

sunburnt pigs in vivo and in vitro [60]. They observed minimal penetration of ZnO and TiO2,

only into the upper layers of the viable epidermis and found no evidence of systemic

absorption.

While this evidence about safety of ZnO nanoparticles is fairly convincing, particularly for

intact skin, it should be recognised that the results obtained for compromised skin in

relatively common conditions like psoriasis and atopic dermatitis, or in sunburn, are limited.

Further work in this area is warranted, but the enormous benefits of sunscreens in protecting

against skin cancer should not be overlooked when assessing the potential risks of

nanoparticle application to the skin. Relevant to this debate is a recent study by Ryu et al.

[90], who applied up to 1000 mg/kg of a suspension of 29 nm ZnO nanoparticles to the skin

of rats daily for 90 days. No evidence of toxicity was found, based on clinical examination,

haematology, blood and urine biochemistry and histological analysis of tissues. ZnO

accumulation in the liver, intestines and faeces was thought to be due to ingestion.

3.2. Follicular delivery of nanoparticles

In deliberate attempts to promote skin delivery, the Lademann group has used massage to

deliver nanoparticles of optimal sizes of 300 – 600 nm deep within the follicle. They showed

that although an intact and relatively impermeable stratum corneum similar to that of the

interfollicular epidermis covers the acroinfundibulum (the upper portion of the follicle as it

enters the epidermis), this barrier is interrupted in the lower follicular infundibulum. The

corneocytes in this area appear smaller and crumbly, suggesting that the skin barrier is

permeable and provides an opportunity for interactions between topically applied compounds

and the hair follicle epithelium [91]. Interestingly, this particle size matches the cuticula

thickness on human (530 nm) and porcine (320 nm) hair, so the suggestion is that hair

movement during massage allows the cuticulae to engage with the matching particles like a

geared system, driving them deep into the follicle [66, 92, 93]. The ability to target

nanoparticles to different depths in the follicle, depending on their size may allow treatment

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of different site-specific conditions. In addition, 320 nm particles have been shown to reside

in the follicle for up to 10 days, indicating that the follicle could act as a reservoir for long

term storage and release of drugs delivered in association with nanoparticles [94]. Trauer et

al. [95] also used massage and occlusion to enhance follicular penetration of liposomal

systems in ex vivo human skin. Massage was found to enhance the follicular penetration of

rigid and flexible liposomes significantly, while occlusion was favoured only for the rigid

liposomes.

Vogt et al. [96], also from the Lademann group in Berlin, showed that after cyanoacrylate

stripping to remove the contents of human vellus hair follicles followed by massage, 40 nm

particles could be targeted deep within the follicle and through the follicular epithelium, to be

internalized by Langerhans cells surrounding the follicles [97], whereas 750 and 1500 nm

particles remained in the infundibulum and were not internalized, as shown in Figure 10.

These workers suggested that the small nanoparticles could be an efficient delivery system

for vaccine delivery to cutaneous antigen-presenting cells illustrated in Figure 2. They

expanded these concepts in recent work [98], showing that larger 200 nm nanoparticles also

penetrated deeply with massage into human vellus and intermediate hair follicles after a

single cyanoacrylate treatment to remove follicular contents. Moreover, the cyanoacrylate

treatment was associated with clear signs of activation and migration of dendritic cells

indicated by retraction of dendrites, reduction of LC numbers in the epidermis with

concomitant increases in the dermis surrounding the follicles and expression of activation

markers. Their work suggests that follicular delivery combined with immune activation

would be a viable technique to promote uptake of topical antigens.

Other nanodelivery systems investigated for follicular delivery include lipid nanocarriers [95,

99-104], nanoemulsions [105-107] and polymeric vesicles [108-111]. The Hoffman group in

San Diego used a phosphatidylcholine-based liposome system to deliver proteins, genes,

melanin and small molecules selectively to the pilosebaceous unit in mice, with negligible

amounts detected in the epidermis, dermis or systemic circulation [101-104]. These

nanosystems will be discussed in more detail below, with regard to delivery through the

stratum corneum barrier.

4. Colloidal nanocarrier systems

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These have been increasingly exploited for dermatological, cosmetic and personal care

applications. Common colloidal nanosystems include the liquid emulsions (microemulsions

and nanoemulsions) (NE), solid lipid nanoparticles (SLN), nanostructured lipid carriers

(NLC) and advanced nanovesicles such as flexible and ultraflexible liposomes. These

formulations can offer increased efficacy and targeted delivery, improved bioavailability and

stability of the active, reduced irritancy, and facilitate the formulation of lipophilic, poorly

water-soluble compounds.

4.1. Emulsions

Microemulsions were first described scientifically by Hoar and Schulman in 1943 [112].

They noted that coarse macroemulsions stabilized with an ionic surfactant became

transparent on addition of a medium chain length alcohol, a co-surfactant. After originally

referring to these systems as hydrophilic oleomicelles or oleophilic hydromicelles, Schulman

et al. introduced the term „microemulsion‟ in 1959 [113]. It was not until the 1980s that the

potential use of microemulsions as topical formulations for skin delivery was explored [114,

115].

4.1.1. Nanoemulsions (NE) for skin delivery

Nanoemulsions are dispersions of oil and water phases stabilised by surfactants, with mean droplet

diameter in the submicron range [116, 117]. They can be formed by high-energy and low-energy

emulsification techniques [118] and typically contain a relatively low percentage of surfactant [119].

The small droplet size results in a kinetically stable system that resists destabilisation due to

gravitational separation, flocculation and coalescence [119], and is transparent or translucent. The

low surfactant content offers advantages in topical application with reduced irritancy to the skin.

The small droplet size provides a large surface area and uniform distribution on the skin, good

occlusiveness, film formation, aesthetic qualities and skin feel [120, 121]. NEs have wide application

in the pharmaceutical and cosmetic industries for indications including inflammation, anti-aging,

moisturisation and beauty [122]. They have particular utility in the management of chronic skin

diseases such as atopic dermatitis or dry skin, to provide skin protection and hydration, without

irritating or weakening the skin barrier [123]. NEs can enhance permeation into the skin by a

number of mechanisms. First, they provide a high solubilisation capacity for both lipophilic and

hydrophilic compounds, thereby increasing the loading capacity and dose application of the

formulation. Second their large surface area and good skin contact, coupled with their occlusive

nature ensures good surface contact with the stratum corneum surface. Third, their oil and

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surfactant components may have a direct permeation enhancement effect on the stratum corneum

lipid structure [Krielgard ref from main list]. A recent review provides a summary of the

dermatological applications of NEs [122].

We showed significantly enhanced flux of the hydrophilic caffeine and lipophilic naproxen across

human epidermis in vitro [34]. The NE oil phase was composed of the skin penetration enhancers

oleic acid or eucalyptol with the aim of investigating a synergistic effect of the increased surface area

and contact with the skin, enhanced solubility and direct chemical enhancement effects of the oil

excipients within the stratum corneum. There was only a modest increase in caffeine solubility

within the stratum corneum, therefore the increase in skin diffusivity that contributed to the

maximum flux enhancement resulted primarily from the direct effect of the NE excipients reducing

the diffusional resistance for caffeine penetration within the stratum corneum. In contrast,

increased flux of the lipophilic naproxen was primarily due to formulation-increased solubility of

naproxen in the stratum corneum, with only a modest increase in diffusivity.

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4.2. Lipid-based colloidal nanosystems

4.2.1. Solid lipid nanoparticles (SLN)

SLN were developed in parallel in Germany [124] and Italy [125], in the early 1990s. They

are composed of lipids, lipid-like materials or a mixture with a diameter from 10 nm to 10

µm, are solid at ambient temperature and able to incorporate active agents into their solid

core for various delivery applications (e.g. parenteral, enteral, pulmonary and topical)

resulting in a controlled release over a long period of time [126]. They are normally prepared

by high-pressure homogenization and offer the opportunity to formulate hydrophobic actives

such as vitamin A, vitamin E [127] and coenzyme Q10 in cosmetically elegant products,

rather than an ointment with poor aesthetic qualities and consumer acceptability. They can

also increase the stability of compounds such as retinol that are prone to decomposition in the

presence of light and oxygen [55, 128] and provide controlled delivery of actives to the skin.

For example Zhang et al [129] showed higher stratum corneum deposition but lower flux of

aconitine from SLN compared with a nanoemulsion emulsion of similar sized vesicles (70-90

nm). SLNs form a film on the skin surface that combines with the skin lipid film to reduce

water loss, increase skin hydration and help protect the barrier function of the stratum

corneum [130, 131]. Good skin adherence is particularly useful in sunscreen products where

wash off due to sweating and bathing can lead to unforeseen sunburn. Enhanced stratum

corneum permeation is attributed to multiple mechanisms: prolonged contact with the skin

surface; the occlusive nature of the SLN formulation hydrating the skin [8]; and interaction

between formulation lipids and stratum corneum lipids facilitating permeation of lipid soluble

compounds. chler et al [132] used scanning electron microscopy to show the loss of SLN

platelet structure 2h after administration on the surface of pig skin. Evidence of the

interaction between cetyl palmitate SLN formulation lipids and stratum corneum lipids were

demonstrated by shifts of phase transition temperature (differential scanning calorimetry) and

stretching bands (Fourier transform infrared spectroscopy) [133]. Zhang et al [134] observed

that this interaction was highly formulation dependant as prolonged and localized delivery of

betamethasone 17-valerate could be achieved with monostearin SLN but not with other lipids

such as beeswax. The cationic phospholipids 1,2-dioleoyl-3-trimethylammonium-propane

(DOTAP) and dioleoylphosphatidylethanolamine (DOPE) provide the potential for charge

interaction with the negatively charged skin surface. Highly positively charged (51 mV) SLN

using DOTAP, DOPE, Tween 20 as surfactants, tricaprin as a solid lipid core, and

encapsulating plasma DNA, showed enhanced in vitro permeation into mouse skin and

expression of mRNA in vivo in mice after topical application [135].

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4.2.2. Nanostructured lipid carriers (NLC)

The second generation of SLN, nanostructured lipid carriers (NLC) appeared in 1999 with the filing of

the US patent by the German company PharmaSol GmbH. These nanoparticles showed improved

drug loading and stability of long-term incorporation [136]. As with liposomes, the introduction of

NLC was into the cosmetic market, aided by lower regulatory hurdles, with the launch of Cutanova

Cream Nano Repair Q10 and Intensive Serum NanoRepair Q10 by Dr. Rimpler GmbH (Germany) in

October 2005 [137, 138].

NLC are composed of a fluid lipid phase embedded into a solid lipid matrix [126] or localized at the

surface of solid platelets and the surfactant layer [139]. The lipids spatial structure allows greater

drug loading and better stability compared to SLN [140]. For example the sunscreen loading capacity

of 70% in NLC compared to 10-15% in SLN, allows the development of suncare products with higher

sun protection factor (SPF) [141]. Chen et al [142] showed improved stability of Coenzyme Q10 in

NLCs compared to an emulsion or ethanol formulation (decrease of 5.59%, 24.61% and 49.74%

respectively). The accumulation of Co Q10 in rat epidermis from the optimised Q10-NLC formulation

(particle size 151.7 ± 2.31) was also 10 times greater than from the emulsion. NLC and SLN are

reported to have a similar mechanism of permeation enhancement, via occlusion and mixing

between the formulation and stratum corneum lipids, however the liquid lipid component in NLC

increases solubilization and thereby loading of the active, resulting in greater skin deposition.

Improved skin deposition has been demonstrated for a number of compounds including tacrolimus

[143, 144], psoralen [145], methotrexate [146], miconazole nitrate [147], fluocinolone acetonide

[148] and 5-amino levulinic acid [149].

Singh‟s group has investigated surface modification for enhancing delivery and targeting of

NLC to the skin [150-152]. NLC with the cell-penetrating peptide transactivating

transcriptional activator (TAT-NLC) showed greater epidermal deposition of a fluorescent

probe and the lipophilic drug celecoxib, compared to control NLC, that were located mainly

in the hair follicles following topical administration to rat skin in vitro [150, 151]. They

subsequently showed that surface modification of the NLC with a cell penetrating peptide

containing 11 arginines had significantly greater permeation enhancing ability compared to

other polyarginines and TAT peptides [152].

4.2.3. Flexible nanovesicles

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Flexible liposomes or vesicles are composed of materials that will aggregate into bilayer

structures but have the ability to deform in shape. Cevc‟s original Transfersomes® were

composed of phosphatidylcholine with the surfactant sodium cholate included as an „edge

activator‟ to confer flexibility, allowing the spherical shape to elongate and potentially

squeeze through narrow channels relative to the their spherical diameter [153-155]. Based on

the principals of conferring flexibility to the bilayer structure and/or incorporating

components that are known to act as skin penetration enhancers, various vesicle compositions

have been developed and their skin permeation quantified. These include ethosomes

(phospholipids with a high proportion of ethanol) [13, 156-160], niosomes (flexible non-ionic

surfactant vesicles) [161-166], invasomes (phosphatidylcholine, ethanol and a mixture of

terpene penetration enhancers) [167-169], SECosomes (surfactant, ethanol and cholesterol)

[14, 170] and PEVs (penetration enhancer containing vesicles) [171, 172]. Many different

components including chemical penetration enhancers have been investigated including oleic

acid, limonene [171, 173], propylene glycol [156, 174], glycerol [175], transcutol [176].

Flexible nano-vesicles have shown effective skin penetration enhancement of many

compounds that have very poor passive permeation. For example, Dragicevic-Curic and Fahr

[177] recently reviewed the area of flexible vesicle delivery for topical photodynamic therapy

(PDT), which involves the delivery of a photosensitizer compound to cancer cells within the

skin, followed by light activation to kill the cells. Photosensitizer compounds, such as 5-

amino levulinic acid and temoporfin (mTHPC), have very poor passive permeation, and the

aim in PDT is to target the skin whilst minimising systemic uptake. Dragicevic-Curic et al

[178] showed that an enhancement ratio of 3.4 and 2.2 for mTHPC delivery into the skin

could be achieved with invasomes (phosphatidylcholine, ethanol and terpenes) compared to

conventional liposomes and ethanol containing vesicles respectively. They also showed that

the choice of terpene significantly influenced skin deposition, with cineole being superior to

limonene, citral and mixtures of these terpenes. They also showed that the surface charge of

flexible liposomes (composed of neutral, anionic and cationic lipids, and polysorbate 20

surfactant) effects the physicochemical properties, stability and permeation in human

epidermal membranes in vitro [169]. Skin deposition of mTHPC was enhanced by all

flexible vesicles in the following order: cationic > neutral > anionic > conventional

liposomes, with the suggestion that the cationic lipids most effectively inteact with the

negatively charged stratum corneum. In work by Geusens et al. (Figure 11), cationic ultra-

flexible 58 nm nanosomes (SECosomes) were shown to deliver siRNA into the viable

epidermis with deposition monitored by multiphoton tomography in combination with

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fluorescence lifetime imaging microscopy (MPT-FLIM) [14]. SECosomes were composed of

the cationic lipid 1,2-dioleoyl-3- trimethylammonium propane chloride (DOTAP),

cholesterol, sodium cholate and 30% ethanol. Images showed human epidermal delivery of

siRNA after 1h and NADH lifetime changes in the epidermal tissue, that suggest cell

penetration of the fluorescent siRNA.

Bracke et al [170] have applied the SECosome approach to the delivery of siRNA to target

human beta defensin (hBD-2), which is highly up-regulated in psoriasis and has been defined

as a biomarker for the condition. DEFB4-siRNA-containing SECosomes, targeting hBD-2,

was applied in a bioengineered skin-humanized mouse model for psoriasis. The authors

reported a significant improvement in the psoriatic phenotype, normalization of the skin

architecture and a reduction in the number and size of blood vessels in the dermal

compartment. SECosome treatment lead to recovery of transglutaminase activity, filaggrin

expression and stratum corneum appearance that was similar to normal regenerated human

skin. Further support to the work of Geusens was reported in 2016 by Dorrani et al. [179],

who showed that passive application of small (~75 nm) liposome:siRNA complexes could

deposit siRNA as deeply as the upper dermis of human cadaver skin. Significantly, no labeled

liposomes or siRNA was detected in the receptor compartment of the Franz cells in which the

study was done, suggesting that the lipoplexes did not travel beyond the dermis but deposited

their cargo locally. Furthermore, they were able to show that the lipoplexes could internalize

into melanoma cells in culture, knock down BRAF expression and inhibit melanoma cell

proliferation.

There are a number of proposed mechanisms by which nanovesicle systems enhance skin

permeation. One mechanism is that the vesicles disintegrate on the skin surface and their

components penetrate into the stratum corneum, mixing with and modifying the lipid bilayers

and thereby indirectly enhancing permeation [180]. Many of the oil and surfactant

components of flexible vesicles are known to disrupt the stratum corneum lamellae to

facilitate skin transport [181]. Another theory proposed by Cevc [16], and supported by other

colleagues [182], is that the intact flexible vesicles can penetrate into and through the stratum

corneum due to the transepidermal osmotic gradient, carrying their payload into the skin. The

composition of the vesicle must be such that the molecules within the vesicle bilayer can

adjust to allow the vesicle to deform to the shape of a pore within the stratum corneum. This

is achieved because edge-active molecules such as surfactants or short-chain alcohols can

concentrate at the sites of greatest curvature [17, 183]. However, recent evaluations of vesicle

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skin interactions using the high resolution technologies of multiphoton excitation

fluorescence microscopy imaging and cross correlation-raster image correlation spectroscopy

(CC-RICS) [180] and stimulated emission depletion microscopy (STED) and CC-RICS [184]

have failed to show evidence of intact vesicles within the skin. STED provides image

resolution to 20 nm, whilst CC-RICS allows tracking of two fluorescent probes so that intact

vesicles can be resolved. In the latter study, vesicles and flexible vesicles incorporating

sodium cholate (mean diameter 96 nm) were applied unoccluded to freshly excised human

skin for 4-8 h, then imaged. Images showed that for both vesicles and flexible vesicles, a

large number of intact vesicles remained at the skin surface, particularly in the skin furrows,

with very few intact vesicles in the stratum corneum. A much higher proportion of vesicles

than flexible vesicles remained intact on the skin surface, and there was significantly more

fluorescent probe in the deeper layers of the stratum corneum for the flexible vesicles. As the

fluorescent signal was not associated with any vesicle-like shape, the authors concluded that

the flexible vesicle components were contributing to the penetration enhancement. They also

suggested that the penetration of intact flexible vesicles shown in earlier studies was due to

the choice of mouse skin with its thinner stratum corneum.

5. Physical enhancement and combinational methods for facilitated-delivery

Normally, when the stratum corneum is intact and the skin barrier is uncompromised, dermal

and transdermal delivery is limited to molecules that are relatively lipophilic, small and

uncharged. Consequently, many potentially useful therapeutic peptides, proteins, vaccines,

gene fragments or drug-carrying particles are excluded. In efforts to extend the range of

potential skin deliverables, various active technologies that apply physical forces to overcome

the skin barrier, utilizing acoustic, electrical, or electromagnetic energy (e.g. laser radiation or

radiofrequencies) and high pressure have been extensively investigated over several decades

[185, 186]. Techniques include iontophoresis, sonophoresis, thermal and radiofrequency

ablation, laser assisted delivery and jet propulsion systems, as well as microneedles, which

directly breach the stratum corneum. However, despite this level of research interest, FDA

approvals for device assisted transdermal delivery of molecules have been few (5 between

1979 and 2013) [187]. Furthermore, commercial development of transdermal devices has

mainly been in the area of iontophoresis (e.g. Ionsys® and Zecuity®, both FDA-approved for

fentanyl and sumatriptan delivery respectively and others). A few products used other

technologies such as thermal ablation (e.g. the PassPort® patch developed by Altea

Therapeutics, later acquired by Nitto Denko Technicla Corp.), radiofrequency ablation (e.g.

the ViaDerm® system by TransPharma Medical Ltd.), lasers (e.g. the EpitureTM system

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developed by Norwood Abbey), jet propulsion (e.g. Mini-JectTM by Valeritas and the

PowderJect® system) and direct heat application (Controlled Heat-Assisted Drug Delivery

(CHADD™) technology). The SonoPrep® system, developed by Sontra, used low frequency

ultrasound to deliver lidocaine for local analgesia. This was the only low frequency

sonophoresis system on the market but has since been withdrawn.

Further enhancement and even synergistic transdermal delivery has been achieved with a

combination of techniques, including an active technology with chemical permeation

enhancers [188] or two active technologies [189]. Of particular interest is the combination of

microneedle delivery with active technologies such as iontophoresis [190] or sonophoresis

[191]. Here, the goal is to use the microneedles to accurately place the deliverable at a

specific target site and to use the second active technology to enhance its release and

distribution. Donnelly et al. [190] showed enhanced delivery of peptides and proteins with

the combination of iontophoresis and hydrogel-forming microneedle arrays and suggested

that the technique may more useful for macromolecules than small molecules which are

already delivered efficiently by microneedles alone. While the use of combination

technologies may offer enhanced delivery with improved control over delivery rates, along

with some degree of specific targeting in the case of microneedles, they are also currently

associated with greater complexity, cost and difficulty of use. If they are to achieve

widespread acceptance and use, it will be necessary to harness advances in device design and

fabrication to make them more refined and inexpensive.

6. Safety and adverse effects

As discussed in Section 3, there is concern that penetration of biopersistent solid

nanoparticles into the skin could lead to damage to viable epidermal cells or accumulation in

secondary organs following biodistribution [70]. Examples of such materials include gold,

silver, metal oxides such as titanium dioxide and zinc oxide, quantum dots, fullerenes and

carbon nanotubes. Consequently, rigorous risk assessment is required for these [192]. Also

falling into the biopersistent category are the environmental hazards such as clay particles

absorbed though skin of bare feet [80] or skin-absorbed beryllium [64]. There is less concern

over colloidal nanosystems such as emulsions and lipid-based materials because these are

likely to be biodegradable on application to the skin, although the molecular products will

still be assessed in the usual way for irritancy and cytotoxicity. For example, Todosijević et

al. [193] used changes in erythema index, skin hydration and TEWL to assess the skin

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irritancy of different microemulsion formulations containing natural and synthetic non-ionic

surfactants for delivery of a model NSAID and showed that irritancy was strongly associated

with the presence of Polysorbate 80 and Transcutol P. Gürbüz et al. [194] assessed the

cytotoxicity of microemulsions for isotretinoin delivery using the MTT assay with a mouse

fibroblast cell line, comparing the microemulsions to SLS as a positive control. Standard

techniques to assess cell viability with mouse or human cell lines have also been used for

lipid-based vesicles [195, 196] and ultraflexible liposomes [14, 197]. Raju et al. used

multiphoton imaging of fluorescence following staining by acridine orange and ethidium

bromide to demonstrate cell viability or death in response to interventions by delivery devices

such as gene guns [198]. These workers showed that ballistic delivery of gold microparticles

coated with DNA vaccine at densities of 0.16, 1.35 and 2.72 particles per m2 to freshly

excised mouse ear skin resulted in cell death rates of 3.96, 45.91 and 90.52% respectively.

The results raised questions about the safety of this technique and the authors suggested that

successfully optimized transfection with this device would require a compromise between the

payload density and the inevitable level of cell death.

7. Future developments

We are constantly facing new challenges in skin delivery and as we have seen, extensive

research is being undertaken to develop novel and improved delivery systems. For example,

a recent sunscreen formulation uses bioadhesive nanoparticles with limited skin penetration

to prevent epidermal cell exposure to potentially toxic chemical UV filters, at the same time

providing enhanced UV protection [199]. As Vogt et al. have recently pointed out, a

worthwhile nanodelivery system should offer significant advantages, such as easier

application, reduced systemic absorption and enhanced selectivity to diseased skin or specific

cell populations, compared to more traditional methods [200]. We would add that, in our

view, the development of dermal and transdermal nanodelivery systems should be driven by a

clinical or therapeutic need, rather than by the availability of advanced technology. One such

clinical need is the long sought after, but highly elusive goal of non-invasive insulin delivery.

Nanodelivery strategies being explored for this hydrophilic macromolecule include a patch

consisting of a polymeric nanotube backbone to which insulin is electrostatically bound

[201]. An electric potential is then applied to release the bound insulin. The potential is also

hypothesized to drive the drug into the skin, analogous to iontophoresis. Another important

clinical application of nanodelivery systems is likely to be needle free transcutaneous

immunization, particularly by the hair follicle route [202].

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The next generation of nanocarriers will be designed to release their drug payload in response

to certain chemical, physical or biological stimuli, allowing much greater precision in

delivery to particular targets – the so-called “Smart” nanosystems. Much work is already

being carried out on intravenous or orally administered drug or gene delivery systems that are

responsive to temperature, UV or IR radiation, ultrasound or pH [203]. For example, smart

nanocarriers that can accumulate in tumors after intravenous injection have been developed

which combine pH-triggered doxorubicin release and near infrared light-triggered

photosensitization to provide dual chemotherapy and photodynamic therapy [204]. New skin

penetrating nanocarriers containing similar triggers will no doubt be developed to deliver

deeper, faster and better-targeted skin cancer therapy than that currently available with

creams containing aminolevulinic acid (ALA) or its methyl ester (MAL).

As we have already seen, the Lademann group has explored follicular delivery of

nanoparticles and hypothesized that these appendages may act as a reservoir for a drug

payload [92]. Up to now, the difficulty has been how to initiate and control drug release,

rather than relying on passive diffusion. In recent work [205] they are now exploring smart

nanoparticles for follicular delivery that are designed to release a drug after the application of

specific stimuli. Three nanoparticle systems were developed, where model drug release

occurred by passive diffusion through a porous surface, in response to enzymatic degradation

of the encapsulating particle and after photoactivation by infrared radiation. Deep follicular

penetration of the model drugs, as well as uptake by sebaceous glands was seen with the

latter two active release mechanisms.

8. Conclusion

Our observation is that nanodelivery systems show great promise, provided that they

are optimally matched to their designed purpose, rather than simply being seen as “one

technology fits all” applications. With this in mind, it should also be recognised that

nanosystems may not necessarily be the most efficient way to solve a particular problem.

Vogt et al. [200] recently alluded to this, citing the example of treating scalp diseases, where

nanoparticles may not be required for follicular penetration. However, the evolutionary

development in these systems is likely to continue, with a greater focus on solving difficult

skin delivery problems, by developing smart materials to deliver peptides, proteins and

genetic material with accurate targeting to specific cell populations. As nanosystems are

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refined, emphasis will need to be placed on those that can be translated to clinical application

and which provide significant economic and patient benefits.

Acknowledgements and Disclosures

Michael Roberts would like to acknowledge the support by grants from the National Health

and Medical Research Council of Australia (APP1049906; 1002611) and the US FDA grants

(U01FD005226 and U01FD005232). No conflict of interest to be declared.

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Figure Legends

Figure 1. Nanodelivery systems investigated for dermal and transdermal drug delivery.

Figure 2. Structure of human skin, showing major structures and cell types. Skin layer

thicknesses (not drawn to scale) are approximations only and actual values vary according to

the anatomical site and factors such as the degree of hydration.

Figure 3. Properties of nanosystems determining skin absorption and potential routes of

penetration (skin layer thicknesses not drawn to scale).

Figure 4. Gold-based nanoparticle penetration into rat, pig and human skin.

Figure 5. Quantum dots with various external functional groups penetrating to deeper layers

in excised pig skin (A), but remaining on the surface or localized in furrows in excised human

skin (B).

A: Confocal scanning microscopy of porcine skin treated for 8 h with PEG, PEG-amine

(NH2), or carboxylic acid (COOH)-coated QD 565. Coatings are noted at the top of each

column. Top row: confocal-DIC channel only allows an unobstructed view of the skin layers.

Middle row: confocal-DIC overlay with the QD fluorescence channel (green) shows QD

localization within the skin layers. Arrows indicate QD localization in the epidermis or

dermis. Bottom row: fluorescence intensity scan of QD emission. Quantum dots 565 are

localized in the epidermal (PEG and COOH coatings) or dermal (NH2 coating) layers by 8

h. All scale bars (lower right corners) are 50 µm.

J.P. Ryman-Rasmussen et al. Toxicol Sci 91 (2006) 159-165 Reproduced with permission.

[63]

B: LSCM images of untreated intact human skin or skin treated with PEG, PEG-NH2 and

COOH modified QDs at pH 7.0, for 24 h in static Franz cells. The QDs (red) at pH 7.0 can be

seen primarily on the stratum corneum and in skin furrows (arrow heads). Skin reflectance

(green) reveals nuclei as dark ellipsoids, with no QDs within the stratum granulosum or

spinosum. Scale bar indicates 100 mm; the optical section thickness was estimated as 2 mm.

T.W Prow et al. Nanotoxicology 6 (2012) 173-185 Reproduced with permission. [61]

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Figure 6. Confocal microscopic images of skin, showing penetration of FITC-conjugated

dextran beads (red). Images were obtained from the centre of the tissue cross section at a

depth of 10 μm. (A) 1 µm beads were observed at the stratum corneum–epidermal interface

and in the epidermis following 30 min of flexure (blue arrows). (B) 1 µm beads were

observed at the stratum corneum–epidermal interface and in the epidermis after 30 min of

flexure, and in the epidermis and dermis after 60 min (all arrows); beads were found

randomly distributed in the tissue, and occasional clusters of beads were identified (yellow

arrows). (C) 4 µm beads remained on the surface of the skin (tissue depth 13 μm). (D)

Discontinuous stratum corneum permitted entry of a bolus of 0.5 μm beads into the skin

directly under the tear (yellow arrow). Bar = 10 μm for A–D.

S.S. Tinkle et al., Environ Health Perspect 111 (2003) 1202-1208. Reproduced with

permission. [64].

Figure 7. Investigation of penetration of topically applied uncoated and coated ZnO

nanoparticles in the stratum granulosum (SG) layer of in vivo human skin following

application with flexing and massage. Representative FLIM images of SG of human skin

following flexing (top) and massage (bottom). Images are pseudocolored to distinguish

cellular autofluorescence (green) and the ZnO NP signal (violet-yellow-white scale bar

representing intensity of the ZnO NP signal). ZnO is confined mainly to the furrows and is

not detected in the viable epidermis cellular region.

Figure derived from data in V.R. Leite-Silva et al., Nanomedicine (Lond.) 11 (2016) 1193-

1205. [59].

Figure 8. Representative multiphoton images of intact and barrier-impaired in vivo human

skin; (A) without occlusion and (B) with occlusion.

Images were measured 6 h after topical application of the vehicle control (CCT), uncoated or

coated ZnO NP (100 mg.mL−1

; 2 mg.cm−2

) on intact (In) and barrier-impaired (BI) skin

(tape-stripped 20 times), with and without occlusion. The white dotted box shows ZnO NP

signals detected within the BI viable epidermis (VE). The white scale bar represents a length

of 40 μm. Figure derived from data presented in V.R. Leite-Silva et al., Eur J Pharm

Biopharm 104 (2016) 140-147. [1].

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Figure 9. Application of ZnO nanoparticles to A: excised human skin and B-D: lesional and

non-lesional skin of psoriasis patients.

A: Electron micrographs of excised intact human skin showing ZnO nanoparticles present on

the surface of the skin and around desquaming corneocytes. No penetration into the

underlying intact stratum corneum was observed.

S.E. Cross et al. Skin Pharmacol Physiol 20 (2007) 148-154. Reproduced with permission.

[56].

B: Treatment areas in lesional and non-lesional areas of forearms in psoriasis patients.

C: Minimal pixel intensities in lesional and non-lesional skin below the stratum corneum,

derived from the FLIM spectra shown in D.

D: Penetration profile of zinc oxide nanoparticles in skin furrows. Panels a and b show

untreated lesional (a) and zinc oxide nanoparticle containing sunscreen treated lesional sites

(b). The inset in panel b is an electron microscopy image of the individual zinc oxide

nanoparticles with a mean diameter of 35 nm, the bar indicates 100 nm. T.W. Prow et al.,

Adv Drug Deliv Rev 63 (2011) 470-491. Reproduced with permission. [70]

Figure 10. Penetration of particles into hair follicles; effect of massage and hair removal.

A: Superimposed transmission and fluorescent images showed in vitro penetration of a dye-

containing formulation into intact hair follicles of porcine skin with and without massage.

Deeper penetration occured when the dye was in particle form.

J. Lademann et al. Eur J Pharm Biopharm, 66 (2007) 159-164. Reproduced with permission.

[66]

B: After hair removal by application of superglue, followed by massage of the applied

formulations into human hair follicles, 40 nm, but not 750 nanoparticles penetrated via the

vellus hair follicle and were internalized by Langerhans cells in surrounding tissue.

(i, ii) 40 nm nanoparticles penetrated deep into vellus hair follicles. Laser scan microscopy

revealed fluorescent signals in adjacent tissue (*), indicating that nanoparticles penetrated

into and through the follicular epithelium.

Transcutaneously applied (iii, iv) 750 nm fluorescent nanoparticles aggregated in the

infundibulum of human vellus hair follicles. No penetration to deeper parts of the hair

follicles and no penetration into viable epidermis was observed in any of the samples. A. Vogt

et al. J Invest Dermatol, 126 (2006) 1316-1322. Reproduced with permission. [99]

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Figure 11. Penetration of ultraflexible vesicles (SECosomes) through excised human skin.

A: Overlaid TEM and confocal images excited at 639 nm from skin biopsy cross sections.

Untreated skin is shown in (a) and the red fluorescence signal due to the Cy5-siRNA

indicates penetration of complexes with surfactant containing liposomes (b, SCLPX),

ethanolic liposomes (c, ELPX) and ultraflexible SECosomes (d, SECoplexes). The strongest

signal comes from SECoplexes.

B: Multiphoton microscopy images showing enhanced SECoplex penetration around

keratinocytes at a depth of 40 μm (enlarged in C, left).

C: (right) To confirm whether the complexes are taken up by the cells, time-resolved NADH

autofluorescence spectra (free and bound) were obtained using FLIM following excitation at

740 nm. A shift in fluorescence lifetime was observed compared to normal untreated skin

(blue line), confirming the presence of the SECoplexes (red line) inside the cells.

B. Geusens et al. et al. Adv Funct Mater, 20 (2010) 4077-4090. Reproduced with permission.

[14]

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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Figure 11

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Table 1. Scope of the field of topical and cutaneous delivery using nanosystems.

Nanosystem use Dermatologicals, vaccines, diagnostic agents, cosmetics

Target sites Surface, Skin, Systemic

Product range Nanoparticles, nanoemulsions, nanovesicles

Chemical adjuncts Formulation enhancers, Skin pretreatment

Physical adjuncts Electrical, sound, electromagnetic, physical, other technologies

Efficacy Able to facilitate cargo penetrating in sufficient quantities?

Safety Major area of concern from regulators and consumers

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Table 2. Properties of nanodelivery systems in use or under development

Type of Nanosystem Possible mode of action Advantages Disadvantages

Solid nanoparticles

Zinc oxide and titanium

dioxide nanoparticles

Zinc oxide and titanium dioxide nanoparticles are

mainly used in sunscreens because of their ability to

filter UVA and UVB radiations

Nanoparticles < 100nm in size are

invisible, while retaining sun screening

properties; Current evidence suggests

they remain localized in the stratum

corneum of the skin with no permeation

observed in the viable epidermis [1, 2]

In vitro toxicity seen in cell

cultures [3]; Controversy

surrounding skin permeation.

Liquid colloidal systems

Microemulsions Disruption of SC lipid organization due to the

formulation components such as surfactants, co-

surfactants and oils [4].)

Thermodynamically more stable

compared to macroemulsions and

efficient solubilisation of the active in

the formulation.

Stability influenced by factors such

as pH & temperature changes;

Potential for skin irritation due to

high concentration of surfactants.

Nanoemulsions

Solubilization/extraction of stratum corneum lipids

due to the components used in nanoemulsion,

thereby decreasing resistance for drug transport [5].

Isotropic, transparent, kinetically stable;

high solubilization capacity for both

lipophilic & hydrophilic drugs [6]

Careful selection of formulation

components essential to avoid skin

irritation

Vesicular carriers

Solid Lipid nanoparticles Solid lipid nanoparticles reported to deform in shape

and melt after application to skin reducing the skin

barrier and causing occlusion on the skin surface

favoring the penetration of the active [7, 8].

Incorporation of lipophilic and

hydrophilic drugs, low cost compared to

liposomes and ease of scale-up and

manufacturing. Lipid matrix composed

of physiological lipids exhibiting low

toxicity [9].

Poor stability and low drug loading.

Liposomes Numerous studies have been undertaken to elucidate

the mechanism of action of liposomes. A general

mode involves adhesion of the liposomes on the skin

surface followed by release of the active from the

liposomal structure. The phospholipids and

surfactants included in the formulation may act as

Ability to encapsulate a wide range of

hydrophilic or hydrophobic drugs, good

biocompatibility, low toxicity, and

targeted delivery of drugs to the site of

action [11, 12].

Poor stability and limited

penetration in the upper layers of

the stratum corneum [13].

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penetration enhancers [10].

Secosomes

(combination of

DOTAP, Na cholate,

cholesterol, ethanol)

Leaching of stratum corneum lipids leading to

migration of secosomes in the skin fissures and

uptake by keratinocytes [14].

Enhanced skin penetration due to

ultradeformability [14, 15].

Novel delivery system with limited

research undertaken.

Transferosomes Destabilization & increase in deformability by

lowering interfacial tension of lipid bilayers [16].

Elastic vesicles capable of permeating

intact skin [16].

Emulsifiers (edge activators) must

be high purity to avoid risk of skin

irritation & toxicity

Ethosomes Fluidization of the lipid bilayers due to an edge

activator or alcohols which also enhance the

deformability of the vesicles [17].

Increase in stability and enhancement in

transdermal permeation compared to

conventional liposomes. High loading

capacity for lipophilic drugs compared

to liposomes; relatively simple &

inexpensive to manufacture.

Skin irritation due to high

concentration of alcohol and poor

skin penetration.

Microneedles Minimally invasive and act by creating microscopic

aqueous pores through which molecules can gain

access to the dermal circulation without stimulating

the dermal nerves or puncturing the dermal blood

vessels [18].

Obvious advantages over traditional

patches in terms of delivery of actives.

Possibility of delivering larger peptides,

proteins and vaccines non-invasively

through skin [18].

Problems in processing & storage

with sugar-based microneedle

arrays; Thermal treatment during

manufacture can restrict type of

active loaded into arrays;

Evaluation of biocompatibility of

all microneedle materials with skin

required; Regulatory issues should

be considered due to

innovativeness of delivery mode

[19].

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Table 3. Examples of recently published studies reporting the in vivo evaluation of topically

applied nanosystem formulations for the management of skin disorders.

Nanosystem Drug delivered Skin disorder Reference

Lipid-based nanosystems

Liposome Curcumin Melanoma [20]

Ethosome gel Mitoxantrone Melanoma [21]

Quercetin Inflammation [22]

Amphotericin B Fungal infection

(Candida albicans) [23]

Liposome, ethosome &

deformable liposome Ketoconazole

Fungal infection

(Candida albicans) [24]

NLC-based gel Clobetasol propionate Eczema [25]

SLN Artemisone Melanoma [26]

Doxorubicin Squamous cell

carcinoma [27]

Polymer-based nanosystems

Polymeric chitosan NP Hydrocortisone/Hyroxytyros

ol (co-administration) Atopic dermatitis [28, 29]

PLGA NP Pranopofen Inflammation [30]

Lipid-polymer hybrid NP Lidocaine Pain [31]

Metal-based nanosystems

NP Silver Scalp fungal infection

(Malassezia furfur) [32]

Gold Psoriasis [33]

Surfactant-based nanosystems

Niosomes Artemisone Melanoma [26]

Oil-in-water ME Caffeine UVB-induced skin

cancer [34]

Water-in-oil ME Pentoxifylline Inflammation [35]

Ionic liquid-in-water ME Etodolac Infllammation [36]

Hydrogel-thickened ME Ibuprofen Inflammation [37]

ME = microemulsion; NLC = Nanostructured lipid carrier; NP = nanoparticle; PLGA =

poly(lactic-co-glycolic acid); SLN = Solid lipid nanoparticles

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Graphical abstract