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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
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
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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: [email protected]
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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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References
[1] V.R. Leite-Silva, W.Y. Sanchez, H. Studier, D.C. Liu, Y.H. Mohammed, A.M. Holmes, E.M.
Ryan, I.N. Haridass, N.C. Chandrasekaran, W. Becker, J.E. Grice, H.A. Benson, M.S. Roberts,
Human skin penetration and local effects of topical nano zinc oxide after occlusion and
barrier impairment, Eur J Pharm Biopharm, 104 (2016) 140-147.
[2] G.J. Nohynek, E.K. Dufour, M.S. Roberts, Nanotechnology, cosmetics and the skin: is
there a health risk?, Skin Pharmacol Physiol, 21 (2008) 136-149.
[3] P. Kocbek, K. Teskac, M.E. Kreft, J. Kristl, Toxicological aspects of long-term treatment of
keratinocytes with ZnO and TiO2 nanoparticles, Small, 6 (2010) 1908-1917.
[4] M. Kreilgaard, Influence of microemulsions on cutaneous drug delivery, Advanced Drug
Delivery Reviews, 54 (2002) S77-S98.
[5] K.R. Pawar, R.J. Babu, Lipid materials for topical and transdermal delivery of
nanoemulsions, Critical reviews in therapeutic drug carrier systems, 31 (2014) 429-458.
[6] N. Salim, N. Ahmad, S.H. Musa, R. Hashim, T.F. Tadros, M. Basri, Nanoemulsion as a
topical delivery system of antipsoriatic drugs, Rsc Adv, 6 (2016) 6234-6250.
[7] V.B. Junyaprasert, V. Teeranachaideekul, E.B. Souto, P. Boonme, R.H. Muller, Q10-loaded
NLC versus nanoemulsions: stability, rheology and in vitro skin permeation, Int J Pharm, 377
(2009) 207-214.
[8] S.A. Wissing, R.H. Muller, The influence of solid lipid nanoparticles on skin hydration and
viscoelasticity--in vivo study, Eur J Pharm Biopharm, 56 (2003) 67-72.
[9] S.D. Mandawgade, V.B. Patravale, Development of SLNs from natural lipids: application
to topical delivery of tretinoin, Int J Pharm, 363 (2008) 132-138.
[10] K. Vrhovnik, J. Kristl, M. Sentjurc, J. Smid-Korbar, Influence of liposome bilayer fluidity
on the transport of encapsulated substance into the skin as evaluated by EPR,
Pharmaceutical Research, 15 (1998) 525-530.
[11] C. Chen, D. Han, C. Cai, X. Tang, An overview of liposome lyophilization and its future
potential, J Control Release, 142 (2010) 299-311.
[12] M.L. Immordino, F. Dosio, L. Cattel, Stealth liposomes: review of the basic science,
rationale, and clinical applications, existing and potential, Int J Nanomedicine, 1 (2006) 297-
315.
[13] E. Touitou, N. Dayan, L. Bergelson, B. Godin, M. Eliaz, Ethosomes - novel vesicular
carriers for enhanced delivery: characterization and skin penetration properties, Journal of
Controlled Release, 65 (2000) 403-418.
[14] B. Geusens, M. Van Gele, S. Braat, S.C. De Smedt, M.C. Stuart, T.W. Prow, W. Sanchez,
M.S. Roberts, N. Sanders, J. Lambert, Flexible Nanosomes (SECosomes) Enable Efficient
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
25
siRNA Delivery in Cultured Primary Skin Cells and in the Viable Epidermis of Ex Vivo Human
Skin, Adv Funct Mater, 20 (2010) 4077-4090.
[15] E.L. Romero, M.J. Morilla, Highly deformable and highly fluid vesicles as potential drug
delivery systems: theoretical and practical considerations, Int J Nanomedicine, 8 (2013)
3171-3186.
[16] G. Cevc, G. Blume, Lipid vesicles penetrate into intact skin owing to the transdermal
osmotic gradients and hydration force, Biochim Biophys Acta, 1104 (1992) 226-232.
[17] G. Cevc, A. Schatzlein, H. Richardsen, Ultradeformable lipid vesicles can penetrate the
skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM
experiments and direct size measurements, Biochimica et Biophysica Acta (BBA) -
Biomembranes, 1564 (2002) 21-30.
[18] R.F. Donnelly, D.I.J. Morrow, F. Fay, C.J. Scott, S. Abdelghany, R.R.T. Singh, M.J. Garland,
A. David Woolfson, Microneedle-mediated intradermal nanoparticle delivery: Potential for
enhanced local administration of hydrophobic pre-formed photosensitisers, Photodiagnosis
and Photodynamic Therapy, 7 (2010) 222-231.
[19] E. Larrañeta, R.E.M. Lutton, A.D. Woolfson, R.F. Donnelly, Microneedle arrays as
transdermal and intradermal drug delivery systems: Materials science, manufacture and
commercial development, Materials Science and Engineering: R: Reports, 104 (2016) 1-32.
[20] Y. Chen, Q. Wu, Z. Zhang, L. Yuan, X. Liu, L. Zhou, Preparation of curcumin-loaded
liposomes and evaluation of their skin permeation and pharmacodynamics, Molecules
(Basel, Switzerland), 17 (2012) 5972-5987.
[21] X. Yu, L. Du, Y. Li, G. Fu, Y. Jin, Improved anti-melanoma effect of a transdermal
mitoxantrone ethosome gel, Biomed Pharmacother, 73 (2015) 6-11.
[22] C. Caddeo, O. Diez-Sales, R. Pons, X. Fernandez-Busquets, A.M. Fadda, M. Manconi,
Topical anti-inflammatory potential of quercetin in lipid-based nanosystems: in vivo and in
vitro evaluation, Pharm Res, 31 (2014) 959-968.
[23] L. Kaur, S.K. Jain, R.K. Manhas, D. Sharma, Nanoethosomal formulation for skin
targeting of amphotericin B: an in vitro and in vivo assessment, J Liposome Res, 25 (2015)
294-307.
[24] F. Guo, J. Wang, M. Ma, F. Tan, N. Li, Skin targeted lipid vesicles as novel nano-carrier of
ketoconazole: characterization, in vitro and in vivo evaluation, Journal of materials science.
Materials in medicine, 26 (2015) 175.
[25] U. Nagaich, N. Gulati, Nanostructured lipid carriers (NLC) based controlled release
topical gel of clobetasol propionate: design and in vivo characterization, Drug Deliv Transl
Res, 6 (2016) 289-298.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
26
[26] A. Dwivedi, A. Mazumder, L.T. Fox, A. Brummer, M. Gerber, J.L. du Preez, R.K. Haynes, J.
du Plessis, In vitro skin permeation of artemisone and its nano-vesicular formulations, Int J
Pharm, 503 (2016) 1-7.
[27] L.A. Huber, T.A. Pereira, D.N. Ramos, L.C. Rezende, F.S. Emery, L.M. Sobral, A.M.
Leopoldino, R.F. Lopez, Topical Skin Cancer Therapy Using Doxorubicin-Loaded Cationic Lipid
Nanoparticles and lontophoresis, J Biomed Nanotechnol, 11 (2015) 1975-1988.
[28] M.I. Siddique, H. Katas, M.C. Amin, S.F. Ng, M.H. Zulfakar, A. Jamil, In-vivo dermal
pharmacokinetics, efficacy, and safety of skin targeting nanoparticles for corticosteroid
treatment of atopic dermatitis, Int J Pharm, 507 (2016) 72-82.
[29] Z. Hussain, H. Katas, M.C. Mohd Amin, E. Kumolosasi, F. Buang, S. Sahudin, Self-
assembled polymeric nanoparticles for percutaneous co-delivery of
hydrocortisone/hydroxytyrosol: an ex vivo and in vivo study using an NC/Nga mouse model,
Int J Pharm, 444 (2013) 109-119.
[30] G. Abrego, H. Alvarado, E.B. Souto, B. Guevara, L.H. Bellowa, M.L. Garduno, M.L. Garcia,
A.C. Calpena, Biopharmaceutical profile of hydrogels containing pranoprofen-loaded PLGA
nanoparticles for skin administration: In vitro, ex vivo and in vivo characterization, Int J
Pharm, 501 (2016) 350-361.
[31] J. Wang, L. Zhang, H. Chi, S. Wang, An alternative choice of lidocaine-loaded liposomes:
lidocaine-loaded lipid-polymer hybrid nanoparticles for local anesthetic therapy, Drug Deliv,
23 (2016) 1254-1260.
[32] M.F. Anwar, D. Yadav, S. Jain, S. Kapoor, S. Rastogi, I. Arora, M. Samim, Size- and shape-
dependent clinical and mycological efficacy of silver nanoparticles on dandruff, Int J
Nanomedicine, 11 (2016) 147-161.
[33] H. Bessar, I. Venditti, L. Benassi, C. Vaschieri, P. Azzoni, G. Pellacani, C. Magnoni, E.
Botti, V. Casagrande, M. Federici, A. Costanzo, L. Fontana, G. Testa, F.F. Mostafa, S.A.
Ibrahim, M.V. Russo, I. Fratoddi, Functionalized gold nanoparticles for topical delivery of
methotrexate for the possible treatment of psoriasis, Colloids Surf B Biointerfaces, 141
(2016) 141-147.
[34] E. Abd, S. Namjoshi, Y.H. Mohammed, M.S. Roberts, J.E. Grice, Synergistic Skin
Penetration Enhancer and Nanoemulsion Formulations Promote the Human Epidermal
Permeation of Caffeine and Naproxen, J Pharm Sci, (2015).
[35] A.L. Cavalcanti, M.Y. Reis, G.C. Silva, I.M. Ramalho, G.P. Guimaraes, J.A. Silva, K.L.
Saraiva, B.P. Damasceno, Microemulsion for topical application of pentoxifylline: In vitro
release and in vivo evaluation, Int J Pharm, 506 (2016) 351-360.
[36] S. Goindi, R. Kaur, R. Kaur, An ionic liquid-in-water microemulsion as a potential carrier
for topical delivery of poorly water soluble drug: Development, ex-vivo and in-vivo
evaluation, Int J Pharm, 495 (2015) 913-923.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
27
[37] L. Djekic, M. Martinovic, R. Stepanovic-Petrovic, A. Micov, M. Tomic, M. Primorac,
Formulation of hydrogel-thickened nonionic microemulsions with enhanced percutaneous
delivery of ibuprofen assessed in vivo in rats, Eur J Pharm Sci, (2016).
[38] B. Godin, E. Touitou, Transdermal skin delivery: predictions for humans from in vivo, ex
vivo and animal models, Adv Drug Deliv Rev, 59 (2007) 1152-1161.
[39] R.J. Scheuplein, Analysis of permeability data for the case of parallel diffusion pathways,
Biophys J, 6 (1966) 1-17.
[40] R.J. Scheuplein, I.H. Blank, Permeability of the skin, Physiological reviews, 51 (1971)
702-747.
[41] G.B. Kasting, N.D. Barai, T.F. Wang, J.M. Nitsche, Mobility of water in human stratum
corneum, J Pharm Sci, 92 (2003) 2326-2340.
[42] B. Illel, H. Schaefer, J. Wepierre, O. Doucet, Follicles play an important role in
percutaneous absorption, J Pharm Sci, 80 (1991) 424-427.
[43] A.C. Lauer, L.M. Lieb, C. Ramachandran, G.L. Flynn, N.D. Weiner, Transfollicular drug
delivery, Pharm Res, 12 (1995) 179-186.
[44] M.S. Roberts, S.E. Cross, M.A. Pellett, K.A. Walters, Skin transport, in: K.A. Walters (Ed.)
Dermatological and Transdermal Formulations, Marcel Dekker, New York, 2002, pp. 89-196.
[45] M.R. Prausnitz, S. Mitragotri, R. Langer, Current status and future potential of
transdermal drug delivery, Nat Rev Drug Discov, 3 (2004) 115-124.
[46] J. Kreuter, Preface, in: J. Kreuter (Ed.) Colloidal Drug Delivery Systems, Marcel Dekker,
Inc., New York, 1994.
[47] J. Kreuter, Nanoparticles--a historical perspective, Int J Pharm, 331 (2007) 1-10.
[48] J. Kreuter, Nanoparticles and nanocapsules--new dosage forms in the nanometer size
range, Pharmaceutica acta Helvetiae, 53 (1978) 33-39.
[49] J. Kreuter, Nanoparticles, in: J. Swarbrick, J.C. Boylan (Eds.) Encyclopedia of
Pharmaceutical Technology, Marcel Dekker, Inc., New York, 1994, pp. 165-290.
[50] F. Brasseur, P. Couvreur, B. Kante, L. Deckers-Passau, M. Roland, C. Deckers, P. Speisers,
Actinomycin D adsorbed on polymethylcyanoacrylate nanoparticles: Increased efficiency
against an experimental tumor, European Journal of Cancer (1965), 16 (1980) 1441-1445.
[51] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer
chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor
agent smancs, Cancer research, 46 (1986) 6387-6392.
[52] Prescribing Information [Online], 2015, Cited on
[53] V.H. Li, R.W. Wood, J. Kreuter, T. Harmia, J.R. Robinson, Ocular drug delivery of
progesterone using nanoparticles, J Microencapsul, 3 (1986) 213-218.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
28
[54] Z. Cui, R.J. Mumper, Chitosan-based nanoparticles for topical genetic immunization, J
Control Release, 75 (2001) 409-419.
[55] V. Jenning, A. Gysler, M. Schafer-Korting, S.H. Gohla, Vitamin A loaded solid lipid
nanoparticles for topical use: occlusive properties and drug targeting to the upper skin, Eur J
Pharm Biopharm, 49 (2000) 211-218.
[56] S.E. Cross, B. Innes, M.S. Roberts, T. Tsuzuki, T.A. Robertson, P. McCormick, Human skin
penetration of sunscreen nanoparticles: in-vitro assessment of a novel micronized zinc oxide
formulation, Skin Pharmacol Physiol, 20 (2007) 148-154.
[57] B. Gulson, M. McCall, M. Korsch, L. Gomez, P. Casey, Y. Oytam, A. Taylor, M. McCulloch,
J. Trotter, L. Kinsley, G. Greenoak, Small amounts of zinc from zinc oxide particles in
sunscreens applied outdoors are absorbed through human skin, Toxicol Sci, 118 (2010) 140-
149.
[58] H.I. Labouta, D.C. Liu, L.L. Lin, M.K. Butler, J.E. Grice, A.P. Raphael, T. Kraus, L.K. El-
Khordagui, H.P. Soyer, M.S. Roberts, M. Schneider, T.W. Prow, Gold nanoparticle
penetration and reduced metabolism in human skin by toluene, Pharm Res, 28 (2011) 2931-
2944.
[59] V.R. Leite-Silva, D.C. Liu, W.Y. Sanchez, H. Studier, Y.H. Mohammed, A. Holmes, W.
Becker, J.E. Grice, H.A. Benson, M.S. Roberts, Effect of flexing and massage on in vivo human
skin penetration and toxicity of zinc oxide nanoparticles, Nanomedicine, 11 (2016) 1193-
1205.
[60] N.A. Monteiro-Riviere, K. Wiench, R. Landsiedel, S. Schulte, A.O. Inman, J.E. Riviere,
Safety evaluation of sunscreen formulations containing titanium dioxide and zinc oxide
nanoparticles in UVB sunburned skin: an in vitro and in vivo study, Toxicol Sci, 123 (2011)
264-280.
[61] T.W. Prow, N.A. Monteiro-Riviere, A.O. Inman, J.E. Grice, X. Chen, X. Zhao, W.H.
Sanchez, A. Gierden, M.A. Kendall, A.V. Zvyagin, D. Erdmann, J.E. Riviere, M.S. Roberts,
Quantum dot penetration into viable human skin, Nanotoxicology, 6 (2012) 173-185.
[62] J.G. Rouse, J. Yang, J.P. Ryman-Rasmussen, A.R. Barron, N.A. Monteiro-Riviere, Effects
of mechanical flexion on the penetration of fullerene amino acid-derivatized peptide
nanoparticles through skin, Nano Lett, 7 (2007) 155-160.
[63] J.P. Ryman-Rasmussen, J.E. Riviere, N.A. Monteiro-Riviere, Penetration of intact skin by
quantum dots with diverse physicochemical properties, Toxicol Sci, 91 (2006) 159-165.
[64] S.S. Tinkle, J.M. Antonini, B.A. Rich, J.R. Roberts, R. Salmen, K. DePree, E.J. Adkins, Skin
as a route of exposure and sensitization in chronic beryllium disease, Environ Health
Perspect, 111 (2003) 1202-1208.
[65] I.T. Degim, D. Kadioglu, Cheap, suitable, predictable and manageable nanoparticles for
drug delivery: quantum dots, Current drug delivery, 10 (2013) 32-38.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
29
[66] J. Lademann, H. Richter, A. Teichmann, N. Otberg, U. Blume-Peytavi, J. Luengo, B.
Weiss, U.F. Schaefer, C.M. Lehr, R. Wepf, W. Sterry, Nanoparticles--an efficient carrier for
drug delivery into the hair follicles, European journal of pharmaceutics and
biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische
Verfahrenstechnik e.V, 66 (2007) 159-164.
[67] G.J. Nohynek, J. Lademann, C. Ribaud, M.S. Roberts, Grey goo on the skin?
Nanotechnology, cosmetic and sunscreen safety, Crit Rev Toxicol, 37 (2007) 251-277.
[68] F. Larese Filon, M. Mauro, G. Adami, M. Bovenzi, M. Crosera, Nanoparticles skin
absorption: New aspects for a safety profile evaluation, Regul Toxicol Pharmacol, 72 (2015)
310-322.
[69] X.J. Cai, A. Woods, P. Mesquida, S.A. Jones, Assessing the Potential for Drug-
Nanoparticle Surface Interactions To Improve Drug Penetration into the Skin, Mol Pharm, 13
(2016) 1375-1384.
[70] T.W. Prow, J.E. Grice, L.L. Lin, R. Faye, M. Butler, W. Becker, E.M. Wurm, C. Yoong, T.A.
Robertson, H.P. Soyer, M.S. Roberts, Nanoparticles and microparticles for skin drug delivery,
Adv Drug Deliv Rev, 63 (2011) 470-491.
[71] Z. Zhang, P.C. Tsai, T. Ramezanli, B.B. Michniak-Kohn, Polymeric nanoparticles-based
topical delivery systems for the treatment of dermatological diseases, Wiley Interdiscip Rev
Nanomed Nanobiotechnol, 5 (2013) 205-218.
[72] G. Sonavane, K. Tomoda, A. Sano, H. Ohshima, H. Terada, K. Makino, In vitro
permeation of gold nanoparticles through rat skin and rat intestine: effect of particle size,
Colloids Surf B Biointerfaces, 65 (2008) 1-10.
[73] M. Kirillin, M. Shirmanova, M. Sirotkina, M. Bugrova, B. Khlebtsov, E. Zagaynova,
Contrasting properties of gold nanoshells and titanium dioxide nanoparticles for optical
coherence tomography imaging of skin: Monte Carlo simulations and in vivo study, J Biomed
Opt, 14 (2009) 021017.
[74] C. Graf, M. Meinke, Q. Gao, S. Hadam, J. Raabe, W. Sterry, U. Blume-Peytavi, J.
Lademann, E. Ruhl, A. Vogt, Qualitative detection of single submicron and nanoparticles in
human skin by scanning transmission x-ray microscopy, J Biomed Opt, 14 (2009) 021015.
[75] H.I. Labouta, L.K. el-Khordagui, T. Kraus, M. Schneider, Mechanism and determinants of
nanoparticle penetration through human skin, Nanoscale, 3 (2011) 4989-4999.
[76] B. Baroli, Penetration of nanoparticles and nanomaterials in the skin: Fiction or reality?,
Journal of Pharmaceutical Sciences, 99 (2010) 21-50.
[77] L. Panigrahi, S. Pattnaik, S.K. Ghosal, The effect of pH and organic ester penetration
enhancers on skin permeation kinetics of terbutaline sulfate from pseudolatex-type
transdermal delivery systems through mouse and human cadaver skins, AAPS
PharmSciTech, 6 (2005) E167-173.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
30
[78] L.W. Zhang, N.A. Monteiro-Riviere, Assessment of quantum dot penetration into intact,
tape-stripped, abraded and flexed rat skin, Skin pharmacology and physiology, 21 (2008)
166-180.
[79] L.W. Zhang, W.W. Yu, V.L. Colvin, N.A. Monteiro-Riviere, Biological interactions of
quantum dot nanoparticles in skin and in human epidermal keratinocytes, Toxicol Appl
Pharmacol, 228 (2008) 200-211.
[80] G. Blundell, W.J. Henderson, E.W. Price, Soil particles in the tissues of the foot in
endemic elephantiasis of the lower legs, Annals of tropical medicine and parasitology, 83
(1989) 381-385.
[81] M.E. Burnett, S.Q. Wang, Current sunscreen controversies: a critical review,
Photodermatol Photoimmunol Photomed, 27 (2011) 58-67.
[82] What's the story with nanoparticles in sunscreen?, http://www.foe.org.au/whats-story-
nanoparticles-sunscreen, 2012, Cited on
[83] T.G. Smijs, S. Pavel, Titanium dioxide and zinc oxide nanoparticles in sunscreens: focus
on their safety and effectiveness, Nanotechnology, science and applications, 4 (2011) 95-
112.
[84] V. Sharma, S.K. Singh, D. Anderson, D.J. Tobin, A. Dhawan, Zinc oxide nanoparticle
induced genotoxicity in primary human epidermal keratinocytes, J Nanosci Nanotechnol, 11
(2011) 3782-3788.
[85] S. Wang, G. Zhang, H. Meng, L. Li, Effect of Exercise-induced Sweating on facial sebum,
stratum corneum hydration, and skin surface pH in normal population, Skin Res Technol, 19
(2013) e312-317.
[86] S.W. Bian, I.A. Mudunkotuwa, T. Rupasinghe, V.H. Grassian, Aggregation and dissolution
of 4 nm ZnO nanoparticles in aqueous environments: influence of pH, ionic strength, size,
and adsorption of humic acid, Langmuir, 27 (2011) 6059-6068.
[87] A.M. Holmes, Z. Song, H.R. Moghimi, M.S. Roberts, Relative Penetration of Zinc Oxide
and Zinc Ions into Human Skin after Application of Different Zinc Oxide Formulations, ACS
Nano, 10 (2016) 1810-1819.
[88] A.V. Zvyagin, X. Zhao, A. Gierden, W. Sanchez, J.A. Ross, M.S. Roberts, Imaging of zinc
oxide nanoparticle penetration in human skin in vitro and in vivo, J Biomed Opt, 13 (2008)
064031.
[89] L.L. Lin, J.E. Grice, M.K. Butler, A.V. Zvyagin, W. Becker, T.A. Robertson, H.P. Soyer, M.S.
Roberts, T.W. Prow, Time-Correlated Single Photon Counting For Simultaneous Monitoring
Of Zinc Oxide Nanoparticles And NAD(P)H In Intact And Barrier-Disrupted Volunteer Skin,
Pharm Res, 28 (2011) 2920-2930.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
31
[90] H.J. Ryu, M.Y. Seo, S.K. Jung, E.H. Maeng, S.Y. Lee, D.H. Jang, T.J. Lee, K.Y. Jo, Y.R. Kim,
K.B. Cho, M.K. Kim, B.J. Lee, S.W. Son, Zinc oxide nanoparticles: a 90-day repeated-dose
dermal toxicity study in rats, Int J Nanomedicine, 9 Suppl 2 (2014) 137-144.
[91] F. Knorr, J. Lademann, A. Patzelt, W. Sterry, U. Blume-Peytavi, A. Vogt, Follicular
transport route--research progress and future perspectives, Eur J Pharm Biopharm, 71
(2009) 173-180.
[92] J. Lademann, F. Knorr, H. Richter, U. Blume-Peytavi, A. Vogt, C. Antoniou, W. Sterry, A.
Patzelt, Hair follicles--an efficient storage and penetration pathway for topically applied
substances. Summary of recent results obtained at the Center of Experimental and Applied
Cutaneous Physiology, Charite -Universitatsmedizin Berlin, Germany, Skin Pharmacol
Physiol, 21 (2008) 150-155.
[93] J. Lademann, A. Patzelt, H. Richter, C. Antoniou, W. Sterry, F. Knorr, Determination of
the cuticula thickness of human and porcine hairs and their potential influence on the
penetration of nanoparticles into the hair follicles, J Biomed Opt, 14 (2009) 021014.
[94] J. Lademann, H. Richter, U.F. Schaefer, U. Blume-Peytavi, A. Teichmann, N. Otberg, W.
Sterry, Hair follicles - a long-term reservoir for drug delivery, Skin Pharmacol Physiol, 19
(2006) 232-236.
[95] S. Trauer, H. Richter, J. Kuntsche, R. Buttemeyer, M. Liebsch, M. Linscheid, A. Fahr, M.
Schafer-Korting, J. Lademann, A. Patzelt, Influence of massage and occlusion on the ex vivo
skin penetration of rigid liposomes and invasomes, Eur J Pharm Biopharm, 86 (2014) 301-
306.
[96] A. Vogt, B. Combadiere, S. Hadam, K.M. Stieler, J. Lademann, H. Schaefer, B. Autran, W.
Sterry, U. Blume-Peytavi, 40 nm, but not 750 or 1,500 nm, nanoparticles enter epidermal
CD1a+ cells after transcutaneous application on human skin, J Invest Dermatol, 126 (2006)
1316-1322.
[97] M. Pasparakis, I. Haase, F.O. Nestle, Mechanisms regulating skin immunity and
inflammation, Nat Rev Immunol, 14 (2014) 289-301.
[98] A. Vogt, S. Hadam, I. Deckert, J. Schmidt, A. Stroux, Z. Afraz, F. Rancan, J. Lademann, B.
Combadiere, U. Blume-Peytavi, Hair follicle targeting, penetration enhancement and
Langerhans cell activation make cyanoacrylate skin surface stripping a promising delivery
technique for transcutaneous immunization with large molecules and particle-based
vaccines, Exp Dermatol, 24 (2015) 73-75.
[99] M.Y. Alexander, R.J. Akhurst, Liposome-medicated gene transfer and expression via the
skin, Hum Mol Genet, 4 (1995) 2279-2285.
[100] I.A. Aljuffali, C.T. Sung, F.M. Shen, C.T. Huang, J.Y. Fang, Squarticles as a lipid
nanocarrier for delivering diphencyprone and minoxidil to hair follicles and human dermal
papilla cells, AAPS J, 16 (2014) 140-150.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
32
[101] R.M. Hoffman, Topical liposome targeting of dyes, melanins, genes, and proteins
selectively to hair follicles, J Drug Target, 5 (1998) 67-74.
[102] L. Li, R.M. Hoffman, The feasibility of targeted selective gene therapy of the hair
follicle, Nat Med, 1 (1995) 705-706.
[103] L. Li, V. Lishko, R.M. Hoffman, Liposome targeting of high molecular weight DNA to the
hair follicles of histocultured skin: a model for gene therapy of the hair growth processes, In
Vitro Cell Dev Biol Anim, 29A (1993) 258-260.
[104] L. Li, V.K. Lishko, R.M. Hoffman, Liposomes can specifically target entrapped melanin
to hair follicles in histocultured skin, In Vitro Cell Dev Biol, 29A (1993) 192-194.
[105] D. Kumar, J. Ali, S. Baboota, Omega 3 fatty acid-enriched nanoemulsion of
thiocolchicoside for transdermal delivery: formulation, characterization and absorption
studies, Drug Deliv, 23 (2016) 591-600.
[106] M. Ulmer, A. Patzelt, T. Vergou, H. Richter, G. Muller, A. Kramer, W. Sterry, V. Czaika, J.
Lademann, In vivo investigation of the efficiency of a nanoparticle-emulsion containing
polihexanide on the human skin, Eur J Pharm Biopharm, 84 (2013) 325-329.
[107] H. Wu, C. Ramachandran, N.D. Weiner, B.J. Roessler, Topical transport of hydrophilic
compounds using water-in-oil nanoemulsions, Int J Pharm, 220 (2001) 63-75.
[108] M. Lapteva, M. Moller, R. Gurny, Y.N. Kalia, Self-assembled polymeric nanocarriers for
the targeted delivery of retinoic acid to the hair follicle, Nanoscale, 7 (2015) 18651-18662.
[109] M. Lapteva, K. Mondon, M. Moller, R. Gurny, Y.N. Kalia, Polymeric micelle nanocarriers
for the cutaneous delivery of tacrolimus: a targeted approach for the treatment of psoriasis,
Mol Pharm, 11 (2014) 2989-3001.
[110] P. Pan-In, A. Wongsomboon, C. Kokpol, N. Chaichanawongsaroj, S.
Wanichwecharungruang, Depositing alpha-mangostin nanoparticles to sebaceous gland area
for acne treatment, J Pharmacol Sci, 129 (2015) 226-232.
[111] N. Suwannateep, S. Wanichwecharungruang, J. Fluhr, A. Patzelt, J. Lademann, M.C.
Meinke, Comparison of two encapsulated curcumin particular systems contained in
different formulations with regard to in vitro skin penetration, Skin Res Technol, 19 (2013)
1-9.
[112] T.P. Hoar, J.H. Schulman, Transparent water-in-oil dispersions: the oleopathic hydro-
micelle, Nature, 152 (1943) 102-103.
[113] J.H. Schulman, W. Stoeckenius, L.M. Prince, Mechanism of Formation and Structure of
Micro Emulsions by Electron Microscopy, The Journal of Physical Chemistry, 63 (1959) 1677-
1680.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
33
[114] J.C. Wang, B.G. Patel, C.W. Ehmann, N. Lowe, The release and percutaneous
permeation of anthralin products, using clinically involved and uninvolved psoriatic skin, J
Am Acad Dermatol, 16 (1987) 812-821.
[115] M.C. Martini, M.F. Bobin, H. Flandin, F. Caillaud, J. Cotte, [Role of microemulsions in
the percutaneous absorption of alpha-tocopherol], Journal de pharmacie de Belgique, 39
(1984) 348-354.
[116] T.G. Mason, J. Wilking, K. Meleson, C. Chang, S. Graves, Nanoemulsions: formation,
structure, and physical properties, Journal of Physics: Condensed Matter, 18 (2006) R635.
[117] F. Shakeel, S. Baboota, A. Ahuja, J. Ali, M. Aqil, S. Shafiq, Nanoemulsions as vehicles for
transdermal delivery of aceclofenac, AAPS PharmSciTech, 8 (2007) 191-199.
[118] M.N. Yukuyama, D.D. Ghisleni, T.J. Pinto, N.A. Bou-Chacra, Nanoemulsion: process
selection and application in cosmetics--a review, Int J Cosmet Sci, 38 (2016) 13-24.
[119] T. Tadros, P. Izquierdo, J. Esquena, C. Solans, Formation and stability of nano-
emulsions, Adv Colloid Interface Sci, 108-109 (2004) 303-318.
[120] C. Solans, P. Izquierdo, J. Nolla, N. Azemar, M.J. Garcia-Celma, Nano-emulsions, Curr.
Opin. Colloid Interface Sci., 10 (2005) 102-110.
[121] K. Bouchemal, S. Brianc on, E. Perrier, H. Fessi, Nano-emulsion formulation using
spontaneous emulsification: solvent, oil and surfactant optimisation., Int. J. Pharm., 280
(2004) 241-251.
[122] Y. Wu, Y.H. Li, X.H. Gao, H.D. Chen, The application of nanoemulsion in dermatology:
an overview, J Drug Target, 21 (2013) 321-327.
[123] Y. Baspinar, C.M. Keck, H.-H. Borchert, Development of a positively charged
prednicarbate nanoemulsion, International journal of pharmaceutics, 383 (2010) 201-208.
[124] R.H. Muller, R. Shegokar, C.M. Keck, 20 years of lipid nanoparticles (SLN and NLC):
present state of development and industrial applications, Curr Drug Discov Technol, 8 (2011)
207-227.
[125] R.H. Müller, U. Alexiev, P. Sinambela, C.M. Keck, Nanostructured Lipid Carriers (NLC):
The Second Generation of Solid Lipid Nanoparticles, in: N. Dragicevic, I.H. Maibach (Eds.)
Percutaneous Penetration Enhancers Chemical Methods in Penetration Enhancement:
Nanocarriers, Springer Berlin Heidelberg, Berlin, Heidelberg, 2016, pp. 161-185.
[126] R.H. Müller, M. Radtke, S.A. Wissing, Solid lipid nanoparticles (SLN) and
nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations, Advanced
Drug Delivery Reviews, 54, Supplement (2002) S131-S155.
[127] A. Dingler, R.P. Blum, H. Niehus, R.H. Muller, S. Gohla, Solid lipid nanoparticles
(SLN/Lipopearls)--a pharmaceutical and cosmetic carrier for the application of vitamin E in
dermal products, J Microencapsul, 16 (1999) 751-767.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
34
[128] V. Jenning, M. Schafer-Korting, S. Gohla, Vitamin A-loaded solid lipid nanoparticles for
topical use: drug release properties, J Control Release, 66 (2000) 115-126.
[129] Y.T. Zhang, Z.H. Wu, K. Zhang, J.H. Zhao, B.N. Ye, N.P. Feng, An in vitro and in vivo
comparison of solid and liquid-oil cores in transdermal aconitine nanocarriers, J Pharm Sci,
103 (2014) 3602-3610.
[130] H. Chen, X. Chang, D. Du, W. Liu, J. Liu, T. Weng, Y. Yang, H. Xu, X. Yang,
Podophyllotoxin-loaded solid lipid nanoparticles for epidermal targeting, J Control Release,
110 (2006) 296-306.
[131] J. Kuntsche, H. Bunjes, A. Fahr, S. Pappinen, S. Ronkko, M. Suhonen, A. Urtti,
Interaction of lipid nanoparticles with human epidermis and an organotypic cell culture
model, Int J Pharm, 354 (2008) 180-195.
[132] S. Kuchler, M.R. Radowski, T. Blaschke, M. Dathe, J. Plendl, R. Haag, M. Schafer-
Korting, K.D. Kramer, Nanoparticles for skin penetration enhancement--a comparison of a
dendritic core-multishell-nanotransporter and solid lipid nanoparticles, Eur J Pharm
Biopharm, 71 (2009) 243-250.
[133] S. Khurana, P.M. Bedi, N.K. Jain, Preparation and evaluation of solid lipid nanoparticles
based nanogel for dermal delivery of meloxicam, Chem Phys Lipids, 175-176 (2013) 65-72.
[134] J. Zhang, E. Smith, Percutaneous permeation of betamethasone 17-valerate
incorporated in lipid nanoparticles, J Pharm Sci, 100 (2011) 896-903.
[135] S.E. Jin, C.K. Kim, Charge-mediated topical delivery of plasmid DNA with cationic lipid
nanoparticles to the skin, Colloids Surf B Biointerfaces, 116 (2014) 582-590.
[136] R.H. Muller, V. Jenning, K. Mader, A. Lippacher, Lipid particles on the basis of mixtures
of liquid and solid lipids and method for producing same. US 8663692, (1999).
[137] S.A. Wissing, R.H. Müller, Cosmetic applications for solid lipid nanoparticles (SLN),
International Journal of Pharmaceutics, 254 (2003) 65-68.
[138] R.H. Müller, R.D. Petersen, A. Hommoss, J. Pardeike, Nanostructured lipid carriers
(NLC) in cosmetic dermal products, Advanced Drug Delivery Reviews, 59 (2007) 522-530.
[139] K. Jores, A. Haberland, S. Wartewig, K. Mader, W. Mehnert, Solid lipid nanoparticles
(SLN) and oil-loaded SLN studied by spectrofluorometry and Raman spectroscopy, Pharm
Res, 22 (2005) 1887-1897.
[140] M. Uner, Preparation, characterization and physico-chemical properties of solid lipid
nanoparticles (SLN) and nanostructured lipid carriers (NLC): their benefits as colloidal drug
carrier systems, Pharmazie, 61 (2006) 375-386.
[141] Q. Xia, A. Saupe, R.H. Muller, E.B. Souto, Nanostructured lipid carriers as novel carrier
for sunscreen formulations, Int J Cosmet Sci, 29 (2007) 473-482.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
35
[142] S. Chen, W. Liu, J. Wan, X. Cheng, C. Gu, H. Zhou, S. Chen, X. Zhao, Y. Tang, X. Yang,
Preparation of Coenzyme Q10 nanostructured lipid carriers for epidermal targeting with
high-pressure microfluidics technique, Drug Dev Ind Pharm, 39 (2013) 20-28.
[143] P.V. Pople, K.K. Singh, Development and evaluation of colloidal modified nanolipid
carrier: application to topical delivery of tacrolimus, Part II--in vivo assessment, drug
targeting, efficacy, and safety in treatment for atopic dermatitis, Eur J Pharm Biopharm, 84
(2013) 72-83.
[144] P.V. Pople, K.K. Singh, Development and evaluation of colloidal modified nanolipid
carrier: application to topical delivery of tacrolimus, Eur J Pharm Biopharm, 79 (2011) 82-94.
[145] J.Y. Fang, C.L. Fang, C.H. Liu, Y.H. Su, Lipid nanoparticles as vehicles for topical
psoralen delivery: solid lipid nanoparticles (SLN) versus nanostructured lipid carriers (NLC),
Eur J Pharm Biopharm, 70 (2008) 633-640.
[146] N.K. Garg, R.K. Tyagi, B. Singh, G. Sharma, P. Nirbhavane, V. Kushwah, S. Jain, O.P.
Katare, Nanostructured lipid carrier mediates effective delivery of methotrexate to induce
apoptosis of rheumatoid arthritis via NF-kappaB and FOXO1, Int J Pharm, 499 (2016) 301-
320.
[147] S. Singh, M. Singh, C.B. Tripathi, M. Arya, S.A. Saraf, Development and evaluation of
ultra-small nanostructured lipid carriers: novel topical delivery system for athlete's foot,
Drug Deliv Transl Res, 6 (2016) 38-47.
[148] M. Pradhan, D. Singh, S.N. Murthy, M.R. Singh, Design, characterization and skin
permeating potential of Fluocinolone acetonide loaded nanostructured lipid carriers for
topical treatment of psoriasis, Steroids, 101 (2015) 56-63.
[149] A. Qidwai, S. Khan, S. Md, M. Fazil, S. Baboota, J.K. Narang, J. Ali, Nanostructured lipid
carrier in photodynamic therapy for the treatment of basal-cell carcinoma, Drug Deliv, 23
(2016) 1476-1485.
[150] P.R. Desai, P.P. Shah, R.R. Patlolla, M. Singh, Dermal microdialysis technique to
evaluate the trafficking of surface-modified lipid nanoparticles upon topical application,
Pharm Res, 29 (2012) 2587-2600.
[151] R.R. Patlolla, P.R. Desai, K. Belay, M.S. Singh, Translocation of cell penetrating peptide
engrafted nanoparticles across skin layers, Biomaterials, 31 (2010) 5598-5607.
[152] P.P. Shah, P.R. Desai, D. Channer, M. Singh, Enhanced skin permeation using
polyarginine modified nanostructured lipid carriers, J Control Release, 161 (2012) 735-745.
[153] G. Cevc, D. Gebauer, J. Stieber, A. Schatzlein, G. Blume, Ultraflexible vesicles,
transfersomes, have an extremely low pore penetration resistance and transport
therapeutic amounts of insulin across the intact mammalian skin, Bba-Biomembranes, 1368
(1998) 201-215.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
36
[154] G. Cevc, G. Blume, New, highly efficient formulation of diclofenac for the topical,
transdermal administration in ultradeformable drug carriers, Transfersomes, Bba-
Biomembranes, 1514 (2001) 191-205.
[155] G. Cevc, Rational design of new product candidates: the next generation of highly
deformable bilayer vesicles for noninvasive, targeted therapy, J Control Release, 160 (2012)
135-146.
[156] M.M. Elsayed, O.Y. Abdallah, V.F. Naggar, N.M. Khalafallah, Deformable liposomes and
ethosomes: mechanism of enhanced skin delivery, Int J Pharm, 322 (2006) 60-66.
[157] D. Ainbinder, E. Touitou, Testosterone ethosomes for enhanced transdermal delivery,
Drug Deliv, 12 (2005) 297-303.
[158] B. Godin, E. Touitou, Ethosomes: new prospects in transdermal delivery, Critical
reviews in therapeutic drug carrier systems, 20 (2003) 63-102.
[159] A. Ahad, M. Aqil, K. Kohli, Y. Sultana, M. Mujeeb, Enhanced transdermal delivery of an
anti-hypertensive agent via nanoethosomes: statistical optimization, characterization and
pharmacokinetic assessment, Int J Pharm, 443 (2013) 26-38.
[160] R. Rakesh, K.R. Anoop, Formulation and optimization of nano-sized ethosomes for
enhanced transdermal delivery of cromolyn sodium, Journal of pharmacy & bioallied
sciences, 4 (2012) 333-340.
[161] A. Manosroi, R. Chutoprapat, Y. Sato, K. Miyamoto, K. Hsueh, M. Abe, W. Manosroi, J.
Manosroi, Antioxidant activities and skin hydration effects of rice bran bioactive compounds
entrapped in niosomes, J Nanosci Nanotechnol, 11 (2011) 2269-2277.
[162] D. Paolino, R. Muzzalupo, A. Ricciardi, C. Celia, N. Picci, M. Fresta, In vitro and in vivo
evaluation of Bola-surfactant containing niosomes for transdermal delivery, Biomedical
microdevices, 9 (2007) 421-433.
[163] S. Mura, F. Pirot, M. Manconi, F. Falson, A.M. Fadda, Liposomes and niosomes as
potential carriers for dermal delivery of minoxidil, J Drug Target, 15 (2007) 101-108.
[164] M. Tabbakhian, N. Tavakoli, M.R. Jaafari, S. Daneshamouz, Enhancement of follicular
delivery of finasteride by liposomes and niosomes 1. In vitro permeation and in vivo
deposition studies using hamster flank and ear models, Int J Pharm, 323 (2006) 1-10.
[165] M. Manconi, C. Sinico, D. Valenti, F. Lai, A.M. Fadda, Niosomes as carriers for tretinoin:
III. A study into the in vitro cutaneous delivery of vesicle-incorporated tretinoin, Int J Pharm,
311 (2006) 11-19.
[166] M.M. Wen, R.M. Farid, A.A. Kassem, Nano-proniosomes enhancing the transdermal
delivery of mefenamic acid, J Liposome Res, (2014).
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
37
[167] N. Dragicevic-Curic, D. Scheglmann, V. Albrecht, A. Fahr, Temoporfin-loaded
invasomes: development, characterization and in vitro skin penetration studies, J Control
Release, 127 (2008) 59-69.
[168] N. Dragicevic-Curic, D. Scheglmann, V. Albrecht, A. Fahr, Development of different
temoporfin-loaded invasomes-novel nanocarriers of temoporfin: characterization, stability
and in vitro skin penetration studies, Colloids Surf B Biointerfaces, 70 (2009) 198-206.
[169] N. Dragicevic-Curic, S. Grafe, B. Gitter, A. Fahr, Efficacy of temoporfin-loaded
invasomes in the photodynamic therapy in human epidermoid and colorectal tumour cell
lines, Journal of photochemistry and photobiology. B, Biology, 101 (2010) 238-250.
[170] S. Bracke, M. Carretero, S. Guerrero-Aspizua, E. Desmet, N. Illera, M. Navarro, J.
Lambert, M. Del Rio, Targeted silencing of DEFB4 in a bioengineered skin-humanized mouse
model for psoriasis: development of siRNA SECosome-based novel therapies, Exp Dermatol,
23 (2014) 199-201.
[171] S. Mura, M. Manconi, C. Sinico, D. Valenti, A.M. Fadda, Penetration enhancer-
containing vesicles (PEVs) as carriers for cutaneous delivery of minoxidil, International
Journal of Pharmaceutics, 380 (2009) 72-79.
[172] L.R. Hart, S. Li, C. Sturgess, R. Wildman, J.R. Jones, W. Hayes, 3D Printing of
Biocompatible Supramolecular Polymers and their Composites, ACS applied materials &
interfaces, 8 (2016) 3115-3122.
[173] G.M. El Maghraby, A.C. Williams, B.W. Barry, Interactions of surfactants (edge
activators) and skin penetration enhancers with liposomes, Int J Pharm, 276 (2004) 143-161.
[174] M.M.A. Elsayed, O.Y. Abdallah, V.F. Naggar, N.M. Khalafallah, Lipid vesicles for skin
delivery of drugs: Reviewing three decades of research, International Journal of
Pharmaceutics, 332 (2007) 1-16.
[175] M.L. Manca, M. Zaru, M. Manconi, F. Lai, D. Valenti, C. Sinico, A.M. Fadda,
Glycerosomes: A new tool for effective dermal and transdermal drug delivery, International
Journal of Pharmaceutics, 455 (2013) 66-74.
[176] M. Manconi, C. Caddeo, C. Sinico, D. Valenti, M.C. Mostallino, G. Biggio, A.M. Fadda,
Ex vivo skin delivery of diclofenac by transcutol containing liposomes and suggested
mechanism of vesicle-skin interaction, Eur J Pharm Biopharm, 78 (2011) 27-35.
[177] N. Dragicevic-Curic, A. Fahr, Liposomes in topical photodynamic therapy, Expert Opin
Drug Deliv, 9 (2012) 1015-1032.
[178] N. Dragicevic-Curic, S. Grafe, B. Gitter, S. Winter, A. Fahr, Surface charged temoporfin-
loaded flexible vesicles: in vitro skin penetration studies and stability, Int J Pharm, 384
(2010) 100-108.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
38
[179] M. Dorrani, O.B. Garbuzenko, T. Minko, B. Michniak-Kohn, Development of edge-
activated liposomes for siRNA delivery to human basal epidermis for melanoma therapy, J
Control Release, 228 (2016) 150-158.
[180] J. Brewer, M. Bloksgaard, J. Kubiak, J.A. Sorensen, L.A. Bagatolli, Spatially resolved
two-color diffusion measurements in human skin applied to transdermal liposome
penetration, J Invest Dermatol, 133 (2013) 1260-1268.
[181] A.C. Williams, B.W. Barry, Penetration enhancers, Advanced Drug Delivery Reviews, 56
(2004) 603-618.
[182] P.L. Honeywell-Nguyen, G.S. Gooris, J.A. Bouwstra, Quantitative assessment of the
transport of elastic and rigid vesicle components and a model drug from these vesicle
formulations into human skin in vivo, J Invest Dermatol, 123 (2004) 902-910.
[183] H. Heerklotz, Interactions of surfactants with lipid membranes, Q Rev Biophys, 41
(2008) 205-264.
[184] J. Dreier, J.A. Sorensen, J.R. Brewer, Superresolution and Fluorescence Dynamics
Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier, PLoS One, 11
(2016) e0146514.
[185] J.E. Grice, T.W. Prow, M.A.F. Kendall, M.S. Roberts, Electrical and physical methods of
skin penetration enhancement, in: H.A.E. Benson, A.C. Watkinson (Eds.) Topical and
transdermal drug delivery: Principles and practice, Wiley, Hoboken NJ, 2012, pp. 43-65.
[186] S. Mitragotri, Devices for overcoming biological barriers: the use of physical forces to
disrupt the barriers, Adv Drug Deliv Rev, 65 (2013) 100-103.
[187] Future of Transdermal Drug Delivery Systems (TDDS),
http://www.americanpharmaceuticalreview.com/Featured-Articles/163672-Future-of-
Transdermal-Drug-Delivery-Systems-TDDS/, 2014, Cited on 07/25/2016.
[188] S.R. Lahoti, S. Dalal, Synergistic iontophoretic drug delivery of risedronate sodium in
combination with electroporation and chemical penetration enhancer: In-vitro and in-vivo
evaluation, Asian J Pharm, 9 (2015) 171-177.
[189] A. Alexander, S. Dwivedi, Ajazuddin, T.K. Giri, S. Saraf, S. Saraf, D.K. Tripathi,
Approaches for breaking the barriers of drug permeation through transdermal drug delivery,
J Control Release, 164 (2012) 26-40.
[190] R.F. Donnelly, M.J. Garland, A.Z. Alkilani, Microneedle-iontophoresis combinations for
enhanced transdermal drug delivery, Methods Mol Biol, 1141 (2014) 121-132.
[191] T. Han, D.B. Das, Potential of combined ultrasound and microneedles for enhanced
transdermal drug permeation: a review, Eur J Pharm Biopharm, 89 (2015) 312-328.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
39
[192] Zinc oxide (nano form),
http://ec.europa.eu/health/scientific_committees/opinions_layman/zinc-oxide/en/l-
3/6.htm, 2015, Cited on 28/07/2016.
[193] M.N. Todosijevic, M.M. Savic, B.B. Batinic, B.D. Markovic, M. Gasperlin, D.V.
Randelovic, M.Z. Lukic, S.D. Savic, Biocompatible microemulsions of a model NSAID for skin
delivery: A decisive role of surfactants in skin penetration/irritation profiles and
pharmacokinetic performance, Int J Pharm, 496 (2015) 931-941.
[194] A. Gurbuz, G. Ozhan, S. Gungor, M.S. Erdal, Colloidal carriers of isotretinoin for topical
acne treatment: skin uptake, ATR-FTIR and in vitro cytotoxicity studies, Arch Dermatol Res,
307 (2015) 607-615.
[195] C. Caddeo, M. Manconi, M.C. Cardia, O. Diez-Sales, A.M. Fadda, C. Sinico, Investigating
the interactions of resveratrol with phospholipid vesicle bilayer and the skin: NMR studies
and confocal imaging, Int J Pharm, 484 (2015) 138-145.
[196] M. Carboni, A.M. Falchi, S. Lampis, C. Sinico, M.L. Manca, J. Schmidt, Y. Talmon, S.
Murgia, M. Monduzzi, Physicochemical, cytotoxic, and dermal release features of a novel
cationic liposome nanocarrier, Advanced healthcare materials, 2 (2013) 692-701.
[197] D. Cosco, D. Paolino, J. Maiuolo, L.D. Marzio, M. Carafa, C.A. Ventura, M. Fresta,
Ultradeformable liposomes as multidrug carrier of resveratrol and 5-fluorouracil for their
topical delivery, Int J Pharm, 489 (2015) 1-10.
[198] P.A. Raju, N. McSloy, N.K. Truong, M.A. Kendall, Assessment of epidermal cell viability
by near infrared multi-photon microscopy following ballistic delivery of gold micro-particles,
Vaccine, 24 (2006) 4644-4647.
[199] Y. Deng, A. Ediriwickrema, F. Yang, J. Lewis, M. Girardi, W.M. Saltzman, A sunblock
based on bioadhesive nanoparticles, Nat Mater, 14 (2015) 1278-1285.
[200] A. Vogt, C. Wischke, A.T. Neffe, N. Ma, U. Alexiev, A. Lendlein, Nanocarriers for drug
delivery into and through the skin - do existing technologies match clinical challenges?, J
Control Release, (2016).
[201] T.M. Nguyen, S. Lee, S.B. Lee, Conductive polymer nanotube patch for fast and
controlled ex vivo transdermal drug delivery, Nanomedicine, 9 (2014) 2263-2272.
[202] S. Hansen, C.M. Lehr, Transfollicular delivery takes root: the future for vaccine
design?, Expert Rev Vaccines, 13 (2014) 5-7.
[203] M. Karimi, A. Ghasemi, P. Sahandi Zangabad, R. Rahighi, S.M. Moosavi Basri, H.
Mirshekari, M. Amiri, Z. Shafaei Pishabad, A. Aslani, M. Bozorgomid, D. Ghosh, A. Beyzavi, A.
Vaseghi, A.R. Aref, L. Haghani, S. Bahrami, M.R. Hamblin, Smart micro/nanoparticles in
stimulus-responsive drug/gene delivery systems, Chem Soc Rev, 45 (2016) 1457-1501.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
40
[204] S. Wang, W. Yang, J. Cui, X. Li, Y. Dou, L. Su, J. Chang, H. Wang, X. Li, B. Zhang, pH- and
NIR light responsive nanocarriers for combination treatment of chemotherapy and
photodynamic therapy, Biomater Sci, 4 (2016) 338-345.
[205] A. Patzelt, W.C. Mak, S. Jung, F. Knorr, M.C. Meinke, H. Richter, E. Ruhl, K.Y. Cheung,
N.B. Tran, J. Lademann, Do nanoparticles have a future in dermal drug delivery?, J Control
Release, (2016).
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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