Advance Drug Delivery Reviews

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Nanoparticles and microparticles for skin drug delivery

Tarl W. Prow a, Jeffrey E. Grice a, Lynlee L. Lin a, Rokhaya Faye a, Margaret Butler c, Wolfgang Becker d,Elisabeth M.T. Wurm e, Corinne Yoong e, Thomas A. Robertson a,b, H. Peter Soyer e, Michael S. Roberts a,b,a The University of Queensland, School of Medicine, Therapeutics Research Centre, Brisbane, QLD, Australiab The University of South Australia, Therapeutics Research Centre, School of Pharmacy and Medical Science, Adelaide, SA, Australiac Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australiad Becker & Hickl, GmbH, Berlin, Germanye Dermatology Research Centre, The University of Queensland, School of Medicine, Princess Alexandra Hospital, Brisbane, QLD, Australia

a b s t r a c ta r t i c l e i n f o

Article history:

Received 21 September 2010

Accepted 31 January 2011

Available online 23 February 2011

Keywords:

Nanoparticle

Drug delivery

Topical

Skin

Percutaneous penetration

Transdermal delivery

Skin is a widely used route of delivery for local and systemic drugs and is potentially a route for their delivery

as nanoparticles. The skin provides a natural physical barrier against particle penetration, but there are

opportunities to deliver therapeutic nanoparticles, especially in diseased skin and to the openings of hair

follicles. Whilst nanoparticle drug delivery has been touted as an enabling technology, its potential in treating

local skin and systemic diseases has yet to be realised. Most drug delivery particle technologies are based on

lipid carriers, i.e. solid lipid nanoparticles and nanoemulsions of around 300 nm in diameter, which are now

considered microparticles. Metal nanoparticles are now recognized for seemingly small drug-like

characteristics, i.e. antimicrobial activity and skin cancer prevention. We present our unpublished clinical

data on nanoparticle penetration and previously published reports that support the hypothesis that

nanoparticles N10 nm in diameter are unlikely to penetrate through the stratum corneum into viable human

skin but will accumulate in the hair follicle openings, especially after massage. However, significant uptake

does occur after damage and in certain diseased skin. Current chemistry limits both atom by atom

construction of complex particulates and delineating their molecular interactions within biological systems.

In this review we discuss the skin as a nanoparticle barrier, recent work in the field of nanoparticle drug

delivery to the skin, and future directions currently being explored. 2011 Elsevier B.V. All rights reserved.

Contents

1. Skin as a site for particle delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471

1.1. Skin as a natural particle barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471

1.2. Barrier properties of the skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472

1.2.1. Skin structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472

1.2.2. The stratum corneum barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473

1.2.3. Transport routes of exogenous substances across the stratum corneum . . . . . . . . . . . . . . . . . . . . . . . . . . . 473

1.2.4. Acid mantle maintains the stratum corneum barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473

1.2.5. Viable epidermis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473

1.2.6. Hair follicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4741.3. Barrier properties of diseased skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474

1.4. Skin flexing and massage for enhanced delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474

2. Small drug particle delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475

2.1. Anti-inflammatory drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475

2.1.1. Corticosterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475

2.1.2. Flufenamic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476

Advanced Drug Delivery Reviews 63 (2011) 470491

This review is part of the Advanced Drug Delivery Reviews theme issue on Nanodrug Particles and Nanoformulations for Drug Delivery.

Corresponding author at: The University of Queensland, Therapeutics Research Unit, School of Medicine, Princess Alexandra Hospital, Woolloongabba, QLD, 4102, Australia.

Tel.: +61 7 3176 2546.

E-mail address: [email protected] (M.S. Roberts).

0169-409X/$ see front matter 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.addr.2011.01.012

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r
http://dx.doi.org/10.1016/j.addr.2011.01.012http://dx.doi.org/10.1016/j.addr.2011.01.012http://dx.doi.org/10.1016/j.addr.2011.01.012mailto:[email protected]://dx.doi.org/10.1016/j.addr.2011.01.012http://www.sciencedirect.com/science/journal/0169409Xhttp://www.sciencedirect.com/science/journal/0169409Xhttp://dx.doi.org/10.1016/j.addr.2011.01.012mailto:[email protected]://dx.doi.org/10.1016/j.addr.2011.01.012


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2.2. Anti-photoageing drugs and antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478

2.2.1. Retinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478

2.2.2. Tocopheryl acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

2.2.3. Multiphoton imaging for in vivo lipid nanoparticle delivery studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

3. Other topical nanoparticle drug delivery applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

3.1. Antimicrobial agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

3.1.1. Antifungal drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

3.1.2. Silver nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

3.2. Anti-proliferative agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483

3.2.1. 5-aminolevulinic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4833.2.2. Podophyllotoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484

3.3. Other topical particulate delivered drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484

4. Recent advances in particle-based drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485

5. Systemic safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486

6. Limitations of nanoparticle and nanocarrier chemical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486

7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486

Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487

1. Skin as a site for particle delivery

The theory and practical aspects of percutaneous penetration of

drugs, particulate material and contaminants have been covered in a

number of excellent reference texts. For comprehensive treatment of

the topics in this section, the reader is referred to chapters by Roberts

et al. [1], Norlen [2], Monteiro-Riviere and Baroli [3], Mller et al. [4]

and other relevant chapters in these books.

Whilst the skin has historically been used for the topical delivery of

compounds, it is only since the 1970s with the advent of transdermal

patches that it has widely been used as a route for systemic delivery [5].

Nanoparticle delivery to the skin is being increasingly used to facilitate

local therapies. The nanoparticle definition designated by the National

Nanotechnology Initiative has been adopted by the American National

Standards Institute as particles with all dimensions between 1 nm and

100 nm [6,7]. Fig. 1 shows that the potential sites for targeting

nanoparticles include the surface of the skin, furrows, and hair follicles.

A recent review by Baroli discusses nanoparticle penetration largely

from the skin structure perspective.The title of this reviewPenetration

of Nanoparticles and Nanomaterials in the Skin: Fiction or Reality?

highlights the ongoing debate of nanoparticle penetration [8]. This

debate is fuelled by the need for more rigorous, multidisciplinary

approaches to shed light on mechanisms of particle penetration and

interactionswith skin. Likewise,Schneider et al. make a compelling case

in their review on nanoparticle skin interactions for more rigorous

studies on the effects of hydration and mechanical stress on skin with

regard to nanoparticleskin interactions [9].

Particles can interact with skin at a cellular level as adjuvants. This

nanoparticleskin interaction can be used to enhance immune

reactivity for topical vaccine applications [10]. Another example of

nanoparticleskin interactions the topical use of silver nanoparticles

as over the counter antimicrobial agents [11], where the nanoparticles

provide a slow release of silver ions that have wound healing and

antimicrobial properties. The silver ions released from nanoparticle

can inhibit microbial proliferation, but also accelerate wound healing.

This controlled release of silver ions while the nanoparticles remainon the skin surface highlights one of the most successful topical

nanoparticle drug delivery strategies.

Generally, the promise of nanoparticle-mediated drug delivery into

the epidermis and dermis without barrier modification has met with

little success. Where the barrier is compromised, however, such as in

aged or diseased skin, there may be potential for enhanced particle

penetration. Ulcerated squamous cell carcinoma is one example. The

opportunities and obstacles for nanoparticle drug delivery are only just

beginning to be explored in ongoing clinical trials. For instance,

capsaicin loaded nanoparticles are being used to treat the pain

associated with diabetic neuropathy [12]. Advances in particle engi-

neering, formulation science and an improved understanding of

nanoparticleskin interactions will undoubtedly lead to important

clinically relevant improvements in topicaldrug delivery. An overridingconcern is the safety of any applied nanoparticle, recognising the

possibility that non-biodegradable nanoparticles could be taken up and

retained by the reticulo-endothelial system [13]. Inaddition, there isthe

potential for local toxicity, shown by a recent report of nanoparticles

inducing keratinocyte apoptosis, wherethe subtlerelationship between

longer and shorter phosphatidylcholine chain lengths makes the

difference between life and death for keratinocytes [14]. This illustrates

the need for toxicity monitoring in vitro and especially in vivo.

1.1. Skin as a natural particle barrier

It is generally recognised that the potential for nanoparticle and

microparticle skin penetration is negligible [15]. Most environmental

Fig. 1. Sites in skin for nanoparticle delivery. Topical nanoparticle drug delivery takes

place in three major sites: stratum corneum (SC) surface (panel a), furrows

(dermatoglyphs) (panel b), and openings of hair follicles (infundibulum) (c). The

nanoparticles are shown in green and the drug in red. Other sites for delivery are the

viable epidermis (E) and dermis (D).

471T.W. Prow et al. / Advanced Drug Delivery Reviews 63 (2011) 470 491


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nanoparticles, be they viruses, bacteria, dust, allergens or materials,

do not penetrate human skin unless the skin barrier is disrupted

[13,16]. Topical drug delivery with nanoparticles, with the aim of

targeting the nanoparticles into the deeper layers of skin, therefore

has to overcome a significant barrier honed over millions of years of

evolution. Human papilloma virus (HPV) is perhaps the best example

of a nanoparticle able to penetrate barrier compromised skin to cause

skin warts [17]. Today, biomimetic nanoparticle engineering is

applying these principles from nature to develop better nanoparticledelivery systems [18] and progress is being made in the area of

nanoparticle interactions with skin [9]. HPV is also in the same size

range [19] as topical nanoparticles for drug delivery in skin [20] and

both utilize lipids to facilitate payload delivery (Fig. 2).

The benefits of drug delivery resulting from topical penetration of

nanoparticles are offset by their potential for toxicity, as discussed

above. This has led to the topic of environmental and synthetic

particle penetration across human skin becoming a contentious

subject that has attracted the attention of regulatory agencies around

the world.

The likelihood of nanoparticle penetration across the skin has

recently been reviewed by the Scientific Committee on Consumer

Products (SCCP) who conclude that in relation to dermal exposure

[21]:

1) There is evidence of some skin penetration into viable tissues

(mainly into the stratum spinosum in the epidermal layer, but

eventually also into the dermis) for very small particles (less than

10 nm), such as functionalised fullerenes and quantum dots.

2) When using accepted skin penetration protocols (intact skin),

there is no conclusive evidence for skin penetration into viable

tissue for particles of about 20 nm and larger primary particle size

as used in sunscreens with physical UV-filters.

3) The above statements on skin penetration apply to healthy skin

(human and porcine). There is an absence of appropriate

information for skin with impaired barrier function, e.g. atopic

skin or sunburned skin. A few data are available on psoriatic skin.

4) There is evidence that some mechanical effects (e.g. flexing) on

skin may have an effect on nanoparticle penetration.5) There is no information on the transadnexal penetration for

particles under 20 nm. Nanoparticles of 20 nm and above

penetrate deeply into hair follicles, but no penetration into viable

tissue has been observed.

The statement that nanoparticles N20 nm in diameter do not

penetrate into viable tissue is controversial. There have been reports

showing nanoparticles N20 nm penetrating through the stratum

corneum (SC), to the viableepidermis[22,23]. However, our experience

with gold, silver and quantum dots illustrates that these nanoparticles

do consistently penetrate into the SC, but not into the viable epidermis.

The reasons for this important disparity may be due to differences in

nanoparticle constituents, models, and methodologies.

We have also investigated nanoparticle gene, vaccine, siRNA, and

drug delivery in a variety of systems, including skin [2436]. Our past

and present focus in the field of topical therapeutic nanoparticles is

developing a better understanding of nanoparticle disposition and

toxicity [16,3740]. The most common tool used to evaluate

nanoparticle penetration, delivery, toxicity or localization is the

confocal microscope, illustrating that some primary aspects we areinvestigating are spatial and concentration related. In other words,

where are the nanoparticles and how much is there? The Monteiro-

Riviere, Lademann, Guy and Roberts groups are currently investigat-

ing nanoparticle penetration, delivery, toxicity and species differ-

ences. The Monteiro-Riviere group have made significant

contributions towards understanding nanoparticle penetration, tox-

icity and model systems, and hasgenerated an extensivebody of work

that has largely focused on nanoparticle interactions with skin and

skin cells. These reports have illuminated molecular mechanisms of

nanoparticle uptake, toxicity, and bioretention [22,4164]. The

Lademann group has made significant contributions towards a better

understanding of the role hair follicles play as nanoparticle reservoirs

[16,6576]. The Guy group has also contributed to this are and has

investigated nanoparticle versus model drug localization in skin

compartments, revealing valuable information about drug and

nanoparticle distributions in skin [7781]. The Roberts groups is

using novel fluorescence lifetime imaging techniques to simulta-

neously evaluate NAD(P)H lifetime changes with nanoparticle

penetration [40,82,83].

1.2. Barrier properties of the skin

Among the multiple, complex functions of mammalian skin, one of

its major roles is to prevent invasion of the organism by acting as a

defensive barrier to threats from the external environment. The skin

has evolved defensive mechanisms which give it physical, immuno-

logical, metabolic and UV-protective barriers to allow it to inhibit

attacks by microbes, toxic chemicals, UV radiation and particulate

matter (including nanoparticles, which may occur in the naturalenvironment)[84]. Ontheotherhand, the skincan beusedas a portof

entry for therapeutic substances such as drugs and vaccines if the

mechanisms that confer the barrier properties are understood and

exploited.

1.2.1. Skin structure

Skin consists of two main layers. The underlying dermis contains a

variety of cell types, nerves, blood vessels and lymphatics embedded

in a dense network of connective tissue. Above the dermis and

separated from it by the basement membrane, the epidermis is

Fig. 2. Electron microscopy comparison of size and morphology of a virus (panel a, adapted from [254]) that infects human skin, human papilloma virus, and nanoparticles (panels b

and c) for topical drug delivery are in similar size ranges. The human papilloma virus is 40 nm in diameter, the same size range as smaller drug delivery vehicles like poly(q-

caprolactone)-block-poly(ethyleneglycol) nanoparticles, 40 nm, designed to deliver minoxidil to hair follicles (panel b, adapted from [130]). Solid lipid nanoparticles like these

developed for penciclovir can be several times larger at an average diameter of 250 nm (panel c, adapted from [255]). The bar indicates 200 nm for all panels.

472 T.W. Prow et al. / Advanced Drug Delivery Reviews 63 (2011) 470 491
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composed mostly of layers of stratified keratinocytes, where the SC

cells or corneocytes are bathed in a protein-rich envelope with an

outer lipid envelope, surrounded by an extracellular lipid matrix [85].

Keratinocytes undergo a process of keratinisation, in which the cell

differentiates and moves upward from the basal layer (stratum

basale), through the stratum spinosum and stratum granulosum, to

the outermost layer, the stratum corneum (SC or horny layer). On

reaching the SC, cells become anucleated and flattened and are

eventually sloughed off [86]. Interspersed amongst the keratinocytesin theviable epidermisare cells with roles such as melanin production

(melanocytes), sensory perception (Merkel cells) and immunological

function (Langerhans and other cells). In addition to the structured

cellular components of skin, there are appendages including the

pilosebaceous units (hair follicles and associated sebaceous glands),

apocrine and eccrine sweat glands.

1.2.2. The stratum corneum barrier

TheSC represents themain physical barrier of theskin,so that fora

substance permeating across the skin, diffusion through the SC is the

rate limiting step [1]. Conversely, the SC is also the main barrier for

diffusionof water out of theskin [87]. The flattened, anuclear, protein-

rich corneocytes of the SC are densely packed within the extracellular

lipid matrix which is arranged in bilayers [88]. Thisis often referred to

as a bricks and mortar arrangement [89]. The corneocytes are held

together by corneodesmosomes, which help to form a tough outer

layer by maintaining cellular shape and regular packing. Eventual

degradation of the corneodesmosomes by proteolytic enzymes leads

to desquamation [90].

Transport of substances across the SC occurs mainly by passive

diffusion and based on the dual-compartment bricks and mortar

structure of the SC, interrupted by appendages, is considered to occur

via three possible routes. These are the transcellular, the intercellular

and the appendageal routes.

Covering the corneocytes on the SC surface is a thin (0.410 m),

irregular and discontinuous layer consisting of sebum secreted by the

sebaceous glands, along with sweat, bacteria and dead skin cells. This

layer is considered to have a negligible effect as an additional barrier

to permeation through the SC [91].

1.2.2.1. Inter-cellular spacing. For most penetrants, the intercellular

route is favoured. Small molecules are able to move freely within the

inter-cellular spaces and diffusion rates are governed largely by their

lipophilicity, but also physicochemical properties such as molecular

weight or volume, solubility and hydrogen bonding ability [92].

However, the free movement of macromolecules or particles may be

physically restricted within the lipid channels, which have been

estimated by van de Merwe et al. to be 19 nm [93] and by Baroli et al.

to be 75 nm [23]. This suggests that for such materials, the SC could

present an additional barrier that is not present for small molecules.

1.2.2.2. Skin turnover as a moving barrier. The outer layers of the SC

(the so-called stratum dysjunctum) are subject to desquamation,allowing the SC to be completely turned over in a period of about

14 days in humans, depending on anatomical site and age [94]. Thus,

these cells might be regarded as a moving, constantly renewable

barrier, providing an inherent mechanism for preventing foreign

bodies from penetrating the skin and gaining a foothold [95]. The

continuous upward migration and sloughing of corneocytes from the

surface might assist in eliminating pathogens, cancerous cells or solid

particulate matter.

1.2.3. Transport routes of exogenous substances across the stratum

corneum

Polar and non-polar solutes were originally thought to permeate

through the SC via separate routes [96], with polar solutes taking a

transcellular routeand morelipophilic solutes goingvia the intercellular

lipids. However a perception of the difficulty of repeated partitioning

between lipophilic and hydrophilic compartments in the SC led to this

pathway being regarded as unlikely in most cases. This was supported

by histochemical [87] and theoretical [97,98] evidence showing that

diffusion through intercellular lipids was more likely for most solutes.

Despite a recent reaffirmation of importance of the transcellular route,

even for lipophilic solutes, by Wang et al. [99] the transcellular route

remains controversial.

With most work focussing on the hair follicles, delivery of drugs orparticles via the appendageal route is regarded as a realistic

alternative to delivery across the SC, despite the relative sparseness

of these features on the skin surface [100]. The follicles extend deep

into the skin, the thickness of the SC layer is progressively reduced as

it extends into the structure and there is a rich capillary blood supply

available to transport solutes diffusing out of the follicle. There is

considerable interest in targeted follicular delivery with tailored drug

formulations [98] or nanoparticle-bound drugs [101]. There have also

been suggestions that partitioning of drugs into sebum itself may be a

potential delivery route [100].

1.2.4. Acid mantle maintains the stratum corneum barrier

The surface of theskin haslong been recognised to be acidic, with a

pH of 4.25.6 measured in humans by Blank over 70 years ago [102].

This acidic skin surface is described as the acid mantle. The surface pH

is influenced by sex and anatomical site [103], sweat, sebum and

hydration [104]. There isa sharp gradientacross the SC, with the pH in

the upper viable layers in the stratum granulosum approaching

neutral. Using two-photon fluorescence lifetime imaging of a pH

dependent fluorophore applied to the SC of hairless mice, Hanson et

al. [103] identified acidic microdomains in the extracellular matrix,

which became less frequent away from the surface towards the viable

epidermis. This, they suggested, accounted for the pH gradient,

although specific biochemical mechanisms have been proposed to

account for the acidification per se, including generation of trans-

urocanic acid by histidase-catalysed degradation of histidine [105].

The acid mantle hasa numberof functions, includingantimicrobial

defence [106], the maintenance of the permeability barrier by effects

on extracellular lipid organisation and processing [107], the preser-vation of optimal corneocyte integrity and cohesion, regulated by pH-

sensitive proteolytic enzymes [108] and restriction of inflammation

by inhibiting therelease of pro-inflammatory cytokines [109]. There is

a clear association between elevated skin pH and diseases such as

atopic dermatitis [102], with significant pH differences between

affected and unaffected skin in individual patients [110].

Interestingly, theacidic pH of theskin surface mayalso support the

skin's barrier to nanoparticle penetration in some cases, as carbox-

ylated polystyrene nanoparticles were found to aggregate due to

decreasing electrostatic forces as the solution pH was lowered [111].

Aggregates would be less likelyto penetratethe SC.On theotherhand,

by maintaining the integrity and cohesion of the SC, embedded

particles would be less likely to be sloughed off during desquamation.

Further, for certain nanoparticles, such as zinc oxide, the acidic pHmay greatly affect the nanoparticle aggregation and dissolution

kinetics, thereby confounding experiments on skin penetration

[112,113].

1.2.5. Viable epidermis

1.2.5.1. Tight junctions. The existence of functional tight junctions has

been demonstrated in mammalian stratum granulosum [114,115],

although many constituent tight junction proteins have been

identified in other epithelial layers, as well as follicles [116]. Tight

junctions are regarded as important elements of the epidermal barrier

system and localization and expression of tight junction proteins have

been shown to be altered in diseases characterised by a compromised

skin barrier, such as psoriasis [117,118].



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1.2.5.2. Skin deactivation of nanoparticles by skin metabolism and other

mechanisms. As well as acting as a physical barrier, the skin functions

as a chemicalor metabolic barrier, with enzymes mainlylocated in the

basal layer of the viable epidermis [119], as well as the extracellular

spaces of the SC [120] and the appendages in the dermis [119]. A

number of nanoparticles are biodegradable through hydrolysis,

enzyme activity and physical forces causing, for instance, liposomes

to merge with intercellular lipids [86]. Zinc oxide nanoparticles

hydrolyse at neutral pH and even more so at acidic pHs, leading totheir conversion to zinc ions [37,112,113], which is accelerated on

exposure to light [112]. This hydrolysis can be, however, complex and

affected by the local environment. Cross et al. showed that there was

no significant difference in the levels of zinc ions passing through the

human epidermis from nanoparticle zinc oxide formulations relative

to controls [37]. More recently, Gulson and colleagues showed

increased recovery of 68Zn in blood and urine following application

of 68Zn-enriched zinc oxide particles to human volunteers and

concluded that zinc was able to penetrate the skin barrier. They

were unable, however, to distinguish between the penetration of solid

or solubilised zinc oxide,or zinc ions [121]. Skin metabolism following

topical delivery has often been referred to a first-pass effect,

analogous to that seen in the liver [84]. With nanoparticles, a similar

phenomenon applies but arises from the destruction of nanoparticles

not only by enzymatic processes but also by physical and chemical

processes as discussed above. In the same way that skin metabolism

may be exploited in a pro-drug or soft-drug approach to delivery

[122], there is a potential for similar exploitation of nanoparticles in

drug delivery. Indeed, the effective delivery of drugs to the skin by

nanometer scale (b100 nm) liposomes, formed from engineered lipid

nanovesicles, hasbeen suggested to involve the release of drugs in the

intercellular lipid layer [36].

There is also a wide range of protein transporters present in skin

[123] which may assist in active transport of drugs or other

compounds. Nanoparticle uptake in keratinocytes is, however, more

likely to occur through other specialised processes. For instance,

Zhang et al. have shown that 18 nm quantum dots (QD) with a

carboxylic acid surface coating are taken up in human epidermal

keratinocytes by endocytosis on recognition by lipid rafts [57].Transport involved internalizing into early endosomes before trans-

ferred to late endosomes or lysosomes. The uptake of solid lipid

nanoparticles (b180 nm) into keratinocytes has also been studied

with it being suggested that they easily traverse the cell membrane,

distribute throughout the cytosol and localize in the perinuclear

region without any toxic effects [124].

Delivery systems based on drugs, pro-drugs, soft-drugs or particle-

bound drugs may need to be designed to exploit, or to overcome, the

complex array of enzymes and transporters awaiting them in the skin

[125,126]. Particle bound drugs must be able to reach a site where

they can be released and as we have seen, the barriers to particle

penetration of the skin are considerable. One possibility of doing this

is to target the follicular route, to be discussed below.

1.2.6. Hair follicles

Whereas hair follicles were regarded as insignificant as potential

routes for drug delivery, covering only 0.1% of the human skin surface

area, their complex vascularisation and deep invagination with a

thinning SC has led to a reappraisal of this view [101]. Work has been

done on assessing the contribution of the follicular route to drug

penetration [127,128], as well as targeted delivery [98]. Follicular

penetration of solid particles, including liposomes [129], minoxidil-

loaded [130], and fluorescent polystyrene [78] nanoparticles has been

demonstrated. Lademann discussed the finding that 300600 nm

particles penetrated follicles best on massage as a consequence of the

distance between the scales on the hairs, and suggested that the

movement of the hair acted as a geared pump to push the particles

into the follicle [70]. They viewed follicles as an efficient reservoir for

nanoparticle-based drug delivery [101], havingpreviously shown that

titanium dioxide particles did not penetrate beyond the follicle [131]

and would be eliminated in time by outward sebumflow. It is possible

also that some follicles may be blocked by a plug of sebum or closed

leading to particle penetration being impeded. Significant penetration

of 40 nm nanoparticles beyond the follicles into epidermal cells can

occur when the hair sheath has been pulled out [75].

1.3. Barrier properties of diseased skin

As stated in the SCCP nanotechnology report [21], there is limited

data on skin penetration of nanoparticles through diseased skin.

Topical nanoparticle drug delivery for local effects is likely to be used

on diseased skin. Among the most common adult skin diseases are

atopicdermatitis (6.9%) and psoriasis(6.6%) [132]. The effects of these

barrier altering diseases on the penetration of nanoparticles are

unknown.

A report by the Australian Government, Department of Health and

Ageing, Therapeutic Goods Administration (TGA) identifies a single

unpublished report that assessed systemic zinc levels in psoriatic

patients and foundno evidence of an increase in systemic zinc [133].

We have carried out pilot clinical studies with the aim of quantifying

nanoparticle penetration in psoriatic and atopic dermatitis lesions.

We and others have investigated the penetration of nanomaterial

contained in sunscreens, the most commonly applied source of

nanoparticles, [16,134137], but there is a gap in the clinical literature

where the penetration of nanoparticles has not been quantified in

subjects with clinically relevant skin diseases like psoriasis and

dermatitis. Hypothetically, nanoparticles could penetrate these

lesions more efficiently due to an altered SC, inflammation and

increased keratinocyte turnover.

Our investigation included zinc oxide nanoparticle (35 nm)

penetration profiling by quantifying second harmonic generation of

the nanoparticles with non-invasive multi-photon imaging using

time-correlated single photon counting (Fig. 3). There is a low level of

background signal that can be seen in untreated skin images, taken en

face, that may be due to melanin, keratin aggregates, or collagen

(Fig. 3a). We observed concentrated zinc oxide nanoparticle signals inlesion furrows (Fig. 3b, red, Furrow and in Fig. 3c, F). The

nanoparticles penetrated laterally from the furrows into the SC, but

remained outside of the viable epidermis in non-lesional and lesional

tissue (Fig. 3c). There was an intense zinc oxide nanoparticle signal

from alltreated skin optical biopsies,but the imagesshow a punctuate

distribution on the SC. Compared to the signal obtained from the

untreated non-lesional (0.230.17 mg/ml at 0 m) and lesional sites

(0.23 0.13 mg/ml at 0 m), no higher signal for zinc oxide

nanoparticles could be detected within the viable epidermis of

sunscreen treated non-lesional (0.17 0.10 mg/ml at 0 m) or

lesional skin (0.220.09 mg/ml at 0 m). These results are consistent

with previous reports [133] and the lack of nanoparticle penetration

may be a mechanism that explains why applying zinc oxide

sunscreens to subjects with psoriasis does not give rise to an increasein systemic zinc levels.

1.4. Skin flexing and massage for enhanced delivery

Nanoparticle skin penetration has the potential for highly

controlled drug delivery in therapeutically relevant concentrations.

The penetration enhancement by skin flexing and massage could

impact topical nanoparticle drug delivery and nanotoxicology. Tinkle

et al. investigated the potential for mechanical stress on skin to

enhance particle penetration [138]. The confocal microscopy results

showed that human skin treated with 1 m beads and then flexed for

60 min resulted in dermal penetration of the 1 m beads. They also

showed rapid penetration through a tear in the skin and significant

penetration into the deeper layers of the SC using scanning electron



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microscopy of SC tape strips. Later, detailed studies by the Monteiro-

Riviere group showed that mechanical flexion in porcine skin

increased the rate of nanoparticle penetration of 3.5 nm modified

fullerenes [48]. However, we replicated Tinkle'sflexing apparatus and

conditions, using excised human skin treated with 35 nm zinc oxide

nanoparticles, and did not find penetration (unpublished work).

Likewise, Lademann et al. has shown no penetration of nanoparticles

into the SC after massaging [72]. Studies on non-massaged and

massagedporcine skinrevealed that massaging 400

700 nm diameterparticles resulted in the highest levels of follicular penetration (Fig. 4)

[72]. An alternative explanation has been advanced by Lekki et al. as

they found 20 nm nanoparticles penetrated as deep as 400 mintothe

follicle [139], a process inconsistent with the much larger sizes of

nanoparticles used by Lademann et al. to support a gearing

mechanism. They suggest that a mechanical process is involved and

is formulation dependent so that certain formulations may be enabling

the mechanical pushing of products into follicles or that inhomoge-

neous formulations are involved. Noting Lademann's other work

showing that 40 nm particlesdo penetratedeep into follicleswhenthe

hair shaft is removed [66], we propose that massaging may push the

hair shaft to one side increasing the available area and depth of the

opening (infundulum). As a consequence, greater penetration of

nanoparticles may be enabled in a formulation, skin type and massage

mechanism dependent manner. Togetherthese data suggest thatthere

may be substantial differences between the penetration and interac-

tions of nano- and micro-particles that depend greatly on the skin

type, nature of applied flexion and massage and formulation used.

2. Small drug particle delivery

2.1. Anti-inflammatory drugs

Anti-inflammatory drugs represent a broad range of molecules,

many with potential for topical delivery. Reports on nanoparticle-

delivered drugs with anti-inflammatory properties for topical use

include: aceclofenac [140], betamethasone-17-valerate [141], cele-

coxib [142], clobetasol propionate [143,144], corticosterone

[145,146], flufenamic acid [147,148], flurbiprofen [149,150], glycyr-

rhetic acid [151], ketoprofen [152,153], naproxen [153155], nime-

sulide [156], prednicarbate [141,157,158], and triptolide [159]. These

drugs can be divided into steroids, e.g. corticosterone, and non-

steroidal anti-inflammatory drugs (NSAIDs), e.g. naproxen. Corticos-

teroids work to reduce inflammation by binding glucocorticoid

receptors, whereas NSAIDs work through inhibiting cyclooxygenase.

2.1.1. Corticosterone

In 2008, Kuntsche et al. investigated the potential for solid lipid

nanoparticles (SLN) (tripalmitate), smectic nanoparticles (cholesteryl

myristate and cholesterylnonanoate),and cubic nanoparticles (glycerol

monooleate)to deliver corticosterone to the skin[146]. TheSLN,smectic

particles, and cubic microparticles were an average of 94 nm, 106 nm,and 361 nm, respectively. The nanoparticles were highly characterized

with electron microscopy, particle sizing, small angle X-ray diffraction,

Fig. 3. Penetration profile of zinc oxide nanoparticles from 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.

FLIM measurements were used to differentiate skin autofluorescence (green) from zinc

oxide nanoparticles (red). The intensity profile of the autofluorescence and zinc oxide

nanoparticles is shown in Panel c and is derived from the white lines in panels a and b.

The nanoparticles aggregate in the furrow region (F, Furrow) that is bordered by the

stratum corneum (SC), that protects the viable epidermis (VE). The bar in panel b

indicates 50 m.



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and differential scanning calorimetry. Nanoparticle skin interactions

were monitored with fluorescence microscopy. Storage of these

nanoparticles for N15 months did not dramatically changethe diameter

or polydispersity of any of the formulations. Permeation studies were

carried out in excised human skin and a rat epidermal keratinocyte

organotypic culture model mounted in Franz type diffusion cells.

Cumulative corticosterone in the receptor was measured over 48 h.

These results showed that the PBS control had the highest cumulative

permeationat 1.5%, cubic nanoparticles at 1.0%, smectic nanoparticles at

0.4%, and SLN at 0.1% in human epidermis. In the rat epithelial

keratinocyte organotypic culture the cumulative permeation was 0.7%

for PBS, 0.7% for cubic nanoparticles, 0.6% for smectic nanoparticles, and0.2% forSLN. Thepermeability coefficientswere alsocalculated and only

the cubic nanoparticles showed a distinct improvement over the PBS

control, 4.3107 cm/s versus 1.8107 cm/s. Fluorescence micros-

copy only detected fluorescence with the cubic nanoparticle group. In

conclusion, exposure of skin to the largest particle (361 nm) resulted in

the highest level of drug delivery. This highlights the importance of

interactions of the materials over that of shape and size.

A recent report by Jensen et al. describes topical corticosterone

delivery with SLN using distearate [145]. A series of corticosteroids in

distearate (hydrocortisone, hydrocortisone-12-acetate, hydrocorti-

sone-17-butyrate, hydrocortisone-17-valerate, and hydrocortisone-

21-caprylate) with calculated log P from 1.28 to 4.16 were tested. The

mean diameters of these particles ranged from 180 5 to 220 28 nm.

In vitro release assays using cellulose membranes showed that

permeation was inversely related to lipophilicity. At 12 h, the

cumulative amount of drug released was 23.0% for hydrocortisone,

27.2% for hydrocortisone-12-acetate, 18.3% for hydrocortisone-17-

butyrate, 7.5% for hydrocortisone-17-valerate, and 4.9% for hydrocor-

tisone-21-caprylate.

Although both Kuntsche et al. and Jensen et al. both used SLN

encapsulated corticosteroids for topical delivery, each used different

lipids to construct the nanoparticles. The nanoparticles themselves

were similar in size and both were capable of releasing corticoster-

oids. One possibility for improvement in corticosteroid delivery would

be to explore the glycerol monooleate nanoparticle formulation [146]

to deliver the hydrocortisone derivatives [145] for optimal controlledanti-inflammatory delivery. The major differences in permeation

models limit further comparison.

2.1.2. Flufenamic acid

Flufenamic acid (FFA) is an anti-inflammatory drug that was

chosen as a model lipophilic drug for testing topical nanoparticle drug

delivery by the Schaefer and Lehr group from Saarbrucken, Germany

[147,148]. This model drug also hasfluorescence properties when in a

non-polar environment and excited at 420 nm. This enables micro-

scopic localization studies in addition to traditional pharmacokinetic

studies. In 2006, Luengo et al. described topical FFA delivery with

328 nm poly-lactic-co-glycolic acid (PLGA) particles loaded with FFA

[147]. The particles were characterized with atomic force microscopy

(Fig. 5). Release experiments showed that after 6 h, both the free and

Fig. 4. Particle accumulation inhair folliclesaftermassage. Massaged skin is shown treated with particulatedye andnon-particulate dyein panelsa andb, respectively.Panels c andd

show skin, without massage, that has been treated with particulate dye (panel c) and non-particulate dye (panel d). Panel e and f show the scales on hair follicles for human and

porcine skin which serves as a basis for a geared pump delivery of nanoparticles deep into the opening on massaging. Adapted from [70,72].



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gel particulate formulations reached 100% FFA release using human

skin. Cumulative penetration (area under the penetration curve) was

determined for free and gel particle formulations. After 24 h the

particulate group showed significantly more FFA (6.7 g/cm2) in the

deep skin layers than the non-particulate control (3.8 g/cm2) for a

1.8 fold increase.

Santander-Ortega et al. investigated novel starch derivatives, with

two different substitution groups for nanoparticle encapsulation of FFA

[148]. The particles were fully characterized in addition to stability and

pharmacokinetic experiments. Thesize of the particlesranged from 150

to 182 nm. The size remained at 180 nm after 25 days at room

temperature. The encapsulation efficiency for both starches was N95%.

A total of 14.8% of drug was released and 4.4 g/cm2 permeated within

the first 12 h.

The FFA loaded PLGA particles had 6.7 g/cm2 permeated after 24 h

and the starch nanoparticles had 8.3g/cm2 permeated at the same time

point. These data show a substantial improvement when using PLGA or

starch particles to deliver FFA to skin over non-particle controls.

Fig. 5. AFM images from Luengo et al. showing FFA loaded PLGA nanoparticles for topical anti-inflammatory drug delivery [147]. Two formulations are shown, an aqueous

nanoparticle suspension (Panel a) and a gel formulation (Panel b). Multi-photon microscopy images of human skin treated with drug-free nanoparticle gel (Panel c) and FFA

containing nanoparticle gel (Panel d) are shown where the drug is present in the furrows (white arrow heads)." to "AFM images from Luengo et al. showing FFA loaded PLGA

particles for topical anti-inflammatory drug delivery [147]. Two formulations are shown, an aqueous particle suspension (Panel a) and a gel formulation (Panel b). Multi-photon

microscopy images of human skin treated with drug-free particle gel (Panel c) and FFA containing particle gel (Panel d) are shown where the drug is present in the furrows (white

arrow heads).

Table 1

Particle-delivered anti-photoageing drugs and antioxidants. Not determined (ND).

Drug(s) Nanoparticle

material

Diameter range, potential Dose, vehicle Model Ref.

Tretinoin SLN 173406 nm, 2347 mV 0.05%, Hydroxylethyl-cellulose (0.8%) Rhino mice for skin irritation [162]

Tr et inoin SLN 5 2.0 20 .8 t o 6 5.7 28.0 nm, ND 0 .0 5% , C ar bopol Ult rez 1 0 ( 1% , w/w ) Exc ised hairless a bd ominal r at skin [163]

Tretinoin SLN 344 nm, ND 0.05%, Carbopol ETD 2020 Excised hairless abdominal rat skin [166]

Tretinoin Silica 166.5261.6 nm,52.035.6 mV 0.05%, Lecithin or oleylamine in medium

chain triglycerides

Franz type diffusion cells with cellulose

acetate membrane and porcine skin

[160,161]

Tretinoin CaCO3 ND 0.10.4%, Study subjects [167]

Tretinoin CaCO3 1316 nm, ND 0.1%, aqueous ddY mouse [171]

Isotretinoin SLN 31.3 to 50.0 nm,14.1 to17.9 mV 0.06%, Tween 80 and soybean lecithin Excised abdominal rat skin [168]

Retinol SLN 224 nm, 58.10.6 mV 0.5%, glycerol, water and xanthin gum Cellulose membrane in Franz type

diffusion cells

[172,173]

Vitamin A palmitate SLN 350 nm, ND 0.25%, Carbopol 940 (1%) Cadaver skin using Keshary Chien cells [169]



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Importantly, these well controlled and informative studies correlate well

despite having been published 4 years apart thus allowing comparison.

2.2. Anti-photoageing drugs and antioxidants

2.2.1. Retinoids

Retinoids arecommonly usedin topicalacne andanti-ageingproducts

and are one of the most studied drug groups in terms of topical

nanoparticle and microparticle delivery (Table 1) [160

173]. Retinoidswork through nuclear hormone receptors (retinoic acid receptors) and

retinoid X receptors. The molecular responses to receptor binding are

diverse, but in the context of skin disease, topical retinoid treatment

initially reduces inflammation, helps to reduce comedones and wrinkles.

Retinoids are used clinically to treat acne vulgaris [174] and cosmetically

to treat photoageing [175]. Tretinoin (all-trans retinoic acid) is the first

generation retinoid. Instability in the presence of light and oxygen is a

drawback for using Tretinoin and is one of the major focus points for

improvement by nanoparticle encapsulation. In the late 1990s, third

generation retinoids, i.e. adapalene and tazarotene, overcame this

drawback and were approved for clinical use [176], but have not been a

focus for nanoparticle delivery. Theother major issue for retinoid therapy

is local erythema, peeling, dryness, and pain. Skin irritation side effects

have been mitigated by nanoparticle encapsulation, where the mecha-

nism of action is likely to be controlled release.

Improved stability and controlled release have been the primary

focal points of nanoparticles for retinoid delivery. Castro et al. used solid

lipid nanoparticles to encapsulate Tretinoin withan efficiency of 94% by

using a lipophilic amine (stearylamine) [162]. They exploited ion

pairing between Tretinoin and the lipophilic amine to increase the

lipophilicity of the Tretinoin, thus enabling dramatic improvement in

encapsulation efficiency. The microparticles varied in diameter from

2283 to 68226 nm depending on the surfactant to lipid ratio

(0.40.1). Storage of these microparticles at 25 C for 90 days revealed

that samples with stearylamine maintained higher encapsulation

efficiency than SLN without, 975% versus 552%. Skin irritation

studieswere doneby comparing the SLNwith stearylamine to a placebo

gel and a marketed Tretinoin cream containing a completely different

formulation utilizing liposomes. Castro et al. used a rhino mouse modelto test irritation. Although the engineered SLN performed better than

the commercialformulation,the lack of stearylamine controls precludes

further interpretation. Likewise, the lack of skin retention and flux data

hampers comparison of this SLN formulation withother similar studies.

Others including the Patravale group have used SLN to deliver

Tretinoin to skin with similar success [163,166]. In both studies, 0.05%

Tretinoin was formulated with Carbopol to form gels with favourable

spreadability, viscosity, and flow. Two distinct SLN sizes were tested

in these studies: b100 nm [163] and N300 nm [166]. Both reports

utilized excised skin from the abdomen of hairless rats. Both the

smaller and larger SLN had similar, but not identical skin retention at

4.2% and 6.4%, respectively. The encapsulation efficiency of the small

and large SNL was similar at 46% and 49%, respectively. The flux

differed with the larger SLN being exactly twice that of the smallermicroparticle at 75.6 ng/cm2 h versus 37.7 ng/cm2 h. These were

compared to a commercial formulation with a flux of 64.5 ng/cm2 h,

which contained a completely different formulation but had the same

Tretinoin level (0.05%) as the SNL formulations. It is not possible to

identify any potential stability benefits of nanoparticle encapsulation

between the nanoparticles (b100 nm) and microparticles (N300 nm)

because photostability was only investigated with the N300 nm SLN.

Inorganic microparticles have also been used as Tretinoin carriers,

including silica [177] and calcium carbonate [167,171]. Tretinoin coated

silica microparticles were developed by the Prestidge laboratory and

found to be 166 to 261 nm in diameter depending on whether the

microparticles were formulated with lecithin or oleylamine, respec-

tively. The zeta potential of the microparticles was 50 and +34 mV

for lecithin and oleylamine, respectively. Neither the diameter nor the

zeta potential was dramatically altered by oil or water phase

formulation. The microparticle formulations were compared to lecithin

or oleylamine formulations and the controlled release properties were

investigated. The silica microparticle groups showed a significant

reduction in the steady state flux to 90% for lecithin emulsions and

50%for oleylamine emulsions. The72 h cumulative release was13.4 and

7.4 g/cm2 for lecithin and oleylamine emulsions with silica micro-

particles, respectively. However, the skin integrity after 72 h of

treatment is questionable. There was a maximum of 30% and 76%reduction in the cumulative release by the silica microparticle groups

compared to the non-microparticle containing controls. The 12 h skin

retention with silica microparticle formulated with lecithin in oil phase

wassimilar to that ofb100 nm SLNat 3.9% and4.2%,recognizingthat the

silica studies were done with porcine skin [177] and the SLN studies in

rat skin [163]. Clinical studies [167] have been carried out in Japanwith

CaCO3 nanoparticles (1315 nm) coated with Tretinoin at high doses

(0.1%). These studies do not include skin retention or flux measure-

ments, but do describe favourable murine [171] and clinical outcomes.

The lack of pharmacokinetic data, among other discontinuities with the

more recent literature, restricts comparisons with silica and SLN

delivered Tretinoin, however.

Tretinoin derivatives, isotretinoin [168], retinol [172,173], and

vitamin A palmitate [169], have also been delivered to skin with SLN.

The isotretinoin loaded SLN described by Liu et al. [168]were in the

same size range as the tretinoin loaded SLN described by Mandaw-

gade et al. [163], 3050 nm and both studies used excised abdominal

rat skin to investigate the pharmacokinetics. Isotretinoin loaded

nanoparticles were capable of encapsulation efficiencies of up to

99.7% depending on the surfactant concentration (8% soybean lecithin

and 4.5% Tween 80). Stability wastested over 3 monthsat 28 C with

no changes in isotretinoin levels noted. The steady state flux for the

non-SLN control (0.06% isotretinoin) was 0.76 0.3 g/cm2 h, but

there was no isotretinoin found in the receptor chambers after 8 h.

However, the skin retention of the control (2.81 g) and maximum

SLN (3.65 g) formulation were comparable. This was an increase of

30% from the non-SLN control.

Retinol was also delivered by drug loaded SLN, but these studies

utilized particles N220 nm in diameter [172,173]. The skin retentionof retinol was 0.68% (over 6 h) in porcine skin, an improvement of

0.9 g over a nanoemulsion control group. The in vitro flux from the

retinol loaded SLN was 150 ng/cm2 over 6 h, or 25 ng/cm2 h.

Vitamin A palmitateis converted into retinol in theskin andfinally to

tretinoin. Vitamin A palmitate has been formulated into SLN of

approximately 350 nm in diameter by Pople and Singh [169]. The

pharmacokinetics of thisformulationwas exploredusing humancadaver

skin with Keshary Chien cells. After 24 h the SLN drug release was 67.5%

and the gel control was 54.4%, implying that the flux was unusually

higher with the SLN formulation, SLN formulation usually results in

lower flux with longer term release. Pople and Sing also evaluated the

effects of treatment on SC thickness in rats. SLN formulation treatment

resulted in an increasein SC thickness of almost 3 times comparedto the

conventional gel. The conventional gel control was a formulation thatcontainedthe samecomponentsas theSLNformulations. Thisincrease in

SC thickness is likely to be due to a hydration effect.

In summary, a number of SLN and inorganic particles have been

used to improve retinoid pharmacokinetics and stability. SLN were the

most common particles tested and two major size ranges were tested,

b100 nm and N200 nm. This could be interpreted as a broad

comparison of nanoparticle versus microparticle retinoid delivery,

given the definition of nanoparticles as b100 nm in diameter. Both

nanoparticles and microparticles could be formulated with near 100%

encapsulation efficiency and both showed improved stability and

pharmacokinetics (depending on controls used). Together, no real

benefits of nanoparticle(b100 nm) formulation are evident compared

to microparticle (N100 nm) data, but encapsulation appears to have

advantages over free drug formulations.



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2.2.2. Tocopheryl acetate

Zhao et al. explored thebenefitsof a hydrofluoroalkane foam to aid

tocopheryl acetate release from lipid nanoparticles upon contact with

the skin [178]. The uncharged lipid nanoparticles were formulated

with an oil-in-water emulsion and the size range of the loaded

nanoparticles was from 53 1 to 57 0 nm. Maximal encapsulation

efficiency was 90.13.7% and 100% of the drug was recoverable.

Delivery to human skin was evaluated by treating skin with 400 g/

cm

2

tocopheryl acetate for 24 h. Saturated silicone oil was comparedto the aqueous nanosuspension and nanoparticle containing foam. No

tocopheryl acetate was found in the receptor fluid for any group and

the estimate for skin retention was 1.70.4% for the oil control and

0.70.4% for the foam with nanoparticles, while no drug was

delivered to the SC by aqueous nanoparticles. These data suggest no

major differences between the groups and no major benefit of

nanoparticle formulations in terms of total delivery. However, studies

showed improved stability over a 4 week trial with the foam

formulation.

Similar studies from the same group used solid lipid nanoparticles

to deliver tocopheryl acetate with hyalauronic acid gels [179,180],

with similar results. The nanoparticles in this study were comparable

in size to those described by Zhao et al., at 50 1 nm. No tocopheryl

acetate was released over 24 h when permeability tests were carried

out with synthetic membranes treated with SLN formulations [180].

Synthetic membrane permeation studies revealed that the saturated

silicone oil formulation gave a maximal flux of 836.2 137.9 g/

cm2 h. Penetration studies were carried out with porcine and human

skin using tape stripping to extract material from the skin surface.

These experiments showed that porcine skin treated with silicone oil

saturated with tocopheryl acetate or SLN gel retained 1.320.57%

and 1.650.90% respectively of the total dose applied. Interestingly,

in human skin, the saturated silicone oil delivered 1.700.36%, while

tocopheryl acetate could not be detected after SLN gel treatment.

These species differences could be due to a number of reasons

including differences in SC lipid orientation and thickness, supporting

the hypothesis that human skin is less permeable than pig skin [181].

Another difference described by Lademann was the shrinking of hair

follicles in human skin which may prevent penetration of nanopar-ticles [71]. These well controlled and coordinated studies show no

substantial delivery benefit of nanoparticle formulation delivered

tocopheryl acetate over saturated silicone oil, regardless of vehicle.

2.2.3. Multiphoton imaging for in vivo lipid nanoparticle delivery studies

Photoageing severity is significantly associated with skin cancer

(pb0.05) [182]. Photoageing can result in collagen damage, DNA

damage and metabolic changes due to reactive oxygen species and

inflammation. Epidermal and dermal antioxidant enzymes are

powered by NAD(P)H. NAD(P)H is an endogenous fluorophore that

can be investigated with fluorescence lifetime imaging. These data

have direct implications for the metabolic state of the skin.

Our hypothesis was that commercial topical products containing

lipid nanoparticle components and tretinoin, niacinamide, ubiqui-none and folic acid as active agents would alter the NAD(P)H

fluorescence lifetime. This is especially relevant for niacinamide

containing products, as niacinamide is a precursor to NAD(P)H. Other

products include folic acid which is a form of vitamin B that is

essential for cell growth and metabolism.

We applied Nivea Visage Q10 Plus Day cream (Product A,

ubiquinone), Nivea Visage DNage Day cream (Product B, folic acid),

L'Oreal Revitalift Day cream (Product C, retinol) and Olay Regenerist

Microsculpting Cream (Product D, niacinamide) at 2 mg/cm2 every

day for up to 6 days. In vivo NAD(P)H changes pre- and post-

treatment were investigated with multiphoton microscopy using

fluorescence lifetime imaging microscopy on days 2 and 6.

All multiphoton images were collected using DermaInspect system

(JenLab GmbH, Jena, Germany). Excitation emission of 740 nm was

used, and theaverage incident optical power was30 mW at therear of

the objective. The light going to the FLIM detector was filtered with a

band pass filter transmitting 350450 nm light for NAD(P)H. Images

were analysed with SPCImage 2.9.4 software. Two component decay

matrices were calculated and the region of interest of each image was

selected for analysis. The mean value of each NAD(P)H component

(i.e. 1, 2, a1%, a2% and a1%/a2%) were analysed to assess any

metabolic changes. Stratum basale treated with folic acid (Product B)

for 6 days is shown in Fig. 6.The a1%/a2% ratio is inversely related to the metabolic rate and so

was used to evaluate any anti-ageing cream induced changes. At

stratum granulosum and stratum spinosum, all treated sites were

observed to have decreases in relative amounts of free NAD(P)H over

the course of the experiment. Interestingly, ubiquinone-, folic acid-

and retinoic acid-treated sites had increases in free NAD(P)H at the

stratum basale level, indicating lower metabolic states. The only

significant increases in NAD(P)H levels were found in the retinol

treated group on day 2 in the stratum spinosum (Fig. 7, *).

3. Other topical nanoparticle drug delivery applications

3.1. Antimicrobial agents

There are several recent studies thatdescribe topical nanoparticles for

anti-microbialdelivery applications [11,183190]. These studiesexamine

two types of nanoparticles, SLN for imidazole anti-fungal drugs

[183,188190] and silver nanoparticles [11,184187] that have broad

anti-microbial applications. The most prominent topical antimicrobial in

consumer productsis nanoparticle formulated silver.Silver nanoparticles

possesses antimicrobial properties [191,192] and the mechanism by

which silver functions as a disinfectant is not yet fully understood, but

may be related to silver ion induced metabolic inhibition. Alternately,

imidazole antifungal agents inhibit the synthesis of ergosterol, an

important part of fungal cell membranes [193].

3.1.1. Antifungal drugs

Econazole nitrate was loaded into SLN by Sanna et al. and the

particles characterized and permeation studies done to validate thesystem [189]. The particles had diameters ranging between 14013

and 154 5 nm and the encapsulation efficiency ranged between 97

and 102%. The cumulative econazole release from non-particle

containing gel into porcine skin was 124.7221.6 g/cm2 after 24 h

and the particle containing gel was 48.460.8 g/cm2. This could be

explained by the drug leaving theouter particle surface, leaving a high

concentration of drug in the core [20,194,195].

Passerini et al. have also investigated SLN made of glycerol

palmitostearate loaded with econazole nitrate for topical delivery,

but importantly this study compared SLN to solid lipid microparticles

using identical formulations [188]. The microparticles hada size range

of 18.03.17 to 44.75.16 m and the SLN had the same size range

described above (150 nm). Permeation experiments conducted with

porcine skin revealed a flux of 2428 g/cm2

/h1/2

for microparticlesand 1520 g/cm2/h1/2 for SLN, with no statistically significant

difference between the groups. The cumulative amount of econazole

nitrate that permeated after 24 h was 124.20.12 g/cm2 for non-

particulate econazole compared to 78121 g/cm2 for microparticles

and 4881 g/cm2 for SNL. These results show that there is no

significant difference between micro and nano-particle formulation

and econazole delivery kinetics in porcine skin, suggesting no size

dependent effects on drug delivery.

3.1.2. Silver nanoparticles

Topical silver nanoparticles, having antibacterial and antifungal

effects, are some of the most studied therapeutic nanoparticles

[11,184187,192], but important questions remain regarding topical

use. Silver nanoparticles are similar to solid drug nanoparticles in that



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the active agent appears to be the breakdown product of the particle.

Silver nanoparticles exhibit minimal penetration into skin and are

consequently considered safe. Studies of long term occupational

exposure to silver ions and silver nanoparticles conclude that they are

relatively non-toxic. The sub-milligram levels of silver present in burn

dressings are considered low risk [196].

Samberg et al. recently described an in vivo study in porcine skin

[61]. The skin was dosed topically with 2080 nm silver nanoparticles

for 14 days. The authors explored the effects of washing the

nanoparticles and carbon coating the nanoparticles prior to applica-

tion. Their results showed that none of the groups penetrated the SC

and that the sites of application showed focal inflammation. Finally,

Fig. 6. Metabolic state of stratum basale treated with folic acid cream for 6 days. In vivo multiphoton images show untreated (left) and folic acid-treated (right, Product B) stratum

basale. Free NAD(P)H lifetime contributionover protein-bound NAD(P)H contribution ratios (a1%/a2%)are inversely related to the metabolic rate.The 1 component is related to free

NAD(P)H in the cytosol, while the 2 component changes when NAD(P)H protein-binding changes.



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the authors showed through in vitro and in vivo systems that washed

or carbon coated silver nanoparticles were the least toxic form of

silver nanoparticles.

Tian et al. investigated the relationship between topical silver

nanoparticle delivery and wound healing in mouse models. The silvernanoparticles in thewounddressings were 14 10 nm in diameter,as

measured by transmission electron microscopy. Silver nanoparticle

impregnated dressings were changed daily, making treatment

continuous for the course of the experiments. Thermal injury took

35.41.29 days to heal without intervention and took 9 days less

with silver nanoparticle treatment (26.50.93 days). Interestingly,

injury treated with the same concentration of silver, but without

nanoparticles (silver sulfadiazine) took 37.43.43 days to heal, or

10 days longer than with the nanoparticles. Tian et al. also saw

improvements in cosmesis in thermal injury, improved healing time

in diabetic mice, and protection from microorganisms in wound areas.

All of these experiments were conducted in mouse models.

There is a knowledge gap in topical silver nanoparticle treatment in

study subjects. We investigated silver nanoparticle penetrationin intact

and tape stripped skin, while simultaneously monitoring NAD(P)H

metabolic rate. We also used reflectance confocal microscopy to

visualize silver nanoparticle aggregate bio-retention in intact and tape

stripped skin. An aqueous commercial product containing silver

nanoparticles with a size range of 1345 nm based on 100 nanoparticlemeasurements (Fig. 8a) was applied under occlusive conditions for 4 h

(180 l, containing 0.040.045 mg/ml silver). The silver nanoparticles

were characterized by transmission electron microscopy, FLIM, and

reflectance confocal microscopy. Skin barrier integrity was assessed by

TEWL.

Our results show that the commercial topical silver nanoparticle

spray was composed of silver nanoparticles b50 nm and that 740 nm

excitation could be used to excite the silver nanoparticle and NAD(P)

H simultaneously (Figs. 8a, b and 9). Skin penetration of silver

nanoparticles was not dramatically enhanced by tape stripping 20

times compared to silver nanoparticle treated intact skin (Fig. 8c).

This is likely to be due to the incomplete removal of the stratum

corneum. While silver nanoparticle treatment for four hours de-

creased metabolism, as determined by increased a1%/a2%, no

Fig. 7. Summary of NAD(P)H effects from antioxidants delivered with lipid nanoparticle formulations in commercial products. The total NAD(P)H fluorescence was measured by

FLIM photon counting by summing the free and protein-bound NAD(P)H signals weighted by intensity after 2 and 6 days of daily anti-ageing cream treatment formulated with lipid

particles. Optical sections were taken of stratum granulosum, stratum spinosum andstratumbasalefromthe volar forearms ofthreestudysubjects. Theactive ingredients areshown

on the x-axis of each graph.Initial increases in NAD(P)H at 2 days were not observed after 6 days of treatment. Two-tailed Students t-test revealed statistical significance (*)for only

the Retinol treated stratum spinosum after 2 days of treatment.



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statistically significant differences were detected (Fig. 8d.). Differ-

ences between untreated and silver treated groups decreased with

depth, suggesting a depth dependent effect. Fig. 9 shows a1% color

coded images illustrating the difference between the NAD(P)H

(green/yellow, a1% 4585) and silver nanoparticle second harmonic

generation (orange/red, a1% 90100).

Silver nanoparticle aggregates (Fig. 10, arrow heads) were tracked

in furrows up to 10 days after treatment with reflectance confocal

microscopy (Fig. 10, Video 1). Aggregate formation could be due to

protein/nanoparticle binding [197]. After 10 days, no silver nanopar-

ticle aggregates could be detected in intact skin, but aggregates

persisted in tape stripped skin. Similar results were observed in hair

follicles (data not shown). These results show that even relatively

brief treatment with silver nanoparticles can last longer than 10 days

on treated skin, agreeing with previous work by Lademann et al. [71].

This suggests that the treatment benefits of silver nanoparticle

treatment may last longer in damaged skin and that toxicity issues

may need to be studied over longer time courses.

Fig. 8. Characterization of silver nanoparticles and in vivo human skin penetration. Nanoparticles were characterized with TEM (panel a) and MPM (panel b) for size and second

harmonic generation characteristics, respectively. A study subject with intact (non-Tape) and tape stripped (Tape) skin was exposed to a commercial silver nanoparticle containing

antimicrobial solution under occlusive conditions for 4 h prior to MPM imaging. FLIM was used to isolate and measure the silver nanoparticle second harmonic signal. Non-furrowcontaining sites were analysed for silver nanoparticle signal (Integrated Density) and depth plots were generated from FLIM z-stacks. Silver nanoparticle signal was simultaneously

measured with NAD(P)H fluorescence. Fluorescence lifetime analysiswas used to measure the free/protein bound NAD(P)Hratio (a1/a2). This ratio is thought to be inversely related

to the metabolic rate [256,257]. Application of silver nanoparticles was associated with an depth dependent increase in the free/protein bound NAD(P)H contribution which is

consistent with previous reports of silver inhibition of metabolism. However, this trend was not statistically significant when compared to the untreated data.



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3.2. Anti-proliferative agents

Hyperproliferative skin disease is not limited to cancer, but also

precancerous lesions. It can also be caused by inappropriate

inflammatory responses. Several anti-cancer and anti-proliferation

drugs have been delivered with dendrimers and nanoparticles,

including 5-aminolevulinic acid (ALA), 5-fluorouracil (5FU), paclitax-

el, podophyllotoxin, and Realgar [198

204].

3.2.1. 5-aminolevulinic acid

Photodynamic therapy is based on the delivery of photosensitive

drugs to skin lesions followed by targeted light exposure. ALA and

methylaminolevulinate (MAL) are commonly used as they both

induce the production of the photosensitiser protoporphyrin IX in

skin cells. Photodynamic therapy is now being used for an increasing

number of skin conditions, including actinic keratosis and non-

melanoma skin cancers. Topical delivery has not been optimized.

Fig. 9. In vivo z-stack of FLIM images color coded from a1% 0 to 100%, blue to red, respectively. The images are 214214 m2 and show untreated and silver nanoparticle treated skin

thatwas intact (No tapestrip) or tape stripped. The yellow/green indicates NAD(P)H autofluorescence and orange/red indicates silver nanoparticle second harmonic generation. Bar

indicates color code and 50 m.



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There are significant gaps in knowledge regarding the relationship

between photodynamic response and dose parameters. Both drugshave

poor skin penetration profiles (0.26% methylaminolevulinate penetra-tion/24 h). MAL is faster acting at 3 h versus ALA at 46 h. Importantly,

19%of those treated do notrespond [Australian clinical trial,PC T305/99]

and MAL (Metavix, US$225/g) is nine times more expensive than ALA.

Therefore, enhanced ALA delivery could improve non-responder rates,

recurrence rates, and decrease the cost of therapy.

Battah et al., from the MacRobert group, have used dendrimers

conjugated to ALA for improved efficacy by mitigating the hydrophilic

nature of ALA and characterized porphyrin production and photo-

sensitivity [198,200]. However, these compounds have not been

reported for topical use, only reports of systemic application are

available [205]. The practical problem with systemic ALA application

is that the entire person would become photosensitive and much

more drug would likely be needed, thus eliminating any benefit over

methylaminolevulinate. The lack of topical data in this area could bedueto insufficient skin penetration levels of the large dendrimers-ALA

molecules or premature ALA cleavage by esterases in the skin. This

issue may be resolved with the development of a peptide-ALA

conjugatethat hasbeen shown to work well in an excised porcine skin

model [206]. Treatment efficacy of this prodrug has the potential to be

enhanced by nanoparticle delivery in the future.

3.2.2. Podophyllotoxin

Podophyllotoxin is a topical antiproliferative drug that is extracted

from the roots and rhizomes of Podophyllum species [207]. This toxin

is used clinically to treat HPV induced warts in a 0.5% gel called

Condylox. Typical therapy would include twice daily application for

three days and then no treatment for four days before treating again.

No more than 10 cm2

should be exposed and a maximum of 0.5 ml

applied per day. Although this drug is a first-line treatment for genital

warts, there are severe side effects and systemic absorption can be

dangerous [208]. This toxicity issue led Chen et al. to explore thepotential for SLN-podophyllotoxin delivery to the skin surface [199].

SLN were prepared from tripalmitin, soybean lecithin, poloxamer 188,

and polysorbate 80 in an aqueous solution. The control tincture

contained podophyllotoxin in 75% ethanol and water. The resulting

SLN had two populations of particles with mean diameters of 44 nm

and 194 nm and all had negative zeta potentials that ranged from

48 to 17 mV. Atomic force microscopy, differential scanning

calorimetry, and X-ray diffraction were used to further characterize

the formulations. Podophyllotoxin is fluorescent and can be excited at

290 nm with emission at 633 nm, so the endogenous fluorescence

was used to visualize podophyllotoxin localization after treating

excised porcine ear skin (Fig. 11). Podophyllotoxin fluorescence

accumulation can be seen in a furrow and hair follicle, in a manner

similar to the zinc oxide nanoparticles in Figs. 3, 9 and Video 1, FFAloaded PLGA microparticles are shown in Fig. 5d and the silver

nanoparticles in Figs. 9, 10 and Video 1. Encapsulation of podophyllo-

toxin in SLNresulted in an increase in thecumulative podophyllotoxin

compared to the tincture, 23.380.55 g and 6.080.31 g, respec-

tively. However, no podophyllotoxincould be detected in thereceptor

chamber of SLN treated skin, suggesting that the majority of the drug

was contained in the SLN on the outer layers of the skin. The

podophyllotoxin activity was not measured, so the therapeutic value

of SLN delivery cannot be assessed.

3.3. Other topical particulate delivered drugs

The field of nanoparticle drug delivery to the skin is fragmented in

terms of the drugs investigated. Some examples of molecules and

Fig. 10. In vivo RCM images of untreated and silver nanoparticle treated skin that was intact (No tape strip) or tape stripped. Silver nanoparticle aggregates (arrow heads) were seen

2 days and 6 days (data not shown) after application but not at 10 days after application in intact skin. In contrast, silver nanoparticle aggregates were still seen 10 days after

application, but only in discrete areas. Each image is 0.5 0.5 mm2.

http://localhost/var/www/apps/conversion/tmp/scratch_6/image%20of%20Fig.%E0%B1%B0


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combinations of molecules that have been investigated for topical

nanoparticle delivery that have not been discussed are: diethyltolua-

mide and ethylhexyl p-methoxycinnamate [209]; fludrocortisone

acetate and flumethasone pivalate [210]; hinokitiol, glycyrrhetinic acid

and6-benzylaminopurine [211]; tolterodine tartrate [212]; JSH18[213];

acyclovir [214]; amlodipine [215]; ascorbyl palmitate [216]; benzylnicotinate [217]; buprenorphine [218]; calcipotriol with methotrexate

[219]; ceramide-3 [220]; coenzyme Q10 [221]; cyclosporin A [222,223];

cyproterone acetate [224]; frusemide [225]; insulin [226,227]; lidocaine

[228]; methoxycinnamate[77]; minoxidil [229,230]; nitrendipine

[231,232]; oxybenzone [233]; progesterone [234]; psoralen [235]; RU

58841-myristate [236]; temoporfin [237]; thrombin [238]; and triam-

cinolone acetonide acetate [239]. These reports describe the use of

several different nanoparticle carriers: chitosan [214]; invasomes [237];

iron oxide [238]; lipid [218220,223,230]; micro- and nano-emulsions

[210,212,215,217,218,227]; non-ionic surfactantvesicles(proniosomes)

[225]; polymer [77,211,226,240]; solid drug [221]; and solid lipid

nanoparticles [209,213,216,222,224,228,229,231236,239]. We recog-

nize that many reports use more than one type of nanoparticle material

and some of these carrier categories overlap. This integration ofnanoparticle drug delivery technologies has helped to cross validate

studies.

4. Recent advances in particle-based drug delivery

Materials advances are critical for the advancement of topical

nanoparticle drug delivery and have the potential to dramatically

improve drug delivery kinetics, but also reveal new biological

information on skin function. Poly(amidoamine) or PAMAM dendri-

mers have been used as effective delivery devices for some time,

reviewed by Jain et al. and Nishiyama et al. [241,242]. Dendrimers are

considered to be nanoparticles, but are composed of highly branched

polymers. Recent advances by Venuganti et al. have shown that

engineering the surface charge on dendrimers can regulate dendrimer

skin penetration for enhanced small drug delivery [202,203].

Venuganti et al. manipulated the surface charge of the dendrimers

by the addition of multiple amine groups (x16, 64, and 256),

carboxylic acid (x64), and hydroxyl (x64) groups, with isopropyl

myristate as the control. The antiproliferative drug 5FU was used as

the model for skin permeation studies in separated porcine ear skin.Dendrimers were used to pre-treat skin, at 0.1 to 10 mM, with 1 mM

as the chosen concentration, for 24 h. Interestingly, all dendrimer

groups had significantly higher cumulative amounts of penetrated

5FU (ranging from 1952 to 2597 g/48 h) than the control (1421 g/

48 h). The flux of the dendrimer groups (96236 g/cm/h) also

increased well above the isopropyl myristate control (67 g/cm/h).

Pre-treatment with the hydroxyl modified dendrimer resulted in the

highest 5FU levels in skin (23.7 g/mg skin), more than twice that of

the isopropyl myristate pre-treated group (11.9 g/mg skin). Togeth-

er, these data suggest that engineering dendrimer surface chemistry

to match the drug and desired skin penetration characteristics has the

potential to improve nanoparticle drug delivery.

Dendrimer drug delivery is currently being explored on many fronts

and the majority of the knowledge about nanoparticle drug deliverysystems focuses on solid lipid nanoparticles. Kuchler et al. from the

Shfer-Korting group in Germany, have reported the use of SLN for a

v

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