From nanoemulsions to nanostructured lipid carriers- A relevant development in dermal deli

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Journal of Drug Delivery Science and Technology 32 (2016) 100e112
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Journal of Drug Delivery Science and Technology
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From nanoemulsions to nanostructured lipid carriers: A relevant
development in dermal delivery of drugs and cosmetics
Lucia Montenegro a, *, Francesco Lai b, Alessia Offerta a, Maria Grazia Sarpietro a,
Lucia Micicch�e a, Anna Maria Maccioni b, Donatella Valenti b, Anna Maria Fadda b
a Dept. Scienze del Farmaco, University of Catania, Catania 95125, Italy
b Dept.Scienze della Vita e dell'Ambiente e Sezione di Scienze del Farmaco, CNBS, University of Cagliari, Cagliari 09124, Italy
a r t i c l e i n f o
Article history:
Received 7 July 2015
Received in revised form
2 October 2015
Accepted 2 October 2015
Available online 9 October 2015
Keywords:
Nanocarriers
Skin delivery
Microemulsions
Solid lipid nanoparticles
Nanostructured lipid carriers
* Corresponding author. Dept. Scienze del Farmaco
Doria 6, 95125 Catania, Italy.
E-mail address: lmontene@unict.it (L. Montenegro
http://dx.doi.org/10.1016/j.jddst.2015.10.003
1773-2247/© 2015 Elsevier B.V. All rights reserved.
a b s t r a c t
Recent advances in nanotechnology have led to the development of nano-scale drugs and delivery
systems to improve drug therapeutic effectiveness. Between the end of 050 and the beginning of 060, the
first colloidal systems in the nano-metric range were achieved by chance. Several research highlighted
the usefulness of these nano-carriers as drug delivery systems to overcome biological barriers later on.
Since few drugs are effective after their topical application, due to the barrier function of the skin,
colloidal systems have being widely explored as carriers to improve drug skin permeation. In particular, a
great deal of attention has been paid to delivery systems based on highly biocompatible and biode-
gradable components such as lipids and phospholipids. As a result, different types of nano-carriers such
as liposomes, microemulsions, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC)
have been developed. This review will focus on the nano-carriers arising from the first colloidal systems
consisting of water, lipids and surfactants, i.e. microemulsions and their consequent improvement
through the development of SLN and NLC. The properties of these nano-carriers will be discussed along
with their applications as skin delivery systems both in pharmaceutical and cosmetic fields.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
1.1. Background
Nanotechnology is a science devoted to creation, modification
and utilization of materials, devices, and systems in the nano-meter
size range. It exploits the physical, chemical, and biological prop-
erties of materials that improve or radically differ from those of
bulk materials, just because they are on a nano-metric scale [1,2].
Nanotechnologies are already used in several commercial
products and industrial applications such as electronics, foods, fuel
and solar cells, batteries, chemical sensors, etc. Moreover,
numerous applications of nanotechnology in the pharmaceutical
and cosmetic have revolutionized the administration of drugs and
cosmetics. Indeed, the use of nano-carriers have led to the defini-
tion of nanomedicine, a multidisciplinary subject area including
many scientific disciplines, which has been defined as the
, University of Catania, v.le A.
).
application of nanotechnology for the prevention, treatment,
diagnosis, monitoring, and control of biological systems [3]. The
main nanomedicine research areas have been classified as:
� Nanotechnology-based diagnostics including imaging (molecu-
lar diagnostics, imaging with nanoparticles, biosensor etc.)
� Nano-pharmaceuticals (targeted drug delivery, nanotechnology-
based drug, nano-pumps and nano-coated stents, etc.)
� Regenerative Medicine and Nano-surgery (nano-biotechnology
scaffolds, nano-laser surgery, etc.)
� Nanorobotics (vascular surgery by nano-robots, nano-robots for
detection and destruction of cancer, etc.)
Nano-carriers for medical application include several engi-
neered constructs, assemblies, architectures, and particulate sys-
tems with distinct physicochemical characteristics, whose unifying
feature is the size between 1 and 1000 nm (as commonly defined in
pharmaceutical sciences). Diagnostic or/and therapeutic agents can
be encapsulated, incorporated into such nano-carriers or covalently
attached or adsorbed onto their surface. Different classes of nano-
carriers such as liposomes, nanocrystals, lipid and polymeric
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L. Montenegro et al. / Journal of Drug Delivery Science and Technology 32 (2016) 100e112 101
nanoparticles, block copolymer micelles, gold nanoparticles, den-
drimers, etc. have been used to improve drug and gene delivery,
targeted therapy, diagnostics and some of them have already a
routine clinical use [1,3].
Several studies have shown that nano-carriers are advantageous
in several pharmaceutical and cosmetic applications and, above all,
vesicular carriers, microemulsions, and lipid nanoparticles are the
most studied and used. Microemulsions and lipid vesicles (lipo-
somes) were described for the first time almost simultaneously in
the 60s of the past century. Indeed, in 1959 Schulman et al. visu-
alized the existence of small emulsion-like structures by electron
microscopy and subsequently coined the term “microemulsions”
while liposomeswere discovered by Sir Alec Bangham in 1961 [4,5].
Successively, these colloidal carriers were proposed in topical drug
delivery and lipid nanoparticles were developed starting from
microemulsions. In this review, wewill focus on the evolution from
microemulsions to lipid nanoparticles, for dermal application in the
pharmaceutical and cosmetic field.
To understand better the potential of these carriers as skin Drug
Delivery Systems (DDS), at first, general issues regarding cutaneous
permeation will be introduced. Indeed, the skin can offer several
advantages as a route of drug administration although its barrier
nature makes it difficult for most drug to penetrate into and
permeate through it.
1.2. Skin anatomy and physiology
The skin is the largest organ in the human body and acts as a
main target as well as a principal barrier for dermal and trans-
dermal drug delivery. Indeed, because of its easily accessible large
surface area, it has received a great research interest as a non-
invasive alternative route to conventional oral or injectable
administration of drugs. Indeed, drug delivery into/through the
skin offers different advantages that include improved bioavail-
ability of drugs that suffer the gastrointestinal environment and/or
hepatic first pass effects, potential of delivering drugs for a pro-
longed period at a constant rate, reduced side effects, and improved
patient compliance. However, percutaneous drug delivery is still
challenging. Indeed, even now, there is the need to overcome in-
dividual variability among the different locations on the skin, and
for the effective barrier that this organ forms between the organism
and the environment. Definitely, the primary function of the skin is
to act as a barrier in order to protect the human and mammalian
body, thus, preventing invasion of pathogens and protecting from
chemical and physical assaults, as well as from unregulated loss of
water and solutes. This important role is due to the architecture of
the skin, which is composed of three functional layers, namely
epidermis, dermis,and hypodermis (also known as subcutaneous
fat layer) [6].
The protective properties are provided by the epidermis, the
outermost skin layer, in spite of its very low thickness, which varies
from 0.02 mm from up to 5 mm depending on the location of the
skin. The epidermis is a stratified squamous epithelial layer, con-
taining keratinocytes organized in four main different strata (i.e.
stratum corneum, granular layer, spinous layer, basal layer). The
physical barrier is primarily localized in its uppermost layer, known
as horny layer or stratum corneum (SC, 10e20 mm thick), consisting
in 15e25 flattened, stackened, hexagonal, and cornified cells (cor-
neocytes) embedded in lipid-enriched intercellular domains [6].
SC thickness varies greatly being particularly thick in the palms
of the hand and soles of the feet. The SC barrier properties are partly
related to its high density (1.4 g/cm3), its low hydration (15e20%) in
comparison with common body tissues (70%), and its low surface
area for solute transport. The barrier properties are further sup-
ported by continuous desquamation of the horny layer with its
complete turnover occurring every 2e3 weeks [6]. All SC cells
originate in the deepest epidermal stratum, the basal layer, and
undergo many morphologic, biochemical and physiological
changes as they move from the basal lamina to the superficial skin
layer under the pressure of the newly produced keratinocytes. In
the epidermal basal layer, also melanocytes, Langerhans cells, and
Merkel cells are present together with the keratynocites.
Corneocytes are mainly composed of insoluble packed keratins
(70%) and lipids (~20%) enclosed in a cornified envelope, while the
intercellular region consists of lipids and desmosomes, which allow
corneocyte cohesion. The lipid intercellular domain consists of
lamellar sheets composed of approximately equimolar concentra-
tions of free fatty acids, cholesterol, and long chain ceramides.
Below the SC is the viable epidermis (50e100 mm), which has an
important function of regeneration of the SC [7].
The stratum corneum barrier function is not only dependent on
one single component but also on its total architecture, described
by Elias as the “bricks and mortar” model where the bricks are
the corneocytes and the mortar refers to the lipid rich matrix
(Fig. 1) [8].
The nucleated epidermis also contributes to the barrier through
tight, gap and adherent junctions, as well as through desmosomes
and cytoskeletal elements. During epidermal differentiation, lipids
are synthesized in the keratinocytes and extruded into the extra-
cellular domains, where they form extracellular lipid-enriched
layers. The cornified cell envelope, a tough protein/lipid polymer
structure, resides below the cytoplasmic membrane on the exterior
of the corneocytes. Ceramides A and B are covalently bound to
cornified envelope proteins and form the backbone for the subse-
quent addition of free ceramides, free fatty acids, and cholesterol in
the lipid matrix of the SC. The lipids are organized as multiple lipid
bilayers, which form regions of semi-crystalline gel and liquid
crystals domains [7].
The dermis (1e2 mm) is directly alongside the viable epidermis
and provides the mechanical properties of the skin. The dermis is
made up of collagen, elastins and glycosaminoglycans, collectively
called the extracellular matrix, as well as fibroblasts that extend the
extracellular matrix. The highly vascularized dermis also contains
the pilo-sebaceous glands, sweat glands, dermal adipose cells, mast
cells and infiltrating leucocytes [9].
1.3. Percutaneous absorption
Dermal and transdermal drug delivery requires efficient pene-
tration of active compounds through the skin barrier basically by a
passive diffusion process. A molecule applied on the skin surface
may use two diffusional routes to penetrate: the transappendageal
and the transepidermal routes (Fig. 1). The transappendageal route
includes transport via the skin shunts, i.e. sweat glands and hair
follicles with associated sebaceous glands. Although these routes
were traditionally considered of minor importance because of their
relatively small area (0.1e1%), recent research has indicated that
the pilo-sebaceous units may contribute significantly to topical
drug delivery by acting as low resistance pathway for nanoparticles
to enter the stratum corneum [10,11]. As known, this route has also
been considered as potential transport route for ions and large
polar molecules [7]. Moreover, the relative surface area of the
shunts may be of greater significance in areas of the body such as
the scalp, where the density and size of hair follicles are much
greater than in other location on the skin such as on the back [12].
In addition, the hair follicles and sebaceous glands are associated
with various dermatological disorders such as acne, alopecia, and
several skin tumours. Therefore, there is a great interest in the pilo-
sebaceous units as targets for localized drug delivery, as well as
shunts for transdermal delivery, even if the specific role of the
Fig. 1. The brick and mortar model for stratum corneum (from ref. 14: J. Hadgraft and M. Lane, Skin: the ultimate interface, Phys. Chem. Chem. Phys., 2011, 13, 5215e5222).
L. Montenegro et al. / Journal of Drug Delivery Science and Technology 32 (2016) 100e112102
follicular pathway in dermal drug absorption is difficult to elucidate
due to the lack of an adequate animalmodel to distinguish follicular
to non-follicular transport [13].
However, the main route to penetrate the skin barrier is
considered the transepidermal passage, following which molecules
can cross the intact, unbroken horny layer by using two different
pathways: the transcellular, across the corneocytes and the lipid
matrix (2A, Fig. 1), and the intercellular (2B, Fig. 1) across the lipid
domains between the corneocytes [14]. Structure and barrier
function of the skin have been extensively described in the litera-
ture [13,15] and it is generally accepted that the tortuous but
continuous intercellular route provides the principal pathway for
the permeation of most drugs [16,17]. However, hydrophilic com-
pounds would preferably follow the transcellular route because of
the aqueous environment due to the great amount of hydrated
keratin inside the corneocytes.
Percutaneous absorption has been studying by in vitro and
in vivo techniques and several recommendations regarding these
methodologies have been collected by regulatory bodies to produce
guidelines.
Academia and industry have been used extensively in vitro
techniques to assess skin penetration and permeation because they
are appropriate to predict human dermal penetration, give results
quickly, are time- and cost-saving, and generally show better
reproducibility of results [12,17]. Moreover, these experiments can
be performed using either human or other mammalian skin sam-
ples. However, the experiments should be performed following the
“OECD Guideline for the Testing of Chemicals. Draft New Guideline
428: Skin Absorption in vitro method” [18].
The most common methods for evaluating in vitro skin pene-
tration employ diffusion cells, and a rich literature confirms the
suitable performance of these experiments. A potential disadvan-
tage of the in vitro studies is the lack of information regarding ef-
fects of blood flow on drug permeation, since the in vivo sink
conditions cannot be completely reproduced [12,17].
Diffusion cell design may vary from a simple two compartment
“static” or a more complex “flow-through” system. They are made
of inert material, generally glass although stainless and Teflon are
also used. The static cells are composed of two compartments, the
donor and the receiver and are usually vertical (Franz cells) or side-
by-side. They can vary in size, with receiver chamber of about
2e10 ml, and diffusional surface generally ranging between 0.2 and
2 cm2.
Excised skin specimens are sandwiched as a barrier between the
two compartments, with the SC sidefacing the donor compartment
and the formulation is applied on the skin surface. The receiver
contains an appropriate fluid that simulates the blood flow and is
continuously mixed by a stir bar. The ideal receiver fluid should
well simulate the in vivo situation of permeation and guarantee
sink conditions. The most commonly used fluids are phosphate-
buffered saline (PBS) and saline, although often not appropriate.
Indeed, it is generally recognized that if the drug water solubility is
less than 10 mg/ml, the water receiver fluid must be added of sol-
ubilizers such as alcohol, albumin, or cyclodextrin. The receiver
fluid must be thermostated to ensure that the skin surface tem-
perature is kept at the in vivo conditions (32 ± 1 �C). The drug
permeating from the donor to the receiver is determined as a
function of time by receptor fluid removal from the sampling port
at regular intervals. To ensure sink conditions, the removed solu-
tion must be replaced with an equivalent amount of fresh receptor
fluid.
A rich literature regarding diffusion cell design and use is
available [12,17].
Flow-through cells can be useful when the permeant has a very
low solubility in the receptor medium. Here, a pump forces the
release medium through the receiver chamber to collect repeatedly
the perfusate [12].
2. Microemulsions and nanoemulsions
2.1. Definition
In 1943, Hoar and Schulman [19] hypothesized the existence of
microscopic emulsion-like structures in a transparent mixture of
L. Montenegro et al. / Journal of Drug Delivery Science and Technology 32 (2016) 100e112 103
oil, alcohol, water and a cationic surfactant.
About fifteen years later, Schulman et al. [20] confirmed the
presence in these systems of small emulsion-like structures by
electron microscopy and coined the term “microemulsion” to
define a system consisting of water, oil and surfactants, which is a
transparent, optically isotropic and thermodynamic stable Newto-
nian non-viscous liquid. Actually, the term microemulsions to
describe these vehicles is a misnomer as they have droplet size in
the nanometric scale (generally in the range of 10e100 nm).
Therefore, they could be better qualified as “nanoemulsions”, a
definition that is being increasingly preferred in the last decade
[21]. However, recently, the term nanoemulsions has been used
specifically for systems having droplet diameter smaller than
250 nm that are in a metastable state compared with micro-
emulsions [22]. In a recent review, McClements [23] pointed out
the main differences between microemulsions and nanoemulsions
stating that the first are thermodynamically stable, form sponta-
neously (or with very low energy input) but require a greater
amount of surfactant compared to emulsions, which may lead to an
increase of their irritation potential. On the contrary, nano-
emulsions are kinetically but not thermodynamically stable and
their preparation requires expensive, high-energy input methods,
being formed with smaller amounts of surfactants.
2.2. Structure and composition
To form a microemulsion or a nanoemulsion, the system has to
contain microstructures that involve the presence of a boundary
between the oil andwater phases at which the surfactant is located.
These colloidal systems are usually formed only in a specific and
narrow range of concentrations for a given surfactant- oil-water
composition. The relationship between the phase behavior of
different mixtures and their composition is generally depicted us-
ing a pseudo-ternary phase diagram (Fig. 2), where a corner will
represent a binary mixture of two components such as surfactant/
co-surfactant, water/drug or oil/drug. Outside the microemulsion
region, the amount of surfactant is too low to allow the formation of
a single microemulsion phase, thus leading to existence of multi-
phase systems.
Therefore, colloidal systems based on nano-droplets can reveal
the existence of different structures and phases. According to
Fig. 2. Example of a pseudo-ternary phase diagram of a simple four-component
microemulsion, at constant temperature and pressure. (1 ɸ): one phase; (2 ɸ): two
phases.
Winsor [24], four different types of microemulsions can be
distinguished:
1. dispersion of oil in water (O/W) in contact with essentially oil;
2. dispersion of water in oil (W/O) in contact with essentially
water;
3. both O/W and W/O dispersions are simultaneously present in
the same domain in mixed state and a continuous network of
water and oil is separated by a film of surfactant (Winsor III);
4. a homogeneous single phase of dispersion either O/W or O/W
not in contact with any other phase (Winsor IV).
By varying the proportion of constituents, inter-conversion
among Winsor phases can occur. The single-phase systems (Win-
sor IV) are generally used as drug delivery systems. To stabilize
these systems, both non-ionic and ionic (cationic, anionic and
zwitterionic) surfactants are often used in combinations to increase
the extent of the microemulsion region. When surfactants are
dispersed in water or oil, they self-associated into different equi-
librium phases, depending on both inter- and intra-molecular
forces and entropic factors. On incorporation of surfactants into
immiscible mixtures of oil and water, the location of surfactants at
the oil/water interface is thermodynamically favorite. Several fac-
tors are involved in determining the formation of O/W or W/O
systems. Schulman et al. [20] provided some guidelines to predict
microemulsion formation, which include the production of a very
low interfacial tension at the water/oil interface, the formation of a
very fluid surfactant film at the interface and the interaction of the
oil molecules with the surfactant film. The flexibility of the sur-
factant film is a key parameter in determining the possible struc-
tures and the ability of inter-conversion among Winsor phases in a
given microemulsion system. Transitions from O/W to W/O
microemulsions can involve the formation of different phases
(lamellar, hexagonal, bicontinuous).
Microemulsions based on non-ionic surfactants are
temperature-sensitive due to the solubility decrease of these sur-
factants as temperature increases, making the phase inversion
temperature (PIT) an important parameter in determining the
system stability. On the contrary, microemulsions based on ionic
surfactants have little or no sensitivity to temperature. To select the
most suitable surfactant to form a microemulsion system, two
empirical parameters are generally taken into account: the
hydrophilic-lipophilic balance (HLB) and the critical packing
parameter (CPP). The HLB value of a surfactant depends on the
relative contribution of the hydrophilic and lipophilic portions of its
molecule. Surfactants with HLB values ranging from 3 to 6 generally
favor the formation of W/O systems while HLB values in the range
8e18 lead to O/W systems. Ionic surfactants with HLB values
greater than 20 are generally associated with a co-surfactant to
reduce their hydrophilicity, thus allowing microemulsion forma-
tion. The CPP is regarded as a useful tool to predict the structure of
the type of aggregates that could be formed depending on the
molecular geometry of the surfactant. As reported in the literature
[25], cone-shaped surfactants tend to form curved interfaces such
as droplets and micelles, while worm-like micelles or lamellar
structures are formed using surfactants whose geometry can be
represented by truncated cones or rectangular blocks.
Examples of non-ionic surfactants used to form micro- and
nano-emulsions include polyoxyethylene surfactants or sugar es-
ters such as sorbitan esters. Among ionic surfactants, the anionic
sodium bis-2-ethylhexylsulphosuccinate (AOT) has been widely
used because of its ability to stabilize W/O microemulsions
[26e30]. Microemulsions based on AOT has been proposed as ve-
hicles for different drugs including vitamin K and steroids such as
hydrocortisone, prednisolone and betamethasone [27,31].
L. Montenegro et al. / Journalof Drug Delivery Science and Technology 32 (2016) 100e112104
As cationic surfactants, quaternary ammonium alkyl salts such
as hexadecyltrimethyl-ammonium bromide (CTAB) [32,33] and
didodecylammonium bromide (DDAB) [34,35] have been largely
investigated to form microemulsions.
Due to their high biocompatibility, biodegradability and safety,
phospholipids are the main class of zwitterionic surfactants used to
form colloidal systems based on nano-droplets for drug or cosmetic
delivery [36,37]. Trotta et al. [38] showed that the extent of the
microemulsion region increased using alkanol phosphocholines as
co-surfactants in lecithin-based microemulsions.
Different oils have been used to obtain microemulsions and
nanoemulsions for pharmaceutical and cosmetic use. The most
widely used are medium chain triglycerides and fatty esters (iso-
propyl myristate, isopropyl palmitate, ethyl or methyl esters of
lauric, myristic and oleic acid) [27,31,39e42].
2.3. Characterization
Despite their ease of preparation, colloidal carriers based on
nano-droplets require different complementary techniques to
achieve a full characterization of their structure. At macroscopic
level, useful information can be gathered by viscosity, conductivity
and dielectric measurements. The presence of structures such as
rod-like or worm-like reverse micelles can be evidenced by
measuring the viscosity of the system [43] while the type of
external phase can be determined from the system conductivity
[30]. A deep and detailed insight of structure and dynamic of these
colloidal systems can be obtained by dielectric measurements [30].
At microscopic level, transmission electron microscopy (TEM) al-
lows visualizing themorphology of nano-droplets and the presence
of lamellar structures. In particular, the freeze-fracture electron
microscopy (FFEM) has been successfully used to evidence bi-
continuous as well as lamellar structure formation in W/O micro-
emulsions depending on components ratio [44]. Asmicroemulsions
and nanoemulsions are clear and isotropic liquid, they can take
advantage of spectroscopic techniques for their characterization.
While pulsed NMR has been widely used to obtain information
about nano-droplet mobility and self-diffusion coefficients of the
components [33,43,45,46], scattering methods such as dynamic
and static light scattering [33,46,47], small-angle neutron scat-
tering (SANS) [45,48] and small-angle X-ray scattering (SAXS)
[45,49], provide a useful tool to evaluate the structure of the system
and to develop models of the potentially existing structures.
2.4. Microemulsions and nanoemulsions as dermal delivery systems
In recent years, microemulsions and nanoemulsions has been
regarded as an attractive strategy to improve cutaneous delivery of
both hydrophilic and lipophilic active ingredients compared to
conventional vehicles or other colloidal carriers [50e55].
Several factors may affect the skin permeation of an active
ingredient from colloidal systems based on nano-droplets,
including its partition between the oil and aqueous phase and its
solubility in the system components, nano-droplet size, presence of
the ingredient at the interface, use of components that can act as
penetration modifiers, site or path of absorption [56,57].
When microemulsions are obtained using low volume fraction
of dispersed phase, the systemwill contains essentially oil or water
droplets and its viscosity will be very low. The resulting formula-
tion will be suitable for many administration routes (parenteral,
oral, ocular) but its topical applicationwould not be convenient. For
vehicles designed for skin delivery, generally systems that are more
viscous are preferred. Therefore, micro- and nano-emulsions con-
taining cubic or hexagonal phases in the bi-continuous region,
micelles or cylindrical, worm-like structures are studied to improve
active ingredient cutaneous permeation. At low water content, the
use of lecithin can lead to such structures in microemulsions and its
usefulness to improve transdermal delivery of different drugs such
as scopolamine, broxaterol, propranolol and aromatic tetramidines,
has been evaluated both in vitro and in vivo [58,59].
An alternative approach to obtain microemulsions with a suit-
able viscosity is the addition of specific non-interacting gelling
agents [60,61].
Several investigations have been focused on the effect of
microemulsion composition on active ingredient release and skin
permeation. A study aimed at evaluating the effects of phase
transformation on indomethacin release from microemulsions
prepared using isopropyl myristate, lecithin, lysolecithin and
ethanol, revealed that drug release was too fast to obtain a
controlled delivery [62].
The effects of oil phase lipophilicity on in vitro release of drugs
(idebenone, naproxen) and cosmetic ingredients (butylmethox-
ydibenzoylmethane, BMBM) was evaluated from topical O/W
microemulsions. This study pointed out that the choice of proper
combinations of oil phase lipids and emulsifiers may allow
achieving drug controlled delivery from O/W microemulsions [63].
A study on the influence of microemulsion composition on both
in vitro release and skin permeation of octylmethoxycinnamate
(OMC, sunscreen agent) evidenced that both lipophilicty and
structure of the lipid used as internal phase were relevant to pro-
vide controlled skin delivery [64].
Microemulsions with different ratios between watereisopropyl
myristateeTween 80, Span 80, 1,2-octanediol were assessed for
their percutaneous and cutaneous delivery of 8- methoxsalen [65].
From saturated vehicles, a 5- and 8-fold enhancement of percuta-
neous delivery of 8-methoxsalen was observed compared to neat
oil andwater, respectively. The greatest increase of skin permeation
was observed for microemulsions with the highest surfactant
content. Topical nanoemulsions containing 8-methoxypsoralen
were prepared using soybean oil as oil phase and different phos-
pholipids as emulsifiers. In vitro permeation study through
newborn pig skin on these formulations showed that 8- methox-
ypsoralen was mainly localized in the skin layers with poor skin
permeation [66].
A comparison of in vitro skin permeation of tretinoin from
nanoemulsions and nanosuspensions highlighted the ability of
nanosuspensions to localize this drug into the different skin layers
while nanoemulsions provided an improvement of skin perme-
ation [67].
The ability of microemulsions to improve drug percutaneous
absorption was further evidenced in a study on azelaic acid
permeation through hairless mouse skin from viscous O/W
microemulsions as these vehicles showed an increase of azelaic
acid flux through the skin [68].
3. Lipid nanoparticles
3.1. Historical background and definitions
The history of lipid nanoparticles begins in 1990, with the first
experiments performed in parallel in the academic labs of M. R.
Gasco in Turin/Italy, and R. H. Müller/Berlin and J. S. Lucks, both at
this time in Kiel/North Germany. These carriers showed a structure
similar to nano-emulsions, but, differently, they were constituted
by a solid lipid matrix and, for this reason, the inventors decided to
call them solid lipid nanoparticles (SLN) [69]. After only one year, in
1991, these innovative carriers were presented to the scientific
community, while first publications appeared shortly [70e72]. SLN
immediately attracted the attention of research groups all over the
world since they had shown the capability to combine advantages
Fig. 3. Structure of SLN made from a solid lipid (left) with almost perfect crystalline
structure limiting the drug loading, NLC made from blend of solid lipid and liquid lipid
with many imperfections (right) [130].
L. Montenegro et al. / Journal of Drug Delivery Science and Technology 32 (2016) 100e112 105
of other nanometric carriers while minimizing the problems
associated with these vehicles [73,74]. Main advantages of lipid
carriers over other traditional drug carriers are good biocompati-
bility, lower cytotoxicity,good production scalability, modulation of
drug release, avoidance of organic solvents in the preparation
process and wide potential application spectrum (oral, dermal,
intravenous, etc). Technically, they are submicron colloidal carriers
and their size typically ranges from 40 to 1000 nm. The matrix
consists of a single solid lipid or mixtures of lipids such as tri-
glycerides, partial glycerides, fatty acids, steroids and waxes (see
Table 1). SLN represent the first step of the technological research
applied to lipid nanocarriers. In 1999, in fact, was developed the 2.0
version of lipid nanocarriers, the so called “nanostructured lipid
carriers” or more simply NLC. This second-generation carrier was
introduced to overcome the potential difficulties with SLN [75]. In
particular, they were formulated using blends of solid and liquid
lipids (oils) that induce a melting point depression compared to the
pure solid lipid while still being solid at body temperature. The oil
incorporation in the solid matrix of lipid nanoparticles avoids the
crystallization process, producing imperfections in the lattice to
load a higher amount of active ingredient in comparison to the
“old” SLNs (Fig. 3). Furthermore, the NLC system minimizes drug
expulsion on storage and high water content of SLN dispersions.
Therefore, we can conclude that these features make the NLC more
physically stable than the SLN.
3.2. Preparation techniques
The conventional method for the production of lipid nano-
particles (both SLN and NLC) is represented by high pressure ho-
mogenization (HPH). This method is subdivided into hot and
cold techniques with the last one recommended for preparing
lipid nanoparticles containing highly temperature-sensitive com-
pounds [76,77].
Table 1
Main ingredients used for the preparation of lipid nanoparticles for topical application.
Solid lipids
Beeswax
Carnauba wax
Cetyl alcohol (Lorol® C16)
Cetyl palmitate (Precifac® ATO 5, Cutina® CP)
Glyceryl behenate (Compritol® 888 ATO)
Glyceryl Cocoate (and) Hydrogenated Coconut Oil (and) Ceteareth-25 (Softisan® 601)
Glyceryl monostearate (Imwitor® 900, Geleol®)
Glyceryl palmitostearate (Precirol® ATO 5)
Glyceryl Trimyristate (Dynasan® 114)
Glyceryl Tripalmitate (Dynasan® 116)
Glyceryl Tristearate (Dynasan® 118)
Hard fat (Witepsol® E 85, Suppocire® NA 150)
Hydrogenated Coco-Glycerides (Softisan® 142)
Hydrogenated Palm Oil (Softisan® 154)
PEG-8 Beeswax (Apifil®)
Stearic acid
Stearyl alcohol
Oil
Caprylic/Capric Triglyceride (Miglyol® 812)
Castor oil
Squalene
Oleic acid
Surfactants
Poloxamer 188 (Lutrol® F68, Pluronic® F68)
Polysorbate 80 (Tween® 80)
Polysorbate 20 (Tween® 20)
Tyloxapol
Polyglyceryl-3 Methylglucose Distearate (TEGO® CARE 450)
Sodium cholate
Phosphatidylcholine (Epikuron® 200, Phospholipon® 80/H)
Soybean lecithin (Lipoid® S75
According to Gasco, the lipid nanoparticles can be produced
using the microemulsion technique [78e80]. It consists in the
addition of a hot o/w microemulsion into a cold water solution. In
these conditions, the emulsion is broken and the SLN are
precipitated.
The method is very cheap and fast and no special equipment is
required. Unfortunately, the technique provides a strong dilution of
the particle dispersion and so very low concentrated particle sus-
pensions are obtained. This could be a problem for a potential in-
dustrial use, since an excessive amount of water should be removed
L. Montenegro et al. / Journal of Drug Delivery Science and Technology 32 (2016) 100e112106
from the final product.
Other important methods are based on the use of organic sol-
vents and are useful to encapsulate thermosensitive drugs since the
preparations are realized at mild operating temperatures. The sol-
vent injection method (or solvent displacement) is the simplest one
and is based on dissolving the lipid in a water miscible organic
solvent and injecting this solution through a syringe needle in
water under stirring with the lipid precipitating in the form of
nanoparticles on contact with water [81]. Other solvent diffusion
methods belonging to the solvent-based approaches are the
emulsification-solvent evaporation and the solvent diffusion methods.
In these techniques, the lipophilic phase is dissolved in a water-
immiscible organic solvent that is emulsified in an aqueous
phase. After the solvent evaporation or the dilution of the organic
solvent, a nanoparticle dispersion is formed by precipitation of the
lipid in the aqueous medium [82].
Scientific literature reports other important techniques based
on the use of large mechanical forces such as high shear homoge-
nization and ultrasonication which are employed, often in associa-
tion, to overcome the formation of lipid particles larger than
desired [83]. Notwithstanding they are valid dispersing methods,
they show some drawbacks such as a certain polydispersity which
could compromise the quality of nanoparticle dispersions [84].
More recent techniques are based on PIT (phase inversion
temperature) and membrane contactor methods [85,86].
3.3. Characterization
The characterization of nanoparticles is a necessity for the
assessment of their quality.
The colloidal size of the nanoparticles as well as the complexity
of the system render the characterization a serious challenge.
The most important parameters that have to be taken in
consideration, mainly because of their effect on the SLN stability
and release, are particle size and zeta-potential, degree of crystal-
linity and lipid modification, which are deeply correlated with drug
incorporation and release rate, and coexistence of additional
colloidal structures and time scale of distribution processes.
Several techniques can be used for nanoparticles analysis.
Photon correlation spectroscopy (PCS) and laser diffraction (LD)
are routinely used for particle size measurements. PCS is used for
determination of the average particle size in a range from 3 to
3000 nm. Using LD technique a broad size range (from nanometers
to millimeters) can be covered [73].
Electron microscopy such as scanning electron microscopy
(SEM), transmission electron microscopy (TEM), and atomic force
microscopy (AFM) are widely used to determine the particle size
and morphology of lipid nanoparticles [87].
The measurement of zeta-potential gives information on the
storage stability of dispersion. In general aggregation of charged
particles is less likely to occur because of the electrical repulsion.
X-ray scattering is used to investigate the length of the long and
short spacing of the lipid lattice. Nuclear magnetic resonance and
electron spin resonance can study dynamic phenomena and the
characteristics of nano-compartments in the nanoparticles.
Differential scanning calorimetry (DSC) [88,89], X-ray diffrac-
tion, Raman spectroscopy and Fourier-transform infrared spec-
troscopy are usually used for the solid-state analysis of the
nanoparticles.
Some results obtained with DSC will be described. In a study in
which the potential use of SLN and NLC as carriers for OMC was
evaluated, DSC data pointed out the key role of the inner oil phase
of NLC in stabilizing the particle architecture and in increasing the
solubility of OMC as compared with SLN [90].
Recently, Laserra et al. prepared and characterized a new SLN
formulation containing lipoylememantine codrug to increase its
solubility in gastrointestinal fluids and favor its intestinal absorp-
tion and employed DSC to demonstrate that the production process
they used permitted to obtain solid lipid nanoparticles [91].
DSC was used to study the organization and distribution of the
different ingredients originating indomethacin-loaded SLN and
NLC. It was demonstrated that NLC systems are constituted by oil
nano-compartments incorporated into a solid matrix and that
these nano-compartments contained a higher amount of indo-
methacin [92].
DSC analysis carried out on unloaded and diethyltoluamide-
and/or OMC-loaded solid lipid nanoparticles highlighted that
diethyltoluamide and OMC modified the temperatureand the
enthalpy change associated to the calorimetric peak of SLN. The
concurrent presence of the two compounds in the SLN caused a
synergic effect, indicating that the lipid matrix of nanoparticles
guaranteed a high encapsulation of both diethyltoluamide and
OMC [93].
DSC studies on benzocaine and lidocaine SLN and benzocaine
and lidocaine loaded NLC, revealed a different behavior of SLN and
NLC toward the two anesthetics. The analysis of calorimetric pro-
files of SLN and NLC allowed to suppose that the inner oil phase of
NLCs plays a significant role in stabilizing the particle architecture
and increasing the drug solubility. It was hypothesized that NLC
solid lipid matrix is characterized by oil “clusters” in which drugs
are entrapped in such a way that do not modify NLCs architecture.
Vice versa, from the analysis of SLN calorimetric curves, drugs
seemed to affect significantly lipid matrix thermal behavior [94].
DSC was also used to suggest that cutina located in the core of
the SLN had been successfully solidified by the phase-inversion
temperature method, used to prepare SLN [85].
The interactions between SLN ingredients and loaded suncreens
(OMC and BMBM) was studied. DSC analyses showed that OMC
distributed inside the SLN causing a decrease of the lipid matrix
molecules cooperativity while no change of SLN calorimetric
behavior was observed after loading BMBM. Furthermore, when
OMC and BMBM were loaded together into these SLN, an interac-
tion between BMBM and OMC occurred. OMC could haveworked as
vehicle that solubilized BMBM and the resulting interaction be-
tween these two components could have led to the observed
changes of SLN calorimetric curves [95].
DSC was employed to evaluate the effects of three different
emulsifiers and different amounts of idebenone loaded on the
thermotropic behavior of SLN and to investigate how the drug was
arranged into these nanoparticles. SLN contained cetyl palmitate as
solid lipid and isoceceth-20, ceteth-20 and oleth-20 as emulsifiers.
DSC results showed that in ceteth-20 SLN and oleth-20 SLN ide-
benone was arranged in the SLN core interacting with the cetyl
palmitate molecules. On the contrary, oleth-20, due to its unsatu-
rated chain, led to a less ordered structure causing idebenone
mainly localization at the cetyl palmitate/surfactant interface and
as the loaded amount of idebenone increased the cooperativity of
the molecules at the interface decreased [96].
Recently, DSCwas used also to study the interaction of solid lipid
nanoparticles, unloaded and loaded with idebenone, with bio-
membrane models with the aim to have information on the inter-
action of SLN with cell membranes. The results put in evidence the
ability of the SLN under investigation to penetrate into the phos-
pholipid bilayers of MLV, used as model of biological membranes
and provided clear evidence of the entry of the SLN into the
phospholipid bilayer and of a likely localization of these SLN in the
outer bilayers of MLV. As the time of contact between SLN and
phospholipid bilayers increased, SLNmoved from the outer bilayers
to the inner bilayers, maintaining almost unchanged their struc-
ture. Loading idebenone into these SLN facilitated idebenone
L. Montenegro et al. / Journal of Drug Delivery Science and Technology 32 (2016) 100e112 107
penetration into the bilayers while free IDE showed only a low
ability to interact with this model of biomembranes [97].
3.4. Application of SLN and NLC in cosmetics and dermal
pharmaceuticals
Topically applied SLN and NLC show a number of advantages
over conventional formulations. They are composed of physiolog-
ical and biodegradable lipids and due to their small size, upon
dermal application, they adhere to the lipid film of the stratum
corneum increasing and/or modulating the amount of active sub-
stance penetrated into the skin. Their occlusive properties guar-
antee an increased skin hydration effect and normalize the living
conditions for the cells underneath. They are well suited for use on
damaged or inflamed skin because they are based on nonirritant
and non-toxic lipids [98]. Furthermore, both SLN and NLC are able
to enhance the chemical stability of compounds sensitive to light,
oxidation and hydrolysis, solving therefore, important issues con-
cerning the cosmetics and the dermal pharmaceuticals [84,99].
Over the past ten years, lipid nanoparticles have gained
increasing attention as novel carrier systems intended for dermal
application of active substances. This increasing interest is outlined
by the high number of published articles regarding this topic, due
to the work of many research groups from all over the world. A
detailed review of the Italian contribution to this topic is given
below, with some examples introducing the importance of these
systems in the topical delivery of drugs and active substances.
3.4.1. Lipid nanoparticles in topical drug delivery
Nonsteroidal anti-inflammatory drugs (NSAIDs) are an impor-
tant class of drugs used in the treatment of different disorders of
the musculoskeletal apparatus. Lipid nanoparticles have been
studied as vehicles for NSAID topical administration in order to
increase their local soft tissue and joint concentration, while
reducing their systemic distribution to avoid side effects. In an
interesting scientific work of 2005, Ricci and coworkers evaluated
the percutaneous absorption of indomethacin (IND), a powerful
NSAID used topically for the treatment of dermatitis and rheumatic
diseases, from NLC-based formulations [83]. The results of the
study showed lower drug fluxes through excised human skin
membranes from NLC-based formulations in comparison with gel
control and a more prolonged anti-inflammatory activity of IND in
in vivo trials.
The same group of investigators evaluated the percutaneous
absorption of ketorolac, an analgesic drug belonging to NSAID
family, using the approach based on lipid nanoparticles [100]. The
results of the present work are in linewith the evidences previously
described, outlining a special feature of NLCs, again more appro-
priate for sustained release due to the possible formation of a drug
reservoir into the skin.
Ketoprofen and naproxen are other two widely studied NSAIDs
used for the treatment of chronic inflammatory pathologies such as
the osteoarthritis and the rheumatoid arthritis. Puglia and co-
workers formulated NLC loaded with these anti-inflammatory
compounds and determined their permeation profiles through
the skin [101]. Nanoparticle behavior on human skin was assessed,
in vitro, to determine the drug percutaneous absorption (Franz cell
method) and, in vivo, to establish the active localization (tape-
stripping technique) and the controlled release abilities (UVB
induced erythema model). The results demonstrated that the par-
ticles were able to reduce the penetration of the drugs with respect
to the reference forms, increasing yet the accumulation in the
stratum corneum of ketoprofen and naproxen. The accumulation
and the consequent formation of a drug depot were responsible of
an interesting anti-inflammatory prolonged effect.
More recently, Cirri and coworkers revisited the NLC strategy for
the topical delivery of ketoprofen [102]. The researchers formulated
a delivery system based on drug cyclodextrin (CD) complexation
and loading into NLC, trying to put together the features of both
carriers in order to optimize the therapeutic efficacy of ketoprofen.
The authors evaluated different CD-NLC based forms containing
ketoprofen and the best one formulated into a xanthan hydrogel,
exhibited drug permeation properties better than those of the
reference drug suspension or the plain drug loaded NLC.
SLN and NLC were formulated in order to optimize the topical
application of flufenamic acid, a NSAID successfully used in the
treatment of rheumatoid arthritis [103]. In vitro experiments
showed excellent skin permeation and penetration properties for
flufenamic acid once loaded into lipid nanoparticles.
Antifungal agentsare widely used to treat skin superficial in-
fections and the topical therapy is often the best choice since it
limits drastically the systemic effects. SLN and NLC have been
investigated for topical delivery of different drugs belonging to this
therapeutic group.
Sanna et al., for instance, formulated SLN for topical adminis-
tration of econazole nitrate [104]. The authors incorporated the
lipid nanoparticles into hydrogels and carried out ex vivo drug
permeation tests using porcine stratum corneum and in vivo
percutaneous absorption studies based on the tape stripping
technique. The results showed that SLN were able to control in vitro
the drug permeation through the stratum corneumwhile, in in vivo
experiments, they promoted a rapid penetration of econazole ni-
trate. The same authors studied more deeply the mechanisms of
topical delivery of econazole nitrate, evaluating a new formulation
strategy based on the spray congealing method [105]. The method
was suitable to produce solid lipid microparticles (SLM) containing
econazole nitrate, although a significant difference in terms of drug
permeation profile was observed with respect to SLN, which
guaranteed the best performance.
More recently, Sanna and coworkers tried to increase the cuta-
neous permeation of econazole nitrate formulating lipid nano-
particles loaded with fatty esters having different chain length
[106,107]. The permeation profile of the antifungal compound
was strongly affected by the fatty esters and the drug flux increased
as their chain length increased. This result suggests that these
formulations may constitute a potential carrier for topical delivery
of econazole nitrate.
Ketoconazole is a broad-spectrum antifungal agent whose use in
therapy is strongly compromised by its poor water solubility and by
chemical degradation phenomena. Paolicelli and coworkers studied
a new system based on SLN entrapped into polysaccharidic
hydrogels to be employed as modified delivery system of ketoco-
nazole in topical formulation [108]. The results showed that SLN
formulations were able to protect the drug from UV degradation
and that the incorporation of drug loaded SLN into dextran
hydrogels gave to the formulation the suitable characteristics for
the topical application. Furthermore, the antifungal activity of the
systemwas successfully tested, in an in vitromodel, against Candida
albicans.
Reactive oxygen species (ROS) are directly involved in the onset
several human pathologies. Therefore, antioxidants have gained
utmost importance because of their potential as prophylactic and
therapeutic agents in many skin diseases. Unfortunately, they show
limited percutaneous absorption profiles and some issues that
strongly compromise their application in therapy. Literature re-
ports some examples of lipid nanoparticles application to overcome
these unfavorable aspects. Chirio and coworkers formulated SLN
loaded with curcumin, a natural polyphenol with antitumor, anti-
oxidant and anti-inflammatory properties and evaluated the effects
of a co-formulation with a and g cyclodextrins on the photo-
L. Montenegro et al. / Journal of Drug Delivery Science and Technology 32 (2016) 100e112108
stability and the percutaneous absorption of the drug [109]. The
results showed that cyclodextrins role was not relevant for curcu-
min photo and chemical stability, while skin uptake studies
revealed an increase in the skin accumulation of the drug when
curcumin was loaded in SLN and cyclodextrin derivatives.
Lutein is a natural carotenoid with antioxidant properties but
characterized by a poor chemical stability representing a practical
limitation to its use. Paolino et al. formulated NLC able to ensure a
controlled release of lutein and to improve its permeability across
the skin [110]. The drug loaded nanocarriers were formulated using
safflower oil and were checked for their physical stability by using
Turbiscan accelerated analysis. In in vivo experiments, the lutein-
loaded NLC were able to improve the photo-protective effects of
the antioxidant compared to a marketed product (Fig. 4). More
recently, Mitri and coworkers investigated the role of many lipid
nanocarriers (nanoemulsion, SLN and NLC) for dermal delivery of
lutein [111]. In vitro penetration study with an artificial membrane
showed highest values for the nanoemulsion, while lower values
for SLN and NLC (8 and 19%, respectively). Instead, permeation
studies with fresh pig ear skin showed that the active remained
mainly in the skin, showing a scarce penetration. Finally, the
nanocarriers were able to protect lutein against UV degradation.
The best results were observed with SLN while the nanoemulsion
gave the worst photoprotection.
Coenzyme Q10 is a well-acknowledged antioxidant showing the
ability to reduce the photoaging in vivo with a mechanism related
to the ability to increase the production of basal membrane com-
ponents, fibroblast proliferation and to the protection of cells
against oxidative damage. The effective delivery of coenzyme Q10
to the skin has several benefits in therapy for different pathologies.
Unfortunately, instability issues compromise the topical applica-
tion of coenzyme Q10, requiring the use of a valid delivery strategy.
Gokce and coworkers formulated Q10 loaded liposomes and
SLNs and carried out biocompatibility/cytotoxicity studies by
means of human fibroblast cell culture under oxidative conditions
[112]. The protective effect of these nanosystems against ROS pro-
duction were evaluated by cytofluorometry studies. The results
demonstrated the capability of Q10 loaded liposomes to protect the
cells against ROS accumulation. Surprisingly, lipid nanoparticles
seem to offer no advantages in terms of effective delivery of Q10 to
skin for antioxidant purposes. Bruge and coworkers [113] obtained
Fig. 4. Protective effects of lutein-loaded NLC against erythema induced on human
skin (NLC20: 4% of lutein; NLC33: 6.66% of lutein; NLC50: 10% of lutein). Data represent
the mean for twelve subjects [110].
different results. The researchers formulated NLC loaded Q10 and
demonstrated, in in vitro experiments, that the system was able to
counteract efficiently the oxidative phenomena inside cell mito-
chondria. More recently, Schwarz et al. formulated ultra-small NLC
for dermal delivery of coenzyme Q10 and demonstrated that the
decrease of particle size improved significantly the permeation and
the physicochemical stability of this active substance [114].
Lipid nanoparticle strategy has been used also for the topical
application of idebenone, a coenzyme Q10 derivative endowed
with a potent antioxidant activity that could be beneficial in the
treatment of skin oxidative damages [115]. Montenegro and co-
workers formulated idebenone loaded SLN using cetyl palmitate as
solid lipid and different non-ionic surfactants. The authors
demonstrated that idebenone penetration into the different skin
layers depended on the type of SLN used while no idebenone
permeation occurred from all the SLN under investigation.
Resveratrol has received considerable attention as a polyphenol
with anti-oxidant, anti-carcinogenic, and anti-inflammatory ef-
fects. Unfortunately, some instability issue counteracts its use in
topical therapy. Carlotti and coworkers investigate the possibility of
producing SLN as protective vehicle of resveratrol for topical de-
livery [116]. The results of the study demonstrated a significant
reduction of resveratrol photo-degradation once entrapped in SLN
matrix. Furthermore, SLN were able to increase the antioxidant
activity of resveratrol and to optimize in vitro its permeation
through porcine skin. Gokce and coworkers evaluated both SLN and
NLC as potential carriers for resveratrol topical administration
[117]. In vitro and ex vivo studies revealed that NLC were more
efficient in carrying resveratrol to the epidermis compared to SLN,
while both vehicles were able to counteract the production of ROS
in cell culture experiments.
Herpes simplex virus (HSV) is oneof the most common viral
diseases in humans. Infections of this virus can cause a wide range
of signs and symptoms varying from muco-cutaneous lesions to
life-threatening encephalitis. Recently Lai and coworkers, formu-
lated SLN loaded with the essential oil of Artemisia arborescens
[118], which had proven, in a previous study, to possess an inter-
esting antiviral activity against intracellular HSV-1 [119]. SLN con-
taining the essential oil were tested in vitro against HSV-1, while the
effect of essential oil incorporation into SLN on the permeation
through the skin was investigated by using in vitro diffusion ex-
periments through newborn pig skin. The results evidenced the
SLN ability to improve the oil accumulation into the skin while
maintaining the anti-herpetic activity of the essential oil.
Local anesthetics are widely used to alleviate pain after surgery,
trauma or medical procedures and although they show a rapid
action, they are characterized by a short effect compared with the
potential duration of pain. Lipid nanoparticles have been evaluated
as topical delivery system to release the anesthetic in a prolonged
fashion at the site of action and to reduce the risk of systemic
toxicity. Puglia and coworkers, for instance, formulated NLC loaded
with two popular anesthetics such as benzocaine and lidocaine
[94]. The release pattern of the two anesthetics was evaluated
in vitro determining their percutaneous absorption through excised
human skin and in vivo by the radiant heat tail flick test effected in
mice. Both experimental studies demonstrated that benzocaine and
lidocaine could be released in a prolonged fashion when incorpo-
rated in NLC.
3.4.2. Application of lipid nanoparticles in cosmetics
Both NLC and SLN have many features that are advantageous for
cosmetic application. Firstly, they are composed of highly
biocompatible lipids and consequently they satisfy the major safety
issues characterizing all the cosmetic products. In the last years, in
fact, the scarce experience about the interaction between nano-
L. Montenegro et al. / Journal of Drug Delivery Science and Technology 32 (2016) 100e112 109
sized material and body has unleashed a witch-hunt against
everything that is nano. The complete biodegradation of lipid
nanoparticles has secured them the title of ‘nano-safe carriers’ and
besides has defined them the prototype of modern cosmeceuticals.
Lipid nanoparticles are able to enhance the chemical stability of
compounds sensitive to light, oxidation and hydrolysis. Enhance-
ment of chemical stability after incorporation into lipid nano-
carriers was proven for many cosmetic actives. Sapino and co-
workers, for instance, demonstrated that SLN were able to protect
retinyl palmitate from the photo-degradation induced by UVA and
UVB radiations [120,121]. The authors hypothesized that this pro-
tection effect could be due to the light scattering properties of the
lipid nanoparticles. Trombino and other researchers demonstrated
that stearyl ferulate based SLN improved the stability of b-carotene
and a-tocopherol, two popular antioxidant used to protect the skin
against UVA induced damages [122]. More recently, Cerreto and
coworkers formulated SLN loaded with parabens as effective
reservoir systems for long-term preservation of multi-dose
cosmetic formulations [123]. Parabens are in fact a class of prod-
ucts characterized by antimicrobial activity and a certain chemical
instability that often compromises the formulation of cosmetic
products, particularly the multi-dose formulations.
The authors formulated paraben loaded SLN able on one hand to
protect the active from instability phenomena and on the other to
slowly release the active substance avoiding the contamination by
consumers during their use.
The occlusion effect is another important feature reported for
both SLN and NLC. Occlusion is responsible of a reduced skin water
loss and of a consequent increase in skin hydration.
Esposito and coworkers evaluated the hydration power of
different nano-systems by means of a corneometer, namely a
Fig. 5. UV absorption spectra of OMC-loaded formulations before and a
device able to measure the skin electrical capacitance before and
after the application of a topical formulation [124]. The results
demonstrated an interesting skin hydrating effect for viscosized
SLN.
The prolonged release of actives with a scarce penetration
through the skin is particularly required by cosmetic market. Mo-
lecular UV blockers, perfumes and repellents are typical examples
of products for whom a prolonged release is required.
Puglia and coworkers, for instance, evaluated the skin perme-
ation of OMC, a well-known UVB filter, from SLN and NLC [90]. The
results of in vitro percutaneous absorption study demonstrated that
OMC, when incorporated in viscosized NLC dispersions (OMC-NLC),
exhibited a lower flux with respect to viscosized SLN dispersions
(OMC-SLN) and other reference formulations. Furthermore, the
photo-stability studies evidenced that NLC were the most efficient
carriers at preserving OMC from UV-mediated photo-degradation
(Fig. 5). This important feature of NLC has been confirmed in a
recent work of the same research group regarding the optimization
of the topical application of different and widespread UVA and UVB
filters, such as ethylhexyltriazone (EHT), diethylamino hydox-
ybenzoyl hexyl benzoate (DHHB), bemotrizinol (Tinosorb S), OMC
and BMBM [125]. The results were in accordance with the evi-
dences obtained in the previous work. In particular, when incor-
porated in NLC, the skin permeation abilities of the sun filters were
drastically reduced, remaining mainly on the surface of the skin.
Finally, the photo-stability studies showed that EHT, DHHB and
Tinosorb S still retain their photo-stability when incorporated in
these carriers.
Diethyltoluamide (DEET) is an ingredient of insect repellent
products whose use is often compromised by its percutaneous
absorption. Lipid nanoparticle approach demonstrated to be a valid
fter UVA exposure, followed by extraction with ethyl acetate [90].
L. Montenegro et al. / Journal of Drug Delivery Science and Technology 32 (2016) 100e112110
strategy to locate the active ingredient onto the skin surface and to
reduce its skin permeation [93].
Lipid nanoparticles showed to possess the characteristics of
physical UV blockers. This feature is very important in order to
formulate sun care products containing a lower amount of chemical
sun filters but preserving their sun protection factor (SPF). Nikolic
and coworkers formulated different NLC based formulations con-
taining a mix of sun filters, namely EHT, bisethylhexyloxyphenol
methoxyphenyl triazine and OMC [126]. The authors demonstrated
that SPF values were strongly influenced by the type of lipid chosen
to formulate NLC and further they pointed out an active role of
these nano-carriers in the sun protection.
Recently, lipid nano-carriers demonstrated to be valid carrier for
topical application of cosmeceuticals. Like cosmetics, cosmeceut-
icals are applied topically but differ in that they contain potent
ingredients that can influence the biological function of the skin
and deliver nutrients to promote healthy skin. Scalia and coworkers
formulated emulsions containing lipid nanoparticles to improve
the topical delivery of quercetin [127]. The skin penetration of
quercetin was investigated in vivo on human volunteers by tape
stripping. The results evidenced a higher flavonoid accumulation in
the human horny layer following the topical application of quer-
cetin loaded lipid nanoparticles with respect to the control emul-
sion. In another recent manuscript, SLN were successfully used to
optimize the skin permeation profile of caffeine, an active ingre-
dient widely used in cosmetic field [128]. Caffeine loaded SLN
generated, in an in vitro study, a significantly faster permeation
than a control formulation over 24 hs of monitoring. In another
work, the SLN strategy was used again to modulatethe topical
delivery of stearyl glycyrrhetinate (SG), a cosmetic ingredient
endowed with lenitive activity [129]. Percutaneous absorption has
been studied in vitro, using excised human skin membranes and
in vivo, determining SG anti-inflammatory aka lenitive activity.
Both in vitro and in vivo results evidenced SLN capability to produce
an interesting delayed and sustained release of SG.
4. Conclusion
The search for successful skin delivery systems starting from
microemulsions has led to development of solid lipid nanoparticles
and nanostructured lipid carriers. These last ones, being the most
recent evolution of colloidal vehicles consisting of oil, water and
surfactants, allow overcoming most of the limits and drawbacks of
the older systems. Increased drug loading and higher stability are
the most relevant features that make NLC a more versatile delivery
system compared to SLN. Several researches have evidenced the
ability of all these carriers to improve skin permeation and/or to
achieve cutaneous targeting of a large variety of active ingredients,
depending on both vehicles composition and active ingredient
physical-chemical properties. Because of the progress in under-
standing the properties and the actual potential of these delivery
systems, new pharmaceutical and cosmetic products based on
these colloidal nano-carriers could be expected to be marketed in
the next future.
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	From nanoemulsions to nanostructured lipid carriers: A relevant development in dermal delivery of drugs and cosmetics
	1. Introduction
	1.1. Background
	1.2. Skin anatomy and physiology
	1.3. Percutaneous absorption
	2. Microemulsions and nanoemulsions
	2.1. Definition
	2.2. Structure and composition
	2.3. Characterization
	2.4. Microemulsions and nanoemulsions as dermal delivery systems
	3. Lipid nanoparticles
	3.1. Historical background and definitions
	3.2. Preparation techniques
	3.3. Characterization
	3.4. Application of SLN andNLC in cosmetics and dermal pharmaceuticals
	3.4.1. Lipid nanoparticles in topical drug delivery
	3.4.2. Application of lipid nanoparticles in cosmetics
	4. Conclusion
	References