Targeted cutaneous delivery of ciclosporin A using micellar nanocarriers and the possible role of inter-cluster regions as molecular transport pathways

Article abstract of DOI:10.1016/j.jconrel.2014.09.021

Oral administration of ciclosporin A (CsA) is indicated in the treatment of severe recalcitrant plaque psoriasis. However, CsA is both nephro- and hepatotoxic and its systemic administration also exposes the patient to other severe side effects. Although

Full text of DOI:10.1016/j.jconrel.2014.09.021

Journal of Controlled Release 196 (2014) 9–18
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Targeted cutaneous delivery of ciclosporin A using micellar nanocarriers
and the possible role of inter-cluster regions as molecular
transport pathways
Maria Lapteva, Verena Santer, Karine Mondon, Ilias Patmanidis, Gianpaolo Chiriano, Leonardo Scapozza,
Robert Gurny, Michael Möller, Yogeshvar N. Kalia ⁎
School of Pharmaceutical Sciences, University of Geneva and University of Lausanne, 30 Quai Ernest Ansermet, 1211 Geneva, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 June 2014
Accepted 23 September 2014
Available online 30 September 2014
Oral administration of ciclosporin A (CsA) is indicated in the treatment of severe recalcitrant plaque psoriasis.
However, CsA is both nephro- and hepatotoxic and its systemic administration also exposes the patient to
other severe side effects. Although topical delivery of CsA, targeted directly to psoriatic skin, would offer signifi-
cant advantages, there are no topical formulations approved for dermatological use. The aim of this work was to
formulate CsA loaded polymeric micelles using the biodegradable and biocompatible MPEG-dihexPLA diblock
copolymer and to evaluate their potential for delivering the drug selectively into the skin without concomitant
transdermal permeation. Micelle formulations were characterised with respect to drug content, size and mor-
phology. Micelle and drug penetration pathways were subsequently visualised with confocal laser scanning mi-
croscopy (CLSM) using fluorescein labelled CsA (Fluo-CsA) and Nile-Red (NR) labelled copolymer. Visualisation
studies typically use fluorescent dyes as “model drugs”; however, these may have different physicochemical
properties to the drug molecule under investigation. Therefore, in this study it was decided to chemically modify
CsA and to use this structurally similar fluorescent analogue to visualise molecular distribution and transport
pathways. Molecular modelling techniques and experimental determination of log D served as molecular scale
and macroscopic methods to compare the lipophilicity of CsA and Fluo-CsA. The spherical, homogeneous and
nanometre-scale micelles (with Zav from 25 to 52 nm) increased the aqueous solubility of CsA by 518-fold.
Keywords:
Psoriasis
Ciclosporin A
Skin topical delivery
Polymeric micelles
Confocal laser scanning microscopy
Transport pathway
2
Supra-therapeutic amounts of CsA were delivered to human skin (1.4 ± 0.6 μg/cm , cf. a statistically equivalent
2
1
.1 ± 0.5 μg/cm for porcine skin) after application of the formulation with the lowest CsA and copolymer
content (1.67 ± 0.03 mg/ml of CsA and 5 mg/ml of copolymer) for only 1 h without concomitant transdermal
permeation. Fluo-CsA was successfully synthesised, characterised and incorporated into fluorescent NR-MPEG-
dihexPLA micelles; its conformation was not modified by the addition of fluorescein and its log D, measured
from pH 4 to 8, was equivalent to that of CsA. Fluo-CsA and NR-MPEG-dihexPLA copolymer were subsequently
visualised in skin by CLSM. The images indicated that micelles were preferentially deposited between
corneocytes and in the inter-cluster regions (i.e. between the clusters of corneocytes). Fluo-CsA skin penetration
was deeper in these structures, suggesting that inter-cluster penetration is probably the preferred transport
pathway responsible for the increased cutaneous delivery of CsA.
©
2014 Elsevier B.V. All rights reserved.
1
. Introduction
angiogenesis and the presence of inflammatory cells, e.g. dendritic cells,
macrophages and T cells in the dermis. Some T cells can also be found
in the epidermis [2]. High-potency corticosteroids are the first-line
therapeutics used for the topical treatment of psoriasis because of
their anti-inflammatory, immunosuppressive and anti-proliferative
effects. However, they have many side effects including cutaneous
atrophy, formation of telangiectasia, steroid rosacea, perioral dermatitis
and increased risk of skin infections to cite but a few [3]. Since the
inflammation in psoriasis is strongly related to an excessive immune re-
sponse, immunosuppressants, such as ciclosporin A (CsA), have shown
good efficacy. CsA is a cyclic macrolide undecapeptide that targets
T cells present in the dermis and epidermis. It inhibits calcineurin
Psoriasis is a common skin disease affecting approximately 25
million people in North America and Europe [1]. It is an auto-immune
mediated inflammatory disorder in which the mitotic rate of the basal
keratinocytes is abnormally high, resulting in erythematous plaques
with silvery scales at the skin surface, a thickened epidermis, increased
⁎
Corresponding author at: School of Pharmaceutical Sciences, University of Geneva, 30
Quai Ernest Ansermet, 1211 Geneva 4, Switzerland. Tel.: +41 22 379 3355; fax: +41 22
79 3360.
E-mail address: Yogi.Kalia@unige.ch (Y.N. Kalia).
3
http://dx.doi.org/10.1016/j.jconrel.2014.09.021
168-3659/© 2014 Elsevier B.V. All rights reserved.
0

10
M. Lapteva et al. / Journal of Controlled Release 196 (2014) 9–18
phosphatase, which is responsible for the translocation of the nuclear
factor of activated T cells (NFAT) from the cytoplasm to the nucleus
where it promotes the transcription and expression of interleukin-2
and other cytokines.
using chick embryo chorioallantoic membrane (CAM) model for both
the copolymer only and polymeric micelles up to copolymer concentra-
tions of 20 mg/ml [15].
Ocular administration of CsA using MPEG-dihexPLA micelles also
showed promising results [17,18]. However, skin and cornea possess
very different histological structures and different barrier properties;
therefore, the ability of MPEG-dihexPLA micelles to deliver CsA into
the skin must be determined experimentally and this was the objective
of the first part of the present study. The second part involved an inves-
tigation into the cutaneous localisation of the drug and the micelles and
a visualisation of transport routes in the skin. Confocal laser scanning
microscopy (CLSM) studies are very useful to achieve this goal. Small,
low molecular weight dyes are generally employed as “model drugs”
but it is not always possible to accurately replicate the properties
of the drug of interest. Furthermore, these “model drugs” are inappro-
priate in our case as both the copolymer and incorporated drug are
of higher molecular weight. Therefore, in order to investigate the
localisation of the copolymer and CsA in skin using CLSM, both were
chemically coupled to fluorescent dyes—Nile Red and fluorescein,
respectively.
CsA was the first immunosuppressant to reach the market in 1983
under the trade name of Sandimmune® and later Neoral®. It is currently
indicated in the treatment of severe recalcitrant psoriasis when admin-
istered orally [4]. Liu et al. [5] demonstrated in rats in vivo that CsA
applied topically using a bicontinuous microemulsion yielded 30-fold
higher drug deposition in skin as compared to oral administration of
Neoral®. Moreover, CsA levels in blood, liver and kidney were much
lower following topical administration. Thus, the results confirmed
that topical administration was an effective means to increase CsA
levels in the skin while decreasing systemic exposure.
Despite the strong pharmacological rationale for its use, clinical
studies have shown that psoriasis patients display considerable variabil-
ity in their response to CsA [6,7]. Hirano et al. [6] measured the CsA
concentration that gave 50% inhibition (IC50) of mitogen-stimulated
peripheral blood mononuclear cells (PBMC) proliferation in 33 psoriasis
patients. They observed that the IC50 ranged from 0.1 to 120.6 ng/ml and
divided the population into two groups—good and poor responders
(
51.5% and 48.5% of the population, respectively). Umezawa et al. [7]
2. Materials and methods
subsequently showed that more hepatopathies and nephropathies
were reported in psoriatic patients with low sensitivity to CsA since
these patients obviously required higher systemically administered
doses. Therefore, in order to avoid unnecessary systemic exposure,
a targeted and effective topical therapy would be preferable to oral
administration of CsA.
However, efficient delivery of poorly water soluble drugs such as CsA
into the stratum corneum is a considerable challenge since formulation
excipients used to solubilise the drug frequently reduce thermodynamic
activity. In addition to physicochemical considerations, physiological
factors must also be taken into account—psoriatic lesions can have
thickened stratum corneum and epidermis. Moreover, from a patient
compliance perspective, topical formulations should ideally have a
once-daily (or even lower) application frequency, be easy to apply,
have quick absorption and absence of greasiness [2].
2.1. Materials
Ciclosporin A was purchased from Hangzhou Dayangchem
(Hangzhou Zhejiang, PR China). The novel amphiphilic MPEG-dihexPLA
copolymer (methoxy-poly(ethylene glycol) di-(hexyl-substituted poly-
lactide)) and its Nile Red labelled analogue were synthesised in-house
as described previously [14,19]. Their respective structures are presented
in Fig. 1. The MPEG-dihexPLA copolymer had a Mn of 6080 g/mol and
a polydispersity index (P.I.) of 1.15 according to GPC measurements
(the GPC setup consisted of a Waters Styragel HR1-4 column, Waters
717 autosampler, Waters 515 HPLC pump and a Waters 410 differential
refractometer (Waters; Baden-Dättwil, Switzerland)). The analysis was
carried out using polystyrene (PS) with different molecular weights as
calibration standards (PSS; Mainz, Germany).
Numerous efforts have been made to improve topical delivery of CsA
into skin with a strong emphasis on the development of colloidal nano-
structured vehicles including microemulsions [5,8,9], monoolein liquid
crystalline formulations [10,11], amphiphilic gels [12] and solid lipid
nanoparticles [13]. The challenge is to increase cutaneous bioavailability
without producing a concomitant increase in transdermal permeation
and, hence, systemic exposure in vivo.
The starting materials, 4-vinylaminobenzene, and Boc-anhydride
(di-tert-butyldicarbonate) used to synthesise Fluo-CsA were purchased
from TCI Europe (Eschborn, Germany) and Fluka (Buchs, Switzerland),
respectively. The second generation Grubbs catalyst, trifluoroacetic
a)
The objective of the present study was to investigate the incorpo-
ration of CsA into polymeric micelles and so develop simple, single
component aqueous formulations for topical delivery into skin to treat
psoriasis. Micelles are colloidal systems usually composed of amphiphil-
ic molecules, able to self-assemble above the critical micelle concentra-
tion (CMC). These spherical structures possess a hydrophilic shell and a
hydrophobic core. In the case of polymeric micelles, the amphiphilic
compound is a block copolymer with lipophilic/hydrophilic component
blocks. Trimaille et al. [14] developed a methoxy-poly(ethylene glycol)-
poly(hexylsubstituted lactides) (MPEG-hexPLA) diblock copolymer:
the methoxy-poly(ethylene glycol) chain provided the hydrophilic
O
O
O
H C
45
O
11 OH
3
O
O
b)
O
“head”, whereas the lipophilic “tail” came from the hexyl substituted
N
polylactide component. The presence of hexyl groups resulted in an
O
O
N
−
6
increased lipophilicity of the core and a low CMC (1.6 × 10 M) com-
pared to the standard MPEG-PLA [14] facilitating the incorporation of
highly lipophilic drugs. CsA-loaded MPEG-dihexPLA micelles (dihexyl
substituted derivative) were first developed and tested for parenteral
use [15]. It was shown that micelles produced a significant increase in
CsA water solubility and that the formulations were stable for up to
O
O
H C
45
O
11
O
O
3
O
1
year at 4 °C. After a forced degradation, the copolymer formed two
non-toxic compounds: 2-hydroxyoctanoic acid and PEG [16]. Moreover,
Fig. 1. a) Structure of MPEG-dihexPLA copolymer and b) structure of Nile Red labelled
no toxicity was demonstrated in vitro in various cell lines and in vivo
MPEG-dihexPLA copolymer.

M. Lapteva et al. / Journal of Controlled Release 196 (2014) 9–18
11
acid, tetrahydrofuran, octanol, acetone, tetrabutylammonium hy-
drogen sulphate (TBAHS), acetic acid, formic acid, hydrochloric
acid, citric acid and sodium chloride were supplied by Sigma-Aldrich
(
Steinheim, Germany). NHS-fluorescein was purchased from Thermo
Scientific (Dreieich, Germany). Silica gel (SiliaFlash P60 with a particle
size of 40–63 μm) was bought from Parc-Technologique BLVD (Quebec
City, Canada). Sodium hydroxide solution (40 M), potassium chloride
and sodium hydrogen phosphate were obtained from Fluka (Buchs,
Switzerland). Triethylamine (TEA), potassium dihydrogen phosphate
and potassium hydrogen phosphate were supplied by Acros Organics
(
Geel, Belgium) and propylene glycol by Hänseler AG (Herisau,
Switzerland). Bovine serum albumin (BSA) was purchased from
Axon Lab (Baden-Dättwil, Switzerland). All other solvents (methanol,
n-hexane, ethyl acetate, dichloromethane, chloroform, acetonitrile
(
MeCN)) were HPLC grade (HiPerSolv Chromatonorm: Darmstadt,
Germany). Ultra-pure water (Millipore Milli-Q Gard 1 Purification
Pack resistivity N 18MΩcm; Zug, Switzerland) was used to prepare all
solutions.
2
.2. Synthesis and characterisation of a fluorescent analogue of ciclosporin
A (Fluo-CsA)
2
.2.1. Synthesis of a fluorescent analogue of ciclosporin A (Fluo-CsA)
CsA was labelled with the fluorescent dye, NHS-fluorescein, using
-vinylbenzylamine (4-VB) as a linker using a four-step procedure,
4
adapted from Gaali et al. [20]. An overview of the four steps is shown
in Fig. 2. The primary amine of 4-VB (①) was first Boc-protected
(②) and then attached to CsA (③) by a cross-metathesis catalysed by
the Grubbs second generation catalyst to yield (④), which was then
deprotected and labelled with the fluorescent dye, NHS-fluorescein
(⑥) to produce the final fluorescent analogue of CsA: Fluo-CsA (⑦).
The detailed methods used during the different steps are described
in the Supplementary Data. In order to confirm the success of the differ-
ent reaction steps, the intermediate products were analysed by mass
spectroscopy (API 150EX, AB Sciex; Framingham, USA) and NMR
(
Varian Gemini 300 BB, 300 MHz, Varian; Palo Alto, USA). CDCl
3
or
DMSO-d were used as solvents (Supplementary Data). The final prod-
6
uct, Fluo-CsA, was characterised by high resolution mass spectroscopy
and its purity was tested consecutively by HPLC, the product appeared
to be 98% pure. The total yield of the four reaction steps was 61%.
2
.2.2. Structural comparison of CsA and Fluo-CsA
In order to examine the possible conformational changes of CsA
Fig. 2. Synthesis of fluorescein-labelled CsA (Fluo-CsA, ⑦).
when compared to Fluo-CsA, a structural analysis was performed by
using computational methods. The analysis was carried out using
the Maestro programme, a tool from the Schrödinger 2013 package
and 0.5 ml of drug-in-octanol solution. The two immiscible phases
were stirred for 24 h at RT. Finally, CsA and Fluo-CsA were quantified
in the octanol phase after appropriate dilution. The concentration in
the aqueous phase was subsequently determined and log D as a func-
tion of pH calculated according to Eq. (1).
(
Maestro; Schrödinger, LLC: Portland, OR, 2009). Energy minimisations
were carried out in water by applying 10,000 iterations with a conjugat-
ed gradient of 0.05 kcal/mol and using the OPLS-2005 force-field.
CsA was used as the reference structure [PDB code: 1CYA]. As
fluorescein carries ionisable groups, Fluo-CsA was submitted to the
minimisation steps considering both of its protonation states: neutral
and deprotonated. The conformation of the backbone of the compounds
was investigated. The structures were represented using the VMD
program [21]. Finally, the lipophilicity potential surface of the CsA and
Fluo-CsA was analysed using Sybyl 2.1.1 programme (SYBYL-X 2.1.1,
Tripos International, St. Louis, Missouri, USA).
ꢀ
ꢁ
½
CsA or Fluo‐CsAꢀoctanol
LogDoctanol=buffer pH x ¼ Log
ð1Þ
½
CsA or Fluo‐CsAꢀbuffer pHx
2
.2.3. Determination of the distribution coefficient of CsA and Fluo-CsA
The distribution coefficients (log D) of Fluo-CsA and CsA were deter-
2.3. Analytical methods
mined experimentally using the shake flask method in order to compare
their lipophilicity as a function of pH (4–8) [22]. Solutions of each com-
pound (100 μg/ml) were prepared in octanol (organic phase). Buffer so-
lutions (pH 4–8) constituted the aqueous phase. Buffer solutions were
saturated with octanol and vice versa for 24 h prior to measurement.
For the experiment, glass vials were filled with 1 ml of buffer solution
Both CsA and Fluo-CsA were quantified using high performance
liquid chromatography (HPLC). The HPLC system consisted of a P680
HPLC pump, an ASI-100 automated sample injector and a TCC-100
thermostatted column compartment and UV and fluorescent detectors,
UVD170U/RF 2000, respectively (formerly Dionex AG, now Thermo
Fisher Scientific AG; Reinach, Switzerland).

12
M. Lapteva et al. / Journal of Controlled Release 196 (2014) 9–18
2
.3.1. Quantification of CsA
2.6. Micelle CsA and Fluo-CsA content determination
For the quantification of CsA, a reverse phase column (Lichrospher
00, RP 18, 125*4.0 mm, 5 μm) was thermostated at 40 °C. The mobile
1
CsA or Fluo-CsA loading in the micelles was quantified as described
above (2.3.1 and 2.3.2, respectively). To ensure complete micelle de-
struction and release of the incorporated substance, 1:20, 1:50 1:100
dilutions were made for each formulation in either acetonitrile or
methanol for CsA or Fluo-CsA loaded micelles, respectively. The drug
content, drug loading and incorporation efficiency were calculated
using Eqs. (2)-(4), respectively:
phase consisted of a mixture of MeCN:phosphoric acid 0.1% buffer,
pH 3 (75:25) at a flow rate of 1 ml/min. CsA was detected by its UV
absorbance at 230 nm. All standards and samples were prepared in
acetonitrile. The retention time of CsA was 6.2 min and the method
was validated (Supplementary Data) according to ICH and FDA guide-
lines [23,24]. The limits of detection (LOD) and quantification (LOQ)
were 0.57 and 1.72 μg/ml respectively.
Drug content ðmg drug= ml formulationÞ
mass of drug in the formulation ðmgÞ
2
.3.2. Quantification of Fluo-CsA
Quantification and purity testing of the newly synthesised com-
¼
ð2Þ
ð3Þ
ð4Þ
Volume of the formulation ðmlÞ
pound were performed using a Jupiter 5 μm, C4, 300 Å 150 × 4.60 mm
column heated at 30 °C. The mobile phase, comprised a 70:30 mixture
of MeOH:pH 8 buffer (5 mM K HPO , 5 mM TBAHS) and the flow rate
2 4
was 1 ml/min. The excitation and emission wavelengths of the fluo-
rescence detector were set at 490 nm and 514 nm, respectively. The
method was validated (Supplementary Data) according to ICH and
FDA guidelines [23,24]. The LOD and LOQ were 0.11 and 0.35 μg/ml,
respectively.
Drug loading ðmg drug=g copolymerÞ
drugꢀin the formulation ðmg=mlÞ
Copolymerꢀin the formulation ðg=mlÞ
½
¼
½
Incorporation efficiency ð%Þ
mass of drug incorporated into micelles ðmgÞ
¼
ꢁ 100
mass of drug introduced ðmgÞ
2
2
.4. Micelle preparation
.4.1. Preparation of CsA loaded micelles
2.7. Skin preparation
Micelles were prepared using the solvent evaporation method
[
25,26]. Briefly, CsA (8–32 mg) and the copolymer (20–80 mg) were
Porcine ear skin was used for the in vitro skin transport studies
27–29]. Ears were purchased from a local abattoir (CARRE, Rolle,
dissolved in 2 ml of acetone. This organic solution was then added
dropwise under sonication (Branson digital Sonifier® S-450D; Carouge,
Switzerland) into 4 ml of ultra-pure water. Acetone was then evapo-
rated with a rotary evaporator (Büchi RE 121 Rotavapor; Flawil,
Switzerland). The final copolymer concentration ranged from 5 to
[
Switzerland). After washing under running cold water, skin samples
with a thickness of ≈0.75 mm were harvested using a Zimmer® air
dermatome (Münsingen, Switzerland). Hairs were excised from the skin
surface using clippers. Discs with a diameter of 32 mm, corresponding
to the formulation application area, were punched out using a puncher
2
0 mg/ml. After equilibration overnight, the micelle solution was
centrifuged at 10,000 rpm for 15 min (Eppendorf Centrifuge 5804;
Hamburg, Germany) to remove non-incorporated drug and the super-
natant was collected.
(Berg & Schmid HK 500, Urdorf, Switzerland) Skin samples were frozen
at −20 °C for a maximum period of 3 months. Prior to the experiment,
skin samples were thawed at room temperature and placed for 15 min
in 0.9% saline solution for rehydration.
Human skin samples were collected immediately after surgery from
the Department of Plastic, Aesthetic and Reconstructive Surgery, Geneva
University Hospital (Geneva, Switzerland). The study was approved by
the Central Committee for Ethics in Research (CER: 08-150 (NAC08-
2
.4.2. Preparation of Fluo-CsA loaded micelles
In the case of Fluo-CsA loaded micelles, the copolymer mixture
consisted of MPEG-dihexPLA and the fluorescent NR-MPEG-dihexPLA
copolymers (7:3) in 2 ml of acetone. Fluo-CsA (6.5 mg) was added to
this mixture. The micelles were prepared using the solvent evapora-
tion method described above. The final copolymer concentration was
0
51); Geneva University Hospital). Hypodermis and fatty tissue were
removed and discs corresponding to the permeation area were punched
out (Berg & Schmid HK 500; Urdorf, Switzerland). The skin discs (again
with a diameter of 32 mm) were subsequently horizontally sliced with a
Thomas Stadie-Riggs slicer (Thomas Scientific; Swedesboro, NJ, USA) to
obtain a thickness of ~0.8 mm. The skin was stored in a biobank at
5
mg/ml.
2
2
.5. Micelle formulation characterisation
.5.1. Size determination
The hydrodynamic diameter (Zav), polydispersity index (P.I.), volume-
−
20 °C for a maximum period of 3 months.
weighted and number-weighted diameter (d
measured using dynamic light scattering (DLS) with a Zetasizer HS
000 (Malvern Instruments Ltd.; Malvern, UK). Parameters were ob-
tained after three runs of ten measurements at an angle of 90° and a
temperature of 25 °C.
v n
and d ) of micelles were
2
.8. In vitro delivery experiments
3
Skin samples were mounted in standard Franz diffusion cells
2
(
area = 2 cm , Milian; Meyrin Switzerland). 500 mg of micelle formu-
lation or control were added to the donor compartment. The receptor
compartment (PBS pH 7.4 + 1% BSA, 10 ml) was stirred at 250 rpm at
room temperature during 1, 4, 8, 12 or 24 h. At the end of the experi-
ment, 1 ml of receptor phase was withdrawn to quantify permeation
of CsA. After centrifugation at 10,000 rpm for 15 min permeation sam-
ples were analysed by HPLC. The diffusion cells were subsequently
dismantled and the skin samples were carefully washed for 5 s under
running water to remove the residual formulation from the stratum
corneum surface. The wash procedure was validated (Supplementary
Data). Skin samples were then cut into small pieces and deposited CsA
was extracted by soaking the skin in 4 ml of acetonitrile or methanol,
respectively, for 4 h with continuous stirring at room temperature.
2
.5.2. Morphology determination
Micelle morphology was characterised with transmission electron
microscopy (TEM) (Technai G2 20) using the negative staining method.
5
2
μl of the micelle solution was applied onto an ionised carbon-coated
00 mesh copper grid (0.3 Torr, 400 V for 20 s). The grid was then de-
posited for 1 s onto a 100 μl drop of a saturated uranyl acetate aqueous
solution and then onto a second 100 μl drop for 30 s. Excess staining
solution was removed and the grid was dried at room temperature
prior to insertion into the sample holder and TEM analysis. TEM images
were processed using Image J software (Image J 1.45 s).

M. Lapteva et al. / Journal of Controlled Release 196 (2014) 9–18
13
The extraction samples were centrifuged at 10 000 rpm for 15 min
and quantified by HPLC. The extraction procedure was also validated
of Fluo-CsA was significantly lower (1.68 ± 0.02 vs. 2.08 ± 0.11).
Although this may have been a statistical anomaly, fluorescein has mul-
tiple ionisable groups [31]. In strongly acidic conditions it exists as a
neutral compound, increasing pH results in deprotonation of the car-
boxylic acid moiety to yield the mono-anion and finally in weakly acidic
conditions, the phenol is deprotonated (pKa ~6.4) to produce the di-
anion (Fig. 4). Therefore, at pH 8, the fluorescein moiety of Fluo-CsA
carries a double negative charge and this tends to render the compound
more hydrophilic, lowering its log D. However, given that the skin
surface pH is ~5.0–5.5, that the micelle formulation pH was 5.5–6 and
that, in general, topical formulations usually have a pH ranging from
weakly acidic to neutral [32], the results indicate that chemical modifi-
cation of CsA and the addition of ionisable groups did not significantly
affect its partition properties over the relevant pH range. Therefore,
from a physicochemical point of view, the synthesised fluorescent
analogue of CsA was considered to be a suitable model for subsequent
CLSM visualisation studies.
(
Supplementary Data).
2
.9. Data analysis
Data were expressed as the mean ± SD. Outliers determined using
the Dixon test were discarded. Results were evaluated statistically
using either analysis of variance (ANOVA) or analysis of means by
Student's t-test. Student–Newman–Keuls test was used when necessary
as post-hoc procedure. The level of significance was fixed at α = 0.05.
2
.10. Visualisation of micelle penetration pathways using Fluo-CsA loaded
micelles
Upon completion of skin delivery experiments with fluorescent
micelles, the skin samples were cut in longitudinal sections. Each indi-
vidual sample was then placed on a glass slide with the exposed
cross-section (epidermis to dermis) facing the objective. Visualisation
of delivery pathways was investigated by confocal laser scanning
microscopy (CLSM) (LSM 710, Zeiss, Germany). The excitation wave-
lengths, fluorescence emission wavelengths, laser power, pinhole and
master gain for each dye are presented in Table 1.
The confocal images were obtained with an air Achroplan 20×
objective and acquired using Zen software (Carl Zeiss, Germany). To
ensure accurate comparison, all samples were visualised using the same
parameters. CLSM pictures were finally processed using Image J software
3.3. Micelle preparation and characterisation
3.3.1. CsA loaded micelles
Micelles of various copolymer contents (from 5 to 20 mg/ml of
copolymer) were formulated and characterised (Table 3).
CsA content increased linearly with increasing copolymer content
2
(y = 0.292x + 0.3108; R = 0.9991). CsA loading remained fairly
constant with values comprised between 310.93 ± 9.36 and 333.75 ±
5.56 mgCsA/gcopo. The highest drug loading 333.75 ± 5.56 mgCsA/gcopo
was achieved in Formulation A, which had the lowest copolymer
content (5 mg/ml). Formulation D (with 20 mg/ml of copolymer) was
able to incorporate 311.23 ± 1.16 mgCsA/gcopo, leading to a maximal
CsA content of 6.225 ± 0.223 mg/ml. Considering that the aqueous
solubility of CsA is only 0.012 mg/ml [15], the micelles were able to
increase this by up to 518-fold.
(Image J 1.45 s).
3
. Results and discussion
3
.1. Structural comparison of CsA and Fluo-CsA
The minimized structures and the lipophilicity surface potential of
All micelles were found to have an average hydrodynamic diameter,
CsA and Fluo-CsA in its different protonation states are presented in
Fig. 3.
As shown in Fig. 3a, the superimposition of the three minimised
structures reveals no significant conformational differences between
Fluo-CsA and CsA. This observation is also supported by the root-mean
Zav, between 25 and 52 nm, and a P.I. between 0.238 and 0.699. The size
of the micelles and P.I. increased slightly with copolymer content, indi-
cating that some aggregation might occur at high copolymer contents.
TEM observations (Fig. 5) showed that CsA micelles with low copolymer
content (5 mg/ml; Fig. 5a) were mainly spherical with diameters rang-
ing from 15 to 40 nm, whereas formulations with higher copolymer
content (20 mg/ml; Fig. 5b) showed still small, yet slightly elongated
worm-like micelles. This last observation could explain the high Zav
and P.I. determined by DLS for this formulation. However, overall results
indicated that polymeric micelle nanocarriers with a high CsA loading
and homogeneous size could be formulated.
-square deviation of atomic positions (RMSD) values, which have been
calculated among the three structures (Table 2).
It should also be noted that the attachment of the fluorescent dye did
not induce any significant change in the lipophilicity potential surface
of CsA (Fig. 3b). Therefore, it can be concluded that the fluorescently
labelled CsA is conformationally very similar to native CsA and may be
used as a representative model.
3.3.2. Fluo-CsA loaded micelles
3
.2. CsA and Fluo-CsA log D determination
Fluorescent NR-MPEG-dihexPLA micelles (5 mg/ml of copoly-
mer) were loaded with Fluo-CsA. A loading of 324.79 ± 4.2
mgFluo-CsA/gcopo was achieved. The Fluo-CsA content of the formulation
was 1.62 ± 0.03 mg Fluo-CsA/ml formulation. These results are
comparable to the corresponding values for CsA micelles (333.75 ±
5.56 mgCsA/gcopo and 1.669 ± 0.027 mg/ml, respectively). Fluo-CsA
loaded micelles were found to have an average hydrodynamic diameter
Although the computational studies predicted that Fluo-CsA was
conformationally similar to native CsA and that it had similar lipophilic-
ity, this needed to be confirmed experimentally at the macroscopic
level. Therefore, the distribution coefficients (log D) of both compounds
were determined from pH 4 to pH 8. The log D of both compounds was
between 1.81 ± 0.26 and 2.08 ± 0.11 over the tested pH range (Fig. 4).
These results were similar to the literature data which indicated a log D
of 2.73 for CsA independent of pH [30].
Zav of 27 nm, a P.I. of 0.328. TEM observations (Fig. 5c) showed that the
micelles were spherical with sizes ranging from 19 to 45 nm.
These results indicated that similar amounts of CsA and the fluores-
cent analogue (Fluo-CsA) were incorporated into the micelles. Further-
more, fluorescent micelles presented similar sizes and morphology to
No significant difference (t-test: p b 0.05) was found between the
log D of CsA and Fluo-CsA from pH 4 to 7. However, at pH 8, the log D
Table 1
Settings used for CLSM.
Dye
Excitation
Emission
Laser power (%)
Pinhole (μm)
Master gain
Nile Red
Fluo-CsA
HeNe diode laser at 543 nm
Ar laser at 488 nm
607–674 nm
504–530 nm
15
20
91
100
800
750

14
M. Lapteva et al. / Journal of Controlled Release 196 (2014) 9–18
a)
b)
Neutral
Fluo-CsA
Deprotonated
Fluo-CsA
CsA
Fig. 3. a) Superimposition of CsA and Fluo-CsA structures in its two distinct protonation states. CsA is coloured green, the neutral and the deprotonated states of Fluo-CsA are respectively in
blue and in red, b) lipophilicity potential surface of CsA, neutral and deprotonated states of Fluo-CsA, respectively. The lipophilicity scale ranges from brown (most lipophilic) to blue (most
hydrophilic surface). The minimum/maximum lipophilicity values for the different structures are: CsA (−0.083, 0.113), neutral Fluo-CsA (−0.072, 0.100) and deprotonated Fluo-CsA
(−0.169, −0.095). The decreased lipophlicity of deprotonated Fluo-CsA reflects the impact of the ionised carboxylate moiety.
the CsA loaded micelles. Thus, Nile Red labelling of the copolymer did
not affect the micelle size. As a result, NR-MPEG-dihexPLA micelles
loaded with Fluo-CsA were considered to be an accurate model to inves-
tigate the skin penetration pathways of both the drug and polymeric
micelles.
glycol) was 18-fold lower than that from the micelle formulation (0.9 ±
0.5 and 16.3 ± 3.0 μg/cm , respectively) at equivalent CsA content.
2
Although few studies have been conducted using polymeric micelles
as skin drug delivery systems, several hypotheses have been proposed
to describe the possible interactions between micelles and skin. It has
been suggested that intact micelles might permeate through intact
skin [33], but this is unlikely, given the high molecular weight of the
polymers used for micelle formation. Other studies suggest that the
enhancement can be due to micelle disassembly when in contact with
skin and action of individual polymer chains as chemical penetration
3
3
.4. In vitro delivery experiments
.4.1. Influence of CsA content in micelle formulation on skin delivery
CsA permeation across porcine skin after application of the formula-
tions for 24 h was undetectable by the HPLC-UV method, suggesting
that drug permeation was extremely low and that in vivo, only very
small amounts might reach the systemic circulation, which is primordial
when the disease is limited to skin. The increase in copolymer content
neutral
monoanion
dianion
O
O
OH
O
O
OH
O
O
O
OH
O
O
(
6
5 to 20 mg/ml), and subsequently in CsA content (1.669 ± 0.027 to
.225 ± 0.223 mg/ml), resulted in an increased topical delivery of CsA
O
O
O
2
into the skin ranging from 7.4 ± 1.1 to 16.3 ± 3.0 μg/cm and delivery
efficiencies ranging from 1.00 ± 0.27 to 1.78 ± 0.27% (Fig. 6a). CsA
delivery from the control formulation (6.2 mg/ml solution in propylene
HN
O
HN
O
HN
O
3
.5
2
CsA
CsA
CsA
Fluo-CsA
CsA
2
1
Table 2
.5
1
RMSD values (Å) among the backbone atoms of CsA and Fluo-CsA in its different proton-
ation states.
3
4
5
6
7
8
9
CsA
Neutral Fluo-CsA
Deprotonated Fluo-CsA
pH
CsA
–
0.33
–
0.20
0.14
–
Neutral Fluo-CsA
Deprotonated Fluo-CsA
0.33
0.20
Fig. 4. Comparison of Fluo-CsA and CsA Log D as a function of pH (data are expressed as
0.14
mean ± SD; n = 3).

M. Lapteva et al. / Journal of Controlled Release 196 (2014) 9–18
15
Table 3
Micelle formulation characterisation in terms of drug incorporation and size (data are expressed as mean ± SD; n = 3).
CsA content characterisation
Size characterisation
Zav (nm) P.I.
Formulation
Copolymer
TARGET drug
Drug loading ±
Drug content ±
SD (mgCsA/ml)
Incorporation
efficiency ± SD (%)
d
n
(nm) [%]d
n
v v
d (nm) [%]d
content (mg/ml) loading (mgCsA/gcopo) SD (mgCsA/gcopo
)
A
B
C
D
5
10
15
20
5
400
400
400
400
400
333.75 ± 5.56
327.28 ± 9.26
310.93 ± 9.36
311.23 ± 1.16
324.79 ± 4.2
1.669 ± 0.027
3.273 ± 0.093
4.664 ± 0.140
6.225 ± 0.223
1.626 ± 0.030
83.44 ± 1.39
81.82 ± 2.31
77.73 ± 2.34
77.81 ± 2.79
81. 91 ± 1.05
28
25
41
52
27
0.238 19 [100.0%]
0.164 19 [100.0%]
0.498 20 [100.0%]
0.699 21 [98.9%]
0.328 15 [100.0%]
13 [100.0%]
15 [98.3%]
16 [100.0%]
17 [100.0%]
13 [100.0%]
Fluorescent micelles
enhancers [34]. Superior delivery of econazole nitrate from MPEG-
dihexPLA micelles as compared to a marketed liposomal formulation
was attributed to the small size of the carriers, increased skin contact
area and depot formation at the skin surface or in appendages [26].
Micelles in a formulation applied to the skin, will be subject to ran-
dom Brownian motion in the solution. Some will reach the skin surface
and given the number of CsA molecules present per micelle, this results
in high local concentrations/thermodynamic activity which favours
partitioning into the stratum corneum. Although CsA molecules present
in the control propylene glycol formulation will also enter into contact
with the surface through Brownian motion, the local concentrations
are lower and hence there may be a lower thermodynamic driving
force to partition. Of course, this does not preclude interactions between
copolymer and the upper layers of the stratum corneum that perturb
lipid organisation/barrier function and so facilitate delivery.
3.4.2. Kinetics of CsA skin deposition and validation in human skin
The 5 mg/ml formulation was selected to study the kinetics of CsA
skin deposition over 24 h (Fig. 7a). CsA skin deposition was 1.1 ±
2
0.5 μg/cm after 1 h and increased before reaching a plateau between
2
12 h and 24 h (8.7 ± 1.8 and 7.4 ± 1.1 μg/cm respectively). Similar
behaviour was previously observed with flufenamic acid [35] and
described using a Michaelis–Menten model:
ꢂ
ꢃ
CsAdep; max ꢁ tapp
CsAdep tapp
¼
ð5Þ
tmax=2 þ tapp
where CsAdep (tapp) and CsAdep,max describe the deposition of CsA as
a function of formulation application time (tapp) and the maximum
(plateau) value and tmax/2 is that time at CsAdep reaches half of the
maximum value. It was hypothesised that there were a limited number
of available molecular binding sites and hence the process was satura-
The presence of a linear relationship between CsA delivery and
CsA content in the micelle formulations (y = 2.7345x − 0.2486;
2
2
R = 0.9588) (Fig. 6b) suggested that the increase in CsA content in
ble. Fitting of the data (R = 0.7434) to Eq. (5) as shown in Fig. 7a
2
the formulations was due to the increase in copolymer content, i.e. to
an increased number of micelles having an essentially constant drug
loading (from 333.75 ± 5.56 to 311.23 ± 1.16 mgCsA/gcopo; Table 3).
As argued above, the increased number of micelles would provide bet-
ter skin coverage and increase delivery as compared to the propylene
glycol formulation despite its high drug content (equivalent to that of
the 20 mg/ml copolymer formulation).
It was observed that delivery from the 5 mg/ml copolymer formula-
tion was higher (1.78 ± 0.27% of the applied dose) than that predicted
by the linear model. It should be mentioned that the 5 mg/ml copoly-
mer formulation had a higher drug loading than the other micelle
formulations (Table 3), resulting in a higher thermodynamic activity
of the drug inside the micelle core. Therefore, although fewer micelles
were in contact with skin, the increased thermodynamic driving force,
i.e. the tendency of the drug to “escape” from the micelle was high and
overall delivery was increased and was similar to that of the 10 mg/ml
copolymer formulation.
gave CsAdep,max and tmax/2 values of 10.6 μg/cm and 6.8 h, respectively.
As mentioned above, psoriatic patients possess high variability in
terms of their sensitivity to CsA. For example, in a psoriatic patient
with very low CsA sensitivity, the IC50 was 120.6 ng/ml and IC90 was
1000 ng/ml [6]. When applied to our in vitro model, this IC90 corre-
3
sponds to a minimum drug content in skin of 1 μg per cm of tissue.
The micelle formulation with the lowest copolymer and drug content
3
resulted in a CsA skin concentrations of 14.6 ± 6.6 μg/cm after only 1
3
h of application and 98.6 ± 14.6 μg/cm after 24 h, which exceeds the
IC90 in poor responders by 15- and 99-fold, respectively. When applied
to human skin for 1 h, the MPEG-dihexPLA micelle formulation yielded
2
a statistically similar CsA delivery of 1.4 ± 0.6 μg/cm (Fig. 7b), suggest-
ing that supra-therapeutic levels in human skin could be achieved after
a short 1 h application time. Nevertheless, a validation in diseased
human skin is necessary as the barrier function in psoriatic skin can be
highly modified [2].
Several colloidal formulations including microemulsions [5,8,9],
monoolein liquid crystalline formulations [10,11], amphiphilic gels
[12] and solid lipid nanoparticles [13] have been developed in order to
increase skin topical bioavailability of CsA. However, only studies con-
ducted by Lopes et al. [10,11,36] were performed using porcine skin
and thus are relevant for comparison with the data from the present
study.
From these results it was possible to conclude that CsA delivery from
the micelle formulations into the skin could be fine-tuned by modulat-
ing either the copolymer content or the drug loading. The 5 mg/ml co-
polymer formulation was selected for further experiments as it seemed
to be optimal in terms of thermodynamic activity and presented good
size and morphology characteristics.
a)
b)
c)
Fig. 5. TEM pictures of a) CsA loaded micelles with 5 mg/ml copolymer content, b) CsA loaded micelles with 20 mg/ml copolymer content and c) Fluo-CsA loaded fluorescent micelles with
mg/ml copolymer content. Bar = 200 nm.
5

16
M. Lapteva et al. / Journal of Controlled Release 196 (2014) 9–18
Delivery efficiency
.78 ± 0.27ꢀ 1.00 ± 0.27ꢀ 1.16 ± 0.16ꢀ 1.05 ± 0.19ꢀ 0.06 ± 0.04ꢀ
these liquid crystalline formulations are superior to MPEG dihex-PLA
micelles given the greater CsA content (4%), different experimental
conditions (inclusion of 10% ethanol in the receiver phase) and the
presence of oleic acid.
a)
1
2
1
1
1
1
1
0
8
6
4
2
0
8
6
4
2
0
In another study, water rich hexagonal nano-dispersions of MO and
oleic acid (2% w/w) were formulated with a CsA content of 0.6% [11].
The nanoparticulate nature of this formulation and its CsA content
were closer to that of the micelle formulations used in the present
study and allowed a more accurate comparison. In vitro topical skin
delivery of CsA from hexagonal nano-dispersions was approximately
2
61 μg/cm after application for 12 h, which is higher than that yielded
by the micelles. However, the ethanol content in the receptor solution
(10%) should again be emphasised. The penetration enhancing activity
of MO and oleic acid is due to their linear lipidic structures and to
their low molecular weight (356.54 g/mol and 282.46 g/mol for MO
and oleic acid, respectively) allowing them to interact with stratum
corneum lipids, disrupt their organisation and so improve transport.
In contrast to the MPEG-dihexPLA copolymer (MW N 6000 g/mol), the
diffusion of such small molecules into skin and their interaction with
stratum corneum is relatively facile. As for the micelles used in the pres-
ent study, it was also hypothesised that the nanoparticulate nature of
the MO/oleic acid system resulted in the formation of a depot on the
skin surface and in the appendages.
5
mg/ml 10 mg/ml 15 mg/ml 20 mg/ml Control
Copolymer content
b)
2
1
1
1
1
1
0
8
6
4
2
0
8
6
4
2
0
y = 2.7345x - 0.2486
R² = 0.9588
In conclusion, the CsA loaded micelle formulation appears to be a
better delivery system than bulk MO liquid crystalline formulations
[10,36]—it enables targeted delivery of CsA to skin, avoids its transder-
Control
mal permeation and potential systemic exposure to the drug in vivo.
Although cutaneous delivery of CsA from the water-rich hexagonal
nano-dispersions [11] was superior this may be attributed, in part, to
the effect of ethanol. It should also be noted that, in contrast to these
more complex formulations, the MPEG-dihexPLA micelles have the
advantage of being a “single excipient” carrier.
0
1
2
3
4
5
6
7
Drug content (mg/ml)
Fig. 6. CsA skin deposition from CsA-loaded micelles as a function of a) copolymer content
and b) CsA content. A 6.2 mg/ml CsA solution in propylene glycol served as control (data
are expressed as mean ± SD; n = 6). The delivery efficiencies (dose delivered/dose
applied) are presented in % in a).
3.5. Visualisation of micelle penetration pathways using Fluo-CsA loaded
micelles
CsA deposition after 12 h from a formulation containing 10%
2
monoolein (MO) in propylene glycol (PG) exceeded 100 μg/cm [36].
After the application of fluorescent micelles on porcine skin, the
Although much higher than that obtained with the micelle formulations
described here, the differences can be explained by the high CsA content
in the formulation (4%) and the presence of ethanol, a known penetra-
tion enhancer, in the receptor solution (10%). The latter may have in-
creased skin permeability by diffusing into the tissue from the dermis
as evidenced by the presence of CsA in the receiver compartment. MO
can form various liquid crystalline structures when mixed with water
localisation of both Fluo-CsA and NR-MPEG-dihexPLA copolymer
could be visualised using CLSM (Fig. 8).
The stability of the bond between Nile Red and the MPEG-dihexPLA
copolymer was proven by mass spectroscopy and the labelled copoly-
mer was found to be stable over the duration of the experiment, infer-
ring that the red signal seen in the images originated specifically from
the copolymer and not from the free dye. For the Fluo-CsA, some free
dye was found in skin after the delivery experiment; however, this
amount was considered to be negligible, as it constituted only 4% of
the total fluorescein signal. It appeared that the NR-MPEG-dihexPLA
copolymer remained on the skin surface (Fig. 8a), whereas Fluo-CsA
penetrated into the skin (Fig. 8b and c). Given its high molecular weight
[37] and in a subsequent study, MO reverse hexagonal and cubic phases
were formulated [10] by the addition of 5% oleic acid to the cubic phase.
Skin delivery of CsA from cubic and hexagonal phase formulations was
2
2
approximately 210 μg/cm and 79 μg/cm , respectively; however,
some permeation was again observed. It is difficult to conclude whether
(
Mn of 6080 g/mol), the copolymer could not cross the stratum
corneum barrier and released Fluo-CsA from the skin surface.
a)
b)
It has been suggested that skin structure is not entirely homoge-
neous and corneocytes are organised not only in parallel horizontal
layers, but also in vertical columns [38–40]. A group of such columns
is called a “cluster” and its boundaries form furrows that seem to
match the fine wrinkles seen at the skin surface [40]. This organisation
has been observed in murine [38], porcine [39] and human skins
2
.5
2
15
1
0
5
0
1
0
.5
1
[
41,42]. Confocal microscopy investigations led to the suggestion that
.5
0
the intercellular spaces between clusters are more permeable [38]
than the traditional intercellular route between single corneocytes,
especially to drugs encapsulated in colloidal systems such as vesicles
and liposomes [38,39,41]. It was also recently shown that ZnO nanopar-
ticles of a similar size to the micelles (30 nm) are preferentially depos-
ited into inter-cluster regions (furrows) and into the orifices of the hair
follicles [42].
0
4
8
12
16
20
24
porcine skin
human skin
time (h)
Fig. 7. a) CsA deposition as a function of application time in porcine skin (data are
expressed as mean ± SD; n = 5). b) Comparison of CsA deposition in porcine and
human skin after micelle application for 1 h.

M. Lapteva et al. / Journal of Controlled Release 196 (2014) 9–18
17
a)
d)
b)
c)
e)
HS
z
x
Cluster
C
z
y
x
f)
Cluster
C
z
z
y
x
x
Fig. 8. Optical skin cross-sections (xz plane) with respective deposition of (a) NR-MPEG-dihexPLA copolymer, (b) Fluo-CsA and (c) superimposition of both signals. Figures (d) and
e) represent a three-dimensional reconstruction of skin surface with deposition of NR-MPEG-dihexPLA copolymer and Fluo-CsA. Corneocytes (C) can be clearly observed and appear
(
to be organised in bigger structures denoted as “clusters”. NR-MPEG-dihexPLA copolymer and Fluo-CsA seem to accumulate preferentially in regions between the clusters (inter-cluster
regions) denoted by arrows. A hair shaft (HS) can be seen (e). (f) Results from an optical skin cross-section (xz plane) from (e) (denoted as dotted line): the skin penetration of Fluo-CsA
seems to be deeper directly under the inter-cluster regions (arrow). Bar = 50 μm.
In the present study, CLSM three-dimensional reconstructions of the
skin (Fig. 8d and e) showed several skin structures such as a hair shaft,
single corneocytes and interestingly inter-cluster regions. It seems that
the inter-corneocyte and inter-cluster regions are preferential micelle
deposition sites, which is consistent with the observations found in
the literature [38–42].
Moreover, from Fig. 8f it can be seen that Fluo-CsA penetration into
skin is deeper directly under the inter-cluster regions (arrows). These
findings can explain the increased CsA delivery yielded by MPEG-
dihexPLA micelles in comparison to the control formulations. Like
other nanoparticulate systems [40,42], micelles are probably deposited
preferentially in skin wrinkles, between the corneocyte clusters, where
they leverage this more permeable zone to increase drug delivery. How-
ever, as yet it is not known whether psoriatic skin possesses such histo-
logical structures and if the inter-cluster pathway is still relevant in this
disease.
Fluo-CsA skin penetration was deeper under these latter structures,
suggesting that this might be the preferential transport pathway re-
sponsible for the increased topical delivery of CsA yielded by micelles.
This study confirms that MPEG-dihexPLA micelles are an innovative
nanosized carrier able to increase topical CsA skin delivery. Although,
at the outset, the aim was to develop a topical formulation that might
be of interest in the treatment of psoriasis, it is clear that CsA has
many other potential applications in dermatological therapy [43,44].
However, considering the histo-pathological modifications of psoriatic
skin, the potential of this delivery system has to be confirmed in vivo
with diseased skin.
Acknowledgements
We thank the University of Geneva for teaching assistantships for
ML and VS. We would also like to acknowledge Dr. Christoph Bauer
and Jerôme Bosset at the Bioimaging Centre of University of Geneva
for their help with confocal and electron microscopy.
4
. Conclusion
MPEG-dihexPLA micelles efficiently incorporated and delivered the
poorly water soluble drug ciclosporin A (CsA). The nanometre-scale,
spherical micelles increased the aqueous solubility of CsA by up to
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jconrel.2014.09.021.
5
18-fold. In vitro skin permeation experiments indicated that micelles
significantly increased cutaneous bioavailability of CsA without any
transdermal permeation—thus, yielding a targeted delivery to skin and
minimising the risk of systemic exposure of the drug in vivo. Statistically
equivalent supra-therapeutic amounts of CsA were delivered to porcine
and human skins after application for 1 h of the formulation with the
lowest CsA and copolymer content. A fluorescent analogue of CsA
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