Hyperbranched glycerol-based core-amphiphilic branched shell nanotransporters for dermal drug delivery

Article abstract of DOI:10.1016/j.polymer.2016.04.074

A novel amphiphilic branched shell (ABS) obtained upon reaction of octadecen-1-yl succinic anhydride and methyl poly(ethylenglycol) (mPEG₅₀₀) is herein presented. Through chemical attachment to the functionalized external surfaces of three different glycerol-based hyperbranched polymers (HBP), a set of novel core-ABS nanocarriers that were tested as possible candidates for dermal delivery of active substances was obtained. Pyrene has been studied as a model to evaluate the potential drug transport capability and the critical micelle concentration (CMC) of these amphiphilic systems. Subsequently, the anti-inflammatory steroid drug Dexamethasone and Finasteride, a drug widely used to prevent prostate cancer and androgenic alopecia, were also efficiently loaded into the nanocarriers. In order to promote these systems as possible candidates for dermal drug delivery, tests to evaluate the cytotoxicity on human keratinocyte cells and the penetration of the encapsulated molecules into the different layers of human skin samples were carried out.

Full text of DOI:10.1016/j.polymer.2016.04.074

Accepted Manuscript
Hyperbranched glycerol-based core-amphiphilic branched shell nanotransporters for
dermal drug delivery
Stefano Stefani, Stefan Hönzke, Jose Luis Cuellar Camacho, Falko Neumann, Ashok
K. Prasad, Sarah Hedtrich, Rainer Haag, Paul Servin
PII:
S0032-3861(16)30380-9
10.1016/j.polymer.2016.04.074
JPOL 18674
DOI:
Reference:
To appear in: Polymer
Received Date: 9 March 2016
Revised Date: 27 April 2016
Accepted Date: 28 April 2016
Please cite this article as: Stefani S, Hönzke S, Camacho JLC, Neumann F, Prasad AK, Hedtrich S,
Haag R, Servin P, Hyperbranched glycerol-based core-amphiphilic branched shell nanotransporters for
dermal drug delivery, Polymer (2016), doi: 10.1016/j.polymer.2016.04.074.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Hyperbranched
glycerol-based
core-amphiphilic
branched
shell
nanotransporters for dermal drug delivery
S. Stefani, S. Hönzke, J. L. Cuellar Camacho, F. Neumann, A. K. Prasad, S. Hedtrich, R.
Haag, P. Servin
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glycerol-based core-amphiphilic
nanotransporters for dermal drug delivery
Hyperbranched
branched
shell
Stefano Stefani †, Stefan Hönzke §, Jose Luis Cuellar Camacho §, Falko Neumann §, Ashok K.
Prasad ‡, Sarah Hedtrich Þ, Rainer Haag § and Paul Servin †,*.
§
Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustr. 3, 14195
Berlin, Germany
Þ
Institute of Pharmaceutical science, Freie Universität Berlin, Königin-Luise-Str. 2-4,
14195 Berlin, Germany
†Nanopartica GmbH, Takustr. 3, 14195 Berlin, Germany
‡ Department of Chemistry, University of Delhi, 110007 Delhi, India
1. Abstract
A novel amphiphilic branched shell (ABS) obtained upon reaction of octadecen-1-yl succinic
anhydride and methyl poly(ethylenglycol) (mPEG500) is herein presented. Through chemical
attachment to the functionalized external surfaces of three different glycerol-based
hyperbranched polymers (HBP), a set of novel core-ABS nanocarriers that were tested as
possible candidates for dermal delivery of active substances was obtained. Pyrene has been
studied as a model to evaluate the potential drug transport capability and the critical micelle
concentration (CMC) of these amphiphilic systems. Subsequently, the anti-inflammatory
steroid drug Dexamethasone and Finasteride, a drug widely used to prevent prostate
cancer and androgenic alopecia, were also efficiently loaded into the nanocarriers. In
order to promote these systems as possible candidates for dermal drug delivery, tests to
evaluate the cytotoxicity on human keratinocyte cells and the penetration of the
encapsulated molecules into the different layers of human skin samples were carried
out.
2. Introduction
Engineering polymeric nanostructures such as hyperbranched polymers, dendrimers
and polymeric micelles is a growing area of contemporary biomaterial science, due to
their unique properties and large potential in drug delivery.[1-3] Dendrimers and
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hyperbranched polymers, which are often referred to as dendritic polymers, especially
have a bright future as drug nanocarriers due to their high number of functional groups,
which allow for various modifications, low viscosity and their high intrinsic stability in
harsh conditions i.e. dilution or temperature. [4,5]
One of the most studied and performing classes of dendritic nanocarriers can be
obtained by functionalizing dendritic polyglycerol (dPG) synthesized by the ring-
opening multi-branching (ROMB) polymerization of glycidol.[6-8] Polyglycerol is a
highly hydrophilic biocompatible dendritic macromolecule that exhibits a large number
of hydroxyl end groups, which can be easily modified by a variety of organic
transformations. It has been shown that dendritic polyglycerol itself is able to
solubilize guest molecules; however, its transport properties and stability are
significantly improved after functionalizing it with an amphiphilic shell thereby
forming core–shell architectures.[9-14] By attaching α−ω octadecane dicarboxylic acid
comprising of water-soluble and biocompatible mPEG on one end to the dPG core,
highly versatile core-multishell systems (CMS) able to efficiently encapsulate polar
and unpolar dyes and drugs could be developed.[15,16]
A local drug delivery approach is the favoured treatment option in skin diseases as it
bears a low risk of systemic adverse effects. Various types of nanoparticles have been
designed to overcome the skin barrier more efficiently, with liposomes, lipid
nanoparticles and dendritic carriers having been developed and tested.[17-19] However,
most of them failed to be introduced into the market, often due to limited stability (e.g.
liposomes) or toxicity.[20]
The polyglycerol based core-multishell (CMS) nanotransporters raised interest due to
its potential use in the topical treatment of skin diseases. It has been proven that these
systems enhance skin penetration of loaded lipophilic agents, are devoid of
cytotoxicity in keratinocytes, and thus may offer potential prospects for the treatment
of severe and recalcitrant skin diseases in the near future.[13,21,22]
Given the recognized improvement in loading capacity and its skin penetration properties,
obtained with the core-multishell nanocarriers, a novel branched amphiphilic shell was
recently developed.[23] The reaction of a succinic anhydride bearing a C12 alkenyl chain with
a water-soluble mPEG chain enabled the formation of an amphiphilic molecule characterized
by a central and free carboxyl group that was used as anchor for attaching the amphiphile to
the functionalized surfaces of different hyperbranched polyesters. The biodegradable
triglycerol-based hyperbranched polyesters employed as core molecules proved to be an
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excellent platform for the development of novel core-shell nanocarriers to efficiently
encapsulate non-water-soluble drug molecules; it was in fact possible to tune the
hydrophobicity of the polymeric core by using different lengths of α−ω dicarboxylic acid for
polymerization.
In order to further optimize and maximize the efficiency of these nanocarriers, in the present
manuscript, a more hydrophobic amphiphilic branched shell characterized by a longer C18
alkenyl side chain was developed and attached to the functionalized surfaces of three
glycerol-based hyperbranched polymers. The biocompatible dPG and two biodegradable
polyesters, C4-HBPE (succinic acid-co-triglycerol hyperbranched polyester) and C6-HBPE
(adipic acid-co-triglycerol hyperbranched polyester) (Figure 1) previously developed were
used as core molecules for the synthesis of novel nanotransporter. The development of these
novel core-ABshell nanocarriers is described herein with the aim of further increasing the
loading capacity of poorly water-soluble drug molecules while enhancing the penetration of
the loaded agents into the skin.
3.
Experimental Section
3.1. Materials
All reagents, which are commercially available from Sigma-Aldrich, Acros and Alfa
Aesar, were used without further purification. dPG 10kDa was provided by
Nanopartica GmbH and used for the polymerization step without any purification. The
reactions were carried out under dry argon atmospheres using standard Schlenk-line
technique.
3.2. Characterization
3.2.1. Dynamic Light Scattering (DLS):
The size of the core-ABshell nanoparticles in aqueous solution was obtained using a Zetasizer
Nano ZS analyzer with integrated 4 mW He-Ne laser at wavelength 633 nm with
backscattering detector angle 173° (Malvern Instruments Ltd, UK) at 25 °C and using as
reference the polystyrene refractive index. To measure the size, an aqueous solution of
polymer with different concentrations was prepared in Milli-Q water and vigorously stirred
for 18 hours at room temperature (25 °C). Solutions were filtered via 0.45 µm
polytetrafluoroethylene (PTFE) filters and used for dynamic light scattering measurements.
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Disposable UV-transparent cuvettes (Sarstedt AG & Co, Germany) were used for all the
experiments. The measurements of every sample were repeated 4 times.
3.2.2. Atomic Force Microscopy (AFM):
The morphological characterization of the dPG´s was carried out with an AFM Nanoscope
Multimode from Bruker operated in Tapping mode. Sample preparation was as follows. A
mica substrate of 1x1cm was cleaved with adhesive tape and glued onto circular metal disks.
A volume of 10µl (Conc: 1mg/ml) was deposited and incubated for no longer than 15
minutes. Then 1-3 ml Milli-Q water was used to rinse several times the sample to remove any
unattached material and the sample was mounted on the AFM head. During imaging the
amplitude setpoint was increased in order to reduce damage on the sample surface with the
AFM tip. Scanning rates of 0.6-0.9 Hz were used and 512 points per line were taken during
imaging. AFM tips SNL-10 from Bruker were used with a nominal tip radius of 2nm and
resonant frequency of 65 kHz.
3.2.3. UV-VIS and Fluorescence Measurements:
Absorption spectra were recorded using a Scinco S-3150 UV/VIS spectrophotometer. All
measurements were carried out in Milli-Q water in a thermostated UV-cell (1 cm).
Fluorescence emission spectra were taken with a Jasco FP-6500 spectrofluorimeter equipped
with a thermostated cell holder at room temperature (25 °C). For pyrene, emission spectra
were recorded from 350 to 600 nm after excitation at 317 nm. Both excitation and emission
slits were set at 1 nm.
3.2.4. Gel Permeation Chromatography (GPC):
When water or THF were used as an eluent, weight average molecular weight (Mw) and
number average molecular weight (Mn) of the polymers were determined using a GPC
equipped with an Agilent 1260 organic pump, refractive index detector, and Mixed-C gel
(THF) and Suprema (H2O) columns with a flow rate of 1.0 mL/min. Measurements in water
were carried out using aqueous solution of NaNO3 (0.1 M) as a mobile phase. The molecular
weights were calibrated with pullulan (water) and polystyrene (THF) standards.
Measurements performed using DMF as eluent were carried out using a GPC equipped with a
Nexera XR LC20 AD XR pump, refractive index (RID-10A) detector and 3x PPS Polarsil
columns (particle size: 5µm) with a flow rate of 1.0 mL/min. The eluent used was DMF with
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3 g/L LiBr and 6 g/L acetic acid. The molecular weights were calibrated with polystyrene
standards.
3.2.5. Nuclear Magnetic Resonance (NMR):
NMR spectra were recorded on a Jeol ECX 400 or a Jeol Eclipse 700 MHz spectrometer.
Proton and carbon NMR were recorded in ppm and were referenced to the indicated solvents.
NMR data were reported as follows: chemical shift, multiplicity (s=singlet, d=doublet,
t=triplet, m=multiplet) and integration. Multiplets (m) were reported over the range (ppm) at
which they appear at the indicated field strength.
3.2.6. High Pressure Liquid Chromatography (HPLC):
HPLC measurements for the detection of Dexamethasone were carried out using a Knauer
Smartline-HPLC system with an internal UV absorption detector (λ = 254 nm) and
Chromgate software. A Gemini RP C18 column (Phenomenix, 250 mm × 4.6 mm, particle
Size: 5 ꢀm). Acetonitrile–water (40 : 60) was used as the mobile phase at a flow rate of 1.0
mL min−1 under isocratic regime.
3.3. Synthesis and procedures
3.3.1. Synthesis of the HBPEs:
The synthesis and characterization of the hyperbranched polyesters C4-HBPE and C6-HBPE
were already described in a previously published manuscript and used for the further
functionalization as such.[23]
3.3.2. Synthesis of dendritic polyglycerol 30kDa:
dPG with a molecular weight of about 30000 g/mol was synthesized starting from dPG
10000 g/mol produced by Nanopartica GmbH. dPG (10 KDa) (10 g, 1 mmol) was dissolved
in a few mL of methanol (10 mL) in a three-neck round bottom flask and potassium
hydroxide (7.57 mg, 135 mmol) was added. The mixture was stirred for 30 minutes at room
temperature in order to allow the deprotonation of the hydroxyl groups. The reaction
temperature was then increased at 125°C allowing the complete evaporation of the solvent.
The glycidol (20.0 g, 17.94 mL, 269 mmol) (at 0°C) was then slowly added dropwise to the
reaction mixture at 125°C. After all the glycidol was added, the reaction was further stirred
for 1 hour and then cooled down at room temperature. The polymer was dissolved in
methanol (200 mL) and Dowex® (64 mL) was added and the mixture was stirred overnight.
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The Dowex® was filtered and the solvent evaporated under reduced pressure. The dPG
produced was purified from small oligomers and unreacted materials via precipitation in
acetone (3x) in order to obtain the desired polymer (25.3 g, 0.83 mmol) that was analysed via
1H-NMR, IG 13C-NMR and GPC (ESI).
1H-NMR (DMSO-d6, 700 MHz): δ = 4.75 - 4.47 (several s, CH-OH CH2-OH), 3.60 – 3.25
(several m, dPG backbone), 1.27 (s, 2H, -CH2-CH3 TMP core), 0.80 (s, 3H, -CH2-CH3 TMP
core).
13
IG C-NMR (DMSO-d6, 175 MHz): δ = 80.3 (1C, dPGL1,3), 78.4 (1C, dPGD), , 73.2 (2C,
dPG2xL1,3), 71.5 - 70.5 (2C, dPG2xD, 2xT), 69.0 (2C, dPGL1,2-L1,3), 63.5 (1C, dPGT), 61.4 (1C,
dPGL1,2).
3.3.3. Synthesis of (2-octadecen-1-yl) succinic anhydride:
(Scheme 2) In a high pressure reactor, maleic anhydride (5.0 g, 50.1 mmol) was charged.
Octadecene (32 ml, 100 mmol) was added as a reagent and solvent to the reaction, followed
by a subsequent addition of a catalytic amount of hydroquinone (50 mg, 0.5 mmol).[24] The
reactor was hermetically sealed and immersed into an oil bath at 200 °C. The reaction was
stirred for 24 hours and stopped by cooling at room temperature. The desired product was
purified via silica-gel column chromatography using an ethyl acetate / hexane (1/2) mixture as
a mobile eluent to obtain a colourless powder in 68% yield (11.9 g, 34.0 mmol) that was
characterized by 1H-NMR and 13C-NMR (ESI).
1H-NMR (CDCl3, 400 MHz): δ = 5.58 (m, 1H, =CHS4), 5.28 (m, 1H, -=CHS5), 3.19 (m, 1H,
S3
S6
CHS2) 2.99 - 2.74 (2 m, 2H, CH2S1 -), 2.47 (m, 2 H, CH2 ), 1.99 (m, 2H, CH2 ), 1.40 – 1.20,
26H, alkyl chain), 0.87 (t, 3H, -CH2-CH3) ppm.
S1
13C-NMR (CDCl3, 101 MHz): δ = 173.2 (1C, -CHS2-COO), 169.9 (1C,-CH2 -COO), 137.0
S1
S3
(1C, =CHS4), 122.9 (1C, =CHS5), 40.68 (1C; -CHS2), 33.6 (m, 1C, CH2 ), 33.1 (1C, CH2 ),
S6
32.5 (1C, CH2 ), 29.5 – 29.15 (m, alkyl chain), 22.6 (s, 1C, -CH2-CH3), 14.1 (1C, -CH2-
CH3) ppm.
3.3.4. Synthesis of the C18-amphiphilic branched shell (C18ABS):
(Scheme 2) Poly(ethylene glycol) mono-methyl ether (mPEG-500 Da) (5.0 g, 10.0 mmol) was
dissolved in acetonitrile (60 mL) followed by the addition of (2-octadecen-1-yl) succinic
anhydride (1.05 equiv., 3.68 g, 10.5 mmol) and a catalytic amount of 4-
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dimethylaminopyridine (DMAP) (0.1 equiv., 0.12 g, 1.0 mmol). The reaction mixture was
stirred for 24 hours at room temperature.[25] The solvent was evaporated and the crude product
re-dissolved in chloroform (200 mL) and washed (2x) with saturated brine solution. The
solvent was evaporated and the residue was precipitated in hexane (3 x 200 ml) to obtain the
desired product as pail yellow oil in 91% yield (7.7 g, 9.1 mmol) that was characterized by
1H-NMR (Figure 2) and 13C-NMR (ESI).
1H-NMR (CDCl3, 400 MHz): δ = *(mixture of two isomers) 5.44 (m, 1H, =CHS4), 5.31 (m,
1H, -=CHS5), 4.21 (m, 2H, PEG-CH2-COO-), 3.93 – 3.50 (m, methylene protons of PEG),
S1
S1
3.35 (s, 3H, PEG -OCH3), 2.86 *(m, 1H, CHS2), 2.63 *(m, 1H, CH2 ), 2.45 *(m, 2H, CH2 ,
S3
S3
S6
CH2 -), 2.21 *(m, 1 H, CH2 ), 1.95 (m, 2H, CH2 ), 1.40 – 1.18, 26H, alkyl chain), 0.83 (t,
3H, -CH2-CH3) ppm.
13C-NMR (CDCl3, 101 MHz): δ = *(mixture of two isomers) 175.6 *(s, 1C, -CH-COOH),
174.3 *(s, 1C,-CH2-COOH), 173.5 *(s, 1C,-CH-COO-PEG), 172.2 *(s, 1C, -CH2-COO-
PEG), 134.1 (1C, =CHS4), 126.1 (1C, =CHS5), 72.1 (s, 1C, -CH2-O-CH3), 71.0 – 70.3
(several, methylene carbons of PEG), 59.2 (s, 1C, -CH2-O-CH3), 39.7 (1C; -CHS2), 35.7 (m,
S1
S3
S6
1C, CH2 ), 34.9 (1C, CH2 ), 32.1 (1C, CH2 ), 29.7 – 29.1 (m, alkyl chain), 22.8 (s, 1C, -
CH2-CH3), 14.2 (1C, -CH2-CH3) ppm.
3.3.5. General procedure for the synthesis of the core-ABS HBPs (synthesis of dPG-
C18ABS):
(Scheme 2) In a round bottom flask equipped with a magnetic stirrer, the dPG (30 kDa) (2.4
g, 0.08 mmol), was dissolved in dry DMF (80 mL). The amphiphilic shell (500 wt%, 6.16 g,
7.25 mmol), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) (1
equiv., 1.39 g, 7.25 mmol) and a catalytic amount of DMAP (88.5 mg, 0.725 mmol) were
subsequently added. The mixture was stirred for 24 hours at room temperature.[26] The solvent
was evaporated and chloroform (80 mL) and saturated brine solution (80 mL) were added to
the crude mixture; the corresponding n-acyl isourea obtained as side product was then
extracted. The organic solvent was evaporated and the crude was purified via precipitation in
a diethyl ether / hexane mixture and ultrafiltration in methanol (MWCO = 3000 Da),
affording the desired dPG-C18ABS nanocarrier (1.5 g, 0.021 mmol) in 26% yield that was
analysed via 1H-NMR (Figure 2), IG 13C-NMR and GPC (ESI).
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1H-NMR (DMSO-d6, 700 MHz): δ = 5.42 (m, 1H, =CHS4), 5.29 (m, 1H, =CHS5), 4.80-4.35
S7
(several m, 4H, -CH2-OH, -CH-OH), 4.09 (m, 4H, CH2 ), 3.53 (m, 4H, -O-CH2-CH2-O-
,methylene protons of PEG), 3,70-3.27 (m, 4H, dPG backbone), 3.23 (s, 3H, PEG –O-CH3),
S2
S1
S1
S3
2.79 (m, 1H, CH2 ), 2.57 (m, 1H, CH2 ), 2.43 (m, 1H, CH2 ), 2.26 (m, 2H, CH2 ), 1.92
S6
(m, 2H, CH2 ), 1.40 – 1.15 (m, alkyl chain), 0.84 (t, 3H, -CH2-CH3) ppm.
13
IG C-NMR (DMSO-d6, 175 MHz): δ = 173.5 *(1C,-CH-COO-), 171.3 *(1C, -CH2-COO-),
133.4 (1C, =CHS4), 125.8 (1C, =CHS5), 79.9 (1C, dPGL1,3), 77.9 (1C, dPGD), 79.9 (1C,
dPGL1,3), 72.9 (2C, dPG2xL1,3), 70.5 (2C, dPG2xD, 2xT), 70.5 (2C, dPGL1,2-L1,3), 70.26 (several,
methylene carbons of PEG), 69.6 (s, 1C, -CH2-O-CH3), 63.1 (1C, dPGT), 61.00 (1C, dPGL1,3),
S1
S3
S6
58.07 (s, 1C, -CH2-O-CH3), 40.7 (1C; -CHS2), 34.3 (m, 1C, CH2 ), 31.9 (2C, CH2 ,CH2 ),
29.7 – 28.8 (m, alkyl chain), 22.1 (s, 1C, -CH2-CH3), 13.9 (1C, -CH2-CH3) ppm.
3.3.6. Solubilization of Guest Molecules:
For the encapsulation experiments of the guest molecules (e.g. pyrene), a 20 mM pyrene
solution was freshly prepared by dissolving an appropriate amount of the dye in dry THF.
Aliquots (20 µL) were taken in vials and the organic solvent was removed. 1.5 mL of the
aqueous polymer stock solutions with various concentrations (0,5 – 0,0125 mg/mL) in Milli-
Q water was added and the resulting mixture was stirred at 500 rpm for 18 hours at room
temperature. The mixture was filtered through a syringe filter (PTFE, 0.45 µm) to remove the
insoluble excess of pyrene. The aqueous solutions of the polymers were then analyzed by UV-
Vis and fluorescence spectroscopy. Guest loading capacity was calculated based on the
following the equation 1:
ꢌꢍꢎꢎ ꢏꢐ ꢑꢒꢓꢎꢔ ꢓꢕꢖꢍꢗꢎꢒꢘꢍꢔꢓꢙ
ꢀꢁꢂꢃꢄꢅꢆ ꢇꢂꢈꢂꢇꢄꢉꢊ (ꢀꢋ) =
x 100
(1)
ꢌꢍꢎꢎ ꢏꢐ ꢚꢏꢎꢔ
3.3.7. MTT assay:
Cytotoxicity of the nanocarriers was evaluated via MTT assay.[27] HaCaT cells were seeded in
a 96-well plate at a density of 1 x 104 cells per well. After overnight incubation at 37°C under
5% CO2, the medium from each well was removed and replenished with 100 ꢀL of fresh
medium containing varying concentrations of nanocarriers (0.5 and 0.05 mg/mL). After 24
hours, the medium was removed and replaced by 100 ꢀL of 0.5 mg/mL MTT in 10% FBS-
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containing medium and the cells were incubated in the CO2 incubator at 37°C for 2 hours.
Subsequently, the medium was removed from each well and the reduced MTT dye in each
well was dissolved in 50 ꢀL of DMSO, followed by 10 minutes of orbital shaking.
Absorbance at 570 nm and reference at 630 nm was measured with an InfinitePro M200
Tecan microplate reader. For each treatment hexaplicates were used for statistical analysis.
3.3.8. Skin penetration studies:
To estimate the dermal drug delivery efficiency of dPG-C18ABS, C4-HBPE-C18ABS, C6-
HBPE-C18ABS, validated test procedures for the Franz cell set-up on human skin (obtained
from patient with informed consent) were performed in the finite dose approach [28,29] and the
delivery of the loaded fluorescence dye Nile red in stratum corneum (SC), viable epidermis
(VE) and dermis (D) was evaluated by picture analysis.[30] For appropriate reference,
fluorescence dye was dispersed into paraffin oil droplets and subsequently incorporated into
base cream up to a concentration of 0.001%. Prior to the experiment, human skin was thawed
and discs of a 2.5 cm diameter were punched and mounted onto static-type Franz cells
(diameter 15 mm, volume 12 mL, PermeGear Inc., Bethlehem, PA, USA) with the horny
layer facing the air and the dermis having contact with the receptor fluid phosphate buffered
saline (PBS; pH 7.4) stirred at 500 rpm. After 30 min equilibration, test formulations (35,4 µl)
were applied onto the skin surface. After 6 hours incubation (33.5°C, skin surface temperature
about 32°C), the skin was removed from the Franz cells and surplus formulations were gently
removed by washing the skin using PBS. Nile red loaded reference and copolymers with same
dye concentration were tested in parallel using human skin of the same donor. For
fluorescence evaluation treated skin areas were embedded in tissue freezing medium (Jung,
Nussloch, Germany) and skin discs were cut into slices of 8 µm thickness using a freeze
microtome (Frigocut 2800 N, Leica, Bensheim, Germany). Nile red amounts in the dermal
layers of interest were determined using the fluorescence microscope (BZ-8000, objectives
20x/0.75, zoom 10x, Plan-Apo, DIC N2, Keyence, Neu-Isenburg, Germany) and slices were
subjected to normal light and fluorescence light. Relative dye content in skin layers of three
skin sections per sample were determined by integration of arbitrary pixel brightness units
(ABU) using BZ image analysis software (Keyence, Neu-Isenburg, Germany). For statistical
analysis one-way analysis of variance, followed by Dunnett’s post hoc testing was performed
using GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, USA). * indicates statistical
significance over base cream treated skin samples. p ≤ 0.05 indicates statistical significance
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4. Results and Discussion
4.1. Synthesis of the Core-ABshell nanocarriers
Three different glycerol-based hyperbranched polymers have been selected and used as
core molecules for the synthesis of the novel core-ABshell nanocarriers. The
biocompatible dendritic polyglycerol (dPG) and two biodegradable triglycerol based
hyperbranched polyesters, characterized by different polarity have been selected for
this scope (Scheme 1). The synthesis and the characterization of the two
hyperbranched polyesters (C4-HBPE and C6-HBPE) have been recently described by
our group.[23] Two different dicarboxylic acids, succinic and adipic acid, were reacted
in a 1:1 molar ratio with the triglycerol moiety in order to obtain respectively the C4-
HBPE and the C6-HBPE of an average molecular weight of 30 kDa. In order to gain
similar characteristics to the polyester, dPG 30 kDa was synthesized starting from dPG
10 kDa and further reacted with a predetermined amount of glycidol via ROMB
polymerization in the presence of potassium hydroxide. The molecular weight of the
final product was analyzed via GPC. (Table 1).
HO
OH
O
HO
HO
OH
OH
O
OH
OH
HO
HO
O
O
O
O
O
O
O
OH
O
O
O
O
HO
O
O
OH
OH
HO
OH
O
O
O
O
O
HO
HO
HO
HO
HO
HO
OH
Scheme 1. Schematic representation of the dendritic polyglycerol (left) and the hyperbranched
polyesters (right)
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Table 1. Characterization of the hyperbranched polymers
Solubility
Mw
Polymer
Db
in H2O
(mg/mL)
> 20
(g/mol)a
33108
27758
34761
1.5
2.2
1.9
dPG
> 20
C4-HBPE
10
C6-HBPE
a)Mw and Mn from GPC analysis (ESI). b)Dispersity (Mw/Mn).
13
By analysing the dPG synthesized via inverse gated decoupling C-NMR (Figure 1
ESI), and assigning the characteristic peaks related to the linear, terminal and dendritic
units in the polymeric structure was possible to determine a degree of branching of
55.5 %.
A novel amphiphilic branched shell (C18-ABS), characterized by a longer alkyl side
chain and thus higher hydrophobicity when compared to the previously published C12-
ABS, was developed. The intermediate octadecen-1-yl succinic anhydride was
synthesized via ene reaction for this purpose by reacting, under high pressure condition
at 200 °C, maleic anhydride and an excess of 1-octadecene also used as a solvent.[24] It
was possible to obtain in good yield the desired C18 alkenyl succinic anhydride
(mixture cis-trans isomers). This was further reacted with poly(ethylene glycol)
monomethyl ether (mPEG500) in the presence of a catalytic amount of DMAP to obtain
a mixture of the two isomers of the desired amphiphilic molecule (Scheme 2) that were
used as such for further functionalization.[25]
The amphiphilic molecules synthesized are characterized by a central and free carboxyl
group that can be strategically used as an anchor for its chemical attachment to the
functionalized surface of the hyperbranched polymers. The C18-AB shell was
covalently bound to the hydroxyl functionalized surfaces of different hyperbranched
polymers.
The three different glycerol-based polymers having similar molecular weights (evenly
30 kDa) have been used as a core for this purpose. The accessible hydroxyl groups of
the glycerol moieties preferentially located in the peripheries of the globular
hyperbranched polymers were reacted using a coupling agent with the free carboxylic
acid of the C18-ABS to form ester bonds.
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O
HO
PEG500
O
O
O
O
cis-trans
O
O
O
O
Hydroquinone cat.
mPEG500
200 °C,
high pressure
68 % yield
DMAP cat.
91 % yield
14
O
O
cis-trans
HO
PEG500
O
cis-trans
OH
OH
O
EDC-HCl, DMAP cat.
DMF, 48h, r.t.
HBP
O
OH
OH
O
O
O
O
OH
O
HBP
O
O
O
O
O
OH
Scheme 2. Synthesis of the novel ABshell and its attachment to the hyperbranched polymers
The water-soluble coupling agent 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC-HCl) and a catalytic amount of DMAP were used to enable ester
formation.[26] After 24 hours of reaction at room temperature, the corresponding n-acyl
isourea obtained as a side product that had formed during the reaction was extracted
and the crude mixtures that were purified via precipitation in a diethyl ether / hexane
mixture and ultrafiltration in methanol (MWCO = 3000 Da), affording the desired
core-amphiphilic branched structures dPG-C18ABS, C4-HBPE-C18ABS and C6-
1
13
HBPE-C18ABS (Scheme 2, Figure 1) that were analysed via H-NMR and C-NMR
(ESI).
1
Determination of the molecular mass of the nanocarriers was carried out via H-NMR.
GPC analysis was also carried out using DMF as an eluent with reference to
conventional polystyrene calibration standards of low polydispersity. We observed a
great discrepancy between the two methodologies, however, for this reason we decided
1
to rely on the molecular weight estimation deducted via H-NMR spectroscopy. The
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GPC measurements were, however, useful to ensure the absence, after purification, of
any non-bond ABS molecules.
Figure 1. Schematic representation of the nanocarrier dPG-C18ABS
Figure 2. 1H-NMR spectrum of dPG-HBPE-C18ABS.
1
Figure 2 shows the H-NMR spectra of the core-ABshell nanocarrier dPG-C18ABS
where it is possible to detect the characteristic peaks of the amphiphilic molecules
bound to the polymeric structure. By integrating the signal related to the amphiphilic
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branched shell and the signals characteristic of the polymeric backbone, and knowing
its molecular weight, it was possible to evaluate the percentage of hydroxyl groups that
effectively reacts with the ABshell and thus the number of ABshell and the average
molecular weight of the nanocarriers (Table 3).
13
Further information about the coupling reaction were obtained comparing the IG C-
NMR of the dPG core (Figure 1 ESI) and the core–ABshell molecule (Figure 4 ESI).
Knowing the integration of the terminal (T, 63.1 ppm) and the linear units (L1,3, 72.9
and L1,2, 61.00 ppm) of the dPG structure, was possible to evaluate the abundance
percentage of the different units and was therefore possible to compare them with the
abundance of the different units after reaction with the ABshell. The ratio between the
units did not significantly change after reaction, allowing us to affirm that the coupling
reaction occurred without preference in the three different units containing free
hydroxyl groups. (Table 1 ESI).
Focusing the attention on the two nanocarriers with a polyester core, C4-HBPE-
C18ABS and C6-HBPE-C18ABS, and comparing the results obtained with the
previously reported methodology using a different coupling agent N,N'-
dicyclohexylcarbodiimide (DCC), the improvements obtained regarding the shell
functionalization are evident. [23]
Table 2. Characterization of core-AB-shell HBPEs
N° ABS
Shell
(wt%)a)
115
NMR-Mw
(g/mol)a)
71300
Tot. OH
Funct. (%)a)
Carrier
per core a)
11.1
19.4
17.8
45
46
49
dPG-C18ABS
141
67000
C4-HBPE-C18ABS
C6-HBPE-C18ABS
120
76600
a) Calculated via 1H NMR spectroscopy.
Attaching a more hydrophobic shell (C18-ABS) using EDC-HCl as coupling reagents
enabled us to obtain a functionalization of 19.4 and 17.8% of the total amount of the
polymers’ hydroxyl groups respectively for C4-HBPE and C6-HBPE. By using this
procedure it was thus possible to obtain an increase in functionalization, 40% on
average.
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4.2. Host-Guest Interaction of core-ABS nanocarriers; solubilization of
pyrene
The hydrophobic polycyclic aromatic hydrocarbon pyrene was employed as a model guest in
order to obtain information about the capabilities of the novel core-ABS systems as drug
nanocarriers. Due to its high hydrophobicity that leads to an almost complete insolubility in
water, and given its easy detection via UV-Vis and fluorescence spectrometry, pyrene became
an easy screening molecule for nanotransporters of poorly water-soluble molecules.
Furthermore, the fluorescence spectra of pyrene is very sensitive to the solvent polarity and
for this reason it was useful to understand the microenvironment and binding sites of this
guest molecule once encapsulated into the nanocarriers.
Tests to evaluate the LC (loading capacity) and CMC (critical micelle concentration) were
successfully carried out via fluorescence and UV-Vis spectroscopy (Table 3). To avoid the
formation of excited dimer complexes, a low concentration of pyrene was used for the
encapsulation study. The absorbance and fluorescence spectra of pyrene in different polymer
concentration solutions (dPG-C18ABS) are shown in Figure 3.
Figure 3. UV-Vis spectrum of pyrene in dPG-C18ABS (left) and Fluorescence spectrum of
pyrene in dPG-C18ABS (right).
Table 3. Solubilization data of pyrene in the core-ABshell nanocarriers.
LCa)
wt%
1.35
1.53
1.78
CMCc)
mg/mL
0.12
b)
Polymer
I1/I3
1.25
1.24
1.25
dPG-C18ABS
0.09
C4-HBPE-C18ABS
C6-HBPE-C18ABS
0.07
a)Loading capacity. b) In a 0.15 mg/mL nanocarrier solution. c) Critical micelle concentration
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Figure 4. Extrapolation of the CMC of the nanocarrier dPG-C18ABS using I1/I3 diagrams.
Using the Lambda maximum at 338 nm the concentration of solubilized pyrene was
calculated. LCs of 1.35, 1.53 and 1.78 wt% of pyrene were determined for polymer dPG-
C18ABS, C4-HBPE-C18ABS and C6-HBPE-C18ABS respectively (ESI).
Focusing the attention on the nanocarriers having as a core molecule the hyperbranched
polyester C4-HBPE and C6-HBPE, and comparing the results obtained in the previous
research when a less hydrophobic shell molecule was employed (C12-ABS), the
improvements in LCs obtained by using the C18-ABS are apparent. It was even possible to
double the pyrene loaded into C4-HBPE-C18ABS and to increase it by 37% in C6-HBPE-
C18ABS.
Due to its strong response to the polarity of the environment directly surrounding pyrene, the
fluorescence spectrum of the pyrene monomer has been used to obtain information about the
internal dynamics of macromolecules and to deduct the critical micelle concentration of
surfactants.[31,32] Plotting the ratio of the first band (I1 = 374 nm) over that of the third band (I3
= 385 nm), with the solution concentration of the nanocarrier it is possible to obtain a curve
from where, by detecting the curve’s flex point, the CMC of the nanocarrier can be extracted
(Figure 4). The fluorescence spectroscopy studies indicated that the CMC of the pyrene-
loaded nanocarriers were 0.12, 0.09 and 0.07 mg/mL respectively for the polymers dPG-
C18ABS, C4-HBPE-C18ABS and C6-HBPE-C18ABS (Table 3) (ESI).
As previously mentioned the I1/I3 ratio can be used as an indicator of hydrophobicity of the
environment in which the pyrene is located and studies carried out by Winnik et. al. shows
that this value is large in a polar solvent (water : 1.87) and small in an apolar solvent (hexane :
0.58).[33] When pyrene is loaded into the three different nanocarriers at a fixed concentration
of 0.15 mg/mL, the fluorescence spectroscopy displays a similar I1/I3 of averagely 1.25. This
value is comparable to the pyrene I1/I3 ratio when dissolved in chloroform confirming that
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after encapsulation into the nanocarriers, the molecules of pyrene are sitting in a relatively
hydrophobic environment. It is therefore possible to conclude that pyrene is entrapped into the
nanocarriers due to hydrophobic interactions, especially in the case of dPG-C18ABS, C4-
HBPE-C18ABS, characterized by a hydrophilic core, that are derived from the presence of the
long aliphatic C18 chains of the amphiphilic branched shell.
Nevertheless, from the data collected, the lipophilicity of the polymeric core displayed its
fundamental importance in enabling higher drug loading capacity. In a 1 g/L solution of C6-
HBPE-C18ABS it was possible to solubilize over 17 mg/L of pyrene; this value is 125 times
higher than the solubility of pyrene in water reported in literature of 0.135 mg/L. at 25°C.[34]
4.3. Size and aggregation behavior of the nanocarriers
The size and the aggregation behaviour of the novel core-ABshell nanocarriers in
aqueous solution were investigated by means of DLS (Dynamic Light Scattering) and
AFM (Atomic Force Microscopy). With this purpose the self-aggregation behaviour of
the different loaded and unloaded carriers was taken into consideration. In all cases the
nanocarriers displayed the presence of relatively big aggregates of about 20-50 nm in
the intensity distribution of the DLS measurements. These greater aggregates were not
observed in the volume and number distribution and were therefore considered as a
minor pronounced species (Table 4).
Table 4. DLS data of the non-loaded and loaded core-shell nanocarriers.
UNLOADED
LOADED
Polymer
Carrier Conc.
rH volume
rH number
(nm) a)
rH volume
rH number
(nm) a)
(mg/mL)
(nm)a)
(nm) a)
1
1
1
12.1 ± 1.6
9.7 ± 2.5
11.7 ± 1.2
7.4 ± 2.4
8.9 ± 3.4
5.8 ± 2.0
dPG-C18ABS
16.6 ± 2.1 10.7 ± 1.8 11.5 ± 1.9
10.0 ± 1.1 7.1 ± 2.1 8.8 ± 2.4
C4-HBPE-C18ABS
C6-HBPE-C18ABS
a)rH volume and rH number are the average hydrodynamic radii calculated by volume and number
given as means ± standard deviation.
Primarily, different concentrations of unloaded nanocarriers in water were studied;
focusing attention on the volume and number size distribution (Table 4) it is possible
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to affirm that the different core-shell nanocarriers display a similar behaviour when
dissolved in aqueous media. Small aggregates having hydrodynamic radii between 10
and 16 nm, hypothetically composed of a few macromolecules, are detected in all the
different nanocarriers’ solutions. Concentrating on the systems having the
hyperbranched polyesters C4-HBPE and C6-HBPE as core molecules, and comparing
the results obtained in our previous research when a less hydrophobic shell molecule
was employed (C12-ABS) it is possible to see that the new systems, bearing a more
hydrophobic PEGylated shell, have an identical behaviour when dissolved in aqueous
media.[23]
In order to confirm the results obtained by DLS measurements, an aqueous solution of
the carrier C6-HBPE-C18ABS at a concentration above CMC was analyzed via atomic
force microscopy to obtain more reliable information about the aggregation behaviour.
Figure 5 shows the AFM image of the nanocarrier deposited from a 1 mg/mL solution
of C6-HBPE-C18ABS that confirmed the presence of small aggregates with an average
hydrodynamic radius of 8 nm in the solution.
Figure 5. AFM height image of the nanocarriers C6-HBPE-C18ABS (left) with a cross section
profile (right).
As the size of the aggregates formed by the nanocarriers in solution has a significant
influence on its uptake into skin and cells, the behaviour of the loaded (pyrene)
nanocarriers in solution was also studied. It is visible from table 4 that the loaded
nanocarriers’ aggregates slightly decrease their size by an average of 3 nm when the
hydrophobic dye was encapsulated. The presence of the hydrophobic guest that forms
hydrophobic interaction with the aliphatic shell chain can be the cause of the shrinking
of the hydrodynamic radii of the aggregates in solution.
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4.4. Solubilization of Dexamethasone and Finasteride
To investigate the suitability of the novel nanocarriers as possible drug delivery
systems, it is of importance to exploit them using non water-soluble drug molecules in
order to understand the transport capacity of the different systems. Two widely used
and poorly water-soluble drugs, the anti-inflammatory steroid drug Dexamethasone
and Finasteride, a drug widely used to prevent prostate cancer and androgenic alopecia,
were selected for this purpose.
Both DOX and FNS were encapsulated into the nanocarriers adopting a general film
method. An excess of drug (50 wt% -wt of polymer) was dissolved in acetone and
subsequently dried under vacuum in order to obtain a uniform layer (film) of the drug
in the flask. Aqueous nanocarrier solutions (1 mg/mL) were added and the samples
were stirred for 18 hours at 500 rpm.
Both drugs are partially soluble in water and for this reason particular attention was
paid in order to evaluate the amount of drug effectively encapsulated into the polymer
from the free drug. With this aim, a blank sample was prepared to evaluate the drug
solubility in water, by which the drug was stirred in water without the nanocarrier. The
solubility of the drug in water obtained from the blank sample was subtracted in order
to calculate the effective loading capacity of the nanocarriers.
After encapsulation, the samples and the blanks were filtered (PTFA, 0.45 µm), and
each solution was diluted with acetonitrile and directly analyzed via HPLC using a
water/acetonitrile mixture (60/40 for Dexamethasone and 40/60 for Finasteride) as an
eluent. Calibration curves related to the drugs were prepared (ESI) in order to evaluate
the solubility of DXM and FNS in water and the loading capacity of the nanocarriers.
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Figure 6. Solubility of the drugs in water (Blank) and in a 1 mg/mL nanocarrier solutions
measured via HPLC chromatography.
Table 5. Solubilization data of DXM and FNS in the core-ABshell HBPEs.
Dexamethasone
DXM a)
Finasteride
FNS a)
b)
b)
Polymer
LCDXM
LCFNS
solubilized
(mg/mL)
solubilized
(mg/mL)
(wt%)
2.5
(wt%)
2.3
0.105
0.128
0.076
0.098
dPG-C18ABS
C4-HBPE-
C18ABS
4.8
4.3
C6-HBPE-
C18ABS
0.142
6.2
0.158
10.3
a) In 1 mg/mL nanocarrier solution. b) Effective LC after subtraction of the blank.
Figure 6 displays solubility of the drugs in water (Blank) and into 1 mg/mL
nanocarrier solutions measured via HPLC analysis (ESI). Similar to a literature
reported value we determined a solubility of Dexamethasone in water of 0.08 mg/mL
[35] and of 0.05 mg/mL for the Finasteride.
As displayed in Table 5, we have been able to encapsulate 2.5 and 4.8 wt% of DXM
respectively into a 1 mg/mL polymer solution of dPG-C18ABS and C4-HBPE-
C18ABS. The loading capacity of the nanocarrier C4-HBPE-C18ABS is over 50%
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higher than in the nanocarrier previously reported where C4-HBPE was functionalized
with a less hydrophobic ABshell.
As expected, the nanocarrier characterized by a more hydrophobic core (C6-HBPE-
C18ABS) displayed the best drug carrier capabilities, allowing to encapsulate 6.2 wt%
of DXM.
The experiment to evaluate the loading capacity of the drug Finasteride, demonstrate
that the best loading performance was once again displayed by the nanocarrier C6-
HBPE-C18ABS; it became possible to almost triple the solubility of FNS in water
using a 1 mg/mL nanocarrier solution corresponding to a loading capacity of 10.3 wt%.
4.5. Studies to evaluate the cytotoxicity of the nanocarriers: MTT assay
In order to be used as drug transporters, it is of great importance for the polymeric
systems synthetized to display no cytotoxicity against human cells. The cytotoxicity
profiles of all the novel core-shell nanocarriers were thus evaluated by using HaCaT
cells (human keratinocyte cell line) over a time period of 24 hours by MTT assay
applied to two nanocarrier concentrations (0.5 and 0.05 mg/mL). The metabolic
activity of HaCaT cells was detected after incubation in a culture medium containing
the nanocarrier solutions by evaluating the chemical reduction of the dye MTT 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan by
means of UV-Vis spectroscopy.
Figure 7 shows the cell viability diagram of different concentrations of nanocarriers in
relation to the control sample where the cells were incubated in absence of the
nanocarriers. As can be seen, no cytotoxicity was detected in any of the systems
investigated at a concentration of 0.05 mg/mL. At a carrier concentration of 0.5 mg/mL
the systems seems to inhibit the metabolism of the HaCaT cells. The toxicity shown by
dPG-C18ABS and C4-HBPE-C18ABS can be considered non relevant, in fact over
80% of the cells’ metabolic activities are maintained after 24 hours of exposure.
Inexplicably, the nanocarrier C6-HBPE-C18ABS has shown a considerable toxicity at
a concentration of 0.5 mg/mL where over 60% of the cells’ metabolic activity was
inhibited by the presence of the nanocarrier.
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Figure 7. Cell viability assay by the MTT method of HaCaT calls treated with aqueous
solutions of dPG-C18ABS, C4-HBPE-C18ABS and C6-HBPE-C18ABS.
4.6. Skin penetration studies
In order to investigate the ability of the novel core-ABshell nanocarriers to transport
hydrophobic guest molecules into the skin, we compared the skin penetration profile of
Nile red (0.001 wt%) loaded into dPG-C18ABS, C4-HBPE-C18ABS and C6-HBPE-
C18ABS. Conventional base cream with same amount of dye served as reference.
To estimate the dermal drug delivery efficiency of dPG-C18ABS, C4-HBPE-C18ABS,
C6-HBPE-C18ABS compared to base cream, we performed a validated test procedure
for the Franz cell set-up in the finite dose approach and the skin absorption of the
loaded fluorescence dye Nile red in stratum corneum (SC), viable epidermis (VE) and
dermis (D) was evaluated by picture analysis.[28,29,30] Therefore, 0.001 wt% of Nile red
was encapsulated into 5 mg/mL aqueous nanocarriers solutions and the amounts of dye
loaded were detected via UV-Vis spectroscopy.
The samples were applied on human skin slices and after 6 hours of incubation
(33.5°C, skin surface temperature about 32°C), the samples were gently washed with
PBS solution from the surplus formulation and the Nile red amounts in the dermal
layers of interest were determined using a fluorescence microscope.
The microscopy evaluation showed that the nanocarriers with the more lipophilic core
significantly enhance the uptake of Nile red in the stratum corneum and transport the
dye even in deeper layers (Figure 8). In contrast, following the application of the
conventional cream, Nile red was almost exclusively detected in the SC.
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Figure 9 displays a diagram of the fluorescence intensity abundance in the different
skin layers. The core-ABshell system C6-HBPE-C18ABS displayed the best
performance; compared to the conventional base cream this nanocarrier increased the
abundance of Nile red in the SC by over 100% and significantly eleven-fold in the
viable epidermis.
The preliminary results collected are useful to achieve a better understanding of the
skin penetration behaviour of the reported nanocarriers and demonstrate once more that
structural variation between nanoparticles, such as polarity of the polymeric core, may
significantly affect the drug delivery efficiency.
Further extended analysis and investigations have to be carried out in order to
understand and to possibly diminish the toxicity of the systems and to prove the
enhancement penetration ability of hydrophobic drugs even at lower nanocarrier
concentrations.
Figure 8. Representative fluorescence microscopy images of human skin following incubation
with conventional base cream or 5 mg/ml nanocarriers in aqueous solution, both loaded with
the model drug Nile red (0.001 wt%).
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Figure 9. Fluorescence intensity (arbitrary pixel brightness unit, ABU) quantified in the
respective skin layers.
4. Conclusions
The synthesis of a set of novel nanocarriers characterized by a glycerol-based
hyperbranched core and an amphiphilic branched shell has been successfully
developed and described. These systems have been primarily tested as potential
nanocarriers for poorly water-soluble active molecules and the results obtained were
compared with a previously reported system in which a less hydrophobic branched
shell was employed. By increasing the hydrophobicity of the amphiphilic shell, it was
possible to significantly increase the loading capacity of these systems; in fact the
pyrene loading capacity in the system C4-HBPE-C18ABS, when compared with the
previously reported system employing the same hyperbranched core molecule, was
even doubled. Subsequently, the drugs Dexamethasone and Finasteride were also
efficiently loaded into the nanocarriers, obtaining a loading capacity respectively of 6.2
and 10.3 wt% into the nanocarrier C6-HBPE-C18ABS.
The cytotoxicity of the systems was also tested on human keratinocyte cells; all the
systems display no cytotoxicity at a concentration of 0.05 mg/mL. However, increasing
the concentration to 0.5 mg/mL, one nanocarrier C6-HBPE-C18ABS appeared to be
toxic for the HaCaT cells employed for the experiment.
Initial skin penetration tests show that the core-ABshell nanocarriers are able to
efficiently transport Nile red as a model therapeutic agent through this complex
biological barrier. It was possible to increase - approximately eleven-fold - the
transport of the guest molecule into the viable epidermis, compared to the ability of a
classical base cream.
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Acknowledgements
This work was supported by the IGSTC (Indo-German Science and Technology Centre),
BMBF (Bundesministerium für Bildung und Forschung) and the SFB1112
(Sonderforschungsbereich 1112 - Nanocarrier). Additionally we would like to thank
Dendropharm GmbH for the GPC measurements.
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ACCEPTED MANUSCRIPT
Highlights
Hyperbranched glycerol-based core-amphiphilic branched shell
nanotransporters for dermal drug delivery
S. Stefani, S. Hönzke, J. L. Cuellar Camacho, F. Neumann, A. K. Prasad, S. Hedtrich,
R. Haag, P. Servin
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The synthesis of novel core-shell nanocarriers is presented
Nanocarriers are composed by a hyperbranched polymeric core and an
amphiphilic branched shell
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Pyrene, Dexamethasone and Finasteride were efficiently encapsulate
The nanocarriers were tested as potential dermal drug delivery systems.