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

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

DOI:

10.1016/j.polymer.2016.04.074

Reference:

JPOL 18674

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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Graphical Abstract

Hyperbranched

glycerol-based

core-amphiphilic

branched

shell

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nanotransporters for dermal drug delivery S. Stefani, S. Hönzke, J. L. Cuellar Camacho, F. Neumann, A. K. Prasad, S. Hedtrich, R.

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Haag, P. Servin

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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.

§

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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

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†

Nanopartica GmbH, Takustr. 3, 14195 Berlin, Germany

Department of Chemistry, University of Delhi, 110007 Delhi, India

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‡

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

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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

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concentration (CMC) of these amphiphilic systems. Subsequently, the anti-inflammatory steroid drug Dexamethasone and Finasteride, a drug widely used to prevent prostate

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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 -1-

ACCEPTED MANUSCRIPT 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

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obtained by functionalizing dendritic polyglycerol (dPG) synthesized by the ringopening 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

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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

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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

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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.

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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

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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 -2-

ACCEPTED MANUSCRIPT 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

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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

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(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

the loaded agents into the skin.

Experimental Section 3.1. Materials

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3.

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loading capacity of poorly water-soluble drug molecules while enhancing the penetration of

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

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technique.

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reactions were carried out under dry argon atmospheres using standard Schlenk-line

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. -3-

ACCEPTED MANUSCRIPT 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

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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

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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

resonant frequency of 65 kHz.

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imaging. AFM tips SNL-10 from Bruker were used with a nominal tip radius of 2nm and

3.2.3. UV-VIS and Fluorescence Measurements:

Absorption spectra were recorded using a Scinco S-3150 UV/VIS spectrophotometer. All

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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

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slits were set at 1 nm.

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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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ACCEPTED MANUSCRIPT 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.

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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,

which they appear at the indicated field strength. 3.2.6. High Pressure Liquid Chromatography (HPLC):

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t=triplet, m=multiplet) and integration. Multiplets (m) were reported over the range (ppm) at

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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 µm). Acetonitrile–water (40 : 60) was used as the mobile phase at a flow rate of 1.0 mL min−1 under isocratic regime.

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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

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were already described in a previously published manuscript and used for the further functionalization as such.[23]

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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. -5-

ACCEPTED MANUSCRIPT 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 H-NMR, IG 13C-NMR and GPC (ESI).

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H-NMR (DMSO-d6, 700 MHz): δ = 4.75 - 4.47 (several s, CH-OH CH2-OH), 3.60 – 3.25

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(several m, dPG backbone), 1.27 (s, 2H, -CH2-CH3 TMP core), 0.80 (s, 3H, -CH2-CH3 TMP core). IG

13

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,

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dPGL1,2).

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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

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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). H-NMR (CDCl3, 400 MHz): δ = 5.58 (m, 1H, =CHS4), 5.28 (m, 1H, -=CHS5), 3.19 (m, 1H,

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1

CHS2) 2.99 - 2.74 (2 m, 2H, CH2S1 -), 2.47 (m, 2 H, CH2S3), 1.99 (m, 2H, CH2S6), 1.40 – 1.20, 13

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26H, alkyl chain), 0.87 (t, 3H, -CH2-CH3) ppm. C-NMR (CDCl3, 101 MHz): δ = 173.2 (1C, -CHS2-COO), 169.9 (1C,-CH2S1-COO), 137.0

(1C, =CHS4), 122.9 (1C, =CHS5), 40.68 (1C; -CHS2), 33.6 (m, 1C, CH2S1), 33.1 (1C, CH2S3), 32.5 (1C, CH2S6), 29.5 – 29.15 (m, alkyl chain), 22.6 (s, 1C, -CH2-CH3), 14.1 (1C, -CH2CH3) 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)

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and

a

catalytic

amount

of

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ACCEPTED MANUSCRIPT 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 H-NMR (Figure 2) and 13C-NMR (ESI).

1

H-NMR (CDCl3, 400 MHz): δ = *(mixture of two isomers) 5.44 (m, 1H, =CHS4), 5.31 (m,

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1

1H, -=CHS5), 4.21 (m, 2H, PEG-CH2-COO-), 3.93 – 3.50 (m, methylene protons of PEG), 3.35 (s, 3H, PEG -OCH3), 2.86 *(m, 1H, CHS2), 2.63 *(m, 1H, CH2S1), 2.45 *(m, 2H, CH2S1,

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CH2S3-), 2.21 *(m, 1 H, CH2S3), 1.95 (m, 2H, CH2S6), 1.40 – 1.18, 26H, alkyl chain), 0.83 (t, 3H, -CH2-CH3) ppm. 13

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C-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-COOPEG), 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, 1C, CH2S1), 34.9 (1C, CH2S3), 32.1 (1C, CH2S6), 29.7 – 29.1 (m, alkyl chain), 22.8 (s, 1C, -

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CH2-CH3), 14.2 (1C, -CH2-CH3) ppm.

3.3.5. General procedure for the synthesis of the core-ABS HBPs (synthesis of dPG-

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C18ABS):

(Scheme 2) In a round bottom flask equipped with a magnetic stirrer, the dPG (30 kDa) (2.4

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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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H-NMR (DMSO-d6, 700 MHz): δ = 5.42 (m, 1H, =CHS4), 5.29 (m, 1H, =CHS5), 4.80-4.35

(several m, 4H, -CH2-OH, -CH-OH), 4.09 (m, 4H, CH2S7), 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), 2.79 (m, 1H, CH2S2), 2.57 (m, 1H, CH2S1), 2.43 (m, 1H, CH2S1), 2.26 (m, 2H, CH2S3), 1.92 (m, 2H, CH2S6), 1.40 – 1.15 (m, alkyl chain), 0.84 (t, 3H, -CH2-CH3) ppm.

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IG 13C-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),

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58.07 (s, 1C, -CH2-O-CH3), 40.7 (1C; -CHS2), 34.3 (m, 1C, CH2S1), 31.9 (2C, CH2S3,CH2S6),

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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

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aqueous polymer stock solutions with various concentrations (0,5 – 0,0125 mg/mL) in MilliQ 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-

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Vis and fluorescence spectroscopy. Guest loading capacity was calculated based on the

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following the equation 1:


    (
) =

      

x 100

(1)

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 µL 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 µL of 0.5 mg/mL MTT in 10% FBS-

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ACCEPTED MANUSCRIPT 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 µL 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.

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3.3.8. Skin penetration studies:

To estimate the dermal drug delivery efficiency of dPG-C18ABS, C4-HBPE-C18ABS, C6HBPE-C18ABS, validated test procedures for the Franz cell set-up on human skin (obtained

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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,

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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

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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

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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,

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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

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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

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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-

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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

HO

OH

O

HO HO

OH

O HO

HO

OH

O

OH

O

O

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final product was analyzed via GPC. (Table 1).

O

O

O

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O O

OH

O

OH

O

HO

O

HO

O

O

OH

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HO HO

O

O

HO

O

O

OH

OH

O

HO

HO

HO

OH

Scheme 1. Schematic representation of the dendritic polyglycerol (left) and the hyperbranched polyesters (right)

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ACCEPTED MANUSCRIPT Table 1. Characterization of the hyperbranched polymers

Solubility Polymer

Mw

in H2O

a

(g/mol)

Db

dPG

> 20

33108

1.5

C4-HBPE

> 20

27758

2.2

C6-HBPE

10

34761

1.9

a)

Mw and Mn from GPC analysis (ESI). b)Dispersity (Mw/Mn).

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(mg/mL)

13

C-NMR (Figure 1

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By analysing the dPG synthesized via inverse gated decoupling

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

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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 C12ABS, was developed. The intermediate octadecen-1-yl succinic anhydride was synthesized via ene reaction for this purpose by reacting, under high pressure condition

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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

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a mixture of the two isomers of the desired amphiphilic molecule (Scheme 2) that were used as such for further functionalization.[25]

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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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ACCEPTED MANUSCRIPT O HO O

O

O

PEG500

O O Hydroquinone cat.

14

O

O

O

cis-trans

mPEG500

200 °C, high pressure 68 % yield

DMAP cat. 91 % yield

13

O 13

cis-trans

O

HO

PEG500

O

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cis-trans

OH

O

EDC-HCl, DMAP cat. DMF, 48h, r.t.

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HBP

OH

O

OH

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OH

O O

O PEG 500 O

OH O

HBP

O

O O OH

13

OP EG O

50 0

13

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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

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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

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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 C6HBPE-C18ABS (Scheme 2, Figure 1) that were analysed via 1H-NMR and

13

C-NMR

(ESI). Determination of the molecular mass of the nanocarriers was carried out via 1H-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 to rely on the molecular weight estimation deducted via 1H-NMR spectroscopy. The - 12 -

ACCEPTED MANUSCRIPT GPC measurements were, however, useful to ensure the absence, after purification, of

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any non-bond ABS molecules.

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Figure 1. Schematic representation of the nanocarrier dPG-C18ABS

Figure 2. 1H-NMR spectrum of dPG-HBPE-C18ABS.

Figure 2 shows the 1H-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 - 13 -

ACCEPTED MANUSCRIPT 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). Further information about the coupling reaction were obtained comparing the IG

13

C-

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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

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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

hydroxyl groups. (Table 1 ESI).

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reaction occurred without preference in the three different units containing free

Focusing the attention on the two nanocarriers with a polyester core, C4-HBPEC18ABS 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

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functionalization are evident. [23]

Table 2. Characterization of core-AB-shell HBPEs

Tot. OH

N° ABS

Shell

NMR-Mw

Funct. (%)a)

per core a)

(wt%)a)

(g/mol)a)

11.1

45

115

71300

C4-HBPE-C18ABS

19.4

46

141

67000

C6-HBPE-C18ABS

17.8

49

120

76600

a)

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dPG-C18ABS

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Carrier

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. - 14 -

ACCEPTED MANUSCRIPT 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

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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

guest molecule once encapsulated into the nanocarriers.

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for this reason it was useful to understand the microenvironment and binding sites of this

Tests to evaluate the LC (loading capacity) and CMC (critical micelle concentration) were

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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

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concentration solutions (dPG-C18ABS) are shown in Figure 3.

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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)

Polymer

I1/I3

b)

wt%

CMCc) mg/mL

dPG-C18ABS

1.35

1.25

0.12

C4-HBPE-C18ABS

1.53

1.24

0.09

C6-HBPE-C18ABS

1.78

1.25

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.

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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-

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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

C18ABS.

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double the pyrene loaded into C4-HBPE-C18ABS and to increase it by 37% in C6-HBPE-

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

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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

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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 pyreneloaded nanocarriers were 0.12, 0.09 and 0.07 mg/mL respectively for the polymers dPGC18ABS, 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 - 16 -

ACCEPTED MANUSCRIPT 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, C4HBPE-C18ABS, characterized by a hydrophilic core, that are derived from the presence of the long aliphatic C18 chains of the amphiphilic branched shell.

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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 C6HBPE-C18ABS it was possible to solubilize over 17 mg/L of pyrene; this value is 125 times

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higher than the solubility of pyrene in water reported in literature of 0.135 mg/L. at 25°C.[34]

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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

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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).

Polymer

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Table 4. DLS data of the non-loaded and loaded core-shell nanocarriers.

UNLOADED

LOADED

rH volume

rH number

rH volume

rH number

(mg/mL)

(nm)a)

(nm) a)

(nm) a)

(nm) a)

dPG-C18ABS

1

12.1 ± 1.6

9.7 ± 2.5

11.7 ± 1.2

7.4 ± 2.4

C4-HBPE-C18ABS

1

16.6 ± 2.1

10.7 ± 1.8

11.5 ± 1.9

8.9 ± 3.4

C6-HBPE-C18ABS

1

10.0 ± 1.1

7.1 ± 2.1

8.8 ± 2.4

5.8 ± 2.0

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Carrier Conc.

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 - 17 -

ACCEPTED MANUSCRIPT 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

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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]

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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.

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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

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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. - 18 -

ACCEPTED MANUSCRIPT 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

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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

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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

were stirred for 18 hours at 500 rpm.

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in the flask. Aqueous nanocarrier solutions (1 mg/mL) were added and the samples

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

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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

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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

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the solubility of DXM and FNS in water and the loading capacity of the nanocarriers.

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ACCEPTED MANUSCRIPT

measured via HPLC chromatography.

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Figure 6. Solubility of the drugs in water (Blank) and in a 1 mg/mL nanocarrier solutions

Table 5. Solubilization data of DXM and FNS in the core-ABshell HBPEs.

Dexamethasone DXM a)

Polymer

LCDXM

TE D

solubilized (mg/mL)

dPG-C18ABS

C18ABS

AC C

C6-HBPEC18ABS

b)

(wt%)

FNS a)

solubilized (mg/mL)

LCFNS b) (wt%)

0.105

2.5

0.076

2.3

0.128

4.8

0.098

4.3

0.142

6.2

0.158

10.3

EP

C4-HBPE-

Finasteride

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-HBPEC18ABS. The loading capacity of the nanocarrier C4-HBPE-C18ABS is over 50% - 20 -

ACCEPTED MANUSCRIPT 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-HBPEC18ABS) displayed the best drug carrier capabilities, allowing to encapsulate 6.2 wt% of DXM.

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The experiment to evaluate the loading capacity of the drug Finasteride, demonstrate that the best loading performance was once again displayed by the nanocarrier C6HBPE-C18ABS; it became possible to almost triple the solubility of FNS in water

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using a 1 mg/mL nanocarrier solution corresponding to a loading capacity of 10.3 wt%.

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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

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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-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan by means of UV-Vis spectroscopy.

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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

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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

4.6. Skin penetration studies

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solutions of dPG-C18ABS, C4-HBPE-C18ABS and C6-HBPE-C18ABS.

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-

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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

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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

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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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ACCEPTED MANUSCRIPT 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.

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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.

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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

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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

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respective skin layers.

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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

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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

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previously reported system employing the same hyperbranched core molecule, was even doubled. Subsequently, the drugs Dexamethasone and Finasteride were also

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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. - 24 -

ACCEPTED MANUSCRIPT

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

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Dendropharm GmbH for the GPC measurements.

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ACCEPTED MANUSCRIPT

Highlights

Hyperbranched glycerol-based core-amphiphilic branched shell

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nanotransporters for dermal drug delivery S. Stefani, S. Hönzke, J. L. Cuellar Camacho, F. Neumann, A. K. Prasad, S. Hedtrich,

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R. Haag, P. Servin

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.

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•