Correlation between the chemical composition of thermoresponsive nanogels and their interaction with the skin barrier

    Correlation between the chemical composition of thermoresponsive nanogels and their interaction with the skin barrier Michael Giulbudagian, Fiorenza Rancan, Andr´e Klossek, Kenji Yamamoto, Jana Jurisch, Victor Colombo Neto, Petra Schrade, Sebastian Bachmann, Eckart R¨uhl, Ulrike Blume-Peytavi, Annika Vogt, Marcelo Calder´on PII: DOI: Reference:

S0168-3659(16)31043-4 doi:10.1016/j.jconrel.2016.10.022 COREL 8513

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

21 July 2016 12 October 2016 23 October 2016

Please cite this article as: Michael Giulbudagian, Fiorenza Rancan, Andr´e Klossek, Kenji Yamamoto, Jana Jurisch, Victor Colombo Neto, Petra Schrade, Sebastian Bachmann, Eckart R¨ uhl, Ulrike Blume-Peytavi, Annika Vogt, Marcelo Calder´on, Correlation between the chemical composition of thermoresponsive nanogels and their interaction with the skin barrier, Journal of Controlled Release (2016), doi:10.1016/j.jconrel.2016.10.022

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ACCEPTED MANUSCRIPT

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Correlation between the chemical composition of

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thermoresponsive nanogels and their interaction with

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the skin barrier

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Michael Giulbudagian ‡,a, Fiorenza Rancan ‡,b,*, André Klossek a, Kenji Yamamoto a, Jana Jurisch b, Victor Colombo Neto b, Petra Schrade c, Sebastian Bachmann c, Eckart Rühl a,*, Ulrike

Freie Universität Berlin, Institute for Chemistry and Biochemistry, Takustrasse 3, 14195 Berlin,

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a

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Blume-Peytavi b, Annika Vogt b, and Marcelo Calderón a,*

b

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Germany

Clinical Research Center of Hair and Skin Science, Department of Dermatology and Allergy,

Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany c

Institute for Anatomy, Charite-Universitatsmedizin Berlin, Charitéplatz 1, 10117 Berlin,

Germany ‡

These authors contributed equally to this work

*

Corresponding authors

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ACCEPTED MANUSCRIPT Author information Corresponding authors Prof. Dr. Marcelo Calderón, Freie Universität Berlin, Institute of Chemistry and Biochemistry,

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*

Takustrasse 3, 14195 Berlin, Germany, Tel.: +49 (0)30 838 593 68 Fax: +49 (0)30 838 459 368,

Dr. Fiorenza Rancan, Clinical Research Center of Hair and Skin Science, Department of

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*

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E-mail: [email protected]

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Dermatology and Allergy, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany, Tel.: +49 (0)30 450 518 347 Fax: +49 (0)30 450 518 952, E-mail: fiorenza.rancan

*

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@charite.de

Prof. Dr. Eckart Rühl, Freie Universität Berlin, Institute of Chemistry and Biochemistry,

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Email: [email protected]

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Takustrasse 3, 14195 Berlin, Germany, Tel.: +49 (0)30 838 523 96, Fax +49 (0)30 838 527 17,

ABSTRACT: In this paper we present a comprehensive study for the ability of thermoresponsive nanogels (tNG) to act as cutaneous penetration enhancers. Given the unique properties of such molecular architectures with regard to their chemical composition and thermoresponsive properties, we propose a particular mode of penetration enhancement mechanism, i.e. hydration of the stratum corneum. Different tNG were fabricated using dendritic polyglycerol as a multifunctional crosslinker and three different kinds of thermoresponsive polymers as linear counterpart: poly(N-isopropylacrylamide) (pNIPAM), p(di(ethylene glycol) methyl ether methacrylate - co - oligo ethylene glycol methacrylate) (DEGMA-co-OEGMA475), and poly(glycidyl methyl ether - co - ethyl glycidyl ether) (tPG). Excised human skin was

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ACCEPTED MANUSCRIPT investigated by means of fluorescence microscopy, which enabled the detection of significant increment in the penetration of tNG as well as the encapsulated fluorescein. The morphology of

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the treated skin samples was thoroughly investigated by transmission electron microscopy and stimulated Raman spectromicroscopy. We found that tNG can perturbate the organization of

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both proteins and lipids in the skin barrier, which was attributed to tNG hydration effects.

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Interestingly, different drug delivery properties were detected and the ability of each investigated

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tNG to enhance skin penetration correlated well with the degree of induced stratum corneum hydration. The differences in the penetration enhancements could be attributed to the chemical

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structures of the nanogels used in this study. The most effective stratum corneum hydration was detected for nanogels having additional or more exposed polyether structure in their chemical

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composition.

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KEYWORDS: drug delivery; nanogels; skin barrier; stimulated Raman spectromicroscopy; stratum corneum; thermoresponsive

1. Introduction

The dermal application of therapeutic molecules provides significant advantages over other routes of administration and at the same time requires the consideration of aspects different from those commonly addressed for the intravenous route. Skin, being the largest organ of our body, is a very efficient barrier and protects the organism from the penetration of harmful substances. In many skin diseases, however, skin barrier integrity is lost resulting in rapid permeation of low molecular weight, moderately hydrophobic drugs like corticosteroids and consequent rapid clearance of the drug from the site of action.[1] On the other hand, for high molecular weight

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ACCEPTED MANUSCRIPT drugs, like calcineurin inhibitors, topical administration using conventional formulations still results in sub-therapeutic drug levels. Enhancement of percutaneous absorption can be achieved

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using different penetration enhancers despite the fact that the mechanisms of enhancement are only partially understood. The different penetration enhancers can be divided roughly into two

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groups of chemical and physical enhancers.[2] Among the chemical enhancers, surfactants

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(which can be found in a high amount in microemulsions), alcohols, sulfoxides, and urea

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derivatives are commonly used.[3-5] Broadly applied physical enhancers, besides massaging and skin occlusion techniques, are ultrasound treatment, small electric fields (iontophoresis),

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electroporation, laserporation, and microneedles.[6-9] But above all these, water remains to be one of the most effective and safe penetration enhancers.[3, 10-13] Considering the typical water

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content of the stratum corneum (SC) of about 15-20%, clearly the penetration of hydrophilic

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drugs is enhanced by increased solubility in hydrated skin. However, the mechanism of action by

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which water enhances the absorption of hydrophobic drugs remains unclear. Nevertheless, hydration of the SC plays a crucial role in the modification of the organization of the lipids in the SC extracellular matrix. Such rearrangement was observed to be effective for the percutaneous absorption of hydrophilic as well as hydrophobic drugs.[2] The research in the field of nanoparticle-based topical delivery of therapeutic molecules has been significantly developed in the past 20 years. Nanocarrier penetration and accumulation in the SC might be a crucial step for the effective and sustained delivery of drugs into the skin. Most research in this field covers the application of lipid-based carriers, polymeric (non-cross-linked), or inorganic nanoparticles.[14, 15] Commonly, it is accepted that nanoparticles above the diameter of 10-20 nm are not capable of penetrating deep in the SC and to translocate into the viable tissue.[16] However, with increasing reports of nanoparticles of larger dimensions which

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ACCEPTED MANUSCRIPT were found to penetrate through the SC, it seem that not only the size but also the surface and mechanical properties of nanoparticles have to be considered.[17, 18] In fact, most of

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nanoparticles applied on skin surface accumulated in the upper part of the SC, in skin furrows, and in the hair follicle canals.[19, 20] The penetration depth is influenced not only by the size

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and the shape of the particulate formulation but also by the hydrophilicity or hydrophobicity of

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the nanoparticles.[21] This significant impact is obvious when considering the SC as the main

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barrier of absorption, excluding the skin appendages like hair follicles and sweat glands.

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Nanogels are macromolecular architectures made of polymeric units cross-linked to each other in a supramolecular framework. These nanoparticles are ideal nanocarriers because they can

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incorporate water soluble drugs or macromolecules.[22, 23] Their ability to encapsulate water up

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to 90% of their weight becomes highly relevant for topical applications where nanogels may act as hydrating agents and penetration enhancers. In addition, nanogels exhibit a high chemical

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stability on the one hand, but are also highly elastic and deformable particles on the other hand.[24, 25] Given the unique characteristics of such cross-linked polymeric nanoparticles, a distinct mode of interaction with the SC is expected. In particular, thermoresponsive nanogels (tNGs) are of our interest in this study for their ability to selectively expel the encapsulated water and the incorporated cargo upon a thermal trigger. This process is accompanied by the shrinkage of the nanogels and a drastic change in their physico-chemical properties. From a highly swollen and hydrophilic state, the nanogels expel the inner water and adopt a rather hydrophobic nature. For the above mentioned reasons, we believe that tNGs provide outstanding properties favoring the delivery of therapeutic moieties into the skin. Their ability to hydrate the skin is complemented with the hydrophilic / hydrophobic balance of the polymeric materials which should confer them optimal distribution in the different layers of the SC. Such balance could be

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ACCEPTED MANUSCRIPT achieved by the utilization of dendritic polyglycerol (dPG) being a hydrophilic macromolecule which acts as a macro-crosslinker and allows the growth of multiple thermoresponsive polymer

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chains.

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To confirm our hypothesis and investigate the effects of different subunits on the interaction of

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tNGs with the SC, we compared three kinds of thermoresponsive polymers which have significant structural differences but at the same time preserve their thermoresponsive nature:

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poly(N-isopropylacrylamide) (pNIPAM), di(ethylene glycol) methyl ether methacrylate - co -

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oligo ethylene glycol methacrylate (DEGMA-co-OEGMA475), abbreviated as OEG, and poly(glycidyl methyl ether - co - ethyl glycidyl ether), a thermoresponsive linear polyglycerol

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derivate (tPG).

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Fluorescence microscopy studies using tNGs tagged with indodicarbocyanine (IDCC) dye and

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loaded with fluorescein (FL) allowed not only to track the nanocarrier penetration after their topical application on excised human skin, but also to follow the release and skin penetration of the encapsulated model dye. Given the heterogeneity of human skin, our study presents a comprehensive analysis of several skin sections from three different donors. Further, transmission electron microscopy (TEM) and stimulated Raman spectroscopy (SRS) were used to analyze effects of the tNGs on SC morphology as well as on lipid and protein supramolecular organization. 2. Material and Methods 2.1 Synthesis of dendritic polyglycerol-IDCC conjugate dPG-(IDCC)1% In a 50 mL round bottom flask IDCC-COOH (6.7mg, 8.2 mmol) was dissolved in 1 mL of dimethylformamide (DMF) and activated by the addition of 4-dimethylaminopyridine (DMAP)

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ACCEPTED MANUSCRIPT (2 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (6.5 mg, 0.034 mmol) and hydroxybenzotriazole (HOBt) (4.5 mg, 0.034 mmol). While stirring the solution for 30 min, an

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aliquot of the methanolic dPG-(NH2)5% solution (Supporting Information, Experimental Section) (50 mg, 6.8 nmol) was mixed with 1 mL DMF. After removing methanol under reduced pressure

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the dPG-(NH2)5% solution was added to the dye solution via a syringe and the resulting reaction

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mixture was stirred at room temperature for 15 h. The solution was cooled down to 0 °C,

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transferred to a dialysis membrane (MWCO = 2 kDa), and dialyzed against Milli-Q water for 1 day. Finally the solution was concentrated, purified by a Sephacryl S-100 column, and stored at 4

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°C. An aliquot was taken for concentration determination and 1H-NMR analysis. Loading was determined by UV/Vis at absorbance maximum of 654 nm. 1H-NMR (400 MHz, MeOD): 𝛿 =

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7.98 (s, 0.06 H, IDCC), 7.73-7.64 (m, 0.65 H, IDCC), 7.30-7.20 (m, 0.68 H, IDCC), 4.00-3.40

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(m, 5 H, polymer backbone), 2.99 (s, 0.33 H, IDCC), 2.86 (d, 0.29 H, J = 0.7 Hz, IDCC) ppm.

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2.2 Synthesis of dPG-(IDCC)1%-(Ac)10%

In a 10 mL round bottom flask dPG-(IDCC)1% (25 mg, 33.8 nmol) was dissolved in 1.5 mL DMF. After adding triethylamine (TEA) (9.4 µL, 67.6 nmol) the solution was cooled down to 0 °C. Acryloylchloride (4.1 µL, 50.6 nmol) was injected dropwise via syringe and the solution was stirred for 5 h. Quenching with water and dialysis (MWCO = 2 kDa) against Milli-Q water for 2 days yielded the product as an aqueous solution. 1H-NMR (400 MHz, MeOH-D4): 𝛿 = 6.60-6.00 (m, 0.3 H, R-O-CO-CH=CH2), 4.10-3.40 (m, 5 H, polymer backbone) ppm. 2.3 Synthesis of dPG-(BCN)10% In a 10 mL round bottom flask DMF (2.5 mL) was placed and an aliquot of a methanolic dPG(NH2)10% (50 mg, 101 nmol) was added. The methanol was removed under reduced pressure and TEA (100 µL) was added via syringe. Following, (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl

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ACCEPTED MANUSCRIPT (4-nitrophenyl) carbonate (BCN) (32 mg, 101 nmol) was dissolved in 1 mL DMF, added slowly to the reaction mixture, and stirred at room temperature for 1.5 h. the product was purified by

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dialysis (MWCO = 1 kDa) against MeOH for 1 day. 1H-NMR (400 MHz, MeOD): 𝛿 = 4.10-3.40

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(m, 5 H, polymer backbone), 2.6-0.6 (m, 0.8 H, cyclooctyne) ppm. 2.4 Synthesis of pNIPAM based nanogels

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dPG-(Ac)10% (30 mg), NIPAM (67 mg), sodium dodecyl sulfate (SDS) (1.8 mg), and ammonium

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persulfate (APS) (2.8 mg) where dissolved in a total volume of 5 mL water. The solution was degassed by applying an argon stream to the stirred solution for 15 min. Heating to 55 °C for 6

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min was followed by activation with N,N,N′,N′-tetramethylethane-1,2-diamine (TEMEDA) (120 µL). After 3 min of constant stirring dPG-(IDCC)1%-(Ac)10% (3 mg) was added to the solution.

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The reaction mixture was then stirred at 250 rpm and 55 °C for 2.5 h. Cooling to room

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temperature was followed by dialysis (MWCO = 50 kDa) against water for 2 days. The nanogels

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were characterized by 1H-NMR, DLS, and UV/Vis spectroscopy. The cloud point temperature (Tcp) was determined by temperature-dependent UV-Vis transmission measurements. 2.5 Synthesis of OEG based nanogels In a 50 mL flask, dPG-(Ac)10% (14.3 mg), di(ethylene glycol) methyl ether methacrylate (DEGMA) (121.5 mg), oligo ethylene glycol methacrylate (OEGMA475) (13.5 mg), SDS (1.8 mg), and APS (2.8 mg) were dissolved in a total volume of 5 mL water. The solution was degassed by applying an argon stream to the stirred solution for 15 min. Heating for 3 min to 55 °C was followed by activation with TEMEDA (120 µL). After 3 min of constant stirring dPG(IDCC)1%-(Ac)10% (1 mg) was added to the reaction mixture. The solution was stirred at 250 rpm and 55 °C for 3.5 h. After cooling to room temperature the solution was dialyzed (MWCO = 50

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ACCEPTED MANUSCRIPT kDa) against water for 2 days. The nanogels were characterized by 1H-NMR, DLS, and UV/Vis. The Tcp was determined by temperature-dependent UV/Vis-transmission measurements.

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2.6 Synthesis of thermoresponsive polyglycerol based nanogels For tPG based nanogels 20 mL of Milli-Q water in a glass vial were heated in an oil bath to 55

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°C. In parallel N3-tPG-N3 (15 mg) and dPG-(BCN)10% (5 mg) were dissolved separately in DMF

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(0.5 mL). The solutions were cooled down in an ice bath to 0 °C. Both solutions were mixed

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together under cooling and injected via a syringe into the water at 55 °C. After keeping the reaction at 55 °C for 6 h the unreacted alkynes were quenched with IDCC-N3 and let to react for

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another 2 h. Following the nanogel solution was dialyzed (MWCO 50 kDa) against water for 1 day, concentrated and purified by a Sephacryl S-100 column. The nanogels were characterized

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transmission measurements.

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by 1H-NMR, DLS, and UV/Vis. The Tcp was determined by temperature-dependent UV/Vis-

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2.7 Encapsulation and in vitro release of fluorescein The lyophilized nanogels or alternatively a concentrated solution of tPG based nanogels were swollen in a solution of sodium fluorescein (50 wt. %). The mixture was stirred overnight under exclusion of light. Following, the tNGs with encapsulated dye were separated from the free dye by washings five times with a centrifugal filtering device. For the release kinetics, 1 mL of the tNG solution was placed in a Float-A-Lyzer dialysis device (MWCO 100 kDa) and dialyzed against 6 mL of PBS (pH = 7.4). At certain time points, the outer solution was replaced with fresh media and the collected fractions were analyzed by Microplate Spectrophotometer (Tecan, Infinite M200Pro). 2.8 Skin penetration experiments

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ACCEPTED MANUSCRIPT Human excised skin was received a few hours after plastic surgery. The donors were informed healthy volunteers who had signed a consent form. The study had the approval of the Ethics

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Committee of the Charité – Universitätsmedizin Berlin (approval EA1/135/06, renewed on July 2015) and was conducted following the Declaration of Helsinki guidelines.

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Skin from three different donors was used selecting regions without lesions or redness. Skin

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pieces of about 2x2 cm were cut and stretched on Styrofoam blocks by means of needles. About

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0.5 cm of subcutaneous tissue was kept. To prevent skin drying, the samples were placed in a box with wet towels and the incubation took place in a humidified incubator.

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The nanogel suspensions were applied on 1 cm2 large area leaving about 0.5 cm of untreated skin as margins to avoid side penetration. Each nanogel suspension was diluted with distilled H2O to

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a final concentration of 5 mg/mL and 20 µL/cm2 were applied resulting in a final concentration

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of 100 µg/cm2. Because the three different tNG suspensions had different amounts of loaded FL

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(Table 1), for the nanocarrier-free FL control, we decided to use the same amount of FL corresponding to that contained in the tPG-tNG, which had the highest loading. FL was applied at a concentration of 0.2 µg/cm2, which correspond to the amount of dye encapsulated in 100 µg of tNG_dPG_tPG. Skin controls were prepared applying 20 µL of sterile 0.9% NaCl solution. Skin was incubated for 4 h at 37 °C, 5% CO2, and 95% humidity. After incubation, the nonpenetrated suspension was removed from skin surface with cotton swabs. The margins were then removed, skin was placed dermis upright and cut in four pieces of 0.5 x 0.5 cm. The tissue was then frozen in liquid nitrogen and stored at -20 °C. To prepare cryosections, skin was immersed in tissue freezing medium (Leica Microsystems, Germany) and skin sections of 6 µm thickness were prepared using a microtome (2800 Frigocut-N, Reichert-Jung, Heidelberg, Germany). Skin sections were observed at a magnification of x200 with a confocal laser microscope (LSM 700,

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ACCEPTED MANUSCRIPT Zeiss, Germany) and a fluorescence microscope (Olympus BX60F3) equipped with 470-490 nm as well as 545-580 nm bright pass filters and a 610 nm long pass filter. Pictures of at least 20

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sections per donor were taken with a charge coupled device (CCD) camera using always the same settings. Pictures were then analyzed using the ImageJ software. The mean fluorescence

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intensity (MFI) of areas in SC, viable epidermis, and dermis was measured. A total of 20 MFI

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values for each samples and control from the three donors were calculated. Averages and

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standard deviations were reported in the diagrams using Microsoft Excel. One way ANOVA and

2.9 Transmission Electron Microscopy

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two-tailed unpaired student T-tests were used for data statistical analysis.

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After 2 h of incubation with distilled H2O and tNG, the skin was cut with a razor blade in 1x1

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mm pieces which were fixed for 2 h at room temperature using 2.5% glutaraldehyde in 0.1 M

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sodium cacodylate buffer. Samples were then post-fixed with 1% OsO4, 0.8% K4[(Fe(CN)6] in 0.1 M sodium cacodylate buffer and dehydrated in a series of washes with increasing ethanol percentages (50, 70, 80, 95, and 100%). Samples were then immersed for 1 h in mixtures of propylene oxide and Epon resin (SERVA Electrophoresis, Heidelberg, Germany) with ratios of 2:1, 1:1 and 1:2, and then overnight in pure Epon resin. The resin was let polymerize at 60 °C for 48 h. Sections of 300 nm were prepared and stained with a Richardson stain. Areas of interest were identified and 70 nm sections were prepared and placed on copper grids. The sections were then stained with Reynolds lead citrate (Merck, Darmstadt, Germany). The prepared sections were examined with a Zeiss EM906 (Zeiss, Oberkochen, Germany) microscope at a voltage of 80 kV. 2.10 Stimulated Raman Spectromicroscopy

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ACCEPTED MANUSCRIPT Stimulated Raman spectromicroscopy (SRS) studies were performed using an IX83 Olympus Microscope, equipped with a 50x NIR objective (LCPLN50XIR Olympus). The samples were

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excited by a PicoEmerald laser system APE (Berlin), with a pulse width of 6 ps and a repetition rate of 80 MHz. This laser emits two pulses of which one was kept at fixed wavelength (Stokes

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pulse, 1064.2 nm), whereas the wavelength of the preceding pump pulse was adjusted according

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to the desired Raman transition using the signal wave of the optical parametric oscillator (OPO).

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Specifically, we used for probing proteins the transitions at 2934 cm-1 [26], which corresponds to the wavelength of the pump pulse of 811 nm. Lipids were probed at 2850 cm-1 [27], requiring

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that the pump pulse was adjusted to 816.6 nm.

Measuring the SRS process was realized in Stimulated Raman-Loss (SRL) detection

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technique.[28] The Stokes beam was modulated by an electrooptical modulator (EOM) to 20

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MHz. Modulations of the transmitted pump beam were detected by a fast photo diode (Thorlabs

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DET36A) and a lock-in amplifier (HF2TA + HF2LI Zurich Instruments). In order to avoid radiation damage of the samples the laser power was limited to 10 mW and 20 mW for pumpand Stokes-beam, respectively. Stimulated Raman maps were performed by scanning the microscope stage with a dwell time at one spot of 160 ms. Possible unwanted backgrounds from cross-phase modulation or other effects [29] are reduced by subtracting background maps performed at 2500 cm-1, corresponding to a pump beam wavelength of 840.5 nm, where no Raman signal is emitted from skin samples. 3. Results and Discussion 3.1 Synthesis and Characterization of the Nanogels

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ACCEPTED MANUSCRIPT The choice of thermoresponsive nanogels relies on previously reported work on their potential for topical application.[30, 31] In general, all nanogels used in this study take the advantage of

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dPG being a biocompatible hydrophilic macromolecule, while creating a diffusion barrier for the encapsulated moieties.[32, 33] The multifunctional surface of dPG allowed to use it as a macro-

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crosslinker and the growth of multiple thermoresponsive polymer chains from a single core

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(Figure 1). pNIPAM is used due to its robust thermoresponsive behavior as a type I polymer

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which preserves its Tcp in various environments.[34, 35] OEG based nanogels are used due to the ability to fine tune the transition temperature by modifying the ratio between the two co-

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monomers: DEGMA and OEGMA475.[36] Moreover, the monomers contribute additional polyether structure by the oligo ethylene glycols attached to the backbone of the polymer. And

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finally, nanogels based on tPG follow a quite different synthetic approach, where the

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presynthesized and functionalized macromolecules are crosslinked upon bioorthogonal click

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reaction.[37] These nanogels present the combination of the dendritic polyether structure of the dPG with the linear polyether backbone of the thermoresponsive polyglycerol of a defined length.

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Figure 1. Schematic representation of the tNGs and their building blocks. The tNGs shrink upon a thermal trigger, while the encapsulated moieties diffuse out of the crosslinked network.

The synthesis of the tNGs relies on previously reported methods with minor modifications which allowed the chemical conjugation of the IDCC.[31, 34, 37] In the case of pNIPAM and OEG based nanogels, where radical precipitation polymerization was applied as a method of nanogel fabrication, the IDCC dye was initially conjugated to the macromolecular crosslinking precursor (dPG) and used in combination with the unlabeled dPG. The conjugation reaction was performed via an amide bond formation between dPG-(NH2)5% and IDCC-COOH, using EDCI, HOBt, and

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ACCEPTED MANUSCRIPT DMAP as catalysts. The precipitation polymerization reaction time and temperature were optimized to 4 h and 50 °C to prevent the degradation of the dye. Alternatively, for the synthesis

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of tPG based nanogels, following the thermonanoprecipitation and the crosslinking reaction, the unreacted alkyne groups were quenched with azide modified IDCC. The nanogels were fully

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characterized in terms of their hydrodynamic radius, cloud point temperature (Tcp), and the

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amount of the labeled dye (Table 1, Figure S1, S2). The Tcp of the nanogels was tuned to occur

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below the incubation temperature, which was set to 37 °C to simulate the temperature of inflamed skin.[38] Based on previously reported studies [31] as confirmed in this work, the tNGs

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improved significantly the skin penetration of the encapsulated molecule which could be detected in higher amounts in the SC when the temperature of incubation was above the Tcp

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(Figure S3).

Nanogel

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Table 1. Characterization of the synthesized tNGs used for skin penetration studies. Wt. % dPGa

of

Size (nm)b

Tcp (°C)c

Coupled IDCCd (wt. %)

Encapsulatedd FL (wt. %)

(PDI)

tNG_dPG_pNIPAM

33

95±1.9 (0.3)

34.6

0.00413

0.0256

tNG_dPG_OEG

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112±1.4 (0.2)

28.1

0.0027

0.0071

33

156±0.7 (0.1)

29.9

0.093

0.23

tNG_dPG_tPG

a

Determined by 1H-NMR (400 MHz, D2O).

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Size and polydispersity index (PDI) by dynamic light scattering (DLS) in water at 25 °C. Measurements were performed in triplicate; intensity average mean value presented. c

Cloud point temperature determined as the temperature of 50% transmission at λ = 500 nm.

d

Determined by UV-Vis absorption in PBS (pH = 7.4). For IDCC at λmax = 654 nm, and for FL at λmax = 482 nm with the corresponding extinction coefficients ε = 250000 L·mol-1·cm-1 and ε = 70568 L·mol-1·cm-1.

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ACCEPTED MANUSCRIPT As a model for therapeutic molecule, fluorescein was encapsulated within the nanogels. The choice of fluorescein was due to its absorption spectrum which does not overlap with the

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conjugated dye IDCC (Figure S2). This allowed us to simultaneously follow both dyes during the skin penetration studies, i.e the pathway of the tNGs and that of the encapsulated dye after its

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release. In addition, the LogP of fluorescein is positive (3.4), being FL a lipophilic substance

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which can easily permeate skin. Thus, it can serve as a model for drugs such as dexamethasone

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and betamethasone (LogP = 1.83 and 1.94, respectively). In vitro release studies of FL confirmed our hypothesis of prolonged release kinetics, when encapsulated within the tNGs. Studied by

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dialysis, the encapsulated FL was released from the tNGs over a period of 24 h, compared to the free FL which was completely released to the outer dialysis solution after 3 h (Figure S4). In

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addition, the prepared tNGs revealed no toxicity towards the keratinocyte cell line HaCaT (data

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not shown) and the microscopic analysis of the Richardson-stained semi-thin sections (Figure

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S5) revealed not morphological changes of the viable epidermis and dermis, underlining the biocompatibility of this material.

3.2 Fluorescein Delivery and Nanogel Skin Penetration after Topical Application on Human Excised Skin

The skin penetration of the tNGs as well as the penetration of released FL to the different skin layers have been investigated by means of fluorescence microscopy. The fluorescent dye IDCC, which was covalently conjugated to the tNG subunits, served to localize tNGs within the skin sections, whereas FL served as a model molecule to test the drug delivery capacity of the investigated tNGs. In Figure 2 (a-e) representative confocal laser microscopy (CLSM) images are shown. Pictures were taken-up with the same camera settings and resulted in low fluorescence signals for the untreated control skin, corresponding to the skin background

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ACCEPTED MANUSCRIPT fluorescence. IDCC and FL fluorescence intensities were appreciable for all three tNGs, with tNG_dPG_tPG having the highest signals, probably due to the better conjugation and loading

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capacity obtained for this tNG.

Figure 2. Release and penetration of fluorescein (FL) in excised human skin 4 h after topical application of three different nanogels: tNG_dPG_pNIPAM, tNG_dPG_OEG, tNG_dPG_tPG. Skin explants were treated with the investigated tNGs (100 µg/cm2) as well as FL (0.2 µg/cm2) and saline. Representative images of skin sections are shown in (a)-(e), where (a) is untreated skin, (b) skin treated with FL, (c) tNG_dPG_pNIPAM, (d) tNG_dPG_OEG, and (e) tNG_dPG_tPG samples. Scale bar = 50 µm. The mean fluorescence intensity (MFI) of FL in SC, viable

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ACCEPTED MANUSCRIPT epidermis (E), and dermis (D) of at least 20 different skin sections per donor was analyzed using the Image J software. Results from three different donors are shown in diagrams (f), (g), and (h).

Especially for tNG_dPG_tPG, it could be visualized that the nanoparticles penetrated down to

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the deepest layers of the SC (Figure 2 (e)), whereas the released FL diffused further to the viable

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epidermis and dermis. In order to avoid subjective interpretation of the images, the analysis of

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the pictures was performed using an image processing software (ImageJ). The pictures were taken from sections of skin samples prepared in three independent experiments with skin from

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three different donors. In total, for each tNG at least 60 images were analyzed. The analysis of FL penetration for the three different donors are shown in Figure 2 (f)-(h). Acceptable variations

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between the results obtained from different donors were found. For all three tNGs, the highest fluorescence intensity was detected in the SC. Because it is visible that tNGs are localized in the

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SC (Figure 2 (c)-(e)), the FL signal measured in this layer is probably due to both encapsulated

released dye.

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and released dye. On the contrary, FL signal detected in the viable epidermis is due mainly to the

In skin treated with free FL, a weaker signal was detected than for the tNG samples after 4 h of incubation. The higher FL signal in the epidermis of skin treated with tNG can be explained by a positive effect that tNGs exert on the skin penetration of FL. Such effect has already been reported in literature and different explanations have been proposed, even if the mechanism by which nanoparticles improve drug penetration has not been elucidated so far.[39-42] For tNG_dPG_tPG and tNG_dPG_OEG higher FL signals were measured in the SC than for the tNG_dPG_pNIPAM samples. A significant FL signal was observed also in the epidermis but only for the tNG_dPG_OEG and tNG_dPG_tPG samples. Thus, these two tNGs seem to have special characteristics allowing to create high local concentration of FL in the viable epidermis.

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ACCEPTED MANUSCRIPT On the contrary, tNG_dPG_pNIPAM delivered FL less efficiently or at a different rate. Surprisingly, although the amount of FL encapsulated in tNG_dPG_OEG is 30 times less than

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that loaded on the tNG_dPG_tPG nanocarrier, similar signal intensities were detected for the two tNGs in the SC as well as in the viable epidermis and dermis. On the other hand, OEG based

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tNGs were loaded with 3.5 times less FL than tNG_dPG_pNIPAM but achieved a better FL skin

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delivery. This good performance of tNG_dPG_OEG is not due to its size or Tcp. In fact, the

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hydrodynamic diameters of OEG and pNIPAM based tNGs are quite similar (112 and 95.5 nm, respectively, Table 1) and the Tcp of OEG and tPG based tNGs are comparable (28.1 and 29.9

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°C, respectively).

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The analysis of tNG skin penetration (Figure 3) shows that tNG_dPG_OEG and tNG_dPG_tPG

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have similar fluorescence intensity values in the SC, despite the different amounts of conjugated IDCC (Table 1), indicating that tNG_dPG_OEG can penetrate the SC better than tNG_dPG_tPG.

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This suggests that the physico-chemical characteristics of tNG_dPG_OEG favor the nanocarrier penetration in the SC and the delivery of the cargo to the epidermis. The chemical structure of the used thermoresponsive polymers might also have an influence on the penetration profile of the different tNGs. In all cases dPG was used as the macromolecular crosslinker, however the themoresponsive polymers differ substantially regarding their chemical structure. The polyether structure is introduced to pNIPAM based nanogels via the dPG alone. In contrast, OEG and tPG based nanogels consist of additional polyether structures; in the side chain for OEG and in the polymer backbone for tPG. The introduced polyethers have a rather amphiphilic nature, which might favor the interaction with different skin components.

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ACCEPTED MANUSCRIPT In accordance with previous studies on tNG, significant amount of tNG signal was also detected in the epidermis.[17, 31] The detected fluorescent signal was substantially higher for tPG and

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OEG based tNG with respect to the pNIPAM based ones. This suggests that the physicochemical properties of these soft nanocarriers influence not only the delivery of the cargo to the

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skin but also its own penetration across the SC barrier. The fact that the tNG penetration profiles

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in the SC are quite similar to those of FL, could indicate that most of the FL in SC is still

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encapsulated in the nanocarriers. However, the measured fluorescence could be of both encapsulated and released FL. The in vitro release kinetics show that 50% of FL is released 4 h

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after incubation. Nevertheless, because of the different environment, in the SC the kinetics of FL release and diffusion seems to be completely different. This indicates that tNG accumulated in

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SC layers, as well as in furrow and hair follicle canals, can act as depot able to release drugs over

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hours. We are currently performing drug release kinetics using the excised human skin model in

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order to confirm these findings.

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Figure 3. Detection of conjugated IDCC in excised human skin 4 h after topical application of three different nanogels: tNG_dPG_pNIPAM, tNG_dPG_OEG, tNG_dPG_tPG. Skin explants were treated with the investigated tNGs (100 µg/cm2) and saline as control. The IDCC intensity of SC, viable epidermis (E), and dermis (D) of at least 20 different skin sections per donor were analyzed by means of ImageJ software. Results from three different donors are shown in diagrams a, b, and c. * p < 0.05, ** p < 0.01, *** p < 0.001.

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3.3 Effects of Nanogels on the Stratum Corneum In several images of skin treated with the tNG samples, and especially with tNG_dPG_tPG, we

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noticed an alteration of the SC morphology, with a slightly swollen aspect. We hypothesize that

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tNGs can penetrate and interact with the SC components modifying its hydration status and, thus,

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creating channels allowing for an enhanced skin penetration of both FL and tNGs. To verify this hypothesis, we used transmission electron microscopy (TEM) to obtain high resolution images of

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the SC treated with pure water and an aqueous suspension of tNG_dPG_tPG (Figure 4).

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ACCEPTED MANUSCRIPT Figure 4. Representative TEM images of skin treated with water ((a), (c), (e)) and an aqueous suspensions of tNG_dPG_tPG ((b), (d), (f)). The pictures at different magnifications show overviews of the epidermis ((a), (b)) and details of the SC (c-f). The SC of skin treated with the tNG_dPG_tPG sample is visibly thicker than the control skin treated with the vehicle only. Most of corneocytes in the sample appear as swollen (d), whereas the space between corneocytes is enlarged and the lipid organization is altered (f).

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Warner et al. have already reported that incubation of SC for 24 h with water can alter its

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ultrastructure, with dilation of spaces between corneocytes, keratinocytes swallowing, formation

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of expansions between corneocytes (cisternae) as well as changes of keratin and lipid structures

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(e.g. roll up of lamellar lipids).[43]

All these changes were found also in skin treated with tNGs, but already after 2 h of incubation.

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In addition, hydration and morphological changes of SC in skin treated with tNGs were clearly more significant than in control skin treated with water only. The corneocytes appeared much

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more swollen. The spatial organization of the keratin filaments in the tNG_dPG_tPG-treated

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sample appeared deeply altered in most of observed corneocytes, whereas in control skin keratin

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had a more compact pattern. The space between corneocytes, which is normally filled with the lipid extracellular matrix, was dilated in both control and sample. However, such expansions were significantly bigger in the tNG_dPG_tPG treated skin sample, indicating a much higher SC hydration degree in the tNG-treated skin than in water-treated skin. Thus, the TEM analysis confirms that tNGs can rapidly hydrate the SC, disrupting its compact structure and suggests that this might be the mechanism by which tNGs increase the skin penetration of carried substances. It has been reported that SC hydration would result in the perturbation of the supramolecular organization of both keratin filaments and lipid lamellae.[44] In order to detect these changes, we performed measurements by means of stimulated Raman spectromicroscopy. SRL images were performed at 2934 cm-1 and at 2850 cm-1, respectively. These energies are related to the symmetric CH3 stretch vibration (sCH3) [45] of proteins and the symmetric CH2 stretch

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ACCEPTED MANUSCRIPT vibration (sCH2) [46] of lipids, respectively. These two groups of molecules are the most important skin components within the SC. Therefore, they are a suitable indicator for induced

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changes by the application of tNGs.

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Representative skin regions for SRL were selected from fluorescence and optical microscopy

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images. An example for this is shown in Figure 5(a), where one can see the SRL region marked within a red frame. Normalized spectra of the SC (black) and the viable epidermis (red) are

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added for better understanding and spectral orientation (Figure 5(b)). The corresponding SRL

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(middle) and lipid distribution (right).

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maps are shown in Figure 5(c), where the optical microscopy (left) is compared to the protein

Figure 5. Example for SRL images of the protein and lipid distribution for skin treated with pNIPAM based tNG: (a) Skin region for SRL marked as red frame in the fluorescence microscopy image; (b) SRL spectra of SC and viable epidermis; (c) optical transmission image (left), distribution of proteins (SRL 2934 cm -1) (middle), and lipids (SRL 2850 cm-1) (right).

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ACCEPTED MANUSCRIPT The distribution of lipids exhibits a high concentration only within the SC (see Figure 5(c) right) in contrast to proteins, which are more or less homogenously distributed within the entire skin

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region (see Figure 5(c) middle). Please note that Figure 5 is an example to demonstrate the basic imaging technique for further analysis. All other skin samples treated with different nanogels

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exhibit comparable distributions of proteins and lipids (Figure S6). From such images we

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extracted the protein-to-lipid ratio (2934 cm-1 / 2850 cm-1), which gives reliable information

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about the samples, neglecting inhomogeneities and individual structures of the selected skin regions. This procedure of analyzing SRL data was previously utilized for the investigation of

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brain tumors and Meibomian gland function.[47, 48] However, to our knowledge, the interpretation of the SRL data for the detection of structural modifications of the skin barrier was

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not reported so far. Three different regions from every donor where investigated for further

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improvement of the reliability and comparability with the fluorescence studies. The results are

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summarized in Figure 6, where one can see the median protein-to-lipid ratio of the SC for control skin as well as skin treated with FL and the investigated tNGs. A significant decrease of the protein-to-lipid ratio can be observed for the samples which were treated with the three nanogels, whereas the skin treated with FL only shows no changes compared to the control groups.

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Figure 6. Median protein (2934 cm-1) to lipid- (2850 cm-1) ratio of the SC for different skin samples: skin treated with saline (control), FL, and the three investigated tNGs. Same skin samples which were used for dye and tNG penetration were investigated here. Statisticla analysis showed significant differences to control with p < 0.05 for tNG_dPG_pNIPAM and p < 0.001 for tNG_dPG_OEG and tNG_dPG_tPG.

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Deeper layers of skin (e.g. the viable epidermis) exhibit no significant deviation of the proteinto-lipid ratio in SRL between the samples. It is important to emphasize that no relative changes in the SRL signal between the measured energies were detected for FL solution [49] and the tNG suspensions, indicating that the detected changes are exclusively due to changes in the proteinto-lipid signal ratio within the SC. These results show that OEG and tPG based nanogels can significantly modify the protein-to-lipid SRL signal ratio with respect to skin treated with saline, FL, or tNG_dPG_pNIPAM. These changes might in principle be due either to an increase of the lipid signal or a decrease of protein signal. The present results point to the conclusion that there is rather a decrease of protein signal. This might be explained by the fact that in the hydrated samples keratin has lost its compact structure, as shown in the TEM images, resulting in a lower concentration of proteins and, thus, in a decrease of the SRL signal. This corresponds to a

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ACCEPTED MANUSCRIPT decrease of the protein-to-lipid ratio. TEM images also show an enhancement of roughness of the lipid lamellar bilayers of the SC. Such changes in morphology can affect the SRL signal

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since Raman spectroscopy is a scattering based technique. It has also been shown that in hydrated SC lipids lose their lamellar organization and tend to form vesicles.[44] This might also

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contribute to a decrease of the protein-to-lipid ratio.

In summary, the stimulated Raman microscopy studies indicate that there are significant changes

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in the morphological ultrastructure of the SC, which are induced by tNGs. These occur as

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modifications in the spatial organization of both lipids and proteins. Interestingly, the decrease of the SRL signal correlates well with the FL delivery efficiency of the tNG, with tNG_dPG_OEG

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having the most noticeable effects on the SRL signal in SC and also the best delivery

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performance. This indicates that tNG-mediated perturbation of SC organization is the mechanism by which tNG enhance FL skin penetration. The label-free SRL is complementary to both label-

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based fluorescence microscopy and TEM investigations. On the one hand, fluorescence microscopy was used to detect the penetration of drugs but was not sensitive to changes of the skin. On the other hand, while TEM provided a qualitative measurement of SC morphological changes, Raman spectroscopy allowed to perform quantitative measurements and statistical analysis of ultrastructural changes between samples and controls.

4. Conclusion In this study, we compared three different tNGs with regard to their interaction with the SC and drug delivery properties. Such thermoresponsive nanocarriers have unique properties that make them attractive as drug delivery systems for dermatological applications. They are water soluble

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ACCEPTED MANUSCRIPT at temperatures below the Tcp but become hydrophobic, and thus more compatible with the SC environment, at temperatures above Tcp. In addition, they carry not only drugs but also

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considerable amounts of water that have permeabilizing effects on the SC.

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The investigated tNGs were found to enhance the penetration of the fluorescent dye FL, used as

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model molecule to simulate a moderately lipophilic drug. Similar results have been found also for other type of nanoparticles and cargos, but the mechanisms behind this effect remain unclear.

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In this study, the interactions and effects of tNGs on the SC were investigated by means of high

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resolution microscopy and SRL spectroscopy. These techniques revealed that tNGs act as penetration enhancers by increasing considerably the hydration of the SC with consequent

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perturbation of the lipid and protein supramolecular organization.

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We believe that the differences in the penetration enhancements can be attributed to the chemical

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structures of the nanogels used in this study. This statement is based directly on the fact that the penetration enhancement property of the tNG studied by CLSM could be directly elucidated by the SRL measurements, correlating enhanced skin penetration with perturbation of SC organization. All nanogels had comparable sizes, Tcp, and architecture, nevertheless they differ in the chemical structure of the thermoresponsive polymers. The better performance of OEG and tPG based nanogels is directly induced by the additional polyether structure and their accessibility on the side chain for OEG and on the polymer backbone for tPG. On the contrary, pNIPAM based nanogels had polyether structure as part of the macro-crosslinker alone. In conclusion, this study revealed that the chemical composition of thermoresponsive nanogels can influence the degree of SC perturbation, which is an important parameter that has to be considered for the development of carriers with optimal skin delivery characteristics.

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Supporting Information. The supporting Information contains information on the used chemicals, Tcp measurement of the

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tNGs, UV-Vis spectra, representative fluorescent microscopy imaged of skin treated with tNGs

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with and without IR irradiation.

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Acknowledgments

We gratefully acknowledge financial support from the Sonderforschungsbereich 1112, projects

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A04, B02 and C04, and the Bundesministerium für Bildung und Forschung (BMBF) through the NanoMatFutur award (13N12561, Thermonanogele). Victor Colombo Neto thanks the Federal

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Government of Brazil for the scholarship 'Ciências sem Fronteiras'.

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Graphical abstract

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