Skin Penetrating Peptide as a Tool to Enhance the Permeation of

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SKIN PENETRATING PEPTIDE AS A TOOL TO ENHANCE THE PERMEATION OF HEPARIN THROUGH HUMAN EPIDERMIS Chiara G. M. Gennari, Silvia Franzé, Sara Pellegrino, Emanuela Corsini, Giulio Vistoli, Luisa Montanari, Paola Minghetti, and Francesco Cilurzo Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01524 • Publication Date (Web): 01 Dec 2015 Downloaded from http://pubs.acs.org on December 9, 2015

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SKIN PENETRATING PEPTIDE AS A TOOL TO ENHANCE THE PERMEATION OF HEPARIN THROUGH HUMAN EPIDERMIS Chiara G.M. Gennari1, Silvia Franzè1, Sara Pellegrino1, Emanuela Corsini2, Giulio Vistoli1, Luisa Montanari1, Paola Minghetti1 and Francesco Cilurzo1 1

Department of Pharmaceutical Sciences – Università degli Studi di Milano, Via Giuseppe

Colombo, 71 – 20133 Milan (Italy) 2

Department of Pharmacological and Biomolecular Sciences – Università degli Studi di Milano, Via

Giuseppe Balzaretti, 9 – 20133 Milan (Italy)

*Corresponding author [email protected] +390250324635

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ABSTRACT

This study aimed to identify a new skin penetrating peptide (SPP) able to enhance unfractionated heparin (UFH) permeation through human epidermis by screening a phage display peptide library. The effects of the synthetized heptapeptide (DRTTLTN) on human stratum corneum organization were investigated by ATR-FTIR spectroscopy and molecular dynamics simulation. The DRTTLTN penetration within the human epidermis caused both a fluidization of the stratum corneum lipids and the extension of keratins due to the increase of the contribution of α-helices. The co-administration of DRTTLTN with UFH resulted ineffective in increasing skin penetration due to UFH affinity for keratins. The conjugation of DRTTLTN to UFH by N-(3Dimethylaminopropyl)-N′-ethylcarbodiimide

hydrochloride

and

sodium

N-

hydroxysulfosuccinimide led to an increase of the flux of 24-36 fold with respect to raw UFH, depending on the adopted synthetic procedure. The new compounds showed a decrease of the antifactor Xa activity of about 4-5 times. DRTTLTN also permitted to increase the fluxes of small model molecules. In conclusion, these data support the use of SPP to enhance the skin penetration of poorly absorbed compounds even in the case of macromolecules as polysaccharides.

KEYWORDS skin penetrating peptide; human epidermis; heparin; phage display; ATR-FTIR spectroscopy.

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INTRODUCTION Skin penetration enhancers represent a long-standing approach for improving transdermal delivery of drugs. They are usually small molecules able to perturb the organization of stratum corneum, which represents the main barrier for the penetration of xenobiotics. In recent years, peptides able to diffuse through the epidermis layers have been also tested to improve the transcutaneous delivery of drugs1. As an example, polyarginine has been shown to carry molecules across the stratum corneum and into the viable epidermis and dermis2. Magainin, a naturally occurring pore-forming peptide, was found to increase skin permeability by direct interaction with and disruption of stratum corneum lipids3,4. Among the possible approaches to screen new skin penetrating peptides, the use of phage display peptide libraries is gaining a growing interest since they allow identifying a specific peptide able to penetrate the skin and to carry filamentous bacteriophages through the stratum corneum5-7. The first application of this technique to the transdermal delivery was due to Chen and coworkers, who identified a 9-mer peptide that increased the transdermal absorption of insulin in diabetic rats through hair follicles5. Using the same approach, Kumar et al. selected a 6-mer peptide (LVGVFH) which improved the penetration of 5-fluorouracil through porcine and mouse skin6. However, the most exhaustive study in this field was carried out by Hsu and Mitragotri that provided the first evidence that skin penetrating peptides selected by phage display are able to enhance the penetration of macromolecules through human skin7 and favor the partition of nanocarrier within the epidermis8. Altogether, these data revealed that the use of skin penetrating peptides could improve the challenging transdermal delivery of large hydrophilic molecules, overcoming the limits of the conventional chemical enhancers1. Unfractionated heparin (UFH) has been the anticoagulant and antithrombotic drug of choice for several decades, even if nowadays its use is restricted to the hospital where it is possible a careful clinical monitoring of the patients. Indeed, the intravenous administration of UFH can be associated with serious complications such as bleeding risk, thrombocytopenia, effect on bone metabolism, along with pain and poor patient compliance9. The delivery of the drug by an alternative, non3 ACS Paragon Plus Environment

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invasive route of administration may alter the pharmacokinetic/pharmacodynamic profiles and, therefore, potentially minimize the adverse effects, increasing at the same time patient compliance10. In particular, topical application appears as the most attractive alternative to UFH injection since it allows a sustained release of the drug with possibility to have both a systemic (transdermal) and local effect10. In fact, the cutaneous administration of UFH has also a clinical relevance in the treatment of the symptoms associated to peripheral vascular disorders, requiring however the application of huge doses of drug to reach the desired antithrombotic effect11. The skin penetration of UFH is very poor due to its unfavorable physico-chemical features, namely high Mw, hydrophilicity and ionization12 and, therefore, a suitable method to improve the UFH diffusion through the skin is desired. This study aimed to identify a new skin penetrating peptide able to enhance the UFH penetration through human epidermis. The selection was performed by screening a random 7-mer phage display peptide library. The possible interactions with the main stratum corneum components, namely keratins and ceramides were studied by ATR-FTIR spectroscopy and molecular dynamic simulations. Moreover, its effect on the permeation of small molecules was also determined. Lidocaine and propranolol-HCl were selected as model drugs since they are used for local anesthesia and hemangiomas, respectively13, 14.

EXPERIMENTAL SECTION

Materials. The Ph.D.-7 Phage Display Peptide Library and Escherichia coli K12 strain ER2738 were obtained from New England Biolabs. Luria Broth, agar, NaCl, Blue/white select, polyethylene glycol

8000

(PEG),

tris-HCl,

sodium

azide,

trypsin,

N-(3-Dimethylaminopropyl)-N′-

ethylcarbodiimide hydrochloride (EDC), N-Hydroxysulfosuccinimide sodium salt (NHS), propranolol-HCl and lidocaine were purchased from Sigma-Aldrich (Italy). Tetracycline was purchased from Fluka Analytical. Sodium unfractionated heparin (activity 195 IU/mg) was kindly 4 ACS Paragon Plus Environment

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provided by LDO S.p.A. (Milan, Italy). Kit Coatest® Heparin was purchased by Chromogenix Instrumentation Laboratory S.p.A. (Milan, Italy). Fmoc Rinkamide resin, Fmoc-protected amino acids, HBTU, HOBT and DIEA were purchased from Iris Biotech Gmbh (Germany). Solvents, piperidine and all cell culture reagents were also obtained from Sigma-Aldrich.

Preparation of human epidermis samples. The epidermis used in the permeation studies was obtained from the abdominal skin of donors, who underwent cosmetic surgery (30-50 year old, Eurasian female). The full-thickness skin was sealed in evacuated plastic bags and frozen at -20 °C after removal. Prior to the experiments, the skin was thawed at room temperature and the excess of fat was carefully removed and the sheet was cutted in square section. The epidermis was then isolated by immersing the skin section in water at 60 °C for 1 min, and gently separating from the remaining tissue with forceps. Finally, the stratum corneum sheets were prepared from isolated epidermis by trypsin digestion (0.1% w/v in phosphate-buffered saline solution (PBS), pH 7.4), after overnight incubation at 37°C. The stratum corneum sheets were then rinsed twice with saline solution before use. The integrity of all tissue samples was assessed measuring their electrical impedance (voltage: 250 mV, frequency: 100 Hz; Agilent 4263B LCR Meter, Microlease, Italy). All samples were hydrated and sandwiched between the donor and receptor compartments of a modified Franz diffusion cell (Permegear, USA) with an effective penetration area and a receptor volume of 0.636 cm2 and 3 mL, respectively. The donor and receiver compartments were filled with physiologic solution. The system was kept at 37 ± 1 °C by means of a circulating water bath so that the skin surface temperature was at 32 ± 1 °C and the receiver medium was continuously maintained under stirring with a magnetic bar. Before the measurement, the samples were equilibrated for 30 min. Samples with an electrical impedance higher than 30 KΩ·cm2 were used for the in vitro permeation experiments.

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Phage Display Library Screening. Physiologic solution containing 0.01 % w/v sodium azide as a preservative was used as receiver medium. Phage library (2 × 1013 pfu, 50 µL) in 0.45 mL TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl) was placed in the donor compartment. After 24 h, the receiver medium was withdrawn and 1 mL was added to 20 mL of a 1∶100 diluted overnight culture of Escherichia coli ER2738 and grown for 4.5 h to amplify the phages. Then, the phages were purified by PEG⁄NaCl precipitation and resuspended in TBS. 0.5 mL amplified phages suspension were then used in the second round of screening. The number of phages placed in the donor compartment, 2 × 1013 pfu, was held constant for both rounds of screening. After the second round of screening, phages that permeated the human epidermis consistently were separated on an agar plate. A 10 µL aliquot of the receiver solution was added to 10 µL of Escherichia coli strain ER2738 and plated on IPTG/Xgal plates. 20 plaques were randomly selected and the DNA from each individual plaque, representing a single type of phage, was extracted and sent for DNA sequencing (BMR Genomics, I).

Peptide synthesis and characterization. The peptide selected by phage display was synthetized by microwave assisted solid phase peptide synthesis using the Fmoc-Chemistry (Liberty Microwave Peptide Synthesizer, CEM Corporation)15.The peptide was C-terminally amidated and N-terminally acetylated using Rink amide resin with a loading of 0.5 mmol g-1 and on a 0.1 mM scale. Single couplings were performed with Fmoc-amino acid (5 eq) activated in situ with HBTU (5 eq), HOBt (5 eq) dissolved in DMF, and DIPEA 1M in NMP (10 eq); each coupling was achieved by using microwave irradiation (5 min, 75 °C, 20W). Fmoc cleavage was accomplished by treating the peptidyl-resin with 20% piperidine in DMF (3 min, 75 °C, 40 W). N-acetylation was performed manually using acetic anhydride (10 equiv.) and DIPEA (10 equiv.) in DCM for 30 min at room temperature. The peptide was manually cleaved from the resin with a mixture of TFA/water/TIS (90:5:5 v/v) for 6 h at room temperature. The crude peptide was precipitated from ice-cold diethyl ether and recovered by centrifugation at 4°C for 5 minutes (4500 rpm). The crude peptide was 6 ACS Paragon Plus Environment

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dissolved in DMSO and diluted in ACN/water (50:50 v/v) solution. The purification was performed by preparative RP-HPLC (Jasco BS-997-01 equipment) using a DENALI C-18 column from GRACE VYDAC (10µm, 250 x 22 mm) and a combination of two mobile phases: A = 95% Water, 5 % ACN, 0.1 % TFA, B = 95% ACN, 5% Water, 0.1% TFA. 2 mL of peptide solution were injected at a flow rate of 20 mL/min. The gradient was: 95% A for 5 min., then 95-30% A over 20 minutes. UV detection was made at 220 nm. The purified peptide was freeze-dried and stored at 0°C. The conformational profile of the synthetic peptide was investigated by circular dichroism (CD). Peptide stock solution was prepared in HPLC-quality water (500 µM, 1.5 mL). Spectra were recorded on a J-810 spectropolarimeter, using a quartz cuvette of 0.01 cm path length (Hellma Suprasil), from 195 to 250 nm with a 0.1 nm step and 1 s collection time per step, taking three average. The spectrum of the solvent was subtracted to eliminate interference from cell, solvent, and optical equipment. The CD spectra were plotted as mean residue ellipticity θ (degree x cm2 x dmol-1) versus wavelength λ (nm). Noise-reduction was obtained using a Fourier-transform filter program from Jasco.

Synthesis of UFH-peptide conjugates. Two conjugates of UFH with the identified peptide were prepared using EDC and sulfo-NHS as activating agents16. In the case of the first conjugate (UFHSPEH1), UFH (average Mw 15,000 Da) and a mixture of EDC and sulfo-NHS (2:1 w/w) were dissolved in MilliQ-water on ice. A 5-fold molar excess of activating agent with respect to UFH carboxylic acid (-COOH) groups was used. In parallel, the peptide was dissolved in MilliQ-water (UFH: peptide 1:1 molar ratio) and kept on ice until its use. After vigorous mixing, the UFH and EDC/sulfo-NHS solution was incubated in ice (approx. 6-8 °C) for 15 min to activate the free carboxylic group of UFH. Subsequently the peptide solution was added to the activated UFH. After vortexing and incubation for 15 min at 4 °C, the reaction mixture was maintained at 25°C under

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continuous stirring for 4 h. Afterwards, the reaction product was purified by ultrafiltration (membrane filters Microcon/Amicon, cut-off 3KDa) and the recovered conjugate was freeze-dried. In the case of UFH-SPEH2 synthesis, the procedure was slightly modified since the SPEH and the other reagents were added to the cooled UFH solution at the same time.

In Vitro Skin Permeation Studies. The experiments were performed using modified Franz diffusion cell under occlusive conditions. The experimental conditions of the performed experiments are summarized in Table 1. At predetermined interval times, samples (0.2 mL) were withdrawn from the receptor chamber and an equivalent volume of fresh receiver medium was added. Each experiment was performed in triplicate for the small molecules and at least in quadruplicate in the case of the polysaccharides, using randomized epidermis samples from two different donors.

Drug assays. The amounts of propranolol and lidocaine that permeated through human skin were assayed by HPLC, using an Agilent 1100 HPLC system (1100 autosampler, 1100 quaternary pump with degasser, 1100 thermostated column compartment, and 1100 diode array detector) (Agilent, Palo Alto, CA). SPEH determination – A C18 Reversed-Phase column (4.6 x 250 mm, 300 A, 5µ, DIONEX, I) was used as stationary phase and a combination of water and acetonitrile at the ratio of 90 to 10, both acidified with 0.05% phosphoric acid, was used as mobile phase. The flow rate was set at 1.8 mL/min; the injection volume was 20 µL; the wavelength was 208 nm; the column temperature was set at 40°C. The retention time of SPEH was about 5.4 min. A calibration curve was generated in the 10-500 µg/mL range with a correlation coefficient of 0.99997. Lidocaine determination - The analyses were performed in the following experimental conditions: column: 5 µm C-8, 150 mm x 4.6 mm (Supelco, USA); mobile phase: acetonitrile/water (adjusted to pH 2.5 by 85% ortophosphoric acid) (50/50 v/v); flow rate: 1 ml/min; UV detector: 224 nm; 8 ACS Paragon Plus Environment

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injection volume: 20 µl; temperature: 20°C. The retention time was about 5.0 min. A calibration curve from 0.2 to 50 µg/ml was calculated based on peak area measurements. It was generated with a correlation coefficient of 0.99990. Propranolol determination - A reverse-phase column was used as stationary phase (Bondclone C18, 10 µm, 3.9 x 300 mm, Phenomenex, USA) and the mobile phase was a mixture of acetonitrile/0.2% v/v phosphoric acid at 40/60 v/v ratio. The flow rate was set at 1.2 mL/min. The injection volume was 20 µL. The retention times of propranolol was 8 min. The drug concentration was determined from a standard calibration curve in the 0.2-20 µg/mL range (correlation coefficient: 0.99996). UFH determination- UFH concentration was determined by measuring the anti-factor Xa activity using the Coatest® kit (Chromogenix, Instrumentation Laboratory S.p.A., I). The method relies on the ability of UFH-antitrombin III (AT) complex to react with an excess of factor Xa (FXa). Unreacted FXa is hydrolyzed by a chromogenic substrate and read spectrophotometrically at 405 nm. The absorbance is related to UFH or UFH-peptide conjugate concentration by an exponential relationship. The compound concentrations were estimated by calibration curves built in the following ranges: UFH 0.03-0.50 µg/mL; SPEH-UFH1 and SPEH-UFH2 0.15-5.00 µg/mL.

Fourier Transform Infrared (FTIR) Spectroscopy. ATR-FTIR spectra were recorded between 4,000 and 450 cm−1 using a Bruker Alpha-P spectrometer (128 scans; 4 cm−1 resolution), equipped with an attenuated total reflection diamond crystal accessory (Bruker Optics Inc., Germany). Human epidermis was soaked in demineralized water or in a solution of the peptide (0.25 or 0.5 mg/mL) in water for 1 h, then allowed to dry at room temperature. The spectra were recorded on epidermis sheets incubated with and without the tested compound, thus, each epidermis sample acted as its own control at each time. All measurements were performed at room temperature (25 ± 2°C). The spectrum was automatically corrected by the ATR correction function of OPUS software (Bruker Optics Inc., Germany) and analyzed by Origin Pro (Origin Lab, USA). The maximum 9 ACS Paragon Plus Environment

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absorbance of peaks in the 2950 cm−1 and 2800 cm−1 region was assigned by second derivative. Fourier self-deconvolution of the overlapping band components of CHx stretching bands in the 2950 cm−1 and 2800 cm−1 region as well as the hidden peaks of amide I bands in the 1690-1590 cm−1 region were resolved by the second-order derivative with respect to the wavelength after smoothing with a Savitsky−Golay function. Deconvolution was performed using Gaussian line shape. A nonlinear least-square method was used to take the reconstituted curve as close as possible to the original deconvoluted spectra. The fitting results were further evaluated by examining the residual from the differences between the fitted and the original curve and accepted when the regression coefficient was higher than 0.9998. The presented data are the means of determinations performed at least on four different donors.

Molecular dynamic simulation. The analysis of the putative interactions with keratin was performed by following a computational strategy similar to that utilized by Kumar et al.17 In detail, the here proposed peptide was compared with two known peptides included in the above mentioned study, namely SPACE and DLP, taken as representative of good and poor keratin binders, respectively. The initial conformation of the four simulated peptides was generated by FUGUE, an online server which produces reliable results even for such short peptides. After adding side-chains and hydrogen atoms, the obtained models were minimized keeping the backbone atoms fixed to preserve the predicted folding. The resolved structure of keratin (PDB Id: 3TNU) was completed by adding hydrogen atoms and minimized by keeping the backbone atoms fixed to preserve the experimentally resolved folding. Docking simulations were then carried out by using PLANTS, a program which produces reliable ligand poses by ant colony optimization algorithms. To optimize the obtained results the entire protein structure was involved in docking searches, while the peptides were considered as rigid due to their large sizes and to speed up the simulations. For each ligand, 20 poses were generated and scored by the ChemPLP scoring function using a speed equal to 1. The so

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obtained best complexes were finally minimized by keeping all atoms fixed outside a 20 Å radius sphere around the bound peptide and utilized to calculate the docking scores. Similarly, the analysis of the interaction capacity with the hydrophobic components of the skin was performed by exploiting a computational approach recently proposed by us and based on a basket composed of four CER 6 molecules symmetrically arranged to define a rather hydrophobic internal cavity. It should be emphasized here that such a basket was not designed to reproduce the fine architecture of the human epidermis, but to generate a simplified host structure by which the ceramide-peptide interactions can be simulated at an atomic level. The three above mentioned peptides in their previously generated best conformations underwent docking simulations with the basket structure using PLANTS with the same docking parameters above described.

Cell culture and cytotoxicity assessment. Two commercially available human cell lines , namely the keratinocyte cell line NCTC2544 and the promyelocytic cell line THP-1, were used to evaluate the tolerability of the synthetic peptide. Lactate dehydrogenase (LDH) leakage and MTT reduction were used to assess cell viability. LDH is a well-known indicator of cell membrane integrity and cell viability, while the MTT assay measures the enzymatic conversion of the vital dye MTT (3(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide), into a blue formazan salt that is quantitatively measured after extraction from cells18. In details, THP-1 cells, obtained from Istituto Zooprofilattico di Brescia (Italy), were diluted to 106 cells/mL in RPMI 1640 containing 2 mM L-glutamine, 0.1 mg/mL streptomycin, 100 IU/mL penicillin, 50 µM 2-mercaptoethanol, supplemented with 10 % heated-inactivated foetal calf serum (culture media) and cultured at 37°C in 5% CO2 incubator. To assess cytotoxicity 0.1 ml of cell suspension was seeded in 96-well plate and 0.1 ml of increasing concentrations (0.0012-1.25 mg/ml) of peptide diluted in culture media were added for 24 h. Each concentration was tested in triplicate (n= 3 wells). At the end of the incubation time, plates were centrifuged at 800 rpm for 5 min and supernatants transferred to a new 96-well plate for LDH assessment. 100 µl/well of MTT 11 ACS Paragon Plus Environment

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solution 0.75 mg/ml in culture medium was added to cells. Cells were incubated for 3 h at 37 °C, plates were then centrifuged, culture medium was discard and cells lysed in 100 µl/well of a mixture of HCl 1 N and isopropanol (1:24). The absorbance of the resulting solutions was read at a wavelength of 595 nm in a microplate reader (Molecular Devices). Results are expressed as % of viable cells compared to control cells. LDH activity was determined in cell-free supernatants using a commercially available kit (Takara Bio Inc., Japan). Results are expressed as % of LDH leakaged from control cells. NCTC 2544 cells (Istituto Zooprofilattico di Brescia, Brescia, Italy) were cultured in RPMI 1640 containing 2 mM L-glutamine, 0.1 mg/mL streptomycin, 100 IU/mL penicillin, 10 µg/ml gentamycin supplemented with 10% heated-inactivated foetal calf serum (medium) and cultured at 37°C in 5% CO2 incubator. Cells were seeded in 96-well plate at a cell density of 5 x 105/ml (0.1 ml/well in 96-well plate). After overnight adherence, cells were exposed to increasing concentrations of the peptide (0.0012-1.25 mg/ml) dissolved in medium. Each concentration was tested in quadruplicate (n= 4 wells). After incubation medium was collected for the measurement of LDH leakage, and 100 µl/well of MTT solution 0.75 mg/ml in medium was added. Cells were incubated for 3 h at 37 °C, medium was discard and cells lysed in 100 µl/well of a mixture of HCl 1 N and isopropanol (1:24). The absorbance of the resulting solutions was read at a wavelength of 595 nm in a microplate reader (Molecular Devices). Results are expressed as % of viable cells compared to control cells. LDH activity was determined in cell-free supernatants using a commercially available kit (Takara Bio Inc., Japan). Results are expressed as % of LDH leakaged from control cells.

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RESULTS

Identification of Skin Penetrating Peptide. After the two rounds of screening by in vitro phage display, the 7-mer peptide that consistently diffused through the human epidermis resulted to be DRTTLTN. Indeed, the DNA sequencing revealed that 66% phages expressed such peptide on their surface. The identified sequence (skin penetration enhancer heptapeptide, SPEH) was thus chemically synthesized by microwave assisted solid phase peptide synthesis and acetylated at the N-terminus using protocols previously optimized15.

Figure 1 - CD spectra of SPEH

The CD spectrum, shown in Figure 1, is characterized by the presence of a minimum at 198 nm and a negative shoulder around 220 nm, indicating that the peptide did not possess a preferred conformation even if the different secondary structures (i.e. 310 helix and β) were present.

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The presence of a β-structure was outlined also by ATR-FTIR analysis since a peak at 1635 cm-1 in the amide I region was detected, together with a shoulder at around 1663 cm-1 that indicated also a 310 helix contribution (Figure 2).

Figure 2 - ATR-FTIR spectra of UFH, SPEH and UFH-SPEH conjugate.

As depicted in Figure 3, SPEH was able to penetrate into the skin even when removed from the phage, regardless of its concentration. Indeed, the SPEH resulted detectable in the receiver medium after 30 min, even if it resulted quantifiable only at the highest concentration. Furthermore, when 5 mg/mL peptide solution was applied on the epidermal sheets, the flux was 59.32 ± 15.22 µg/cm2/h, about 5-times higher than that obtained with the lowest peptide concentration (8.73±1.27 µg/cm2/h). In the latter case, no significant differences were observed comparing the permeation profiles through epidermal or isolated stratum corneum sheets, suggesting that the epidermis layer does not influence the permeation of SPEH or, in other word, the viable epidermis layer does not represent a 14 ACS Paragon Plus Environment

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limiting barrier for SPEH diffusion. Moreover, the permeability properties of SPEH resulted to be sequence-specific since when a scrambled peptide (LDTNTRT) was synthetized and tested (at the concentration of 0.5 mg/mL, n=4), the permeant was detected in the receiver compartment only in one diffusion cell after 24 hours.

Figure 3 - Skin permeation profiles a) 5 mg/mL SPEH through human epidermis (○); 0.5 mg/mL SPEH through human epidermis (●) or stratum corneum (▲).

The analyses of ATR-FTIR spectra revealed that SPEH determined some alterations of spatial organization of lipidic lamellae in stratum corneum. As reported in Table 2, the second derivative of the CHx stretching region (2988-2828 cm-1) in the ATR-FTIR region evidenced four different peaks of which the asymmetric (υasymCH2) and symmetric (υsymCH2) stretching bands centred at about 2920 and 2850 cm-1 are the most relevant. In the case of the epidermis sample treated with 0.5 mg/mL SPEH, these bands shifted to higher wavenumbers (p