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Selective Peptide-Mediated Enhanced Deposition of Polymer Fragrance Delivery Systems on Human Hair Kemal Arda Günay, Damien Loïc Berthier, Huda A Jerri, Daniel Benczédi, Harm-Anton Klok, and Andreas Herrmann ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06569 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 27, 2017

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ACS Applied Materials & Interfaces

Selective Peptide-Mediated Enhanced Deposition of Polymer Fragrance Delivery Systems on Human Hair Kemal Arda Günay,1 Damien L. Berthier,2 Huda A. Jerri,3 Daniel Benczédi,2 Harm-Anton Klok1* and Andreas Herrmann2* 1. École Polytechnique Fédérale de Lausanne (EPFL), Institut des Matériaux and Institut des Sciences et Ingénierie Chimiques, Laboratoire des Polymères, Bâtiment MXD, Station 12, CH1015 Lausanne, Switzerland 2. Firmenich SA, Division Recherche & Développement, Route des Jeunes 1, CH-1211 Genève, Switzerland 3. Firmenich Inc., R&D Division, 250 Plainsboro Road, Plainsboro NJ 08536, USA

CORRESPONDING AUTHOR FOOTNOTE [email protected], ORCID: 0000-00033365-6543, Tel: + 41 21 693 4866; [email protected], ORCID: 0000-00016997-3458, Fax: + 41 22 780 3334, Tel: + 41 22 780 3474

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Abstract The deposition of fragrance delivery systems onto human hair from a shampoo formulation is a challenging task, as the primary function of shampoo is to cleanse the hair by removing primarily hydrophobic moieties. In this work, to tackle this challenge, phage display identified peptides that can bind to human hair under shampooing conditions are first identified and subsequently used to enhance the deposition of model fragrance delivery systems. These delivery systems are either based on poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) copolymers as a representative for polymeric profragrances or polyurethane/urea-type core-shell microcapsules as a model physical fragrance carrier. The incorporation of a hair binding peptide enhanced the deposition of PHPMA copolymers with a factor of 3.5-5.0 depending on the extent of peptide incorporation, whereas 10 w/w% surface functionalization of microcapsules with the peptide led to a 20-fold increase in their deposition. In a final experiment, treatment of the hair samples under realistic application conditions with the peptide-functionalized microcapsules resulted in an increase fragrance release from the hair surfaces. Keywords: Surface deposition, fragrance delivery, functional polymers, post-polymerization modification, microcapsules, bioconjugation, phage display.


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INTRODUCTION The design of fragrance delivery systems that can efficiently deposit from a personal care product onto e.g. hair or skin to provide a long lasting perception of the volatiles is a challenging task. First of all, fragrances are highly volatile compounds that easily evaporate during application.1-3 Second, many fragrances, such as aldehydes, are prone to oxidation under ambient conditions, which can lead to a rapid loss of their activity and may also generate degradation products that can act as contact allergens.4,5 Some of these challenges can be addressed using polymer profragrances or polymer nanoparticles or microcapsules as fragrance delivery systems.6-10 The use of profragrances in which the volatile is attached to a polymer carrier via a pH, temperature or light-sensitive labile bond7,8 or the encapsulation of fragrances in polymer nano- or micrpcapsules can prevent degradation of the active compound and avoid premature evaporation and allow controlled release.9,10

While much of the work that has been done on polymer-based fragrance delivery systems has addressed issues related to the volatility, stability and controlled release of fragrances, the deposition of these systems on the surface of interest is another important parameter that determines product performance. To optimize the olfactive impact and benefits of fragrance delivery systems, deposition and substantivity after rinsing must be maximized. Fragrances are among the most expensive materials formulated into many consumer products and while encapsulation can provide chemical stability and allows to control the release of the precious and volatile oil payload, tuning the interactions of these delivery systems with the substrate of interest is equally important. Enhancing surface deposition will reduce the amount of unadhered fragrance oil payload, which is otherwise washed away. In particular, the efficient deposition of



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fragrances and fragrance delivery systems from a shampoo medium onto hair is a challenging task. This is due to the specific functional and physio-chemical characteristics of shampoos. The primary function of a shampoo is to cleanse the hair surface11-13 by removing hydrophobic molecules such as non-covalently bound lipids14 and dust particles. The removal of these hydrophobic molecules is typically achieved using anionic surfactants, as they can effectively entrap these molecules by forming micelles. These micelles can be easily rinsed off with water owing to the electrostatic repulsions between the anionic head group of the surfactants and the hair surface.15 Since most fragrances are hydrophobic, they will predominantly localize inside the micelles rather than in the aqueous phase.16 Consequently, they are rinsed off together with these surfactants. Enhancing the affinity for and deposition of the fragrance delivery systems on the substrate of interest could reduce this loss and contribute to a longer lasting perception.

Shampoo formulations are often comprised of a cationic cellulose derivative or a quaternary ammonium functionalized copolymer (polyquaternium material) and an anionic surfactant, which via non-specific electrostatic interactions deposit on hair as a coacervate during rinsing upon dilution below the critical micelle concentration (CMC). The deposition of these coacervates can reduce combing friction, improve the appearance of the condition of hair and has also been used to enhance the delivery of silicones.17-24 This manuscript will present a conceptually alternative approach to enhance surface deposition of polymer-based fragrance delivery systems on hair. Instead of relying on non-specific electrostatic interactions as in the case of coacervate mediated surface deposition, the strategy presented here involves the use of short, cyclic peptide ligands, which have been selected via phage display and are able to bind to hair in shampoo formulations. It will be shown that incorporation of these peptide ligands in a model polymer profragrance (Figure 1A) or decoration of polymer microcapsules with these peptides (Figure 1B) enhances 4 ACS Paragon Plus Environment

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the deposition on hair of these delivery systems. In a recent report, we have demonstrated the feasibility of this strategy to enhance the deposition of fragrance delivery systems from a laundry softener formulation onto cotton.25 While laundry softener formations are designed to enhance surface deposition, shampoo serves to remove matter from hair, i.e. prevent deposition. As a consequence, enhancing or improving surface deposition from shampoo is a challenge that is fundamentally different (and more complicated) as compared to improving deposition from laundry softener on cotton fabric. This manuscript consists of three parts. The first part will describe the identification of hair binding cyclic peptide disulfides via phage display under shampoo conditions. After that, the feasibility of these peptides to enhance the deposition of two model, polymer-based fragrance delivery systems, viz. polymeric profragrances and polymer microcapsules, will be explored. These two technologies are complementary and depending on the particular circumstances found in application either one is used. The two systems represent the two major approaches to achieve fragrance long-lastingness in application. Typically, if the aim is to pronounce one particular note of a perfume, the profragrance approach is chosen. If the objective is to deliver a more complex perfume an encapsulation system would be preferable. Not all systems work under all circumstances, e.g. stability or dispersibility in formulation might be an issue for preferring either one of the systems. As a first example, the peptides will be used to construct poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA)-peptide conjugate based model polymer profragrances. Fluorescence intensity measurements revealed that the incorporation of small amounts of the phage display identified peptides enhances the deposition of these polymers 3.5 - 5.0 fold onto hair under shampoo conditions. As a second example, peptide-functionalized polyurethane/urea-type core-shell microcapsules containing a model perfume will be presented. The peptide functionalization of these microcapsules results in approximately a 10-fold enhancement of their deposition onto human hair according to HPLC measurements. Finally, it 5 ACS Paragon Plus Environment


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will be shown via dynamic headspace sampling measurements that the amount of fragrance released from peptide functionalized microcapsules is significantly higher than that of nonfunctionalized control microcapsules as a result of increased deposition of the former.

Figure 1: Peptide (cyclo(CNHQHQKGC))-mediated deposition of (A) PHPMA-based model polymer profragrances and (B) core-shell microcapsules on hair.

EXPERIMENTAL MATERIALS All chemicals were used as received unless otherwise described. The preparation of pentafluorophenyl methacrylate (PFMA),26 reversible addition-fragmentation chain transfer (RAFT) polymerization of PFMA27 as well as the removal of the dithioester end-groups of the synthesized PPFMA polymer28 (Mn,NMR = 42000 g/mol, DP = 167 and Mw/Mn = 1.20) were performed according to previously described protocols. Chloramphenicol was received from BioChimica. Tween® 20, bovine serum albumin (BSA), 1-amino-2-propanol (93.0%), dansyl 6 ACS Paragon Plus Environment

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cadaverine (> 97.0%), propargylamine (97%), triethylamine (TEA) (97.0%), piperidine (99.0%), trifluoroacetic acid (TFA) (≥ 99.0%) N-methyl-2-pyrrolidone (NMP) (≥ 98.0%), N,Ndiisopropylethylamine

(DIEA)

(98.0%),

triisopropylsilane

(TIS)

(99%),

tris(3-

hydroxypropyltriazolylmethyl)amine (THPTA) (95%), aminoguanidine hydrochloride (99%), guanidine carbonate (99%), CuSO4·5H2O (97%), sodium ascorbate (NaAsc) (98%), dansyl chloride (98%) and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) (≥ 97%) were received from Sigma Aldrich. N-hydroxysuccinimide (NHS) was obtained from Alfa Aesar. ULTRA-TMB ELISA solution was received from Thermo Scientific. Anti-M13-HRP monoclonal antibody was received from Abcam. All Fmoc-amino acids except Sacetaminomethyl (Acm) protected cysteine (Fmoc-Cys(Acm)), Oxyma-Pure and Fmoc Rink Amide resin were received from Iris Biotech. Fmoc-Cys(Acm) (99%) and 5-azidopentanoic acid (5-AzPOH) (97%) were obtained from Bachem. N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1yl)uronium hexafluorophosphate (HBTU) (98%) was received from Fluorochem. Thallium trifluoroacetate (Tl(CF3COO)3) (97%) was obtained from TCI. Texapon® N70 NA and Salcare® SC 60 were obtained from BASF. Mirataine® BET C-30 was obtained from Solvia. Takenate® D110N was obtained from Mitsui Chemicals. Poly(vinyl alcohol) KC506 was obtained from Kuraray. Acetonitrile (AcN) (HPLC grade) was obtained from VWR international. Dansyl Novatag resin was received from Merck Millipore. Tenax® TA (poly(2,6-diphenyl-p-phenylene oxide), 35-60 Mesh) was obtained from Agilent Technologies. Cartridges used for headspace sampling were filled with 100 mg of the polymer. Cyclic peptide disulfide Pep1-COOH (≥ 95.0%) was received from ChinaPeptides. The synthesis of the N-terminal azide functionalized peptide N3-Pep1 was carried out using a previously published protocol.29



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A model shampoo formulation for the phage display experiments was prepared by adding sodium lauryl ether sulfate (SLES, Texapon® N70 NA, 12.0 g) and cocamidopropylbetaine (CAPB, Mirataine® BET C-30, 3.0 g) to a citrate buffer (50 mM, 1 L). The final pH was adjusted to 6.0 by slowly adding 0.1 M NaOH solution. For the testing of functionalized microcapsules, a model cleanser was formulated with SLES (17.2 g), CAPB (10.0 g), Salcare® SC 60 (1 w/w% in water, 50.0 g) and deionized water (22.8 g), which was meant to be a simplification of typical shampoo and shower gel cleansing formulations. The pH was adjusted with a citric acid solution (50%) to 5.5. Net untreated brown Caucasian hair swatches (0.5 g, 10 cm) with an average diameter of 0.4 mm were purchased from International Hair Importers and used as received. Affinity selections were carried out using a combinatorial phage library displaying 9-mer cyclic peptide disulfides, with 7 amino acid moieties between two cysteine (C) residues, the latter of which close the ring by forming a disulfide link (C7C). These phage libraries were a gift from Prof. Christian Heinis and their construction was described in a previous publication.30

METHODS Fluorescence intensity and absorbance measurements were performed with a Tecan Infinite Pro 200 reader. Fmoc solid phase peptide synthesis was carried out using a CEM Liberty automated microwave synthesizer. HPLC was performed on a Shimadzu Prominence system containing LC20AP pumps, a FRC-10A fraction collector, a CTD-20AC column oven and a SPD-M20A diode array detector coupled to a LCMS-2020 liquid chromatography mass spectrometer. The peptides were characterized using an analytical Grace Vydac 218TP54 C18 column and purified using a Grace Vydac 218TP15 preparative column. 1H-NMR spectra were recorded on a Bruker (ARX400) 400 MHz spectrometer at room temperature using a relaxation time (t1) of 10 s. Chemical shifts are reported relative to the residual proton signal of the solvent. Scanning electron 8 ACS Paragon Plus Environment

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microscopy (SEM) was performed on a JEOL 6010-LA instrument with an accelerating voltage of 4 kV, a working distance of 10 mm and a spot size of 40. 500 mg Hair swatches were prepared using the deposition protocol. After dosing and rinsing the model surfactant mixture (loaded with microcapsules C2), the hair was left to air dry. Treated hairs were then cut and mounted onto aluminum stubs using carbon tape. The samples were sputter-coated with a gold-palladium plasma for 45 s to facilitate imaging of non-conductive samples. PROCEDURES Identification of hair binding peptides under shampoo conditions by phage display: A combinatorial C7C phage library was produced via incubation of chloramphenicol resistant Escherichia coli (E. coli) TG1 cell lines having an optical density (OD) of 0.1 in 500 mL 2YT media containing 0.1 mg.mL-1 chloramphenicol at 30 °C. After 16 h, the bacteria pellet was precipitated by centrifugation and the supernatant, which contained the phages, was mixed with 190 mL of 2.5 M aqueous NaCl solution containing 20 w/w% of polyethylene glycol (PEG). The phages were isolated by centrifugation and subsequently resuspended in 8 mL of the shampoo formulation. The concentration of the input phages was approximately 2 x 1012 plaque forming units pfu.mL-1 in the beginning of each round of selection. Knotted hair samples (9 - 11 mg) were prewashed once with H2O/isopropanol 16 : 1 v/v% and three times with MilliQ water and allowed to dry for 2 h prior to incubation with the phage solution. Next, the hair samples were incubated with 8 mL of the shampoo formulations containing the phages for 20 min at 37 °C with gentle agitation in falcon tubes. Then, the hair samples were removed from the falcon tubes, inserted into new tubes and washed 8 times either with 10 mL tris-buffered saline solution (TBS) (pH 7.4) containing 0.1 v/v% Tween® 20 at each round of selection (Experiment #1) or with TBS buffer containing 0.1, 0.2, 0.3, 0.4 and 0.5 v/v% 9 ACS Paragon Plus Environment


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Tween® 20 in the 1st, 2nd, 3rd, 4th and 5th round of selection (Experiment #2), respectively, to remove weakly bound phages. The hair samples were washed twice more with 10 mL TBS solution that did not contain any Tween® 20, removed from the falcon tubes and dried. Each washing step was performed for 5 min with gentle agitation and the samples were transferred into new falcon tubes four times throughout the 10 washing steps in order to minimize the selection of background binders associated with the polystyrene tube. Following washing, the infection step was performed by directly immersing the hair samples into 30 mL 2YT medium containing fresh TG1 cells with an OD of 0.4. The infection was allowed to continue for 90 min at 37 °C. Following infection, the solutions were centrifuged, the supernatants were discarded and the bacterial pellets were redissolved in 1 mL 2YT and subsequently plated into chloramphenicol containing agar plates. The plates were incubated overnight at 37 °C and scraped off to recover the infected bacteria. Aliquots of solutions were taken following the infection step to determine the output phage titers after each selection round. In total, 2 independent affinity selection experiments were performed up to the 5th round, which were are referred to as Experiment 1 and Experiment 2 depending on whether washing condition #1 or #2 (vide supra), was used. The evolution of the output phage titers with respect to number of selection rounds in these experiments is summarized in Figure S1. Individual colonies of phage infected TG1 cells were picked from the 3rd, 4th and 5th rounds of selection from these 2 experiments, their plasmids were extracted using a QIAGEN spin Miniprep kit and these plasmids were subsequently sent to sequencing analysis. Phage enzyme-linked immunosorbent assay (ELISA): The individual phages, which were identified as potentially affine hair binders and the combinatorial C7C phage library were produced similar to the protocol described in the previous section. The phages were resuspended 10 ACS Paragon Plus Environment

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in 2 mL model shampoo formulation and the initial concentrations (input titer) of the phages were calculated from the average of three independent phage titrations. Before incubation, the surfaces of 1.5 mL polystyrene Eppendorf tubes were blocked with 500 µL of a phosphate-buffered saline solution (pH 7.4) containing 0.1% Tween® 20 (PBST 0.1%) and 2% BSA for 30 min. Next, the wells were washed three times with 500 µL MilliQ water and air dried. Prewashed hair samples (mass = 9 - 11 mg) were introduced to the wells and 250 µL model shampoo formulation with phage concentrations ranging from 2 x 109 to 5 x 1012 pfu.mL-1 was added. Phage ELISA experiments were carried out using both the individual phages that were identified as potentially affine binders as well as with the combinatorial phage library, which was used as a reference. In addition to these experiments, blank measurements were also performed by incubating the hair samples with the model shampoo formulation that did not contain any phage. Incubations were carried out for 1 h at 37 °C and the hair samples were washed three times with 250 µL PBST 0.1% buffer (pH 7.4) and once more with PBS (pH 7.4) (washing cycle). Another blocking step was performed by introducing 250 µL PBST 0.1% buffer containing 2 w/w% of BSA and the residual BSA was washed off by subsequently performing another washing cycle. Following the second washing cycle, the samples were incubated with 250 µL PBST 0.1% buffer containing 2% BSA as well as 1/5000 dilution of a 40 µg.mL-1 stock solution of Anti-M13-HRP antibody for 1 h and another washing cycle was performed to remove the unbound antibody. The hair samples were allowed to dry and subsequently transferred into new Eppendorf tubes. 250 µL of an ULTRA-TMB solution was added to these tubes, which led to the evolution of a colorimetric response. The evolution of the colorimetric response was quenched by adding 250 µL 2 M H2SO4 after 15 min. 200 µL fractions of the quenched solutions were transferred into a 96-well plate and the absorbance in the wells was measured at 450 nm. The normalized absorbances associated with the amount of hair-bound phage were calculated by taking the difference between the 11 ACS Paragon Plus Environment


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absorbance values obtained from the sample and the blank measurements. All of the phage ELISA experiments were performed three times. The apparent binding strengths of the phages were calculated using Serizawa’s method by fitting the normalized absorbance as a function of phage concentration according to a Langmuir-type adsorption model.31 Synthesis of cyclic peptide disulfides Dansyl-Pep1, Dansyl-Pepx and Pep1-Dansyl: General solid-phase peptide synthesis (SPPS) protocol: N-terminal dansylated peptides Dansyl-Pep1 and Dansyl-Pepx were prepared on a Rink-amide resin with a loading capacity of 0.71 mmol/g on a 0.25 mmol (0.352 g) scale. A dansyl Novatag resin (loading capacity 0.44 mmol/g, 0.10 mmol scale) was employed for the preparation of C-terminal dansylated peptide Pep1-Dansyl. Deprotection of Fmoc groups, coupling with the amino acid and washing was performed before and after the incorporation of each amino acid. A two-step Fmoc deprotection was performed at 75 °C using 40 W microwave power for 30 s and 54 W microwave power for 6 min, respectively, with 5 mL of a DMF/piperidine = 4 : 1 solution containing 0.1 M Oxyma pure. Coupling of the amino acids was carried out by subsequent addition of amino acids (0.2 M) in DMF (5 mL), HBTU (0.5 M) and Oxyma-Pure in DMF (2 mL, activator) and DIEA (2 M) in NMP (1 mL, activator base) for 6 min using a microwave power of 24 W (T = 60 °C). Washing of the resin before and after the addition of each amino acid was performed using DMF (10 mL). Following the SPPS, the resin was transferred to a reaction vessel, consecutively washed with 50 mL of DMF, DCM, MeOH and DCM and dried under a flow of N2. Peptide cyclization: Peptide cyclization was performed according to the protocol described by Andresen and coworkers.32 Briefly, cyclization was carried out by first swelling the resin in anhydrous DMF (2.5 mL) under N2. In parallel, 2 equiv. Tl(CF3COO)3 with respect to the resin bound peptide (0.5 and 0.2 mmol for Rink-amide and Dansyl Novatag resin, respectively) was 12 ACS Paragon Plus Environment

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dissolved in anhydrous DMF (2.5 mL) under a flow of N2 and this solution was cannula transferred to the reaction vessel. The reaction was allowed to continue for 2 h at room temperature with gentle agitation. Following this first step, the resin was washed extensively with DMF and once more with DCM to remove residual thallium ions and dried under a flow of N2. N-terminal dansyl functionalization: Dansyl chloride (269 mg, 1.00 mmol, 4 equiv. with respect to Rink-amide bound peptide) was dissolved in anhydrous DMF (5 mL) in a round bottom flask and subsequently DIEA (348 µL, 2.00 mmol, 8 equiv. with respect to Rink-amide bound peptide) was added under a flow of N2. In parallel, the resin (0.25 mmol peptide) was swollen in anhydrous DMF (5 mL) and the mixture that contained the dansyl chloride and DIEA was transferred to the reaction vessel via a cannula under a flow of N2. The reaction was allowed to continue for 2 h at room temperature with gentle agitation. In the final step, the resin bound peptides were washed with DMF (3 x 50 mL) and once more with DCM (50 mL) and dried. Cleavage and purification: Cleavage of the peptides from the resin was achieved by treating the resin with 5 mL of a solution of TFA/TIS/H2O = 95 : 2.5 : 2.5 v/v% for 2 h at room temperature under gentle agitation. The cleaved peptides were precipitated in Et2O 4 times, dissolved in H2O (20 mL) and lyophilized. Following the lyophilization, the peptides were purified via preparative HPLC using a gradient from 95% H2O / 5% AcN (0.05% TFA) to 65% H2O / 35% AcN (0.05% TFA) in 30 min and the pure fractions were collected and lyophilized. The yields were between 9 - 27%. The purities of Dansyl-Pep1 and Dansyl-Pepx were greater than 95%, and the purity of Pep1-Dansyl was 82%. Analytical HPLC elution profiles as well as the corresponding ESI-MS spectra of the purified peptides are provided in Figure S2.



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Synthesis of PHPMA-peptide conjugates: PHPMA-peptide conjugates P1Pep1 (yield = 90%) and P2Pep1 (yield = 81%) as well as the alkyne modified PHPMA PC (yield = 63%) were prepared according to a literature procedure from a PPFMA precursor with a Mn,NMR = 42000 g/mol (DP = 167).29 1H-NMR spectra of the polymers are provided in Figure S3, S4 and S5. Surface deposition measurements of polymer-peptide conjugates and dansylated peptides: The same protocol was used to assess the surface deposition of the PHPMA-peptide conjugates and dansylated peptides, except for two differences. First, while the polymer-peptide conjugates were investigated over a range of concentrations varying from 0.01 – 0.2 mg.mL-1, 0.5 to 15.0 µM of the peptides were incubated with hair samples to assess their extent of deposition. Secondly, apart from shampoo conditions, the extent of deposition of the peptides onto hair was also assessed in 50 mM citrate buffer at pH 6.0 without the presence of SLES and CAPB in order to determine the influence of the stringency of the shampoo medium on the binding of the peptides. For the surface deposition experiments, first prewashed hair samples (9 - 11 mg) were inserted into 1.5 mL polystyrene Eppendorf tubes. Then, 500 µL of a model shampoo formulation or citrate buffer solution containing varying concentrations of PHPMA-peptide conjugates or dansylated peptides were introduced to the tubes. In parallel, the same solutions were also introduced into tubes that did not contain any hair (blank measurements). The incubation was carried out for 1 h at 37 °C with gentle agitation. Following incubation, 100 µL of the solutions were transferred into a 96-well plate. The solutions were diluted 3-fold with model shampoo formulation or citrate buffer solution and the fluorescence intensities of the samples (FIhair) were recorded at λem = 530 nm using λex = 335 nm. Separately, the fluorescence intensity of the solutions introduced to the wells that do not contain any cotton (FIblank) were also diluted 3-fold 14 ACS Paragon Plus Environment

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in a new 96-well plate and measured. The percentage deposition of the conjugates at each concentration was calculated using the following equation: % Deposition = (FIblank – FIhair) / (FIblank) x 100. Figures S6 and S7 show the % deposition as a function of peptide concentration for the dansylated peptides and for different polymer conjugates, respectively. Fluorescence intensity measurements of polymer-peptide conjugates were performed 9 times, whereas the measurements carried out using the dansylated peptides were repeated 6 times. Preparation of polyurethane/urea core-shell microcapsules (C0): In a beaker, a mixture of a model perfume (consisting of Romascone®, Verdox®, Vertenex, (Z)-2-Phenylhex-2-enenitrile and Cyclamen aldehyde, 7.60 g each for a total of 38.00 g, Chart 1), a UV-tracer (Uvinul A+, 2.00 g) and an isocyanate (Takenate® D-110N, 5.10 g, 12 mmol NCO, Chart S1) was added to an aqueous solution (49.00 g) of poly(vinyl alcohol) KC506 at 2 w/w % in water and stirred with an Ultra-Turrax at 24000 rpm for 4 min. The pH of the resulting emulsion was adjusted to about 10.5 - 11.0 with a solution of sodium hydroxide (30 w/w%). The emulsion was then transferred into a Schmizo reactor (250 mL) equipped with an anchor and a mechanical stirrer and stirred at 350 rpm. Then, a solution of guanidine carbonate (0.90 g, 20 mmol NH2) in water (5.00 g) was added dropwise at room temperature during 1 h. After that, the reaction mixture was heated to 70°C during 1 h and kept at the same temperature for 2 h to afford C0 (average diameter: 10 µm, solid content: 45.7 w/w%, amount of encapsulated model perfume in the dispersion: 37.0 w/w%, in the capsule: 80.9 w/w%).



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Chart 1: Structures of the ingredients of the model perfume.

Preparation of peptide-modified polyurethane/urea core-shell microcapsules C1 and C2: Microcapsules C0 were washed by decanting the capsule dispersion and removing the aqueous phase. The resulting dispersion was diluted with water (20 mL) twice and the aqueous phase removed each time. Finally, a washed dispersion of C0 containing 33.2 w/w% capsules was obtained (as shown by thermogravimetry). The washed dispersion of capsules C0 (5.80 g) was placed in a Schmizo reactor (250 mL), equipped with an anchor and a mechanical stirrer and diluted with water (80 g) at pH 10.50. Then, a solution of cyclic peptide Pep1-COOH (0.04 g = 1 w/w%), EDC (0.029 g, 0.19 mmol), NHS (0.017 g, 0.15 mmol) in water (14 g) prepared in a beaker at pH 5.02 was added and the reaction mixture stirred at room temperature for 24 h to afford a white dispersion. The obtained dispersion was decanted and the aqueous phase removed to afford C1 (solid content: 20.5 w/w%, amount of encapsulated model perfume in the dispersion: 16.6 w/w%). The HPLC elution profile of the residual aqueous phase of C1 dispersion did not reveal the presence of Pep1-COOH, which indicates quantitative incorporation of the peptide onto the surface of the capsules (Figure S8). 16 ACS Paragon Plus Environment

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Similarly, the washed dispersion of C0 (2.60 g), diluted with water (7.50 g) at pH 9.00, was modified as described above using cyclic peptide Pep1-COOH (0.17 g = 10 w/w%), EDC (0.13 g, 0.84 mmol), NHS (0.07 g, 0.61 mmol) to afford C2 as a white dispersion (solid content: 20.9 w/w%, amount of encapsulated model perfume: 16.9 w/w%). The HPLC elution profile of the aqueous phase of C2 dispersion revealed the presence of residual Pep1-COOH, which indicates that the majority of the peptide was incorporated onto the surface of the capsules (Figure S8). Characterization of the core-shell microcapsules: The solid content and amount of model fragrance

encapsulated

into

core-shell

microcapsule

dispersions

were

assessed

by

thermogravimetric analysis on a TGA/SDTA851e instrument (Mettler-Toledo) equipped with a microbalance having an accuracy of 1 µg and a 35 mL oven. Samples (ca. 12 mg) were introduced into an aluminum oxide crucible (70 µL) and their mass was measured as a function of temperature and time under a constant flow of nitrogen (20 mL.min-1). Measurements were carried out at temperatures from 25°C to 50°C, at a rate of 5°C.min-1, then at 50°C for 4 h (Figure S9). Stable core-shell microcapsules displayed characteristic plateaus corresponding to the mass of solid in the dispersion. Deposition of core-shell microcapsules on hair: Core-shell microcapsules C0, C1 and C2 were deposited onto human hair swatches from a shampoo cleanser application using the following test protocol (Figure S9). (a) Hair swatches (0.5 g) were wetted with warm tap water (39 °C) and (b) gently squeezed out. (c) The capsule-loaded (0.5 w/w%, encapsulated oil basis) model cleanser formulation (0.1 mL) was then applied and the surfactant mixture was distributed with (d) 10 horizontal and (e) 10 vertical passes. As a reference, and to track the mixture homogeneity and extraction efficiency of the microcapsules, an aliquot of each formulation applied to hair was loaded into a scintillation vial to serve as the 100% deposition control. (f) The hair swatch was



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then rinsed with tap water (100 mL) and the excess water was gently squeezed out. (g) For the quantification of the functionalized microcapsule deposition, the hair swatch was then cut into a pre-weighed 20 mL scintillation vial. The process illustrated in Figure S10 was repeated in triplicate and then the vials containing the cut hair were completely dried in a vacuum oven at 50 °C, weighed to determine the mass of the hair in the vials and finally prepared for solvent extraction of the tracer using 200 proof ethanol (4 mL) under sonication for 1 h. Controls were also prepared in triplicate. After sonication, the samples were filtered through a 0.45 µm PTFE filter and the extracted tracer masses for the applied and control samples were determined by HPLC using UV detection. The quantified tracer masses, extraction efficiency controls and measured mass of hair per swatch were then used to calculate the mass of deposited oil per gram of hair, which can also be reported as percentage of oil mass deposited on the hair swatches after rinsing. Dynamic headspace analysis: Dispersions of polyurethane/urea core-shell microcapsules C0 (0.13 g) or C2 (0.39 g) were added to the model cleanser formulation to make up for 10.01 g and to contain the same amount (0.48 w/w%) of model perfume in the final formulation. The samples were manually shaken for 10 min. The core-shell microcapules with the model perfume were then deposited onto the hair swatches following the protocol described above (steps (a)-(f), Figure S9). The uncut hair swatches were line-dried for 3.5 h (210 min) and analyzed by dynamic headspace sampling. For the dynamic headspace measurements one of the hair swatches was placed in a homemade headspace sampling cell (inner volume ca. 160 mL), which was thermostatted at 25 °C. A constant air flow (ca. 200 mL.min-1), which was filtered through activated charcoal and bubbled through a saturated solution of NaCl to ensure a constant humidity of the air of ca. 75%) was aspirated through the headspace sampling cell.33,34 To equilibrate the system, the volatiles evaporating from the hair surface were adsorbed onto a waste 18 ACS Paragon Plus Environment

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Tenax® cartridge for 130 min. Then, a clean Tenax® cartridge was placed in the system and the volatiles were collected over a period of 20 min. The end of the sampling corresponded to a total of 6 h of drying. The hair swatch was then placed back for line-drying and re-analyzed 24 h later (end of sampling) as described above (equilibrating for 130 min with a waste cartridge and sampling for 20 min on a clean cartridge). The waste cartridges were discarded and the clean cartridges thermally desorbed on a Perkin Elmer TurboMatrix 350 desorber coupled to an Agilent Technologies 7890A gas chromatograph equipped with a HP-1 capillary column (30 m, i.d. 0.25 mm, film 0.25 µm) and coupled to an Agilent Technologies 5975C inert MSD mass spectrometer. The volatiles were analyzed using a temperature gradient starting at 100 °C for 1 min, then going to 220 °C at 10 °C.min-1. Headspace concentrations (in ng.L-1 air) were obtained by external standard calibration using five different concentrations of the model perfume to be released in ethanol. Each calibration solution was injected onto a clean Tenax® cartridge, which was desorbed and analyzed under the same conditions.

RESULTS AND DISCUSSION Identification, synthesis and characterization of hair binding peptides. To identify peptide ligands that can be used to enhance the deposition of model polymer profragrances and microcapsules onto hair under shampoo conditions, the phage display technique was used. Phage display is a powerful in vitro selection technique that allows the identification of substrate selective, highly affine short peptide ligands.35-38 Phage display has already been used to identify hair binding peptide ligands in a hair conditioning medium.39 Hair conditioner and shampoo, however, have fundamentally different physiochemical characteristics. While conditioners are positively charged, shampoo media are negatively charged. Since phage



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display is very sensitive to the medium in which the selection experiments are performed and since it is known that small variations in the selection medium can lead to the identification of different peptides, phage display experiments were carried out in a model shampoo formulation. The identification of hair binding peptide sequences was carried out using a combinatorial phage library that displays 9-mer cyclic peptide disulfides (C7C).30 A cyclic peptide library was utilized rather than a linear one as it is known that conformationally constrained libraries often lead to the isolation of peptide binders with higher affinities compared to their linear counterparts.40,41 Selection experiments were carried out by incubating hair samples in a model shampoo formulation consisting of 50 mM citrate buffer (pH 6) containing 1.2 w/w% sodium lauryl ether sulfate (SLES) as the anionic surfactant and 0.3 w/w% cocamidopropylbetaine (CAPB) as the amphoteric foaming agent. Two independent phage display experiments were performed in parallel using two different washing conditions. For each of these experimental conditions, five of rounds of selection were performed. In the first experiment (referred to as Experiment #1), washing was performed using a surfactant (Tween® 20) concentration of 0.1 w/w% in each round. In the second experiment (Experiment #2), the washing stringency was gradually enhanced by stepwise increasing the surfactant concentration from 0.1 w/w% (Round 1) to 0.5 w/w% (Round 5). Output phage titers were measured after each round of selection in order to determine the enrichment of the phage library towards the hair samples. Figure S1 shows that the output phage titers gradually increase from ~ 106 pfu.mL-1 in the 1st round to 1-5 x 108 pfu.mL-1 in the 4th round of selection in both experiments, indicating a 102 - 103 fold enrichment of the phage library towards hair. In the 5th selection round, however, a slight decrease in the output phage titers in Experiment #2 was observed, illustrating the effect of the increased stringency of the washing step towards the enrichment of the phage libraries. Owing to this decrease in the output phage titers in the 5th round, the affinity selection experiments were not continued for a 6th 20 ACS Paragon Plus Environment

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round. Subsequently, 32 clones were picked from the phages that resulted from the 4th and 5th rounds of selection and subjected to sequencing analysis (Table S1). Table 1 lists the amino acid sequences as well as their relative frequency of occurrence in the selection of phage clones, which were investigated by sequence analysis. The identified sequences have in common that they are primarily composed of cationic (lysine (K), histidine (H)) and polar (asparagine (N), glutamine (Q) and serine (S)) amino acids with a net charge between +1.5 to +3.5 at the pH of the shampoo formulation used (6.0). As the hair surface is known to have a net negative charge,15 these results suggest that the recognition of the hair surface with these peptides predominantly relies on the electrostatic interactions.

Table 1: List of potentially affine hair binding peptides identified by phage display, their frequency of occurrence in the sequencing analysis, net charge at pH 6.0 as well as their apparent binding strength (Kapp) and maximum relative affinity (RAmax), which were calculated from phage ELISA experiments. Sequence ID

Sequence

Frequency / Total frequency

Net charge at pH 6.0

Kapp / 1010 M-1

RAmax

Seq1

CNHQHQKGC

10/32

+ 3.09

108

37.23

Seq2

CKSKNHPSC

5/32

+ 3.53

21.0

18.44

Seq3

CQNAHQKGC

4/32

+ 1.54

2.4

4.18

Seq4

CQSHKNNKC

2/32

+ 3.53

N.A.

N.A.

Seq5

CGHNKNKDC

2/32

+ 2.54

N.A.

N.A.

Seq6

CHDKQSKKC

2/32

+ 3.54

N.A.

N.A

In the next step, phage enzyme-linked immunosorbent assay (ELISA) experiments were performed in order to determine the apparent binding strength (Kapp) and relative affinity (RAmax) of selected phage clones in comparison to the random phage library. These experiments were carried out with Seq1, Seq2, and Seq3, which were most frequently identified in the sequencing analysis (See Table 1). Figure 2 shows the results of these ELISA experiments for phage clones 21 ACS Paragon Plus Environment


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Seq1 and Seq2. The corresponding apparent binding strengths (Kapp) and relative affinities (RAmax), which can be obtained from these analyses, are included in Table 1. The phage ELISA experiments revealed that phage clones Seq1 (CNHQHQKGC) and Seq2 (CKSKNHPSC) had relative affinities to hair that are respectively 37 and 18 times higher as compared to the random phage library. Seq3 (CQNAHQKGC) also showed selectivity towards the hair surfaces, but to a lower extent (RAmax = 4.18).

Figure 2: (A) Evolution of the colorimetric response and (B) the relative affinity as a function of phage concentration in the phage ELISA assay of Seq1 (●), Seq2 (▲) and the combinatorial phage library (■). The error bars represent the standard deviation of 9 independent measurements.


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In order to explore peptide ligands that are based on Seq1 to enhance surface deposition on hair under shampoo conditions, a number of cyclic peptide disulfides was prepared, which incorporate this sequence and which also contain functional groups that allow their attachment to model polymer profragrances or microcapsules. The structures of these peptides are shown in Chart 2. Two cyclic peptide derivatives based on Seq1 were prepared. A first peptide sequence derived from Seq1 was functionalized with an N-terminal azide group (N3-Pep1) in order to allow incorporation into alkyne containing poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA)-based polymer profragrances via copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction. A second Seq1 peptide derivative, which was prepared, contains a C-terminal carboxylic acid group (Pep1-COOH) and was used to attach the peptide to the surface amine groups of polyurethane/urea microcapsules via an EDC/sulfo-NHS coupling reaction. The preparation and characterization of N3-Pep1 have been reported previously,29 and therefore, will not be described here in detail. Furthermore, three different peptides containing fluorescent dansyl groups were also prepared in order to carry out peptide deposition studies. The first two peptides were Seq1 derivatives that contain either an N- or a C- terminal dansyl group and are referred to as DansylPep1 and Pep1-Dansyl, respectively. Dansyl-Pepx is a highly cationic, N-terminal dansylated peptide that shares sequence similarity with Seq2 and which was used as a control in peptide deposition studies.



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Chart 2: Schematic illustration of the peptides used in the study. N- or C-terminal dansylated peptides Dansyl-Pep1, Pep1-Dansyl and Dansyl-Pepx were used to assess the peptide deposition onto hair. N-terminal azide functionalized peptide N3-Pep1 was incorporated onto PHPMA-based model polymer profragrances. C-terminal carboxylic acid modified Pep1-COOH was grafted onto polyurethane/urea core-shell microcapsules.

Peptide deposition studies Prior to the preparation of the polymer conjugates and peptide-modified microcapsules, the deposition properties of Pep1 were studied using three dansylated analogues, which are shown in Chart 2. The aims of these experiments were threefold. The first aim was to validate the results of the phage ELISA experiments. A second aim was to investigate the potential influence of Nor C-terminal modification of the peptides on the deposition properties, which would be important in the design of the chemistry used to conjugate the peptide to a model polymer 24 ACS Paragon Plus Environment

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profragrance or microcapsule. To this end, both N- and C-terminal dansylated variants of Pep1, which are referred to as Dansyl-Pep1 and Pep1-Dansyl, were used. The third aim was to study the influence of the shampoo medium on the deposition properties of the peptide. To address this question, deposition experiments were carried out both in a shampoo formulation and in a 50 mM citrate buffer at pH 6.0. The only difference between the shampoo and the citrate buffer was the presence of surfactants in the former medium, whereas both the pH and the buffer strength were identical. Dansyl-Pepx was used as a control in the deposition experiments.

Table 2, Figure 3 and Figure S6 summarize the results of the deposition studies. Table 2 and Figure 3 show the extent of deposition when 15 µM of the peptides were exposed to 10 mg of hair both in shampoo and citrate buffer. First of all, these results demonstrate the selective binding of Pep1 towards hair as the extent of deposition of the N- and C-terminal dansylated Pep1 derivatives were at least 3 - 5 times higher than Dansyl-Pepx both in the shampoo and the citrate buffer. Even though affinity selection results suggest that peptide-hair interactions are electrostatic in nature, this result highlights the importance of sequence specificity, since both sequences have similar net charge at pH 6.0 (+3.09 and +3.53 for Pep1 and Pepx, respectively) but have a 3-5-fold difference in their extent of hair deposition. Second, it is evident from these experiments that the presence of surfactants in the shampoo formulation significantly limits the extent of Pep1 deposition, irrespective of the location of the incorporated dansyl groups. Third, the comparison of N- and C-terminal dansylated Pep1 first revealed that the extent of deposition of Pep1-Dansyl (5.1%) and Dansyl-Pep1 (4.8%) were almost the same under shampoo conditions (p = 0.78). In citrate buffer, in contrast, the extent of deposition of Pep1-Dansyl (26.7%) was more than two-fold as compared to Dansyl-Pep1 (12.9%). As there was no significant difference between the deposition of Dansyl-Pep1 and Pep1-Dansyl under shampoo 25 ACS Paragon Plus Environment


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conditions, both C-terminal and N-terminal conjugation strategies have been used to prepare the corresponding model fragrance delivery systems (vide infra). Model polymer profragrances were prepared by incorporation of N-terminal azide functionalized peptide N3-Pep1 onto an alkyne containing PHPMA precursor polymer via CuAAC reaction. Poly(urethane-urea) microcapsules were modified by the grafting of the C-terminal carboxylic acid derivative of Seq1 (Pep1COOH) onto the microcapsule surface, which contains amine groups, via a EDC/NHS coupling reaction.

Figure 3: The extent of deposition of dansylated peptides at a peptide concentration of 15 µM onto hair from a shampoo formulation (white bars) and from a 50 mm citrate buffer at pH 6.0 (grey bars). Error bars represent the standard deviation of 6 independent measurements. *** represents a p value smaller than 0.001 in a two-sided Student’s t-test that compares the means of two independent measurements. The difference between the extent of deposition of Dansyl-Pep1 and Pep1-Dansyl was not statistically significant under shampoo conditions (p = 0.78).


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Table 2: The extent of deposition of 15 µM of dansylated peptides onto 10 mg hair in model shampoo formulation and in pH 6.0, 50 mM citrate buffer.

Deposition (%) Peptide name

Peptide sequence

Under shampoo conditions

In 50 mM citrate buffer, pH 6.0

Dansyl-Pepx

CKSRHNPKC

1.3 ± 1.1

4.8 ± 0.4

Dansyl-Pep1

CNHQHQKGC

4.8 ± 2.2

12.9 ± 2.2

Pep1-Dansyl

CNHQHQKGC

5.1 ± 1.5

26.7 ± 3.9

PHPMA-peptide conjugate model polymer profragrances PHPMA-peptide conjugate model polymer profragrances that incorporate Pep1 were prepared via post-polymerization modification of a poly(pentafluorophenyl methacrylate) (PPFMA, Mn NMR

= 42000 g/mol) precursor following a previously published strategy (Scheme 1).29 This

strategy first involved a two-step active ester aminolysis of pentafluorophenyl ester groups with 5 mol% propargylamine and 1 mol% dansyl cadaverine with respect to pentafluorophenyl ester groups followed by quantitative conversion of the remaining active ester groups into N-(2hydroxypropyl)methacrylamide (HPMA) units to yield a water soluble alkyne side chain functionalized polymer, which is referred to as PC. The dansyl groups were incorporated in order to allow monitoring the deposition of the conjugates via fluorescence intensity measurements. In the final step, N-terminal azide functionalized hair binding cyclic peptide disulfide N3-Pep1 was incorporated via copper-catalyzed azide/alkyne cycloaddition (CuAAC) using the protocol described by Finn and coworkers.42 Two PHPMA-peptide conjugates were prepared, which incorporated 0.3 (P1Pep1) and 1.6 (P2Pep1) mol% of N3-Pep1, respectively, in order to assess the influence of the extent of peptide incorporation on the deposition of polymer conjugates. Table 3 lists the peptide contents of the different conjugates. 27 ACS Paragon Plus Environment


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Scheme 1: Schematic illustration of the synthesis of PHPMA-peptide conjugates P1Pep1 and P2Pep1.

Table 3: Composition of the PHPMA-peptide conjugates and their extent of deposition onto hair under shampoo conditions. Fluorescence intensity measurements

Peptide Polymer name

Peptide sequence

Feed (mol%)

Incorporated (mol%)a

Extent of polymer deposition (%)

Relative deposition with respect to PC

PC

-

-

-

1.7 ± 1.1

-

P1Pep1

CNHQHQKGC

5.0

0.3

5.8 ± 0.4

3.4 ± 0.9

P2Pep1

CNHQHQKGC

5.0

1.6

8.6 ± 1.3

5.1 ± 0.9

a Calculated from 1H-NMR analysis of the conjugates in methanol-d4.


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In order to assess their deposition on hair under shampoo conditions, the different polymer conjugates as well as the non-peptide modified polymer PC at a concentration range between 0.01 to 0.2 mg.mL-1 were incubated with or without 10 mg hair in 500 µL of a model shampoo formulation. The fluorescence intensities of the solutions that were incubated with or without hair were measured and referred to as FIhair and FIblank, respectively. The percentage deposition of the conjugates at each concentration was calculated using the following equation: % Deposition = (FIblank – FIhair) / (FIblank) x 100. Figure S7 plots the % deposition for the different polymer conjugates as a function of the concentration of the polymers in the medium. The results indicate that the % deposition does not significantly vary with the concentration of the polymer conjugates, indicating that the amount of polymer that is deposited is proportional to its concentration and that not all available binding sites are occupied. Figure 4 compares the % deposition of PC, P1Pep1 and P2Pep1 at a concentration of 0.2 mg.mL-1. The results in Figure 4 indicate that while the deposition of PC was only 1.7%, 0.3 mol% incorporation of N3-Pep1 (P1Pep1) already led to a 3.4-fold enhancement of the deposition of the PHPMA-peptide conjugates (5.8%). When the N3-Pep1 content was 1.6 mol% (P2Pep1), the deposition of the conjugates further increased to 8.6%. These results show that the incorporation of Pep1 enhanced the deposition of the PHPMA-peptide conjugates onto hair, and its extent can be tuned with the amount of Pep1 incorporated into these polymers.



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Figure 4: The extent of deposition of PHPMA-peptide conjugates. The reported depositions were measured following the incubation of 0.2 mg/mL of the polymers to 10 mg of hair. Error bars represent the standard deviation of 9 independent measurements. *** and ** represent a p value smaller than 0.001 and 0.01, respectively, in a two-sided Student’s t-test that compares the means of two independent measurements.

Peptide functionalized polyurethane/urea core-shell microcapsules based model fragrance carriers Polyurethane/urea microcapsules were prepared via interfacial polymerization of water soluble guanidine carbonate and an oil soluble trifunctional isocyanate (Takenate® D-110N) (Chart S1).43,44 Additionally, a perfume consisting of Romascone®, Verdox®, Vertenex, (Z)-2Phenylhex-2-enenitrile and Cyclamen aldehyde at an equivalent weight ratio (Chart 1) as well as 10 w/w% of Uvinul® A+ as a UV-tracer for the deposition measurements (Chart S1) was added to the oil phase. These capsules are referred to as C0.


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The fragrance loaded microcapsules were then modified with Pep1-COOH using an EDC/NHS mediated coupling reaction using the surface amino groups on the microcapsules that resulted from the hydrolysis of residual isocyanate groups. Two different Pep1 modified microcapsules were prepared by either using 1 or 10 w/w% of Pep1-COOH with respect to the mass of C0 during the EDC/NHS coupling reaction. These capsules are referred to C1 and C2, respectively. The aqueous phases of the microcapsule dispersions (C1 and C2) were analyzed by HPLC to detect and quantify any residual free peptide. Almost quantitative conversions were achieved for the grafting in both cases (Figure S8).

To determine the deposition efficiency following conjugation of Pep1-COOH, a test protocol was followed using a model cleanser formulation containing anionic surfactant (sodium lauryl ether sulfate (SLES), 12 w/w%), amphoteric surfactant (cocamidopropyl betaine (CAPB), 3 w/w%) and a conditioning film-forming, cationic polymer Salcare® SC-60 (0.5 w/w%) and subsequently the pH of the solution was adjusted to 5.5. The protocol for the microcapsule deposition experiments is provided in the Experimental Section. Briefly, tracer-loaded capsules were incorporated into the shampoo formulation (0.5 wt% equivalent encapsulated oil), dosed onto miniature hair swatches (0.1 mL of shampoo formulation onto 0.5 g, 10 cm net virgin Caucasian hair swatches), distributed evenly and rinsed with 100 mL of warm water. Hair treatments and controls (to establish formulation homogeneity and tracer extraction efficiency) were repeated in triplicate. The tracer was finally extracted from the deposited microcapsules using sonication in solvent and quantified by HPLC to determine the perfume oil mass in the control samples (equivalent to 100% deposition) as well the oil mass deposited onto the hair swatches after rinsing. The ratio of the oil mass extracted from the rinsed hair swatches to the



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controls yielded the calculated percentage of oil mass deposited onto the hair swatches after rinsing, i.e. ‘percent deposition’.

Figure 5 shows the extent of deposition of C0, C1 and C2 microcapsules onto hair at a microcapsule concentration of 0.5 w/w% encapsulated oil. The results of these experiments are also summarized in Table S2. First of all, these results show that 1 w/w% of Pep1-COOH grafting (C1) did not enhance the deposition of microcapsules. However, when the Pep1-COOH content of the capsules was increased to 10 w/w%, an almost 20-fold enhancement in deposition was achieved. While the deposition of C0 was only 0.7 ± 0.5%, this increased to 13.5 ± 0.3 for C2. Figure 6 shows a SEM image of a capsule C2 deposited onto hair following a specific and preferential adhesion event. It is a visual demonstration of specific binding, which is effective even for larger sized materials (i.e. going from molecules (on the nm scale) to capsules (on the µm scale)). It is not intuitive that substantive materials on their own can bring large microcapsules (at least 2 to 3 orders of magnitude larger) sticking to the surface. Taken together, these results show that Pep1 can be used to significantly enhance the deposition of fragrance loaded microcapsules onto hair, however, only after relatively high grafting of the peptide onto the surface of the microcapsules. This enhancement at high grafting density can be attributed to the multivalency of the Pep1 on the capsule surfaces, as multivalency effects were previously shown to significantly improve the binding strength of peptide-grafted nanoparticles.45,46


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Figure 5: Average percentage of oil deposited onto hair from a cleanser formulation containing 0.5 w/w% of polyurethane/urea core-shell microcapsules (on an equivalent encapsulated oil lading weight basis) after rinsing. The average percentage of oil deposited from peptidefunctionalized microcapsule C1 (1 wt% Pep1-COOH) and C2 (10 wt% Pep1-COOH) is benchmarked against the unmodified control capsules, C0.

Figure 6: Scanning electron micrograph of hair after deposition of capsules C2 modified with 10 w/w% of Pep1-COOH as deposition aid.



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Following the demonstration that grafting of Pep1-COOH to the surface of core-shell microcapsules significantly increased their deposition on hair under realistic shampoo cleansing application conditions, the final step was to investigate whether this improved deposition also resulted in increased headspace concentrations of the model perfume released from the capsules on the hair surface. Dispersions of microcapsules C0 and C2 were thus added to the model cleanser to contain the same total amount of model perfume in the final formulation. Hair samples were then washed with the model cleanser under the same conditions described for the deposition measurements. The uncut hair samples were then line-dried and the evaporation of the ingredients of the model fragrance analyzed by dynamic headspace analysis after 6 and 24 h.

Figure 7 and Figure S11 shows significantly increased fragrance release from the hair surfaces treated with C2 microcapsules compared to C0 both after 6 and 24 h. Furthermore, while the headspace concentrations of the fragrances released from C0 decreased with increasing drying time, those released from C2 remained constant or even slightly increased with time. The relatively large difference between each independent experiment in Figure 7 (Figure S11 plots the average of these two experiments for each condition) was attributed to the fact that the measurements were performed under realistic application conditions, in which several physiochemical parameters such as ambient temperature and relative humidity during drying, and amongst many others, and not strictly controlled.33,47 Table S3 summarizes the headspace concentrations for the release of each of the fragrance in the perfume composition encapsulated in C0 or C2 after 6 and 24 h. These data demonstrate that our approach to increase the deposition of polymer fragrance carriers onto hair by using a selective cyclic peptide tag indeed allowed improving the performance of fragrance delivery under realistic rinse-off shampoo cleansing conditions. Enhanced deposition of these fragrance delivery systems usually translates directly 34 ACS Paragon Plus Environment

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into improved olfactive intensity and hence more effective delivery technologies. An order of magnitude improvement in adhesion and retention was thus expected to lead to appreciable improvements in the sensorial impact of the functionalized microcapsules.

Headspace concentration (ng L-1)

(A)

1.6

1.2

0.8

0.4

0.0 Romascone®

(B) Headspace concentration (ng L-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Verdox®

Vertenex

Cyclamen Phenylhexene aldehyde nitrile

Verdox®

Vertenex

Cyclamen aldehyde

1.6

1.2

0.8

0.4

0.0 Romascone®


Phenylhexene nitrile


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Figure 7: Headspace concentrations measured for the different ingredients of the model perfume released from microcapsules C0 (white bars) and C2 (grey bars) on hair after a shampoo application and drying for (A) 6 h and (B) 24 h. Each bar represents an independent headspace sampling measurement. Refer to Table S3 for the numerical data.

CONCLUSIONS Phage display identified peptides offer a solution to tackle the modest deposition of fragrance delivery systems onto hair under shampoo formulations. As a proof-of-concept, first, the feasibility of identifying hair binding peptide ligands under highly stringent shampoo medium conditions was demonstrated. In the next step, hair binding peptide ligands were incorporated into PHPMA conjugates and the peptide incorporation was found to enhance the deposition of these polymers onto hair surfaces with a factor of 3.5 to 5.0. As a final example, polyurea coreshell type microcapsules containing a model perfume were synthesized and functionalized with the hair-binding peptide. 10 w/w% grafting of the peptide was found to increase the deposition of the capsules onto hair approximately with a factor of 20. This enhanced deposition led to higher release of the model perfume from the hair surfaces even after 24 h under realistic application conditions as evidenced by dynamic headspace measurements.

ACKNOWLEDGEMENTS We thank Prof. Christian Heinis from the Laboratory of Therapeutic Proteins and Peptides (LPPT) at EFPL for providing the phage libraries. We thank Geraldine León, Christopher Hansen and Alain Trachsel (Firmenich SA) for technical assistance.


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SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website; detailed summary of the sequence and the frequency of the hair-binding peptides obtained in different rounds of affinity selection experiments and evolution of the phage titers; HPLC elution profiles and ESI-MS spectra of the synthesized peptides; 1H-NMR spectra of the model polymeric profragrances and chemical structures of the monomers as well as the UV tracer used during the preparation of polyurethane/urea core-shell microcapsules; Percent deposition of peptides, polymer profragrances and average deposition of peptide-functionalized microcapsules as well as the results of dynamic headspace measurements.

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TOC Figure.


Selective Peptide-Mediated Enhanced Deposition ... - ACS Publications

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