Cytocompatibility of Molecularly Imprinted Polymers for Deodorants

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Cytocompatibility of Molecularly Imprinted Polymers for Deodorants - Evaluation on Human Keratinocytes and Axillary-Hosted Bacteria Alejandra Mier, Sofia Nestora, Paulina Medina-Rangel, Yannick Rossez, Karsten Haupt, and Bernadette Tse Sum Bui ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00388 • Publication Date (Web): 03 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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ACS Applied Bio Materials

Cytocompatibility of Molecularly Imprinted Polymers for Deodorants Evaluation on Human Keratinocytes and Axillary-Hosted Bacteria Alejandra Mier#, Sofia Nestora#, Paulina X. Medina Rangel, Yannick Rossez, Karsten Haupt*, and Bernadette Tse Sum Bui* Sorbonne Universités, Université de Technologie de Compiègne (UTC), CNRS Enzyme and Cell Engineering Laboratory, Rue Roger Couttolenc, CS 60319, 60203 Compiègne Cedex, France

Alejandra Mier# and Sofia Nestora# : both contributed equally to the work Corresponding authors: [email protected] (B. Tse Sum Bui); [email protected] (K. Haupt) ORCID Alejandra Mier : 0000-0002-7669-880X Sofia Nestora : 0000-0001-6842-6268 Paulina X. Medina Rangel : 0000-0003-2919-7575 Yannick Rossez : 0000-0001-5435-1373 Karsten Haupt : 0000-0001-6743-5066 Bernadette Tse Sum Bui : 0000-0002-4170-2303

ABSTRACT Molecularly imprinted polymers (MIPs), often dubbed ‘synthetic antibodies’ can recognize and bind their target molecule with a high affinity and selectivity, making them serious competitors vis-à-vis biological antibodies. Recently, MIPs have gained popularity in various clinical applications, and have even been applied in vivo. However, only a few studies on the biocompatibility of MIPs have been reported. Herein, we investigate on the example of a MIP which has proved its efficacy as an active agent to suppress body odors in cosmetic formulations, its effect on the viability and irritation potential of human epithelial cells. Since body odors are caused by bacteria present on the skin, bactericides are regularly added to deodorants sold on the market. However, there is growing anxiety

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concerning these bactericides as they can generate resistant bacteria, a problem for human and animal health. Therefore, we also assessed whether the MIP perturbs the resident skin bacteria, which was isolated from human sweat. Our results show that MIPs do not affect bacterial growth when cultured in liquid media, suggesting that they will not affect the skin flora which protects the body from dangerous pathogens. This thorough in vitro toxicological assessment shows the biocompatibility of MIPs, and constitutes a step further in their future consideration within cosmetic or pharmaceutical formulations for skin applications.

KEYWORDS Biocompatibility, cytokine, cytotoxicity, molecularly imprinted polymer, skin bacteria

INTRODUCTION Molecularly imprinted polymers (MIPs) are macromolecular receptors in which specific binding sites for a target molecule are molded.1-3 They are obtained by polymerization of a template molecule (target or structural analogue) in the presence of functional and cross-linking monomers. The subsequent removal of the template from the polymer will reveal specific recognition cavities which perfectly match the template’s shape, size and functional groups (Figure S1), enabling the future recognition of the target molecule with a high specificity and affinity. Research on MIPs has kept growing exponentially mostly as from 1993 when in an immunoassay for determining drugs in human serum, the group of Mosbach found comparable results with both MIPs and antibodies, whereafter MIPs were neologized ‘antibody mimics’.4 However, MIPs are more advantageous as they are easier to prepare, do not require killing of animals, are cost-effective and are (bio)chemically, physically and thermally more stable. During the past 25 years, MIPs have proved their usefulness in affinity separation,5,6 chemical sensors,7,8 immunoassays,9,10 drug delivery,11,12 bioimaging,13,14 among others. Nevertheless, reports of MIPs for purely biological applications or applications in vivo15-19 or for


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therapy,20-22 though starting to emerge, are still scarce, and consequently biocompatibility studies on MIPs are not frequently reported. We propose herein a complete set of tools for studying the biocompatibility of MIPs, which could eventually serve for other MIPs. A MIP which was recently applied as an active ingredient in a deodorant, for selectively trapping molecules that cause body odors,23,24 was used for demonstration. Body odors are mainly due to the presence of volatile mediumchain fatty acids, namely (E)-3-methyl-2-hexenoic acid (3M2H) and 3-hydroxy-3-methyl-hexanoic acid (3H3MH).25 They are produced from odorless glutamine conjugate precursors, cj3H3MH and cj3M2H, naturally secreted from axillary sweat glands, by a hydrolytic enzyme, N-acylglutamine aminoacylase of Corynebacterium spp. residing on the skin26-28 (Figure 1A). To prevent the formation of unpleasant odors, cosmetic industries either use anti-perspirants based on aluminium salts to stop the flow of sweat, or deodorants containing broad-spectrum non-specific bactericides (triclosan, chlorhexidine,…) as active agents, to limit bacterial growth. However, the extremely wide use of these antibacterials can perturb the fragile skin microbiota, and with time can even generate resistant bacteria.26 We hypothesized that by using a MIP which can sequester the glutamine conjugate precursors, they will no longer be available for enzymatic hydrolysis and therefore no odoriferous acids will be produced. Indeed the MIP, blended in a dermocosmetic deodorant formulation could bind cj3H3MH and cj3M2H present in human sweat at 37 °C,23,24 hence could act as an active deodorant ingredient, offering promising alternatives to classical bactericides. Nevertheless, the potential of skin toxicity and irritation of the MIP was not addressed. Herein, we use human keratinocytes (HaCaT) as an in vitro cell model system for the study of epidermal toxicity. These immortalized non-tumorigenic skin cells possess the advantage of providing intra- and inter-laboratory reproducibility.29 MIP cytotoxicity was assessed by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) and neutral red assays combined with epifluorescence microscopy of cells stained by propidium iodide (PI), a dye which can only cross the membrane of non-viable cells. Irritation was assessed by quantifying the amount of pro-inflammatory cytokines produced, IL-1, IL-6 and IL-8, known biomarkers of


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cutaneous inflammation.30,31 These data as well as the impact of MIPs on the dynamics of three bacterial strains isolated from human sweat (Corynebacterium striatum, Staphylococcus epidermidis and Micrococcus luteus), are described.

EXPERIMENTAL SECTION Reagents and instrumentation. All chemicals and solvents were of analytical grade and purchased from Sigma-Aldrich (France) or VWR International (France), unless otherwise stated. HPLC solvents were purchased from Biosolve Chimie (Dieuze, France). Glycine and paraformaldehyde (PFA) were from Applichem (France). Human adult low calcium high temperature (HaCaT) cells were obtained from Cell Lines Service (Eppelheim, Germany). Glass cover slips, cell culture flasks, 24 and 96 well plates, phosphate-buffered saline (PBS) pH 7.4, penicillin/streptomycin, Dulbecco's Modified Eagle Medium, high glucose (DMEM), fetal bovine serum (FBS) and trypsin-EDTA (0.05%) with phenol red, Hoechst 33342, trihydrochloride, trihydate (10 mg/mL water) and propidium iodide (1 mg/mL water) were from Thermo Scientific (Illkirch, France). Culture media for bacterial growth were obtained from Sigma-Aldrich. They were prepared according to the manufacturer’s instructions, sterilized in an autoclave at 121 °C for 30 min and kept in the dark at room temperature until use. Human sweat was collected from the armpit of healthy volunteers prone to develop armpit odors. The volunteers had not used any deodorant products three weeks prior to sweat collection. The sweat was pooled, aliquoted and immediately frozen to preserve the composition. Human IL-1, IL-6 and IL-8 (CXCL8) standard 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) enzyme-linked immunosorbent assay (ELISA) development kits and ABTS ELISA buffer kit were from PeproTech (Neuilly-Sur-Seine, France). Microscope slides for cell specimens were from Roth Sochiel E. U. R. L. (Lauterbourg, France). Water was purified using a Milli-Q system (Millipore, Molsheim, France).

MIP synthesis. In a 30-mL glass vial, 0.4 mmol (98 mg) of the template N-hexanoyl glutamic acid and 0.8 mmol (199.2 mg) of the functional monomer, (4-acrylamidophenyl)(amino)methaniminium


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acetate (AB) were dissolved in 20 mL of ethanol/H2O (4/1), under sonication in an ultrasonic bath. 8 mmol (1.51 mL) of the cross-linker, ethylene glycol dimethacrylate (EGDMA) and 0.084 mmol (33.5 mg) of the initiator, Bis(tert-butylcyclohexyl)peroxydicarbonate (Perkadox 16), were then added. The glass vial was closed with an airtight septum and nitrogen gas was bubbled for 10 min in the mixture, on ice. Polymerization proceeded for 15h in a water bath at 40 °C. The resulting polymers were transferred to 50 mL centrifuge tubes and washed for 1 h with 15 mL of C2H5OH/CH3COOH (9/1) (x2), water (x1), C2H5OH/300 mM NH3 (x2), water (x2) and C2H5OH (x2), at 40 °C. Finally, the polymers were vacuum-dried for 15h. Non-imprinted polymers (NIPs), i.e. control polymers, were synthesized as described for the MIPs, but no template was added. Physicochemical characterization. The morphology and size of MIPs were examined by scanning electron microscopy (SEM) imaging on a Quanta FEG 250 scanning electron microscope (Eindhoven, Netherlands). Polymer particles were sputter coated with gold prior to measurement. Thermogravimetric analysis (TGA) was performed on 4.5 mg of MIP using a SDT Q600 TA Instruments analyzer under N2 atmosphere, with a heating rate of 1 °C/min over a temperature range of 19 – 240 °C. Binding of cj3M2H and cj3H3MH in human sweat by MIP, in a deodorant formulation. The experimental procedure was previously described.23,24 First, MIP and NIP (2, 4, 8 mg) were weighed in 1.5 mL Eppendorf safe-lock tubes. 200 µL of deodorant emulsion formulation (prepared as described in Supplementary Information) was pipetted in the tubes, followed by 200 µL of human sweat. A mixture of 200 µL deodorant emulsion and 200 µL human sweat, without polymers, served as control. After vortexing, the tubes were agitated on a tube rotator (SB2, Stuart Scientific) for 15 h 37 °C, followed by centrifugation at 40,000g for 45 min. Finally, a 100 µL aliquot of the aqueous part of the supernatant was withdrawn and analysed on a DIONEX Ultimate 3000 HPLC (Thermo Scientific), connected to evaporative light scattering (380-ELSD, Agilent Technologies) and diode array detectors set at 235 and 203 nm (see details in Supporting Information). This allows to measure the amount of unbound conjugates; the amount bound to the polymers was obtained by subtracting


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the amount of the unbound conjugates from the amount of the conjugates present in the mixture of deodorant formulation and human sweat, i.e. from zero-polymer control sample. Cell culture. HaCaT cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, hereafter referred as complete medium, at 37 °C, 5% CO2 and 100% humidity. Cells were passaged when confluent, using trypsin-EDTA 0.05% in PBS for cell detachment. Cell viability assays. Indirect contact method. Suspensions of MIPs (0.025 – 5 mg/mL) were prepared in 10 mL of complete medium in glass vials fitted with screw caps, and incubated for 72 h at 37 °C without agitation to extract any leachable components from the polymer. After incubation, the contents of the vials were filtered through Acrodisc Syringe Filters 0.2 µm (Supor® Membrane, Merck Millipore) and the supernatants were kept. HaCaT cells were grown to confluency and trypsinized as described above. Cells (200 µL) were seeded in complete medium at a density of 15 x 103 cells/well in 96-well microplates. After overnight incubation, the wells were washed with PBS and the cells were incubated with 200 µL MIP extracts or Triton X-100 (1%), 6 replications each (n=6) in complete medium, for 24, 48 and 72 h. Cells incubated in complete medium without any addition served as control. After incubation at the defined time-period, the cells were subjected to MTT and neutral red assays. Direct contact method. A stock suspension of MIP (10 mg/mL) was prepared in PBS, homogenized by ultrasonication with the microtip of a Branson sonifier 250 and sterilized in an autoclave for 20 min, 121 °C. The MIP was then left for the same time period as for the indirect method, i.e. 72 h at 37 °C. After this time, MIP suspensions (0.025 – 1 mg/mL) were prepared in complete medium and were examined on the cells, as reported above, and subjected to MTT and neutral red assays. MTT assay. After incubation at the defined time-period, the wells were washed 3 times with PBS and 200 µL of a solution containing 90% of complete medium and 10% MTT reagent (stock solution 5 mg/mL, prepared in PBS) were added to each well followed by 3 h incubation. The produced


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formazan salt was dissolved using 200 µL DMSO. The absorbance was measured on a Spark 10M multimode microplate reader (Tecan Trading AG, Switzerland) at 570 and 620 nm, where 570 nm is the absorbance wavelength of the soluble formazan and 620 nm the reference corresponding to the impurities. Cell viability was calculated by considering the signal of non-treated cells as the 100% viability and was determined by dividing the absorbance (620 - 570 nm) measured for treated cells by that of the non-treated controls. Neutral red assay. A stock solution of neutral red (4 mg/mL) was prepared in PBS. After incubation at the defined time-period, the wells were washed 3 times with PBS and 150 L of neutral red (40 g/mL) in complete medium was added to each well followed by 2 h incubation at 37 °C. The neutral red medium was removed and the wells were washed with PBS and the dye was extracted with 150 L of a solution containing 50% ethanol, 49% water and 1% acetic acid, for 10 min with constant agitation. The absorbance of the dye at 540 nm was measured on a Spark 10M multimode microplate reader (Tecan Trading AG, Switzerland). Cell viability was calculated by considering the signal of non-treated cells as the 100% viability and was determined by dividing the absorbance at 540 nm measured for treated cells by that of the non-treated controls. Cell imaging by epifluorescence microscopy. HaCaT cells were grown to confluency and trypsinized as described above. 100 µL of cells at a density of 1 x 105 were cultured in 24-well plates equipped with round glass cover slips (diameter 12 mm). After 3 h incubation, 1 mL of complete medium was added to each well and left to grow to confluency for 24 h. Each cover slip was then washed with PBS and incubated with MIP (1000 µg/mL) or Triton X-100 (1%), 3 replications each (n=3) in complete medium, for 24 h. Cells incubated in complete medium without any addition served as control. After incubation, the wells were washed 3 times with PBS. For staining the cell nucleus, a solution of Hoechst (10 µg/mL) was prepared in PBS and 1 mL of the solution was added per well and incubated for 10 min in the dark. The wells were then washed 3 times with PBS and 1 mL of a solution of propidium iodide (1 mg/mL) was added per well to stain the DNA of dead cells, followed by 10 min incubation in the dark. The wells were washed 3 times with PBS and then cells were fixed


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at room temperature for 10 min in 600 µL paraformaldehyde (3% w/v) in PBS. To stop fixation, each cell sample was incubated 3 times with 1 mL 20 mM glycine in PBS for 20 min at room temperature and finally they were washed 3 times with 1 mL PBS. Afterward, cover glasses were mounted for fluorescence microscopy imaging on a microscope slide with 5 µL mounting medium. The mounting medium consisted of 0.5 mL water, 0.5 mL 1 M Tris-HCl buffer pH 8.0 and 9 mL glycerol. Epifluorescence images were captured with a Leica DMI 6000B microscope, filter sets A4 and TX2, with 40x and 63x magnification. Images were captured using exactly the same settings concerning light intensity and exposure time in 16-Bit Tiff format. For each sample, at least 3 images were captured with the Leica Application Suite (LAS) software. Cytokines quantification. The inflammatory response of MIPs on HaCaT cells was determined by the quantification of cytokines in the cells supernatants after 48 h incubation. A stock suspension of MIP (10 mg/mL) was prepared in PBS, ultrasonicated with the microtip of a Branson sonifier 250 and sterilized in an autoclave for 20 min, 121 °C. A stock solution of DNCB (5 mM), the positive control, was prepared in ethanol. Cells were grown to confluency and trypsinized as described above. Cells (200 L) were seeded in complete medium at a density of 15 x 103 cells/well in 96-well microplates. After overnight incubation, the wells were washed with PBS and the cells were incubated with MIPs (10 – 1000 g/mL) or DNCB (5 M), 6 replications each (n=6), in complete medium, for 48 h. Cells incubated in complete medium without any addition served as control. After incubation, the different test media were transferred to 1.5 mL-polypropylene microcentrifuge tubes, centrifuged at 300g for 5 min and the supernatants were subjected to IL-1, IL-6 and IL-8 quantification. Cytokine levels were determined using the human IL-1, IL-6 and IL-8 ABTS ELISA Kits according to the manufacturer’s protocol. The cytokines standards were diluted to construct calibration curves (25-250 pg/mL) for IL-1, (50-1500 pg/mL) for IL-6 and (10-1000 pg/mL) for IL-8, in a final volume of 500 L. The absorbance was measured at 405 nm on a Spark 10M multimode microplate reader (Tecan Trading AG, Switzerland). Each experiment was done with three replications and repeated twice. ACS Paragon Plus Environment

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Isolation of bacteria from human sweat - identification by MALDI-TOF mass spectrometry. The protocol used is as previously described.24 A 50 L-aliquot of human sweat, kept at -20 °C was thawed and streaked on blood agar and Lysogeny Broth (LB) plates. After an incubation of 72 h at 37 °C, single colonies were observed and the bacterial species were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Fresh colony material was spread on a MALDI target plate (MSP 96 target polished steel BC) (Bruker Daltonik GmbH, Germany) using a toothpick, mixed with 1 L of a saturated -cyano-4-hydroxy-cinnamic acid matrix solution in acetonitrile 50% - trifluoroacetic acid 2.5%, and dried in air at ambient temperature. Mass spectra were acquired and analyzed (see details in Supporting Information) on a microflex LT/SH mass spectrometer (Bruker Daltonik) using a Bruker’s MALDI Biotyper software reference database library of 3,995 entries, version 3.1.2.0 and default parameter settings, as reported.32 Four microorganisms, Corynebacterium striatum, Micrococcus luteus, Staphylococcus epidermidis and Staphylococcus hominis, were identified. Growth of bacteria in presence of MIPs. Brain heart infusion (BHI) medium was used for bacterial growth, as all three bacteria can grow in this medium. MIP suspensions in BHI were homogenised in an ultrasonic water bath (VWR ultrasonic cleaner) and sterilized in an autoclave for 30 min at 121 °C. Silver nitrate prepared in BHI, was sterilized by passing the solution through Acrodisc Syringe Filters 0.2 m (Supor® Membrane, Merck Millipore). In separate Falcon 15 mL polypropylene centrifuge tubes, were pipetted 5 mL of BHI medium containing either 0.5 mg/mL of MIP or 0.5 mg/mL of AgNO3 (positive control), or blank BHI medium without any addition (negative control). To monitor growth, a volume of inoculum corresponding to 1 x 106 cells of S. epidermidis, C. striatum and M. luteus was used. After inoculation in aseptic conditions, the samples were incubated at 37 °C with reciprocal shaking at 200 rpm. This rotation speed was sufficient to prevent deposition of the MIP particles during incubation. At time points of 3, 6, 9, 12, 24, 32, 48 and 56 h, the bacterial concentrations were measured by reading their absorbance at 600 nm. Samples were diluted with BHI medium so as to obtain absorbance values < 1. The absorbance was measured in disposable


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polystyrene cuvettes on a Cary 60 UV-Vis spectrophotometer, using BHI medium as blank. The bacterial growth curves were plotted taking into account that the absorbance of 1 corresponds to a concentration of 1 x 109 cells per mL.

RESULTS AND DISCUSSION MIP’s synthesis and characterization. Human sweat contains a multitude (over 60) of chemical compounds secreted from sweat glands (salts in particular NaCl, amino acids, vitamins, urea, fatty acids,…)33 and sebaceous glands (triglycerides, squalene, wax esters,…).34 For our application where the MIP must capture selectively the target analytes in a particularly complex environment (mixture of human sweat and deodorant formulation (2-phenoxyethanol, polydimethylsiloxane, pentane-1,2diol, cetylstearyl alcohol, ceteareth-33, PPG-15 stearyl ether)), interactions involving strong associations were mandatory. Hence, we employed an amidine-based monomer, (4acrylamidophenyl)(amino)methaniminium (AB) acetate, which can form stoichiometric interaction with a high binding constant for carboxyl groups (Ka = 9.4 x 103 M-1 in CD3OD/D2O).23,24 Thus, the MIPs were prepared by using N-hexanoyl glutamic acid as template, AB as functional monomer (Figure S2) and EGDMA as cross-linker, in a molar ratio of 1 : 2 : 20. Figures 1B-C show the equilibrium binding isotherms of MIP and NIP for cj3H3MH and cj3M2H in a mixture of human sweat and deodorant formulation. The NIP is a control polymer and was prepared in the same way as the MIP but without template. We can observe the high specificity of the MIP (blended in a dermocosmetic formulation), as it could bind both the glutamine precursors present in human sweat, to a higher extent than the NIP, indicating the creation of imprinted sites. Hence, we propose a new route towards odor control and the potential application of MIPs in deodorants.


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Figure 1. (A) Formation of volatile malodorous fatty acids, 3-hydroxy-3-methyl-hexanoic acid (3H3MH) and (E)-3-methyl-2-hexenoic acid (3M2H), from their non-odorant glutamine conjugates by Corynebacterium spp. in axillary sweat; (B,C) MIP and NIP binding to cj3H3MH and cj3M2H in human sweat, at 37 °C. The MIP and NIP were homogeneously suspended in a deodorant formulation. Data are the mean from 2 independent experiments with three analyses for each point. Amount of cj3H3MH and cj3M2H in human sweat were 135 ± 6.6 nmol/mL and 17 ± 1 nmol/mL, respectively.

The thermal stability of the MIPs was investigated by thermogravimetric analysis (TGA). Figure 2A shows that the MIP sample showed a weight loss of 1% when heated from 19 °C to 105 °C, which is due to the loss of water, indicating that the MIP is stable and will not decompose when used at body temperature. Moreover, the overall weight loss of the polymer is only 7% when heated from 19 °C to 240 °C. The mean particle diameter was  600 nm, as deduced from a scanning electron micrograph (Figure 2B) and meets the criterium for skin application, as this large size prevents its passage through the skin.


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Figure 2. (A) Thermogravimetric analysis of MIP from 19 to 240 °C. (B) SEM image of MIP, scale-bar 2 µm.

Cell viability – MIP’s cytotoxicity evaluation. Cell viability in presence of MIPs was measured with the MTT 35 and neutral red36 assays. Two assay systems were used as they addressed different metabolic pathways of the cell. MTT is based on the reduction of the yellow dye, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, to purple formazan by mitochondrial dehydrogenases present in metabolically active cells. The amount of formazan is read spectrophotometrically and correlates with the number of viable cells. The neutral red assay is based on the ability of viable cells to incorporate the dye in the lysosomes. The amount of retained dye is proportional to the number of viable cells. Indirect contact method. The effect of MIPs was tested via an indirect method, i.e. with MIP extracts, according to the international standards ISO 10993-537 and ISO 10993-12.38 The ISO standards set guidelines for samples’ preparation, for instance, test with extracts tries to mimic or amplify the conditions practised in clinical applications, to determine the potential effects of chemical leachables. In this study, the extraction vehicle was complete culture medium and the conditions of extraction were 72 h at 37 °C, simulating an extreme time of residence of deodorants on skin. After incubation, the samples were filtered to remove the MIPs and the potential toxicity of the supernatant was tested on the cells. Figures 3A-B show that high concentrations (0.025 – 5 mg/mL) of MIPs extracts, were not cytotoxic indicating that the constituent materials were safe for contact. Both MTT and neutral red assays generated similar results. ACS Paragon Plus Environment

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Direct contact method. Initially, MIPs suspensions were tested in direct contact with the cells, as commonly reported for the majority of MIPs studied so far. The direct contact approach aims to test the material without any modification in the cytotoxicity assays. For this, a stock suspension of MIPs was sterilized in an autoclave for 20 min at 121 °C. The MIP was then incubated for 72 h at 37 °C and after this time, different concentrations (0.025 – 1 mg/mL) were prepared in culture medium and directly added to the cells. Figures 3C-D show that the MIP starts to show cytotoxicity at a concentration of 250 µg/mL, based on the fact that a material is considered toxic when cell viability drops below 70%,39 according to ISO 10993-5.37 However, it has to be noted that the MIP particles tend to heavily aggregate on the cells, as observed under a microscope (Figure S3); this might prevent the cells from feeding on the culture nutrients, and might be the cause of the increased cytotoxic effects observed and not due to the ‘toxic’ properties of the MIP. Previous studies have shown that low concentrations (1 - 27 µg/mL) of 400 nm-sized acrylamide/AB/EGDMA MIPs19 and (10 – 50 µg/mL) of ~200 nm-sized methacrylate-based MIPs40 were not cytotoxic on HaCaT cells, as assayed by MTT. Therefore, our results from the direct contact assay are consistent with other reports, when using low concentrations of polymers. However, it is worth noting that one has to take care that largesized solid particles do not interfere so as to avoid false-positive results.


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Figure 3. Cell viability using MTT (A, C) and neutral red (B, D) assays of MIP extracts/indirect contact (A, B) and MIP suspensions/direct contact (C, D). Positive control: 1% Triton X-100. Data are mean values ± S.D. of six independent experiments with ***P < 0.0001 and **P < 0.01, with respect to the controls.

Another method to assess cell viability is to use propidium iodide (PI), a membrane impermeant dye that is excluded from viable cells but can penetrate dead cells with damaged membrane to intercalate with the nucleic acid to give a red fluorescence signal, when excited at 535nm.41 Cells were stained with Hoechst (blue), a cell-permeable nuclear stain, prior to the addition of PI. Epifluorescence micrograph of cells incubated for 24 h with MIP (1 mg/mL), the highest concentration tested in the direct contact method, showed that the cell morphology was unaffected and cells were viable, as propidium iodide (red) was not visualized (Figure 4C). Untreated cells served as negative control (Figure 4A). On the other hand, Triton X-100 as positive control, caused membrane damage and permeation to PI (red), indicating the presence of dead cells (Figure 4B).


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Figure 4. Epifluorescence micrographs of cells at 24 h. (A) untreated (control), (B) Triton X-100 (1%)-treated and (C) MIPs (1000 µg/mL)-treated. Staining of nucleus in blue (Hoechst) and of apoptotic cells in red (propidium iodide). MIPs are seen as black aggregates.

Irritation potential of MIPs - Release of proinflammatory cytokines. Irritancy by monitoring cytokines is an adequate prescreening tool of in vivo toxicity.29,42 2,4-dinitrochlorobenzene (DNCB), a chemical allergen of keratinocytes is known to release proinflammatory cytokines, IL-1, IL-6, IL8, and was used as positive control.31 Release of IL-1, IL-6 and IL-8 was assessed after 48 h (Figure 5), based on their calibration curves (Figure S4). The basal, control levels of IL-1 IL-6 and IL-8 in the culture medium at 48 h were determined to be 12.44 ± 0.17, 113.84 ± 0.31 and 236.34 ± 6.3 pg/mL respectively. In the presence of MIPs (10 to 1000 µg/mL), the levels of IL-1, IL-6 and IL-8 were not significantly increased and no dose-dependent behaviors were observed, whereas there was 13, 2.3 and 2.9-fold increase of the respective cytokines in presence of 5 µM of DNCB. These results indicate that the MIPs will not cause an inflammatory reaction to human skin.


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Figure 5. Effect of MIPs (10-1000 g/mL) on cytokines’ release at 48 h. (A) IL-1, (B) IL-6 and (C) IL-8. DNCB (5 M) served as a positive control. Data are mean values ± S.D. of six independent experiments with ***P < 0.0001, **P < 0.01 and *P < 0.05. Influence of MIP on the growth of bacteria isolated from human sweat. Bacterial species from human sweat (isolated from single colonies grown on LB and blood agar plates) were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS).32 Four bacterial species, namely Corynebacterium striatum, Staphylococcus epidermidis, Staphylococcus hominis and Micrococcus luteus, commonly belonging to the resident skin flora,27,28 were identified. Of these Corynebacterium spp. and Staphylococcus spp. dominate the axillary flora.26,27 Since S. hominis belongs to the same phylum as S. epidermidis, only the last three species were studied. S. epidermidis, C. striatum and M. luteus were grown in Brain Heart Infusion (BHI) medium, supplemented with a homogeneous suspension of MIP prepared in BHI. Medium supplemented with silver nitrate prepared in BHI, and blank medium without any addition, served as positive and negative controls, respectively. Silver ions and silver-based nanoparticles are popular antibacterials and are commonly added to deodorants to inhibit the growth of skin commensal bacteria responsible for body odors.43,44 Bacterial concentrations at 3, 6, 9, 12, 24, 32, 48 and 56 h were determined by measuring their optical density at 600 nm. The bacterial growth curves (Figure 6) show that the incorporation of MIPs in the culture medium do not affect the bacterial population number and growth rates, as they were similar to the blank samples. Contrarily, the presence of silver nitrate prevented bacterial growth, for all three microorganisms tested.


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Figure 6. C. striatum, S. epidermidis and M. luteus were cultivated in the absence (open circles) and presence of 0.5 mg/mL MIP (full circles) or 0.5 mg/mL silver nitrate (squares). Values represent the mean of two independent experiments.

CONCLUSION MIPs which can suppress body odors in deodorants, were evaluated for their cytotoxic effect and irritancy potential on HaCaT cells. Cell viability was investigated by two assay systems addressing different specific metabolic pathways, namely MTT (mitochondrial activity) and neutral red (lysosomal activity). Two types of test samples, prepared according to international standard methods were examined: the indirect (MIP extracts in solution) and the direct contact (MIP suspensions). Both MTT and neutral red assays show that MIP extracts were not cytotoxic, indicating that the constituents of the polymer were safe for contact. With the direct contact method, polymers tend to agglomerate on the cells, blocking nutrients for cells to feed on, and could generate ‘false’ cytotoxic ACS Paragon Plus Environment

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effect. This is comforted by the fact that no dead cells were observed in the presence of a high MIP concentration, under epifluorescence microscope when staining with propidium iodide, indicating that the 600-nm sized MIP was located extracellularly and did not compromise the cell membrane. Taken together, these results suggest that the MIPs are biocompatible. Interestingly, the MIPs do not appear to cause skin irritation as pro-inflammatory cytokines stayed at a basal level in their presence. Furthermore, MIP does not affect the population number and the growth rates of three bacteria species isolated from human sweat, in liquid culture, suggesting that it will not disturb the skin microbiota which helps to defend our skin against harmful pathogens. Contrarily to unspecific antibacterials used in deodorants, we can expect that MIP will not contribute to the generation of resistant bacteria. In summary, the MIPs offer promising alternatives to bactericides in deodorants as a result of their non-cytotoxicity, non-irritancy and non-perturbation of the skin microbiota. Though in vitro biocompatibility studies predict that MIPs are ‘safe’, they will need to be confirmed by appropriate in vivo studies.

ASSOCIATED CONTENT Supporting Information Preparation of deodorant formulation, details of HPLC conditions and identification of bacteria from human sweat, by Bruker’s MALDI Biotyper reference library using MALDI-TOF mass spectrometry, figures representing the molecular imprinting principle, the proposed complex formation between the template N-hexanoyl glutamic acid and AB, the microscope image of HaCaT cells treated with MIP suspension and the calibration curves of the cytokines.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We acknowledge funding from the Ministry of National Education Research and Technology (PhD fellowship of SN), the Region of Picardy and the European Union (cofunding of equipment under ACS Paragon Plus Environment

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CPER 2007-2020). AM and PXMR thank the Mexican National Council for Science and Technology (CONACYT), the Instituto de Innovacion y Transferencia de Tecnologia de Nuevo Leon and the Instituto para el Desarrollo de la Sociedad del Conocimiento del Estado de Aguascalientes, for PhD scholarships. We thank Franck Merlier from CNRS Enzyme and Cell Engineering Laboratory-UTC for help in LC-UV-ELSD analysis; Frederic Nadaud from the Physicochemical Analysis LaboratoryUTC for SEM measurements; Dr. Benoît Ménart from the Microbiology Department at the Hospital Center of Valenciennes for MALDI-TOF MS analysis; Bruno Dauzat from Integrated Transformations of Renewable Matter Laboratory-UTC for TGA analysis.

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Cytocompatibility of Molecularly Imprinted Polymers for Deodorants

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