# Mimicking the Chemistry of Natural Eumelanin Synthesis: The KE Sequence in Polypeptides and in Proteins Allows for a Specific Control of Nanosized Functional Polydopamine Formation

Camille Bergtold, Daniel Hauser, Alain Chaumont, Salima El Yakhlifi, Mihaela Mateescu, Florent Meyer, Marie-Hélène Metz-Boutigue, Benoît Frisch, Pierre Schaaf, Dris Ihiawakrim, et al.

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Camille Bergtold, Daniel Hauser, Alain Chaumont, Salima El Yakhlifi, Mihaela Mateescu, et al. Mimicking the Chemistry of Natural Eumelanin Synthesis: The KE Sequence in Polypeptides and in Proteins Allows for a Specific Control of Nanosized Functional Polydopamine Formation. Biomacromolecules, 2018, 19 (9), pp.3693-3704. 10.1021/acs.biomac.8b00818. hal-02185304

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Article

# Mimicking the chemistry of natural eumelanin synthesis: the KE sequence in polypeptides and in proteins allows for a specific control of nanosized functional polydopamine formation

Camille Bergthold, Daniel Hauser, Alain Chaumont, Salima El Yakhlifi, Mihaela Mateescu, Florent Meyer, Marie-Hélène Metz-Boutigue, Benoit Frisch, Pierre Schaaf, Dris Ihiawakrim, Ovidiu Ersen, Christophe A. Monnier, Alke Petri-Fink, Barbara Rothen-Rutishauser, and Vincent Ball

Biomacromolecules, **Just Accepted Manuscript** • DOI: 10.1021/acs.biomac.8b00818 • Publication Date (Web): 30 Jul 2018

Downloaded from http://pubs.acs.org on July 30, 2018

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Mimicking the chemistry of natural eumelanin
synthesis: the KE sequence in polypeptides and in
proteins allows for a specific control of nanosized
functional polydopamine formation

Camille Bergtold ¹,²,*, Daniel Hauser ³,*, Alain Chaumont ⁴, Salima El Yakhlift ², Mihaela Mateescu ¹, Florent Meyer ¹,², Marie-Hélène Metz-Boutigue ¹,², Benoît Frisch ⁵, Pierre Schaaf ¹,⁶, Dris Ihiawakrim ⁷, Ovidiu Ersen ⁷, Christophe A. Monnier ³, Alke Petri-Fink ³, Barbara Rothen-Rutishauser ³ & Vincent Ball ¹,²,#

1: Université de Strasbourg, Faculté de Chirurgie Dentaire, 8 rue Sainte Elisabeth, 67000 Strasbourg, France.

2: Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 1121, 11 rue Humann, 67085 Strasbourg Cedex, France.

3: Adolphe Merkle Institute, University of Fribourg, 4 Chemin des Verdiers, CH-1700 Fribourg, Switzerland.

4 : Faculté de Chimie, Chimie de la Matière Complexe, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7140, 4 rue Blaise Pascal, 67081, Strasbourg, France.

5: Université de Strasbourg, Faculté de Pharmacie, Laboratoire de Conception et application de molécules bioactives.

6: Centre National de la Recherche Scientifique, Institut Charles Sadron, Unité Propre 22, 23 rue du Loess, 67034 Strasbourg, France.
7: Centre National de la Recherche Scientifique, Institut de Physique et de Chimie des Matériaux, Unité Mixte de Recherche 7504, 23 rue du Loess, 67034, Strasbourg, Cedex 2, France.

KEYWORDS: oxidation of dopamine, polydopamine, nanoparticles, specific amino acids, molecular dynamics.

ABSTRACT

The oxidation of dopamine and of other catecholamines leads to the formation of
conformal films on the surface of all known materials and to the formation of a precipitate in
solution. In some cases, it has been shown that the addition of additives in the dopamine
solution, like certain surfactants or polymers, polyelectrolytes and certain proteins allow to get
polydopamine nanoparticles of controlled size and the concomitant decrease, in an
additive/dopamine dependent manner, in film formation on the surface of the reaction beaker.
However, the mechanism behind this controlled oxidation and self-assembly of catecholamines
is not known. In this article, it is shown that a specific diad of amino acids in proteins, namely
KE, allows for specific control in the oxidation-self assembly of dopamine to obtain
polydopamine@protein core shell nanoparticles which are biocompatible. The interactions
between dopamine and the adjacent KE amino acids potentially responsible for the size control
of polydopamine aggregates was investigated by molecular dynamics simulations. The obtained
core-shell nanoparticles display the biological activity of the protein used to control the self-
assembly of PDA. The photon to heat conversion ability of PDA is conserved in the
PDA@protein particles.
Eumelanin in the skin is a hierarchical material made of grains a few hundred nanometers in diameter and the melanins from the cuttlefish *Sepia Officinalis* also display a hierarchical structure. ¹,² These black grains allow for an extremely efficient photoprotection of the skin ³,⁶ Melanins are also important for the pigmentation of the eyes and the hairs. Neuromelanins appear in the substantia nigra of the central nervous system where they play a central role in the protection of neurons against toxic species like metal cations. ⁷⁻⁹ Natural eumelanin is synthesized by the oxidative self-association of L-3,4-dihydroxyphenylalanine (L-DOPA), a non-proteinogenic amino acid obtained through enzymatically catalyzed hydroxylation of L-tyrosine. ¹⁰,¹¹ The eumelanin grains obtained in a biological environment are known to be surrounded by a layer of proteins. ¹² On the other hand *in vitro* synthesis of synthetic eumelanins from L-DOPA solutions yields huge, often polydisperse aggregates. ¹³ Some attempts have been made to get monodisperse eumelanin like particles without adjuvants by playing on the pH of the solution¹⁴ and by adding some co-solvents to water, like ethanol. ¹⁵,¹⁶ The synthesis in the presence of ethanol and ammonia allows to obtain spherical particles 260 nm in diameter. ¹⁵,¹⁶

The properties and size of eumelanin (brown-black) or pheomelanin (yellow-reddish) particles also depend markedly on the nature of used monomers to be oxidized ¹⁷ as well as on the oxidation pathway either auto-oxidation in the presence of dissolved oxygen or enzymatically catalyzed oxidation. ¹⁸ In the latter case the formation of particles around 100 nm in final diameter seems to be a four step hierarchical self-assembly process. ¹⁹

The discovery that adjuvants like poly(vinylalcohol) ²⁰, surfactants ²¹, short tripeptides containing L-tyrosine, L-phenylalanine and L-aspartic acid ²², human serum albumin (HSA) ²³ or
the whole protein pool of egg white<sup>24</sup> allow to considerably reduce the size of the eumelanin like aggregates changed the perspectives on these eumelanin like colloids which may find numerous applications in biomedical applications, materials science<sup>25</sup> and bioelectronics<sup>6,26,27</sup>.

An interesting approach to investigate synthetic analogues of eumelanin is to oxidize dopamine yielding a material conveniently referred as “polydopamine (PDA). When charged surfactants are added to the dopamine solution during oxidation, the deposition of “polydopamine” (PDA) films on solid substrates, a universal coating method<sup>28-30</sup>, was strongly inhibited whereas the size of the particles in solution progressively decreased from a few µm in hydrodynamic diameter to a few nm when the concentration of surfactants (sodium dodecyl sulfate, SDS, and cetyltrimethylammonium bromide, CTAB) was increased above their critical micellar concentration.<sup>21</sup> The size of the obtained PDA particles was found to be only slightly larger than the size of surfactant micelles, suggesting that the production of the eumelanin like PDA material maybe favored in the surfactant micelles.<sup>21</sup>

In the case of Human Serum Albumin (HSA), the obtained results suggest a different mechanism for the control of quite monodisperse PDA formation.<sup>23</sup> HSA is a particular protein being negatively charged at pH = 8.5 at which the PDA nanoparticles were synthesized. In the case of the whole protein extract from egg white, biomelanin nanoparticles from the oxidation of 5,6-dihydroxyindole as small as 15-65 nm in diameter could be obtained but without a detailed knowledge of the proteins implied in the size control from such a complex mixture.<sup>24</sup> However the use of tyrosine containing tripeptides, able to undergo self-assembly during tyrosinase mediated oxidation of L-tyrosine showed the importance of the amino acid sequence in the size and morphology of the eumelanin like aggregates.<sup>22</sup> Hence, it is useful to know if other proteins differing in their size, composition in amino acids (and hence in their isoelectric points) could
play a similar role in controlling the size and in particular if specific sequences encoded
information is required to ensure such a size/shape control of the obtained eumelanin like
particles. To solve such an enigma instead of proceeding in a trial and error manner,
bioinspiration could be of high interest. Indeed, it is the composition of the mussel foot proteins,
rich in L-DOPA and in L-Lysine, which was at the origin of using dopamine to coat the surface
of almost all known materials with so called “polydopamine” films.³¹ In the present
investigation, the inspiration not only came from the protein eumelanin composite particles in the
skin or in *Sepia Officinalis* but also from the chromaffin vesicles secreted by the adrenal
medullary chromaffin cells. Chemical and biochemical analysis revealed that the chromaffin
vesicles are simultaneously rich in catecholamines and in proteins like chromogranin A (CgA)
and B (CgB) ³² which mature upon controlled proteolysis to produce a large repertoire of
regulatory peptides having a broad range of biological activities among them antimicrobial
peptides. ³³ Most often catecholamines are protected from oxidation in such vesicles and the
interactions between CgA and catecholamines govern exocytosis.³⁴ But occasionally in specific
metabolic conditions, an oxidative stress for instance, the vesicles turn brownish implying
oxidation of the present catecholamines.³⁵ The possibility to control the oxidation of
catecholamines leads to the assumption that among the pool of peptide sequences issued from
CgA some of them should play a role in the control of oxidation processes and self-assembly of
catecholamines into eumelanin like nanoparticles. The identification of such short oligo-peptides
(10-20 amino acids) should greatly help in understanding the role played by proteins in the
controlled self-assembly of eumelanin like materials in nano-sized particles.

We will show herein that a specific diad of amino acid in such CgA derived peptides is
sufficient to obtain controlled oxidation of dopamine in a PDA@peptide conjugate. The validity
of this assumption was tested on a set of larger soluble proteins, namely bovine fibrinogen (Fib, pHi= 5.8, Mw=340000 g.mol⁻¹), alkaline phosphatase from porcine mucosa (Alp, Mw= 140000 g.mol⁻¹), human hemoglobin (Hb, pHi=6.95, Mw=64000 g.mol⁻¹), hen egg white lysozyme (HEL, pHi=11.1, Mw=14600 g.mol⁻¹) bovine milk alpha lactalbumin (αLact, pHi=4.3, Mw=14300 g.mol⁻¹) and a mixture of glucose oxidase (GOX, pHᵢ=4.2, Mw= 160000 g.mol⁻¹) and horseradish peroxidase (POX, Mw= 44000 g.mol⁻¹). It could be shown that among these proteins, those that allow to inhibit the deposition of PDA on the walls of the reaction beaker and to simultaneously reduce the size of PDA particles, in a protein/dopamine dependent ratio, all contain the same diad of amino acids, -KE- (K: lysine, E: glutamic acid) than the active peptides derived from CgA. The obtained PDA@protein nanoparticles, with sizes down to 20-30 nm in hydrodynamic diameter, are of the core shell type, non-cytotoxic for different cell types even at unusually high concentrations and can be used as catalysts of biochemical reaction after deposition in films using the Layer-by-layer deposition method.³⁶⁻³⁸

## 2. Materials and methods

### 2.1. Chemicals

Dopamine hydrochloride (ref. H8502), Human serum albumin (ref. A9511), alpha lactalbumin (ref. L5385), bovine fibrinogen (ref. F8630), alkaline phosphatase from bovine intestinal mucosa (ref. P7640), human hemoglobin (ref. H7379), glucose oxidase from *Aspergillus Niger* (GOX, ref. G7141) and Horseradish peroxidase (POX, ref. 8375) were purchased from Sigma Aldrich and used without purification. Hen egg white lysozyme (ref. 5933-D) was purchased from Euromedex (Schiltigheim, France). Poly(allylamine hydrochloride) (PAH, ref 283223, 56000 g.mol⁻¹ as given by the furnisher) was dissolved at 1 mg.mL⁻¹ in 50
Tris(hydroxymethyl) aminomethane buffer (T-1503, Sigma Aldrich). The pH of all solution was measured with a HI221 pH meter (Hanna Instruments).

All aqueous solution were made from Milli Q water (p=18.2 MΩ.cm, RO system from Millipore).

## 2.2 Synthetic peptides and their interactions with dopamine in oxidizing conditions

Chromofungin (Chr) and Catestatin (Cat) are two chromogranin A (CgA)-derived peptides corresponding respectively to the sequence of bovine CgA47-66 (RILSILRHQNLLKELQDLAL) and CgA344-364 (RSMRLSFRARGYGFRGPGLQL). They were provided from ProteoGenix (Schiltigheim, France). Five short peptides including K and E residues were provided by Pepmic (Suzhou, China). They correspond to the sequence KE (peptide 1), GKEG (peptide 2), GGKEGG (peptide 3), GGKGEGG (peptide 4) and GGKGGEGG (peptide 5). These sequences were validated by automated Edman sequencing and by Matrix Assisted Laser Desorption-Time Of Flight (MALDI-TOF) mass spectrometry at the Laboratoire de Spectrométrie de Masse Bio-Organique, UMR7178 (CNRS-UDS) Strasbourg, France. The mass measurements were carried out on a Bruker BIFLEX MALDI-TOF spectrometer (Bruker Daltonics, USA) according to the procedure previously reported.³⁹

## 2.3. Treatment of the peptides with dopamine and HPLC-SEC analysis

For each peptide we used a molar ratio dopamine/peptide of 10 and the reaction was realized in 50 mM Tris-HCl pH 8.5 during 16 h at room temperature before analysis by HPLC-SEC (SEC holds for size exclusion chromatography).

After incubation, the mixture was analyzed by using a Dionex HPLC system (Ultimate 3000; Sunnyvale, CA USA) and a Tosoh Bioscience column (Interchim, Montluçon, France) TSK Gel G3000PWXL (7.8 mm x 300 mm; particle size 7 µm). Absorbance was monitored at 214 nm,
and the eluent consisted in 50 mM Tris-HCl pH 8.5. Material was eluted at a flow rate of 0.7 mL.min⁻¹ by an isocratic program.

## 2.4 Antimicrobial assays

Antibacterial activity of CgA-derived peptides Chr and Cat was tested by measuring the inhibition of bacterial growth.⁴⁰ The Gram positive bacteria (*Micrococcus luteus A270*) was precultured aerobically at 37 °C in Mueller-Hinton Broth Medium (Becton-Dickinson Microbiology Company, Sparks, USA) at pH=7.4. The bacterial strain was plated on the agar plates and cultivated for 24 h at 37°C. After this incubation, one isolated colony was transferred to 10 mL of the Mueller-Hinton Broth medium and incubated for 22 h at 37°C to reach the stationary growth phase. Then the bacteria were suspended and diluted with Mueller-Hinton Broth medium to have a bacterial solution in the first phase of bacterial growth (A620 nm of 0.001; mid logarithmic phase). Finally, we have tested 10 µM to 100 µM of Chr (10 µl) and Cat (10µl) in 90 µl of diluted bacterial solution by using 96-well plates (Sarstedt AG and Co., Nümbrecht, Germany). We used Tetracycline (10µg/ml) and Cefotaxime (0.1µg/ml) as positive controls of growth inhibition. Microbial growth was assessed by the increase of the absorbance at 620 nm after 24 h of incubation at 37°C. The absorbance value control cultures growing without peptide corresponded to 0% of bacterial growth inhibition, while the culture with antibiotics was taken as 100%. Each assay was performed three times.

## 2.5. Synthesis of composite protein-PDA particles and their characterization

The used protein was dissolved at a given concentration in 50 mM Tris buffer (pH = 8.5) and after equilibration to ensure complete solubilization of the protein, this solution was slightly
added to dopamine (H8502 from Sigma Aldrich) to yield a catecholamine solution at 2 mg.mL⁻¹ (10.6 mM). This solution was gently shaken at (25 ± 2)°C during 24 h at 300 rpm with a magnetic stirrer. This procedure may well induce some protein denaturation but is required to allow constant refreshing of dissolved oxygen which is mandatory to oxidize dopamine. The reaction time of 24h was found sufficient to reach a plateau value in the absorbance of the solution at all wavelengths between 250 and 800 nm.²³ Small aliquots of the dopamine + protein mixture were regularly removed and diluted 20 fold with Tris buffer before measurement of the UV-vis spectra. Those spectra were acquired with a double beam mc² spectrophotometer from Safas (Monaco) using Tris buffer in the reference cuvette. The obtained particles will be called PDA@protein particles. The eventually unbound protein and non oxidized dopamine were removed from the reaction medium by dialyzing 25 mL of the suspension against 1L of Tris buffer at 4°C using dialysis membranes with a molecular weight cut off at 300 kDa (Spectrapor) allowing for the diffusion of all the proteins excepted bovine fibrinogen (340 kDa) out of the suspension in the dialysate. The dialysis was done at 4°C in the dark and was repeated twice yielding a total dilution of the dialysate by a factor of 1600. The dialysis step is important to remove eventually unbound proteins from the medium in order to ensure that any biological activity attributed to the proteins originated only from PDA bound proteins. However, the PDA@fibrinogen suspensions were not dialyzed owing to the impossibility to get dialysis membranes with large enough pores.

The undialyzed and dialysed PDA@protein containing solutions were characterized by dynamic light scattering using a Nano ZS device from Malvern working in the backscattering mode to allow the determination of the size distribution of the particles. The intensity autocorrelation function was analyzed with the Contin algorithm.
## 2.6. CryoTEM

Before analyses, a drop of the dialyzed PDA@protein solution was deposited on an electron microscopy grid covered by a hydrophobic carbon membrane. The drop size was progressively reduced in order to obtain a thin film covering the whole membrane. The grid was subsequently plunged into ethane at liquid nitrogen temperature. By maintaining the specimen at this temperature, the grid was transferred on the cryo-holder and inserted in the column of the electron microscope. The specimens have been analyzed in a JEOL 2100F (S)TEM microscope working at 200 kV and equipped by a probe aberration corrector, an EELS (Gatan Tridiem) spectrometer and an EDX (Si-Li) detector. This set-up allows to reach resolutions of 2 Å and 1.1 Å under TEM and STEM modes, respectively. For limiting the irradiation damage, the images were acquired by using a low density of the electron beam.

## 2.7. Molecular dynamics simulations (MD)

The dopamine + peptide systems were simulated by classical molecular dynamics “MD” using the AMBER.14 GPU software in which the potential energy U is empirically described by a sum of bond, angle and dihedral deformation energies and a pair wise additive 1-6-12 (electrostatic + van der Waals) interactions between non bonded atoms.
$$U = \sum_{\text{bonds}} k_b (r-r_0)^2 + \sum_{\text{angles}} k_\theta (\theta-\theta_0)^2 + \sum_{\text{dihedrals n}} \sum_{n} V_n (1+\cos(n\varphi-\gamma)) \\ + \sum_{i<j} \left[ \frac{q_i q_j}{R_{ij}} - 2\varepsilon_{ij} \left( \frac{R_{ij}^*}{R_{ij}} \right)^6 + \varepsilon_{ij} \left( \frac{R_{ij}^*}{R_{ij}} \right)^{12} \right] \quad [1]$$

Each simulated systems is composed of one -GGXGG- peptide, one dopamine molecule and about 3500 water molecules. Force field parameters for the peptide and for the dopamine molecule were taken from the AMBER ff14SB force field. 41,42 Partial charges (Figure SI 2) used for the dopamine molecule were generated by performing RESP fitting 43 of the electrostatically potential computed at the HF 6-31G* level using the GAUSSIAN09 software. The TIP3P model was used for water. 44 Cross terms in van der Waals interactions were constructed using the Lorentz-Berthelot rules. 1-4 van der Waals and 1-4 electrostatic interactions were scaled by a factor of 2. The MD simulations were performed at 300 K starting with random velocities. System temperatures were set using a Langevin thermostat with a virtual collision frequency of 2 ps⁻¹. A time step of 2 fs was used to integrate the equations of motion via the Verlet leapfrog algorithm. The trajectories were analyzed using the cpptraj software 43 and snapshots along the trajectory were taken using the VMD software. 45,46

## 2.8 LBL containing PDA@Alp and PDA@GOX+POX films

30 mL of a aqueous solution of poly(allylamine hydrochloride) at a concentration of 1 mg.mL⁻¹ was prepared in a 50 mM Tris buffer at pH =8.5.

Identically, 30 mL of PDA-Alp or PDA@GOX+POX solution was obtained by adding 20 mL of Tris buffer at pH 8.5 to 10 mL of dyalized nanoparticles, obtained as described previously.
A freshly cleaned Quartz glass slide (ethanol rinsing followed by plasma cleaning during 30 min using an PDC-32G-2 plasma cleaner from Harrick Plasma, USA) was immersed in the PAH solution for 5 min and then rinsed with buffer for 5 min. Next, the glass slide was immersed into the PDA@Alp or PDA@GOX+POX containing nanoparticle solution for 5min followed again with 5min of contact with the Tris buffer. These deposition steps were repeated *n* times to yield (PAH-PDA@Alp)ₙ or (PAH-PDA@GOC+POX)ₙ films. The optimal deposition time was determined by following the deposition process using a E1 quartz crystal microbalance device from QSense (Göteborg, Sweden). The adsorption substrate was a silica coated quartz crystal (QSense). The polyelectrolyte, nanoparticles or buffer solutions were injected by means of a peristaltic pump at a constant flow rate of 250 µL.min⁻¹. The reduced frequency changes, Δf<sub>v</sub>/v, as well as the dissipation changes, ΔD<sub>v</sub>, at the 3<sup>rd</sup> (v=3), 5<sup>th</sup> (v=5) and 7<sup>th</sup> (v=7) overtone were followed as a function of time. When the reduced frequency changes were smaller than 0.5 Hz per minute, the adsorption process of each polyelectrolyte was considered to be finished.

The deposition of PDA or PDA@protein conjugates in alternation with PAH on the surface of
freshly cleaned silicon slides (Siltronix France) was also followed by means of ellipsometry (PZ
2000, Horiba, Longjumeau-France) operating at a constant angle of incidence of 70° and at
λ=632.8 nm. The silicon slides were regularly removed from the reaction medium rinsed with
distilled water and dried under a stream of nitrogen. The film thickness was determined on 5
independently chosen location on 1 cm² coated silicon slide. The ellipsometric angles were
transformed in film thickness assuming a model of a uniform PDA film with a refractive index of
1.73 ± 0.02i at λ=632.8 nm.⁴⁷

The morphology of the (PAH@PDA@Alp)g and (PAH@PDA@GOX+POX)g films was
investigated directly on the quartz crystals used for the QCM-D experiments. The coated face of
the quartz crystal was rinsed with distilled water (to avoid for the deposition of salt crystals issued from the buffer solution) and gently dried under a stream of nitrogen before gluing the crystal on the J piezoelectric scanner of the used Atomic Force Microscope (Nanoscope III, Veeco). The surface was imaged in the dry state in contact mode at a frequency of 1Hz applying the lowest possible deflection set point and using an MLCT-C cantilever with a nominal spring constant of 0.1 N.m⁻¹.

## 2.9. Enzymatic assays on the PDA@Alp and PDA@GOX+POX containing films

The activity of the (PAH@PDA@Alp)₈ films was measured by immersing the quartz slide covered with the film in 4 mL of paranitrophenyl phosphate (PNP) solution at 3 x 10⁻⁵ mol.L⁻¹ in the presence of 50 mL Tris buffer at pH = 8.5. The absorbance increase due to the formation of paranitrophenol was measured at λ = 405 nm with respect to a reference cuvette containing PNP at the same concentration (in order to compensate for the spontaneous hydrolysis of PNP).

Coupled enzymatic reactions used in this study consist in conversion of glucose and O₂ by GOX in D-glucono-∂ lactone and H₂O₂. Then, POX ,using, the H₂O₂ produced from the hydrolysis of glucose, oxidizes the fluorescent peroxidase substrate coloring in a purple colored product.

To that aim, the glass slides covered with (PAH-PDA@GOX+POX)₈ films were introduced in the plastic cuvette containing 4 mL of 1mg.mL⁻¹ glucose in 50 mM Tris buffer (pH 8.5) and 80µL of fluorescent peroxidase substrate. The reaction was followed by monitoring the absorbance at λ=570 nm, during 1h, at room temperature.
At the end of experiment, the quartz lamella was intensively washed with buffer and kept at 4°C before the next measurement of enzymatic activity (after 7 days or 14 days of storage).

## 2.10. Evaluation of the cytotoxicity of protein-PDA particles

J774A.1 mouse monocyte/macrophages and MRC-5 human fibroblasts were cultured in RPMI (Gibco) supplemented with 10% FBS (Gibco), 1% penicillin / streptomycin (Gibco) and 1% L-Glutamine (Gibco) or Modified Eagle Medium (Gibco) with added 10% FBS (Gibco), 1% penicillin / streptomycin (Gibco), 1% L-Glutamine (Gibco) and 1% non-essential amino-acids (Gibco) respectively. The cell lines were incubated at 37°C and 5% CO₂.

To assess possible cytotoxicity, either 5 x 10⁴ fibroblasts or macrophages were seeded in each well of an 8-well slide (BD Falcon™ 8 Chamber Polystyrene Vessel Tissue Culture Treated Glass Slide). To take the substantially slower proliferation of fibroblasts into account, the incubation was prolonged to 96h, whereas the fast-growing macrophages were exposed after 24h. The supernatant was then replaced with 480 µl media containing different concentrations of PDA@HSA nanoparticles and the slides were incubated again for 24h. Subsequently, the supernatant was collected and stored for the quantification of lactate dehydrogenase (LDH) activity, and 2.2 mg.mL⁻¹ resazurin solution was added instead. After additional 3h of incubation, the amount of resorufin, a metabolic product of resazurin, was measured by its specific absorption.

## 2.11. Illumination of the PDA@HSA suspensions with UV-light and measurement of temperature changes.

To measure the PDA@HSA nanoparticles' ability of heat generation by light irradiation, a common property of eumelanin particles¹⁶,⁴⁸, the following arrangement was used: different
amounts of PDA@HSA nanoparticles in water were added to a cuvette. The cuvette was placed in between an UV Detector (Hönle UV technologies) and an UV laser (Hönle UV technologies, bluepoint 4), equipped with a filter allowing only light with a λ of 320 – 390 nm to pass. After set up mounting, the surrounding box was closed due to safety reasons and the laser was activated for 100 s, irradiating the sample with 0.9W.cm⁻². Meanwhile, an IR camera (Optris PI) facing the cuvette recorded the temperature increase and the UV detector monitored the absorption (Absorption<sub>Sample</sub> = 0.9W/cm² – irradiation measured by detector for mounted sample).

### 3. Results and discussion

After identifying a target sequence from CgA derived peptides to control the synthesis of PDA nanoparticles, some model peptides containing this sequence and some variants thereof were tested. The molecular origin of the control exerted by the selected amino acid diad will then be investigated by means of molecular dynamics simulations. Proteins containing or not this diad will then be investigated for their ability to control the self-assembly of PDA and the structure – properties of those PDA@protein nanoparticles will be investigated in the last section.

3.1 Identification of a specific amino acid diad able to control the formation of PDA particles.

When dopamine is mixed with different peptides issued from the controlled proteolysis of CgA, its oxidation yields a brown-black solution in all cases. However in the presence of some peptides issued from the proteolysis of CrgA, the occurrence of huge aggregates issued from the
oxidation of the catecholamine is detected by means of visual observation of the solution (Figure 1) whereas for other peptides from the same origin, the suspension remains stable for days. For example, in the presence of bovine chromofungin (Chr, RILSILRHQNLLKELQDLALQGAK) some aggregates including dopamine elute with a large peak (14-18 min) detected at 280 nm and corresponding to a molecular weight of 120.000 to 70.000 Da (Figure 1A). In contrast, in the presence of bovine catestatin (Cat, RSMRLSFRARGYGFRGPGLQL) the aggregates including dopamine were not detected meaning that they are eluted in the free volume or too large to travel along the size exclusion column (Figure 1A).
A

Chr: RILSILRHQNLLKELQDLALQGAK

Cat: RSMRLSFRARGYGFRGPGLQL

<figure><img src="image_2.png" /></figure>

B

a

% of inhibition

<figure><img src="image_3.png" /></figure>

b

% of inhibition

<figure><img src="image_4.png" /></figure>
**Figure 1:** A: Size exclusion chromatography (SEC) profiles of dopamine mixed with Tris buffer (pH = 8.5) alone and in the presence of Chr and Cat. The inset digital pictures show that PDA formation occurs in all cases after 16h in the presence of O₂ from the air. The detection wavelength was set at 280 nm.

B: Evolution of bacterial growth inhibition as a function of the peptide concentration (Cat in row a, Chr in row b) in the absence and in the presence of dopamine (the peptide / dopamine ratio was constant and equal to 1/10 in all experiments). The ** symbol means that the inhibition is statistically not different from zero.

In addition, we investigated if the interaction of the 2 antimicrobial peptides Cat and Chr combined with dopamine may modulate their antimicrobial activities against *Micrococcus luteus*. For Chr that allows for the production of stable PDA suspensions we noted the disappearance of its antibacterial activity (Figure 1Ba). In contrast, for Cat (the peptide not able to control the aggregation process) the antimicrobial activity (lytic effect) was maintained (Figure 1Bb). These findings strongly suggest that the active peptides in the control of PDA formation are not available to contact the bacteria, most probably due to their strong interactions with the forming PDA in oxidizing conditions at pH = 8.5. There is hence strong evidence that Chr either loses its activity when PDA formation occurs or more probably that it is bound to PDA particles in a non-active manner. Indeed proteins are known to bind to quinone groups of PDA via a nucleophilic attack of their amino groups. 48,49 On the other hand, Cat remains fully active, strongly suggesting that it does not interfere with the formation of PDA.

Additional experiments were then performed with the single model peptides GKEG, GKGEG and GKGGEG peptides in which the K and E amino acids were directly adjacent to each other or separated with one or two glycine residues, glycine acting as a spacer amino acid.
Glycine was chosen as a spacer because of its low steric constraints and because it will not affect the solubility of the peptide in water. By using size exclusion chromatography (SEC) with detection at 214 nm, the peptides GGKEGG, GGKGEGG and GGKGGEGG were eluted at 26 min, 23.5 min and 19.5 min respectively (Figure SI1). After incubation with dopamine, only the peptide GGKEGG controls the PDA aggregation with the formation of a complex eluted at 20.5 min. Indeed, it appears that a single glycine residue (G) between K and E was critical for the production of stable PDA suspensions (a low difference in the time of elution 23.5 min/22.5 min). This data was validated by the peptide GGKGGEGG including 2 G between K and E and the absence of variation in the time of elution for the peptide alone (19.5 min) and the peptide treated with dopamine (19.5 min). We also tested the single dipeptide KE (dissolved at 1 mM) in its ability to control the size of PDA nanoparticles : it plays a role producing however larger particles , in the 300-500 nm diameter range, than the GKEG peptide at the same concentration. In this latter case the hydrodynamic diameter of the PDA particles is in the 100-200 nm range. This means that the positively charged amino and negatively charged carboxyl end groups of the KE containing peptides have to be sufficiently far away from the central KE diad for allowing an optimized control in the size of PDA (data not shown). It has to be noted also that an equimolar mixture (1 mM each) of K and E amino acids is inefficient in controlling the size of PDA: in this control experiment a precipitate of PDA was obtained from a 2 mg.mL⁻¹ dopamine solution after 24 h of oxidation as in the absence of amino acids. This shows that K and E have to be covalently bound to exert a templating effect in the assembly of PDA particles.

## 3.2 Molecular dynamics

To give some physical insight into the probable role of KE containing sequences in the control of PDA self-assembly in oxidizing conditions, we performed molecular dynamics (MD)
simulations to estimate how dopamine itself interacts with such sequences. We arbitrarily select dopamine as a target molecule and not 5,6 dihydroxyindole (DHI) because the later one undergoes oligomerization whose description would require ab-initio MD simulations which is beyond the scope of this study. These simulations are hence only able to describe the first events occurring when dopamine and the target peptides are mixed and totally neglect the subsequent (and fast) oxidation of dopamine.

We will focus on a set of small model peptides of the N-GGXGG-C type with X = KE, EK, KD or KGE (as those issued from the degradation of ChrA). All molecular dynamics simulation started with the dopamine molecule at about 20 Å away from the center of mass of the peptide. During the dynamics we observe that the dopamine molecule is found in close contact with the peptide about 20 – 30 % of the time. The average time during which this dopamine – peptide pair is formed *increases* in the order N-GGEKGG-C < N-GGKDDGG-C < N-GGKGEGG-C < N-GGKEGG-C. (0.131 *ns*, 0.201 *ns*, 0.219 *ns* and 0.246 *ns* respectively). Correspondingly the maximum lifetime during which dopamine interacts with the peptide follows the same trend (7.66 *ns*, 9.85 *ns*, 12.81 *ns* and 23.85 *ns*, respectively), pointing to the fact that the dopamine molecule interacts the strongest with the N-GGKEGG-C peptide.

A closer look at the specific interactions between the dopamine molecule and the various peptides reveals the hydrogen bond formation between the –OH or –NH₃⁺ protons of the dopamine molecule with the different carboxylic functions of the peptides situated either on the central E or D residues or on the C-terminus. Hydrogen bonds via the dopamine –NH₃⁺ group protons happens however less than 0.5 % of the time and hence will not be discussed anymore here.
In the case of the N-GGKEGG-C peptide, the dopamine molecule seems to favor the interaction with the carboxylic group situated on the central glutamic acid (E) residue over the carboxylic group situated on the C-terminus. (10.5 % vs. 5.8 % of the time). The reason for the preference of the dopamine molecule interacting with the central glutamate residue certainly comes from the fact that when coordinated to this carboxylic group, the $NH_3^+$ group of the nearby lysine residue moves towards the benzene ring of dopamine molecule which then further stabilizes this close contact pair via cation-π interactions (Figure 2).

One reason for the lack of cation-π interactions in the case of the GGKGEGG compared to the GGKEGG system may be due to entropy effects. In fact, adding a G residue between residues K and E increases the flexibility of the system and in particular increases the amplitude of the movement between the L-lysine and L-glutamic acid residues. However, to be able to form cation-π interactions the $NH_3^+$ group of the L-lysine residues has to be close to the region where the dopamine molecule docks on to the $COO^-$ group of the glutamic acid. This is more likely the case for the less flexible GGKEGG than for the GGKGEGG peptide, hence the chances of forming cation-π interactions are higher in the case of the GGKEGG peptide than for the GGKGEGG peptide.

On the other hand in the case of the 3 remaining peptides (N-GGEKGG-C, N-GGKDGG-C and N-GGKGEGG-C) the dopamine is seen to interact more often with the $COO^-$ group on the C-terminus than with the one on the central E or D residues. (2.8 % vs. 7.1 %, 8.2 vs. 13.5 % and 6.2 % vs. 8.4 %, respectively). This shift in tendency stems from various reasons. In the case of the N-GGEKGG-C peptide we observe the formation of hydrogen bonds of the central glutamic acid with the terminal N-terminal $NH_3^+$ group, making it less accessible to the dopamine molecule. In the case of the N-GGKDGG-C peptide the shorter alkyl chain (compared
to the E residue) makes it less flexible and the COO⁻ group less accessible to the dopamine molecule and hence favors the hydrogen bond with terminal COO⁻ group. Finally, in the case of the N-GGKGEGG-C peptide, we do no longer observe cation-π interactions and hence interactions with the central group are no longer favored.

<figure><img src="image_5.png" /><figcaption>**Figure 2:** Top: Population analysis in the case of (N-GGKEGG-C (**left**) and N-GGKGEGG-C (**right**), as a function of the distance between the center of mass (CoM) of the benzene ring and the N atom of the NH₃⁺ group of the lysine residue and between the C atom of</figcaption></figure>
the COO⁻ of the glutamic acid (E) residue and the center of mass of both -OH groups of dopamine. Green colored regions correspond to populations that are often observed, blue colored regions corresponds to populations that are seldomly observed, white colored regions population that are never observed.

Bottom: Typical snapshot of the dopamine – N-GGKEGG-C pair corresponding to the 2 most observed populations.

In summary we can say, that there is a tendency for the dopamine molecule to interact via hydrogen bonding between the -OH protons of dopamine with the central E (or D) residue or with the terminal carboxylic group. The former interaction are further stabilized by cation – π interaction of the terminal $NH_3^+$ group of the lysine with the benzene ring of dopamine in the case of the N-GGKEGG-C peptide.

### 3.3 Influence of the KE diad present in proteins to control the formation of PDA particles.

These qualitative experimental results and interaction energies-residence times data from MD simulations incited us to investigate a series of proteins to control the aggregation process of PDA, in addition to HSA which has already been shown to allow for a concentration dependent inhibition of PDA deposition on the walls of the reaction beaker and a reduction of the particles hydrodynamic diameter.²³ It has to be noted that HSA contains 2 KE motifs in its amino acid sequence (see Figure 7 in the Supporting Information).

Among the investigated proteins, two categories emerge:

(i) those which do not influence the PDA deposition on surfaces and its assembly in solution (Hen-egg white lysozyme and α-lactalbumine) as shown in Figure 3 and 4 in the Supporting Information.
(ii) and those which simultaneously inhibit film deposition on surfaces and allow for a reduction in particle size after 24 h of oxidation in the presence of dissolved air at pH 8.5. Among this second category of proteins: human fibrinogen (Figure 5 in the Supporting Information), alkaline phosphatase (Figure 3) and a mixture of glucose oxidase and horseradish peroxidase (Figure 6 in the Supporting Information). A close look at the amino acid sequence of those proteins shows that they all contain the KE diad of amino acids as the CgA derived peptides (Figure 7 in the Supporting Information). The case of human hemoglobin is surprising: even if it contains the KE diad (Figure 7 in the Supporting Information) exposed to the solvent, this protein does not exert the expected controlled inhibition of PDA deposition on the walls of the reaction beaker and the expected reduction in the size of PDA aggregates (Figure 8 in the Supporting Information). However, a closer examination reveals that the K residue in the unique present KE diad is postranslationally modified in this protein, making the KE diad sterically unattainable to dopamine. This important result means that the KE diad has not only to be exposed to the solvent to exert control in the synthesis of PDA but also that the amino acids have to be chemically unmodified.

All our findings are summarized in Table 1:
<table><caption>Table 1: Summary of the role played by diverse proteins in the size control of PDA particles.</caption><thead><tr><th>Protein</th><th>Number of KE diads in the protein</th><th>Ability to control the size of the PDA particles</th></tr></thead><tbody><tr><td>Human serum albumin</td><td>2</td><td>Yes</td></tr><tr><td>Alkaline phosphatase from porcine mucosa</td><td>3</td><td>Yes</td></tr><tr><td>Mixture of Glucose oxidase and peroxidase</td><td>1 KE in each protein</td><td>yes</td></tr><tr><td>Bovine fibrinogen</td><td>2</td><td>yes</td></tr><tr><td>Human Hemoglobin</td><td>1 but postranslationally modified</td><td>no</td></tr><tr><td>Hen egg white lysozyme</td><td>0</td><td>no</td></tr><tr><td>bovine milk alpha lactalbumin</td><td>0</td><td>no</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>**Figure 3:**</figcaption></figure>

A: Deposition of PDA on quartz plates monitored at λ=500 nm as a function of the oxidation time at pH = 8.5: in the absence of Alp (●) and in the presence of Alp at 0.1 (○) and 0.5 (■) mg.mL⁻¹. Alp was also preadsorbed during 1h from a solution at 1mg.mL⁻¹ before the beginning of dopamine oxidation but in the absence of ALP (●).
B: Evolution of the hydrodynamic radius of PDA particles after 24 h of oxidation at pH = 8.5 in the presence of Alp at different concentrations. The initial dopamine concentration was of 2 mg.mL⁻¹ in all experiments.

C: Representative TEM micrographs of the PDA@Alp particles obtained after 24 h of oxidation at pH= 8.5 from a 2 mg.mL⁻¹ dopamine solution in the presence of ALP at 0.5 and 2.0 mg.mL⁻¹.

D: TEM micrograph PDA@Alp particles obtained after 24 h of oxidation at pH= 8.5 from a 2 mg.mL⁻¹ dopamine solution in the presence of Alp at 2.0 mg.mL⁻¹.

E: Size distribution of the PDA@Alp nanoparticles (n=322) obtained after 24 h of oxidation at pH= 8.5 from a 2 mg.mL⁻¹ dopamine solution in the presence of Alp at 2.0 mg.mL⁻¹. The average size is of (36.1 ± 4.5)nm.

F: High resolution image of an individual PDA@Alp nanoparticle.

The obtained PDA-Alp composite nanoparticles display a core-shell morphology as shown by cryo-TEM images in the case of PDA@ALP nanoparticles (Figure 3F).

When then coming back to one of our selected biomimetic systems, the eumelanin grains in the skin which are known to be associated with proteins, it comes out that those proteins also contain KE diads. ¹² Based on all these results from the CgA derived peptides, from MD simulations and from globular proteins, we have strong evidence that the KE diad of amino acids interacts specifically with dopamine to control its oxidation and subsequent self-assembly process and that this diad of amino acids may be required to control the self assembly of PDA from dopamine. At this stage of our research we may however not exclude that other specific sequences of amino acids could control the self-assembly of PDA from dopamine solutions. Note that proteins can conjugate to quinone groups of PDA via nucleophilic groups like those on L-lysine residues. ⁴⁹ But in this investigation, dopamine is added in the protein solution and the proteins are not put in contact with already formed PDA. Hence we believe that the control of PDA size is exerted by the interactions between the KE diads with dopamine at the beginning of the oxidation process
and not via conjugation of the proteins on already formed PDA aggregates. This is our model but further proofs are required.

The influence of the KE containing peptides and proteins on the size of PDA particles is clearly dependent on the KE/dopamine ratio mixture (Figure 3B and Figure 5 and 6 in the Supporting Information). Above a critical ratio we assume that most of the dopamine molecules and their oxidation products interact with the introduced peptide/protein and are not available anymore to produce a PDA coating at the surface of the reaction vessel.

### 3.4 Applications and biocompatibility of PDA@protein nanoparticles.

Those protein capped PDA nanoparticles can be co-immobilized with polycations like poly(allyl amine hydrochloride) to yield polyelectrolyte multilayer films<sup>36-38</sup> displaying a linear growth with the number of deposition cycles of PDA@protein and PAH (Figure 4 A), a smooth morphology (Figure 4B) and stable enzymatic activity upon storage at 4 °C (Figure 4C). Even more interesting, when dopamine is oxidized in the simultaneous presence of glucose oxidase (GOX) and peroxidase (POX), the PDA@GOX+POX nanoparticles (Figure 6 in the Supporting Information) immobilized in polyelectrolyte multilayers can be used as a glucose sensor in the simultaneous presence of Glucose and gaïacol (Figure 4D). As for PDA@Alp, the enzymatic activity is very robust upon storage with only 30% of decrease in the relative enzymatic activity after 14 days of storage (Figure 4D).
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

**Figure 4:** Deposition of PDA nanoparticles capped with enzymes (Alkaline phosphatase or a mixture of Glucose oxidase and peroxidase) in polyelectrolyte multilayer films and demonstration of long term conservation of their enzymatic activity.

A: Deposition of PDA@ALP nanoparticles (made in the presence of 1 mg.mL⁻¹ enzyme during 24 h, the excess enzyme is removed by dialysis) with an average diameter of 100 nm (Figure 3B) in alternation with poly(allylamine hydrochloride) (PAH) to yield a polyelectrolyte multilayer film. The deposition process was followed by means of QCM-D. The green and pink vertical lines correspond to the injection of PDA@Alp and PAH respectively.
B: Surface morphology of a (PAH-PDA@Alp)₈ film obtained by AFM in the dry state and in contact mode. The rms roughness over this 10 µm x 10 µm topography is of 39 nm.

C: Initial hydrolysis rate of paranitrophenolphosphate (PNP) put in contact with a (PAH-PDA@Alp)₈ film as a function of the storage time: day 4 (○), day 9 (●), day 12 (●), day 15 (●), day 19 (◆) and day 21 (◆). The straight lines correspond to linear regressions to the data. The slope of those lines is divided by the slope obtained immediately after film preparation to yield the relative enzymatic activity (in the inset).

D: initial rate of oxidation of the peroxidase substrate, gaiacol, measured on a (PAH-PDA@GOX+POX)₈ polyelectrolyte multilayer film when the film is put in contact with glucose (1 mM) and the peroxidase substrate (1mM) after different storage times: day 0 (○), day 7 (■) and day 14 (▲). The straight lines correspond to linear regressions to the data. The slope of those lines is divided by the slope obtained immediately after film preparation to yield the relative enzymatic activity (in the inset).

Note that PDA nanoparticles containing ALP or GOX+POX display the activity of the used enzyme whereas PDA nanoparticles prepared in the presence of the antimicrobial chromofungin do not display the expected activity. The difference between both situations may come from the difference in size of the used capping agents: the pretty small chromofungin peptide lies probably flat of the surface of the PDA nanoparticles with its antimicrobial sequence not available to the bacteria in contact with the nanoparticles whereas the large enzymes are in contact with the surface of the nanoparticles with only a small fraction of their amino-acids, almost non affecting their average tertiary structure.

Finally the biocompatibility and heat-induction upon UV light activation of PDA@HSA nanoparticles (synthesized during 24 h in the presence of 1 mg.mL⁻¹ protein) was tested in order to evaluate their potential for biomedical applications. The particles do not display any
measurable cytotoxicity for two cell types tested as mouse monocyte/macrophages (i.e. J774.A1 cell line) and human fibroblasts (i.e. MRC-5 cell line) (Figure 5B). Two different assays have been used, the LDH and the MTT assay, in order to exclude an unspecific interference of one assay with the nanoparticles. In addition, the PDA@HSA nanoparticles are able to convert UV light into heat in a particle concentration dependent manner as expected for a material closely related to eumelanin (Figure 5C). Hence the presence of the proteins does not hinder the photothermal effect already characterized for PDA nanoparticles (25-43 nm in diameter) synthesized in reverse microemulsions.⁵⁰

<figure><img src="image_11.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_16.png" /></figure>
**Figure 5:**

A,B, C, D: Cytotoxicity evaluation of PDA@HSA nanoparticles in macrophages and human fibroblasts as a function of the PDA@HSA concentration (as indicated on the right) by LDH (A, B) and MTT assay (C, D). The positive control Triton X-100 induced significant cytotoxicity in the LDH assay in both cell types.

E: The PDA@HSA suspension heats up in a concentration-dependent manner as shown by quantification with an IR camera.

F: Temperature increase of PDA@HSA containing solutions upon exposure to UV-light as a function of the concentration in nanoparticles.

The PDA@HSA nanoparticles were produced by oxidizing dopamine (2 mg·mL⁻¹) in the presence of HSA at 1 mg·mL⁻¹ during 24 h in the presence of 50 mM Tris buffer. The nanoparticles were dialyzed against Tris buffer before use.

**Conclusions**

Stable Polydopamine nanoparticles can be easily synthesized in the presence of proteins containing the KE diad of amino acids in their primary structure. The importance, but probably not the exclusivity of these two amino acids, has been highlighted with small peptides issued from the proteolytic cleavage of CrgA issued from the chromaffin rich vesicles of the surrenal gland as a biomimetic system. In addition small synthetic peptides containing the KE diad allow for a similar control in the oxidation of dopamine and its subsequent self-assembly. Molecular dynamics simulations have provided a molecular explanation for the control exerted by the KE sequence: the hydroxyl groups of the catechol moiety in dopamine form hydrogen bonds with the
carboxylic group of glutamate (E) whereas the protonated amino group of the lysine (K) forms a cation-π interaction with the aromatic ring of dopamine. These interactions are strongly decreased when a single glycine residue is introduced as a spacer between the lysine and glutamate residues.

The importance of the protein-dopamine interactions are manifested by a protein concentration dependent reduction in the size of the obtained core shell-nanoparticles as well as by a progressive inhibition in the deposition of a polydopamine film on the wall of the reaction beaker. The presence of proteins on the shell of the particles is demonstrated by the enzymatic activity of the obtained nanoparticles in the case of alkaline phosphatase and the glucose oxidase-peroxidase mixture. At this level of our research we cannot exclude that some proteins are also present in the polydopamine rich core of the obtained nanoparticles. Those particles are non cytotoxic up to unusually high concentrations and are able to convert UV light in heat as expected for a polydopamine, eumelanin like, material. We will exploit the biological potential of the presence of specific proteins in the shell of the particles and the eumelanin photon to heat conversion potential of the core in forthcoming work.

## ASSOCIATED CONTENT

**Supporting Information.** Size exclusion (SEC) chromatograms of the GGKEGG, GGKGEGGG and GGKGGEGGG peptides and of the same peptides with added dopamine in the presence of Tris buffer (pH = 8.5), schematic representation of the dopamine molecule and the RESP charges used during MD simulation, influence of different proteins (lysozyme, α-lactalbumin, human fibrinogen, glucose oxidase + peroxidase and human hemoglobin) on the absorbance at λ=500
nm of dopamine solutions after 24 h of oxidation at pH = 8.5 and amino acid sequence of the
used proteins where the KE diads are highlighted in red.

**Corresponding Author**

*vball@unistra.fr

ACKNOWLEDGMENT

This study was supported by the Swiss National Science Foundation through the National Center
of Competence in Research Bio-Inspired Materials, the Adolphe Merkle Foundation and the SNF
visitor grant (IZK0Z2_161420 / 1) for VB. AC thanks the HPC - Center of the Universié de
Strasbourg for computing time.

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Oxidation of dopamine at pH 8.5 +
peptides/proteins containing **KE**

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

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polydopamine
nanoparticles

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