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3D arrays on quantum dots embedded within a bio-inspired template studied by SAXS

Etienne Henry, Aurélien Dif, M. Schmutz, Loïc Legoff, François Amblard,

Valérie Marchi, Franck Artzner

To cite this version:

Etienne Henry, Aurélien Dif, M. Schmutz, Loïc Legoff, François Amblard, et al.. 3D arrays on quantum dots embedded within a bio-inspired template studied by SAXS: SWING beamline Crystallization of fluorescent quantum dots within a 3D bio-organic template of actin filaments and lipid membranes. 2011, pp.34-35. hal-04411481

Generating new physical properties using the crystallization of nanoparticles is highly challenging. The design of such new materials with unexpected physical properties is thus the major motivation in the investigation of new strategies for controlling the crystallization of nanoparticles [1]. In this context, biological molecules and molecular self-assemblies are promising templates to organize well-defined inorganic nanostructures [2]. This approach allows the spontaneous formation of structures of well-defined shapes and monodisperse characteristic sizes [3]. The SWING Beamline have been designed and is powerful to study these bottom-up nanomaterials by Small Angle X-ray Scattering, SAXS. SWING is a highly versatile setup whose the investigated distances can vary from 0.5 to 500 nanometers. For example, we recently studied by SAXS on SWING a library of 17 peptides forming nanotubes of monodisperse diameter ranging from 9 to 36 nm [4].

Here we report on the ability of a self-assembled threedimensional crystal template of helical actin protein filaments and lipids bilayers to generate a hierarchical self-assembly of quantum dots (Figure 1). With this goal, functionnalized tricystein peptidic quantum dots (QDs) are incorporated during the dynamical self-assembly of this actin/lipid template through electrostatic interaction. This slow mixing results in the formation of crystalline fibers (Figure 2). This process ensures the high quality of the crystal. We demonstrate the formation of a very well-defined 3D crystal of QDs by SAXS performed with Florian Meneau on SWING (Figure 3). The crystal parameters, 26.5 x 18.9 x 35.5 nm 3 , are imposed by the membrane thickness, the diameter, and the pitch of the actin self-assembly. In a first simple approach, we used only lipid multilayers to insert water soluble functionalized QDs [5] and the 3D order was lost. The inclusion of extraneous objects providing additional well-defined length scales such as actin is necessary to create 3D order. The fluorescence properties of the 3D crystals of quantum dots demonstrate a direct effect of the nanostructuration of the quantum dots within the crystal.

Such a formation of 3D arrays of nanoparticles opens new route toward optical materials that are easily prepared by using self-assembling of vesicles, proteins, and hydrophilic nanoparticles in aqueous environment. This strategy could be extended to any kind of hydrophilic nanoparticles with various morphologies. Furthermore, the range of characteristic lengths can be extended by using other biological materials.

3D arrays on quantum dots embedded within a bio-inspired template studied by SAXS

SWING beamline

Crystallization of fluorescent quantum dots within a 3D bio-organic template of actin filaments and lipid membranes E. Henry, A.

Figure 1 :

 1 Figure 1: Schematic view of the structure. Strong electrostatic interactions between anionic microfilaments (red) and cationic lipids results in large undulations of the lipid bilayers (blue). Quantum Dots (Green) are embedded between actin as well as lipid membranes.

Figure 2 :

 2 Figure 2: Self-assembly mechanism: (i) the electrostatic adsorption of anionic quantum dots QD and actin proteins onto the cationic membranes; (ii) the actin polymerisation and (iii) the rupture of cationic vesicles by adhesion onto the anionic filaments.

Figure 3 :

 3 Figure 3: Powder diffraction observed by SAXS experiments performed with a 3m sample-detector distance at SWING@SOLEIL and the corresponding peak indexations in a 3D centered orthorhombic lattice. The HWHM of the Bragg peaks (<0.004Å -1 ) shows a positional order larger than 3000Å.


Said Jebors

Coline Pinese

Benjamin Nottelet

Karine Parra

Muriel Amblard

Ahmad Mehdi

Jean Martinez

Gilles Subra
email: gilles.subra@univ-montp1.fr

Turning peptides in comb silicone polymers §

Keywords: hydroxysilyl peptides, peptide materials, silicone, comb-polymers, polysiloxane

We have recently reported on a new class of silicone-peptide`biopolymers obtained by polymerization of di-functionalized chlorodimethylsilyl hybrid peptides. Herein, we describe a related strategy based on dichloromethylsilane-derived peptides, which yield novel polymers with a polysiloxane backbone, comparable with a silicone-bearing pendent peptide chains. Interestingly, polymerization is chemoselective toward amino acids side-chains and proceeds in a single step in very mild conditions (neutral pH, water, and room temperature). As potential application, a cationic sequence was polymerized and used for antibacterial coating.

Introduction

Polysiloxanes, well known as 'silicones', are biocompatible, bioinert, and biodurable materials extensively used since the 1960s in medicine [START_REF] Willoughby | Silicones today[END_REF] especially as medical device components for the fabrication of catheters, drains, shunts, orthopedics, and implants and also as injectable fluids for reconstructive surgery [START_REF] Chasan | The history of injectable silicone fluids for soft-tissue augmentation[END_REF]. Despite their outstanding properties such as resistance to temperature and chemicals, high flexibility and elasticity, there is still a need for functional silicones whose properties can be adjusted by the addition of a bioorganic moiety. Sugar-silicon [START_REF] Beppu | Synthesis of sugarpolysiloxane hybrids having rigid main-chains and formation of their nano aggregates[END_REF] or poly(L-lactic acid)silicon hybrids [START_REF] Maeda | Control of silicon species released from poly(lactic acid)-polysiloxane hybrid membranes[END_REF] have been reported for cell recognition and bone tissue regeneration [START_REF] Colilla | Amino-polysiloxane hybrid materials for bone reconstruction[END_REF]. The association of silicone and peptides has also been proposed for improved cell-material interactions or detection purposes, but it has been mostly limited to functionalize silicone surface with short peptides [START_REF] Davis | Immobilization of RGD to < 1 1 1 > silicon surfaces for enhanced cell adhesion and proliferation[END_REF][START_REF] Sam | Peptide immobilisation on porous silicon surface for metal ions detection[END_REF]. To date, only few works report on the direct synthesis of peptide-silicon hybrids by a bottom-up approach. To the best of our knowledge, the only published study deals with silicones incorporating random homopolypeptide sequences obtained by ring opening polymerization of amino acids NCAs initiated by a poly(dimethylsiloxane) [START_REF] Kania | Preparation of poly(dimethylsiloxane)-polypeptide block copolymers[END_REF].

We recently reported on a strategy yielding siloxane polymers of well-defined peptide sequences starting from hybrid dimethylhydroxysilane-derivatized peptides [START_REF] Jebors | A new way to silicone based peptide polymers[END_REF]. In contrast to linear peptide polymers, which were obtained via N-terminal and C-terminal modifications, comb-peptide polymers were synthesized by addition of a lysine residue at the N-terminus, which was derivatized at its α and ε primary amino groups by isocyanopropyl dimethylchlorosilane. We suggest here a simple way to obtain comb-polymers of peptide sequences using methyldihydroxysilyl derivatives (Figure 1).

Material and Methods

Reagents

All solvents and reagents were used as supplied. Solvents used for Liquide Chromatography/Mass Spectrometry (LC/MS) were of HPLC grade. Fmoc-amino acid derivatives, HBTU, and Fmoc-Rink amide ChemMatrix resin were purchased from Iris Biotech (Marktredwitz, Germany). DIEA and DMF, were obtained from Sigma-Aldrich (St. Louis, MO, USA). ICMDCS was obtained from Sikemia (Montpellier, France).

Synthesis

Example of solid phase peptide synthesis

The synthesis of H-Arg-Arg-Leu-Leu-Leu-NH 2 was performed by Fmoc/tBu SPPS [START_REF] Amblard | Methods and protocols of modern solid phase peptide synthesis[END_REF]. H-Rink amide ChemMatrix Resin 0.49 mmol/g was swollen for 15 min in DCM. Fmoc deprotection was carried out with 20% piperidine in DMF for 20 min. Beads were washed with DMF (three times). About 2.5 ml of a 0.2-M solution of Fmocamino acid in DMF (5 eq.), 1 ml of a 0.5-M solution of HBTU (5 eq.) in DMF, and 0.5 ml of a 2-M solution of DIEA in DMF (10 eq.) were successively added to the resin. The coupling was carried out for 2 h. The resin was washed three times with DMF and once with DCM. After stepwise assembly of the peptide, it was cleaved from the resin with neat TFA for 1 h. After filtration, the solution was concentrated under reduced pressure. The peptide was precipitated with diethyl ether, filtered and dried under high vacuum, and characterized by LC/MS. Procedures for the synthesis of other peptides are described in the Supporting Information Section.

Synthesis of silylated monomers 1-5

Commercially available 3-isocyanatopropyl dichloromethylsilane is used for the introduction of the chlorodimethylsilyl functionality onto peptides via their N-terminus.

Briefly, to the solution of peptide (0.5 mmol) in 500 μl of DMF were added DIEA (6 eq.) and 3-isocyanatopropyl dichloromethylsilane (1.25 eq.). The reaction mixture was allowed to stir for 2 h at room temperature. After 120 min, the reaction was monitored by HPLC. Ether (50 ml) was poured into the reaction mixture to cause precipitation. The precipitate was suspended in ether and recollected. This procedure was repeated three times to remove 3-isocyanatopropyl dichloromethylsilane and DIEA. In the case of hybrid peptide 2, an aliquot was deprotected in TFA/H 2 O/TIS (92.5/5/2.5, v/v/v) solution to yield hybrid peptide 3, which was precipitated by addition of diethylether (Supporting Information). All crude compounds were analyzed by analytical HPLC and LC/MS. It is worth noting that dichlorosilyl derivatives were hydrolysed during workup (or LC/MS analysis), yielding dihydroxysilyl hybrid peptides.

Polymerization

Hybrid peptide blocks 1-5 generated poly[methyl(peptido)siloxane] P1-P5 through condensation mechanism between two SiOH groups with the release of water. Hybrid blocks were solubilized in a 1/1000 v/v TFA/water solution g/ml). Phosphate buffer (pH 7.4) was added dropwise under stirring to slowly increase the pH of the reaction mixture above 7. Condensation occurred during buffer addition and induced precipitation of the polymer. The medium was freeze dried, and polymer was obtained as a white powder or a sticky gel depending on the peptide sequence. The resulting polymers were insoluble in most solvents (i.e. DCM, MeOH, THF, EtOH, and chloroform), whereas P1, P2, and P5 were soluble in DMF.

General Methods

LC/MS analyses

All compounds were analyzed on a Quattromicro (Micromass, Manchester, UK) triple-quadrupole mass spectrometer fitted with an electrospray interface. Chromatography was performed with a C18 reversed-phase column (VWR chromolith column, 50 mm × 3.9 mm): 100% A to 100% B linear gradient over 5 min at a flow rate of 3 ml/min (A: 0.1% TFA in H 2 O; B: 0.1% TFA in CH 3 CN). Detection was performed at 214 nm. Positive ion electrospray mass spectra were acquired at a solvent flow rate of 204 μl/min. Nitrogen was used for both the nebulizing gas and the drying gas. Data were obtained in a scan mode ranging from 100 to 1000 m/z in 0.1 s intervals; 10 scans were summed up to obtain the final spectrum.

Steric exclusion chromatography

Molecular weight of the polymers was determined by size exclusion chromatography (SEC) using (Waters corporation, USA) equipment fitted with a 5-μm mixed C PLgel, 5-μm MIXED-D (300 × 7.5 mm) (Agilent Technologies, USA), and SHODEX RI 101 refractometric detector (Shodex, China). The mobile phase was DMF at 0.8 ml/min flow. Typically, the polymer (10 mg) was dissolved in DMF (2 ml), and the resulting solution was filtered through a 0.45-μm Millipore filter before injection of 100 μl of a filtered solution. Mn and Mw were expressed according to calibration using polystyrene standards.

Cytotoxicity and Antibacterial Assays

Cytotoxicity

Polymers were uniformly spread at the bottom of culture's well. Cytoxicity was evaluated by lactate dehydrogenase (LDH) release measurement. LDH leakage into the culture medium as a result of fibroblasts L292 membrane lysis was evaluated with a cytotoxicity detection kit (LDH cytotoxicity Kit II, PromoKine, Heidelberg, Germany) according to the manufacturer's protocol. The LDH activity was determined spectrophotometrically after 30-min incubation at 25 °C of 10-μl culture medium and 100-μl reaction mixture, by measuring the oxidation of NADH at 450 nm in the presence of pyruvate, according to the manufacturer's kit instructions.

Antibacterial activity

Antibacterial activity of polymers was investigated according to the standard ISO 22196 with minor modifications. Polymers were uniformly spread on glass strips. About 20 μl of a 10 5 UFC ml À1 bacterial suspension was dropped on glass stripcoated polymers. Coverslips were dropped off into the suspension in order to spread it, and samples were incubated at 37 °C. After 24 h, bacteria were detached from supports by washing with 10 ml of sterile saline solution. Samples were vortexed vigorously for 30 s and sonicated for 3 min. The viable cells in solution were then serially diluted, and 100-μl aliquots were spread on gelose tripticase soja plates for CFU determination. ) were estimated by steric exclusion chromatography (SEC) in DMF, using polystyrene as standard. Mn allows the calculation of degree of polymerization (DP).

TURNING PEPTIDES IN SILICONE POLYMERS

Result and Discussion

Five hybrid peptides were prepared (Table 1). Hybrid dipeptide 1 was first used as a model sequence to demonstrate the feasibility of the polymerization. Sequences 3 and 4 were selected as small cationic peptides containing arginine residues, which could yield polymers able to interact and potentially disrupt bacterial membranes. Noteworthy, a silylated analog of the Ahx-Arg-Arg-NH 2 tripeptide was already used in our group to graft a glass surface, providing interesting antibacterial properties [START_REF] Jebors | From protected trialkoxysilyl-peptide building blocks to bioorganic-silica hybrid materials[END_REF]. Hybrid peptide 2 was the Pbf-protected analog of 3. Sequence 5 is the cyclic peptide RGDfK derivative, a ligand of integrin receptors, which has been used to improve cell adhesion to surfaces [START_REF] Kämmerer | Immobilisation of linear and cyclic RGD-peptides on titanium surfaces and their impact on endothelial cell adhesion and proliferation[END_REF][START_REF] Hersel | RGD modified polymers: biomaterials for stimulated cell adhesion and beyond[END_REF][START_REF] Kantlehner | Surface coating with cyclic RGD peptides stimulates osteoblast adhesion and proliferation as well as bone formation[END_REF]. After the synthesis of the peptides sequences either in solution or on solid support (Figure 2) and subsequent silylation using ICPMDCS, methyldichlorosilyl hybrid peptides were obtained and solubilized in DMF. Polymerization of hydrolyzed methyldihydroxysilyl hybrid peptides started by dropwise addiction of a pH 7.4 buffer at room temperature. Condensation of hydroxysilane groups proceeded quickly, and the polysiloxane-peptide precipitated within 2 h without use of any chemical reagent. Polymers were analyzed by SEC to determine the average molecular weight (Mn) according to calibration using polystyrene standards. Glass transition temperatures (Tg) were determined by differential scanning calorimetry analyses, which were performed from À150 °C to 110 °C (Table 1). In contrast to polydimethylsiloxanes, all tested polymers exhibited no Tgs in this range. This could be a consequence of hyperbranched and hybrid structure of the comb-peptide polymers. SEC chromatograms showed P1 had a significant molecular weight of ≈11600 corresponding to an average polymerization degree of 25, whereas P2 Mn (P2) ≈16950, and P5 Mn(P5) ≈8993 gmol À1 (Table 1), corresponded, respectively, to polymerization degrees of ca. 15.4 and 12. This difference could be attributed to the structure of monomers 2 and 5 composed of more bulky organics parts, which could slow down the polymerization process. Dispersity Ð was found to be close to 1, demonstrating that, for a given polymer, chain lengths are similar, see Figure 3 Amorphous polymers P3, P4, and P5 were dropped off without solvent and spread as a layer on a glass surface previously activated by piranha treatment (H 2 SO 4 /H 2 O 2 4/1 v/v). They were incubated overnight at 40 °C in order to immobilize the polymer on the glass surface.

Cytotoxicity was evaluated by LDH release measurement. After 24 h contact with polymers, fibroblasts (Figure 4) showed a low level of LDH (OD 450 = 0.92) close to fibroblasts level on tissue culture polystyrene (TCPS) control (OD 450 = 0.84). In comparison, lysed fibroblasts exhibited a high LDH level (OD 450 = 2.42). This first result shows that polymers are non-cytotoxic and can be used in a biomedical application, alone or, interestingly, in combination with polydimethyl siloxane units.

As a proof of concept, we evaluated our polymers for their antibacterial effects against Escherichia coli. Indeed, in most cases, infections are initiated by the adherence of planktonic bacterial cells onto the biomaterial surface. Silicone medical devices such as catheters, laryngeal masks, or septums may be prompted to progressive colonization and biofilm formation. It is therefore important for potential biomedical application and further development that combcationic polymers prepared by our methodology possess bactericidal properties. Thus, we selected the three polymers (P3, P4, and P5) that display guanidine groups in their sequences.

As shown in Figure 5, no bacteria were observed after 24-h culture on polymers P3-P5. The antimicrobial effect results in an eight-log reduction of living bacteria upon incubation. Surprisingly, no significant difference of activity was observed between P3, P4 (Arg-Arg derivatives), and P5 (cRGDfK). All of them exhibit significant antimicrobial properties toward E. coli.

Conclusion

In this study, we propose a new class of bioorganic polysiloxanes obtained by oligomerization of well-defined hybrid peptide sequences bearing a dimethylchlorosilane moiety. Straightforward, hydrolytic condensation occurs in water at room temperature and does not require any catalyst or chemical reagent. As a proof of concept, we prepared comb-polymers displaying cationic peptides, which proved to be efficient against E. coli. We believe that this type of polymerization in water can easily be scaled up and can find several applications to advantageously add dedicated biological properties lacking to conventional silicones.

Figure 1 .

 1 Figure 1. Comb-polymers from peptide sequences. (A) using N-terminally modified lysine with dimethyl hydroxysilyl groups [9]. (B) New strategy involving methyldihydroxysilyl N-terminal peptides.

Figure 2 .

 2 Figure 2. Synthesis of dichloromethylsilyl hybrid peptide 4.

Figure 3 .Figure 4 .

 34 Figure 3. SEC chromatogram (DMF, LiBr 1%) of polymer P2 (UV detection at 254 nm).

Figure 5 .

 5 Figure 5. Escherichia coli number per cm 2 after 24 h of contact with polymers.

Table 1 .

 1 Hybrid peptides (HO) 2 SiMe-(CH 2 ) 3 NHCO-peptide and comb-peptide polysiloxanes [OSiMe-(CH 2 ) 3 NHCO-peptide] n

	Monomer

a Dispersity Ð and the average molecular weight Mn (gmol À1

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Supporting Information

Additional supporting information may be found in the online version at the publisher's web site.

TURNING PEPTIDES IN SILICONE POLYMERS