EP3642348A1

EP3642348A1 - Redox cleavable comb-like copolymer for controlled adhesion between cells and substrates

Info

Publication number: EP3642348A1
Application number: EP18731864.7A
Authority: EP
Inventors: Christophe Tribet; Emmanuelle MARIE BEGUE; Louise HESPEL
Original assignee: Centre National de la Recherche Scientifique CNRS; Sorbonne Universite; Universite Paris Sciences et Lettres
Current assignee: Centre National de la Recherche Scientifique CNRS; Sorbonne Universite; Universite Paris Sciences et Lettres
Priority date: 2017-06-22
Filing date: 2018-06-22
Publication date: 2020-04-29
Also published as: WO2018234564A1

Abstract

La présente invention concerne un copolymère clivable qui est un copolymère de type peigne comprenant un squelette polymère polycationique et des chaînes latérales polymères hydrophiles greffées sur le squelette polymère polycationique par l'intermédiaire d'un lieur lié de manière covalente au squelette polymère polycationique et à la chaîne latérale polymère hydrophile : - le squelette polymère polycationique étant un polymère linéaire ou ramifié comprenant au moins 30 % en moles de motifs récurrents structuraux cationiques, ledit motif récurrent structural cationique étant sous une forme cationique à un pH compris entre 5 et 9 ; - les chaînes latérales polymères hydrophiles étant chacune une chaîne linéaire ou ramifiée de monomères non ioniques solubles dans l'eau ; le lieur étant un groupe chimique contenant un pont disulfure (-S-S-) ; et le rapport de greffage étant situé entre 10 % en mole et 70 % en mole par rapport à la quantité molaire des motifs récurrents structuraux cationiques. La présente invention concerne également un kit ou une composition comprenant au moins un tel copolymère clivable et au moins un copolymère non clivable, ainsi qu'un procédé de revêtement d'un substrat par un tel mélange de copolymères, le substrat revêtu obtenu et l'utilisation correspondante pour la culture cellulaire.The present invention relates to a cleavable copolymer which is a comb-type copolymer comprising a polycationic polymer backbone and hydrophilic polymeric side chains grafted onto the polycationic polymer backbone via a linker covalently bonded to the polycationic polymer backbone and the polycationic polymer backbone. hydrophilic polymer side chain: the polycationic polymer backbone being a linear or branched polymer comprising at least 30 mol% of cationic structural repeating units, said cationic structural repeating unit being in a cationic form at a pH of between 5 and 9; the hydrophilic polymer side chains each being a linear or branched chain of nonionic monomers soluble in water; the linker being a chemical group containing a disulfide bridge (-S-S-); and the grafting ratio being between 10 mol% and 70 mol% relative to the molar amount of the cationic structural repeating units. The present invention also relates to a kit or a composition comprising at least one such cleavable copolymer and at least one non-cleavable copolymer, as well as a process for coating a substrate with such a mixture of copolymers, the coated substrate obtained and the corresponding use for cell culture.

Description

REDOX CLEAVABLE COMB-LIKE COPOLYMER FOR CONTROLLED ADHESION BETWEEN CELLS AND SUBSTRATES

The present invention deals with the general field of surface coating by biocompatible polymers that are used to control cell cultures, specifically promoting and/or remotely controlling cell-surface recognition while minimizing non-specific adhesion.

More particularly, the present invention relates to a redox cleavable comb-like copolymer, a kit and a composition comprising such a copolymer, advantageously in association with a non-cleavable copolymer, a method for coating the surface of a substrate with said copolymer or association of copolymers, notably under the form of patterns, and uses thereof for cell culture.

Bioengineering is rapidly progressing toward in vitro functional tissue and organ reconstitutions from cell cultures. One needs thus to manage complex assemblies of cells on artificially designed scaffolds.

In addition to promising therapeutic application, the preparation of specific assemblies of cells on the surface of scaffolds such as microplates is desirable as models for diagnostic and/or for drug screening. Such substrates are useful for diagnostic devices, in vitro cell cultures, and implants [Mendes, Chemical Society Reviews 2008, 37(1 1 ), 2512-2529 ; Patel, Zhang, Organogenesis 2013, 9(2), 93-100 ; Higuchi et al., Progress in Polymer Science 2014, 39(9), 1585-1613].

For preparing such complex assemblies of cells, bioengineers need tools to manipulate the surface of such scaffolds during co-deposition of different cell lines, and/or to orient migration and growth on relevant micrometric to millimetric spatial resolutions. Finally, among many parameters that affect cell behavior onto solid substrates, the chemical nature of the surfaces is critical for specificity, i.e. targeting specific cell types. Reaching the technological needs in this field comes down to the spatio-temporal control of presentation on the top of surfaces of either a bio-repellent layer or bio-adhesive chemical moieties such as peptides and proteins. To this aim, surfaces are generally "decorated" with polymer-based adhesion micropatterns in order to match with the above requirements and to influence cell fates and growth [Nakanishi et ai, Anal. Sci. 2008, 24(1 ), 67-72; Yu, L. M. Y. et al., Materials Today 2008, 11 (5), 36-43; Cimetta, E.; et al., Biomedical Microdevices 2009, 11 (2), 389-400; Phillips, J. E.et al., Acta Biomaterialia 2010, 6 (1 ), 12-20; Guex, A. G. et al., Acta Biomaterialia 2012, 8 (4), 1481 -1489]. Presentation of a large variety of polymers and optimization of composition in the polymer layer are major technological challenges.

Common approaches involve covalent attachment of hydrophilic polymer chains on the surfaces of interest [see ref. above], and accordingly propose different strategies and different chemistries each one being adapted to particular substrate's surface chemistry (metals, glass, plastics such as Petri dishes or PDMS microdevices). Modification of surface composition is accordingly based on specialized chemistries and surface handling procedures that are usually not available in biology or biomedical laboratories, (e.g. living radical polymerizations, surface metallizing, molecular monolayer transfers, etc.). In addition, combination of polymers of different chemical natures in mixed layers of adjustable composition is a heavy task when covalent chemistries are implemented. Standardized and externalized productions of substrates are possible, though it suffers from a lack of versatility. Indeed, fine tuning of surface properties is required to match with variability and diversity of experimental needs (variety of cell lines and culture media, shape and size of patterns). A major issue is to modulate the chemical nature of surfaces, and to control densities of bioactive groups on these surfaces (e.g. charge density, peptide attachment, thickness and density of hydrophilic repellent layer).

There exists thus a need for new methods for surface coating with biocompatible polymers which are not based on specialized chemistries so as to be easily implemented in biology or biomedical laboratories which have not the required equipment; which can be implemented on various substrates commonly used in this field; and which allows a greater flexibility and control during cell culture and for cell adhesion/deadhesion with minimized non-specific adhesion.

The inventors of the present invention have thus developed a method for surface coating of a substrate which allows responding to these issues. For a redox cleavable copolymer, advantageously in mixture with a non-cleavable copolymer, is used.

This method implies to deposit layer(s) of redox cleavable copolymers or mixed layer(s) of redox cleavable copolymers and non-cleavable copolymers on the surface of a substrate to be coated, such copolymers having various terminal units with repellent or adhesive properties towards living cells for example. This coating imparts surfaces with gentle control of cell adhesion. Indeed, the surface composition can be modified by short incubation in the presence of a non-toxic reducing agent which will cleave the cleavable copolymers and remove its terminal units. Depending on the nature of the terminal units present on the redox cleavable copolymers and on the optional non-cleavable copolymers, the surface can thus change from a repellent nature to an adhesive nature towards living cells or inversely.

The coating of the substrate is based on spontaneous adlayer formation by simple bath application of aqueous solutions of cationic polymers. Adsorption takes place onto substrates having an anionic surface charge in aqueous solutions, notably at pH near neutrality. Such a coating can be performed onto flat substrates, e.g. for in vitro cell culture, but also onto colloid beads and substrates of various shapes used for immuno-assays, microfluidic cell sorting, or implants.

The copolymers having a comb-like structure comprises:

1 ) a polycationic polymeric backbone,

2) hydrophilic polymer side chains grafted on said backbone that may be functionalized on their extremity with either a biomolecule or a biorthogonal reactive group (e.g. azide), and

3) in the case of the redox cleavable copolymer, a cleavable linker between the backbone and the side chains that can be cleaved in reducing conditions.

The cationic backbone serves to bind the polymer onto surfaces of opposite ionic charges via coulombic attractions but this backbone has also non-specific adhesive properties for living cells. Grafted side chains allow preventing or decreasing the non-specific attractions, in particular with proteins, of the coated substrate while they cover it by a more or less dense brush-like layer (this property is also referred as bio- or cell-repellency, protein-resistant, non-adhesiveness, or else biopassivation). When said grafted side chains carry a biomolecule end-group, such as a peptide, specific recognition may thus occur which allows capturing cells or proteins from solutions. When present, the bio-orthogonal reactive azide group can be used to attach the biomolecule in situ, such as a peptide or other more complex or fragile biomolecules that cannot be introduced prior to coating, such as antibodies, enzymes, or fragment of matrix proteins (collagen, elastin, fibronectin, laminin). The redox sensitive linker allows for on demand cleavage of the side chains in the copolymer, and retrieval of a merely polycationic backbone adsorbed on the surface of interest. In the case of functionalized side chains, their detachment releases the links between the substrate and cells thus weakening cell-substrate adhesiveness. In the case of non- functionalized layers, it removes the biorepellent protective grafts thus triggering non- specific adsorption of proteins and cell adhesion onto the "newborn" polycationic adlayer.

Redox cleavable copolymer

The present invention relates thus to a redox cleavable copolymer which is a comb-like copolymer comprising a polycationic polymeric backbone and hydrophilic polymeric side-chains grafted onto the polycationic polymeric backbone via a linker covalently bound to the polycationic polymeric backbone and the hydrophilic polymeric side-chain, wherein:

- the polycationic polymeric backbone is a linear or branched, preferably linear polymer comprising at least 30 mol%, notably at least 50 mol%, preferably at least 80 mol%, in particular 100 mol% of cationic structural repeat units, a cationic structural repeat unit according to the invention being under the form of a cation (ionized form) at pH = 7, notably in the range of pH between 6 and 8, in particular between 5 and 9,

the hydrophilic polymeric side-chains are each a linear or branched, preferably linear chain of water-soluble non-ionic monomers;

the linker is a chemical group containing a disulphide bridge (-S-S-); and wherein the grafting ratio is between 10 mol% and 70 mol%, preferably between 30 mol% and 50 mol% with respect to the molar amount of the cationic structural repeat unit.

The polycationic polymeric backbone aims to adhere on an anionic surface of a substrate with no need for covalent attachment. Thus, when the comb-like copolymer is adhered on a surface, the polycationic polymeric backbone will be electrostatically bound to the surface and will lay below a layer formed by the hydrophilic polymer side- chains grafted on the said backbone and protruding away from the surface. The hydrophilic polymeric side-chains will be the first accessible moieties on the top of the coating, the backbone being "hidden" by said side chains. Thus, the optimal grafting ratio shall not be too high to preserve the cationic density of the backbone but shall be high enough to provide a minimal density of hydrophilic grafts in order to allow effective resistance to non-specific adsorption. Typically, it can be varied from one graft every ten to one graft every three cationic structural repeat units.

The polycationic polymeric backbone have non-specific adhesive properties for living cells, whereas the hydrophilic polymeric side-chains have repellent properties for living cells. Moreover, as mentioned below, biomolecules can be bound to the end of these hydrophilic polymeric side-chains rendering these side-chains adhesive for any or specific living cells / proteins depending on the nature of the biomolecule.

In the presence of a reducing agent (i.e. redox conditions), the disulphide bridge (present on the linker which bound the hydrophilic polymeric side-chains to the polycationic polymeric backbone) will be reduced which will allow cleaving the linker and releasing the hydrophilic polymeric side-chains from the polycationic polymeric backbone. It is why this copolymer is named in the present invention "redox cleavable copolymer" or "cleavable copolymer".

Thus, when the copolymer is cleaved, the surface nature of the coated substrate will change. If no biomolecule is grafted on the side chains, the surface will shift from a repellent nature (hydrophilic polymer) to an adhesive nature (polycationic backbone) for living cells. Now, if a biomolecule is grafted on the side chains, living cells can be adhered. It is then possible to release these adhered living cells when the copolymer is cleaved.

Polycationic polymeric backbone:

The polycationic polymeric backbone comprises cationic structural repeat units. The cationic structural repeat units can be alkylene amines, bis(alkylene) amines, tris(alkylene) amines (e.g. respectively -CH2CH2NH2, (-ChbCI-b^N H and (- ChbChb^N units constitutive of the poly(ethylenimine) backbone), amino-acids selected from the group consisting of ornithine, lysine, histidine and arginine, or combinations thereof. Preferably said cationic structural repeat unit is an amino-acid, such as a lysine.

By "alkylene amine", "bis(alkylene) amine", and "tris(alkylene) amine" is meant in the present invention respectively a group of the following formula:

-R1 NH2 (-Ri )(-R₂)N H (-Ri )(-R₂)(-Rs)N alkylene amine bis(alkylene) amine tris(alkylene) amine (monovalent group) (divalent group) (trivalent group) wherein Ri to R3 represent, independently of one another, a (Ci-Ce)alkylene group. It can be for example -CH₂CH₂NH₂, (-CH₂CH₂)₂N H-, (-CH₂CH₂)₃N, -CH2CH2CH2NH2, (- CH2CH₂CH2)2NH, (-CH2CH₂CI-l2)3N . It can be in particular ethylene amine, bis(ethylene) amine, tris(ethylene) amine (i.e. -CH2CH2NH2, (-ChbChb^NH and (-CH2CH2)3N units constitutive of the poly(ethylenimine) backbone).

By "(Ci-C6)alkylene" is meant in the present invention a divalent straight, cyclic, or branched saturated hydrocarbon chain containing from 1 to 6, notably 1 to 4, carbon atoms including, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl, and the like.

The cationic structural repeat units can be issued from identical or different monomers. Advantageously, the cationic structural repeat units are issued from identical monomers and are preferably an amino-acid, such as a lysine.

Thus, the polycationic polymeric backbone can be polyornithine, polylysine, polyhistidine or polyarginine, and preferably polylysine (PLL).

The polycationic polymeric backbone comprises advantageously between 20 and 10 000 cationic structural repeat units, preferably between 50 and 1 000 cationic structural repeat units.

The polycationic polymeric backbone can further comprise additional structural repeat units in addition to the cationic structural repeat units. These additional structural repeat units can be polar non-ionic structural repeat units and/or anionic structural repeat units such as amino acids selected from the group consisting of glutamic acid, aspartic acid, serine, threonine, glutamine, and tyrosine, oligo(ethylene oxide), oligo(propylene oxide) or combinations thereof, preferably polar non-ionic structural repeat units, with the proviso that the molar ratio of anionic structural repeat units to cationic structural repeat units is inferior to 10 mol%. Preferably, no additional structural repeat unit is present so that the polycationic polymeric backbone is made only of cationic structural repeat units.

In particular, the polycationic polymeric backbone will have a number average molecular weight between 5 000 and 150 000 g.mol^"1, preferably between 10 000 and 50 000 g.mol^"1.

Hydrophilic polymeric side-chains:

The hydrophilic polymeric side-chains are made of water-soluble non-ionic monomers.

The water-soluble non-ionic monomers are advantageously selected from the group consisting of ethylene glycol, oxazoline, N-isopropylacrylamide, vinylpyrrolidinone, glycidol and a combination thereof. More particularly, the water- soluble non-ionic monomers are identical and are preferably ethylene glycol. The hydrophilic polymeric side-chains can be chosen from among polyoxazoline, poly(N-isopropylacrylamide), poly(vinylpyrrolidone), polyglycidol, and polyethyleneglycol (PEG). Preferably, the hydrophilic polymeric side-chains are polyethyleneglycol (PEG).

The longest linear chain of monomers in each hydrophilic polymeric side-chain advantageously comprises between 20 and 500, preferably between 60 and 200 monomers.

Each hydrophilic polymeric side-chain will have advantageously a number average molecular weight between 1 000 and 20 000 g.mol^"1, preferably between 3 000 and 10 000 g/mol.

Part or all of the hydrophilic polymeric side-chains can be terminated by a bioorthogonal reactive group or a biomolecule.

The bioorthogonal reactive group can be an azido group (N3) or a fluorophore group such as rhodamine. Preferably, it is an azido group.

The biomolecule can be for example a peptide such as a cell adhesion peptide or an enzyme; a growth factor; an antibody; a fragment of antibody such as scFv (single chain variable fragment) or a diabody; or an antibody mimic such as a monobody.

The azido group is useful for grafting another unit comprising an alkyne group by Click chemistry. The fluorophore is useful for imaging by fluorescence. The biomolecule is useful notably for cell culture.

Linker:

The polycationic polymeric backbone and the hydrophilic polymeric side-chains are linked together by means of a linker which comprises a disulphide bridge This is this disulphide bridge which allows the resulting copolymer to be cleavable in redox conditions i.e. in the presence of a reducing agent.

The linker will be more particularly covalently bound at one of its extremity to an amino group of a cationic structural repeat unit of the polycationic polymeric backbone and at its other extremity to a hydrophilic polymeric side-chain.

Advantageously, the linker is bound to the cationic structural repeat unit of the polycationic polymeric backbone via a linking group which can be a urea (N-CO-N), urethane (N-CO-O), amide (N-CO) or triazole group, preferably an amide group.

Advantageously, the linker is bound to a hydrophilic polymeric side-chain via a linking group which can be a urea (N-CO-N), urethane (N-CO-O), carbonate (0-CO-O), amide (N-CO), thioether (S) or triazole group, or via its disulphide bridge. Preferably, the linker is bound to a hydrophilic polymeric side-chain via its disulphide bridge.

The chemical nature of the linker is not a critical point, except for the presence of the disulphide bridge. Indeed, it aims only to link a hydrophilic polymeric side-chain to the polycationic polymeric backbone and to be cleavable in redox conditions thanks to its disulphide bridge. For example, the chain of the linker between the two linking groups can be a linear or branched saturated hydrocarbon chain comprising 1 to 10 carbon atoms, in which one or several, for example 1 or 2, carbon atoms are replaced with a phenyl ring or an amide group (NH-CO), said chain further comprising the disulphide bridge if it is not used as a linking group.

Method of synthesis:

The cleavable copolymers according to the present invention can be prepared by coupling methods well-known to the one skilled in the art.

For that, a bifunctional organic compound comprising two reactive groups, optionally in a protected form, is used to introduce the linker between the polycationic polymeric backbone and the hydrophilic polymeric side-chains. The first reactive group aims to react with a reactive group such as an amino group present on a cationic structural repeat unit of the backbone, whereas the second reactive group aims to react with a hydrophilic polymer bearing also a reactive group on one of its extremity. The bifunctional organic compound will also comprise a disulphide bridge or one of its reactive group will be able to form such a disulphide bridge.

According to a first preferred alternative, the bifunctional organic compound is first reacted with the polycationic polymeric backbone in order to graft several molecules of this organic compound on cationic structural repeat units all along the polycationic polymeric backbone. Then, the resulting product is reacted with the hydrophilic polymer bearing a reactive group in order to graft hydrophilic polymeric side-chains on the polycationic polymeric backbone via a linker. In this method, it can be necessary to protect the second reactive group of the bifunctional organic compound to avoid side reactions during the first coupling step. In this case, the second reactive group has to be deprotected before performing the second coupling reaction.

According to a second alternative, the bifunctional organic compound is first reacted with the hydrophilic polymer bearing a reactive group so as to graft one organic compound on the extremity of the hydrophilic polymer. Then, the resulting product is reacted with the polycationic polymeric backbone in order to graft the hydrophilic polymeric side-chains all along the polycationic polymeric backbone via a linker. In this method, it can be necessary to protect the first reactive group of the bifunctional organic compound to avoid side reactions during the first coupling step. In this case, the first reactive group has to be deprotected before performing the second coupling reaction.

The nature of the reactive groups on the bifunctional organic compound will depend on the nature of the linking groups to be formed.

Typically, the reactive group intended to react with the cationic structural repeat unit of the polycationic polymeric backbone (i.e. with an amino group NH) can be a succinimidyloxycarbonyl or a sulfo-succinimidyloxycarbonyl. However, any other group which can react with an amino group to form a urea (N-CO-N), urethane (N-CO-O), amide (N-CO) or triazole group can be used.

The reactive group intended to react with a hydrophilic polymer bearing a reactive group will depend on the nature of this terminal reactive group and on the nature of the linking group to be formed. The one skilled in the art knows how to perform such coupling reactions.

If the linking group with the hydrophilic polymeric side chains is the disulphide bridge, then the reactive group present on said hydrophilic polymer can be a thiol (SH) and the reactive group of the bifunctional organic compound can be a pyridyl-dithio group (such as 2-pyridyl-dithio). In this case, which is the preferred one, the bifunctional organic compound used can be for example N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), succinimidyl-6-[3(2-pyridyldithio)propionamido] hexanoate (LC-SPDP), sulfo-succinimidyl-6-[3(2-pyridyldithio)propionamido]hexanoate (sulfoLC-SPDP) or 4-succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio) toluene (SMPT). Advantageously, it is SPDP.

Kit and composition

The present invention relates also to a kit or a composition comprising:

at least one cleavable copolymer, and

optionally at least one non-cleavable copolymer.

Preferably, the kit or composition according to the invention comprises:

at least one cleavable copolymer, and

at least one non-cleavable copolymer. The cleavable copolymer is the redox cleavable copolymer according to the invention as defined previously.

The non-cleavable copolymer corresponds to a redox cleavable copolymer as defined previously but without a linker including a disulphide bridge so that the non- cleavable copolymer cannot be cleaved in redox conditions (i.e. in the presence of a reducing agent).

In consequence, the non-cleavable copolymer according to the invention is a comb-like copolymer comprising a polycationic polymeric backbone and hydrophilic polymeric side-chains covalently grafted onto the polycationic polymeric backbone, wherein:

- the polycationic polymeric backbone is as defined previously, and

- the hydrophilic polymeric side-chains are each as defined previously, and wherein the grafting ratio is between 10 mol% and 70 mol%, preferably between 30 mol% and 50 mol% with respect to the molar amount of the cationic structural repeat unit.

According to a preferred embodiment the hydrophilic polymeric side-chains of the non-cleavable copolymer will be different from the hydrophilic polymeric side-chains of the cleavable copolymer.

The kit and the composition can also comprise several different cleavable copolymers and/or several different non-cleavable copolymers.

The composition can be used to coat the surface of a substrate with copolymers, notably by dipping the substrate in the composition or depositing said composition on the surface to be coated of the substrate. Thus, the composition is preferably liquid. It can be a solution or dispersion of the at least one cleavable copolymer optionally in mixture with the at least one non-cleavable copolymer , preferably an aqueous solution or dispersion.

The pH of the composition will be advantageously between 5 and 9 so that the polycationic polymeric backbone be in a cationic form. The pH will be preferably between 6 and 8, more preferably the pH is neutral.

Coating methods

1^st Coating method

The present invention relates to a first method for coating an area on the surface of a substrate comprising the successive steps of: (1 ) forming anionic charges onto said area, and

(2) contacting the charged area resulting from step (1 ) with a composition according to the present invention in the form of a solution or a dispersion, so that the polymers contained in said composition are deposited onto the area of the surface of the substrate.

The present invention relates thus also to a coated substrate obtainable by the 1 ^st coating method according to the present invention. The substrate to be coated, and more particularly its surface, can be made of various materials, such as:

- glass, silica, quartz,

metal, such as titanium or stainless steel,

metal oxide, such as iron oxide or T1O2,

- a polymer material, such as polystyrene, poly(methyl methacrylate) (PMMA), polypropylene, polydimethylsiloxane (PDMS) or polyethylene,

a semi-conductor, such as a silicon wafer or ITO (indium tin oxide),

and mixtures thereof.

The substrate can be a solid flat support for cell culture such as petri dishes (e.g. in polystyrene). It can be also a microfluidic device (e.g. in PDMS) or an implant. It can be also particles and microbeads (e.g. silica, iron oxide magnetic beads, poly(styrene) and other polymer latex).

Step (1):

The formation of anionic charges on the area of the surface of the substrate to be coated can be performed by methods well-known to the one skilled in the art.

For example, in the case of silicon wafers and inorganic oxides, including glass and silica, the surface to be coated can be ionized by incubation in a solution of sodium hydroxide (> 1 mol/L) and eventually rinsed with water. In the case of polymer materials (e.g. polystyrene petri dishes, PDMS microfluidic devices), the surface can be treated with oxygen or air plasma in order to introduce weak acid groups that confer an anionic character to surfaces at near neutral pHs. In the case of particles and microbeads (e.g. silica, iron oxide magnetic beads, poly(styrene) and other polymer latex), they can directly be obtained under a negatively charged form prior to use and they can be subjected to purification, for example in water by ultrafiltration, size exclusion chromatography, or several cycles of centrifugation / redispersion.

Preferably, the area of the surface to be coated will be cleaned before performing step (1 ).

Step (2):

The contact of the charged area resulting from step (1 ) with a composition comprising at least one cleavable copolymer and optionally at least one non-cleavable copolymer in the form of a solution or dispersion allows spontaneous adsorption of the copolymers and monolayer formation.

The contact duration can be comprised between 1 min and 60 min and notably be less than 10 minutes. It can be performed at room temperature, i.e. between about 15 and 30°C.

The concentration of the polymer is not critical and can be for example between 0.5 and 10 g/L.

The composition will be more particularly an aqueous solution or dispersion of the at least one cleavable copolymer and optionally the at least one non-cleavable copolymer.

As small ions may compete with coulombic binding of the polymer layer, ionic strength of the solutions should be preferably as low as possible, and the presence of high concentration (> 1 mol/L) of small ions should be preferably avoided.

The contact can be performed by dipping the substrate or at least the area of the surface to be coated in the composition. However, drops or droplets of the composition can also be deposited onto the area of the surface to be coated.

Advantageously, the coated surface will be rinsed after the deposition of the polymers, preferably with water.

Step (3):

Step (2) can be performed notably with a composition containing at least one cleavable copolymer or non-cleavable copolymer wherein part or all of the hydrophilic polymeric side-chains are terminated by an azido group (N3). In this case, step (2) can be followed by an additional step (3) of grafting a biomolecule, on said hydrophilic polymeric side-chains terminated by an azido group (N3), by Click chemistry, provided that said biomolecule bears an alkyne group, preferably a terminal alkyne group (-C≡CH), such as a BCN (bicyclo[6.1 .0]-nonyne) moiety.

Click chemistry involves a reaction between an azide function (-IM3) and an alkyne function (preferably a terminal alkyne function -C≡CH), also called azide-alkyne Huisgen cycloaddition. The azide and alkyne functions react together to form a 1 ,2,3- triazole by a 1 ,3-dipolar cycloaddition.

The cycloaddition can be performed in various solvents, such as alcohols (such as tert-butanol), dimethylsulfoxyde (DMSO), Ν,Ν-dimethylformamide (DMF), acetone, water or mixtures thereof. Preferably, the reaction will be performed in water.

The reaction can be carried out at room temperature, i.e. between about 15 and

30°C.

Step (4):

The method according to the present invention can comprise an additional step

(4) after step (2), said step (4) consisting in cleaving the cleavable linker by a reducing treatment. In these conditions, only the hydrophilic polymer side chains of the optional non-cleavable copolymer will remain onto the coated area.

The reducing treatment will be performed in the presence of a reductive agent, such as glutathione (GSH), tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol (DTT) or mercaptoethanol, the reductive agent being advantageously GSH, DTT or TCEP, more advantageously TCEP.

The duration of the reducing treatment is advantageously between 30 s and 60 min, notably between 1 min and 30 min, in particular between 1 min and 10 min.

2^nd Coating method

The present invention relates also to a second method for coating an area on the surface of a substrate comprising the successive steps of:

(A) forming anionic charges onto said area,

(B) contacting the charged area resulting from step (A) with a composition comprising a polycationic polymer optionally in mixture with a non-cleavable copolymer in the form of a solution or a dispersion, so that the polycationic polymer and the optional non-cleavable copolymer contained in said composition are deposited onto the area of the surface of the substrate, (C) reacting the polycationic polymer deposited onto the area of the surface of the substrate in step (B) with a bifunctional organic compound in order to form an activated polycationic polymer onto the area of the surface of the substrate, and

(D) reacting the activated polycationic polymer obtained in step (C) with a hydrophilic polymer bearing a reactive group on one of its extremity so as to form a redox cleavable copolymer according to the invention onto the area of the surface of the substrate, optionally in mixture with a non-cleavable copolymer,

wherein the non-cleavable copolymer is as defined previously,

wherein the polycationic polymer is a linear or branched, preferably linear polymer comprising at least 30 mol%, notably at least 50 mol%, preferably at least 80 mol%, in particular 100 mol% of cationic structural repeat units, a cationic structural repeat unit according to the invention being under the form of a cation (ionized form) at pH = 7, notably in the range of pH between 6 and 8, in particular between 5 and 9,

wherein the hydrophilic polymer bearing a reactive group on one of its extremity is a linear or branched, preferably linear chain of water-soluble non-ionic monomers, said chain bearing a reactive group on one of its extremity, and

wherein the bifunctional organic compound is an organic compound comprising two terminal reactive groups, optionally in a protected form, and comprising a disulphide bridge or one of its reactive group is able to form such a disulphide bridge.

The polycationic polymer aims to form the the polycationic polymeric backbone of the redox cleavable copolymer as defined previously.

The hydrophilic polymer bearing a reactive group on one of its extremity aims to form the hydrophilic polymeric side-chains of the redox cleavable copolymer and is as defined previously.

The bifunctional organic compound aims to form the linker containing a disulphide bridge of the redox cleavable copolymer which links the hydrophilic polymeric side-chains to the polycationic polymeric backbone. Thus, the bifunctional organic compound comprises two reactive groups, optionally in a protected form, wherein the first reactive group aims to react with a reactive group such as an amino group present on a cationic structural repeat unit of the polycationic polymer, whereas the second reactive group aims to react with the hydrophilic polymer bearing also a reactive group on one of its extremity. The bifunctional organic compound is as defined previously. It can be SPDP for example. The present invention relates thus also to a coated substrate obtainable by the 2^nd coating method according to the present invention.

The substrate to be coated is as defined previously for the 1^st coating method.

Step (A): see step (1 ) of the 1 ^st coating method. Step (B):

The contact of the charged area resulting from step (A) with a composition comprising a polycationic polymer optionally in mixture with a non-cleavable copolymer in the form of a solution or a dispersion allows spontaneous adsorption of the polycationic polymer and optionally the non-cleavable copolymer and monolayer formation.

The pH of the composition will be advantageously between 5 and 9 so that the polycationic polymer be in a cationic form. The pH will be preferably between 6 and 8, more preferably the pH is neutral.

The concentration of the polymer is not critical and can be for example between

0.5 and 10 g/L.

The composition will be more particularly an aqueous solution or dispersion of the polycationic polymer optionally in mixture with a non-cleavable copolymer.

Step (C):

The coupling reaction between the polycationic polymer deposited onto the area of the surface of the substrate in step (B) and a bifunctional organic compound aims to graft several molecules of this organic compound on cationic structural repeat units all along the polycationic polymer. It can be performed by methods well-known to the one skilled in the art depending on the nature of the involved reactive groups.

In this step, the second reactive group of the bifunctional organic compound can be advantageously in a protected form in order to avoid side reactions during the coupling reaction of step (B). In this case, the second reactive group will be deprotected before performing the coupling reaction of step (C).

Step (D):

The coupling reaction between the activated polycationic polymer obtained in step (C) and the hydrophilic polymer bearing a reactive group on one of its extremity can be performed by methods well-known to the one skilled in the art depending on the nature of the involved reactive groups. Step (E):

Step (D) can be performed notably with a hydrophilic polymer bearing a reactive group on one of its extremity comprising also an azido group (N3) on its other extremity or in the presence of a non-cleavable copolymer wherein part or all of the hydrophilic polymeric side-chains are terminated by an azido group (N3). In this case, step (D) can be followed by an additional step (E) of grafting a biomolecule on the azido groups (N3), by Click chemistry, provided that said biomolecule bears an alkyne group, preferably a terminal alkyne group (-C≡CH), such as a BCN (bicyclo[6.1 .0]-nonyne) moiety.

The preferred reaction conditions of Click chemistry are presented in step (3) of the 1 ^st coating method.

Step (F):

The method according to the present invention can comprise an additional step (F) after step (D), said step (F) consisting in cleaving the disulphide bridge of the redox cleavable copolymer by a reducing treatment. In these conditions, only the hydrophilic polymer side chains of the optional non-cleavable copolymer will remain onto the coated area.

The preferred conditions of the reducing treatment are presented in step (4) of the 1 ^st coating method. 3^rd Coating method

The present invention relates also to a third method for coating an area on the surface of a substrate comprising the successive steps of:

(a) forming anionic charges onto said area,

(b) contacting the charged area resulting from step (a) with a composition comprising an activated polycationic polymer optionally in mixture with a non- cleavable copolymer in the form of a solution or a dispersion, so that the activated polycationic polymer and the optional non-cleavable copolymer contained in said composition are deposited onto the area of the surface of the substrate, and

(c) reacting the activated polycationic polymer deposited onto the area of the surface of the substrate in step (b) with a hydrophilic polymer bearing a reactive group on one of its extremity so as to form a redox cleavable copolymer according to the invention onto the area of the surface of the substrate, optionally in mixture with a non-cleavable copolymer,

wherein the non-cleavable copolymer is as defined previously,

wherein the activated polycationic polymer is a polycationic polymer coupled to a bifunctional organic compound,

wherein the bifunctional organic compound is an organic compound comprising two terminal reactive groups and comprising a disulphide bridge or one of its reactive group is able to form such a disulphide bridge.

The hydrophilic polymer bearing a reactive group on one of its extremity aims to form the hydrophilic polymeric side-chains of the redox cleavable copolymer and is as defined previously. The bifunctional organic compound aims to form the linker containing a disulphide bridge of the redox cleavable copolymer which links the hydrophilic polymeric side-chains to the polycationic polymeric backbone. Thus, the bifunctional organic compound comprises two reactive groups, wherein the first reactive group aims to react with a reactive group such as an amino group present on a cationic structural repeat unit of the polycationic polymer, whereas the second reactive group aims to react with the hydrophilic polymer bearing also a reactive group on one of its extremity. The bifunctional organic compound is as defined previously. It can be SPDP for example.

The activated polycationic polymer is formed by coupling the polycationic polymer with bifunctional organic compounds so as to graft several molecules of this organic compound on cationic structural repeat units all along the polycationic polymer. It can be performed by methods well-known to the one skilled in the art depending on the nature of the involved reactive groups. For that, the second reactive group of the bifunctional organic compound can be advantageously in a protected form in order to avoid side reactions during the coupling reaction. In this case, the second reactive group will be then deprotected to form the activated polycationic polymer.

The present invention relates thus also to a coated substrate obtainable by the 3^rd coating method according to the present invention.

Step (a): see step (1 ) of the 1 ^st coating method.

Step (b):

The contact of the charged area resulting from step (A) with a composition comprising an activated polycationic polymer optionally in mixture with a non-cleavable copolymer in the form of a solution or a dispersion allows spontaneous adsorption of the activated polycationic polymer and optionally the non-cleavable copolymer and monolayer formation.

The contact duration can be comprised between 1 min and 60 min and notably be less than 10 minutes. It can be performed at room temperature, i.e. between about 15 and 30°C. The pH of the composition will be advantageously between 5 and 9 so that the activated polycationic polymer be in a cationic form. The pH will be preferably between 6 and 8, more preferably the pH is neutral.

The composition will be more particularly an aqueous solution or dispersion of the activated polycationic polymer optionally in mixture with a non-cleavable copolymer.

Step (c):

The coupling reaction between the activated polycationic polymer deposited onto the area of the surface of the substrate in step (b) and the hydrophilic polymer bearing a reactive group on one of its extremity can be performed by methods well- known to the one skilled in the art depending on the nature of the involved reactive groups. Step (d):

Step (c) can be performed notably with a hydrophilic polymer bearing a reactive group on one of its extremity and an azido group (N3) on its other extremity or in the presence of a non-cleavable copolymer wherein part or all of the hydrophilic polymeric side-chains are terminated by an azido group (N3). In this case, step (c) can be followed by an additional step (d) of grafting a biomolecule on the azido groups (N3), by Click chemistry, by Click chemistry, provided that said biomolecule bears an alkyne group, preferably a terminal alkyne group (-C≡CH), such as a BCN (bicyclo[6.1 .0]- nonyne) moiety.

The preferred reaction conditions of Click chemistry are presented in step (3) of the 1 ^st coating method. Step (e):

The method according to the present invention can comprise an additional step (e) after step (c), said step (e) consisting in cleaving the disulphide bridge of the redox cleavable copolymer by a reducing treatment. In these conditions, only the hydrophilic polymer side chains of the optional non-cleavable copolymer will remain onto the coated area.

The preferred conditions of the reducing treatment are presented in step (4) of the 1 ^st coating method. 4^th Coating method

The present invention relates also to a fourth method for coating an area on the surface of a substrate comprising the successive steps of:

(i) forming anionic charges onto said area,

(ii) contacting the charged area resulting from step (i) with a composition comprising a polycationic polymer optionally in mixture with a non-cleavable copolymer in the form of a solution or a dispersion, so that the polycationic polymer and the optional non-cleavable copolymer contained in said composition are deposited onto the area of the surface of the substrate, and

(iii) reacting the polycationic polymer deposited onto the area of the surface of the substrate in step (ii) with an activated hydrophilic polymer so as to form a redox cleavable copolymer according to the invention onto the area of the surface of the substrate, optionally in mixture with a non-cleavable copolymer,

wherein the non-cleavable copolymer is as defined previously,

wherein the activated hydrophilic polymer is a hydrophilic polymer bearing a reactive group on one of its extremity coupled to a bifunctional organic compound,

wherein the hydrophilic polymer bearing a reactive group on one of its extremity is a linear or branched, preferably linear chain of water-soluble non-ionic monomers, said chain bearing a reactive group on one of its extremity, and wherein the bifunctional organic compound is an organic compound comprising two terminal reactive groups and comprising a disulphide bridge or one of its reactive group is able to form such a disulphide bridge. The polycationic polymer aims to form the the polycationic polymeric backbone of the redox cleavable copolymer as defined previously.

The bifunctional organic compound aims to form the linker containing a disulphide bridge of the redox cleavable copolymer which links the hydrophilic polymeric side-chains to the polycationic polymeric backbone. Thus, the bifunctional organic compound comprises two reactive groups, wherein the first reactive group aims to react with a reactive group such as an amino group present on a cationic structural repeat unit of the polycationic polymer, whereas the second reactive group aims to react with the hydrophilic polymer bearing also a reactive group on one of its extremity. The bifunctional organic compound is as defined previously. It can be SPDP for example.

The activated hydrophilic polymer is formed by reacting the reactive group of the hydrophilic polymer bearing a reactive group on one of its extremity with the second reactive group of the bifunctional compound. This coupling reaction can be performed by methods well-known to the one skilled in the art depending on the nature of the involved reactive groups. For that, the first reactive group of the bifunctional organic compound can be advantageously in a protected form in order to avoid side reactions during the coupling reaction. In this case, the first reactive group will be then deprotected to form the activated hydrophilic polymer.

The present invention relates thus also to a coated substrate obtainable by the 4^th coating method according to the present invention.

The substrate to be coated is as defined previously for the 1 ^st coating method. Step (i): see step (1 ) of the 1 ^st coating method. Step (ii): see step (B) of the 2^nd coating method. Step (Hi):

The coupling reaction between the polycationic polymer deposited onto the area of the surface of the substrate in step (ii) and the activated hydrophilic polymer can be performed by methods well-known to the one skilled in the art depending on the nature of the involved reactive groups.

Step (iv):

Step (iii) can be performed notably with an activated hydrophilic polymer comprising an azido group (N3) or in the presence of a non-cleavable copolymer wherein part or all of the hydrophilic polymeric side-chains are terminated by an azido group (N3). In this case, step (iii) can be followed by an additional step (iv) of grafting a biomolecule on the azido groups (N3), by Click chemistry, provided that said biomolecule bears an alkyne group, preferably a terminal alkyne group (-C≡CH), such as a BCN (bicyclo[6.1 .0]-nonyne) moiety.

Step (v):

The method according to the present invention can comprise an additional step (v) after step (iii), said step (v) consisting in cleaving the disulphide bridge of the redox cleavable copolymer by a reducing treatment. In these conditions, only the hydrophilic polymer side chains of the optional non-cleavable copolymer will remain onto the coated area.

The preferred conditions of the reducing treatment are presented in step (4) of the 1 ^st coating method.

In 1 ^st, 2^nd, 3^rd and 4^th coating methods presented above, the area to be coated can be the whole surface of the substrate but most preferably will be under the form of patterns. For that, several methods can be envisaged such as a local deposition of the copolymers or an etching of patterns, which will remove locally the copolymer layer.

Local deposition: Several patterning techniques can be used to locally deposit the polymers, such as inkjet printing and spotting. Stamping is another common technique to patterning that is compatible with deposition of the polymers. In practice, a soft plastic stamp (e.g. PDMS textured scaffold) is brought in close contact with the surface of interest and solution or dispersion of the polymer composition to be deposited is introduced through microchannels to reach the uncovered regions of the surface. Functionalization steps can follow (see for example steps (C) and (D) of the 2^nd coating method; step (c) of the 3^rd coating method; step (iii) of the 4^th coating method). After removing of the stamp, the whole surface can be dipped for example into a second polymer composition to be deposited to form a continuous layer. Functionalization steps can also follow (see for example steps (C) and (D) of the 2^nd coating method; step (c) of the 3^rd coating method; step (iii) of the 4^th coating method). Another alternative is photolithography. In this case, areas are covered with a protective organic resin formed upon UV exposure, and post-UV polymer depositions occurs onto non-exposed areas.

Etching: When it is feasible, in particular for glass, silica and silicon surfaces, the preferred patterning procedure is UV etching. In this approach, a patterned mask with UV-transparent regions and UV-blocking ones is squeezed against the pre-coated surface of interest. Exposure to deep UV light etches the corresponding regions that become accessible to a second deposition process, if any.

Steps (1 ) and (2) and optional steps (3) and (4) for the 1 ^st coating method; steps (A) to (D) and optional steps (E) and (F) for the 2^nd coating method; steps (a) to (c) and optional steps (d) and (e) of the 3^rd coating method; and steps (i) to (iii) and optional steps (iv) and (v) of the 4^th coating method can be performed at least a second time on a distinct area so as to obtain more complicated patterns.

Application

The coated substrate according to the present invention, in particular when it is a solid flat support, can be used for cell culture.

More generally, the coated substrates according to the invention can be used for biological experiments.

One application is to pattern substrates with a non-adhesive layer whose repellent microdomains change on demand to display biomolecules which were previously hidden. The change on demand is obtained by a reducing treatment. To obtain this property, the cleavable copolymer could comprise hydrophilic polymeric side chains with long chains and no terminal group, whereas the non-cleavable copolymer comprises hydrophilic polymeric side chains with shorter chains terminated by a biomolecule. Thus, before the reducing treatment, the biomolecule will be hidden by the longer hydrophilic chains of the cleavable copolymer. The reducing treatment will cleave these long hydrophilic chains so as to render accessible the biomolecules.

Another application is the cell detachment upon mild redox-triggered cleavage of the supporting layer. This can be performed notably when the biomolecule is present on the cleavable copolymer. The cells can be adhered to the substrate thanks to the biomolecules. The cells are then detached upon reducing treatment which cleaved the hydrophilic polymeric side chains bearing the biomolecules.

Other applications with more complexed mixtures of copolymers or pattern substrates could be envisaged.

The examples and figures that follow illustrate the invention without limiting its scope in any way.

FIGURES

Figure 1 represents the reaction scheme to synthesize DSPP.

Figure 2 represents zetapotential variation of DSPP-coated silica beads in PBS 1 x upon addition of DTT or GSH reducing agent at time zero at various concentrations. Figure 3 represents epifluorescence images of a glass plate patterned with 6- micrometer large stripes coated with 100% DSPP-rhodamine before (A) and after (B) a GSH treatment.

Figure 4 represents fluorescence intensity along a horizontal line of a glass plate patterned with 6-micrometer large stripes coated with 100% DSPP-rhodamine before (A) and after (B) a GSH treatment.

Figure 5 represents microscope imaging of HeLa cells adherent to DSPP:PLL-PEG layers on glass plates before (A) and after (B) a DTT treatment (pictures are representative of random picking on the plate).

Figure 6 represents microscope imaging of HeLa cells deposited on RGD-displaying azidoDSPP:PLL-PEG layers on glass plates before (A) and after (B) TCEP treatment.

EXAMPLES

The following abbreviations have been used:

BCN : bicyclo[6.1 .0]-nonyne

DMEM : Dulbecco's Modified Eagle's medium DMSO dimethylsulfoxide

DTT dithiothreitol

GSH gluthation

HPLC high performance liquid chromatography

MWCO molecular weight cut-off

NMR nuclear magnetic resonance

PBS phosphate buffered saline

RGD Arginine-Glycine-Aspartic acid tripeptide

TCEP tricarboxyethylphosphine

This chapter describes:

1 ) the synthesis of cleavable copolymers, with the example of poly(lysine) derivatives DSPP (illustrated on Figure 1 ) and DSPP-azide,

2) the coating with DSPP layer of silica microbeads whose surface property is characterized by zeta potential measurements in the absence or presence of reducing agent to characterized in-layer cleavage,

3) the characterization of the detachment of PEG side chains by a reducing treatment from micropatterned flat layers,

4) the deposition and spreading of mammalian cells on flat glass plates coated with various copolymers and mixture of copolymers, with a comparison of cell spreading and attachment on these layers before and after exposure to reducing conditions, 5) the coating of a glass surface according to the 2^nd coating method of the invention. 1. DSPP and DSPPazide synthesis

Synthesis (illustrated on Figure 1 for DSPP) was completed in two steps. Firstly, the poly(lysine) (PLL) backbone is modified by conjugation with 3-(2- pyridyldithio)propionate (SPDP) to yield a reactive intermediate PLL-PDP. Secondly, o thio,(jo-methoxy-poly(ethylene glycol) (MeO-PEG-SH) is reacted with the PLL-PDP to obtain the DSPP upon disulfide bridge formation.

Synthesis of PLL-PDP. PLL with an average molecular weight of 20 kDa (9.5mg of the hydrobromide form, 0.0475mmol, 1 Eq Lysine) was dissolved in 0.5mL of sodium tetraborate-HCI buffer (50 mM, pH= 8.4). After complete dissolution, SPDP in 0.3 mL of anhydrous DMSO (4.2 mg, 0.013mmol, 0.28Eq Lysine) was added to the PLL solution. The mixture was stirred overnight under inert atmosphere at 35°C. Finally, the solution was dialyzed against water for one day with five changes of the dialysis bath (Slide-A- Lyzer Thermo Scientific, MWCO 7 kDa). The solution was freeze-dried yielding PLL- SPDP as a white powder (near quantitative). The percentage of modified lysine residue was determined by ¹H-NMR analysis in D2O from the peak intensity ratio of the PLL backbone's proton to the pyridyl protons of 3-(2-pyridyldithio)propionyl groups.

1H-NMR (D₂0, 300 MHz): δ (ppm) 8.4 (m, pyridyl, 1 H) 7.8 (m, pyridyl 2H), 7.25 (m, pyridyl, 1 H), 4.3(m, PLL backbone's proton, 4.5H), 3.14-3.0 (m, CH2-SS-pyridyl, a- amide grafted residue, oamine free residue, 13H), 2.65 (m, CH2-CH2-SS-pyridyl, 2H), 1 .7 (m, aliphatic PLL side chains, 34H)

Synthesis of DSPP. PLL-PDP (6,6 mg, 0.0259mmol, 1 Eq lysine) was dissolved in 1 .5mL of PBS (pH = 7.4) under inert atmosphere. Then MeO-PEG2000-SH (16.6 mg, 0.008 mmol, 0.32Eq lysine) was added. The mixture was stirred overnight under inert atmosphere at room temperature, prior to extensive dialysis against pure water (Slide- A-Lyzer Thermo Scientific, MWCO 7kDa) for one day with five changes of the dialysis bath. Freeze-drying yield DSPP as a white powder (near quantitative). The percentage of modified lysine residue was determined by ¹H-NMR analysis in D2O from the peak intensity ratio of the PLL backbone's chiral proton to the methoxy end group of the PEG chains.

¹H-NMR (D₂0, 300 MHz): δ (ppm) 4.3(m, PLL backbone's proton, 4H), 3.7 (m, PEG, -200H), 3.39 (s, methoxy group, 3H) 3.2 (m, CH2-SS, 2H), 3 (m, a-amide grafted residue, a-amine free residue 1 1 H), 2.68 (m, CH2-CH2-SS ,2H), 2-1 .2 (m, aliphatic PLL side chains, 30H)

Synthesis of DSPPazide. The procedure is similar to the synthesis of DSPP, though the corresponding a-thio,oo-azido-poly(ethylene glycol) chains is not commercially available and has to be prepared from a-amino-oo-azido-poly(ethylene glycol). Briefly, N3-PEG-NH2 with a molecular weight of 3kDa (50 mg, 0.017 mmol, 1 Eq) was first dissolved in 2mL of anhydrous dichloromethane. Traut's reagent (1 1.6 mg, 0.084 mmol, 5Eq) in solution of 0.2mL of MeOH was added. Then, the mixture was stirred for 2h under argon atmosphere at room temperature in the dark. After evaporation of the solvent, the product was dissolved in 1 mL of milliQ water, transferred to a dialysis tube (cut off 0.5-1 kDa) and dialyzed overnight. Finally, the solution was freeze-dried yielding a white powder.

¹H-NMR (D₂0, 300 MHz): δ (ppm) 3.7(m, PEG, -200H), 3.2 (m, CH2 -SH, 2H), 2.44 (m,CH2-CH2-SH, 2H) 2. Coating of DSPP layer on silica microbeads

In this example, silica beads were coated with DSPP and zeta potential was measured prior and after supplementation of their solution with a reducing agent (DTT or GSH) to characterize surface properties of the beads. Namely, the negative zeta potential of the beads prior to coating (ca. - 40 mV) is screened by adsorption of the copolymers and becomes slightly positive (+8mV) due to a slight excess of PLL cations in the layer compared to silica anionic surface charges. A gradual detachment of the PEG side chains by reaction of the disulphide bridge of DSPP with a thiol reducing agent translates into a gradual shift of the potential with increasing incubation times.

This experiment shows that 10 min incubation at mM concentrations of reducing agent efficiently cleaves the links between PLL backbone and the PEG side chains. In addition, this effect and control of surface properties are studied on micrometer beads showing the versatility of the system allowing to coat colloidal particles, as well as flat plates (see examples 3 & 4).

DSPP coating. Silica beads of 1 μηη diameter were sonicated for 15 min in 1 mol/L NaOH prior to dialysis against pure milliQ water for 3h (MWCO 3.5 kDa). The dispersion of beads was concentrated by centrifugation (10 min, 7000 rpm, 4xg) and the pellet was diluted in water to reach 76 mg of beads in 1 mL. Coating with DSPP was obtained as follows: 44 μί of bead dispersion was mixed with 294 μί of 3.4 g/L DSPP solution in water and incubated for 1 h at room temperature. The excess unbound DSPP was removed by three cycle of centrifugation (10 min, 7000 rpm, 4xg), and redispersion of the pellet in 1 mL PBS (1 x, pH 7.4). Final bead concentration was 3.3 g/L.

Zeta potential measurement. A dispersion of 3.3 g/L beads in PBS 1x was mixed with a solution of the reducing agent (1 mM-10 mM) in PBS 1 x up to ten fold dilution, and measurements were started at the time of mixing. Zetameter was a Malvern zetasizer Nano Z 90. Results are summarized in Figure 2. In the presence of DTT, the increase of zeta potential from +8 up to +20 mV indicates the release of PEG chains that diminishes the screening of PLL charges. In the presence of GSH, the potential first increases on similar magnitude as with DTT, and then decreases.

3. Release of PEG side chains from DSPP-rhodamine micropatterns

In this example, a flat glass plate was coated with stripes of DSPP-azide and then incubated in a solution of BCN-rhodamine to "click" the fluorescent dye on the azide- presenting stripes (the resulting copolymer is named DSPP-rhodamine). Then, the plate was imaged by epifluorescence with an aqueous solution deposited on the top of the plate. The intensity and contrast of surface fluorescence report on the presence of stripe-bound rhodamine, i.e. on preservation of uncleaved DSPP.

Plate coating. PLL-PEG was adsorbed on a microscope glass plate that has been cleaned and preconditioned (by cleaning with HPLC grade ethanol, 15 min sonication in 1 M NaOH and rinsing with milli-Q water). One drop (20μΙ_) of stock solution of PLL- PEG (1 g/L in water) was deposited on a hydrophobic film of PARAFILM® and brought in close contact with the clean glass for 30 min. The plate was rinsed with water, dried with N2. For the following deep-UV etching step, a chromium quart photomask (Delta ask, Enschede, The Netherlands) was exposed to UV light for five minutes. Then, the PLL-PEG-coated plate was placed on the mask with ~2μί of water to allow for tight capillary adhesion. The masked substrate was exposed to deep-UV for 15 min in order to etch stripes of width w = 6μηι separated between them by d = 25 μηη. Then, one drop (20μί) of aqueous solution of DSPPazide (1 g/L in water) was deposited on a hydrophobic film of PARAFILM® and brought in close contact with the PLL-PEG- coated glass for 30 min. The plate was rinsed with water, dried with N2. Finally, one drop (20μί) of solution of BCN-rhodamine (50μΜ in water) was deposited on a hydrophobic film of PARAFILM® and brought in close contact with the coated glass for 60 min. The plate was rinsed with water, dried with N2.

Epifluorescence imaging. The patterned plate was observed by epifluorescence microscopy at an excitation wavelength of 525 nm (emission 578 nm) prior and after bath application of a solution of a reducing agent (here 25 mM GSH). The loss of both intensity and contrast after exposure to reducing condition indicates the release of fluorescently labelled PEG chains from the stripes.

Figures 3 and 4 illustrates the patterned plate before (A) and after (B) GSH treatment.

4. Cell adhesion on mixed PLL-g-PEG:DSPP layers.

In this example, a flat microscope glass plate was coated with DSPP:PLL-PEG mixed layers and used to capture HeLa cells for increasing incubation times. The cell attachment and spreading on surfaces were determined for varying layer compositions and on plate treated or not with a reducing agent (DTT).

Plate coating. Cleaned and preconditioned microscope glass plates (rinsed with HPLC grade ethanol, 15 min sonication in 1 M NaOH, and rinsing with milli-Q water) were coated by application for 30 min of one drop (-20 μί) of a mixed solution of DSPP and PLL-PEG at a fixed total concentration of 1 g/L and DSPP/PLL-PEG weight ratios adjusted between 0 and 100% (another coating made with a solution of poly(lysine), PLL, was used as a reference for highly adhesive layer). The plates were rinsed with water and dried under N2.

Cell adhesion. To prepare DTT-treated coatings, the dried plates were immersed in a solution of 10mM DTT for 30min, then rinsed with water. DTT-treated, or non-treated plates were then immersed in wells containing cell culture medium (DMEM high glucose (Sigma), calf serum 10%). 30 000 HeLA cells (wild type) were seeded on each well, incubated at 37°C under CO2 atmosphere for 3.5 h to 24h. Average number of sedimentated round-like and of spreaded adherent cells were determined by random observations on a total of 0.16 mm² area. Figure 5 represents microscope imaging of the cells deposited on plates that have been either DTT-treated or not. Based on data reported in Tables 1 and 2 below, the following conclusions are established:

1 ) The composition of the mixed layer markedly affects cell attachment. In particular, increasing the %PLL-PEG gradually decreases the propensity for adhesion.

2) The treatment with DTT favors both cell adherence (total number of adherent cells) and spreading.

3) The contrast between adhesion prior and after DTT treatment depends on the mixed layer composition, and is NOT reached at 100% DSPP or at 100%PLL-PEG (that correspond to almost no adhesion).

Table 1. HeLa cell adhesion at 37°C on DSPP:PLL-PEG mixed layers deposited on glass plates, and increasing incubation time as quoted in columns, (in each column, the number on the left indicates the average number of cells in 0.16 mm² ± the standard deviation; the % number on the right is the % of cells that were tightly bound and appeared as spreaded on the substrate)

Coating t=3.5h t=3.5h + DTT t=7h t=7h + DTT

PLL 2,0±1 .9 17% nd 7,8±3.2 64% nd

PLL-PEG

0,0±0 0% 0,0±0 0 0,2±0.4 5% 0,0±0 0% (100%)

DSPP:PLL- PEG 1 ,0±1 .2 6% 20±14 34% 4,6±2.4 38% 13.5±5.4 68%

30/70

DSPP:PLL- PEG 1 ,0±1 .4 5% 5,6±2.6 34% 7,6±4.3 62% 12,3±3.8 81 %

50/50

DSPP

2,0±1 .9 18% 10,6±3.8 51 % 8,0±2.2 55% 12.0±2.5 79% (100%)

Table 2. Same as Table 1 , except that the layer also contained a small amount of PLL

(as obtained by preexposure of the mixed layer to a solution of 1 g/L PLL for 20 s)

Coating t=3.5h t=3.5h + DTT t=7h t=7h + DTT

PLL 2,0±2.0 17% nd 7,80±3.2 64% nd

PLL-PEG

0,0±0 0% 0,0±0 0% 0,4±0.5 6% 1 ,2±1 .3 31 % (100%)

DSPP:PLL- PEG 0,4±0.5 4% 3,4±0.5 44% 3,0±1 .6 23% 8,0±1 .9 74%

30/70

DSPP:PLL- PEG 2,2±0.4 24% 3,8±1 .8 43% 5,2±2.2 68% 12,0±3.9 78%

50/50

DSPP

2,8±1 .8 28% 2,8±2.8 30% 10,8±4.0 59% 14,2±4.2 86% (100%)

5. Cell detachment from a mixed PLL-PEG :RGD-DSPP layer.

In this example, a flat microscope glass plate was coated with DSPP-azide:PLL-PEG mixed layer and used to capture HeLa cells after grafting of a RGD peptide on the azido function by "click" chemistry. After cell attachment and spreading on surface, the bath application of the reducing agent TCEP induces cell detachment as detected by loss of their spread configuration and transition to round-shaped cells. Plate coating. Cleaned and preconditioned microscope glass plates (rinsed with HPLC grade ethanol, 15 min sonication in 1 M NaOH, and rinsing with milli-Q water) were coated by application for 30 min of one drop (-20 μΙ_) of a mixed solution of DSPP- azide and PLL-PEG at a fixed total concentration of 0.1 g/L and DSPP-azide/PLL-PEG weight ratios adjusted to 10%. The plates were rinsed with water and prior to bath application for 30 min of a 100 μΜοΙ/L solution of BCN-RGD to "click" the cell-adhesive RGD peptide on the top of the layer. Then the plates were rinsed again with water and then immersed in DMEM high glucose (Sigma), calf serum 10%.

Cell adhesion. The immersed plates in a well containing cell culture medium (DMEM high glucose (Sigma), calf serum 10%) were seeded with 30 000 HeLA cells (wild type) on each well, incubated at 37°C under CO2 atmosphere for 4 h. At time 4 h, the medium was gently removed by aspiration, and replaced by fresh, cell-free DMEM. Random observation of the plates confirmed that a predominant cell fraction was spread on the glass (Figure 6A). After addition of TCEP solution in the wells, to reach a final concentration of 5 mM, cells were observed 12 min after TCEP treatment by optical microscopy to undergo a transition to rounded shapes, which is indicative of their detachment (Figure 6B which is representative of images taken at random).

6. In-situ synthesis of ad layers of DSPP and DSPPazide

The synthesis recapitulates the same steps as for bulk synthesis, but onto adsorbed PLL layers. It was completed in three steps:

First, poly(lysine) (PLL) is deposited on a plasma-cleaned glass surface by contact with an aqueous solution of poly(lysine) hydrobromide. The PLL-coated surface is flushed with milli-Q water, then an aqueous solution of an amine-reactive pyridyldithio reagent (ex: 3-(2-pyridyldithio)propionate, SPDP) is applied to yield the reactive intermediate PLL-PDP. After flushing again with milliQ-water, a solution of othio,oo-azido- poly(ethylene glycol) (N3-PEG-SH) (or a mixture of N3-PEG-SH with a-thio,oo-methoxy- poly(ethylene glycol)) is reacted with the surface-bound PLL-PDP to obtain the DSPP adlayer.

Example of a detailed conditions of synthesis.

PLL with an average molecular weight of 20 kDa was dissolved at 0.1 g/L in water or PBS 1 x buffer pH 7.2. Clean glass plates were prepared by first 5 min. bath in ethanol, followed by treatment in oxygen plasma (400 mbar, 1 min. )- The PLL solution was applied to the glass surface at 4°C, for 30 min., before flushing out the excess PLL with milliQ water. A 20 mM solution of succinimidyl 3-(2-pyridyldithio)propionate (SPDP, Aldrich) in anhydrous DMSO was diluted in PBS buffer down to a final concentration of 0.3 mM and immediately applied to the PLL-coated glass for 30 min. at 25°C. The excess reactant was then rinsed out with milliQ water. Eventually, a solution of thiol- ended PEGs (a-thio,oo-azido-poly(ethylene glycol) Mw = 5000 g.mol ¹, from NANOCS; 10 g. L¹ in PBS 1 X) was applied for 30 min. at 25° C yielding the final DSPP adlayer. Determination of the relative PEG density in DSPP layer.

The relative surface density of azido groups attached on surfaces was characterized by fluorescence measurement after clicking Cy3 photochromes onto the DSPP adlayer. Plate A: Layer of DSPPazide were prepared as described above using 100% a-thio,oo- azido-poly(ethylene glycol). After rinsing with milliQ water, the layer was UV etched to form parallel stripes of polymer adlayers next to bare glass ones. A solution of DBCO- Sulfo-Cy3 (Aldrich; at 100 μΜ in water) was applied onto the surface (30 min., room T), and eventually the surface was flushed 3 times with 0.1 M NaCI solution in water followed by milliQ water to remove all non-reacted Cy3. Optical microscopy fluorescence imaging was performed to measure the intensity of Cy3-labelled stripes vs non-labelled bare glass.

Plate B: a reference layer was obtained by adsorption of DSPP from solution as described in the paragraph "coating method" (using here DSPPazide at 0,1 g.L-1 in PBS 1 X , 30 min. 4°C). Similar to above, parallel stripes were UV-etched to form polymer layer alternated with bare glass, and polymer stripes were labelled with DBCO-Sulfo-Cy3 and eventually imaged by fluorescence.

The ratio of fluorescence intensity (intensity of stripes on plate A / intensity on plate B) reached a mean value of 2.5, showing the marked enhancement of PEG-azide attachment by the in-situ synthesis.

Claims

1. A comb-like copolymer comprising a polycationic polymeric backbone and hydrophilic polymeric side-chains grafted onto the polycationic polymeric backbone via a linker covalently bound to the polycationic polymeric backbone and the hydrophilic polymeric side-chain, wherein:

the polycationic polymeric backbone is a linear or branched, preferably linear polymer comprising at least 30 mol%, notably at least 50 mol%, preferably at least 80 mol%, in particular 100 mol% of cationic structural repeat units, a cationic structural repeat unit being under the form of a cation at pH = 7;

the hydrophilic polymeric side-chains are a linear or branched, preferably linear chain of water-soluble non-ionic monomers;

the linker is a chemical group containing a disulphide bridge (-S-S-); and wherein the grafting ratio is between 10 mol% and 70 mol%, preferably between 30 mol% and 50 mol%, with respect to the molar amount of the cationic structural repeat units.

2. The comb-like copolymer according to claim 1 , wherein said cationic structural repeat unit is selected from the group consisting of an alkylene amine, a bis(alkylene) amine, a tris(alkylene) amine, an amino-acid selected from the group consisting of ornithine, lysine, histidine and arginine, and a combination thereof; preferably said cationic structural repeat unit is an amino-acid, such as lysine.

3. The comb-like copolymer according to claim 1 or 2, wherein the polycationic polymeric backbone comprises between 20 and 10 000 cationic structural repeat units, preferably between 50 and 1 000 cationic structural repeat units.

4. The comb-like copolymer according to any one of claims 1 to 3, wherein the polycationic polymeric backbone further comprises polar non-ionic structural repeat units and/or anionic structural repeat units, preferably polar non-ionic structural repeat units, with the proviso that the molar ratio of anionic structural repeat units to cationic structural repeat units is inferior to 10 mol%.

5. The comb-like copolymer according to any one of claims 1 to 4, wherein said water-soluble non-ionic monomer of the hydrophilic polymeric side-chain is selected from the group consisting of ethylene glycol, oxazoline, N-isopropylacrylamide, vinylpyrrolidinone, glycidol and a combination thereof, and is preferably ethylene glycol.

6. The comb-like copolymer according to any one of claims 1 to 5, wherein the longest linear chain of monomers in each hydrophilic polymeric side-chain comprises between 20 and 500 monomers.

7. The comb-like copolymer according to any one of claims 1 to 6, wherein the polycationic polymeric backbone is polylysine (PLL) and the hydrophilic polymeric side- chains are polyethyleneglycol (PEG).

8. The comb-like copolymer according to any one of claims 1 to 7, wherein part or all of the hydrophilic polymeric side-chains are terminated by a bioorthogonal reactive group or a biomolecule, wherein:

- the bioorthogonal reactive group is an azido group (N3) or a fluorophore group such as rhodamine, preferably an azido group; and

the biomolecule is advantageously a peptide such as a cell adhesion peptide or an enzyme; a growth factor; an antibody; a fragment of antibody such as scFv (single chain variable fragment) or a diabody; or an antibody mimic such as a monobody.

9. The comb-like copolymer according to any one of claims 1 to 8, wherein the linker is covalently bound at one of its extremity to an amino group of a cationic structural repeat unit of the polycationic polymeric backbone via an urea, urethane, amide or triazole group, preferably an amide group; and is covalently bound at its other extremity to a hydrophilic polymeric side-chain via an urea, urethane, carbonate, amide, thioether or triazole group or via its disulphide bridge, preferably via its disulphide bridge.

10. A kit or composition comprising:

■ at least one cleavable copolymer, which is a comb-like copolymer according to any one of claims 1 to 9; and

■ optionally at least one non-cleavable copolymer, which is a comb-like copolymer comprising a polycationic polymeric backbone and hydrophilic polymeric side- chains covalently grafted onto the polycationic polymeric backbone, wherein: the polycationic polymeric backbone is as defined in any one of claims 1 to 4 and 7,

the hydrophilic polymeric side-chains are each as defined in any one of claims 1 and 5 to 8, and

wherein the grafting ratio is as defined in claim 1.

11. The composition according to claim 10, wherein it is an aqueous solution or dispersion with a pH between 5 and 9, preferably between 6 and 8, more preferably the pH is neutral.

12. A method for coating an area of the surface of a substrate comprising the steps of:

(1 ) forming anionic charges onto said area,

(2) contacting the charged area resulting from step (1 ) with a composition according to claim 10 or 1 1 in the form of a solution or a dispersion, so that the polymers contained in said composition are deposited onto the area of the surface of the substrate,

(3) optionally, when step (2) is performed with a composition containing at least one cleavable copolymer or non-cleavable copolymer wherein part or all of the hydrophilic polymeric side-chains are terminated by an azido group (N3), grafting a biomolecule bearing an alkyne group by Click chemistry to the azido groups, and

(4) optionally cleaving the linker under reducing conditions, preferably by treatment with a reductive agent selected from the group consisting of glutathione (GSH), tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol (DTT) and mercaptoethanol.

13. The method according to claim 12, wherein steps (1 ) and (2) and optionally steps (3) and (4) are performed at least a second time on a distinct area.

14. A method for coating an area of the surface of a substrate comprising the steps of:

(A) forming anionic charges onto said area,

(B) contacting the charged area resulting from step (A) with a composition comprising a polycationic polymer optionally in mixture with a non-cleavable copolymer in the form of a solution or a dispersion, so that the polycationic polymer and the optional non-cleavable copolymer contained in said composition are deposited onto the area of the surface of the substrate,

(C) reacting the polycationic polymer deposited onto the area of the surface of the substrate in step (B) with a bifunctional organic compound in order to form an activated polycationic polymer onto the area of the surface of the substrate,

(D) reacting the activated polycationic polymer obtained in step (C) with a hydrophilic polymer bearing a reactive group on one of its extremity so as to form a comb-like copolymer according to any one of claims 1 to 9 onto the area of the surface of the substrate, optionally in mixture with a non-cleavable copolymer,

(E) optionally, when step (D) is performed with a hydrophilic polymer bearing a reactive group on one of its extremity and an azido group (N3) on its other extremity or in the presence of a cleavable copolymer or non-cleavable copolymer wherein part or all of the hydrophilic polymeric side-chains are terminated by an azido group (N3), grafting a biomolecule bearing an alkyne group by Click chemistry to the azido groups, and

(F) optionally cleaving the disulphide bridge of the comb-like copolymer according to any one of claims 1 to 9 formed in step (D) under reducing conditions, preferably by treatment with a reductive agent selected from the group consisting of glutathione (GSH), tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol (DTT) and mercaptoethanol,

wherein the non-cleavable copolymer is as defined in claim 10,

15. The method according to claim 14, wherein steps (A) to (D) and optionally steps (E) and (F) are performed at least a second time on a distinct area.

16. A method for coating an area of the surface of a substrate comprising the steps of:

(a) forming anionic charges onto said area,

(b) contacting the charged area resulting from step (a) with a composition comprising an activated polycationic polymer optionally in mixture with a non- cleavable copolymer in the form of a solution or a dispersion, so that the activated polycationic polymer and the optional non-cleavable copolymer contained in said composition are deposited onto the area of the surface of the substrate,

(c) reacting the activated polycationic polymer deposited onto the area of the surface of the substrate in step (b) with a hydrophilic polymer bearing a reactive group on one of its extremity so as to form a comb-like copolymer according to any one of claims 1 to 9 onto the area of the surface of the substrate, optionally in mixture with a non-cleavable copolymer,

(d) optionally, when step (c) is performed with a hydrophilic polymer bearing a reactive group on one of its extremity and an azido group (N3) on its other extremity or in the presence of a cleavable copolymer or non-cleavable copolymer wherein part or all of the hydrophilic polymeric side-chains are terminated by an azido group (N3), grafting a biomolecule bearing an alkyne group by Click chemistry to the azido groups, and

(e) optionally cleaving the disulphide bridge of the comb-like copolymer according to any one of claims 1 to 9 formed in step (c) under reducing conditions, preferably by treatment with a reductive agent selected from the group consisting of glutathione (GSH), tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol (DTT) and mercaptoethanol,

wherein the non-cleavable copolymer is as defined in claim 10,

wherein the activated polycationic polymer is a polycationic polymer coupled to a bifunctional compound,

The method according to claim 16, wherein steps (a) to (c) and optionally steps and (e) are performed at least a second time on a distinct area.

18. A method for coating an area of the surface of a substrate comprising the steps of:

(i) forming anionic charges onto said area,

(ii) contacting the charged area resulting from step (i) with a composition comprising a polycationic polymer optionally in mixture with a non-cleavable copolymer in the form of a solution or a dispersion, so that the polycationic polymer and the optional non-cleavable copolymer contained in said composition are deposited onto the area of the surface of the substrate,

(iii) reacting the polycationic polymer deposited onto the area of the surface of the substrate in step (ii) with an activated hydrophilic polymer so as to form a comblike copolymer according to any one of claims 1 to 9 onto the area of the surface of the substrate, optionally in mixture with a non-cleavable copolymer,

(iv) optionally, when step (iii) is performed with an activated hydrophilic polymer comprising an azido group (N3) or in the presence of a cleavable copolymer or non-cleavable copolymer wherein part or all of the hydrophilic polymeric side- chains are terminated by an azido group (N3), grafting a biomolecule bearing an alkyne group by Click chemistry to the azido groups, and

(v) optionally cleaving the disulphide bridge of the comb-like copolymer according to any one of claims 1 to 9 formed in step (c) under reducing conditions, preferably by treatment with a reductive agent selected from the group consisting of glutathione (GSH), tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol (DTT) and mercaptoethanol,

wherein the non-cleavable copolymer is as defined in claim 10, wherein the polycationic polymer is a linear or branched, preferably linear polymer comprising at least 30 mol%, notably at least 50 mol%, preferably at least 80 mol%, in particular 100 mol% of cationic structural repeat units, a cationic structural repeat unit according to the invention being under the form of a cation (ionized form) at pH = 7, notably in the range of pH between 6 and 8, in particular between 5 and 9,

wherein the activated hydrophilic polymer is a hydrophilic polymer bearing a reactive group on one of its extremity coupled to a bifunctional compound,

19. The method according to claim 18, wherein steps (i) to (iii) and optionally steps (iv) and (v) are performed at least a second time on a distinct area.

20. A coated substrate obtainable by the method according to any one of claims 12 to 19.

21. The use of the coated substrate according to claim 20 for cell culture.