WO2021013992A1

WO2021013992A1 - Injectable polymer compositions with hyaluronic acid cross-linked by boronate esters and functionalised by rgd peptides for cell and tissue engineering

Info

Publication number: WO2021013992A1
Application number: PCT/EP2020/070958
Authority: WO
Inventors: Rachel Auzely-Velty; Tamiris VILAS BOAS FIGUEIREDO; Marlène RIPPE
Original assignee: Centre National De La Recherche Scientifique; Universite Grenoble Alpes
Priority date: 2019-07-24
Filing date: 2020-07-24
Publication date: 2021-01-28
Also published as: FR3099163A1

Abstract

The application relates to a polymer composition comprising a mixture of: a) a hyaluronic acid polymer at least one carboxylate function of which is grafted with 3-aminophenylboronic acid, and b) a hyaluronic acid polymer at least one carboxylate function of which is grafted with 1-amino-1-deoxy-D-fructose or with gluconamide, on the condition that at least one of the aforementioned polymers is grafted with an RGD peptide, and the uses of said polymer composition for cell culture, cell transplantation, cell therapy, as printing ink or for tissue engineering.

Description

TITLE: Injectable polymeric compositions of hyaluronic acid crosslinked by boronate esters and functionalized by RGD peptides for cell and tissue engineering

The present invention relates to injectable polymeric compositions of hyaluronic acid crosslinked by boronate esters and functionalized by RGD peptides for cell and tissue engineering.

Application WO 2014/072330 describes a polymer composition comprising:

- a hyaluronic acid polymer of which at least one hydroxyl function is grafted with phenylboronic acid, and

- A hyaluronic acid polymer, at least one hydroxyl function of which is grafted with the hydroxyl of a group comprising a cis-diol, preferably maltose.

It is the hydroxyl functions of hyaluronic acid that are grafted. The preparation of these polymers requires the grafting of spacer arms ("linkers" in English) carrying a thiol group on phenylboronic acid and on maltose, and the grafting of groups carrying an alkene function on hyaluronic acid. These grafts complicate the process for preparing the polymers.

Application WO 2018/024793 describes a polymer composition comprising:

- a hyaluronic acid polymer of which at least one carboxylate function is grafted with a group comprising a boroxole and

- a hyaluronic acid polymer, at least one carboxylate function of which is grafted with the amine function of a compound comprising a diol.

This application distances the person skilled in the art from using a hyaluronic acid polymer grafted with phenylboronic acid. Indeed, it teaches that a boronate hemiester has a greater affinity towards the diols than phenylboronic acid, and that the gels obtained from a boronate hemiester have a higher modulus of conservation G ', and properties of improved gel. The polymeric composition described thus has an exacerbated self-repairing power compared to the polymeric compositions in which the hyaluronic acid polymer is grafted with phenylboronic acid instead of boroxole. The inventors of the present application have however observed that the high affinity of the boronate hemiester towards the diols has drawbacks: the gels according to WO 2018/024793 can be difficult to sterilize because the hyaluronic acid polymer of which at least one carboxylate function is grafted by a group comprising a boroxole is not stable under autoclaving conditions (probable degradation of the polymer), so that it no longer allows the formation of a gel when mixed with the acidic polymer hyaluronic grafted with a diol compound. When these polymeric compositions comprise an aqueous phase, they form hydrogels. These resemble the natural extracellular matrix, and therefore have the advantage of being usable in biomedical applications. However, the spreading and viability of the cells incorporated into these hydrogels are not sufficient.

There is a need to provide polymeric compositions capable of containing cells, allowing control of the behavior of cells, in particular their spreading and their proliferation, and adaptable according to the type of cells used, while maintaining the injectable and self-contained nature. -reparing polymeric compositions described above.

To this end, according to a first object, the invention relates to a polymeric composition comprising a mixture of:

a) a hyaluronic acid polymer of which at least one carboxylate function is grafted with 3-aminophenylboronic acid ("HA-PBA-xRGD" below), and

b) a hyaluronic acid polymer, at least one carboxylate function of which is grafted with 1 -amino-1 -deoxy-D-fructose (“HA-fructose-yRGD” below) or with gluconamide (“HA-gluconamide-zRGD ”Below),

with the proviso that at least one of said polymers is grafted with an RGD peptide.

In the polymeric composition, the HA-PBA-xRGD polymer and the HA-fructose-yRGD polymer, or the HA-PBA-xRGD polymer and the HA-gluconamide-zRGD polymer, are linked to each other by boronate ester functions between the boronic acid of the HA-PBA-xRGD polymer and the saccharide unit either fructosamine (1 -amino-1 -deoxy-D- fructose) of the HA-fructose-yRGD polymer, or gluconamide (glucose in its open form ) HA-gluconamide-zRGD polymer.

By "hyaluronic acid" or "HA" is meant hyaluronic acid in neutral or salt (hyaluronate) form, regardless of its charge and the nature of its counterion (s). It can, for example, be sodium hyaluronate. Hyaluronic acid can be of animal or non-animal origin. Sources of non-animal origin include yeasts and preferably bacteria which are used to produce hyaluronic acid biotechnologically. The mass average molar mass (Mw) of hyaluronic acid is typically in the range of 1 to 3000 kg / mol, but other mass average molar masses are possible.

The polymeric composition comprises a hyaluronic acid polymer, at least one carboxylate function of which is grafted with 3-aminophenylboronic acid, called the HA-PBA-xRGD polymer. 3-Aminophenylboronic acid is grafted to the acid hyaluronic by an amide bond formed between the amine of 3-aminophenylboronic acid and a carboxylate function of hyaluronic acid.

The polymeric composition also comprises a hyaluronic acid polymer of which at least one carboxylate function is grafted either by 1 -amino-1 -deoxy-D-fructose (HA-fructose-yRGD polymer), or by gluconamide (HA-gluconamide polymer). zRGD).

Gluconamide is grafted to hyaluronic acid via a spacer arm which links a carboxylate function of hyaluronic acid to the amine function of gluconamide, preferably via amide bonds. Preferably, this spacer arm has the formula -NH-L- in which:

- the terminal group -NH forms, with a carboxyl function of hyaluronic acid, an amide bond (formed by reaction of a carboxylate function of HA and amine),

- L represents a carbon chain comprising from 2 to 10 carbon atoms and optionally interrupted by one or more oxygen atoms (typically from 1 to 5 oxygen atoms). L represents in particular a group - (CH ₂ ) _n - in which n represents an integer from 1 to 10, preferably from 1 to 4, or a group - (CX-CX-OJ _m -CX-CX- in which m represents an integer of 1 to 4, preferably 1 or 2.

For example, the spacer arm has the formula -NH- (CH ₂ -CH ₂ -0) ₂ -CH ₂ -CH ₂ -, the terminal group -NH forming, with a carboxyl function of hyaluronic acid, an amide bond (formed by reaction of a carboxylate function of HA and of the amine), and the -ex terminal group being linked to the -NH- terminal group of the gluconamide. 1 -amino-1 -deoxy-D- fructose is grafted to hyaluronic acid by an amide bond formed from the amine of 1 -amino-1 -deoxy-D-fructose and the carboxylate function of hyaluronic acid.

According to a first alternative, the polymeric composition comprises an HA-PBA-xRGD polymer and an HA-fructose-yRGD polymer, the HA-PBA-xRGD polymer and / or the HA-fructose-yRGD polymer being grafted with a peptide RGD. Such a polymer composition is also called “HA-PBA-xRGD / HA-fructose-yRGD” below. This alternative is particularly preferred because the bonds between HA-PBA-xRGD and HA-fructose-yRGD are very strong, which could be explained by the fact that the boronic acid of the polymer HA-PBA-xRGD becomes complex in three dots on the 1 -amino-1 -deoxy-D-fructose saccharide unit, whereas in most of the polymeric compositions described in the prior art, the boronic acid grafted onto the first hyaluronic acid only becomes complex in two dots on the cis-diol group grafted onto the second hyaluronic acid. In this first alternative, the polymeric composition is generally free of HA-gluconamide-zRGD polymer. According to a second alternative, the polymeric composition comprises an HA-PBA-xRGD polymer and an HA-gluconamide-zRGD polymer, the HA-PBA-xRGD polymer and / or the HA-gluconamide-zRGD polymer being grafted with a peptide RGD. Such a polymeric composition is also called “HA-PBA-xRGD / HA-gluconamide-zRGD” below. In this second alternative, the polymeric composition is generally free of HA-fructose-yRGD polymer.

Whatever the alternative considered, the inventors have observed that the polymeric compositions comprising an HA-PBA polymer and either an HA-fructose polymer, or an HA-gluconamide polymer make it possible to obtain more stable gels than those obtained with compositions. Polymers comprising an HA-PBA polymer and an HA- [saccharide derivative other than fructosamine or gluconamide] polymer.

By way of illustration, [Fig 4] Figure 4 shows that a polymer composition comprising

- a hyaluronic acid polymer of which at least one carboxylate function is grafted with 3-aminophenylboronic acid ("HA-PBA"), and

- a hyaluronic acid polymer of which at least one carboxylate function is grafted with glucose blocked in its cyclic form,

is not a gel (G "> G").

This improved stability and / or this possibility of obtaining a gel could be explained by the existence of particularly strong ester boronate bonds between the boronic acid of the HA-PBA-xRGD polymer and the saccharide unit, namely fructosamine (1 -amino-1 -deoxy-D-fructose) of the HA-fructose-yRGD polymer, or gluconamide (glucose in its open form) of the HA-gluconamide-zRGD polymer. Without wishing to be bound by a particular theory, the inventors assume that these two specific saccharide derivatives are capable of complexing with boronic acid at three points thanks to the non-cyclic “open” form of the saccharide which exhibits very great flexibility, with formation of a tricovalent boronate ester, while the other saccharide derivatives generally form a bicovalent boronate ester with boronic acid, therefore less stable.

The degree of substitution (DS) is the average mole number of substituting for every 100 disaccharide repeating units of hyaluronic acid, this substituent being phenylboronic acid (hereinafter “PBA”) for the HA-PBA- polymer. xRGD, fructosamine for HA-fructose-yRGD polymer, and gluconamide for HA-gluconamide-zRGD polymer. For each of the three polymers (independently of one another), the degree of substitution is generally from 5 to 70 mol%, preferably from 10 to 50 mol%. It has been observed experimentally that these DS ranges make it possible to obtain a “gel” type behavior by analysis of the rheological properties in dynamic mode (storage modulus (G ')> loss modulus (G ”) over the entire frequency range explored (0.01 -10 Hz)). The degree of substitution can be measured by NMR.

In the polymeric composition, the molar ratio:

- PBA groups of the HA-PBA-xRGD polymer relative to the fructosamine of the HA-fructose-yRGD polymer, or else

- PBA groups of the HA-PBA-xRGD polymer relative to the gluconamide group of the HA-gluconamide-zRGD polymer,

is generally from 10/1 to 1/10, in particular from 5/1 to 1/5, preferably from 1/3 to 3/1, particularly preferably from 1/2 to 2/1, ideally from 0.8 at 12. The number of moles of PBA grafted onto the HA-PBA-xRGD is preferably as close as possible either to the number of moles of fructose of HA-fructose-yRGD, or to the number of moles of gluconamide of HA-gluconamide-zRGD, in order to 'optimizing the formation of ester boronate bonds and obtaining optimum complexation by mixing solutions of HA-PBA-xRGD and either HA-fructose-yRGD or HA-gluconamide-zRGD in equal volume.

By "grafted with an RGD peptide" is meant that the hyaluronic acid polymer is grafted with at least one group carrying a peptide comprising the arginine-glycine-aspartic acid (RGD) sequence. This peptide helps promote cell adhesion. The peptide can include other amino acids. The "RGD peptide" typically comprises 3 to 20 amino acids, especially 4 to 15 amino acids, preferably 5 to 15 amino acids, with the proviso that at least three of them together constitute the RGD sequence. The amino acids contiguous to the RGD sequence are chosen independently of one another from natural amino acids. Preferably, at least 50% of the amino acids contiguous to the RGD sequence are polar residues chosen from Gly, Ser, Thr and Tyr. For example, hyaluronic acid can be grafted with a GRGDY or GRGDS peptide.

At least one of the polymers of the polymeric composition is grafted with an RGD peptide. The HA-PBA-xRGD polymer comprises an average number of moles "x" of RGD peptides per 100 disaccharide repeat units of hyaluronic acid. The HA-fructose-yRGD polymer comprises an average number of moles "y" of RGD peptides per 100 disaccharide units of hyaluronic acid. The HA-gluconamide-zRGD polymer comprises an average number of moles “z” of RGD peptides per 100 disaccharide units of hyaluronic acid x, y and z are also called “RGD peptide density”. x, y and z are, independently of one another, a number generally comprised from 0 to 50 mol%, in particular from 0 to 40 mol%, typically from 0 to 30 mol%, preferably from 0 to 20 mol%. Since at least one of the polymers of the polymeric composition is grafted with an RGD peptide:

- when the polymer b) is the HA-fructose-yRGD polymer, x and y do not both represent 0,

- when the polymer b) is the HA-gluconamide-zRGD polymer, x and z do not both represent 0.

When the HA-PBA-xRGD polymers and either HA-fructose-yRGD or HA-gluconamide-zRGD are mixed in equal amounts, the average number of moles "c" of RGD peptides per 100 disaccharide repeat units of hyaluronic acid of the polymeric composition is equal to:

(x + y) / 2 when the polymer b) is HA-fructose-yRGD, or

(x + z) / 2 when the polymer b) is HA-gluconamide-zRGD.

It is therefore easy to modulate the number of average moles "c" of RGD peptides per 100 disaccharide repeating units of hyaluronic acid of the polymeric composition according to the invention by adjusting that of each of the polymers which it contains (by adjusting x and either y or z).

In a first embodiment, the HA-PBA-xRGD polymer is grafted with an RGD peptide, and either the HA-fructose-yRGD polymer or the HA-gluconamide-zRGD polymer is free of RGD peptide. In this embodiment, x is different from 0 and either y is 0 or z is 0. c is then equal to x / 2.

In a second embodiment, either the HA-fructose-yRGD polymer or the HA-gluconamide-zRGD polymer is grafted with an RGD peptide, and the HA-PBA-xRGD polymer is free of RGD peptide. In this embodiment, either y or z is different from 0 and x is 0. c is then equal to y / 2 when the polymer b) is the HA-fructose-yRGD polymer, or c is equal to z / 2 when the polymer b) is the HA-gluconamide-zRGD polymer.

In a third embodiment, the HA-PBA-xRGD polymer is grafted with an RGD peptide, and either the HA-fructose-yRGD polymer or the HA-gluconamide-zRGD polymer is grafted with an RGD peptide. In this embodiment, x and either y or z are different from 0. c is then equal to (x + y) / 2 when the polymer b) is the HA-fructose-yRGD polymer, or c is equal to (x + z) / 2 when the polymer b) is the HA-gluconamide-zRGD polymer.

Most often, the polymeric composition comprises an aqueous solution. It is then in the form of a hydrogel.

The sum of the concentrations of HA-PBA-xRGD and HA-fructose-yRGD polymers in the aqueous solution, or the sum of the concentrations of HA-PBA-xRGD and HA-gluconamide-zRGD polymers in the aqueous solution, generally ranges from 1 to 100 mg / ml, in particular 2 to 50 mg / ml, preferably 5 to 30 mg / ml, particularly preferably 10 to 30 mg / ml.

The pH of the aqueous solution of the polymeric composition is generally from 7.0 to 10.0, especially from 7.0 to 9.0, preferably from 7.0 to 8.0, particularly preferably from 7.0 to 7.5.

The aqueous solution is typically a buffer solution, for example, phosphate buffered saline (0.15 M NaCl) (whose pH is 7.4), DPBS (Dulbecco's phosphate buffer), or cell culture medium, which is adapted depending on the cell type of the polymeric composition, for example DMEM (Eagle's minimum essential medium) culture medium for fibroblasts.

The high affinity of the saccharide units of the HA-fructose-yRGD polymer or of the HA-gluconamide-zRGD polymer for the PBA of the HA-PBA-xRGD polymer makes it possible to obtain a “gel” type behavior of the HA-PBA polymer composition. - xRGD / HA-fructose-yRGD or HA-PBA-xRGD / HA-gluconamide-zRGD. The “gel” type behavior is deduced from rheological measurements in dynamic mode and is defined by the fact that the storage modulus (G ') is greater than the loss modulus (G ”) on all frequencies from 0.01 to 10 Hz The rheological properties of the polymer composition are little impacted by the functionalization with an RGD peptide, and therefore by the values of x, y, z and c.

The dynamic nature of the boronate ester bonds between the hyaluronic acid chains of the HA-PBA-xRGD polymer and the HA-fructose-yRGD or HA-gluconamide-zRGD polymer gives the hydrogels a self-repairing character. By way of illustration, the hydrogels can be injected through a needle of small diameter (typically with a diameter of 0.1 to 0.4 mm) and rapidly recover its mechanical properties after injection. When the polymer is pushed into the needle of a syringe, the reversible bonds formed between the PBA of the HA-PBA-xRGD polymer and the saccharide units of the HA-fructose-yRGD or HA-gluconamide-zRGD polymer break down and then break down. reform once the polymer is ejected from the needle. The polymer is advantageously malleable until the bonds reform. In this case, advantageously, the kinetics of the reactions initiated are sufficiently slow to allow the polymeric composition to be shaped in a reproducible manner, and sufficiently fast to maintain this shape. This makes the polymeric composition particularly suitable for uses as ink, in particular as a bio-ink, for printing, in particular 3D printing, in particular 3D bioprinting, as detailed below.

The polymeric composition can comprise cells, in particular fibroblasts, for example human fibroblasts. Usually cells are present at its surface and / or are dispersed within the polymeric composition. By adjusting the RGD peptide density (i.e. c, and therefore x and either y or z), it is advantageously possible to control the behavior of the cells, in particular the degree of cell spreading and proliferation in the polymer composition.

The polymeric composition can comprise one or more therapeutic agent (s).

The polymeric composition may further comprise a sodium alginate, which is a polysaccharide capable of gelling in the presence of calcium ions. It then comprises a mixture of three polymers: HA-PBA-xRGD / HA-fructose-yRGD / sodium alginate or HA-PBA-xRGD / HA-gluconamide-zRGD / sodium alginate. The concentration of sodium alginate in the polymer composition is generally 0.01 to 5.0% (w / v), preferably 0.1 to 2.0% (w / v)). The presence of sodium alginate has little impact on the rheological properties of the polymer composition. Such a polymer composition is particularly suitable as a bio-ink for printing, as explained below. Such polymeric compositions can be shaped together with cells.

According to a second object, the invention relates to a process for preparing the polymer composition defined above comprising the mixture:

a) a hyaluronic acid polymer of which at least one carboxylate function is grafted with 3-aminophenylboronic acid, and

b) of a hyaluronic acid polymer of which at least one carboxylate function is grafted with 1 -amino-1 -deoxy-D-fructose or with gluconamide

with the proviso that at least one of said polymers is grafted with an RGD peptide, in an aqueous solution the pH of which is from 7.0 to 10.0, in particular from 7.0 to 9.0, preferably from 7.0 to 8.0, particularly preferably 7.0 to 7.5.

The mixing is therefore carried out at a pH close to physiological pH.

Usually, an aqueous solution of the HA-PBA-xRGD polymer and either an aqueous solution of HA-fructose-yRGD or an aqueous solution of HA-gluconamide-zRGD are mixed.

When the polymeric composition to be prepared contains cells, these can be introduced into the aqueous solution of the HA-PBA-xRGD polymer and / or either into the aqueous solution of HA-fructose-yRGD, or into the aqueous solution of HA- gluconamide-zRGD before their mixtures.

The almost instantaneous preparation process for polymeric compositions by simply mixing HA-PBA-xRGD polymers and either HA-fructose-yRGD or HA-gluconamide-zRGD, allows cells to be easily encapsulated without damaging them and without them having time to settle. The cell adhesion properties of the polymeric compositions can easily be modulated as a function of the RGD peptide density of the polymeric composition (therefore as a function of c), which depends on the RGD peptide densities (x, y and z) of the HA polymers. -PBA-xRGD, HA-fructose-yRGD and HA-gluconamide-zRGD it contains. It is thus possible to easily vary the density of RGD peptide in the polymeric composition by adapting x of HA-PBA-xRGD and either y or z of HA-fructose-yRGD or of HA-gluconamide-zRGD, which is essential to control the behavior of cells of the polymeric composition.

According to a third object, the invention relates to a kit comprising:

a) a first container comprising a hyaluronic acid polymer, at least one carboxylate function of which is grafted with 3-aminophenylboronic acid, and

b) a second container comprising a hyaluronic acid polymer, at least one carboxylate function of which is grafted with 1 -amino-1 -deoxy-D-fructose or with gluconamide, provided that at least one of said polymers is grafted with a peptide RGD.

This kit is suitable for preparing the polymeric composition defined above by mixing the two polymers. The embodiments described above are applicable. For example, the first container can further comprise an aqueous solution. Likewise, the second container may further comprise an aqueous solution.

According to a fourth object, the invention relates to the use of the polymeric composition defined above, for cell culture, cell transplantation or cell therapy.

These applications require the polymeric composition to contain or be added to cells. Depending on the cells used, one of the HA-PBA-xRGD / HA-fructose-yRGD or HA-PBA-xRGD / HA-gluconamide-zRGD compositions may be more suitable than the other.

The cellular response of the cells of the polymeric compositions according to the invention is improved compared to that of cells of the polymeric compositions obtained from polymeric compositions comprising identical polymers except that they are not grafted with an RGD peptide. For example, the cells spread out in the polymeric compositions according to the invention, while they remain round in the polymeric compositions comprising identical polymers except that they are not grafted with an RGD peptide.

The optimum density of RGD peptide (“c” defined above, which depends on x and either on y or on z) of the polymeric composition depends on the type of cells used. This is because the optimum density of RGD peptide in a hydrogel varies depending on the cell type. It is therefore particularly advantageous to have hydrogels with peptide densities Modular RGDs. Advantageously, depending on the type of cells used, the RGD “c” peptide density of the polymeric composition can be adapted on a case-by-case basis, by simply adjusting x and / or either y or z, to optimize cell behavior as a function of specific cells used.

According to a fifth object, the invention relates to the use of the polymeric composition defined above, in the embodiment in which it comprises a sodium alginate, as ink, in particular as bio-ink, for printing, in particular 3D printing, in particular 3D bioprinting, or for tissue engineering.

As an essential component of the extracellular matrix, hyaluronic acid is a biopolymer of choice for the development of models of biological objects, such as tissues, by bioprinting. However, its use in the gel state involves its crosslinking. The crosslinking method conventionally used in tissue engineering consists in modifying the hyaluronic acid with methacrylate groups allowing its gelation by UV irradiation. However, the use of UV light can be damaging to cell survival. On the contrary, the use of the polymeric composition according to the invention as a bio-ink does not require UV irradiation.

According to a sixth object, the invention relates to a method of preparing an object comprising the steps of:

i) shaping the polymeric composition as defined above, in particular by extrusion, preferably by a 3D printer,

ii) bringing the shaped polymeric composition into contact with an aqueous solution comprising calcium ions.

The method makes it possible in particular to prepare an object having a defined geometric shape using a 3D printer.

By "3D printer" is meant a system for programmatically depositing a quantity of polymeric composition at a point defined by its coordinates in the three dimensions of space. The 3D printer can print by extrusion, laser or inkjet.

There is a strong demand in the field of bioprinting for printable biocompatible and biodegradable hydrogels, and also compatible with several printing methods (extrusion, laser, inkjet, stereolithography). Being able to 3D print a hydrogel-based object having a geometric shape defined automatically using a printer is now a challenge. This is called “bioprinting”. The hydrogel is a key element in bioprinting technologies, not only playing a structural role in the fabrication of the object but also biological, by transmitting biochemical signals to cells so that they survive and develop in the object formed. While there are currently several types of bioprinters available on the market, the development of bioprinting is nevertheless limited by the lack of biocompatible hydrogels, customizable and modifiable according to the tissues / organs targeted and suitable for production at large scale. The polymeric compositions according to the invention advantageously make it possible to carry out bioprinting and have all the desired advantages.

The step of shaping by a 3D printer typically comprises the deposition, layer by layer, of the polymeric composition (preferably containing cells).

The contacting in step ii) is generally carried out by immersing the shaped object in an aqueous solution comprising calcium ions. Step ii) obtains the object. Step ii) leads to the formation of an interpenetrating network doubly crosslinked by boronate ester bonds between the HA-PBA-xRGD and HA-fructose-yRGD polymers or else HA-PBA-xRGD and HA-gluconamide-zRGD, and by ionic crosslinks of calcium alginate. HA-PBA-xRGD polymers and either HA-fructose-yRGD or HA-gluconamide-zRGD provide high print fidelity, while sodium alginate is used as a structural component, ensuring the object in form good mechanical stability.

According to a seventh object, the invention relates to an object capable of being obtained by the method defined above. The invention also relates to an object comprising: a) a hyaluronic acid polymer of which at least one carboxylate function is grafted with 3-aminophenylboronic acid,

b) a hyaluronic acid polymer of which at least one carboxylate function is grafted with 1 -amino-1 -deoxy-D-fructose or with gluconamide,

provided that at least one of said polymers a) and b) is grafted with an RGD peptide, c) a calcium alginate, and

d) optionally cells.

This object can in particular be a tissue, for example a dermal tissue, or an organ.

When the object contains cells, advantageously, their cell viability is high, cell proliferation is high, and the degree of cell spreading is high.

FIGURES

[Fig 1] Figure 1 represents the elastic modulus (G ') (solid symbols) and the viscous modulus (G ”) (empty symbols) (in dynamic mode - in Pa) of three HA-PBA-xRGD / HA polymer compositions -fructose-yRGD in the form of hydrogels depending on the frequency (in Hz). The three HA-PBA-xRGD / HA-fructose-yRGD polymeric compositions have different densities c of RGD peptides:

- c = 0 mol%, x = 0 mol%, y = 0 mol% (polymers not grafted by RGD peptides - comparative) (round),

- c = 5 mol%, x = 5 mol%, y = 5 mol% (squares),

- c = 12.5 mol%, x = 10 mol%, y = 15 mol% (triangles).

[Fig 2] Figure 2 represents the elastic modulus (G ’) (solid symbols) and the viscous modulus (G”) (empty symbols) (in dynamic mode and in Pa) of two polymeric compositions in the form of hydrogels:

- HA-PBA-xRGD / HA-fructose-yRGD (free of alginate - triangles),

- HA-PBA-xRGD / HA-fructose-yRGD / sodium alginate (stars),

each with a RGD peptide density c = 12.5 mol%, x = 10 mol%, y = 15 mol%, as a function of frequency (in Hz).

[Fig 3] Figure 3 provides three photographs of HA-PBA-xRGD / HA-fructose-yRGD / sodium alginate polymeric compositions in PBS shaped by extrusion:

- at the end of the extrusion forming step and before the immersion step in an aqueous solution of CaCh (images A and B), and

- when coming into contact with an aqueous solution of CaCh (image C).

[Fig 4] Figure 4 represents the elastic modulus (G ’) (solid symbols) and the viscous modulus (G”) (empty symbols) (in dynamic mode and in Pa):

- of an HA-PBA / HA-glucose polymeric composition (free of RGD groups and free of alginate - gray symbols),

- hyaluronic acid (unmodified) (black symbols),

as a function of frequency (in Hz).

EXAMPLES

Example 1: Preparation of polymeric compositions

1 .1. Synthesis of HA-gluconamide-zRGD polymer (with z = 0)

Has a solution of Boc-1 -amino-DOOA (0.113 g, 0.45 mmol) in DMF (25 mL), a solution of D-glucono-1, 5-lactone (0.081 g, 0.45 mmol) in DMF (20 mL) is added dropwise over about 3 h. After stirring overnight at room temperature, the solvent is evaporated off under reduced pressure and the resulting transparent syrup is dried in a vacuum oven (60 mbar, 40 ° C) in order to obtain Boc-1 -amino-DOOA-gluconamide (0.178 g, 92%).

¹ H NMR (400 MHz, D20, 25 ° C) of Boc-1 -amino-DOOA-gluconamide: 5H (ppm) 4.35 (1H, H2), 4.15 (1H, H3), 3, 88 to 3.72 (10H, H4-H6 and Hb-Hd), 3.65 (2H, He), 3.55 (2H, Ha), 3.32 (2H, Ht), 1.5 (9H, Hg).

Removal of the Boc protecting group is then carried out by acid treatment of Boc-1 -amino-DOOA-gluconamide (0.055 g, 0.13 mmol) in TFA (0.6 mL, 7.77 mmol) for 5 min at ambient temperature. The reaction medium is neutralized by adding 1M NaOH dropwise, at a temperature between 0-4 ° C., and the solvent is then removed under reduced pressure. Complete deprotection of the amine group making it possible to obtain 1 -amino-DOOA-gluconamide is confirmed by ¹ H NMR analysis.

HA-gluconamide-zRGD is then synthesized by an amidation reaction between HA (pharmaceutical grade, of non-animal origin, supplied by CONTIPRO, M _w = 400-500 kg / mol) and 1 -amino-DOOA -gluconamide, using DMTMM (4- (4,6-Dimethoxy-1, 3,5-triazin-2-yl) -4-methylmorpholinium chloride) as a coupling agent. The reaction consists in adding the DMTMM to a solution of HA in a water / DMF mixture (3/2, v / v). After 15 min of stirring at room temperature, 1 -amino-DOOA-gluconamide is added, the pH of the reaction medium is adjusted to 6.5 with a 0.5 M NaOH solution and the reaction is left under stirring for 65 h at room temperature. Once the reaction is complete, the HA-gluconamide-zRGD conjugate (with z = 0) is purified by diafiltration and the product is recovered by lyophilization.

Under these conditions, the HA-gluconamide-zRGD (with z = 0) is obtained with a Degree of Substitution (DS) of 10 mol% determined by ¹³ C NMR analysis and from the reaction kinetics (Table 1). This method consists in quantifying the level of free primary amines in the reaction medium as a function of time using the TNBS reagent (2,4,6-trinitrobenzenesulfonic acid). [Chem 1]

HO H

oc- -am no- Drip HO, _in 7

(3 hrs + 1 night)

DMF, t.a. Boc-1 -amino-DOOA-gluconamide

HA-gluconamide

Scheme 1: Reaction scheme illustrating the synthesis of the HA-gluconamide-zRGD polymer (with z = 0).

1.2. Synthesis of HA-PBA-xRGD (with x = 0) and HA-fructose-yRGD (with y = 0) polymers

The HA-PBA-xRGD (with x = 0) and HA-fructose-yRGD (with y = 0) polymers (Scheme 2) are respectively synthesized by a peptide coupling reaction between HA and GARBA (3-aminophenylboronic acid) or fructosamine (1 -amino-1 -deoxy-D-fructose), containing free primary amine groups. For this, GARBA or fructosamine are added to a water / DMF (3/2, v / v) mixture containing DMTMM and HA according to the conditions in Table 1, and the pH is adjusted to 6.5 with a solution of 0.5 M NaOH. After stirring for 24 h at room temperature, the polymers are purified by diafiltration and the products are recovered by lyophilization. This procedure makes it possible to synthesize HA-PBA-xRGD (with x = 0) and HA-fructose-yRGD (with y = 0) with a DS of 15 mol% determined by ¹ H NMR analysis or from the reaction kinetics. using TNBS (Table 1).

[Chem 2]

HA-PBA HA-fructose

Diagram 2: Chemical structures of HA-PBA-xRGD polymers with x = 0 and HA-fructoseyRGD with y = 0.

[Table 1]

aDS determined by ¹ H NMR (10 mol% accuracy).

bDS estimated from reaction kinetics using TNBS.

^C DS determined by ¹³ C NMR (20 mol% accuracy).

Table 1: Reaction conditions and DS for HA-PBA-xRGD, HA-fructose-yRGD and HA-gluconamide-zRGD polymers. 1.3. Synthesis of HA-PBA-xRGD polymers with x ¹ 0, HA-fructose-yRGD with y ¹ 0 and HA-gluconamide-zRGD with z ¹ 0

The HA-PBA-xRGD, HA-fructose-yRGD and HA-gluconamide-zRGD polymers are synthesized by peptide coupling from RGD peptides (containing a primary amine function) using the DMTMM coupling agent (Scheme 3 ). For this, the GRGDS peptide used as an example is added in a solution of water / DMF (3/2, v / v) containing the DMTMM and the polymer HA-PBA-xRGD with x = 0, the polymer HA-fructose- yRGD with y = 0 or the HA-gluconamide-zRGD polymer with z = 0 previously solubilized (Table 2). The pH of the reaction medium is adjusted to 6.5 by adding 0.5 M NaOH solution and the reaction is left under stirring at room temperature for 24-30 hours. Once the reaction is complete, the HA-PBA-xRGD, HA-fructose-yRGD and HA-gluconamide-zRGD polymers are purified by diafiltration and the polymers are recovered by lyophilization.

The method described above makes it possible to control the rate of grafting of RGD peptides on the polymers, so as to obtain values of x, y and z of between 1 and 30 mol% determined by ¹ H NMR and also from the reaction kinetics using TNBS (Table 2).

[Chem 3]

-g uconam e-

Scheme 3: Reaction scheme illustrating the synthesis of HA-PBA-xRGD, HA-fructose-yRGD and HA-gluconamide-zRGD polymers by grafting of GRGDS peptide.

[Table 2]

Ratio Molar ratio

molar DMTMM / HA

x, y or z Coupling Efficiency

RGD- polymer (mol%) (%) (%)

NH2 /

DMTMM

HA-PBA-xRGD,

HA-fructose- yRGD or

0.5 0.02-0.06 1 -30 ^ab 50-70> 80

HA- gluconamide- zRGD ^a x, y or z determined by ¹ H NMR (10 mol% precision).

b x, y or z estimated from reaction kinetics using TNBS.

Table 2: Reaction conditions for the syntheses of HA-PBA-xRGD, HA-fructose-yRGD and HA-gluconamide-zRGD polymers.

1 .4. Preparation of a polymeric composition comprising cells for cell culture

The HA-PBA-xRGD, HA-fructose-yRGD or HA-gluconamide-zRGD polymers are solubilized at 15 g / L in DPBS (Dulbecco's Phosphate-Buffered Saline) with stirring for 2-4 hours.

Polymeric compositions useful for cell culture are prepared as follows. The aqueous solution of HA-PBA-xRGD is first deposited in inserts placed in 24-well culture plates.

An aqueous solution of HA-fructose-yRGD seeded with cells is prepared. An aqueous solution of HA-gluconamide-zRGD seeded with cells is prepared.

Next, the polymeric compositions comprising cells (total polysaccharide concentration = 15 g / L; PBA / saccharide molar ratio = 1/1) are directly formed in each well by adding an aqueous solution of HA-fructose-yRGD or HA-gluconamide-zRGD seeded with cells. The mixture of HA-PBA-xRGD and HA-fructose-yRGD polymers, or else of HA-PBA-xRGD and HA-gluconamide-zRGD polymers within each well results in the formation of the polymeric composition in hydrogel form.

1 .5. Preparation of a polymeric composition comprising sodium alginate and cells for bioprinting

The alginate comes from Alfa Aesar (A18565).

Solutions :

- HA-PBA-xRGD polymer containing sodium alginate,

- HA-fructose-yRGD polymer containing sodium alginate,

- HA-gluconamide-zRGD polymer containing sodium alginate,

were prepared by stirring for 2-4 h in DPBS (Dulbecco's Phosphate-Buffered Saline) or in DMEM culture medium (polysaccharide concentration between 10-30 g / L). The solutions have a final sodium alginate concentration between 0.1 and 2.0% (w / v)). Once the cells are seeded in the solution of the HA-fructose-yRGD polymer or in the solution of the HA-gluconamide-zRGD polymer, the HA-PBA-xRGD / HA-fructose-yRGD / sodium alginate or HA-PBA polymer compositions -xRGD / HA- gluconamide-zRGD / sodium alginate (total polysaccharide concentration = 10-30 g / L; PBA / fructosamine molar ratio or PBA / gluconamide molar ratio = 1/1 or 1/2 or 2/1) useful as bioinks are directly formed in cartridges for extrusion.

1 .6. Rheological behavior in dynamic mode according to the densities of RGD peptide c, x, y and z.

The rheological behavior of the various polymeric compositions in the form of hydrogels was studied with a conical plate rheometer (AR2000EX from TA Instruments). All rheological measurements in dynamic mode were checked against strain to ensure that experiments were performed in the linear viscoelastic region. A cone having a diameter of 2 cm and an angle of 4 ° was used for the viscoelastic samples. To prevent water evaporation, the measuring system was surrounded by low viscosity silicone oil (50 mPa.s) carefully added to the edges of the cone.

[Fig 1] Figure 1 provides the elastic modulus (G ') and viscous modulus (G ”) of three HA-PBA-xRGD / HA-fructose-yRGD polymeric compositions in hydrogel form, with average mole numbers of RGD peptides per 100 disaccharide repeat units of hyaluronic acid:

- c = 0 mol%, x = 0 mol% and y = 0 mol% (comparative, polymeric composition in which the two polymers are free from RGD)

- c = 5 mol%, x = 5 mol%, y = 5 mol%, or

- c = 12.5 mol%, x = 10 mol%, y = 15 mol%.

[Fig 1] FIG. 1 shows that the RGD peptide densities (therefore c, x, y and z) have very little impact on the rheological behavior of the polymer composition.

1 .7. Rheological behavior in dynamic mode in the absence and presence of sodium alginate

The measurement method is identical to that of the previous paragraph.

[Fig 2] Figure 2 provides the elastic modulus (G ’) and viscous modulus (G”) of two polymeric compositions in hydrogel form:

- HA-PBA-xRGD / HA-fructose-yRGD / sodium alginate,

- HA-PBA-xRGD / HA-fructose-yRGD, with in both cases c = 12.5 mol%, x = 10 mol% and y = 15 mol%.

The presence of alginate has little impact on the rheological properties of polymeric compositions in hydrogel form.

Example 2: Spreading of fibroblasts (MEF) in hydrogels having different densities of RGD peptides (c = 0 mol%, 5.0 mol% or 12.5 mol%)

The spreading of fibroblasts (MEF) was determined by carrying out microscopic images of fibroblasts seeded in the HA-PBA-xRGD / HA-fructose-yRGD hydrogels after 7 days of cell culture at 37 ° C (labeling of the living cells with the calcein).

In the comparative RGD-free polymeric composition (c = x = y = 0, or c = x = z = 0), cells observed by confocal microscopy were round. On the contrary, in the polymeric composition according to the invention (with a polymeric composition in which c = 5 mol%, x = 5 mol% and y = 5 mol% and a polymeric composition in which c = 12.5 mol%, x = 10 mol% and y = 15 mol%, confocal microscopy images showed that the cells have proliferated and are elongating, demonstrating the adhesion and spreading of fibroblasts in 3D.

Example 3: Printing using the polymeric composition as ink by the extrusion technique

3.1. Preparation of printed objects

The polymeric hydrogel composition of Example 1 .4 formed in cartridges for extrusion was extruded through a 0.2mm diameter needle to form extruded objects ([Fig 3] Fig 3, images A and B). The extruded objects were immersed in an aqueous solution containing 0.5% (m / v) CaCh ([Fig 3] Figure 3, Image C)

3.2. Cell viability in printed objects

The cell viability before and after printing of fibroblasts (NIH-3T3 eGFP) seeded in the polymeric compositions in the form of hydrogels (bio-inks) after 7 days of cell culture at 37 ° C was tested before and after printing (Live test - Dead carried out using an epi-fluorescence microscope for cell counting: labeling of living cells with calcein and dead cells with propidium iodide). [Table 3]

Table 3: Cell viability (%) of fibroblasts (NIH-3T3 eGFP) on D7 in the polymeric composition in hydrogel form before printing and in the shaped object 5.3. Adhesion / spreading of fibroblasts in printed objects

The spread of human fibroblasts was determined by taking epi-fluorescence microscopic images of human fibroblasts in the printed objects HA-PBA-xRGD / HA-fructose-yRGD / calcium alginate obtained from the polymeric compositions in the form of HA-PBA-xRGD / HA-fructose-yRGD / sodium alginate hydrogels in which the fibroblasts were seeded after 7 days of cell culture at 37 ° C (labeling of living cells with calcein).

In the object obtained from the comparative polymeric composition free of RGD (HA-PBA-xRGD / HA-fructose-yRGD / calcium alginate with c = x = y = 0, or else HA-PBA-xRGD / HA- gluconamide-zRGD / calcium alginate with c = x = z = 0), cells observed by epi-fluorescence microscopy were round.

On the contrary, in the object obtained from the polymeric composition according to the invention (from a polymeric composition in which c = 5 mol%, x = 5% and y = 5 mol% and from a polymer composition in which c = 12.5 mol%, x = 10 mol% and y = 15 mol%), the images showed the adhesion and spreading of the fibroblasts in 3D.

Claims

1. Polymeric composition comprising a mixture of:

2. Polymeric composition according to claim 1, in which:

- the hyaluronic acid polymer of which at least one carboxylate function is grafted with 3-aminophenylboronic acid comprises an average number of moles x of RGD peptides per 100 disaccharide repeating units of hyaluronic acid,

- the hyaluronic acid polymer of which at least one carboxylate function is grafted with 1-amino-1 -deoxy-D-fructose comprises an average number of moles y of RGD peptides per 100 disaccharide repeating units of hyaluronic acid,

- the hyaluronic acid polymer of which at least one carboxylate function is grafted with gluconamide comprises an average number of moles z of RGD peptides per 100 disaccharide repeating units of hyaluronic acid,

where x, y and z are independently of each other from 0 to 50 mol%, in particular from 0 to 40 mol%, typically from 0 to 30 mol%, preferably from 0 to 20 mol%,

with the proviso that x and y do not both represent 0, or that x and z do not both represent 0.

3. Polymeric composition according to claim 2, in which:

- x is different from 0 and either y is 0, or z is 0, or

- x is 0 and either y is different from 0, or z is different from 0, or

- x is different from 0 and either y is different from 0, or z is different from 0.

4. A polymeric composition according to any one of claims 1 to 3, comprising cells.

5. A polymeric composition according to any one of claims 1 to 4, comprising sodium alginate.

6. Kit including: a) a first container comprising a hyaluronic acid polymer of which at least one carboxylate function is grafted with 3-aminophenylboronic acid, and

7. Use of the polymeric composition according to any one of claims 1 to 5, for cell culture, cell transplantation or cell therapy.

8. Use of the polymeric composition according to claim 5, as ink for printing, in particular 3D printing, in particular 3D bioprinting, or for tissue engineering.

9. Method for preparing an object comprising the steps of: i) shaping the polymeric composition according to claim 5, in particular by extrusion, preferably by a 3D printer,

10. Object comprising:

a) a hyaluronic acid polymer of which at least one carboxylate function is grafted with 3-aminophenylboronic acid,

d) optionally cells.