Process for synthesizing degradable hydrogels

Abstract

Process for the synthesis of degradable hydrogels. The present invention relates principally to a process for preparing a crosslinked hydrogel with controlled degradability, comprising at least the steps of: Having aqueous droplets comprising at least a mixture of at least two crosslinkable biopolymers of different degrees of substitution (DS), exposing said droplets to an effective crosslinking stimulus to achieve simultaneous crosslinking of the two biopolymers, and recovering the crosslinked hydrogel, in which one of the biopolymers has a degree of substitution less than or equal to 0.2. Figure for the abstract: none.

Description

Title of the invention: Process for the synthesis of degradable hydrogels Technical field

[0001] The present invention relates to the field of hydrogels useful for encapsulating and delivering entities of interest, chemical such as for example therapeutic active agents or reagents such as for example a catalyst, or entities of biological interest, such as for example biomolecules, nucleic acids, proteins or antibodies, cells, or viruses and bacteria for therapeutic purposes.

[0002] More particularly, it aims to propose a process for preparing hydrogels capable of releasing in a controlled manner the entity(ies) of interest that they encapsulate. Prior art

[0003] Hydrogels, by definition, are crosslinked three-dimensional networks, insoluble in water due to their crosslinking, and which are capable of absorbing and retaining significant quantities of an aqueous medium, such as water or even a biological fluid. A hydrogel can be classified in particular with regard to its dimensions and formats as a macrogel, microgel or even nanogel.

[0004] Crosslinked microgels based on different polymers, in particular biopolymers such as alginate, chitosan, hyaluronic acid, and in particular dextran have already been described for applications of encapsulation and release of chemical or biological entities of interest.

[0005] These biopolymers offer several advantages, including "bio" properties such as natural synthesis in physiological environments, enzymatic biodegradability properties and sensitivity to external stimuli such as temperature, pH, ionic strength and / or polarity. They can be natively crosslinkable, for example alginate with carboxylic functions, or easily chemically modified to become crosslinkable, for example dextran, chemically modified to be substituted by crosslinkable functions such as methacrylates.

[0006] The use of crosslinked biopolymer hydrogels for the purposes of encapsulation and controlled release of entities of interest is therefore particularly interesting with regard to the aforementioned properties, provided that their stability and degradability can be controlled. It is important that these hydrogels have a sufficient degree of crosslinking to prevent their destabilization to external stimuli but nevertheless compatible with degradation, in particular by enzymatic means, to release the encapsulated entity(ies) of interest.

[0007] The classical methods described in the literature for adjusting the properties of The strength (i.e. mechanical properties) and degradability of a hydrogel are generally based on the adjustment of its molecular weight, its concentration during the crosslinking reaction, or the degree of substitution, DS, of the biopolymer from which it is derived. However, these methods are not entirely effective in achieving this compromise between stability and enzymatic degradability. Thus, studies carried out on dextran nanogels show that if the dextran polymer is crosslinked at a high scale, dextranase cannot digest the nanogel. In addition, controlling the release process of the encapsulated entities, in terms of duration, between 2 and 3 hours in particular, raises an additional difficulty. Statement of the invention

[0008] The present invention has the precise objective of developing a biopolymer hydrogel meeting these expectations.

[0009] In particular, it aims to propose an effective crosslinked hydrogel for the temporary encapsulation of one or more entities of interest.

[0010] It also aims to provide a crosslinked hydrogel stabilized with respect to external stimuli such as in particular temperature, pH and ionic strengths.

[0011] It also aims to propose a crosslinked hydrogel which is degradable by enzymatic means.

[0012] It also aims to propose a crosslinked hydrogel whose enzymatic biodegradability is controlled in terms of duration and in particular is not immediate but completed in less than 3 hours and in particular in less than 2 hours.

[0013] Thus, according to one of its aspects, the present invention relates to a method for preparing a crosslinked hydrogel with controlled degradability comprising at least the steps consisting of: a. Having aqueous droplets comprising at least one mixture of at least two crosslinkable biopolymers with different degrees of DS substitution, b. Exposing said droplets to an effective crosslinking stimuli to achieve simultaneous crosslinking of the two biopolymers and c. Recover the crosslinked hydrogel,

[0014] wherein one of the biopolymers has a degree of substitution, DS, less than or equal to 20%.

[0015] Conventionally, the DS corresponds to the number of polymerizable functions per 100 monomer units. This DS can be determined by 1H NMR, in particular as detailed for a specific dextran biopolymer in .

[0016] Against all expectations and as is apparent from the examples given below, the inventors have found that the simultaneous crosslinking of at least two crosslinkable biopolymers having different degrees of substitution, DS, with only one of them having a low DS and in particular less than or equal to 20%, makes it possible to achieve the desired compromise, namely stability to stimuli known to specifically destabilize these biopolymers and biodegradability nevertheless possible over a controlled period, in particular of the order of less than 3 hours or even less than 2 hours.

[0017] According to a particular embodiment, the biopolymers considered are distinguished from one another only by their degree of substitution, DS.

[0018] In particular, only one of the biopolymers in the mixture has a DS less than or equal to 20%.

[0019] In particular, the biopolymers are chemically modified and in particular substituted by crosslinkable units chosen in particular from thiol, divinylsulfonyl, maleimide, azide, alkynyl, alkenyl, acrylate, methacrylate, aldehyde, norbornenyl, oxyamine, amine, N-hydroxysuccinimide ester units and mixtures thereof, in particular from acrylates and methacrylates.

[0020] In particular, the droplets contain a mixture of two polydextrans of identical molecular weight and chemically modified with units chosen from acrylate, methacrylate, alkenyl and alkynyl and in particular a mixture of two polydextrans of identical molecular weight chemically modified by methacrylate units, DexMet, and DS varying from 10 to 40%.

[0021] In particular, the two biopolymers are derived from dextran and in particular are polydextrans chemically modified by methacrylate units, also called Dex(Met).

[0022] More particularly, the droplets contain a mixture of at least one DexMet with a DS of less than 20% and a DexMet with a DS of greater than 25% and in particular a mixture of a DexMet with a DS equal to 13% + / - 0.5% and a DexMet with a DS equal to 31% + / - 1%.

[0023] According to another of its aspects, the present invention relates to a crosslinked hydrogel with controlled degradation, characterized in that it is derived from the simultaneous crosslinking of at least two polydextrans chemically modified with units chosen from thiol, divinylsulfonyl, maleimide, azide, alkynyl, alkenyl, acrylate, methacrylate, aldehyde, norbornenyl, oxy-amine, amine, N-hydroxysuccinimide ester units and in particular from acrylates and methacrylates, one of which has a DS less than or equal to 20%.

[0024] In particular, the two polydextrans only differ in their DS and more particularly are chemically modified by methacrylate units, DexMet, and their DS varies from 10% to 40%.

[0025] According to a particular embodiment, this hydrogel is obtained by the method according to the invention.

[0026] According to a particular embodiment, this hydrogel further contains at least one entity of interest to be released in a controlled manner.

[0027] According to yet another of its aspects, the present invention relates to the use of a hydrogel in accordance with the invention for cell or bacterial culture.

[0028] According to yet another of its aspects, the present invention relates to the use of aqueous droplets, in particular formed by microfluidics, and comprising at least one mixture of at least two crosslinkable and chemically modified polydextrans with units chosen from thiol, divinylsulfonyl, maleimide, azide, alkynyl, alkenyl, acrylate, methacrylate, aldehyde, norbornenyl, oxy-amine, amine, N-hydroxysuccinimide ester units and mixtures thereof, in particular from acrylates and methacrylates with one of them and in particular only one of them having a DS less than or equal to 20%, for the encapsulation of at least one entity of interest

[0029] Other characteristics, variants and advantages of the objects of the invention will emerge more clearly on reading the description, examples and figures which follow, given for illustrative and non-limiting purposes of the invention.

[0030] In the rest of the text, the expressions “between ... and ...”, “ranging from ... to ...” and “varying from ... to ...” are equivalent and are intended to mean that the limits are included, unless otherwise stated. Brief description of the drawings

[0031] [Fig-1]: It represents the 1H NMR spectra of a) Dextran and b) Dextran-me- thacrylate (0.0625 eq to 0.3 eq of methacrylic anhydride) in D2O at 300 MHz. Detailed description

[0032] As is clear from the above, the invention aims to propose new hydrogels dedicated in particular to encapsulating and releasing entities of chemical and / or biological interest and which combine stabilization to external stimuli and in particular to surfactants with controlled enzymatic biodegradability because it is not immediate but within a short time.

[0033] By definition, the term hydrogel refers to a crosslinked, water-insoluble three-dimensional network that is capable of absorbing and retaining enormous quantities of an aqueous medium, such as water or even a biological fluid. The hydrogel can be a macrogel, microgel or even a nanogel. These water-swellable colloidal networks have typical diameters ranging from 20 to 1000 nm for nanogels, from 1 pm to 800 pm for microgels, and larger for macrogels.

[0034] In the context of the invention, the hydrogels considered are preferably microgels and of typical diameter varying from 10 to 100 μm.

[0035] The droplets can be characterized under a binocular microscope equipped with a camera for image acquisition (10x / 0.25 Olympus, 75 mm lens tube, IDS UI388xCP-M achromatic camera). The size of the pixels making up the image is determined (0.58pm / pixel) and knowing the diameter of a drop by measuring directly under the microscope, reading a plot then allows us to go back to the number of drops per cm2.

[0036] The hydrogels considered according to the invention are formed from biopolymers.

[0037] For the purposes of the invention, the term "biopolymer" designates a polymer derived from renewable natural resources (a material also called "natural"). The biopolymers considered according to the invention have the advantage of possessing, in addition to very good compatibility with human skin, biodegradability as such or even in vivo.

[0038] This term “biopolymer” thus covers natural polymers such as, for example, a protein or a polysaccharide, but also derivatives of natural polymers such as, for example, those which derive from a chemical modification of these natural polymers to make them crosslinkable or compatible with a specific crosslinking method.

[0039] By way of illustration of biopolymers suitable for the invention, mention may in particular be made of polyhydroxy acids, such as, for example, polyglycolic acid (PGA) and polylactic acid (PLA), also known as polylactide; polysaccharides, such as, for example, cellulose, chitin and starch and proteins or peptides, such as, for example, collagen, gelatin, elastins, fibroins, corn zein, silk from various species of silkworms, keratin and their derivatives.

[0040] As specified above, these biopolymers must be crosslinkable to form chemical crosslinking nodes (covalent, ionic, coordination bond) in the three-dimensional network constituting the corresponding hydrogel and furthermore degradable in particular by an enzyme.

[0041] According to a particular embodiment, these biopolymers are derived from compounds chosen from alginates, hyaluronic acid, cellulose, carboxymethylcellulose, chitosan, dextran. Their respective polymers are in fact enzymatically degradable. As representative of the corresponding enzymes, for example, alginate lysase, hyaluronidase, esterases, dextranase, cellulase and chitosanase may be cited.

[0042] According to another embodiment, these biopolymers can be derived from collagen, gelatin, elastin or poly(amino acids) degradable by metalloproteases.

[0043] These biopolymers are crosslinked within the scope of the present invention. Natively, they can be crosslinkable if they carry crosslinkable units such as carboxylic functions such as, for example, alginate.

[0044] Otherwise, these biopolymers are implemented in a so-called chemically modified form to provide them precisely with crosslinkable functional groups. This chemical modification most often consists of introducing into the structure of the polymers to be modified crosslinkable units in particular chosen from thiol, divinylsulfonyl, maleimide, azide, alkynyl, alkenyl, acrylate, methacrylate, aldehyde, norbornenyl, oxy-amine, amine, N-hydroxysuccinimide ester units and in particular among acrylates, methacrylates.

[0045] These functional groups which will allow crosslinking, are introduced with a given degree of substitution, DS.

[0046] In this case, the hydrogels according to the invention derive from the simultaneous crosslinking of at least two crosslinkable biopolymers with a different degree of substitution, one of which is less than 20%.

[0047] According to a particular embodiment, the two crosslinkable biopolymers differ only by their degree of substitution, DS. In other words, they have the same chemical nature and the same molecular weight. In particular, the two crosslinkable biopolymers are derived from dextran.

[0048] A photocrosslinkable polydextran is obtained by chemical modification of a polydextran to introduce photosensitive chemical groups, such as unsaturated functional groups (acrylate, methacrylate, alkenyl, alkynyl, etc.). Acrylates and methacrylates are well known as photopolymerizable groups for functionalizing polysaccharides because they exhibit high photoreactivity for polymerizing in the presence of a photoinitiator.

[0049] According to a preferred embodiment, the droplets considered in the method according to the invention contain a mixture of two polydextrans of identical molecular weight and chemically modified by units chosen from acrylate, methacrylate, alkenyl, alkynyl and their mixtures.

[0050] The method according to the invention considers in particular droplets containing a mixture of two polydextrans of identical molecular weight and chemically modified by methacrylate units, DexMet, and DS varying from 10% to 40%.

[0051] In the process of the invention, these crosslinkable biopolymers are used within aqueous droplets, in particular microdroplets.

[0052] According to a first variant, the aqueous droplets of crosslinkable biopolymers can be obtained by a microfluidic process. This manufacturing method is particularly interesting when it is desired to control the size of the droplets, in particular in the microdroplet state, and the number of entities of interest, if present, per hydrogel (for example 1 bacterium / hydrogel), or if the entities of interest are fragile.

[0053] According to a second variant, these droplets are obtained by water-in-oil emulsification. This technique is generally preferred to obtain a high loading rate of entities of interest, such as for the encapsulation of drugs.

[0054] Advantageously, the droplets are formed by microfluidics. The microfluidic process is a gentle technique. In addition, microfluidic processes make it possible to obtain a very monodisperse population of microdroplets.

[0055] As detailed in the examples below, these microdrops can in particular be formed by a microfluidic flow focusing process using a commercial PDMS chip (EZ Drop (DROPKIT01)).

[0056] The size of the droplets formed within the framework of the invention therefore varies according to the technique used to form them.

[0057] Generally speaking, their average size in number can vary from 10 to 100 pm, in particular from 30 to 50 pm.

[0058] Microdroplets are most often obtained in an oily or organic phase (the aqueous core of the droplet encapsulating the polymer(s)).

[0059] According to a particular embodiment, the method of the invention uses droplets which contain a mixture of at least one DexMet of DS less than 20% and at least one DexMet of DS greater than 25%.

[0060] According to another particular embodiment, the method of the invention uses droplets which contain a mixture of DexMet with DS equal to 13% + / -0.5% and of DexMet with DS equal to 31% + / -1%.

[0061] In particular, these droplets contain a mixture of 75% by weight of DexMet with DS equal to 13% + / - 0.5% and 25% by weight of DexMet with DS equal to 31% + / - 1%. In particular, the two types of DexMet differ only in their DS.

[0062] According to another particular embodiment, the method of the invention uses droplets which contain a 50 / 50 mixture by weight of DexMet with DS equal to 13% + / - 0.5% and of DexMet with DS equal to 31% + / - 1%. In particular, the two types of DexMet differ only in their DS.

[0063] As specified above, the method according to the invention requires that the crosslinking of the two biopolymers present in these droplets be carried out simultaneously.

[0064] This crosslinking can be carried out by conventional methods and its choice is generally conditioned by the nature of the crosslinkable functions. It can thus be chemical or photochemical.

[0065] Due to its ease of use and versatility, chemical photocrosslinking is one of the classic methods used to crosslink hydrogels. Compared to chemical crosslinking by radical polymerization of methacrylate functions, the photochemical route also allows better control of the reaction and does not require a potentially cytotoxic chemical catalyst-type polymerization initiator used in conventional chemical crosslinking methods.

[0066] Thus, the biopolymers present in the droplets are preferably crosslinked photochemically.

[0067] To do this, the droplets also contain at least one crosslinking agent and in particular a photoinitiator.

[0068] This photoinitiator may in particular be chosen from lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LAP, and l-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-l-propan-1-one, in particular marketed under the name Irgacure 2959.

[0069] Thus, the chemical crosslinking of the methacrylate functions of two polydextrans, Dex-Met, of different DS and one of which is less than or equal to 20%, can be obtained when the droplet of aqueous solution comprising them with a water-soluble photoinitiator is exposed to UV light, generally at 365 nm or 405 nm.

[0070] According to this embodiment, the photochemical crosslinking is carried out after the manufacture of the droplets incorporating the two biopolymers, the photoinitiator and if present one or more entities of interest.

[0071] It should be emphasized that in the case of chemical crosslinking, this can also be initiated during the manufacture of the droplets and if considered the encapsulation of the entity(ies) of interest in the core of these droplets. In this case, the crosslinking is carried out by chemical reaction between the polymer and more particularly the reactive group introduced on the polymer with a controlled DS and a crosslinking agent, or between two polymer chains carrying two reactive groups introduced on the polymer with a controlled DS (and capable of reacting with each other).

[0072] According to a preferred variant, the droplets further contain at least one entity of chemical and / or biological interest. This entity may be chosen from chemical therapeutic active agents or chemical reagents such as, for example, a catalyst or biochemical active agents in particular chosen from nucleic acids such as, for example, siRNA, RNAi and DNA, proteins, peptides, enzymes, viruses, bacteria and / or cells.

[0073] According to the method of the invention, the droplets are loaded with at least one entity of interest and the crosslinked hydrogel formed from the droplets is isolated from the non-crosslinked droplets.

[0074] According to a particular embodiment, the crosslinked hydrogel can be isolated from the non-crosslinked droplets by selectively destabilizing them. This selective destabilization can in particular be carried out by bringing the crosslinked hydrogel, mixed with the non-crosslinked droplets, into contact with at least one surfactant. The latter will selectively destabilize the non-crosslinked droplets.

[0075] As is apparent from the examples given below, the hydrogels, in this case the microgels obtained according to the invention, considering in particular specific mixtures of Dex-Met and in particular those detailed above, indeed possess the expected properties, namely stability to surfactants combined with degradability by at least one enzyme.

[0076] These enzymes can be generic to certain families of polymers (for example esterases, metalloproteases, etc.) or specific (alginate lysase for alginate, hyalunoridase for hyaluronic acid, dextranase for dextran, etc.)

[0077] Thus and as illustrated in the examples which follow, the combination of 75% by weight of the mixture of DexMet polymers (DS=13%+ / -0.5%) and 25% by weight of the mixture of Dex-Met polymers (DS=31%+ / -1%), leads to a microgel which is both stable and degradable by dextranase under experimental conditions suitable for the catalytic activity of this enzyme.

[0078] It should also be noted that such a combination of DexMet leads to a stable and degradable microgel corresponding to an average DS of 17.5% (corresponding to (0.75 x 0.13) + (0.25 x 0.31)). However, as also illustrated in the examples, a microgel obtained from a single Dex-Met with DS varying from 17% to 18% is neither stable nor degradable.

[0079] Consequently, the present invention makes it possible to access, via specific combinations of two types of biopolymers to be crosslinked simultaneously, new hydrogels with the expected properties in terms of enzymatic degradability and chemical stability and not accessible from a single biopolymer. Applications

[0080] As is apparent from the above, the hydrogels in accordance with the invention are effective in encapsulating and releasing one or more entities of interest.

[0081] Thus, one of the aspects of the invention also relates to the use of aqueous droplets, in particular formed by microfluidics, and comprising at least one mixture of at least two crosslinkable and chemically modified polydextrans, in particular with units chosen from thiol, divinylsulfonyl, maleimide, azide, alkynyl, alkenyl, acrylate, methacrylate, aldehyde, norbornenyl, oxy-amine, amine, N-hydroxysuccinimide ester and mixtures thereof, in particular among the acrylates and methacrylates with one crosslinkable polydextrans and in particular only one of them having a DS less than or equal to 20%, for the encapsulation of at least one entity of interest, in particular as defined above.

[0082] The hydrogels in accordance with the invention can also be used for cell or bacterial culture, particularly in 2D or 3D.

[0083] The invention will now be described by means of the following figures and examples, given of course for illustrative and non-limiting purposes of the invention. Materials and methods Raw materials:

[0084] Dextran T70 (Dex, Mw = 70,000 g / mol, Pharmacosmos),

[0085] Methacrylic anhydride (94%),

[0086] Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, <95%),

[0087] “Phosphate buffer saline” (PBS) buffer (IX: 137 mM NaCl, 2.7 mM KC1, 10 mM Na2HPO4, 1.8 mM KH2PO4; pH = 7.4) from Sigma-Aldrich.

[0088] Dialysis membranes (12,000-14,000 Da molecular weight cut-off) from Fisher Scientific. Deionized water produced with the Purelab purifier from ELGA-Veolia (18.2 MOhm.cm).

[0089] Dextranase is derived from chaetomiun erraticum (D0443 from Sigma-Aldrich). Apparatus

[0090] Labconco freeze dryer.

[0091] Bruker-300 MHz Spectrometer Example 1 Synthesis of dextran-methacrylate

[0092] The methacrylated dextran, with a defined degree of substitution, used to produce the microgels were synthesized according to the method described in application FR 2 114 492.

[0093] The detailed protocol is as follows:

[0094] Dextran (5 g) was dissolved in 100 ml of distilled water in a beaker. After complete dissolution, different equivalent numbers of methacrylic anhydride (from 0.0625 to 0.5 equivalent) relative to the hydroxyl groups of dextran were added dropwise into the dextran solution to obtain the desired DS. Then, the pH was adjusted using NaOH (3 mol.1-1) to around 9-11 for the entire reaction time (1 h at room temperature, 20-25°C). Finally, the obtained Dex-Met polymer was dialyzed against distilled water for 1 week (membrane 12,000-14,000 Da) with about 14 bath changes to remove impurities, and lyophilized at -46°C, 0.140 mbar for 1 week. The white solid was characterized by 1H NMR and FTIR, and stored at -20°C before use. Characterization of DS

[0095] It is determined from the 1H NMR spectra (300 MHz, in D2O) of each modified biopolymer compared to that of the unmodified biopolymer, presented in [Fig.l].

[0096] Typical Dextran protons carried by carbons C2 to C6 (denoted from 2 to 6) give a signal of 3.5 to 4.2.

[0097] The proton H1 carried by the carbon Cl gives a signal at 5.1 ppm and 5.4 ppm, noted 1, corresponding respectively to the bonds a-1,6 and -1,3.

[0098] A typical value of 4% -1,3 bonding relative to -1,6 bonding was calculated, based on the integrals of the corresponding signals at 5.4 ppm and the signals of the other Dex protons.

[0099] On the spectra of dialyzed and lyophilized Dex-Met polymers, the disappearance of the 5.4 and 5.7 ppm methacrylic acid signals and other trace impurities indicates a well-purified polymer (>0.01%).

[0100] In addition to the Dex protons previously described, protons of the methacrylate group (vinyl protons noted 9, and methyl protons noted 10, with a ratio between vinyl and methyl signals of 2:3) are clearly visible on the spectra of the different Dex-Met, showing the good incorporation of the methacrylate fragments.

[0101] The degree of substitution, defined as the amount of methacrylyl groups per 100 dextran glucopyranose residues, is calculated as follows

[0102] [Math.l] Os* boHS DS - --------------------------------------.....-................................................ (f ppm}+ |2 + |3 + |4 + Js + / 9.96

[0103] Table 1 below details the examples of synthesis of Dext-Met with different DS obtained as a function of the quantity (number of equivalents) of methacrylate anhydride initially introduced into the reaction medium.

[0104] [Tables 1] Dextran Methacrylic Anhydride DS Reaction Yield (%) 5g 0.915 ml-0.0625 eq 13% + / - 0.5% 92 5g 1.83 ml-0.125 eq 26% + / - 1% 90 5g 3.66 ml - 0.25 eq 52% + / - 2% 89 5g 4.39 ml - 0.3 eq 62% + / - 2.5% 85 Example 2

[0105] Preparation of hydrogels not in accordance with the invention because they are based on a single Dext-Met polymer Detailed preparation protocol

[0106] The drop suspension is made by a microfluidic flow focusing process using a commercial PDMS chip (EZ Drop (DROPKIT01)). The principle is that a central flow (polymer) is sheared by a secondary immiscible flow orthogonal to the first, thus forming monodisperse micrometric drops. The flows are controlled by a Fluigent pressure generator. The size of the drops is determined by the geometrical sizes of the chip and by the applied pressures. The microdrops are produced at a frequency between 300 and 5000 Hz depending on the applied pressures. The size of the microdrops is around 45 μm in diameter. The suspension is collected in a 1 ml centrifuge tube. It is composed of microdrops of polymers suspended in oil.

[0107] A Dex-Met polymer solution (5% w / w Dex-Met, 0.1% w / w LAP in PBS 1X) is prepared just before drop formation. The fluid The immiscible polymer is oil mixed with the surfactant (2% DSurf surfactant in 3M™ Novec™ 7500 perfluorinated oil) to prevent coalescence of the formed drops. The chip is positioned under a microscope and a high-speed camera allows the acquisition of images during the formation of the drops. A light-opaque protection is used to protect the polymer and the formed drops, which are photosensitive in the case of Dex-Met.

[0108] Once formulated in the form of microdrops, the Dex-Met polymer solution (5% w / w) comprising the LAP photoinitiator (Sigma-Aldrich) 0.1% w / w is photocrosslinked as follows in order to lead to the microgels.

[0109] 40 pl of the suspension of drops are placed in a cuvette formed by two thicknesses of Gene Frame (Thermo Scientific™ Gene Frame, 25 μl (1.0 x 1.0 cm)) of 0.25 mm thickness each (i.e. 0.5 mm in total) glued on a glass slide. The polymerization of the polymer drops is carried out by UV exposure (405 nm) at 75 mW / cm2 (UVA-Hand lamp from Honle) for 30 seconds. The slide exposed for 30 seconds to UV is placed under the microscope for visualization. 20 μl of surfactant (1 / / ,1 / / ,2 / / ,2 / / -perfluoro-l-octanol (Sigma-Aldrich)) are added to the cuvette and the evolution of the suspension is recorded on the high-speed camera.

[0110] Dex-Met microgels of defined DS (DS = 13%; 15%; 17%; 18%; 20%; 25%; 31%; 37%) were prepared and photocrosslinked by reproducing this protocol. Determination of their stability and degradability

[0111] The stability to surfactants is evaluated by adding to a suspension in oil (40 pL of solution) of the crosslinked polymer considered, 20 pL of 1 / / ,1 / / ,2 / / ,2 / / -perfluoro-l-octanol (Sigma-Aldrich). The destabilization is observed under microscope. Comparatively, it was noted that the destabilization of the droplets of non-crosslinked polymer is very rapid (< 5 minutes).

[0112] The degradability is evaluated with respect to dextranase^ Dextranase is capable of hydrolyzing the (l->6)-alpha-D-glucosidic bonds of dextran.

[0113] To test the degradation of crosslinked microgels by dextranase, 5 μL of dextranase (from chaetomiun erraticum, D0443 from Sigma-Aldrich) are added to 20 μL of a solution of photocrosslinked drops of 45 μm diameter, i.e. approximately 250,000 drops, brought to a temperature of 37°C. Digestion is evaluated by visualizing the drops under a microscope.

[0114] Table 2 below summarizes the results obtained with microgels resulting from the photocrosslinking of a single type of dextran-methacrylate.

[0115] [T ables 2] Microgel Stability to surfactant addition Degradability by dextranase DS=13% No 30 min DS=15.5% No 20 min DS=17% No <lh DS=18% Non <lh ds="25%" oui 4h30>48 h DS=31% Yes >48 h DS=37% Yes >48 h

[0116] From these results, it appears that for a DS greater than or equal to 20%, the microgels resist the addition of the surfactant: the polymer network is sufficiently strong. On the other hand, for a lower DS, the microgels become destabilized upon addition of the surfactant.

[0117] Furthermore, the crosslinked microgels are digested in less than 2 hours only when the DS is less than or equal to 20%. For a higher DS, the enzyme cannot degrade the microgels, or else it can degrade them too slowly (more than 2 hours).

[0118] None of these microgels therefore allows for simultaneous satisfaction in terms of stability and controlled biodegradability. There is in fact no microgel which is both stable upon addition of the surfactant and degradable in less than 2 hours by dextranase at 37°C. Example 3

[0119] Preparation of hydrogels from several mixtures of two methacrylated dextran including a dextran with DS less than 20%

[0120] Several mixtures of methacrylated dextran in proportion 75% by weight of methacrylated dextran with DS lower than 20% and 25% by weight of methacrylated dextran with DS higher are produced.

[0121] Each 75:25 mixture of powdered methacrylated dextran is dissolved in aqueous solution (water or PBS buffer) and then LAP is added to obtain a total by weight for the aqueous phase of 5% methacrylated dextran and 0.1% LAP. This polymer solution is then used to form microdroplets which are photocrosslinked into hydrogels according to the protocol detailed in example 2.

[0122] The stability of the crosslinked hydrogels thus obtained is tested in the presence of surfactant in the oily phase and their degradability by dextranase is evaluated as described in example 2.

[0123] The results obtained are summarized in Table 3 below.

[0124] [Tables3] Hydrogel Stability to surfactant addition Degradability by dextranase EC 75% by weight of a low DS value 25% by weight of a high DS value Weighted average DS 13% 31% 17.% Yes 1h Single dextran of DS=18% pure No 1h Single dextran of DS=20% pure Yes 4h30 15.5% 37% 20.8% Yes 1h

[0125] From these results, it emerges that only the microgels in accordance with the invention are satisfactory. In particular, they make it possible to obtain a weighted average DS range in which stability and controlled degradability are simultaneously observed within less than two hours of the microgels.

[0126] A microgel, obtained from a single polymer whose DS value is close to the weighted average DS value of the microgels according to the invention, is devoid of either the expected stability or degradability. Example 4

[0127] Preparation of a hydrogel from a 50 / 50 mixture of two methacrylated dextrans, one of which has a DS of less than 20%.

[0128] A 50:50 mixture of two methacrylated dextrans, one of DS = 13% (low DS) and the other of DS = 31% (high DS) in powder form is dissolved in aqueous solution (water or PBS buffer) and then LAP is added in order to obtain a total by weight for the aqueous phase of 5% of methacrylated dextran and 0.1% of LAP. This polymer solution is then used to form microdroplets which are photocrosslinked into microgels as described in Example 2. The stability of these microgels in the presence of surfactant in the oily phase and their degradability by dextranase is evaluated as described in Example 2. The results obtained are summarized in Table 4 below which also recalls the results obtained with this same mixture of methacrylated dextran but in a proportion of 75 / 25 and stated in Example 3.

[0129] [Tables4] Hydrogel Stability to surfactant addition Degradability by dextranase CE Qty in Low DS (DS = 13%) Qty in High DS (DS = 31%) Weighted average DS value 50% 50% 22% Yes 2h Single dextran of DS at 20% pure Yes 4h30 75% 25% 17.5% Yes 1h

[0130] From these results, it emerges that the 50:50 DS (DS = 13%), and DS (DS = 31%) mixture reproduces the two expected qualities like the 75:25 low DS (DS = 13%), and high DS (DS = 31%) mixture.< / lh>

Claims

Claims

1. A method for preparing a crosslinked hydrogel with controlled degradability comprising at least the steps of a. Providing aqueous droplets comprising at least one mixture of at least two crosslinkable biopolymers and different degrees of substitution, DS, b. Exposing said droplets to a crosslinking stimuli effective for achieving simultaneous crosslinking of the two biopolymers and, c. Recovering the crosslinked hydrogel, in which one of the biopolymers has a degree of substitution less than or equal to 20%

2. Process according to any one of the preceding claims in which said biopolymers are chemically modified and in particular substituted by crosslinkable units in particular chosen from thiol, divinylsulfonyl, maleimide, azide, alkynyl, alkenyl, acrylate, methacrylate, aldehyde, norbornenyl, oxy-amine, amine, N-hydroxysuccinimide ester units and mixtures thereof, in particular from acrylates and methacrylates.

3. Method according to any one of the preceding claims in which said biopolymers are derived from compounds chosen from alginates, hyaluronic acid, cellulose, carboxymethylcellulose, chitosan, dextran and in particular dextran.

4. A method according to any preceding claim wherein said biopolymers differ only in their degree of substitution, DS.

5. Method according to any one of the preceding claims in which said droplets contain a mixture of at least two polydextrans of identical molecular weight and chemically modified by units chosen from acrylate, methacrylate, alkenyl, alkynyl and their mixtures, in particular a mixture of two polydextrans of identical molecular weight chemically modified by methacrylate units, DexMet, and DS varying from 10 to 40%.

6. A method according to any preceding claim wherein said droplets contain a mixture of at least one DexMet with DS less than 20% and DexMet with DS greater than 25% and in particular a mixture of DexMet with DS equal to 13% + / - 0.5% and DexMet with DS equal to 31% + / -1%.

7. A method according to any preceding claim wherein said droplets contain a 75% by weight mixture of DexMet with DS equal to 13% + / -0.5% and 25% by weight of DexMet with DS equal to 31% + / -1% or a 50 / 50 by weight mixture of DexMet with DS equal to 13% + / -0.5% and 25% by weight of DexMet with DS equal to 31% + / -1%.

8. A method according to any preceding claim wherein the crosslinking is carried out photochemically.

9. Method according to any one of the preceding claims in which said droplets also contain at least one crosslinking agent and in particular a photoinitiator in particular lithium phenyl-2,4,6-trimethylbenzoylphosphinate or 1 - [4-(2-Hydroxyethoxy) -phenyl] -2-hydroxy-2-methyl-1 -propan-1 -one.

10. A method according to any preceding claim wherein said droplets are formed microfluidically.

11. Method according to any one of the preceding claims in which said droplets have a number average size varying from 10 to 100 pm and in particular from 30 to 50 pm.

12. Method according to any one of the preceding claims in which said droplets contain at least one entity of chemical and / or biological interest, in particular chosen from chemical therapeutic active agents, chemical reagents and biochemical active agents, in particular chosen from nucleic acids such as siRNA, RNAi and DNA, proteins, peptides, enzymes, viruses, bacteria and / or cells.

13. A method according to any one of the preceding claims wherein said droplets are loaded with at least one particular entity of interest as defined in claim 12, and in that said crosslinked hydrogel is isolated from non-crosslinked droplets by selective destabilization thereof.

14. Method according to the preceding claim in which the selective destabilization is carried out by bringing the crosslinked hydrogel, mixed with the non-crosslinked droplets, into contact with at least one surfactant.

15. A method according to any preceding claim wherein the hydrogel formed is a macrogel, microgel or nanogel and preferably a microgel.

16. Crosslinked hydrogel with controlled degradation obtained by the method according to any one of the preceding claims.

17. Crosslinked hydrogel with controlled degradation, in particular obtained by the process according to any one of claims 1 to 15, characterized in that it is derived from the simultaneous crosslinking of at least two polydextrans chemically modified with units chosen from thiol, divinylsulfonyl, maleimide, azide, alkynyl, alkenyl, acrylate, methacrylate, aldehyde, norbornenyl, oxyamine, amine, N-hydroxysuccinimide ester units and in particular from acrylates and methacrylates, one of which has a DS less than or equal to 20%, in particular two polydextrans differing only by their DS and more particularly two polydextrans chemically modified by methacrylate units, DexMet, and DS varying from 10% to 40%.

18. Hydrogel according to the preceding claim deriving from the crosslinking of a mixture of 75% by weight of DexMet with DS equal to 13% + / -0.5% and 25% by weight of DexMet with DS equal to 31% + / -1% or of a mixture of 50 / 50 by weight of DexMet with DS equal to 13% + / -0.5% and of DexMet with DS equal to 31% + / -1%.

19. Hydrogel according to claim 17 or 18 containing at least one entity of interest in particular chosen from chemical therapeutic active agents, chemical reagents and biochemical active agents in particular chosen from nucleic acids such as siRNA, RNAi and DNA, proteins, peptides, enzymes, viruses, bacteria, cells and mixtures thereof.

20. Use of a hydrogel obtained according to any one of claims 1 to 16 or according to one of claims 17 to 20 for cell or bacterial culture.

21. Use of aqueous droplets, in particular formed by microfluidics, and comprising at least one mixture of at least two crosslinkable and chemically modified polydextrans with units chosen from thiol, divinylsulfonyl, maleimide, azide, alkynyl, alkenyl, acrylate, methacrylate, aldehyde, norbornenyl, oxyamine, amine, N-hydroxysuccinimide ester units and mixtures thereof, in particular from acrylates and methacrylates with one of them and in particular only one of them having a DS less than or equal to 20%, for the encapsulation of at least one entity of interest, in particular as defined in claim 19.

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