Chelating wound dressing for treating complex wounds

Abstract

The disclosure relates to an aqueous composition, having a Newtonian viscosity between 0.5 Pa.s and 1000 Pa.s, comprising at least one statistical polysaccharide B, having a weight-average molar mass of between 100 kDa and 1000 kDa, of formula I: and optionally a chitosan A, said statistical polysaccharide B being in a mass concentration CB, said chitosan A being in a mass concentration CA, the sum of CB plus CA being of at least 10 g.L-1. The disclosure also concerns the aqueous composition mentioned above for its use in the treatment of complex wounds, such as wounds related to diabetes, burns and wounds containing one or more metals, and to a to a wound dressing comprising said aqueous composition.

Description

DescriptionTitle: Chelating wound dressing for treating complex woundsTechnical field

[0001] The present disclosure concerns treatment of complex wounds. In particular, the present disclosure is directed to a wound dressing comprising a gel forming gel composition. More specifically, the present disclosure is about a modified chitosan hydrogel capable of chelating metals.Background art

[0002] It is known to use gels of chitosan in the field of skin repair (J. Zhao et al., Int. J. Biol. Macro., 2023). This is quite efficient, especially due to the antibacterial effect of the chitosan. However for complex wounds, such as wounds related to diabetes, burns and wounds containing one or more metals, the efficiency may be deeply enhanced.

[0003] In particular, currently, no technology is able to respond to all types of wounds and at each stage of wound development.

[0004] There are approximately 2.5 million people with wounds in France per year (including 35% complex wounds) (E. Lupon et al., Journal de Medecine Vasculaire, 2019) and this number is expected to increase due to the aging of the population associated with an increase in the number of people with diabetes. A study published in 201 1 estimated the cost of treating chronic wounds in France at nearly a billion euros, including €210 million for dressings and compresses alone (A. F. Motte et al., Hospital Pharmacist and Clinician, 2017).

[0005] In order to reduce the cost of healthcare expenses, new dressings must be developed to allow faster and high-quality healing and to limit treatment complications. These future new bioactive dressings are expected to replace current solutions for the most complex wounds.

[0006] In this context, the present disclosure is particularly concerned by complex wounds, such as chronic wounds, like wounds related to diabetes or eschars or arterial ulcers, burns, chemical burns, wounds related to radioactive contaminations and wounds containing one or more metals.

[0007] The present disclosure aims at fulfilling at least one or the following objectives:-01- Proposing a wound dressing having a great exudate absorption capacity.-02- Proposing a wound dressing able to chelate metals so as to reduce oxidative damage and inflammation linked to metals but also toxicity linked to heavy metals linked to metal fragments, particularly in conflict zones.-03- Proposing a wound dressing having an antibacterial effect and chelating properties.-04- Proposing a wound dressing having a modulable biodegradability depending on the type of wound.-05- Proposing a wound dressing able to perfectly fit the shape of the wound.-06- Proposing a wound dressing able to accelerate healing of the wound.-07- Proposing a wound dressing able to improve quality of the resulting scar (better elasticity, better aesthetic appearance)-08- Proposing a wound dressing which is usable under non-aseptically conditions, for example on battle fields, on site of an accident in an industrial environment.Brief summary

[0008] At least one of the above objectives is reached thanks to an aqueous composition, having a Newtonian viscosity between 0.5 Pa.s and 1000 Pa.s, comprising at least one statistical polysaccharide B, having a weight-average molar mass of between 100 kDa and 1000 kDa, of formula I:Formula I in which:- each Rc independently represents a group including a chelating moiety,- each Z independently represents a linkage which may be a single bond or a hydrocarbonbased chain including between 1 and 12 carbon atoms, said chain possibly being linear or branched and possibly including one or more unsaturations and possibly including one or more heteroatoms, preferably chosen from nitrogen, oxygen, sulfur and atoms of the halogen family,- x is between 0. and 0.40,- y is between 0.05 and 0.25,- the ratio y / x being greater than or equal to 0.2, preferably greater than or equal to 1 ,- the sum x + y being greater than or equal to 0.1 , and optionally a chitosan A, said statistical polysaccharide B being in a mass concentration CB, said chitosan A being in a mass concentration CA, the sum of CB plus CA being of at least 10 g.L-1, preferably of at least 30 g.L-1and more preferably of 50 g.L-1.

[0009] The invention also concerns the aqueous composition mentioned above for its use in the treatment of complex wounds, such as chronic wounds, like wounds related to diabetes or eschars or arterial ulcers, burns and wounds containing one or more metals.

[0010] The invention is also directed to a wound dressing comprising the aqueous composition mentioned above.

[0011] In the following, terms as used herein are defined in their meaning.The term “about” or “ca.” has herein the meaning that the following value may vary for ± 20%, preferably ± 10%, more preferably ± 5%, even more preferably ± 2%, even more preferably ± 1 %.Unless otherwise defined, “%” has herein the meaning of weight percent (wt%), also referred to as weight by weight percent (w / w%). the mean molar weight Mw of polysaccharides such as Chitosan A and statistical polysaccharide B are measured by steric exclusion chromatography, the method being described in “Physico-chemical studies of the gelation of chitosan in a hydroalcoholic medium” A. MONTEMBAULT, C. VITON, A. DOMARD, Biomaterials, 26(8), 933-943, 2005.Brief description of the drawingsFig. 1

[0012] [Fig. 1] is an1H NMR spectra of raw chitosan used for the synthesis of MEX-CD2Fig. 2

[0013] [Fig. 2] is a graph showing absorbance at A=295nm as a function of the copper concentration for synthesized MEX-CD2Fig. 3

[0014] [Fig. 3] is a graph showing viscosity of polymer solutions with shear rates from 102to 103s1for four formulations : MEX-CD2 / chitosan mass ratios 1 / 0, 5 / 1 , 2 / 1 and 1 / 1 (mB / mA)Fig. 4

[0015] [Fig. 4] is graphs showing evolution of storage modulus and tan(6) after injection in PBS for three formulations: MEX-CD2 / chitosan 1 / 1 , 2 / 1 and 1 / 0Fig. 5

[0016] [Fig. 5] is a SAXS profile of three formulations comprising a mixing of MEX-CD2 and chitosan : MEX-CD2 / chitosan ratios are either 1 / 0, 2 / 1 , 0 / 1Fig. 6

[0017] [Fig. 6] is a WAXS profile of three formulations comprising a mixing of MEX-CD2 and chitosan : MEX-CD2 / chitosan ratios are either 1 / 0, 2 / 1 , 0 / 1Fig. 7

[0018] [Fig. 7] is a set of pictures of the wounds at day 1 , 3, 5 and 7 of the study for each group of example 4.Fig. 8

[0019] [Fig. 8] is a graph showing evolution of wound areas for all groups of example 4.Fig. 9

[0020] [Fig. 9] is a set of bar diagrams showing Blood lymphocytes (LYM), hemoglobin (HGB), hematocrit (HCT) and granulocytes (GRAN) concentrations at the 7thday of the study for all groups of example 4.Fig. 10

[0021] [Fig. 10] is a set of pictures of the wounds each week of the study for control and MEX-CD2- I groups (pig 2) of example 5.Fig. 11

[0022] [Fig. 11] is a graph showing variation of wounds area relatively to their initial area for control and MEX-CD2-I groups (*p<0.05, **p<0.01 , compared to the respective group) of example 5.Fig. 12

[0023] [Fig. 12] is a set of showing bacterial density score evolution for pig 1 (P1) and pig 2 (P2) of example 5.Fig. 13

[0024] [Fig. 13] is a set of pictures showing histological sections of biopsies taken on day 30 for control (left) and MEX-CD2-I (right) groups of example 5.Fig. 14

[0025] [Fig. 14] is the different hematological parameters for rats experienced acid burns and treated with MEX-CD2-I 5% (w / w) during 7 days with (A) total leukocyte count, (B) eosinophils, (C) hemoglobin concentration in blood (D) erythrocytes count and (E) hematocrit (*p<0.05, **p<0.01 , compared to the respective control group) of example 6.Fig. 15

[0026] [Fig. 15] is a graph showing variation of alkaline burns area relatively to their initial area for control and MEX-CD2-I (*p<0.05) of example 6.Fig. 16

[0027] [Fig. 16] is the different hematological parameters for rats experienced alkaline burns and treated with MEX-CD2-I 5% (w / w) during 7 days with (A) total leukocyte count, (B) monocytes, (C) erythrocytes count (D) hemoglobin concentration in blood (E) mean corpuscular hemoglobin (MCH) amount (*p<0.05 compared to the respective control group) of example 6.Fig. 17

[0028] [Fig. 17] A is a graph showing variation of weight of rats, for control, no treatment and MEX- CD2-I (*p<0.05) of example 9.

[0029] [Fig. 17] B is a graph showing variation of phosphorus burns area relatively to their initial area for no treatment and MEX-CD2-I (*p<0.05) of example 9.

[0030] [Fig. 17] C is a graph showing variation of lymph nodes for control, no treatment and MEX- CD2-I (*p<0.05) of example 9.Detailed descriptionThe aqueous composition

[0031] As mentioned above, the invention concerns an aqueous composition, having a Newtonian viscosity between 0.5 Pa.s and 1000 Pa.s, comprising at least one statistical polysaccharide B, having a weight-average molar mass of between 100 kDa and 1000 kDa, of formula I:Formula I in which:- each Rc independently represents a group including a chelating moiety,- each Z independently represents a linkage which may be a single bond or a hydrocarbon-based chain including between 1 and 12 carbon atoms, said chain possibly being linear or branched and possibly including one or more unsaturations and possibly including one or more heteroatoms, preferably chosen from nitrogen, oxygen, sulfur and atoms of the halogen family,- x is between 0 and 0.40,- y is between 0.05 and 0.25,- the ratio y / x being greater than or equal to 0.2,- the sum x + y being greater than or equal to 0.1 , and optionally a chitosan A, said statistical polysaccharide B being in a mass concentration CB, said chitosan A being in a mass concentration CA, the sum of CB plus CA being of at least 10 g.L1.

[0032] The aqueous composition is preferably in the form of a solution able to become a hydrogel when applied on a wound.The Newtonian viscosity

[0033] According to the invention, the aqueous composition has a Newtonian viscosity between 0.5 Pa.s and 1000 Pa.s.

[0034] In some embodiments, the aqueous composition has a Newtonian viscosity between 10 Pa.s and 200 Pa.s and preferably between 20 Pa.s and 70 Pa.s.

[0035] The range of viscosity is important since it determines the ability of the composition to spread and fit the shape of the wound. A too low viscosity would induce a too broad spread and the composition may leak out of the wound. A too high viscosity would induce a too low spread and the composition may not fit the shape of the wound.

[0036] In addition, depending on the wound to be treated, the viscosity of the aqueous composition may be adjusted.The statistical polysaccharide B

[0037] The polysaccharide according to the invention is composed of three monomer units, namely an N-acetylglucosamine type unit A, a glucosamine type unit B and a glucosamine type unit C functionalized with a chelating moiety (Rc type) linked by a linkage (Z type) to the glucosamine nitrogen.

[0038] The polysaccharide of formula I is a statistical polymer. In other words, the sequence of the various monomer units A, B and type C is random.

[0039] In formula I, x represents the proportion of units A.

[0040] In formula I, y represents the proportion of units C.

[0041] The remaining monomer units are units B. Thus, in formula I, the proportion of units B is equal to 1-x-y.

[0042] As mentioned above, the aqueous composition comprises a statistical polysaccharide B of formula I, having a weight-average molar mass of between 100 kDa and 1000 kDa.

[0043] In some embodiment, the statistical polysaccharide B of formula I has a weight-average molar mass of between 200 kDa and 800 kDa, preferably of between 300 kDa and 600 kDa, more preferably of about 500 kDa.

[0044] As used herein, the term “independently” for the Rc moiety i.e. the chelating moiety, means that each Rc of the statistical polysaccharide B is a chelating moiety that may be different from one to another, or may be identical.

[0045] Hence, it is understood that, in formula I above, several groups Rc may be present in the polysaccharide. These groups Rc may be identical to or different from each other. They are all independently chosen from groups bearing a chelating moiety. For example, each Rc is identical i.e., there is one type of Rc throughout formula I, or there may be more than one type of Rc throughout formula I i.e. two, three, four, five or even n different Rc, n being an integer. The same applies to the linkages Z: several linkages Z may be present, and they may be identical to or different from each other.

[0046] Each of the groups Rc may contain one or more coordination sites. Preferably, the coordination site is a nitrogen or oxygen atom. Advantageously, each of the groups Rc includes between 4 and 8 coordination sites, more advantageously between 6 and 8 coordination sites and even more advantageously each of the groups Rc includes 8 coordination sites.

[0047] The term “coordination site” means a single function that is capable of chelating a metal. For example, an amine function represents a coordination site by formation of a dative bond between the nitrogen atom and the metal, and a hydroxamic acid function also represents a coordination site by formation of a dative bond between the oxygen of the carbonyl unit and by a covalent bond with the oxygen of the N-oxide unit, the coordination site thus forming a five-membered ring.

[0048] In preferred embodiments, the chelating moiety is derived from a chelating agent chosen from the group consisting of 1 ,4,7,10-tetraazacyciododecane-N,N',N",N"'-teracetic acid (DOTA), 1 ,4,7- triazacyclononane-1 ,4, 7-triacetic acid (NOTA), ,4,7-triazacyclononane-1-glutaric-4,7-diacetic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1 ,4,7, 10-tetraazacyclododecan-1-yl)pentanedioicacid (DOTAGA), 1 ,4,7,10-tetrakis(carbamoylmethyl)-1 ,4,7,10-tetraazacyclodecane (DOTAM), 2’4,7,10- tris(2-amino-2-oxoethyl)-1 ,4,7,10-tetraazacyclododecan-1-yl)acetic acid (DO3AM), 1 ,4,7- tetrakis(carbamoylmethyl)-1 , 4,7-triazacyclononane (NOTAM), 1 ,4,7,10-tetraazacyclododecane 1 ,4,7,10-tetrakis(methylene phosphonate (DOTP), 1 ,4,7-tetrakis(methylene phosphonate)-1 , 4,7- triazacyclononane (NOTP), 1 ,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-teracetic acid (TETA), 1 ,4,8, 11-tetraazacyclotetradecane-N,N',N",N"'-tetrakis(carbamoyl methyl) (TETAM), diethylene triaminopentaacetic acid (DTPA) and deferoxamine (DFO), preferably from the group consisting of DOTAGA, DFO, DOTAM and DTPA, more preferably DOTAGA.

[0049] As understood by those skilled in the art, derived from a chelating agent means that said chelating agent is covalently linked to linkage Z. Hence, in preferred embodiments the chelating moiety is selected from the group consisting of, wherein “wavy” represents the link site:

[0050] The term “Z-type linkage” means Z in the polysaccharide of formula I, and the linkages Zi and Z2, when the linkage Z2 is present, in the polysaccharide of formula II.

[0051] Preferably, in formula I, each Z is independently a single bond or a hydrocarbon-based chain including between 1 and 12 carbon atoms, said chain possibly being linear or branched and possibly including one or more unsaturations and possibly including one or more heteroatoms, preferably chosen from nitrogen, oxygen, sulfur and atoms of the halogen family.

[0052] According to one embodiment, in formula I, each Z is independently selected from the group consisting of: a bond, a linear or branched alkyl chain including between 1 and 12 carbon atoms, and a linear or branched alkenyl chain including between 1 and 12 carbon atoms, said alkyl and alkenyl chains possibly being interrupted with one or more C6-C10 aryl groups, and / or with one or more heteroatoms or groups selected from the group consisting of -O-, -S-, -C(O)-, -NR’-, -C(O)NR’-, -NR’-C(O)-, -NR’-C(O)-NR’-, -NR'-C(O)-O-, -O-C(O)NR’, -C(S)NR’-, -NR’-C(S)-, -NR’- C(S)-NR’ said alkyl and alkenyl chains possibly being substituted with one or more groups selected from the group consisting of halogens, -OR', -COOR’, -SR', -NR’2, each R’ is independently H or a C1-C6 alkyl.

[0053] Advantageously, in formula I, each Z is independently selected from the group consisting of: a bond and a linear or branched alkyl chain including between 1 and 12 carbon atoms, said alkyl chain possibly being interrupted with one or more C6-C10 aryl groups, and / or with one or more heteroatoms or groups selected from the group consisting of -O-, -S-, -C(O)-, -NR’-, -C(O)NR’-, -NR’-C(O)-, -C(S)NR’-, -NR’-C(S)-, -NR’-C(S)-NR’, each R' is independently H or C1-C6 alkyl.

[0054] In one particular embodiment, each Z is an alkyl chain including between 1 and 12 carbon atoms.

[0055] In another particular embodiment, each Z is a polyethylene glycol (PEG).

[0056] In a preferred embodiment, in formula I, x is between 0.01 and 0.15 and, preferably about0.05.

[0057] In a preferred embodiment, in formula I, y is between 0.10 and 0.22, preferably between 0.15 and 0.20 and more preferably about 0.17.

[0058] In a preferred embodiment, in formula I, the ratio y / x is greater than 0.5 and preferably greater than or equal to 1 .

[0059] In a preferred embodiment, in formula I, the sum x + y is greater than 0.2 and preferably greater than 0.3.

[0060] In a particularly preferred embodiment, the statistical polysaccharide B is of formula I, wherein x is about 0.05, y is about 0.17, Z is a single and Rc is DOTAGA:

[0061] According to a particular embodiment, the statistical polysaccharide B is of formula II:Formula II in which:Rci and RC2 are different, and are groups including a chelating moiety,Zi and Z2, which may be identical or different, are linkages which may be a single bond or a hydrocarbon-based chain including between 1 and 12 carbon atoms, said chain possibly being linear or branched and possibly including one or more unsaturations and possibly including one or moreheteroatoms, preferably chosen from nitrogen, oxygen, sulfur and atoms of the halogen family,- x is between 0 and 0.40,- y is between 0.05 and 0.25,- the ratio y / x being greater than or equal to 0.2,- the sum x + y being greater than or equal to 0.1 , and z is between 0.5 and 1 .

[0062] In this specific embodiment, the statistical polysaccharide B of formula II may comprise:- only one type of group including a chelating moiety, Rci, when z = 1 , or- two types of groups including a chelating moiety, Rci and Rc2, when 0.5 < z < 1 .

[0063] According to one embodiment, z is between 0.8 and 0.99, and the group Rci is thus largely predominant.

[0064] The above features of Rc, Z, x and y apply to Rci, RC2, Z1 , Z2, x and y in formula II.

[0065] According to one embodiment, for the statistical polysaccharide B of formula II, the group Rci is DOTAGA, and preferably z = 1 .

[0066] According to one embodiment, for the statistical polysaccharide B of formula II, the group Rci is DOTAGA, and the group RC2 is DFO.

[0067] The choice of linkages Z, Z1 and Z2 in formulae I and II depends essentially on the groups Rc, Rci and RC2 and the metal to be chelated. Specifically, notably for steric reasons, the groups Rc, Rci and RC2 may be more or less close to the 6-membered nitrogen ring of the glucosamine unit.The chitosan A

[0068] As mentioned above, the aqueous composition may comprise a chitosan A. The presence of a chitosan A may be advantageous in certain embodiments since chitosan A allows to slow down the biodegradation. In other words, in cases it may be advantageous that the hydrogel, obtained from the aqueous composition, resists longer, it is preferable to add a chitosan A in the aqueous composition.

[0069] According to the present disclosure, “chitosan” means a natural polymer of polysaccharide type of D-glucosamine (GlcN) or co-polysaccharide type, consisting of a random distribution (statistic copolysaccharide) or not (blocks copolysaccharide) of D-glucosamine and N-acetyl-D-glucosamine (GIcNAc), linked by glycosidic bonds of P(1 ->4) type.

[0070] In preferred embodiments, said chitosan A has a degree of acetylation less than 40%, preferably less than 10%, for example between 0% and 10%. The proportion of N-acetyl-D- glucosamine is calculated by using1H NMR, following the Hirai’s methodology (A. HIRAI, H ODANI, A. NAKAJIMA, Polymer Bulletin, 26 (1), 87-94, 1991).

[0071] Advantageously, chitosan A has a mean molar weight Mw between 100 kg / mol and 1000 kg / mol, preferably between 200 kg / mol and 700 kg / mol.The concentrations of the statistical polysaccharide B and the chitosan A

[0072] As set forth above, the viscosity of the aqueous composition is an important parameter. The viscosity may be adjusted at the desired value with the concentration of the statistical polysaccharide B and the concentration of the optional chitosan A.

[0073] According to the invention, said statistical polysaccharide B is in a mass concentration CB, said chitosan A is in a mass concentration CA, the sum of CB plus CA being of at least 10 g.L-1.

[0074] In some embodiments, the sum of CB plus CA is of at least 30 g.L-1and preferably of at least 50 g.L1, the maximum depends on the nature of the statistical polysaccharide B and the chitosan A. Indeed, the viscosity increase along with the increase of the total concentration of the statistical polysaccharide B and of the optional chitosan A, i.e. the sum of CB plus CA, and the Newtonian viscosity must not exceed 1000 Pa.s.

[0075] In embodiments in which the chitosan A is present, in the aqueous composition said statistical polysaccharide B and said chitosan A are preferably in a mass ratio (mA / mB) between 4 / 1 and 1 / 10, preferably between 1 / 1 and 1 / 5.The use of the aqueous composition

[0076] As previously mentioned, the invention is also directed to an aqueous composition, having a Newtonian viscosity between 5 Pa.s and 1000 Pa.s, comprising at least one statistical polysaccharide B, having a weight-average molar mass of between 100 kDa and 1000 kDa, of formula I:Formula I in which:- each Rc independently represents a group including a chelating moiety,- each Z independently represents a linkage which may be a single bond or a hydrocarbon-based chain including between 1 and 12 carbon atoms, said chain possibly being linear or branched and possibly including one or more unsaturations and possibly including one or more heteroatoms, preferably chosen from nitrogen, oxygen, sulfur and atoms of the halogen family,- x is between 0 and 0.40,- y is between 0.05 and 0.25,- the ratio y / x being greater than or equal to 0.2, preferably greater than or equal to 1 ,- the sum x + y being greater than or equal to 0.1 , and optionally a chitosan A, said statistical polysaccharide B being in a mass concentration CB, said chitosan A being in a mass concentration CA, the sum of CB plus CA being of at least 10 g.L-1, preferably of at least 30 g.L-1and more preferably of 50 g.L-1, for its use in the treatment of complex wounds, such as wounds related to diabetes, burns and wounds containing one or more metals.

[0077] Each feature of the aqueous composition is as set forth above.

[0078] One of the advantages of the present invention is the ability of the statistical polysaccharide B to chelate metals. Indeed, in many situations a wound may contain metals which have deleterious effects for the healing of said wound.

[0079] Said metals may come from the wound itself, for instance in cases where the wound results from a knife cut, some metal particles of the knife may stay in the wound. In these cases the metal is qualified as exogen.

[0080] Alternatively said metals may come from the blood. Indeed, blood contains several metal ions, and some, like iron, in these cases, the metal is qualified as endogen.

[0081] It is advantageous to chelate the metals, exogen and endogen, since generally, and specifically iron enhance oxidative damages in the region the wound, what delays the healing.

[0082] As such in some embodiments, the metal contained in the wound is iron.The wound dressing

[0083] One of the purposes of the aqueous composition of the present invention is to be incorporated in a wound dressing.

[0084] Hence, as previously mentioned, the invention also relates to a wound dressing comprising the above detailed aqueous composition.

[0085] The wound dressing may be in several forms. In one embodiment, the wound dressing is the composition itself once in a form a hydrogel after application of the aqueous composition on the wound to be treated. In cases where an additional physical protection could be beneficial, the wound dressing of the present invention may be covered by a classical wound dressing.

[0086] In some other embodiments it may be advantageous to construct a wound dressing in which the aqueous composition is deposited on a support. The support may be microperforated polyurethane adhesive.

[0087] In advantageous embodiments, the wound dressing is biodegradable.

[0088] One of the advantages of the wound dressing according to the invention is its anti- bacteriological properties. Another advantage is its easiness of implementation. As such, the invention also concerns a wound dressing as defined above for its use in non-aseptically condition, for example on battle fields, on site of an accident in an industrial environment. This is advantageous since, wounds happening under those conditions must be quickly protected from the environment and especially bacteriological or fungal attacks.ExampleExample 1 : Synthesis of the functionalized polymer of the invention (MEX-CD2)Synthesis

[0089] 15 L of Milli-Q water were introduced in a 30 L thermostated glass reactor. 225 g of raw chitosan (NAT-0030, Matexcel) were then added in the reactor to solubilize. The mixture was left stirring at 300 rpm for 20 minutes until a cloudy whitish solution was obtained. 187.5 mL of acetic acid were added to the mixture in the reactor and left under stirring at 300 rpm for 5 hours until the medium became clear. 15 L of 1 ,2-propanediol were then introduced into the reactor and left stirring for 1 hour, obtaining a limpid viscous slightly yellowish solution. 450 g of DOTAGA anhydride were then added to the reactor and the mixture was kept stirred at 300 rpm for 18 hours.Purification and lyophilization

[0090] The product obtained after synthesis was purified by tangential filtration using the Sartoflow® Advanced device with a Sartocon® Slice PESU cassette (polyethersulfone membranes; cut-off: 100 kDa; filtration area: 0.1 m2) according to a diafiltration-concentration model against 200 L of a 0.1 M acetic acid solution, followed by 200 L of a 5 mM acetic acid solution. Briefly, 5 L of the unpurified product were introduced in the Sartocon device using a peristaltic pump. The device was then connected to a tank containing 200 L of a 0.1 M acetic acid solution. The filtration was carried out with a maximum pressure of 3 bars and with a constant product volume of 15 L ensured by the controlled addition of a 0.1 M acetic acid solution. The product was then purified against a 5 mM acetic acid solution to avoid precipitation of the product at neutral pH. In the same way, the filtration was carried out at a constant volume of 15 L and the product was reconcentrated to 7 L (5.4 g / L) at the end of the filtration. The purification was followed by size exclusion chromatography coupled with a UV detector until obtaining less than 5% of free DOTAGA.

[0091] The resulting product was freeze-dried in a Buchi L-200 pro freeze-dryer for 60 h.Determination of DA and DS

[0092] The acetylation degree (DA) of raw chitosan for this synthesis was determined by1HNMR. Chitosan powder was redispersed at 1 % (w / v) in a 0.35% (w / w) DCI / D2O solution under stirring until homogenization of the solution.1H Spectra was obtained using a Bruker Ascend 400 MHz NMR spectrometer with a zg30 pulse program, an acquisition temperature of 343 K and 128 scans and is displayed on figure 1. Acetylation degree was determined following the Hirai method (Hirai et al.,Polym. Bull. 26, 87-94, 1991): the integration of the peak resulting from the acetyl CH3 proton and the 2-6 proton are included into the equation obtaining a DA of 5,9%:

[0094] Copper chelation studies have been conducted on freeze-dried product reconstituted in acetate buffer to evaluate the copper extraction capacity as well as to estimate the DOTAGA substitution degree (DS) of the synthesized product. Therefore, samples were prepared in a 0.1 M AcOH / 0.1 M AcONH4 buffer solution made by introducing 0.77 g of ammonium acetate and 0.57 mL of acetic acid into a 100 mL volumetric flask and completed with purified water (pH = 4.53). A suitable volume of buffer solution was introduced into a vial to obtain a sample whose total volume was 2 mL. 200 pL of the synthesized product at 10 g / L were then introduced into each vial containing the buffer solution beforehand and a different amount of a 15 mM aqueous solution of CuCh was added. The vials containing the samples at 1 g / L of MEX-CD2 were then homogenized by stirring using a vortex. Copper concentration ranging from 0 to 1 mM Cu were investigated. Then, the absorbance of all samples at A=295nm was recorded using a UV-Vis spectrophotometer.

[0095] By plotting the absorbance as a function of the copper concentration (figure 2), we find a maximum copper chelation capacity for a concentration of 0.65 mM, which corresponds to a copper absorption capacity of 41 .6 mg / g of synthesized MEX-CD2. The degree of DOTAGA grafting (DS) on chitosan is therefore estimated at 15.3%.EXAMPLE 2: preparation of different formulations of polymer solutions comprising MEX-CD2 and chitosan and assessments of the rheological and nanoscopic properties of the hydrogels obtainedPreparation of polymer solutions

[0096] These mixtures were prepared at 5% (w / w) by addition of an adequate amount of MEX-CD2 and chitosan depending on the desired formulation. In the same way, an adequate quantity of acetic acid (or hydrochloric acid) was added for each formulation in order to solubilize raw chitosan. For example, to prepare the MEX-CD2 / chitosan 2 / 1 formulation, 667 mg of MEX-CD2 and 333 mg of chitosan were dispersed in 18.92 mL of milli-Q water and 71 .5 pL of ultrapure acetic acid were added under mechanical stirring at 100 rpm in a 50 mL reactor. The mixture was left under stirring for 2 hours until complete dissolution and homogenization of the medium. The solution obtained was recovered and introduced into a suitable fluid dispenser, then centrifuged at 4000 rpm for 10 minutes to obtain an air bubble-free solution. The mixture was then introduced into glass syringes and sterilized for 20 min at 121 °C in an autoclave.Viscosity

[0097] Viscosity measurements were conducted on a Thermo Scientific HAAKE RheoStress 600 Sensor Systems using a C35 / 2° Ti L cone plate geometry. Prior to measurement, the zero gap and zero force were calibrated. Samples were then spread out on the tray and the geometry gap was set to 105 pm. The sample’s surplus was removed using a spatula in order to limit the edge effects. The viscosity of each sample was measured with a flow sweep linear study conducted by scanning shearrate from 10-2to 103s-1at a regulated temperature of 25 °C. 10 points per decade were recorded with a measurement time of 10 seconds for each value. The Newtonian viscosity is then defined as the average value of the viscosity measured at the plateau at low shear rates.

[0098] Figure 3 shows the viscosity diagrams of MEX-CD2 / chitosan 1 / 0, 5 / 1 , 2 / 1 and 1 / 1 polymer solutions. The 1 / 0 formulation has a very low Newtonian viscosity (around 1 Pa.s) whereas all other formulations are more viscous (between 10 and 60 Pa.s). Those results seem to show that unmodified chitosan enhances interactions between the polymer chains.Rheology

[0099] To obtain information about gelation of the polymer solutions, approximately 2 mL of the formulation was placed into a round mold with an inner diameter of 25 mm and a height of 3 mm. The mold containing the solution was then immersed in 50 mL of PBS at 10 mmol.L1to start the gelation process of the solution. Storage modulus (G’) and loss modulus (G") of the formed hydrogel were recorded on an ARES rheometer from TA instruments using a 25 mm plate geometry. tan(5) is defined as the ratio between G” and G’. Oscillatory rheological properties were measured on the placed mold containing the hydrogel with a dynamic frequency sweep test from 0.05 rad s-1to 100 rad s-1at a constant strain of 1 % at 25 °C.

[0100] Figure 4 shows the evolution of the storage modulus and tan(6) for MEX-CD2 / chitosan 1 / 1 , 2 / 1 and 1 / 0 formulations (polymer concentration: 5% (w / w)). Gelation is characterized by a strong increase in G’ leading to a value of tan(5) below 1 . While 1 / 1 formulation does not have this increase in storage modulus, both 2 / 1 and 1 / 0 formulations seem to form hydrogels with a fast gelation for 1 / 0 hydrogel and a longer gelation for 2 / 1 . However, the 1 / 0 formulation shows a rebound of tan(6) after 1 h, suggesting hydrogel disintegration.Small-angle X-ray scattering (SAXS) and Wide-angle X-ray scattering (WAXS)

[0101] SAXS and WAXS measurements were performed at the ESRF (European Synchrotron Radiation Facility) on the BM02 beamline.

[0102] Polymer solutions were prepared by addition of MEX-CD2 and chitosan in different ratios (1 / 0, 2 / 1 , 0 / 1) with water and acetic acid (0% of acetic acid (w / w) for 1 / 0, 0.4% of acetic acid (w / w) for 2 / 1 and 0 / 1) in a reactor with a total concentration in polymer of 5% (w / w). Hydrogels were prepared by filling of stainless steel cells (0 = 4 mm, h = 3 mm) covered with kapton on one side with the different polymer solutions; the cells were then immerged in the gelling medium (either 10 mM phosphate buffer, 10 mM phosphate buffered saline or 0.9% sodium chloride solution) for 24h, then sealed with another piece of kapton and finally wrapped in parafilm to ensure the impermeability of the system.

[0103] For WAXS, the crystallite’s sizes were estimated using the Scherrer equation (The Defect Crystal, Bernhard Wunderlich, 1973, p.399):

[0104] Dhk0= ^~

[0105] Where Dhko is the size of the crystallites in the direction perpendicular to the reflecting (hkl) planes and Aqhko'is the full width at half maximum of the WAXS crystallinity peak.

[0106] Figure 5 displays the Small-Angle X-rays Scattering (SAXS) profile of hydrogel formed at 5% (w / w) in a phosphate buffer at pH = 7.4 for unmodified chitosan (MEX-CD2 / chitosan 0 / 1), MEX-CD2 (MEX-CD2 / chitosan 1 / 0) and a 2 / 1 MEX-CD2 / chitosan mixture to define the nanoscale structural properties of resulting materials. For 0 / 1 hydrogel, the scattered intensity pattern (l(q) ~ q4) is typically associated with a structure where two media are separated by a sharp interface resulting in a “particle like” behavior of the system. No inflection points within the MEX-CD2 hydrogel resulting curve can be highlighted, meaning that there is little to no inhomogeneity of small size. Regarding high q domains, the scattered intensity pattern (l(q) ~ q-1) is associated with a rod-shaped structure. At very low q however, the intensity tends to be proportional to q-4, which can reveal the presence of aggregates within the material. The pattern of the SAXS profile resulting from the 2 / 1 ratio hydrogel is intermediate to the profile of each of the two polymers with the scattering intensity pattern (l(q) ~ q2) being characteristic of a polymer gel phase.

[0107] Wide-Angle X-ray Scattering (WAXS) analyses have also been recorded for the same formulation at the same pH as SAXS but this time for hydrogels formed in Phosphate Buffered Saline (PBS) solution at 10 mmol L-1(Ch ions being not as detrimental for WAXS than SAXS) to highlight the possible sub-nanometer-sized structures associated with crystallinity of the formed network. The WAXS profile of unmodified chitosan hydrogel (0 / 1) formed in PBS shows a crystallinity peak at 1 .36 A1which has been identified as the 200 reflections for chitosan crystallites (Figure 6). This peak is also present to a lesser extent on the WAXS profile of the 2 / 1 ratio hydrogel and cannot be seen for MEX-CD2 alone (1 / 0). The ratio of the peaks area between both raw chitosan and 2 / 1 systems reveals a relative crystallinity of 27 % of chitosan hydrogel crystallinity for the 2 / 1 hydrogel which may indicate that the crystallinity is directly dependent on the proportion of unmodified chitosan present in the formulation and that no co-crystallization between MEX-CD2 and chitosan occurred. This finding can further be confirmed by the crystallite size which appears to be the same for both hydrogels formed from crude chitosan and a 2 / 1 mixture and was estimated to be around 230 A.EXAMPLE 3: preparation of the formulation MEX-CD2-I 5% (w / w)

[0108] In this example and in the following examples, “MEX-CD2-I” designates the polymer solution containing 2 / 3 MEX-CD2 and 1 / 3 chitosan (in mass). To obtain MEX-CD2-I 5% (w / w), a 5% (w / w) polymer solution comprising 2 / 3 MEX-CD2 and 1 / 3 chitosan (by mass) was thus prepared. Briefly, 3.015 g of MEX-CD2, 1 .485 g of chitosan, 85.14 mL of ultrapure water and 343 pL of glacial acetic acid were added to a reactor and left under mechanical stirring for 1 h until complete dissolution of the polymers. The solution was transferred into a fluid dispenser, then centrifuged at 4000 rpm for 10 minutes to obtain an air bubble-free solution. The mixture was then introduced into 10 mL glass vials (8 mL of solution per vial) and sterilized for 20 min at 121 °C in an autoclave.EXAMPLE 4: Study of the formulation MEX-CD2-I 5% (w / w) for the treatment of wounds polluted with metals on ratsDesign and protocol of the study

[0109] The goal ofthis study was to investigate the ability of MEX-CD2-I hydrogel as metal scavenger to improve the healing of wounds polluted with metals contained in projectiles after topical application, and to mitigate their harmful effects on the organism. The study was conducted on Wistar female rats 12 weeks old (young). Rats were observed for mortality and signs of gross toxicity daily for 7 consecutive days. During seven days of the acclimatization period, 1 animal was kept per cage. All animals were under observation and only animals without any clinical signs of illness were taken into the study.Dose levels, group division, and sampling

[0110] There were 6 groups in the study (n=4 in each group). The distribution of the groups is described as follows:• group 1 (G1) - "clean" wound: rats received no treatment during the study.• group 2 (G2) - "clean" wound dressed with MEX-CD2-I hydrogel: MEX-CD2-I hydrogel was applied on the wound 1 h after wound performing and thereafter daily for 6 days (7 dressings in total) in the volume approximately 0.1 ml / animal.• group 3 (G3) - "dirty" wound: metallic micro-powder from projectiles was applied on the wound in a dose 40 mg / animal, then rats received no treatment during the study.• group 4 (G4) - "dirty" wound dressed with MEX-CD2-I hydrogel: metallic micropowder from projectiles was applied on the wound in a dose 40 mg / animal, MEX-CD2- I hydrogel was applied in 1 h after metallic micro-powder application and thereafter daily for 6 days (7 dressings in total) in the volume approximately 0.1 ml / animal.• group 5 (G5) - "salty" wound: mixture of salts contained the respective metals in proportions similar to those in projectiles micro-powder was applied on the wound in a dose 100 mg / animal, then rats received no treatment during the study.• group 6 (G6) - "salty" wound dressed with MEX-CD2-I hydrogel: mixture of salts was applied on the wound in a dose 100 mg / animal, MEX-CD2-I hydrogel was applied in 1 h after salts application and thereafter daily for 6 days (7 dressings in total) in the volume approximately 0.1 ml / animal.

[0111] Necropsy with blood and skin samples collection and fixation were performed on the 8thday at the terminal sacrifice. Hematological and serum biochemical parameters (ALT, AST, ALP, LDH, GGT, TP, Creatinine, Urea) measurement were performed on the 8thday of the study as well.Wound perform

[0112] Rats were anesthetized with telazol (5 mg / kg, intraperitoneally) and xylazine hydrochloride (8 mg / kg intramuscular), and fur onto the withers was shaved. Skin layer was preserved for further fixation with sutures. In case of the need to investigate the healing ability of MEX-CD2-I hydrogel, wounds were performed by removal of all skin layers (epidermis, dermis and subcutaneous fat) on the withers (approximately 100 mm2).Wound contamination agent preparation

[0113] Metallic micro-powder from projectiles was obtained by mixing the micro-powder made from projectile shells and projectile cores consisted of Al2O3:Fe+Ba(NO3)2 1 :2. Salt mixture was performed from salts of the respective metals in the proportion corresponded to the content of these metals in projectile micro-powder, i.e. FeSO4*7H2O:Al2(SO4)3*18H2O:BaCl2 4:2:1.Drug formulation, route and volume of administration

[0114] Metallic micro-powder was applied on the wound surface at the dose 40 mg / animal once immediately after wound performing. Salt mixture was applied on the wound surface at the dose 100 mg / animal (which corresponded to the amount of metals contained in metallic micro-powder) once immediately after wound performing. MEX-CD2-I formulation was applied in a dose 0.1 ml / animal.Frequency and duration of application

[0115] Animals were treated with MEX-CD2-I hydrogel once in 1 h after wound performing / contamination and thereafter daily during 7 days. Applications were performed once a day at 9-10 AM.Lethality

[0116] The animals were monitored for mortality immediately after the first compounds administration and daily during the observation period.Wound measurement

[0117] Wounds were photographed every day during the study (from 9 AM to 11 AM) before the dressing application. Their area was measured from the photographs using Imaged software. The wound area change was calculated every day as a percentage change of initial (day 1) wound area for each individual animal.Blood collection and euthanasia

[0118] Terminal bleeding and euthanasia were performed on the 7thday of the study for all groups. Animals were anesthetized with CO2 inhalation. The blood was collected from femoral vein into dry microtainers without anticoagulant. For hematological study, 25 pl of fresh blood was transferred in tubes containing equal volume of K2EDTA solution (0.4 % in saline).

[0119] The tubes with collected blood were kept at room temperature for 15-60 min after blood drawing before centrifugation to form a fibrin clot, and then were centrifuged at 9000 rpm for 20 minutes at +4°C. The collected serum was subjected to biochemical analysis.

[0120] Euthanasia was performed after terminal bleeding using cervical dislocation. All procedures were in accordance with the OECD recommendations.Results

[0121] No mortality was observed during the study, no other signs of toxicity were observed during the study. Rats from all the groups gained weight during the study. No significant bodyweight difference between the groups were observed.Wound area

[0122] Pictures of the wounds are displayed on figure 7 and the dynamics of wound area reduction (wound healing) is depicted at figure 8. Wound absolute area was significantly higher on days 2-7 in G3 (metals) and G5 (salts) groups compared to G1 group (control), indicating that the wound pollution with either metals as a micro-powder or a salt slows down the wound healing. Wound absolute area in G4 (metals + MEX-CD2-I) group was significantly higher compared to G1 (control) only on days 6-7, and no difference in groups G2 (MEX-CD2-I) and G6 (salts + MEX-CD2-I) compared to G1 (control), which might evidence that MEX-CD2-I hydrogel application mitigates the harmful effect of metal contamination and speeds up the healing process. Wound area decrease compared to initial day ( / .e. relative wound area) was significantly lower for G2 (MEX-CD2-I) compared to G1 (control) at 2-7 days of the study, for G4 - at 2-6 days of the study, and for G5 - at 5-7 days of the study. This confirmed the most harmful effect of metallic micro-powder pollution on the process of wound healing, and the ability of MEX-CD2-I hydrogel to mitigate particularly this effect. Then, the effect of metals in the form of micro-powder was more harmful than the effect of metals as ions, as well as the protective effect of MEX-CD2-I hydrogel was more expressed against salts than against micro-powder. This might be explained by additional irritating effect of metallic particles in case of micro-powder.Hematology

[0123] Hematological parameters data are presented in figure 9. Inflammation manifestation occurred as a decrease of LYM and an increase of granulocytes percentages compared to G1 (control) group. Hemoglobin (tendency, p=0.065) and hematocrits (significantly) were decreased in G6 (salts + MEX-CD2-I) group compared to G1 (control), which might evidence some issue with red blood cell population. The observed changes might evidence the inflammatory process in the organism after wound pollution with salt mixture, which is mitigated by MEX-CD2-I hydrogel application.EXAMPLE 5: Study of the formulation MEX-CD2-I for the treatment of wounds on pigsDesign and protocol of the study

[0124] The study was carried out on two female pigs aged around 4 months (40 kg), with an acclimatization period of at least 7 days. The animals were housed in individual pens. The duration of the study was 30 days. For each animal, 8 deep skin wounds were created on either side of the spine: 4 wounds on one side, 4 wounds on the other, separated from each other on the same side by 3 to 4 cm, and each wound measuring 6 cm long and 4 cm wide. The wounds were filled either with Urgo Clean® dressings for the wounds on the left side of the spine or with about 5 mL of the MEX-CD2-I 5% (w / w) polymer solution (for each wound) for the wounds on the right side of the spine. They were then covered with a non-adhesive Opsite® polyurethane films and finally the pigs were wrapped with Tensoplast® adhesive strips to ensure impermeability of the wounds. During thedressing changes, photos, tracings for bacteriological analysis and biopsies (one biopsy on a different pig each week) of the wounds were taken (on day 1 , 7, 14, 21 , 30). The pictures of the wounds were processed using the following methodology. First, wound contours were traced and wound area A (in number of pixels) was measured using Imaged software. Then, for each image, a standard-length L was determined by tracing a segment perpendicular to the spine covering the entire width of the animal's back. In order to compare the images in between them, the ratios A / L were calculated. Bacterial density was assessed by counting the number of colonies present on the tracing made on the wounds and assigning a score from 0 to 5 (0 meaning no bacteria, 5 meaning extensive colonization).Results

[0125] Figure 10 shows the pictures of the wounds each week and for each group; figure 11 shows the evolution of wounds area relatively to their initial area %(AA). For both conditions, there is a decrease in wound area that is significantly faster for wounds treated with MEX-CD2 hydrogel, revealing a faster healing. What’s more, for all timepoints, the standard deviation in wound area variation is higher for the control wounds than for the treated ones: this suggests that wounds treated with MEX-CD2 hydrogel heal more consistently.

[0126] The bacterial density of the wounds is displayed on figure 12. Interestingly, there is an important variation in absolute bacterial density between the two pigs P1 and P2. For both pigs, all bacterial density score mean values (except for D14) are higher for control than for MEX-CD2-treated wounds, which may reflect an antibacterial ability of the hydrogel, even if those differences are nonsignificant. This at least confirms that there is no enhancement of infection that is caused by MEX- CD2-I hydrogel.

[0127] Figure 13 shows histological sections of biopsies taken on day 30 (last day of the study). Bacterial pyogranulomas can be seen in the control group (left), indicating bacterial infiltration of the wound bed, whereas no bacterial pyogranuloma was observed in the MEX-CD2-I group (right), which is another indication of the antibacterial activity of the hydrogel.EXAMPLE 6: Study of the formulation MEX-CD2-I 5% (w / w) for the treatment of burns on ratsDesign and protocol of the study

[0128] The goal of this study was to investigate the ability of MEX-CD2-I 5% (w / w) to improve the healing of chemically-induced skin burns (acidic- and alkali-induced). MEX-CD2-I 5% (w / w) solutions were prepared as described in example 3. Male Wistar rats anesthetized with telazol (5 mg / kg, intraperitoneally) and xylazine hydrochloride (8 mg / kg intramuscular), fur onto the withers was shaved. Gauze disc (2.5 cm diameter, approx. 490 mm2) were soaked with either 10 mol.L1HCI or 3 mol.L1NaOH solutions and applied onto the skin for 10 minutes. There were 4 groups of male Wistar rats:Group 1 (Acidic burn), n=12: rats subjected to acidic (HCI) burn induction and received no treatment;Group 2 (Acidic burn + MEX-CD2-I 5%(w / w)), n=12: rats subjected to acidic (HCI) burn induction, MEX-CD2-I 5% (w / w) dressing was applied on the burn in 1 h after induction and thereafter daily for 20 days;• Group 3 (Alkali burn), n=8: rats subjected to alkali (NaOH) burn induction and received no treatment;• Group 4 (Alkali burn + MEX-CD2-I 5% (w / w)), n=8: rats subjected to alkali burn induction, MEX-CD2-I 5% (w / w) dressing was applied on the burn in 1 h after induction and thereafter daily for 13 days.

[0129] Burns were photographed every day during the study (from 9 AM to 11 AM) before the dressing application. Their area was measured from the photographs using Imaged software. The burn area change was calculated for every day as a percentage change of initial burn area (considered as an area of HCI / NaOH-soaked disc application) for each individual animal. After 7, 14 and 21 days since the burn induction in case of acidic-induced burns, and after 7 and 14 days since the burn induction in case of alkali-induced burns, the animals were euthanized by inhalation of CO2 and cervical dislocation. The blood was collected from the femoral vein into the tube with EDTA anticoagulant. Blood collection was also performed on a group (control) of healthy rats to evaluate standard hematological values. Blood cells indices (red and white blood cell count, platelet count, blood hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) were determined using conventional methods. Briefly, the hemoglobin concentration was measured using colorimeter (KFK- 3, Ukraine) and Hemoglobin Assay Kit (Felicit-Diagnostics, Ukraine). The red blood cells, white blood cells and platelets were counted using hemocytometer and light microscope (Olympus BX-41 , Olympus Europe GmbH, Japan). The hematocrit was measured after centrifugation of blood into micro hematocrit capillary. MCH, MCHC and MCV were calculated according to conventional approach. The differential analysis of leukograms was conducted using blood smears, stained according to Pappenheim, counting at least 100 white blood cells, including basophilic, eosinophilic, and neutrophilic granulocytes, lymphocytes, monocytes using the light microscope Olympus BX-41 (Japan).Results

[0130] Figure 14 shows hematological parameters after 7 days for control group and acidic burn groups with and without MEX-CD2-I 5% (w / w). The white blood cell (leukocytes) count showed a significant increase in rats with acidic-induced cutaneous burns ("acid burn") 7 days after burn induction (Figure 14 A). However, topical application of MEX-CD2-I 5% (w / w) reduced this increase by 18% compared to the "acid burn" group (p=0.020, Figure 14 A). This tendency was also seen in eosinophilic granulocytes (Figure 14 B) where their mean value is higher for acidic burns but reverted to control levels with MEX-CD2-I 5% (w / w), even if these differences are non-significant. After 7 days of MEX-CD2-I 5% (w / w) application to acid-burned skin, a restorative effect on red blood cells was observed. This was evidenced by the normalization of blood hemoglobin concentration (p=0.026)(Figure 14 C), an increase in red blood cell count (p=0.015) (Figure 14 D), and restoration of hematocrit (p=0.045) (Figure 14 E) compared to the "acid burn" group.

[0131] Figure 15 shows the evolution of burn relative area during the study for alkaline burn groups. During the whole study, this relative area is significantly lower than for the rats that had not received MEX-CD2-I 5% (w / w), meaning that the burns heal faster with MEX-CD2-I 5% (w / w).

[0132] Figure 16 displays hematological parameters after 7 days for control group and alkaline burn groups with and without MEX-CD2-I 5% (w / w). As well as for acidic burns, the administration of MEX- CD2-I 5% (w / w) to the affected area following an alkaline burn for 7 days effectively halted the increase in leukocyte count in the blood, with counts comparable to those of the healthy control group (p=0.192), despite being 44% higher (Figure 16 A). Conversely, in the untreated "alkaline burn" group, leukocyte numbers surged by 75% compared to the control (Figure 16 A; p=0.028). The presence of a moderate-strength direct correlation between leukocyte count and the extent of the burn lesion (r=0.673, p=0.017) underscored their active involvement in the burn resolution process. MEX-CD2-I 5% (w / w) also maintained the relative number of monocytes at control levels, while in the "alkaline burn" group, their numbers surged by 2.6 times compared to the healthy control (Figure 16 B; p=0.037). In both groups with alkaline burns, no significant alterations in red blood cell count and blood hemoglobin concentration were noted compared to healthy controls at the 7-day post-injury (Figure 16 C and D). However, MEX-CD2-I 5% (w / w) demonstrated a preserved capacity for erythrocytes to accumulate hemoglobin, as indicated by a 7% increase in MCH compared to the untreated alkaline burn group (p=0.026; Figure 16 E), with MCH exhibiting a moderate-strength direct correlation with the burn injury area in this group (r=0.669, p=0.017).EXAMPLE 7: Evaluation of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of MEX-CD2-IDesign and protocol of the study

[0133] The MIC and MBC concentrations were determined using the method described in CLSI (2008) guideline and ISO 20776-1. Broth dilution methods are used to determine the minimum inhibitory concentration (hereafter MIC) of the test substance, which characterizes its microbiological activity. Minimum Inhibitory Concentration (MIC) is the lowest concentration (expressed as pg / mL or pg / pL) of an antimicrobial agent that inhibits the visible in-vitro growth of microorganisms.

[0134] Antibacterial activity of MEX-CD2-I was tested against reference strains of opportunistic pathogens: Staphylococcus aureus ATCC 25923 (gram-positive bacteria), Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 (gram-negative bacteria), Candida albicans ATCC 885-653 (yeast-like fungi). MEX-CD2-I 5% (w / w) was prepared as described in example 3. To determine the MIC, the twofold dilutions of MEX-CD2-I 5% (w / w) in Muller-Hinton broth were prepared to give a range of concentrations. Each concentration was twice as low as the previous one. The test requires a minimum final volume of 1 mL of each dilution. The concentration range used to determine the MIC of MEX-CD2-I was [1 .25;0.0075] pg / mL. The bacterial suspension was prepared according to the McFarland turbidity standard of 0.5 (~1 .5*108CFU / mL) and then dilutedto 5*105CFU / mL. Then 100 pL of the bacterial suspension was inoculated into each test tube. An additional sample containing bacteria but no test substances was used as a positive control. The tubes were incubated at 37°C for 18 to 24 h. MIC endpoints were defined as the lowest antimicrobial concentration that resulted in the absence of visible bacterial growth, as indicated by the lack of turbidity. At this dilution, the antimicrobial is bacteriostatic. It means that some bacteria may still be alive. The experiments were carried out three times to ensure accuracy and consistency. The Minimum Bactericidal Concentration (MBC) is subjectively defined as the lowest concentration that kills 99.9% of the final inoculum. For the determination of MBC, each test tube with no visible culture growth was inoculated onto an agar plate. After 24 h of incubation at 37°C, the dilution of the solution in the tube with no growth was noted.Results

[0135] Results are presented in table 1 below.Table 1 :

[0136] MIC values show that the most resistant strains were Gram-negative bacteria P.aeruginosa and E. coli, while the most sensitive strains were Gram-positive S. aureus and yeast-like fungi C. albicans. The negative control with no test substance showed regular growth. MBC results were consistent with the MIC data. Indeed, MEX-CD2-I showed an MBC of 0.03 pg / mL against S. aureus, which demonstrates a very high bacteriostatic activity compared to other chitosans. This activity was also very high against C. albicans with an MBC of 0.25 pg / mL. Slightly lower activities were obtained with E. coli and P. aeruginosa with higher MBCs, although these values still demonstrate an important bacteriostatic activity.EXAMPLE 8: Evaluation of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of various MEX-CD2-l / chitosan formulations at 5% w / w.

[0137] As in example 7, the MIC and MBC concentrations were determined using the method described in CLSI (2008) guideline and ISO 20776-1. Broth dilution methods are used to determine the minimum inhibitory concentration (hereafter MIC) of the test substance, which characterizes its microbiological activity. Minimum Inhibitory Concentration (MIC) is the lowest concentration (expressed as pg / mL or pg / pL) of an antimicrobial agent that inhibits the visible in-vitro growth of microorganisms.

[0138] Antibacterial activity of MEX-CD2-l / chitosan formulations were tested against reference strains of Staphylococcus aureus ATCC 25923 (gram-positive bacteria), To determine the MIC, the twofold dilutions of MEX-CD2-l / chitosan 5% w / w formulations in Muller-Hinton broth were preparedto give a range of concentrations. Each concentration was twice as low as the previous one, and the test required a minimum final volume of 1 mL of each dilution. The concentration range used to determine the MIC of MEX-CD2-l / chitosan formulations was [1 .25;0.0075] pg / mL. The bacterial suspension was prepared according to the McFarland turbidity standard of 0.5 (~1 .5x108CFU / mL) and then diluted to 5x105CFU / mL. Then 100 pL of the bacterial suspension was inoculated into each test tube. An additional sample containing bacteria but no test substances was used as a positive control. The tubes were incubated at 37°C for 18 to 24 h. MIC endpoints were defined as the lowest antimicrobial concentration that resulted in the absence of visible bacterial growth, as indicated by the lack of turbidity. At this dilution, the antimicrobial is bacteriostatic: it means that some bacteria may still be alive. The experiments were carried out three times to ensure accuracy and consistency. The Minimum Bactericidal Concentration (MBC) is subjectively defined as the lowest concentration that kills 99.9% of the final inoculum. For the determination of MBC, each test tube with no visible culture growth was inoculated onto an agar plate. Identical MIC and MBC values may be inherent to the protocol involving successive twofold dilutions of samples. After 24 h of incubation at 37°C, the dilution of the solution in the tube with no growth was noted.

[0139] Results are reported in table 2 below:Table 2:

[0140] Interestingly, the formulations that were composed of both polymers (MEX-CD2-l / chitosan) displayed the highest antibacterial activities ( / .e. the lowest MICs and MBCs) towards S. aureus compared to those composed of only one of the two polymers (Table 2). Without being bound by a theory, this enhanced activity may be likely due to the complementary mechanisms of each polymer. Chitosan, as a cationic polymer, directly interacts with bacterial cell surfaces via electrostatic interactions, binding to negatively charged sites on bacterial membranes and possibly blocking membrane proteins such as porins. This direct interaction can disrupt cell integrity and inhibit essential functions, leading to bacterial death. For MEX-CD2-I however, the polyampholytic character of the polymer chain should limit these interactions and thus this mechanism of action. Meanwhile; MEX-CD2-I has a strong affinity for metal ions like Fe(lll) (log K = 29.4 for DOTA), which are crucial for bacterial growth. By chelating these ions, MEX-CD2-I may thus indirectly inhibit bacterial proliferation. When used together, these two mechanisms direct membrane disruption by chitosan and metal ion sequestration by MEX-CD2-I seem to synergize to produce a significantly improved antibacterial effect.EXA PLE 9: In vivo study on burns induced by phosphorus pentoxide

[0141] In vivo protocol was approved by the Institutional Animal Care and Use Committee of Taras Shevchenko National University of Kyiv (Protocol #6 dated 27 / 08 / 2024). The study was conducted on adult male Wistar rats (10-12 weeks old) to evaluate the efficacy of the MEX-CD2-I hydrogel on burns induced by phosphorus pentoxide (P2O5). The experiment was divided into three groups of animals:• Healthy Control Group (n=8).• P2O5Burn Group: Rats with P2O5burns, without treatment (n=10).• P2O5Burn + hydrogel Treatment Group: Rats with P2O5burns treated daily with MEX-CD2-I / chitosan 2:1 hydrogel (n=10).On Day 0, rats were anesthetized, and the skin on their backs was shaved and moistened. Approximately 100 mg of P2O5powder was applied to a 2 cm-diameter area on the shaved skin, moisturized by water to become wet, inducing chemical burns. Two hours after the burn induction, rats in the treatment group (P2O5Burn + hydrogel) received a dressing of the MEX-CD2-l / chitosan hydrogel (about 100 pL per animal), which was subsequently applied daily until the end of the study. The bodyweight of all rats was measured daily starting from Day 1 until Day 14 (figure 17 A) The burn areas were measured on Day 1 , Day 7 and Day 14 (figure 17 B). The exact area of the burns on Day 0 was not recorded, as the full extent of the burn became apparent only on Day 1 . This initial difference in the burn area may reflect varying responses of treated and untreated skin in the first 24 hours post-burn. The study duration was 14 days, with necropsies performed at two time points: Day 7 (half of each group) and Day 14 (rest of the animals). At day 7, burn area was significantly lower for hydrogel treated animals in comparison with control group.Animals were sacrificed at these endpoints to assess burn healing progress and immune response indicators. Blood was collected for hematology analysis and serum separation, skin samples from the margin of burn site were harvested and fixed in 10% neutral buffered formalin. H&E-stained samples were analyzed as described earlier, additionally, epidermis thickness was measured using Imaged software. Lymph nodes (figure 17 C), thymus, and spleen were collected and weighed for comparison among the groups. For lymph nodes, no statistical difference is observed at day 7 for relative mass of the lymph nodes for the three groups. However at day 14, significantly higher relative mass is observed for the phosphorous induced burns group in comparison with health group and hydrogel treated group. It can be explained by immune reaction mitigated in presence of the hydrogel.

Claims

Claims

1. Aqueous composition, having a Newtonian viscosity between 0.5 Pa.s and 1000Pa.S, comprising at least one statistical polysaccharide B, having a weight-average molar mass of between 100 kDa and 1000 kDa, of formula I:Formula I in which:- each Rc independently represents a group including a chelating moiety,- each Z independently represents a linkage which may be a single bond or a hydrocarbonbased chain including between 1 and 12 carbon atoms, said chain possibly being linear or branched and possibly including one or more unsaturations and possibly including one or more heteroatoms, preferably chosen from nitrogen, oxygen, sulfur and atoms of the halogen family,- x is between 0 and 0.40- y is between 0.05 and 0.25,- the ratio y / x being greater than or equal to 0.2,- the sum x + y being greater than or equal to 0.1 , and optionally a chitosan A, said statistical polysaccharide B being in a mass concentration CB, said chitosan A being in a mass concentration CA, the sum of CB plus CA being of at least 10 g.L-1.

2. Aqueous composition according to claim 1 , wherein said statistical polysaccharide B and said chitosan A are in a mass ratio (mA / mB) between 2 / 1 and 1 / 10, preferably between 1 / 1 and 1 / 5.

3. Aqueous composition according to any one of the preceding claims, wherein said chitosan A has a weight-average molar mass of between 100 kg / mol and 1000 kg / mol, preferably between 200 kg / mol and 700 kg / mol.

4. Aqueous composition according to any one of the preceding claims, wherein said chitosan A has a degree of acetylation less than 40%, preferably less than 10%, for example between 0% and 10%.

5. Aqueous composition according to any one of the preceding claims, wherein the chelating moiety is chosen from the group consisting of DOTA, NOTA, NODAGA, DOTAGA,DOTAM, DO3AM, NOTAM, DOTP, NOTP, TETA, TETAM, DTPA, EDTA and DFO, preferably from the group consisting of DOTAGA, DFO, DOTAM and DTPA, more preferably DOTAGA.

6. Aqueous composition according to any one of the preceding claims, wherein the statistical polysaccharide B is of formula I , in which x is about 0.05, y is about 0.17, Z is a single and Rc is DOTAGA:

7. Aqueous composition according to any one of the preceding claims, wherein thein which: ci and C2 are different, and are groups including a chelating moiety,Zi and Z2, which may be identical or different, are linkages which may be a single bond or a hydrocarbon-based chain including between 1 and 12 carbon atoms, said chain possibly being linear or branched and possibly including one or more unsaturations and possibly including one or more heteroatoms, preferably chosen from nitrogen, oxygen, sulfur and atoms of the halogen family,- x is between 0 and 0.40- y is between 0.05 and 0.25,- the ratio y / x being greater than or equal to 0.2,- the sum x + y being greater than or equal to 0.1 , and z is between 0.5 and 1 .

8. Aqueous composition, as defined in any one of claims 1 to 7, molar for its use in the treatment of complex wounds, such chronic wounds, like wounds related to diabetes or eschars or arterial ulcers, burns, chemical burns, wounds related to radioactive contaminations and wounds containing one or more metals.

9. Aqueous composition according to claim 8, wherein the metals are exogenous.

10. Aqueous composition according to claim 8, wherein the metals are endogenous.

11. Aqueous composition according to any one of claims 8 to 10, wherein the metal is iron.

12. Aqueous composition according to any one of claims 8 to 10, wherein the metal is radioactive.

13. Wound dressing comprising the aqueous composition according to any one of claims 1 to 7.

14. Wound dressing according to claim 13, wherein it is biodegradable and / or having anti bacteriological properties.

15. Wound dressing according to any one of claims 13 to 14, for its use in non- aseptically condition, for example on battle fields, on site of an accident in an industrial environment.

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