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Title:
BORONATE ESTER CROSSLINKED NANOGELS BASED ON MODIFIED POLYSACCHARIDES
Document Type and Number:
WIPO Patent Application WO/2019/057920
Kind Code:
A1
Abstract:
The invention relates to a boronate ester crosslinked nanogel based on a mixture of: A) a modified anionic polysaccharide grafted with a group X, B) a modified neutral polysaccharide grafted with a group Y, wherein: X is an aryl boronic acid derivative or a cis-diol derivative wherein said cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose, Y is an aryl boronic acid derivative or a cis-diol derivative wherein said cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose, if one of X or Y is an aryl boronic acid derivative, the other is a cis-diol derivative.

Inventors:
AUZELY RACHEL (FR)
RAVAINE VALÉRIE (FR)
POIROT ROBIN (FR)
Application Number:
EP2018/075652
Publication Date:
March 28, 2019
Filing Date:
September 21, 2018
Export Citation:
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Assignee:
CENTRE NAT RECH SCIENT (FR)
UNIV BORDEAUX (FR)
INST POLYTECHNIQUE BORDEAUX (FR)
International Classes:
A61K9/06; A61K9/51; A61K38/28; A61K47/36; A61P3/10; C08B37/02; C08B37/08; C08L5/02; C08L5/08
Domestic Patent References:
WO2014072330A12014-05-15
WO2014072330A12014-05-15
WO2012066133A12012-05-24
Foreign References:
US20110257033A12011-10-20
Other References:
JUAN LI ET AL: "pH and glucose dually responsive injectable hydrogel prepared by in situ crosslinking of phenylboronic modified chitosan and oxidized dextran", JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY, vol. 53, no. 10, 15 May 2015 (2015-05-15), pages 1235 - 1244, XP055455932, ISSN: 0887-624X, DOI: 10.1002/pola.27556
TARUS, D.; HACHET, E.; MESSAGER, L.; CATARGI, B.; RAVAINE, V.; AUZELY-VELTY, R.: "Readily Prepared Dynamic Hydrogels by Combining Phenyl Boronic Acid- and Maltose-Modified Anionic Polysaccharides at Neutral Ph.", MACROMOL. RAPID COMMUN., vol. 35, 2014, pages 2089 - 2095, XP055440663, DOI: doi:10.1002/marc.201400477
MERGY, J.; FOURNIER, A.; HACHET, E.; AUZELY-VELTY, R.: "Modification of Polysaccharides Via Thiol-Ene Chemistry: A Versatile Route to Functional Biomaterials", J. POLYM. SCI., PART A POLYM. CHEM., vol. 50, 2012, pages 4019 - 4028
NOBBMANN, U.; MORFESIS, A.: "Light scattering and nanoparticles", MATERIALS TODAY, vol. 12, 2009, pages 52 - 54, XP026139268, DOI: doi:10.1016/S1369-7021(09)70164-6
Attorney, Agent or Firm:
REGIMBEAU (FR)
Download PDF:
Claims:
CLAIMS

A boronate ester crosslinked nanogel based on a mixture of:

A) a modified anionic polysaccharide grafted with a group X,

B) a modified neutral polysaccharide grafted with a group Y,

wherein:

X is an aryl boronic acid derivative or a cis-diol derivative wherein said cis- diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

Y is an aryl boronic acid derivative or a cis-diol derivative wherein said cis- diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

if one of X or Y is an aryl boronic acid derivative, the other is a cis-diol derivative.

The nanogel according to claim 1 wherein the modified neutral polysaccharide is grafted with group Y through a spacer.

The nanogel according to claim 2 wherein the spacer is a linear chain, substituted or not, comprising from 3 to 10 atoms of carbon, nitrogen, oxygen or sulfur.

The nanogel according to any of the preceding claims wherein the anionic polysaccharide is hyaluronic acid, heparosan, chondroitin, chondroitin sulfate, heparin, heparan sulfate, alginate or pectin.

The nanogel according to any of the preceding claims wherein the neutral polysaccharide is dextran, dextrin or pullulan.

The nanogel according to any of the preceding claims wherein the aryl boronic acid derivative is an aryl boronic acid or a benzoxaborole.

7. The nanogel according to any of the preceding claims wherein the DS of the modified polysaccharide grafted with a cis-diol derivative is comprised between 0,10 and 0,50.

8. The nanogel according to any of the preceding claims wherein the DS of the modified polysaccharide grafted with an aryl boronic acid derivative is comprised between 0,08 and 0,50.

9. The nanogel according to any of the preceding claims wherein the diameter of the nanoparticles is comprised between 80 and 200 nm.

10. A process for manufacturing a nanogel according to any of the preceding claims comprising a step of mixing:

- an aqueous solution containing a modified anionic polysaccharide A) according to any of the preceding claims at a concentration below 2 g/L, and - an aqueous solution containing a modified neutral polysaccharide B) according to any of the preceding claims at a concentration below 2 g/L, at a pH comprised between 7 and 10 to obtain said nanogel.

1 1 . The nanogel according to any of claims 1 to 9, comprising a drug.

12. The nanogel according to claim 1 1 , wherein the drug is insulin. 13. The nanogel according to claim 1 1 , wherein the drug is chosen in the group consisting of 6-mercaptopurin, fludarabin, cladribin, pentostatin, cytarabin, 5- fluorouracil, gemcitabin, methotrexate, raltitrexed, irinotecan, topotecan, etoposide, daunorubicin, doxorubicin, epirubicin, idarubicin, pirarubicin, mitoxantrone, chlormethin, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, carmustin, fotemustin, streptozocin, carboplatin, cisplatin, oxaliplatin, procarbazin, dacarbazin, bleomycin, vinblastin, vincristin, vindesin, vinorelbin, paclitaxel, docetaxel, L-asparaginase, flutamide, nilutamide, bicalutamide, cyproteron acetate, triptorelin, leuprorelin, goserelin, buserelin, formestane, aminoglutethimide, anastrazole, letrozole, tamoxifene, octreotide, lanreotide, (Z)-3-[2,4-dimethyl-5-(2-oxo-1 ,2- dihydro-indol-3-ylidenemethyl)-1 H-pyrrol-3-yl]-propionic acid, 4-((9-chloro-7- (2,6-difluorophenyl)-5H-pyrimidol(5,4-d)(2)benzazepin-2-yl)amino)benzoic acid, 5,6-dimethylxanthenone-4-acetic acid, 3-(4-(1 ,2-diphenylbut-1 - enyl)phenyl)acrylic acid and mixture thereof.

14. A process for manufacturing a nanogel according to any of claims 1 1 to 13 comprising a step of mixing: - an aqueous solution containing a modified anionic polysaccharide A) according to any of the claims 1 to 9 at a concentration below 2 g/L, and

- an aqueous solution containing a modified neutral polysaccharide B) according to any of the claims 1 to 9 at a concentration below 2 g/L, in the presence of a drug at a pH comprised between 7 and 10 to obtain the drug encapsulated in said nanogel.

15. The nanogel according to claim 1 1 or 12 for use in a method of treatment of diabetes.

16. The nanogel according to claim 1 1 or 13 for use in a method of treatment of cancer.

17. A drug delivery system comprising a nanogel according to claim 1 1 to 13 and 15 to 16.

18. An aqueous nanogels suspension comprising nanogels according to claim 1 to 9, 1 1 to 13 and 15 to 16.

Description:
BORONATE ESTER CROSSLINKED NANOGELS BASED ON MODIFIED

POLYSACCHARIDES

FIELD OF THE INVENTION

The present invention relates to novel boronate ester crosslinked nanogels based on modified polysaccharides which can be used as stimuli-responsive drug carriers.

BACKGROUND OF THE INVENTION

Phenyl boronic acid derivatives (PBA) and cis-diol derivatives are known to form cyclic boronate ester in a reversible manner. The boronate ester bond is sensitive to variation of pH and to addition of cis-diol containing molecules, such as sugars and especially glucose. These properties are very attractive for the formation of stimuli- responsive polymers that could be used as stimuli-responsive drug carriers for the treatment of diseases such as diabetes or cancer.

Indeed, diabetes mellitus is a disorder of glucose regulation, characterized by an accumulation of glucose concentration in blood. It is currently treated by injections of insulin in order to adapt the level of glucose in the blood. This method of treatment implies several drawbacks including the fact that insulin is administered by an invasive method and frequent monitoring of glucose concentration in blood are necessary, potentially followed by administration of insulin. A polymer which could encapsulate insulin and deliver it by glucose stimuli could lead to a major improvement in diabetes treatment. Therefore, development of glucose-responsive polymers has attracted much attention lately. Notably nanoparticles designed to use the strategy of reversible boronate ester bond formation have been investigated. They can be formed by synthesis of micelles, vesicles, or nanogels.

However, the nanosystems previously designed can be tedious to prepare or unstable under physiological conditions. Additionally, the polymers used are generally synthetic, non-biodegradable and non-biocompatible which prevents in vivo applications.

The inventors have already developed hydrogels comprising PBA-grafted hyaluronic acid as described in WO2014/072330. Hyaluronic acid is part of the family of polysaccharides which are biocompatible hydrophilic polymers that can be degraded by enzymes in the organism.

However, obtaining nanoparticles from these hydrogels proved to be problematic as it required complex formulation steps.

Unexpectedly, it has been observed that mixing a modified anionic polysaccharide with a modified neutral polysaccharide, grafted with PBA derivatives and cis-diol derivatives, leads directly to nanogels. The nanogels obtained form a dynamic network based on boronate ester bonds and are pH and carbohydrate-sensitive.

These nanogels according to the invention show several significant advantages: They are made of polysaccharides which are hydrophilic, biocompatible, easily accessible materials, notably by bacterial fermentation, and convenient for in vivo applications.

The fabrication process of nanogels according to the invention is simple, reproductive, versatile and happens in mild aqueous conditions without using any organic solvent.

- As the nanogel formation occurs at a physiological pH at which they are stable, encapsulating fragile biologically active molecules, notably insulin, is possible in mild aqueous conditions and with high efficiency.

The properties of the nanogels can be modulated as a function of the polysaccharide backbone, notably their degradability and biological properties or their encapsulation property.

The properties of the nanogels can be also modulated as a function of the nature of the PBA derivatives and cis-diol derivatives, notably their sensitivity to pH and to the addition of cis-diol containing molecules

SUMMARY OF THE INVENTION

The invention relates to a boronate ester crosslinked nanogel based on a mixture of:

A) a modified anionic polysaccharide grafted with a group X,

B) a modified neutral polysaccharide grafted with a group Y,

wherein:

X is an aryl boronic acid derivative or a cis-diol derivative wherein said cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

Y is an aryl boronic acid derivative or a cis-diol derivative wherein said cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

if one of X or Y is an aryl boronic acid derivative, the other is a cis-diol derivative.

Advantageously, the modified neutral polysaccharide is grafted with group Y through a spacer. In particular, the spacer is a linear chain, substituted or not, comprising from 3 to 10 atoms of carbon, nitrogen, oxygen or sulfur.

Advantageously, the anionic polysaccharide is hyaluronic acid, heparosan, chondroitin, chondroitin sulfate, heparin, heparan sulfate, alginate or pectin.

Advantageoulsy, the neutral polysaccharide is dextran, dextrin or pullulan. In particular, the aryl boronic acid derivative is an aryl boronic acid or a benzoxaborole. In particular, the DS of the modified polysaccharide grafted with a cis-diol derivative is comprised between 0,10 and 0,50.

In particular, the DS of the modified polysaccharide grafted with an aryl boronic acid derivative is comprised between 0,08 and 0,50.

Advantageously, the diameter of the nanoparticles is comprised between 80 and 200 nm.

The invention also relates to a process for manufacturing a nanogel according to the invention comprising a step of mixing:

- an aqueous solution containing a modified anionic polysaccharide A) according to the invention at a concentration below 2 g/L, and

- an aqueous solution containing a modified neutral polysaccharide B) according to the invention at a concentration below 2 g/L,

at a pH comprised between 7 and 10 to obtain said nanogel.

The invention also relates to a nanogel according to the invention, comprising a drug. In particular, the drug is insulin.

Alternatively, the drug is chosen in the group consisting of 6-mercaptopurin, fludarabin, cladribin, pentostatin, cytarabin, 5-fluorouracil, gemcitabin, methotrexate, raltitrexed, irinotecan, topotecan, etoposide, daunorubicin, doxorubicin, epirubicin, idarubicin, pirarubicin, mitoxantrone, chlormethin, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, carmustin, fotemustin, streptozocin, carboplatin, cisplatin, oxaliplatin, procarbazin, dacarbazin, bleomycin, vinblastin, vincristin, vindesin, vinorelbin, paclitaxel, docetaxel, L-asparaginase, flutamide, nilutamide, bicalutamide, cyproteron acetate, triptorelin, leuprorelin, goserelin, buserelin, formestane, aminoglutethimide, anastrazole, letrozole, tamoxifene, octreotide, lanreotide, (Z)-3-[2,4-dimethyl-5-(2-oxo-1 ,2- dihydro-indol-3-ylidenemethyl)-1 H-pyrrol-3-yl]-propionic acid, 4-((9-chloro-7-(2,6- difluorophenyl)-5H-pyrimidol(5,4-d)(2)benzazepin-2-yl)amino) benzoic acid, 5,6- dimethylxanthenone-4-acetic acid, 3-(4-(1 ,2-diphenylbut-1 -enyl)phenyl)acrylic acid and mixture thereof.

The invention also relates to a process for manufacturing a nanogel according to the invention comprising a step of mixing:

- an aqueous solution containing a modified anionic polysaccharide A) according to the invention at a concentration below 2 g/L, and

- an aqueous solution containing a modified neutral polysaccharide B) according to the invention at a concentration below 2 g/L, in the presence of a drug at a pH comprised between 7 and 10 to obtain the drug encapsulated in said nanogel.

The invention relates as well to the nanogel according to the invention, comprising a drug for use in a method of treatment of diabetes.

The invention also relates to the nanogel according to the invention, comprising a drug for use in a method of treatment of cancer. The invention also relates to a drug delivery system comprising a nanogel according to the invention, comprising a drug.

The invention relates as well to an aqueous nanogels suspension comprising nanogels according to the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : illustration of a boronate ester crosslinked nanogel based on a neutral and an anionic polysaccharide according to the invention.

Figure 2: Size distribution measured by DLS at 25 °C of a HA100-PBA (DS=0.17)/HA100-p-Maltose (DS=0.22) mixture, 20 min (A) and 1 h (B) after addition of HA100-p-Maltose to HA100-PBA; of HA-PBA (C); and of Dex-p-Maltose (D) (C p = 1 g/L in PBS, pH 7.4).

Figure 3: Influence of the concentration of fructose in solution on the diameter (Dh) and the light scattering intensity (LSI) of nanogels based on HA100-PBA/Dex200-p- maltose (A) and HA20-PBA/Dex200-p-maltose (B).

Figure 4: Influence of the pH of the solution on the diameter (Dh) and the light scattering intensity (LSI) of nanogel based on HA100-PBA/Dex200-p-maltose.

Figure 5: Synthesis, DLS analysis and SEM images in the dried state of doubly cross-linked nanogels with disulfide and boronate ester links.

DEFINITIONS

In the present invention, the term nanogel refers to a crosslinked network of hydrophilic polymers with a diameter less or equal to 200 nm. Nanogels can be referred to as "hydrogel nanoparticles" which are included in the group of materials named "soft nanoparticles".

The nanogel according to the invention has a sphere-like form. In the present invention, the nanogel diameter (Dh) refers to its average hydrodynamic diameter. In the present invention the term polysaccharide refers to polymeric carbohydrate molecules composed of long chains of mono- or disaccharide repeating units bound together by glycosidic linkages.

In the present invention, the term "a nanogel based on" refers to a nanogel comprising the mixture and/or product of the reaction between different basic components, preferably only the product of the reaction between the different basic components. The basic components comprise a modified anionic polysaccharide grafted with a group X and a modified neutral polysaccharide grafted with a group Y.

Polysaccharides can be classified according to their charge. Notably, they can be neutral or anionic. Among the neutral polysaccharides dextran, dextrin or pullulan can be cited. An anionic polysaccharide is a polysaccharide which comprises carboxylic functions that are not functionalized and can be in the form of salts by reaction with an inorganic base. Among the anionic polysaccharides hyaluronic acid, heparosan, chondroitin, chondroitin sulfate, heparin, heparan sulfate, alginate or pectin can be cited. The anionic polysaccharide can react with inorganic bases such as sodium hydroxide, lithium hydroxide, or potassium hydroxide.

In the present invention, "hyaluronic acid" refers to sodium hyaluronate, hyaluronan, hyaluronic acid or hyaluronate.

In the present invention the term modified polysaccharide refers to a polysaccharide that is grafted with at least one PBA derivative or at least one cis-diol derivative.

In the present invention, "polysaccharide grafted with a group X" refers to a polysaccharide grafted with at least one group X, and "polysaccharide grafted with a group Y" refers to a polysaccharide grafted with at least one group Y.

Preferably, PBA derivative and cis-diol derivative are grafted on at least a hydroxyl or a carboxylate group of the polysaccharide.

In the present invention, the term aryl boronic acid derivatives refers to aryl boronic acids, 2-(hydroxymethyl)phenylboronic acid cyclic monoester and their derivatives (otherwise known as "benzoxaborole" derivatives), that are at least monosubstituted with a functional group suitable for grafting so that they can be grafted on the polysaccharide.

In the present invention, the term aryl refers to an aromatic hydrocarbon group comprising 6 to 10 carbon atoms and comprising at least one fused cycle, such as phenyl or naphtyl. Advantageously, it is a phenyl.

In the present invention, the term cis-diol refers to a molecule containing a cis-diol moiety, such as sugars.

In the present invention, the term cis-diol derivatives refers to a cis-diol which is at least monosubstituted with a functional group suitable for grafting so that it can be grafted on the polysaccharide. In the present invention, the term "compound X" refers to the reactant reacting with an anionic polysaccharide to obtain the modified anionic polysaccharide grafted with group X. Compound X is functionalized with a suitable functional group for grafting. Group X corresponds thus to compound X devoid of a hydrogen atom on its functional group suitable for grafting after reaction with the anionic polysaccharide.

In the present invention, the term "compound Y" refers to the reactant reacting with a neutral polysaccharide to obtain the modified neutral polysaccharide grafted with group Y. Compound Y is functionalized with a suitable functional group for grafting. Group Y corresponds thus to compound Y devoid of a hydrogen atom on its functional group suitable for grafting after reaction with the neutral polysaccharide.

In the present invention, the term spacer refers to a chain of carbon and/or other heteroatoms that links the polysaccharide to the group X or Y.

In the present invention, the DS of a polysaccharide refers to the degree of substitution of the polysaccharide, meaning the average number of substituting groups (for example group X, group Y, or alkene group) per repeating unit.

In the present invention, an aqueous solution is a solution comprising water as the major solvent, i.e. at least 50 wt. % of solvent is water, preferably 75 wt. %, more preferably 90 wt. %, even more preferably the solvent is water.

In the present invention, molecular weight Mw, or molar mass Mw refers to the mass average molar mass of the polymer.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Boronate ester crosslinked nanogel

The first object of the present invention is a boronate ester crosslinked based on a mixture of:

A) a modified anionic polysaccharide grafted with a group X,

B) a modified neutral polysaccharide grafted with a group Y,

wherein

X is an aryl boronic acid derivative or a cis-diol derivative wherein the cis-diol is in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

Y is an aryl boronic acid derivative or a cis-diol derivative wherein the cis-diol is in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

if one of X or Y is an aryl boronic acid derivative, the other is a cis-diol derivative.

By mixing an aqueous solution containing a modified anionic polysaccharide with an aqueous solution containing a modified neutral polysaccharide, each at a concentration in aqueous solution below 2 g/L, a nanogel is obtained by boronate ester bond formation so that it is sensitive to pH and cis-diol containing molecules.

On the contrary, by mixing two modified anionic polysaccharides, no well-defined hydrogel nanoparticles are obtained.

The nanogel obtained according to the invention is thus formed by reversibly crosslinking the modified anionic polysaccharides and the modified neutral polysaccharides via their groups comprising an aryl boronic acid derivative and their groups comprising a cis-diol derivative.

The nanogels according to the invention are dynamic networks that are carbohydrate and pH-sensitive. It means that in the presence of a free cis-diol, in particular free fructose, a competitive displacement occurs. The boronate ester link is as well pH-dependent. In acidic pH, the boronate ester bond is not stable and the nanogel disassembles.

A first embodiment of the present invention is a boronate ester crosslinked nanogel based on a mixture of:

A) a modified anionic polysaccharide grafted with an aryl boronic acid derivative,

B) a modified neutral polysaccharide grafted with a cis-diol derivative wherein the cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose. A second embodiment of the present invention is a boronate ester crosslinked nanogel based on a mixture of:

A) a modified anionic polysaccharide grafted with a cis-diol derivative wherein the cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

B) a modified neutral polysaccharide grafted with an aryl boronic acid derivative.

In particular, the molar ratio [group X]/[group Y] in the nanogel according to the invention is comprised between 0.3 and 2.5, preferably between 0.6 and 2.0, more preferably between 0.8 and 1.5, even more preferably it is 1.

Preferably, the molar ratio [PBA]/[maltose] in the nanogel according to the invention is comprised between 0.3 and 2.5, preferably between 0.6 and 2.0, more preferably between 0.8 and 1.5, even more preferably it is 1. In the invention, including the first and second embodiments above, an anionic polysaccharide A) and a neutral polysaccharide B) are mixed. Each polysaccharide has been modified by grafting a group X or a group Y.

Modified anionic polysaccharide A)

A modified anionic polysaccharide grafted with a group X as described above is a first essential constituent of the nanogel according to the invention.

The anionic polysaccharide intended to be modified by grafting with group X is in particular hyaluronic acid, heparosan, chondroitin, chondroitin sulfate, heparin, heparan sulfate, alginate or pectin. Preferably, the anionic polysaccharide intended to be modified by grafting with group X is hyaluronic acid (noted HA herein).

The anionic polysaccharide intended to be modified by grafting with group X has preferably a molecular weight Mw comprised between 10 and 200 kg/mol, preferably between 15 and 150 kg/mol.

Preferably, the modified anionic polysaccharide A) grafted with a group X is a modified hyaluronic acid grafted with a group X.

Advantageously, the DS of the modified anionic polysaccharide grafted with an aryl boronic acid derivative is comprised between 0.08 and 0.50.

Advantageously, the DS of the modified anionic polysaccharide grafted with a cis- diol derivative is comprised between 0.1 and 0.5, preferably between 0.15 and 0.30.

Modified neutral polysaccharide B)

A modified neutral polysaccharide grafted with a group Y as described above is the second essential constituent of the nanogel according to the invention.

The neutral polysaccharide intended to be modified by grafting with group Y is dextran, dextrin or pullulan. Preferably, the neutral polysaccharide intended to be modified by grafting with group Y is dextran (noted Dex herein).

The neutral polysaccharide intended to be modified by grafting with group Y has preferably a molecular weight Mw comprised between 25 and 500 kg/mol, preferably between 40 and 300 kg/mol, more preferably between 100 and 250 kg/mol.

Preferably, the modified anionic polysaccharide B) grafted with a group Y is a modified dextran grafted with a group Y.

Advantageously, the DS of the modified neutral polysaccharide grafted with an aryl boronic acid derivative is comprised between 0.08 and 0.50.

Advantageously, the DS of the modified neutral polysaccharide grafted with a cis- diol derivative is comprised between 0.1 and 0.5, preferably between 0.15 and 0.30. Group X and group Y

In the nanogel according to the invention, both anionic and neutral polysaccharides are modified. The anionic polysaccharide is grafted with a group X and the neutral polysaccharide is grafted with a group Y.

Group X and group Y can be:

an aryl boronic acid derivative, or

a cis-diol derivative wherein said cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose.

In the nanogel according to the invention, group X and group Y are different. If one is an aryl boronic acid derivative, the other is a cis-diol derivative.

Thereafter, we describe aryl boronic acid derivatives and cis-diol derivatives useful as compound X or Y. Preferably, the aryl boronic acid derivative (noted PBA herein) is an aryl boronic acid or a benzoxaborole at least monosubstituted with a functional group suitable for grafting.

More preferably, the aryl boronic acid derivative is an aryl boronic acid at least monosubstituted with a functional group suitable for grafting.

More preferably, the aryl boronic derivative is an aryl boronic acid substituted with at least one functional group suitable for grafting and optionally zero, one or two substituents which are an halogen atom, a nitro group or a C1 -C4 alkyl group.

In particular, the aryl boronic acid derivative is an aryl boronic acid at least monosubstituted with a functional group suitable for grafting wherein the aryl boronic acid is selected from the group consisting of 3-aminophenylboronic acid, 4- aminophenylboronic acid, 4-amino-3-fluorophenylboronic acid, 4-amino-3- nitrophenylboronic acid, 3-amino-4-fluorophenylboronic acid, 3-amino-4- methylphenylboronic acid, 3-amino-4-chlorophenylboronic acid, 3-mercaptophenylboronic acid, 4-mercaptophenylboronic acid. The functional group suitable for grafting is then an amine or a thiol.

In particular, the cis-diol derivative is a cis-diol at least monosubstituted with a functional group suitable for grafting wherein said cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose. Preferably, said cis-diol is selected in the group consisting of fructose and maltose, more preferably it is maltose.

Group X and group Y can be grafted directly on the polysaccharides or they can be grafted on the polysaccharides through a spacer p. The nanogel according to the invention is based on a mixture of a modified anionic polysaccharide grafted with a group X as described above and a modified neutral polysaccharide grafted with a group Y as described above, wherein group X and group Y are either directly grafted on their corresponding polysaccharide or are grafted on their corresponding polysaccharide through a spacer p.

Preferably, the spacer p linking the anionic or neutral polysaccharide with group X or Y is a linear chain, substituted or not, comprising from 3 to 10 atoms of carbon, nitrogen, oxygen or sulfur.

Preferably, the anionic or neutral polysaccharide is linked via at least one hydroxyl. In particular, the spacer p has the following formula:

-C(0)-L-S-L'- wherein L and L' are linear chains, substituted or not, comprising from 3 to 10 atoms of carbon, nitrogen, oxygen or sulfur, S is a sulfur atom, and p is linked to the polysaccharide (PS) and group X or Y according to the formula:

PS-C(0)-L-S-L'-X or PS-C(0)-L-S-L'-Y.

In particular re:

with R = H or p-X, and R' = OZ or O-X, wherein Z is an alkali metal ion, n is an integer greater than 1 .

Preferably, n is an integer so that the molecular weight Mw of the modified anionic polysaccharide is comprised between 10 and 200 kg/mol, preferably between 15 and 150 kg/mol.

In particular, the modified neutral polymer has the following structure:

with n is an integer greater than 1 , R

Preferably, n is an integer so that the molecular weight Mw of the modified neutral polysaccharide is comprised between 25 and 500 kg/mol, preferably between 40 and 300 kg/mol, more preferably between 100 and 250 kg/mol.

In one embodiment, the neutral polysaccharide which the nanogel according to the invention is based on is modified with group Y through a spacer p as described above.

In this embodiment, the anionic polysaccharide which the nanogel according to the invention is advantageously based on is modified with group X through a spacer p as described above.

Alternatively, the group X is advantageously directly linked to at least a carboxylate group from the anionic polysaccharide. In particular, group X is linked by peptide-like coupling.

Another object of the invention is an aqueous suspension comprising the above described nanogels.

The nanogels suspensions of the present invention have preferably a pH ranging from 7 to 10, preferably from 7 to 9, more preferably from 7 to 8, and even more preferably a physiological pH. Process for manufacturing a boronate crosslinked nanogel and a nanogel suspension

To prepare the modified polysaccharides according to the present invention, a strategy has been developed previously by the inventors wherein polysaccharides are functionalized with PBA or cis-diol through "thiol-ene reaction". The procedure has been described in WO2012/066133. Under mild conditions, a polysaccharide is functionalized with alkene groups by reaction with carboxylic acid anhydride bearing an alkene function, such as pentenoic acid anhydride. A polysaccharide intermediate modified with alkene groups is obtained. The alkene moiety allows further functionalization of the polysaccharide by "thiol-ene coupling". The degree of substitution of the polysaccharide with alkene can be adjusted by varying the ratio of [carboxylic acid anhydride]/[polysaccharide]. In particular, the DS of the polysaccharide intermediate modified with alkene groups is comprised between 0.1 and 0.5, preferentially between 0.15 and 0.35.

Then a compound X or Y functionalized with a thiol as functional group for grafting can be coupled with the polysaccharide intermediate modified with alkene groups.

Or the polysaccharide intermediate modified with alkene groups can be further functionalized by thiol-ene coupling with a thiol bearing a carboxylic acid function to obtain a polysaccharide intermediate modified with carboxylic acid groups.

The latter can then react with compound X or Y bearing an amine as functional group for grafting by peptide-like coupling.

In particular, the DS of the polysaccharide intermediate modified with carboxylic acid groups is comprised between 0,10 and 0,4, preferentially between 0,15 and 0,35.

With R- \ = X or Y, R 2 is a linear chain substituted or not, comprising from 3 to 10 atoms of carbon, nitrogen. Another object of the present invention is a process for manufacturing a boronate ester cross-linked nanogel according to the invention comprising a step of mixing:

- an aqueous solution containing a modified anionic polysaccharide A) grafted with a group X at a concentration below 2 g/L,

- an aqueous solution containing a modified neutral polysaccharide B) grafted with a group Y at a concentration below 2 g/L,

wherein

X is an aryl boronic acid derivative or a cis-diol derivative wherein the cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

Y is an aryl boronic acid derivative or a cis-diol derivative wherein the cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose, if one of X or Y is an aryl boronic acid derivative, the other is a cis-diol derivative, at a pH comprised between 7 and 10, preferentially between 7 and 8 to obtain said nanogel.

Thus by mixing an aqueous solution containing a modified anionic polysaccharide A) grafted with a group X at a concentration below 2 g/L, and an aqueous solution containing a modified neutral polysaccharide B) grafted with a group Y at a concentration below 2 g/L, at a pH comprised between 7 and 10, a boronate ester cross-linked nanogel is obtained. The aqueous suspension according to the invention obtained after mixing an aqueous solution containing a modified anionic polysaccharide A) grafted with a group X at a concentration below 2 g/L, and an aqueous solution containing a modified neutral polysaccharide B) grafted with a group Y at a concentration below 2 g/L has a pH ranging from 7 to 10, preferably from 7 to 9, more preferably from 7 to 8, and even more preferably a physiological pH.

Preferably, the concentration of the aqueous solution containing polysaccharide A) is comprised between 0.5 g/L and 2 g/L, even more preferably of 1 g/L. Preferably, the concentration of the aqueous solution containing polysaccharide B) is comprised between 0.5 g/L and 2 g/L, even more preferably of 1 g/L.

Preferably, the process for manufacturing a boronate ester cross-linked nanogel according to the invention comprises the following steps:

(i) preparing a modified anionic polysaccharide A) grafted with a group X as described above,

(ii) preparing a modified neutral polysaccharide B) grafted with a group Y as described above,

(iii) mixing an aqueous solution containing a modified anionic polysaccharide A) at a concentration below 2 g/L and an aqueous solution containing a modified neutral polysaccharide B) at a concentration below 2 g/L, at a pH comprised between 7 and 10, preferably between 7 and 8 to obtain said nanogel.

In one embodiment of the invention, in step (i) the modified anionic polysaccharide is prepared by peptide-like coupling between the anionic polysaccharide and a compound X bearing an amine as functional group for grafting. In another embodiment of the invention, in step (i) the modified anionic polysaccharide is prepared by first grafting an alkene group to at least one hydroxyl of the anionic polysaccharide to obtain an anionic polysaccharide intermediate modified with alkene groups. Preferably, the anionic polysaccharide intermediate modified with alkene groups is obtained by reaction of the anionic polysaccharide with carboxylic acid anhydride bearing an alkene function, such as pentenoic acid anhydride.

Preferentially the anionic polysaccharide intermediate modified with alkene groups is then coupled with compound X functionalized with a thiol as functional group for grafting by thiol-ene reaction.

Preferably, in step (ii) the modified neutral polysaccharide is prepared by first grafting an alkene group to at least one hydroxyl of the neutral polysaccharide to obtain a neutral polysaccharide intermediate modified with alkene groups. More preferably, the neutral polysaccharide intermediate modified with alkene groups is obtained by reaction of the neutral polysaccharide with carboxylic acid anhydride bearing an alkene function, such as pentenoic acid anhydride.

In one embodiment of the invention, in step (ii) the neutral polysaccharide is modified using a thiol-ene reaction between the neutral polysaccharide intermediate modified with alkene groups and compound Y functionalized with a thiol as functional group for grafting.

In another embodiment of the invention, in step (ii) the modified neutral polysaccharide is prepared by further functionalizing the neutral polysaccharide intermediate modified with alkene groups with a thiol bearing a carboxylic acid by thiol-ene reaction, and then doing a peptide-like coupling reaction with compound Y bearing an amine as functional group for grafting.

In particular, the process for manufacturing a boronate ester cross-linked nanogel according to the invention takes place in a physiological buffer saline, preferably without any organic solvent.

Preferably, the process according to the invention takes place at room temperature, i.e. between 15 °C and 35 °C.

In particular, this process can be regarded as a green formulation process.

Notably, the process according to the invention takes place with a limited energy input, in comparison with methods involving an emulsification step for instance. Boronate crosslinked nanogel comprising a drug

Before or during the formation of the nanogels according to the invention, a drug can be added in order to obtain drug-loaded nanogels according to the invention. This way, a stimuli-responsive drug carrier nanogel able to release the drug in response to pH variation or addition of cis-diol containing molecules can be obtained.

Another object of the invention is therefore a nanogel according to the invention, comprising a drug.

The drug is encapsulated in the nanogel during its formation process.

It can be released by variation of pH or addition of a cis-diol containing molecule. Preferably, the drug is insulin or an anticancer drug.

Preferably, the drug is chosen in the group consisting of 6-mercaptopurin, fludarabin, cladribin, pentostatin, cytarabin, 5-fluorouracil, gemcitabin, methotrexate, raltitrexed, irinotecan, topotecan, etoposide, daunorubicin, doxorubicin, epirubicin, idarubicin, pirarubicin, mitoxantrone, chlormethin, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, carmustin, fotemustin, streptozocin, carboplatin, cisplatin, oxaliplatin, procarbazin, dacarbazin, bleomycin, vinblastin, vincristin, vindesin, vinorelbin, paclitaxel, docetaxel, L-asparaginase, flutamide, nilutamide, bicalutamide, cyproteron acetate, triptorelin, leuprorelin, goserelin, buserelin, formestane, aminoglutethimide, anastrazole, letrozole, tamoxifene, octreotide, lanreotide, (Z)-3-[2,4-dimethyl-5-(2-oxo-1 ,2- dihydro-indol-3-ylidenemethyl)-1 H-pyrrol-3-yl]-propionic acid, 4-((9-chloro-7-(2,6- difluorophenyl)-5H-pyrimidol(5,4-d)(2)benzazepin-2-yl)amino) benzoic acid, 5,6- dimethylxanthenone-4-acetic acid, 3-(4-(1 ,2-diphenylbut-1 -enyl)phenyl)acrylic acid and mixture thereof.

Another object of the invention is a process for manufacturing a boronate ester cross-linked nanogel comprising a drug according to the invention, comprising a step of mixing:

- an aqueous solution containing a modified anionic polysaccharide A) grafted with a group X at a concentration below 2 g/L,

- an aqueous solution containing a modified neutral polysaccharide B) grafted with a group Y at a concentration below 2 g/L,

wherein

X is an aryl boronic acid derivative or a cis-diol derivative wherein the cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose, Y is an aryl boronic acid derivative or a cis-diol derivative wherein the cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

if one of X or Y is an aryl boronic acid derivative, the other is a cis-diol derivative, in the presence of a drug at a pH comprised between 7 and 10, preferably between 7 and 8, to obtain the drug encapsulated in said nanogel.

Preferably, the process for manufacturing a boronate ester cross-linked nanogel comprising a drug according to the invention, comprises the following steps:

(i) preparing a modified anionic polysaccharide A) grafted with a group X as described above,

(ii) preparing a modified neutral polysaccharide B) grafted with a group Y as described above,

(iii) mixing an aqueous solution containing polysaccharide A) at a concentration below 2 g/L and an aqueous solution containing polysaccharide B) at a concentration below 2 g/L at a pH comprised between 7 and 10, preferably between 7 and 8 in the presence of a drug to obtain the drug encapsulated in said nanogel.

Preferably, the concentration of the aqueous solution containing a modified anionic polysaccharide A) is comprised between 0.5 g/L and 2 g/L, even more preferably of 1 g/L.

Preferably, the concentration of the aqueous solution containing a modified neutral polysaccharide B) is comprised between 0.5 g/L and 2 g/L, even more preferably of 1 g/L.

The manufacturing process is simple, reproductive, versatile and happens in mild aqueous conditions without using any organic solvent. It can occur at a physiological pH so that fragile biologically active molecules, notably insulin, can be entrapped in the nanogel according to the invention in mild aqueous conditions and high efficiency.

In one embodiment, the drug is introduced in the aqueous solution containing polysaccharide A) or B) before the step of mixing said aqueous solutions containing polysaccharides A) and B).

In another embodiment, the drug is incorporated during the step of mixing the aqueous solutions containing polysaccharides A) and B) which leads to nanogel formation by self-assembly.

Preferably, the concentration of drug in the resulting aqueous solution is comprised between 0.2 g/L and 1.5 g/L, preferably between 0.2 g/L and 0.8 g/L. Doubly crosslinked nanogel

The nanogels according to the invention can also be doubly cross-linked.

The nanogel is obtained by formation of a boronate ester bond and a disulfide bond.

Another object of the invention is a doubly crosslinked nanogel based on a mixture of:

A) a modified anionic polysaccharide grafted with a group X,

B) a modified neutral polysaccharide grafted with a group Y,

C) a water soluble molecule possessing several thiol groups,

wherein

X is an aryl boronic acid derivative or a cis-diol derivative wherein the cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

Y is an aryl boronic acid derivative or a cis-diol derivative wherein the cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

if one of X or Y is an aryl boronic acid derivative, the other is a cis-diol derivative, at least one of the polysaccharide A) or B) is further grafted with a group comprising a pyridyldisulfide group. Preferably, the water-soluble molecule possessing several thiol groups is a poly(ethylene glycol)-bis(thiol).

Another object of the present invention is a process for manufacturing a doubly cross-linked nanogel according to the invention comprising the following steps:

(i) mixing:

- an aqueous solution containing a modified anionic polysaccharide A) grafted with a group X at a concentration below 2 g/L,

- an aqueous solution containing a modified neutral polysaccharide B) grafted with a group Y at a concentration below 2 g/L,

wherein

X is an aryl boronic acid derivative or a cis-diol derivative wherein the cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

Y is an aryl boronic acid derivative or a cis-diol derivative wherein the cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

if one of X or Y is an aryl boronic acid derivative, the other is a cis-diol derivative, at least one of the polysaccharide A) or B) is further grafted with a group comprising a pyridyldisulfide,

at a pH comprised between 7 and 10, preferentially between 7 and 8 to obtain said nanogel,

(ii) adding an aqueous solution of poly(ethylene glycol)-bis(thiol).

Preferably, the concentration of the aqueous solution containing a modified anionic polysaccharide A) is comprised between 0.5 g/L and 2 g/L, even more preferably of 1 g/L. Preferably, the concentration of the aqueous solution containing a modified neutral polysaccharide B) is comprised between 0.5 g/L and 2 g/L, even more preferably of 1 g/L.

Double crosslinking allows increasing the nanogel long term stability. Before or during the formation of the doubly crosslinked nanogels according to the invention, a drug can be incorporated in order to obtain encapsulation of the drug in the nanogel according to the invention. This way, a stimuli-responsive drug carrier nanogel able to release the drug by pH or carbohydrate stimuli can be obtained with increased long term stability.

Another object of the invention is therefore a doubly crosslinked nanogel according to the invention, comprising a drug.

The drug is encapsulated in the nanogel during its formation process.

It can be released by variation of pH or addition of a cis-diol containing molecule. Preferably, the drug is insulin or an anticancer drug.

Preferably, the drug is chosen in the group consisting of 6-mercaptopurin, fludarabin, cladribin, pentostatin, cytarabin, 5-fluorouracil, gemcitabin, methotrexate, raltitrexed, irinotecan, topotecan, etoposide, daunorubicin, doxorubicin, epirubicin, idarubicin, pirarubicin, mitoxantrone, chlormethin, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, carmustin, fotemustin, streptozocin, carboplatin, cisplatin, oxaliplatin, procarbazin, dacarbazin, bleomycin, vinblastin, vincristin, vindesin, vinorelbin, paclitaxel, docetaxel, L-asparaginase, flutamide, nilutamide, bicalutamide, cyproteron acetate, triptorelin, leuprorelin, goserelin, buserelin, formestane, aminoglutethimide, anastrazole, letrozole, tamoxifene, octreotide, lanreotide, (Z)-3-[2,4-dimethyl-5-(2-oxo-1 ,2- dihydro-indol-3-ylidenemethyl)-1 H-pyrrol-3-yl]-propionic acid, 4-((9-chloro-7-(2,6- difluorophenyl)-5H-pyrimidol(5,4-d)(2)benzazepin-2-yl)amino) benzoic acid, 5,6- dimethylxanthenone-4-acetic acid, 3-(4-(1 ,2-diphenylbut-1 -enyl)phenyl)acrylic acid and mixture thereof. Another object of the invention is a process for manufacturing a doubly cross-linked nanogel comprising a drug according to the invention, comprises the following steps:

(i) mixing:

- an aqueous solution containing a modified anionic polysaccharide A) grafted with a group X at a concentration below 2 g/L,

- an aqueous solution containing a modified neutral polysaccharide B) grafted with a group Y at a concentration below 2 g/L,

wherein

X is an aryl boronic acid derivative or a cis-diol derivative wherein the cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

Y is an aryl boronic acid derivative or a cis-diol derivative wherein the cis-diol is selected in the group consisting of fructose, sialic acid, maltose, cellobiose and lactose,

if one of X or Y is an aryl boronic acid derivative, the other is a cis-diol derivative,

at least one of the polysaccharide A) or B) is further grafted with a group comprising a pyridyldisulfide,

at a pH comprised between 7 and 10, preferentially between 7 and 8, in the presence of a drug to obtain said nanogel,

(ii) adding an aqueous solution of poly(ethylene glycol)-bis(thiol).

In one embodiment, the drug is introduced in the aqueous solution containing polysaccharides A) or B) before the step of mixing said aqueous solutions containing polysaccharides A) and B).

In another embodiment, the drug is introduced during the step of mixing the aqueous solutions containing polysaccharides A) and B).

Preferably, the concentration of the aqueous solution containing polysaccharide A) is comprised between 0.5 g/L and 2 g/L, even more preferably of 1 g/L.

Preferably, the concentration of the aqueous solution containing polysaccharide B) is comprised between 0.5 g/L and 2 g/L, even more preferably of 1 g/L. Uses

Boronate crosslinked nanogels and doubly cross-linked nanogels comprising insulin as described above can be used in a method of treatment of diabetes.

The present invention also relates to a method for treating diabetes comprising administering to a patient in need thereof an effective amount of boronate crosslinked nanogels and/or doubly cross-linked nanogels comprising insulin.

Boronate crosslinked nanogels and doubly cross-linked nanogels comprising at least one anticancer drug such as described above can be used in a method of treatment of cancer. The present invention also relates to a method for treating cancer comprising administering to a patient in need thereof an effective amount of boronate crosslinked nanogels and/or doubly cross-linked nanogels comprising at least one anticancer drug. EXAMPLES

Synthesis of the polysaccharide building blocks:

The strategy for the functionalization of the polysaccharide (PS) with cis-diol moieties relied on a thiol-ene coupling reaction performed between a cis-diol-thiol derivative and an alkene-modified PS in aqueous solution. 1"2 The alkene-modified PS, namely dextran- alkene or HA-alkene, was prepared by reaction of the initial PS with pentenoic anhydride according to a procedure previously developed in CERMAV. 3"4 By varying the [cis-diol- thiol derivative]/[alkene] ratio, we obtained PS-p-cis-diol derivatives with a DS ranging from 0.10 to 0.25 (Table 1 ) .

The thiol-ene coupling strategy was also applied to functionalize dextran with carboxylate groups via a spacer. Dextran modified with carboxylate groups was thus prepared by the radical addition of mercaptopropionic acid (MPA) on Dex-alkene to obtain Dex-COOH. The latter reacted with 3-aminophenylboronic acid (PBA) or fructose derivative under peptide-like coupling conditions to give Dex-p-PBA (Table 2, entry 1 ) or Dex-p-fructose (Table 2, entry 2).

The HA-PBA derivatives were also prepared by a peptide-like coupling reaction between initial HA and 3-aminophenylboronic acid or 4-amino-3-fluorophenylboronic acid (Table 3). By varying the [PBA]/[PS repeating unit] ratio, we obtained PS-PBA derivatives with DS ranging from 0.10 to 0.22.

Table 2

Table 3

Finally, the carboxylate functions on dextran-COOH and those of HA could also be used to functionalize the PS with pyridyldisulfide (PDS) groups (Table 4). The PDS functionality, which is reactive but specific to thiols, allows for the covalent cross-linking of the nanogels via disulfide bond formation under mild conditions. Table 4

Formation of boronate-crosslinked nanogels:

The boronate-crosslinked nanogels were formed by mixing aqueous solutions at physiological pH and room temperature of HA-PBA and Dex-p-cis-diol or HA-p-cis-diol and Dex-p-PBA. These mixtures lead to the formation of nanogels with a diameter in the range of 1 15-162 nm (Table 5). This synthesis pathway can be regarded as a green formulation process because it takes place in water, at room temperature, without any organic solvent and with a limited energy input, in comparison with methods involving an emulsification step for instance. Table 5. Size and polydispersity index of nanogels prepared by mixing solutions of PS-

a concentration in PBS Cp = 1 g/L, pH = 7,4, [PBA]/[maltose] ratio = 1 ; b Hydrodynamic diameter (D h ) in intensity determined by dynamic light scattering (DLS), average value in intensity; c determined by DLS

The formation of nanogels was monitored by dynamic light scattering (DLS) 5 which provides several important parameters: the average hydrodynamic diameter (D h in nm), the polydispersity index of nanogels (Pdl) and the light scattering intensity (LSI in kilocounts per second (kCPS)). The Pdl provides information about the size distribution of nanogels while the LSI gives information about the amount of nanogels. Indeed, the LSI is proportional to the particle number concentration multiplied by the particle size.

Cis-diol derivative

A decrease of one third of the LSI is observed for the HA-PBA Dex-p-Fructose mixture (entry 4) compared to HA-PBA/Dex-p-maltose mixture (entry 2). At the same time, the average nanogel diameter is lower for the HA-PBA Dex-p-Fructose mixture. As the LSI is proportional to the particle number concentration multiplied by the particle size, it can be speculated that the nanogel number concentration is similar when Dextran is functionalized with fructose moieties.

Mw of PS

A 3-fold decrease of the LSI is observed for nanogels prepared from Dextran with a low molar mass (40 kg/mol, entries 5 and 6). As the LSI is proportional to the particle number concentration multiplied by the particle size, it can be speculated that the nanogel number concentration is lower when Dextran with a low molar mass is used. This is particularly apparent for nanogels prepared from Dextran and HA with low molar masses (entry 6) which have an average diameter similar to those prepared from the partners with high molar masses (entry 2). This result may be explained by a decrease of the efficiency of Dex-p-maltose to hold together tightly HA-PBA chains by boronate ester crosslinks. Indeed, the number of crosslinks generated by a given Dex-p-maltose chain is decreased because of the lower Mw of Dextran and consequently, of the lower amount of maltose per Dextran chain. The decrease of HA Mw can have a similar effect. The time required to observe nanogel formation is also longer with dextran and HA with low Mw (1 h for a nanogel according to entry 6 instead of 20 min for a nanogel according to entry 2).

Phenylboronic acid derivative

The DLS data for nanogels according to entries 2 and 7 indicate that similar results in terms of size, polydispersity and number concentration of nanogels are obtained when Dex-p-maltose is combined by HA-PBA or HA-FPBA.

Nature of PS and groups X or Y

Nanogels are formed whatever the polysaccharide nature grafted with PBA or cis-diol derivatives (see entries 2 and 8). The average diameters of nanogels prepared from HA- PBA/Dex-p-maltose and from Dex-p-PBA/HA-p-maltose are similar. A slight decrease of the LSI is observed for the Dex-p-PBA/HA-p-maltose mixture. This indicates that the nanogel number concentration is lower when PBA is grafted on Dex. Since glucopyranose can form cyclic boronates between its 4,6-hydroxy groups, PBA can bind the glucose moieties of dextran which are linked through 1 ,3-glucosidic linkages. This may impair PBA accessibility for ester bond formation with HA-p-maltose, and thereby nanogel formation. Nevertheless, the DS of Dex-p-PBA is not enough high to induce self-assembly of the PS into nanogels as demonstrated by the DLS measurements of Dex-p-PBA alone in aqueous solution in comparison to initial Dextran, and to the Dextran intermediates used for the synthesis of Dex-p-PBA, namely pentenoate-modified Dextran and Dextran modified with mercaptopropionic acid.

Formation of boronate cross-linked hydrogel

Several experiments were done by mixing aqueous solutions at physiological pH and room temperature of HA-PBA and HA-p-Maltose alone in PBS. The DLS measurements showed very heterogeneous size distributions in all cases, indicating that no nanogel was formed from the two negatively charged HA-based partners (see Figure 2). Influence of the [PBA]/[Maltose] ratio on the formation of nanogels

The influence of the [PBA]/[Maltose] ratio was evaluated while keeping the concentration of HA and Dex constant (Table 6).

From the DLS data (Table 6), the best conditions to obtain nanogels at high concentration, with a relatively low PDI and a diameter significantly lower than 200 nm, are obtained for a [PBA]/[Maltose] ratio of 1 . In the cases where [PBA]/[Maltose] > 1 , a significant decrease of the nanogel number concentration is observed. By contrast, for [PBA]/[Maltose] < 1 , the nanogel number concentration is relatively high but the average nanogel diameter is much higher than that obtained for a [PBA]/[Maltose]ratio of 1 . Table 6. Analysis by DLS of the effect of the [PBA]/[Maltose] ratio (with [HA] and [Dex] nearly constant) on the formation of nanogels:

a concentration in PBS Cp = 1 g/L, pH = 7,4 ; b Obtained 1 h after addition of partner 2 to partner 1 ; c average value in intensity. Table 7. Nanogel formulations with different [PBA]/[Maltose] ratios but with [HA] and [Dex] nearly constant.

Cis-diol responsiveness of borona te-crosslinked nanogels:

The introduction of PBA groups was found to endow the nanogels with sensitivity to cis- diol-containing molecules based on formation of boronic esters between PBA and the cis- diol derivatives. Figure 3 shows a progressive decrease of the Light Scattering Intensity (LSI) of nanogels prepared from HA100-PBA/Dex200-p-Maltose and HA20-PBA/Dex200- p-Maltose, with an increase of the concentration of fructose in solution. The DLS measurements indicate a decrease of about 60% of the LSI for an addition of 8 g/L of fructose while the nanogels size remains nearly constant.

Acidic pH responsiveness of boronate-crosslinked nanogels:

Boronate esters are usually stable at physiological pH, and can rapidly hydrolyze at acidic pH. 8"10 The hydrolysis of boronate esters in the HA100-PBA/Dex200-p-Maltose nanogels was investigated at pH varying from 5.8 to 9.5 at room temperature. Progressive decrease of the LSI was observed under mild acidic conditions (pH < 6) from DLS measurements (Figure 4). This result thus demonstrates the sensitivity of the nanogels prepared from HA-PBA/Dex200-p-Maltose. Covalent crosslinking of boronate-crosslinked nanogels:

Doubly crosslinked nanogels combining disulfide and boronate ester crosslinks can be obtained in order to increase the nanogel long-term stability. To this end, PS-BPA derivatives containing pyridyl disulfide (PDS) moieties were synthesized to be crosslinked with a poly(ethylene glycol)-bis(thiol) (PEG-(SH) 2 ) crosslinker via a thiol-disulfide exchange reaction. Doubly crosslinked nanogels were formed by simple mixing the PS- PBA-PDS, Dex-maltose in PBS buffer at room temperature and then adding PEG-(SH) 2 using a [SH]/[PDS] ratio of 1 . The resulting doubly crosslinked nanogels remained stable in PBS for at least one week upon storage at 4 °C. Their formation was confirmed by SEM observation after drying at 40 °C of a droplet of the suspension of nanogels in pure water onto the SEM support film (Figure 5).

Encapsulation properties:

Insulin could be successfully entrapped in the nanogels during their formation process by simply mixing an aqueous solution of HA-PBA and an aqueous solution of Dex-p-maltose containing insulin at physiological pH and room temperature. The diameter of the insulin- loaded nanogels was similar to the empty ones, indicating that insulin did not affect the assembly process. The loading capacity (LC) and entrapment efficiency (EE) calculated from equations (1 ) and (2) indicated efficient encapsulation of the protein in the nanogels compared to other insulin nanocarriers reported in the literature 11 (Table 8). The LC increased with increasing the initial concentration of insulin, while the EE slightly decreased or was not affected.

Table 8. Entrapment capacity (LC) and entrapment efficiency (EE) of boronate

Entry HA-PBA Dex-p- [maltose]/[PBA] Insulin EE LC

maltose (g/L) (%) (%)

1 HA100- Dex200-p- 1/1 0.25 47.4±5.9 1 1.9±1.5 PBA maltose

2 HA100- Dex200-p- 1/1 0.75 48.0±3.9 36.0±2.9 PBA maltose

3 HA20-PBA Dex200-p- 1/1 0.25 54.8±8.7 13.7±2.2 maltose

4 HA20-PBA Dex200-p- 1/1 0.75 47.6±2.4 35.7±1.8 maltose T n / amount of insulin in nanogel inn

LL% = — —— X 1(J(J (1 ) amount of insulin-loaded nanogel amount of insulin in nanogel in\ tit!" /o — ' 7 ~ r. X lUU [ . ) total amount of feeding insulin

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