PHARMACEUTICAL COMPOSITIONS, PREPARATION AND USES THEREOF

Abstract

The present disclosure generally relates to the field of medicine. The present invention more specifically relates to a pharmaceutical composition comprising the combination of (i) at least one biocompatible nanoparticle comprising, or consisting in, at least one natural compound which is an inhibitor of a human CYP enzyme, the longest dimension of said nanoparticle being of at least 4 nm and less than 100 nm, and (ii) at least one compound of interest, typically at least one pharmaceutical compound, to be administered to a subject in need of such at least one compound of interest, wherein the combination of the at least one biocompatible nanoparticle and of the at least one compound of interest potentiates the at least one compound of interest's bioavailability. The at least one biocompatible nanoparticle is to be administered to the subject separately from the at least one compound of interest (preferably before), typically with an interval of between at least about 5 minutes (preferably more than about 5 minutes) and about 72 hours.

Claims

1-16. (canceled)

17. A therapeutic or prophylactic method comprising a step of administering at least one pharmaceutical compound to a subject in need thereof and a distinct step of administering at least one biocompatible nanoparticle to said subject, wherein the at least one biocompatible nanoparticle comprises at least one natural compound which is an inhibitor of a human CYP enzyme, the longest dimension of said at least one biocompatible nanoparticle is at least 4 nm and less than 100 nm, the at least one biocompatible nanoparticle is not used as a therapeutic or prophylactic compound, and said at least one biocompatible nanoparticle is administered separately to the subject between 5 minutes and about 72 hours before the at least one pharmaceutical compound.

8. The method according to claim 17, wherein the at least one biocompatible nanoparticle and the at least one pharmaceutical compound are both administered to the subject through an intravenous (IV), subcutaneous, oral or enteral route.

19. The method according to claim 17, wherein the method further comprises a step of administering a second biocompatible nanoparticle comprising at least one natural compound which is an inhibitor of a human CYP enzyme, the longest dimension of the second biocompatible nanoparticle being of at least 4 nm and less than 100 nm.

20. The method according to claim 19, wherein the at least one (first) and/or the second biocompatible nanoparticle comprises at least one natural compound which is an inhibitor of a human CYP enzyme selected from furanocoumarin, flavonoid, arachidonic acid and/or vitamin A.

21. The method according to claim 20, wherein the at least one (first) and/or the second biocompatible nanoparticle comprises at least one natural compound and said natural compound is encapsulated in, trapped in, absorbed in, adsorbed on, linked on, conjugated to, attached to or bound to the at least one and/or to the second nanoparticle.

22. The method according to claim 19, wherein the first and second biocompatible nanoparticles are administered separately in an additional distinct step, or simultaneously, in a subject in need of the at least one pharmaceutical compound, and before said at least one pharmaceutical compound.

23. The method according to claim 19, wherein only one of the first and second biocompatible nanoparticles is administered to the subject through the same route as the pharmaceutical compound which is an oral or enteral route, the remaining biocompatible nanoparticle being administered through a different route which is selected from a IV, subcutaneous, oral or enteral route.

24. The method according to claim 20, wherein the at least one furanocoumarin of the first and/or second biocompatible nanoparticle(s) is selected from bergamottin, 6′,7′-dihydroxybergamottin (DHB) and 6′,7′-epoxy-bergamottin.

25. The method according to claim 20, wherein the at least one furanocoumarin of the first and/or second biocompatible nanoparticle(s) is a furanocoumarin monomer, dimer or oligomer.

26. The method according to claim 25, wherein the at least one furanocoumarin dimer is a Paradisin.

27. The method according to claim 19, wherein each of the at least one (first) and/or second biocompatible nanoparticles is further covered with a biocompatible coating.

28. The method according to claim 19, wherein the at least one (first) and/or second biocompatible nanoparticle(s) comprise(s) an agent enhancing nanoparticle(s)' recognition by enterocytes and/or by hepatocytes.

29. The method according to claim 28, wherein when the biocompatible nanoparticle is to be administered through an IV or subcutaneous route, the agent enhancing the nanoparticle(s)' recognition by hepatocytes comprises a saccharide and when the biocompatible nanoparticle is to be administered through oral or enteral route, the agent enhancing the nanoparticle(s)' recognition by enterocytes and/or by hepatocytes comprises a saccharide, a lectin or a vitamin.

30. The method according to claim 17, wherein the administration of the biocompatible nanoparticle(s) and of the at least one pharmaceutical compound allows a reduction of at least 20% of the administered at least one pharmaceutical compound therapeutic dose when compared to the standard therapeutic dose of said at least one pharmaceutical compound while maintaining the same bioavailability.

31. The method according to claim 17, wherein the at least one nanoparticle is cleared from the subject to whom it has been administered within a period of one hour and six weeks after its administration to a subject in need of the at least one pharmaceutical compound.

32. The method according to claim 17, wherein the at least one pharmaceutical compound is a substrate of a CYP 450 enzyme selected from CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0062] FIG. 1: Furanocoumarin

[0063] FIG. 2: Schematic synthesis of hyaluronan coated nanoparticles consisting in furanocoumarin (such as bergamottin, DHB, 6′,7′-epoxybergamottin, paradisin, etc.) by self-assembly in water (cf. example 2).

[0064] FIG. 3: Schematic synthesis of hyaluronan coated nanoparticles consisting in furanocoumarin (such as bergamottin, DHB, 6′,7′-epoxybergamottin, paradisin, etc.) by self-assembly in water (cf. example 3).

[0065] FIG. 4: Schematic synthesis of hyaluronan coated nanoparticles consisting in furanocoumarin (such as bergamottin, DHB, 6′,7′-epoxybergamottin, paradisin, etc.) by solvent removal (cf. example 4).

[0066] FIG. 5: Schematic representation of nanoparticles consisting in, or comprising furanocoumarin:

[0067] a. nanoparticle of furanocoumarin

[0068] b. furanocoumarin nanoparticle entrapped in an plain nanoparticle (organic or inorganic; possibly conjugated)

[0069] c. furanocoumarins dispersed in a plain nanoparticle (organic or inorganic)

[0070] d. furanocoumarins entrapped in the aqueous cavity of hollow nanoparticle

[0071] e. furanocoumarins entrapped in the layer of hollow nanoparticle

[0072] f. furanocoumarins adsorbed or conjugated on the surface of a plain nanoparticle (organic or inorganic) or hollow nanoparticle (the conjugation could be performed directly between surface and furanocoumarin or via a linker of different size).

[0073] FIG. 6: 3D chromatogram outputs of HPLC-UV injection of “control” sample (docetaxel), “metabolized” sample (HepaRG cells treated with docetaxel) and “inhibited” sample (HepaRG cells treated sequentially with DHB micelles (example 6) and docetaxel). [0074] In the “control” sample chromatogram, the black arrow corresponds to the docetaxel peak; [0075] In the “inhibited” sample chromatogram, the black arrow corresponds to the docetaxel peak; [0076] In the “metabolized” sample chromatogram, the dotted arrow (retention time 12.25 minutes) corresponds to a metabolite of docetaxel and the black arrow corresponds to docetaxel peak.

[0077] FIG. 7: Schedule of administration of the biocompatible nanoparticles (DHB micelles) and docetaxel in HT-29 tumor model xenografted on MRI nude mice.

[0078] FIG. 8: Kaplan-Meier curves for group 1 (control group: NaCl), group 2 (control group: DHB micelles 0.93 g/L, 5 mg/kg), group 3 (treatment group: docetaxel 2g/L, 10 mg/kg) and group 4 (treatment group: pharmaceutical composition comprising (i) DHB micelles 0.93 g/L, 5 mg/kg injected 24hrs before (ii) Docetaxel 2g/L, 10 mg/kg): [0079] Group 1: Aqueous saline (NaCl 0.9%) on Dl, D2, D3, D6, D9, D10; [0080] Group 2: DHB micelles on Dl, D3, D9; [0081] Group 3: Docetaxel (2g/L) 10 mg/kg on D2, D6, D10; [0082] Group 4: (i) DHB micelles (0.93g/L) 5mg/kg on Dl, D3, D9 and (ii) Docetaxel (2g/L) 10 mg/kg on D2, D6, D10.

[0083] Arrows: injections (clear arrow DHB micelles and dark arrow docetaxel).

EXAMPLES

Example 1

Preparation of Nanoparticles (or nanocrystals) Consisting in Furanocoumarin (Such as Bergamottin, DHB, 6′,7′-Epoxybergamottin and/or Paradisin)

[0084] There are various options to produce nanoparticles in the desired shape and size [Nanocrystal technology, drug delivery and clinical applications. Junghanns J-U A H, Müller R H. International Journal ofNanomedicine, 2008:3(3) 295-309].

[0085] Basically three principles can be used: precipitation methods, milling methods and homogenization methods, as well as any combination thereof.

[0086] Precipitation Methods:

[0087] Furanocoumarins are dissolved in a solvent and subsequently added to a non solvent, leading to a precipitation of finely dispersed furanocoumarin nanoparticles. Alternatively, furanocoumarin may be added directly into water, possibly in the context of a ultrasound treatment, and self-assembling is driven by hydrophobic interactions.

[0088] Milling Methods:

[0089] Milling media (such as ball mills), furanocoumarins and dispersion medium (such as water) are charged into a milling chamber. Shear forces of impact, generated by the movement of the milling media, lead to particle size reduction.

[0090] Homogenization Methods:

[0091] Typically this method requires microfluidizer technology which can generate small particles by frontal collision of two fluid streams under pressures up to 1700 bars. Of note, supercritical fluid methods may also be employed to generate nanoparticles.

Example 2

Preparation of Hyaluronan Coated Nanoparticles Consisting in Furanocoumarin by Self-Assembly in Water (cf. FIG. 2)

[0092] Nanoparticles consisting in furanocoumarin are typically obtained by direct addition of furanocoumarins (such as bergamottin, DHB, 6′,7′-epoxybergamottin and/or paradisin) in water, the mixture being then submitted to a ultrasonication treatment. Hyaluronic acid polymers are subsequently added to the obtained suspension. A polymeric layer of hyaluronic acid is formed onto the nanoparticles' surface.

Example 3

Preparation of Hyaluronan Coated Nanoparticles Consisting in Furanocoumarin by Self-Assembly in Water (cf. FIG. 3)

[0093] Hyaluronic acid polymers are first dissolved in water. Subsequently furanocoumarins (such as bergamottin, DHB, 6′,7′-epoxybergamottin and/or paradisin) are added to the solution, said solution being then submitted to a ultrasonication treatment. A polymeric layer of hyaluronic acid is formed onto the nanoparticles' surface.

Example 4

Preparation of Hyaluronan Coated Nanoparticles Consisting in Furanocoumarin by Solvent Removal (cf. FIG. 4)

[0094] Furanocoumarins (such as bergamottin, DHB, 6′,7′-epoxybergamottin and/or paradisin) are added to a solution of acetone. Hyaluronic acid polymers dissolved in water are subsequently added to the furanocoumarins' solution. Acetone is removed by evaporation above 65° C. A polymeric layer of hyaluronic acid is formed onto the furanocoumarin nanoparticles' surface.

[0095] Of note, in the above examples 2, 3 and 4, the hyaluronic acid polymer can be further cross-linked in water.

[0096] Of note, in the above examples 2, 3 and 4, chitosan polymers, PLGA-hyaluronic acid copolymers, PLGA-PEG copolymers, or any water soluble polymer or co-polymer as described herein above can replace partially or totally hyaluronic acid polymer.

[0097] Of note, in the above examples 2, 3 and 4 the polymer can be formally conjugated with furanocoumarin monomer or dimer.

Example 5

Furanocoumarins Inhibit Human CYP Enzymes

[0098] The below Table 3 summarizes the role of furanocoumarms and of other natural compounds of interest or synthetic analog thereof as inhibitors of human CYP enzymes (cf. Summary of information on human CYP enzymes: Human P450 metabolism data. Rendic S. Drug metabolism reviews, 34(1 & 2), 83-448 (2002)):

[0099] Furanocoumarins inhibit in particular the following human CYP enzymes: CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4.

Example 6

Synthesis and Characterization of Micelles Encapsulating 6′-, 7′-Dihydroxybergamottin (DHB) (i.e. DHB Micelles=Biocompatible Nanoparticle as Defined herein Above in the Detailed Description)

[0100] Micelles of 6′, 7′-dihydroxybergamottin (DHB) (i.e. DHB micelles) were formed by self-assembly, by dissolving a surfactant-ethanol solution in aqueous solution. The surfactant (Polysorbate 80) and anhydrous ethanol were mixed 1:1 (v/v) to form a polysorbate 80-ethanol solution.

[0101] DHB was then weighted and dissolved in the polysorbate 80-ethanol (1:1, v/v) solution to a concentration up to the solubility limit. Fifteen (15) minutes of strong vortexing were subsequently performed to completely dissolve the DHB powder.

[0102] Once the dissolution of DHB was completed, micelles were formed by addition of an aqueous solution (either water of saline water) to the polysorbate 80-ethanol (1:1, v/v) solution containing DHB. Typically, saline water containing NaC1 1% (w/w) was added to the polysorbate 80-ethanol solution containing DHB in a ratio equal to 10:1 (saline water: polysorbate 80-ethanol, v:v). Under these conditions, the resulting final concentration of DHB in micelles solution was 2.5 mM.

[0103] a) Particle Size Characterization:

[0104] DHB micelles in saline water (1% w/w NaCl) were measured by dynamic light scattering. The hydrodynamic diameter of micelles was equal to 11.6 nm (distribution by intensity) with a polydispersity index (PdI) equal to 0,089.

[0105] b) In Vitro Cytochromes P450 Inhibition of Pharmaceutical Compound (Here Docetaxel) by DHB Micelles

[0106] “Control” sample: Docetaxel 1 μM

[0107] A “control” sample corresponding to docetaxel 1 μM was used for high-performance liquid chromatography (HPLC) measurement. Docetaxel powder was dissolved in polysorbate 80-ethanol (1:1, v:v) solution. Saline water was subsequently added in a ratio 1:9 (polysorbate 80-ethanol:saline water, v:v). The obtained suspension was diluted in cell culture down to a concentration of docetaxel of 1 μM. Then, the incubation medium was collected and acetonitrile was added to the medium to precipitate proteins (1:1 v/v).

[0108] “Metabolised” sample: incubation on HepaRG cells of Docetaxel 1 μM-4 hrs

[0109] Docetaxel powder was dissolved in polysorbate 80-ethanol (1:1, v:v) solution. Saline water was subsequently added in a ratio 1:9 (polysorbate 80-ethanol:saline water, v:v). The obtained suspension was incubated at the nontoxic concentration of 1 μM for 4 hours on induced HepaRG cells (HepaRG is human hepatic cell-line of hepatocyte progenitors, cultured and induced according to the manufacturer's protocol). Then, the incubation medium was collected and acetonitrile was added to the medium to precipitate proteins (1:1 v/v).

[0110] “Inhibited” sample: sequential incubation on HepaRG cells of (1) DHB micelles 10 μM-1 h and (2) docetaxel 1 μM-4hs

[0111] Polysorbate 80-ethanol micelles loaded with 6′, 7′-dihydroxybergamottin (i.e. DHB micelles) 10 μM were incubated 1 hour on induced HepaRG cells. Then, cells were rinsed with PBS and incubated with the pharmaceutical compound of interest (here docetaxel known to be a substrate of CYP3A4). Docetaxel was incubated at nontoxic concentration of 1 μM for 4 hours. Then, the incubation medium was collected and acetonitrile was added to the medium to precipitate proteins (1:1 v/v).

[0112] The 3 Samples (“control”, “metabolized”, and “inhibited” samples) were vortexed for 30 seconds and centrifuged at 3000 g for 30 minutes. Their supernatants were mixed with ethyl acetate (1:1 v/v) to separate the organic phase from the aqueous phase. The organic phases of each supernatant were dried out in a Rotavapor at 60° C. and re-suspended in methanol. The resulting methanol solutions were injected in the HPLC-UV (Thermo Fisher Scientific Inc.) with an auto-injector. 20 μL of sample were separated on a C18 3 μm, 150 mm×4.6 mm column (Advanced Chromatography Technologies Ltd.) in a gradient of eluent starting at 30% acetonitrile and 70% acidic water (0.1% formic acid), up to 100% acetonitrile in 25 minutes. The chromatograms obtained were extracted at an UV emission wavelength of 230 nm.

[0113] FIG. 6 shows that the sequential treatment of DHB micelles and docetaxel compound on HepaRG cells (i.e. treatment of DHB micelles lh before the treatment of docetaxel) inhibits the metabolization of docetaxel; i.e. the peak corresponding to docetaxel metabolite is no longer present when cells are treated with the sequential administration of DHB micelles and docetaxel when compared to cells treated with docetaxel alone.

[0114] c) In Vivo Cytochromes P450 Inhibition of Pharmaceutical Compounds by DHB Micelles

[0115] This study was performed to investigate the efficacy of the pharmaceutical composition comprising the combination of (i) the DHB micelles (biocompatible nanoparticles) and of (ii) docetaxel as the pharmaceutical compound of interest, in HT-29 tumor model xenografted on NMRI nude mice.

[0116] The human colorectal adenocarcinoma HT-29 cell line was purchased at the ATCC. The cells were cultured in McCoy's 5a Medium supplemented with 10% fetal bovine serum. NMRI female nude mice (NMRI-Foxlnu/Foxnlnu) 6-7 weeks were ordered from Janvier Labs (France). Mice were xenografted with HT-29 cells: 5 million cells were injected in 50 μL subcutaneously in the lower right flank. Tumor volume in mm.sup.3 was measured with a digital calliper, calculated with the formula:

[00001]$\mathrm{Tumor}.Math..Math.\mathrm{volume}.Math..Math.\left({\mathrm{mm}}^3\right)=\frac{\mathrm{length}.Math..Math.\left(\mathrm{mm}\right)\times {\mathrm{width}}^2.Math..Math.\left({\mathrm{mm}}^2\right)}{2}.$

[0117] Mice were randomised into separate cages and identified by a number (pawn tattoo) 2 weeks post xenograft, when the mean tumor volume reached 90 mm.sup.3 (standard deviation 25%). Groups were made of 5 mice [except for the control aqueous saline (NaCl 0.9%) group, 3 mice] (see FIG. 7 for the schedule of administration): [0118] Group 1: NaCl (control group). 3 mice were injected with saline water (NaCl 0.9%) intravenously in the tail vein, on D1 (day 1, corresponding to the first day of treatment), D2, D3, D6, D9, D10. [0119] Group 2: 6′, 7′-dihydroxybergamottin in polysorbate 80-ethanol micelles (DHB micelles) (control group). DHB micelles in saline water (NaCl 1% w:w) at 2.5 mM (0.93 g/L) were injected at a dose of 5 mg/kg intravenously in the tail vein, on D1, D3 and D9. [0120] Group 3: Docetaxel 10 mg/kg (treatment group). Docetaxel (docetaxel anhydrous, Sigma Aldrich, European pharmacopeia) was dissolved in polysorbate 80-ethanol 1:1 (v/v) at 20 g/L. Prior to injection, saline water (NaCl 1%w:w) was added to polysorbate 80-ethanol solution containing the docetaxel compound down to concentration of docetaxel of 2 g/L. The resulting docetaxel suspension was administered intravenously through the tail vein at a dose of 10 mg/kg, on D2, D6 and D10. [0121] Group 4: sequential administration of DHB micelles and Docetaxel 10 mg/kg (pharmaceutical composition group). Group 4 was treated as follows: [0122] Intravenous injection through the tail vein of DHB micelles 2.5 mM at a dose of 5 mg/kg on D1, D3 and D9;

[0123] Intravenous injection through the tail vein of docetaxel suspension prepared as in group 3 herein above (2 g/L), at a dose of 10 mg/kg on D2, D6 and D10.

[0124] Mice were followed up for clinical signs, body weight and tumor size at least twice a week. The tumor volume was estimated from two-dimensional tumor volume measurements with a digital caliper using the following formula:

[00002]$\mathrm{Tumor}.Math..Math.\mathrm{volume}.Math..Math.\left({\mathrm{mm}}^3\right)=\frac{\mathrm{length}.Math..Math.\left(\mathrm{mm}\right)\times {\mathrm{width}}^2.Math..Math.\left({\mathrm{mm}}^2\right)}{2}$

[0125] The overall survival of all animals was followed using the Kaplan-Meyer curves. As illustrated in FIG. 8, 40% of animals in the group treated by the pharmaceutical composition (group 4) survive for at least 15 days more than the group treated by docetaxel 10 mg/kg alone (group 3).

[0126] These results showed an advantageous overall survival when using the pharmaceutical composition of the present invention, when compared to docetaxel alone.

Example 7

Synthesis of Micelles Encapsulating Bergamottin (Bergamottin Micelles)

[0127] Micelles of bergamottin (i.e. Bergamottin micelles) were formed by self-assembly, by dissolving a surfactant-ethanol solution in aqueous solution. The surfactant (Polysorbate 80) and anhydrous ethanol are mixed 1:1 (v/v) to form a surfactant-ethanol solution. Bergamottin was then weighted and dissolved in the polysorbate 80-ethanol (1:1, v/v) solution to a concentration up to the solubility limit. Fifteen (15) minutes of strong vortexing were subsequently performed to completely dissolve the bergamottin powder. Once the dissolution of bergamottin was completed, micelles were formed by addition of an aqueous solution (either water of saline water) to the polysorbate 80-ethanol (1:1, v/v) solution containing bergamottin. Typically, saline water containing NaC1 1% (w/w) was added to the polysorbate 80-ethanol solution containing bergamottin in a ratio equal to 10:1 (saline water:polysorbate 80-ethanol, v:v). Under these conditions, the resulting final concentration of bergamottin in micelles solution was 2.5 mM.

[0128] Particle Size Characterization:

[0129] Bergamottin micelles in saline solution (1% w/w NaCl) were measured by dynamic light scattering. The hydrodynamic diameter of micelles was equal to 13.26 nm (distribution by intensity) with a polydispersity index (PdI) equal to 0.108.

Example 8

Synthesis of Hyaluronic Acid (HA) Nanoparticles Cross-Linked with 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide (EDC) and N-Hydroxysuccinimide (NHS), comprising 6′, 7′-Dihydroxybergamottin (DHB).

[0130] Aqueous solution of HA polymer was prepared by mixing HA polymer in water (2.5 g/L, 5.4 mL) in a 100 mL beaker. Then, 17.0 mL of acetone was added to the flask and stirred with a mechanical agitation for 20 minutes (320 rpm). 0.125 mL of a solution of EDC (50 mg/mL) in water was added to the flask, followed 5 min later by an addition of 0.35 mL of a solution of NHS (27.5 mg/mL) in water. After mixing the solution for 5 min, 21.5 mL of acetone with 0.125 mL of DHB in acetone (10 g/L) were added to the solution and stirring was continued for 15h30 (HA concentration ˜0.30 g/L). Then, the reaction was stopped by dialysis of the solution against reverse osmosis water using dialysis membrane (Regenerated Cellulose (RC), MWCO 12-14 kDa) (minimum 2×4hrs).

[0131] Particles Size Characterization

[0132] Nanoparticles hydrodynamic diameter was measured by DLS (hydrodynamic diameter (distribution by intensity)=92 nm in NaCl (150 mM) and PdI=0,148). Finally, the nanoparticles solution was concentrated with an Amicon® system (Biomax®; 50 kDa; d=25 mm; PES) and stored at 4° C. (Final HA concentration ˜4.00 g/L).

Example 9

Synthesis of Hyaluronic Acid (HA)-Ethylenediamine (EDA) Nanoparticles Cross-Linked with with 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide (EDC) and N-Hydroxysuccinimide (NHS), Comprising 6′, 7′-Dihydroxybergamottin (DHB)

[0133] Aqueous solution of HA polymer was prepared by mixing HA polymer in water (2.5 g/L, 5.3 mL) in a 100 mL beaker. Then, 17 mL of acetone was added to the flask and stirred with a mechanical agitation for 34 minutes (320 rpm). 0.125 mL of a solution of EDC (50 mg/mL) in water was added to the flask, followed 5 min later by 0.35 mL of a solution of NHS (27.5 mg/mL) in water and 0.125 mL of a solution of EDA (15 mg/mL) in water. After mixing the solution for 15 min, 21.5 mL of acetone with 0.180 mL of DHB (10 g/L) in acetone were added to the solution and stirring was continued for 20 min (HA concentration ˜0.30 g/L). Then, the reaction was stopped by dialysis of the solution against reverse osmosis water using dialysis membrane (Regenerated Cellulose (RC), MWCO 12-14 kDa) (minimum 2*4 h).

[0134] Particle Size Characterization

[0135] Nanoparticles hydrodynamic diameter was measured by DLS (hydrodynamic diameter (distribution by intensity)=95 nm in NaCl (150 mM) and PdI=0,136). Finally, the nanoparticles solution was concentrated with an Amicon® system (Biomax®; 50 kDa; d=25 mm; PES) and stored at 4° C. (Final HA concentration ˜4.00 g/L).