Molecular assembly for encapsulating hydrophobic molecules, said assembly having the hydrophobic molecules encapsulated, corresponding manufacturing methods and uses

Abstract

------ Molecular assembly for encapsulating hydrophobic molecules, said assembly having the encapsulated hydrophobic molecules, corresponding manufacturing methods and uses The invention relates to a molecular assembly consisting of paramylon on the chain of which molecular cages are grafted. It also relates to manufacturing methods of said molecular assembly as well as its uses. Figure to be published with the abstract: FIG. 1

Description

Title of the invention: Molecular assembly for the encapsulation of hydrophobic molecules, said assembly having the encapsulated hydrophobic molecules, corresponding manufacturing methods and uses

[0001] The present invention relates to the field of transport and protection of hydrophobic molecules in the chemical and biological context.

[0002] In particular, the present invention relates to a molecular assembly for encapsulating hydrophobic molecules, to said assembly having the encapsulated hydrophobic molecules, to methods of manufacturing said molecular assembly as well as to its uses.

[0003] In the fields of chemistry and biology, there are circumstances in which the problem of the solubility of a hydrophobic molecule in water or an aqueous medium arises. Thus, for example, one may wish to inject a hydrophobic active ingredient into an aqueous saline solution, in which case one seeks to obtain a better concentration of the active ingredient to reduce the quantity of solution required for the given quantity of active ingredient.

[0004] Furthermore, in the pharmaceutical field, there is also an attempt to improve the bioavailability of hydrophobic molecules in biological media. In this case, these hydrophobic molecules can be active ingredients of drugs. The bioavailability of a molecule is understood as the fraction of the molecule released by the pharmaceutical form used for administration and which reaches the systemic blood circulation in unchanged form and also represents the speed at which this process takes place.

[0005] As part of their research to solve all these problems, the present inventors have developed an innovative system called molecular assembly based on paramylon and molecular cages, which assembly has made it possible to encapsulate hydrophobic molecules thus overcoming the problem of low solubility of hydrophobic molecules in water or aqueous media, leading to better bioavailability in the fields of pharmacy and biology, as well as the problem of storage of hydrophobic molecules in water or aqueous media.

[0006] As an example showing the need for better bioavailability, we can mention the case of polyphenols in the pharmaceutical field.

[0007] Polyphenols are a family of organic molecules that have very important effects in the field of health. One of these polyphenols is curcumin. Curcumin has long been used in Chinese medicine for its therapeutic, anti-inflammatory, antibacterial, neuroprotective and antioxidant properties. However, it is not easily absorbed and is quickly degraded due to a very rapid metabolism. As a result, very high doses of curcumin are required to achieve therapeutic effects without having the certainty that the curcumin reaches the target organ.

[0008] Thus, one of the main obstacles to the use of curcumin in therapy is its very low bioavailability, as well as its very rapid metabolism. In the case of oral absorption, curcumin is rapidly metabolized via conjugation mechanisms into glucoronoconjugate and sulfate. These modifications are intended to make curcumin less lipophilic so that it is eliminated more quickly by the body. The liver is the main organ in which these conjugation reactions take place. Competitively during conjugation and especially in the case of intravenous administration, curcumin undergoes reactions by microsomal hepatic enzymes, the reductases. Metabolites are thus formed which are mainly reduced derivatives of curcumin: dihydrocurcumin, tetrahydrocurcumin, hexahydrocurcumin and hexahydrocurcuminol.These main metabolites then undergo conjugation reactions in order to be rapidly eliminated or excreted.

[0009] Nano-technological approaches have been developed, including the incorporation or encapsulation of curcumin into liposomes, polymeric micelles, polymeric nanoparticles, nanogels, nanoemulsions, inclusion complexes, solid lipid nanoparticles, dendrimers, phytosomes, mesoporous silica nanoparticles, metal nanoparticles (Prasad et al., 2014). All these nano-vectors have all been shown to increase the bioavailability and beneficial effects in vitro of curcumin.

[0010] The inventors are particularly aware of the existence of curdlan nanoparticles conjugated to [3-cyclodextrin] inclusion complexes developed by RY Basha and colleagues (Basha et al., 2019, Carbohydrate Polymers 218: 53-62). These curdlan nanoparticles associated with cyclodextrin constitute an example of polymeric nanoparticles that can load and transport hydrophobic molecules, namely anti-tuberculosis drugs, rifampicin and levofloxacin.

[0011] The problem with using curdlan in the context of the invention is that this [3-glucan] has known gelling properties and that it gels in an aqueous medium in the form of nanoparticles.

[0012] Under these circumstances, the in vivo use of such conjugated [3-glucan] nanoparticles raises an underlying problem, namely what is the fate of these nanoparticles once introduced into the human or animal body? The concern As for the storage of nanoparticles in the body, their long-term effect on human or animal health is legitimate, and one can wonder about the effects of the accumulation of nanoparticles in the liver, kidneys, and even bone marrow and lymphoid organs.

[0013] In order to avoid the potential danger of accumulation of nanoparticles in the human or animal organism, the inventors sought to develop a molecular assembly allowing the encapsulation of hydrophobic molecules, said assembly being soluble in order to allow its elimination from the organism.

[0014] The assemblies according to the invention then make it possible to solubilize hydrophobic molecules in an aqueous medium, which in the absence of said assemblies would be unstable and degraded by the surrounding medium. In the biological context, this makes it possible to improve the bioavailability of a hydrophobic molecule in a biological medium.

[0015] The assembly according to the invention is also capable of encapsulating other molecules or active ingredients with very varied therapeutic targets. It can in particular be used in therapeutic, prophylactic, pharmaceutical, cosmetic, nutraceutical, agri-food, phytosanitary or veterinary applications.

[0016] The invention has the advantage of being based on products that are easily accessible or of natural origin. It is thus inexpensive and easy to implement given the accessibility of the compounds, which are all commercially available.

[0017] The invention also has the advantage of remaining without danger of accumulation in the human or animal organism if it is used in vivo given its solubility in aqueous media, in particular biological media, to allow its elimination.

[0018] The present invention therefore firstly relates to a molecular assembly consisting of paramylon onto the chain of which molecular cages are grafted.

[0019] Paramylon is a highly crystalline natural polymer derived from the microalgae Euglenagravillis. It is a linear homopolymer of glucose linked by [3-1,3] bonds, i.e. a polysaccharide composed of linear [3-1,3-glucans. It is approved by the US Food and Drug Administration (FDA) as a food additive.

[0020] Unlike curdlan, paramylon remains insoluble in water and does not tend to gel. Indeed, curdlan - which is also a polysaccharide composed of [3-1,3-glucans - is produced by bacteria, notably Agrobacterium sp. or Alcaligenes faecalis, and gels in the form of nanoparticles in an aqueous medium.

[0021] The molecular cages can be chosen from cyclodextrins, calixarenes, crown ethers, cucurbiturils and their mixtures.

[0022] As cyclodextrin, at least one of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin may be mentioned.

[0023] As calixarene, we can cite at least one of calix[4]arene, calix[5]arene, calix[6]arene, calix[7]arene.

[0024] As crown ether, at least one of 18-crown-6 ether and 9-crown-3 ether may be mentioned.

[0025] As cucurbituril (CB), at least one of cucurbit[5]uril (CB[5]), cucurbit[6]uril (CB[6]), cucurbit[7]uril (CB[7]), cucurbit[8]uril (CB[8]) may be mentioned.

[0026] In particular, the molecular cages are grafted onto the paramylon chain via a grafting agent linked by a covalent bond to an atom of the paramylon chain and by another covalent bond to an atom of the molecular cage.

[0027] The present invention also relates to a method for manufacturing a molecular assembly as defined above, characterized in that the paramylon, the molecular cages and at least one agent for grafting the molecular cages onto the paramylon are mixed in a solvent medium.

[0028] In particular, the above method may comprise the steps of: - dissolve the paramylon and the molecular cage separately; - mix the solutions obtained in the previous step; - add a solution of the grafting agent(s) and stir the reaction mixture; - optionally, return to pH 7 by neutralization; - filter the crude reaction mixture obtained; - optionally, dehydrate the resulting assembly solution.

[0029] The grafting agent(s) may in particular be chosen from epichlorohydrin and its derivatives, 1,4-butanediol diglycidic ethers and their derivatives, epoxyhaloalkanes, triazines and their derivatives.

[0030] The present invention also relates to a molecular assembly as defined above or as prepared as indicated above, in the molecular cages of which at least one hydrophobic molecule is encapsulated by complexation.

[0031] The hydrophobic molecule(s) may be chosen from polyphenols such as curcumin, demethoxycurcumin, bisdemethoxycurcumin and resveratrol, steroids and terpenes.

[0032] The polyphenol may in particular be curcumin. Curcumin may for example be extracted from Curcuma longua. It may also be synthesized. A molecular assembly in which the cages of which curcumin is encapsulated makes it possible to considerably improve the bioavailability of the latter and thus to reduce the dose necessary to produce therapeutic effects, in particular on colorectal cancer cell lines HCT116 and HT-29.

[0033] The present invention also relates to a method for manufacturing a molecular assembly as defined above, characterized in that the hydrophobic molecule(s) to be encapsulated are placed in a solvent capable of solubilizing it(them) and soluble or miscible with water, and the solution obtained is mixed with the assembly(s) as defined above or prepared by the method as defined above.

[0034] The present invention relates to the use of the above assembly or as prepared by the above process in dispersion in water or in an aqueous solvent in therapeutic, prophylactic, pharmaceutical, cosmetic, nutraceutical, agri-food, phytosanitary or veterinary applications.

[0035] To better illustrate the object of the present invention, the following examples will now be described below, by way of illustration and not limitation, in conjunction with the appended drawings.

[0036] The following examples illustrate the present invention without, however, limiting its scope.

[0037] [Fig.l] is a general diagram of the synthesis of the assembly according to the invention followed by the encapsulation of a hydrophobic molecule.

[0038] Figure 2 shows a dynamic light scattering (DLS) graph of the paramylon-[3cyclodextrin assembly ([Fig.2A]) and the curcumin-loaded paramylon-[3cyclodextrin assembly ([Fig.2B]).

[0039] Figure 3 shows MTT assay graphs representing the 24 and 48 hour cell viability of the HCT116 line cells as a function of the curcumin concentration ([Fig.3A]), the paramylon-[3cyclodextrin assembly ([Fig.3B]) and the curcumin-loaded paramylon-[3cyclodextrin assembly ([Fig.3C]).

[0040] Figure 4 shows MTT assay graphs representing the 48-hour cell viability of HT-29 line cells as a function of curcumin concentration ([Fig.4A]), paramylon-[3cyclodextrin assembly ([Fig.4B]) and curcumin-loaded paramylon-[3cyclodextrin assembly ([Fig.4C]).

[0041] Example 1: Synthesis of a paramylon - [3-cyclodextrin] molecular assembly

[0042] A grafting of native [3-cyclodextrin] onto paramylon with a molecular mass of approximately 500 kDa was carried out at room temperature and under magnetic stirring (500 rpm).

[0043] To do this, 400 mg of paramylon and 800 mg (0.705 mmol) of [3-cyclodextrin] were dissolved separately in 100 ml Erlenmeyer flasks each containing 25 ml of NaOH (1 M). After dissolution (~ 30 minutes), the solutions were mixed and homogenized for 5 minutes. Then, 1.2 ml of epichlorohydrin was added to the mixture and the reaction was left under magnetic stirring (500 rpm) at room temperature.

[0044] After 24 hours of reaction, the pH of the reaction medium was adjusted to 7 by adding HCl (1 M). The crude reaction product was filtered through filter paper and dialyzed (MWCO membrane: 2 kDa) for 48 hours in order to remove the excess of [3-native cyclodextrin and epichlorohydrin which had not reacted.

[0045] The experimental protocol was carried out twice in a row to obtain masses of 556 mg the first time and 554 mg the second time.

[0046] After freeze-drying, the product was obtained in the form of a spongy structure and was characterized by DLS ("Dynamic light scattering").

[0047] The DLS results are shown in Figure 2. [Fig.2A] shows the dynamic light scattering pattern for the paramylon-cyclodextrin assembly. The average hydrodynamic diameter is about 70 nm.

[0048] Example 2: Synthesis of a paramylon - a-cyclodextrin molecular assembly

[0049] The procedure was as in Example 1 but with the use of a-cyclodextrin instead of [3-cyclodextrin.

[0050] To do this, 800 mg (0.617 mmol) of α-cyclodextrin and 800 mg of paramylon were reacted. The product resulting from the reaction was obtained with masses of 712 mg and 720 mg.

[0051] After lyophilization, the desired assembly was obtained in the form of a spongy structure. It was characterized as in Example 1.

[0052] Example 3: Synthesis of a paramylon - y -cyclodextrin molecular assembly

[0053] The procedure was as in Example 1 but with the use of y-cyclodextrin instead of [3-cyclodextrin.

[0054] To do this, 400 mg (0.822 mmol) of γ-cyclodextrin and 800 mg of paramylon were reacted. The product resulting from the reaction was obtained with masses of 748 mg and 760 mg.

[0055] After lyophilization, the desired assembly was obtained in the form of a spongy structure. It was characterized as in Example 1.

[0056] Example 4: Obtaining curcumin to be encapsulated in paramylon-cyclodextrin assemblies and preparing curcumin I, demethoxycurcumin (Curcumin II) and bisdemethoxycurcumin (Curcumin III)

[0057] From 0.5 g of curcuminoid powder from Sigma Aldrich, curcumin, called curcumin I, demethoxycurcumin (curcumin II) and bisdemethoxycurcumin (curcumin III) were purified and isolated by preparative thin layer chromatography (CHCl3 / EtOH: 95 / 5).

[0058] These three curcumins were obtained in an amount of respectively 0.33 g (70%, Frontal ratio Rf0.6), 0.095 g (20%, Rf 0.5) and 0.0047 g (10%, Rf 0.4). General encapsulation procedure

[0059] The encapsulation of curcumin by the paramylon-cyclodextrin assemblies was carried out following a complexation reaction between the curcumin and the hydrophobic cavities of the cyclodextrins grafted onto the paramylon.

[0060] To do this, in an Erlenmeyer flask containing 20 mg of the paramylon-cyclodextrin assembly, solubilized in 10 ml of physiological water (NaCl 0.9%), 2 mg of curcumin previously dissolved in 0.2 ml of acetone or ethanol were added using a micropipette. Then, the reaction medium was stirred using an ultrasonic bath. Finally, the solution was left overnight without stirring and protected from light.

[0061] The curcumin-loaded assemblies obtained are purified by filtration with millipore filters (porosity threshold: 0.2 pm). A liquid / liquid extraction of curcumin from 5 ml of complex made it possible to determine, using a UV-Vis assay, the encapsulation rates per dry mass of the assembly alone. The products are characterized by UV-Visible, infrared and DLS.

[0062] Table 1 shows the encapsulation rates of curcumin I in the assembly according to Example 1 relative to the dry mass of the assembly obtained in Example 1. Par / [3-CD (mg) Curcumin I (mg) Encapsulation rate of curcumin I relative to the dry mass of Par / [3-CD 20 2 5% 40 4 5% 50 7.5 5%

[0063] [Table 1] Encapsulation of curcumin by the Paramylon-[3-cyclodextrin assembly of Example 1

[0064] [Fig.2B] shows the dynamic light scattering diagram for the paramylon-cyclodextrin assembly obtained according to Example 1 loaded with curcumin I. The average hydrodynamic diameter is about 98 nm.

[0065] Comparing these results with the DLS results obtained by Basha et al., the dynamic light scattering of the curdlan nanoparticles complexed with [3-cyclodextrin] allowed to observe an average hydrodynamic diameter of approximately 523 nm. The curdlan nanoparticles alone had an average hydrodynamic diameter of approximately 226 nm, already significantly higher than that of the assembly of the present invention whether or not it was loaded with curcumin.

[0066] Example 4: Decreased cell viability of the cancer cell line HCT116

[0067] Cells of the HTC116 cell line were placed in contact with curcumin alone, with an assembly according to Example 1 alone and with an assembly according to Example 1 loaded with curcumin I for a period of 24 hours and a period of 48 hours.

[0068] Curcumin I was introduced at a concentration of 1 pg / ml, 2 pg / ml, 5 pg / ml, 10 pg / ml, 15 pg / ml and 20 pg / ml.

[0069] The assembly alone was introduced at a concentration of 1 pg / ml, 5 pg / ml, 10 pg / ml, 20 pg / ml, 50 pg / ml and 100 pg / ml

[0070] The curcumin I-loaded assembly was introduced at a concentration of 1 pg / ml, 2 pg / ml, 5 pg / ml, 10 pg / ml, 15 pg / ml and 20 pg / ml.

[0071] Cell viability of HTC116 line cells was measured by the MTT tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) test.

[0072] The results are shown in Figure 3, the graph in [Fig.3A] showing cell viability as a function of curcumin concentration after 24 and 48 hours, the graph in [Fig.3B] showing cell viability as a function of assembly concentration alone after 24 and 48 hours and the graph in [Fig.3C] showing cell viability as a function of curcumin I loaded assembly concentration after 24 and 48 hours.

[0073] As can be seen in [Fig.3A], curcumin shows its effect on the decrease in cell viability of HCT116 cells with an almost total disappearance of cells after 48 hours at a concentration of 10 pg / ml. The IC50 concentration is 4.38 pg / ml.

[0074] As can be seen in [Fig.3B], assembly alone has no effect on the viability of HCT116 cells.

[0075] The graph in [Fig.3C] shows that the curcumin-loaded assembly has an effect on HCT116 cells with almost complete disappearance of cells after 48 hours at a concentration of 10 pg / ml. The IC50 concentration is 1.62 pg / ml.

[0076] The lower IC50 of the curcumin I-loaded assembly compared to that of curcumin I alone therefore shows the better bioavailability of the latter compared to curcumin I alone.

[0077] Example 5: Decrease in cell viability of the cancer cell line HT-29

[0078] The procedure in Example 4 was carried out with the cancer cell line HT-29.

[0079] The results are shown in Figure 4.

[0080] The IC50 concentration of curcumin alone is 7.38 pg / m 1 while that of the curcumin-loaded assembly is 6.36%g / ml. Here again the lower IC50 of the curcumin-loaded assembly compared to that of curcumin I alone shows the better bioavailability of the latter compared to curcumin I alone.

Claims

Claims

1. - Molecular assembly consisting of paramylon onto the chain of which molecular cages are grafted.

2. - Assembly according to claim 1, characterized in that the molecular cages are chosen from cyclodextrins, calixarenes, crown ethers, cucurbiturils and their mixtures.

3. - Assembly according to claim 2, characterized in that the cyclodextrins are at least one of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin.

4. - Assembly according to one of claims 1 to 3, characterized in that the molecular cages are grafted onto the paramylon chain by means of a grafting agent linked by a covalent bond to an atom of the paramylon chain and by another covalent bond to an atom of the molecular cage.

5. - Method for manufacturing a molecular assembly as defined in one of claims 1 to 4, characterized in that the paramylon, the molecular cages and at least one agent for grafting the molecular cages onto the paramylon are mixed in a solvent medium.

6. - Method according to claim 5, characterized in that it comprises the steps of: - dissolving the paramylon and the molecular cages separately; - mixing the solutions obtained in the previous step; - adding a solution of the grafting agent(s) and stirring the reaction mixture; - optionally, returning to pH 7 by neutralization; - filtering the crude reaction mixture obtained; - optionally, dehydrating the solution of the assembly obtained.

7. - Process according to one of claims 5 and 6, characterized in that the grafting agent(s) is(are) chosen from epichlorohydrin, diglycidic ethers of 1,4-butanediol, epoxyhaloalkanes, triazines and their derivatives.

8. - Molecular assembly as defined in one of claims 1 to 4 or as prepared in one of claims 5 to 7, in the molecular cages of which at least one hydrophobic molecule is encapsulated by complexation.

9. - Assembly according to claim 8, characterized in that the hydrophobic molecule(s) are chosen from polyphenols such as curcumin, demethoxycurcumin, bisdemethoxycurcumin and resveratrol, steroids and terpenes.

10. - Process for manufacturing a molecular assembly as defined in one of claims 8 and 9, characterized in that the hydrophobic molecule(s) to be encapsulated are placed in a solvent capable of solubilizing it or them and soluble or miscible with water, and the solution obtained is mixed with the assembly(s) as defined in one of claims 1 to 5 or prepared by the process as defined in one of claims 6 and 7.

11. - Use of the assembly according to one of claims 8 and 9 or as prepared by the process according to claim 10 in dispersion in water or in an aqueous solvent in pharmaceutical, cosmetic, nutraceutical, agri-food or phytosanitary applications.

12. - Assembly according to one of claims 8 and 9 or as prepared by the process according to claim 10 in dispersion in water or in an aqueous solvent for its use in therapeutic, prophylactic or veterinary applications.

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