Molecular assembly for the encapsulation of hydrophobic molecules, said assembly having the encapsulated hydrophobic molecules, corresponding manufacturing processes and uses

Abstract

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

Description

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

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

[0004] Furthermore, in the pharmaceutical field, efforts are also underway to improve the bioavailability of hydrophobic molecules in biological environments. In this case, these hydrophobic molecules may 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 that reaches the systemic bloodstream unchanged, and also represents the rate at which this process occurs.

[0005] In the course of their research to solve all these problems, the present inventors have developed an innovative system called a 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 storing 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 constitute 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 Curcumin possesses therapeutic, anti-inflammatory, antibacterial, neuroprotective, and antioxidant properties. However, it is not easily absorbed and is rapidly degraded due to its very fast metabolism. Consequently, very high doses of curcumin are required to achieve therapeutic effects, without any guarantee that the curcumin will reach 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 administration, curcumin is rapidly metabolized via conjugation mechanisms into glucuronide conjugates and sulfate. These modifications aim to make curcumin less lipophilic so that it is eliminated more quickly by the body. The liver is the main organ where these conjugation reactions take place. Competing with conjugation, and especially in the case of intravenous administration, curcumin undergoes reactions by microsomal hepatic enzymes, the reductases. These reactions produce metabolites that 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] Nanotechnological approaches have been developed, including the incorporation or encapsulation of curcumin in liposomes, polymeric micelles, polymeric nanoparticles, nanogels, nanoemulsions, inclusion complexes, solid lipid nanoparticles, dendrimers, phytosomes, mesoporous silica nanoparticles, and metallic nanoparticles (Prasad et al., 2014). All these nanocarriers have increased the bioavailability and beneficial effects of curcumin in vitro.

[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 allow the loading and transport of hydrophobic molecules, namely the antituberculosis 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 aqueous medium in the form of nanoparticles.

[0012] In these circumstances, the in vivo use of such conjugated [3-glucan] nanoparticles raises an underlying problem, namely, what happens to these nanoparticles once introduced into the human or animal organism? 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 question the effects of the accumulation of nanoparticles in the liver, kidneys, and even bone marrow and lymphoid organs.

[0013] In order to eliminate the potential danger of accumulation of nanoparticles in the human or animal body, 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 body.

[0014] The assemblies according to the invention thus 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 environment. In a biological context, this makes it possible to improve the bioavailability of a hydrophobic molecule in a biological environment.

[0015] The assembly according to the invention is also capable of encapsulating other molecules or active ingredients with a wide variety of therapeutic targets. In particular, it can 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 readily available or of natural origin. It is therefore 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 safe from accumulation in the human or animal body if used in vivo, given its solubility in aqueous media, particularly biological media, to allow its elimination.

[0018] The present invention therefore relates firstly 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 a microalga, Euglenagravillis. It is a linear homopolymer of glucose linked by [3-1,3] bonds, in other words, a polysaccharide composed of linear [3-1,3]glucans. It is authorized by the U.S. 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 selected from cyclodextrins, calixarenes, crown ethers, cucurbituriles and mixtures thereof.

[0022] As cyclodextrin, at least one of the following can be cited: α-cyclodextrin, γ-cyclodextrin and γ-cyclodextrin.

[0023] As calixarène, we can cite at least one of the following: calix[4]arène, calix[5]arène, calix[6]arène, calix[7]arène.

[0024] As a crown aether, we can cite at least one among the 18-crown-6 aether, the 9-crown-3 aether.

[0025] As cucurbiturile (CB), we can cite at least one of the following: cucurbit[5]uril (CB[5]), cucurbit[6]uril (CB[6]), cucurbit[7]uril (CB[7]), cucurbit[8]uril (CB[8]).

[0026] In particular, 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.

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

[0028] In particular, the above process may include 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 solution of the assembly obtained.

[0029] In particular, the grafting agent(s) can be chosen from among 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 can be, in particular, curcumin. Curcumin can, for example, be extracted from Curcuma longua. It can also be synthesized. A molecular assembly in which the cages encapsulate curcumin makes it possible to considerably improve its bioavailability and thus reduce the dose required to produce therapeutic effects, particularly on the 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 by the fact that the hydrophobic molecule or molecules to be encapsulated are placed in a solvent capable of solubilizing them and soluble or miscible with water, and the resulting solution is mixed with the assembly or assemblies 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, we will now describe below, by way of illustration and not limitation, the following examples in connection with the attached drawings.

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

[0037] Fig. 1 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 represents a dynamic light scattering (DLS) graph of the paramylon-[3-cyclodextrin assembly ([Fig.2A]) and the curcumin-loaded paramylon-[3-cyclodextrin assembly ([Fig.2B]).

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

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

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

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

[0043] To this end, 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 1 M NaOH. After dissolution (approximately 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 carried out 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 the addition of HCl (1 M). The crude reaction mixture was filtered through filter paper and dialyzed (MWCO membrane: 2 kDa) for 48 h to remove the excess of unreacted native [3-cyclodextrin and epichlorohydrin].

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

[0046] After lyophilization, 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 diagram for the paramylon-cyclodextrin assembly. The mean hydrodynamic diameter is approximately 70 nm.

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

[0049] We proceeded as in Example 1 but with the use of α-cyclodextrin instead of [3-cyclodextrin.

[0050] To achieve this, 800 mg (0.617 mmol) of α-cyclodextrin and 800 mg of paramylon were reacted. The reaction products were obtained with masses of 712 mg and 720 mg.

[0051] After freeze-drying, 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] We proceeded as in Example 1 but with the use of y-cyclodextrin instead of [3-cyclodextrin.

[0054] To achieve this, 400 mg (0.822 mmol) of γ-cyclodextrin and 800 mg of paramylon were reacted. The reaction products were obtained with masses of 748 mg and 760 mg.

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

[0056] Example 4: Obtaining curcumin for encapsulation in paramylon-cyclodextrin assemblies and preparation of curcumin I, demethoxycurcumin (Curcumin II) and bisdemethoxycurcumin (Curcumin III)

[0057] From 0.5 g of Sigma Aldrich curcuminoid powder, curcumin, known as 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 a quantity of respectively 0.33 g (70%, RfR0.6), 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 curcumin and the hydrophobic cavities of the cyclodextrins grafted onto the paramylon.

[0060] To do this, 2 mg of curcumin, previously dissolved in 0.2 mL of acetone or ethanol, was added to an Erlenmeyer flask using a micropipette. The reaction mixture was then 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 using millipore filters (porosity threshold: 0.2 µm). A liquid-liquid extraction of curcumin from 5 ml of the complex was performed to determine, using UV-Vis spectroscopy, the encapsulation rates per unit dry mass of the assembly alone. The products are characterized by UV-Vis, infrared spectroscopy, and DLS.

[0062] Table 1 shows the encapsulation rates of curcumin I in the assembly according to Example 1 in relation to the dry mass of the assembly obtained in Example 1. Par / [3-CD (mg) Curcumin I (mg) Curcumin I encapsulation rate 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 assembly of Paramylon-[3-cyclodextrin 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 mean hydrodynamic diameter is about 98 nm.

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

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

[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 duration of 24 hours and a duration 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 cell line cells was measured by the MTT tetrazolium salt test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide).

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

[0073] As can be seen in [Fig. 3A], curcumin shows its effect on reducing the cell viability of HCT116 cells, with almost total disappearance of the 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 total cell disappearance 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: Decreased cell viability of the cancer cell line HT-29

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

[0079] The results are shown in Figure 4.

[0080] The IC50 concentration of curcumin alone is 7.38 pg / m³, while that of the curcumin-loaded compound is 6.36 g / mL. Here again, the lower IC50 of the curcumin-loaded compound compared to that of curcumin alone demonstrates the greater bioavailability of the latter compared to curcumin alone.

Claims

Demands

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 selected from cyclodextrins, calixarenes, crown ethers, cucurbituriles and mixtures thereof.

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

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

8. - Molecular assembly as defined in any one of claims 1 to 4 or as prepared in any 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 selected from polyphenols such as curcumin, demethoxycurcumin, bisdemethoxycurcumin and resveratrol, steroids and terpenes.

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

11. - Use of the assembly according to any 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, food or plant protection applications.

12. - Assembly according to any 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 use in therapeutic, prophylactic or veterinary applications.

Images