Molecular assemblies for encapsulating hydrophobic molecules, the assemblies having hydrophobic molecules encapsulated, corresponding methods of manufacture and uses

Abstract

The present invention relates to a molecular assembly consisting of a paratope with a molecular cage grafted on its chains. The invention also relates to a process for manufacturing said molecular assembly and to its use.

Description

Technical Field

[0001] This invention relates to the field of transport and protection of hydrophobic molecules in the fields of chemistry and biology.

[0002] Specifically, the present invention relates to a molecular assembly for encapsulating hydrophobic molecules, to said assembly having encapsulated hydrophobic molecules, to a method for manufacturing said molecular assembly, and to the use of said molecular assembly. Background Technology

[0003] In the fields of chemistry and biology, there are situations where the solubility of hydrophobic molecules in water or aqueous media arises. Therefore, for example, it may be necessary to inject a hydrophobic active ingredient into an aqueous salt solution, in which case a higher concentration of the active ingredient is sought to reduce the amount of solution required for a given amount of active ingredient.

[0004] Furthermore, in the pharmaceutical field, there is a desire to improve the bioavailability of hydrophobic molecules in biological media. In this case, these hydrophobic molecules can be the active ingredients of a drug. Molecular bioavailability should be understood as the portion of the molecule released from the drug form intended for administration that reaches the systemic bloodstream in its unaltered form, and it also represents the rate at which this process occurs.

[0005] In research conducted to address all the aforementioned problems, the inventors developed an innovative system called a molecular assembly based on parastarch and molecular cages, which encapsulates hydrophobic molecules, thereby overcoming the problem of low solubility of hydrophobic molecules in water or aqueous media, resulting in better bioavailability in the pharmaceutical and biological fields, and also overcoming the problem of storing hydrophobic molecules in water or aqueous media.

[0006] As an example to illustrate the need for better bioavailability, the case of polyphenols in the pharmaceutical field can be mentioned.

[0007] Polyphenols constitute a class of organic molecules that play a very important role in the health field. One of these polyphenols is curcumin. Curcumin has been used in traditional Chinese medicine for a long time due to its therapeutic, anti-inflammatory, antibacterial, neuroprotective, and antioxidant properties. However, it is poorly absorbed and rapidly degrades due to its very rapid metabolism. Therefore, very high doses of curcumin are required to achieve therapeutic effects, and it is uncertain whether curcumin can even reach the target organs.

[0008] Therefore, one of the main obstacles to the therapeutic use of curcumin is its extremely low bioavailability and its extremely rapid metabolism. In the case of oral absorption, curcumin is rapidly metabolized into glucuronide conjugates and sulfate esters via a conjugation mechanism. These modifications aim to reduce the lipophilicity of curcumin so that it can be cleared from the body more quickly. The liver is the primary organ where these conjugation reactions occur. In a competitive manner with conjugation, especially in the case of intravenous administration, curcumin undergoes a reaction under the action of microsomal liver enzymes, namely reductases. The major metabolites formed are reduced derivatives of curcumin: dihydrocurcumin, tetrahydrocurcumin, hexahydrocurcumin, and hexahydrocurcumol. These major metabolites then undergo conjugation reactions for rapid clearance or excretion.

[0009] Nanotechnology methods have been developed, particularly for incorporating or encapsulating curcumin in liposomes, polymer micelles, polymer nanoparticles, nanogels, nanoemulsions, inclusion complexes, solid lipid nanoparticles, dendritic macromolecules, phytosomes, mesoporous silica nanoparticles, and metal nanoparticles (Prasad et al., 2014). All of these nanocarriers can enhance the bioavailability and beneficial in vitro effects of curcumin.

[0010] The inventors are particularly aware of the existence of gellan gum nanoparticles conjugated with β-cyclodextrin inclusion complexes, developed by RY Basha and colleagues (Basha et al., 2019, Carbohydrate Polymers 218: 53-62). These gellan gum nanoparticles conjugated with cyclodextrin constitute an example of polymeric nanoparticles that allow for the loading and transport of hydrophobic molecules, namely the anti-tuberculosis drugs rifampin and levofloxacin.

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

[0012] In this context, the in vivo application of such conjugated β-glucan nanoparticles raises a fundamental question: what becomes of these nanoparticles once they are introduced into the human or animal body? Concerns about the accumulation of nanoparticles in the body and their long-term effects on human or animal health are justified, and the effects of nanoparticle accumulation in the liver, kidneys, and even bone marrow and lymphatic organs may be questioned. Summary of the Invention

[0013] In order to eliminate the potential danger of nanoparticles accumulating in the human or animal body, the inventors have sought to develop a molecular assembly that allows the encapsulation of hydrophobic molecules, the assembly being soluble so as to allow them to be removed from the body.

[0014] Therefore, the assembly according to the invention allows hydrophobic molecules to be solubilized in aqueous media, whereas in the absence of the assembly, the hydrophobic molecules would be unstable and degraded by the surrounding medium. In a biological context, this allows for improved bioavailability of hydrophobic molecules in biological media.

[0015] The assemblies according to the invention can also encapsulate other molecules or active ingredients having a wide variety of therapeutic targets. They are particularly useful for therapeutic, preventative, pharmaceutical, cosmetic, nutritional, agri-food, phytosanitary, or veterinary applications.

[0016] This invention has the advantage of being based on products that are readily available or of natural origin. Therefore, given that the compounds are all commercially available, this invention is inexpensive and easy to implement.

[0017] The present invention also has the following advantages: if used in vivo, there is no risk of accumulation in the human or animal body, given its solubility in aqueous media (especially biological media) to allow it to be eliminated.

[0018] Therefore, the present invention relates first to a molecular assembly composed of parastarch with molecular cages grafted onto the chains of the parastarch.

[0019] Parastarch is a highly crystalline natural polymer derived from the microalga Euglena filamentosa. It is a linear homopolymer of glucose linked by β-1,3 bonds; in other words, it is a polysaccharide composed of linear β-1,3-glucan. It has been approved by the U.S. Food and Drug Administration (FDA) as a food additive.

[0020] Unlike gellan gum, parastarch remains insoluble in water and does not tend to gel. In fact, gellan gum—which is also a polysaccharide composed of β-1,3-glucan—is produced by bacteria, particularly Agrobacterium or Alcaligenes faecalis, and gels in aqueous media as nanoparticles.

[0021] The molecular cage can be selected from cyclodextrin, calixarene, crown ether, cucurbituril and mixtures thereof.

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

[0023] As calixarene, at least one of the following can be mentioned: calix[4]arene, calix[5]arene, calix[6]arene, and calix[7]arene.

[0024] As a crown ether, at least one selected from 18-crown-6 ether and 9-crown-3 ether can be mentioned.

[0025] As for cucurbituril (CB), at least one of the following can be mentioned: cucurbituril[5] (CB[5]), cucurbituril[6] (CB[6]), cucurbituril[7] (CB[7]), and cucurbituril[8] (CB[8]).

[0026] Specifically, the molecular cage is grafted onto the parastarch chain using a grafting agent. The grafting agent is covalently connected to the atoms of the parastarch chain and to the atoms of the molecular cage via another covalent bond.

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

[0028] Specifically, the above method may include the following steps: - Dissolve the parastarch and the molecular cage separately; - Mix the solution obtained in the previous step; - Add the grafting agent solution and stir the reaction mixture; - Optionally, neutralize to bring the pH back to 7; - The crude reaction mixture obtained by filtration; - Optionally, the obtained assembly solution is dehydrated.

[0029] The grafting agent may be selected from epichlorohydrin and its derivatives, 1,4-butanediol diglycidyl ether and its derivatives, epoxy haloalkanes, triazine and its derivatives.

[0030] The present invention also relates to molecular assemblies as defined above or prepared as described above, wherein at least one hydrophobic molecule is encapsulated in its molecular cage by complexation.

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

[0032] The polyphenol may be, in particular, curcumin. Curcumin can be extracted, for example, from turmeric. It can also be synthesized. Molecular assemblies encapsulating curcumin in their cages can significantly improve their bioavailability, thereby reducing the dose required to produce a therapeutic effect, especially in colorectal cancer HCT116 and HT-29 cell lines.

[0033] The present invention also relates to a method for manufacturing a molecular assembly as defined above, characterized in that the hydrophobic molecule to be encapsulated is placed in a solvent capable of dissolving the hydrophobic molecule and soluble in or miscible with water, and the resulting solution is mixed with an assembly as defined above or prepared by the method defined above.

[0034] This invention relates to the use of the above-described assembly or the assembly prepared by the above method in therapeutic, preventive, pharmaceutical, cosmetic, nutritional, agro-food, phytosanitary or veterinary applications by dispersing it in water or an aqueous solvent. Attached Figure Description

[0035] To better illustrate the subject matter of the invention, the following embodiments are described by way of illustration rather than limitation in conjunction with the accompanying drawings.

[0036] The following examples illustrate the present invention, but do not limit its scope.

[0037] Figure 1 This is a general schematic diagram of the synthesis of the assembly according to the present invention and the subsequent encapsulation of hydrophobic molecules.

[0038] Figure 2 shows the para-starch-β-cyclodextrin assembly ( Figure 2A ) and curcumin-loaded para-starch-β-cyclodextrin assemblies ( Figure 2B Dynamic light scattering (DLS) spectrum of .

[0039] Figure 3 shows the MTT assay results, which indicate the cell viability of the HCT116 cell line at 24 and 48 hours with increasing curcumin concentration ( Figure 3A ), parastarch-β-cyclodextrin assembly ( Figure 3B ) and curcumin-loaded para-starch-β-cyclodextrin assemblies ( Figure 3C (Changes).

[0040] Figure 4 shows the MTT assay results, which indicate the cell viability of the HT-29 cell line at 48 hours with increasing curcumin concentration ( Figure 4A ), parastarch-β-cyclodextrin assembly ( Figure 4B ) and curcumin-loaded para-starch-β-cyclodextrin assemblies ( Figure 4C (Changes). Detailed Implementation

[0041] Example 1: Synthesis of parastarch-β-cyclodextrin molecular assemblies Grafting of natural β-cyclodextrin onto parastarch with a molecular weight of approximately 500 kDa was carried out at room temperature with magnetic stirring (500 rpm).

[0042] For this purpose, 400 mg of parastarch and 800 mg (0.705 mmol) of β-cyclodextrin were dissolved separately in 100 ml Erlenmeyer flasks containing 25 ml of NaOH (1 M). 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 at room temperature with magnetic stirring (500 rpm).

[0043] After 24 hours of reaction, the pH of the reaction medium was adjusted to 7 by adding HCl (1 M). The crude reaction mixture was filtered through filter paper and dialyzed (2 kDa MWCO membrane) for 48 hours to remove excess unreacted natural β-cyclodextrin and epichlorohydrin.

[0044] The experimental protocol was repeated twice to obtain a mass of 556 mg for the first time and 554 mg for the second time.

[0045] After freeze-drying, the resulting product has a sponge-like structure and was characterized by DLS (dynamic light scattering).

[0046] The DLS results are shown in Figure 2. Figure 2A The dynamic light scattering pattern of the parastarch-cyclodextrin assembly is shown. The average hydrodynamic diameter is approximately 70 nm.

[0047] Example 2: Synthesis of parastarch-α-cyclodextrin molecular assemblies The procedure is performed as in Example 1, but α-cyclodextrin is used instead of β-cyclodextrin.

[0048] Therefore, 800 mg (0.617 mmol) of α-cyclodextrin was reacted with 800 mg of parastarch. The resulting reaction products had masses of 712 mg and 720 mg, respectively.

[0049] After freeze-drying, the target assembly was obtained in a sponge-like structure. It was characterized in accordance with the method described in Example 1.

[0050] Example 3: Synthesis of parastarch-γ-cyclodextrin molecular assemblies The procedure is performed as in Example 1, but γ-cyclodextrin is used instead of β-cyclodextrin.

[0051] Therefore, 400 mg (0.822 mmol) of γ-cyclodextrin was reacted with 800 mg of parastarch. The resulting reaction products had masses of 748 mg and 760 mg, respectively.

[0052] After freeze-drying, the target assembly was obtained in a sponge-like structure. It was characterized in accordance with the method described in Example 1.

[0053] Example 4: Preparation of curcumin to be encapsulated in a para-starch-cyclodextrin assembly, as well as curcumin I, demethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin III). Curcumin (referred to as curcumin I), demethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin III) were purified and separated from 0.5 g of curcumin-like powder from Sigma Aldrich by preparative thin-layer chromatography (CHCl3 / EtOH: 95 / 5).

[0054] These three curcumin compounds were used at 0.33 g (70%, inhibitory factor R) respectively. f 0.6), 0.095 g (20%, R) f 0.5 g and 0.0047 g (10%, R) f The amount obtained is 0.4).

[0055] General Encapsulation Program Encapsulation of curcumin via parastarch-cyclodextrin assemblages is based on a complexation reaction between curcumin and the hydrophobic cavities of cyclodextrin grafted onto parastarch.

[0056] To this end, 2 mg of curcumin, pre-dissolved in 0.2 mL of acetone or ethanol, was added to an Erlenmeyer flask containing 20 mg of para-amylose-cyclodextrin assembly (dissolved in 10 mL of physiological saline (0.9% NaCl)) using a micropipette. The reaction medium was then stirred using an ultrasonic bath. Finally, the solution was allowed to stand overnight without stirring and protected from light.

[0057] The obtained curcumin-loaded assemblies were purified by filtration using a Millipore filter (pore size: 0.2 μm). Curcumin was extracted from 5 ml of the complex using liquid / liquid extraction, and the encapsulation efficiency relative to the dry mass of the individual assemblies was determined by UV-Vis assay. The products were characterized by UV-Vis spectroscopy, infrared spectroscopy, and DLS.

[0058] Table 1 shows the encapsulation efficiency of curcumin I in the assembly according to Example 1 relative to the dry mass of the assembly obtained in Example 1.

[0059]

[0060] [Table 1] Encapsulation of curcumin using the para-starch-β-cyclodextrin assembly of Example 1 Figure 2B The dynamic light scattering pattern of the parastarch-cyclodextrin assembly loaded with curcumin I obtained according to Example 1 is shown. The average hydrodynamic diameter is approximately 98 nm.

[0061] By comparing these results with the DLS results obtained by Basha et al., the dynamic light scattering of gellan gum nanoparticles complexed with β-cyclodextrin allowed for the observation of an average hydrodynamic diameter of approximately 523 nm. Individual gellan gum nanoparticles have an average hydrodynamic diameter of approximately 226 nm, which is already significantly larger than the average hydrodynamic diameter of the inventive assemblies, whether or not they are loaded with curcumin.

[0062] Example 4: Decreased cell viability of HCT116 cancer cell line HCT116 cell lines were contacted with curcumin alone, the assembly according to Example 1 alone, and the assembly according to Example 1 loaded with curcumin I for 24 hours and 48 hours, respectively.

[0063] Curcumin I was introduced at concentrations of 1 μg / ml, 2 μg / ml, 5 μg / ml, 10 μg / ml, 15 μg / ml and 20 μg / ml.

[0064] Individual assemblies were introduced at concentrations of 1 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 50 μg / ml, and 100 μg / ml.

[0065] The curcumin I-loaded assemblies were introduced at concentrations of 1 μg / ml, 2 μg / ml, 5 μg / ml, 10 μg / ml, 15 μg / ml and 20 μg / ml.

[0066] Cell viability of the HCT116 cell line was measured using the MTT tetrazolium salt assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).

[0067] The results are shown in Figure 3. Figure 3A The figure shows the changes in cell viability with curcumin concentration after 24 and 48 hours. Figure 3B The figure shows the changes in cell viability with the concentration of individual assemblies after 24 and 48 hours. Figure 3C The figure shows the change in cell viability with the concentration of curcumin I-loaded assemblies after 24 and 48 hours.

[0068] like Figure 3A As can be seen, curcumin reduced the viability of HCT116 cells, with almost complete cell disappearance after 48 hours at a concentration of 10 μg / ml. The IC50 concentration was 4.38 μg / ml.

[0069] like Figure 3B As can be seen, the individual assemblies had no effect on the viability of HCT116 cells.

[0070] Figure 3C The figure shows that the curcumin-loaded assembly had an effect on HCT116 cells, with almost complete cell disappearance after 48 hours at a concentration of 10 μg / ml. The IC50 concentration was 1.62 μg / ml.

[0071] The lower IC50 of the curcumin I-loaded assembly compared to curcumin I alone indicates that the former has better bioavailability than curcumin I alone.

[0072] Example 5: Decreased viability of HT-29 cancer cell line The procedure was performed using the HT-29 cancer cell line, as described in Example 4.

[0073] The results are shown in Figure 4.

[0074] The IC50 concentration of curcumin alone was 7.38 μg / ml, while that of the curcumin-loaded assembly was 6.36 μg / ml. Similarly, the lower IC50 of the curcumin-loaded assembly compared to curcumin I alone indicates that the former has better bioavailability.

Claims

1. A molecular assembly comprising parastarch, wherein molecular cages are grafted onto the chains of the parastarch.

2. The assembly according to claim 1, characterized in that, The molecular cage is selected from cyclodextrin, calixarene, crown ether, cucurbituril and mixtures thereof.

3. The assembly according to claim 2, characterized in that, The cyclodextrin is at least one of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

4. The assembly according to any one of claims 1 to 3, characterized in that, The molecular cage is grafted onto the parastarch chain using a grafting agent. The grafting agent is covalently connected to the atoms of the parastarch chain and to the atoms of the molecular cage through another covalent bond.

5. A method for manufacturing a molecular assembly as defined in any one of claims 1 to 4, characterized in that, The parastarch, the molecular cage, and at least one grafting agent for grafting the molecular cage onto the parastarch are mixed in a solvent medium.

6. The method according to claim 5, characterized in that, It includes the following steps: - Dissolve the parastarch and the molecular cage separately; - Mix the solution obtained in the previous step; - Add the grafting agent solution and stir the reaction mixture; - Optionally, neutralize to bring the pH back to 7; - The crude reaction mixture obtained by filtration; - Optionally, the obtained assembly solution is dehydrated.

7. The method according to any one of claims 5 and 6, characterized in that, The grafting agent is selected from epichlorohydrin, 1,4-butanediol diglycidyl ether, epoxy haloalkanes, triazine and their derivatives.

8. A molecular assembly, as defined in any one of claims 1 to 4 or prepared in any one of claims 5 to 7, wherein at least one hydrophobic molecule is encapsulated in its molecular cage by complexation.

9. The assembly according to claim 8, characterized in that, The hydrophobic molecules 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 to be encapsulated is placed in a solvent capable of dissolving the hydrophobic molecule and soluble in or miscible with water, and the resulting solution is mixed with an assembly as defined in any one of claims 1 to 5 or prepared by a method as defined in any one of claims 6 and 7.

11. The use of the assembly according to any one of claims 8 and 9, or the assembly prepared by the method according to claim 10, in pharmaceutical, cosmetic, nutritional, agri-food, or phytosanitary applications by dispersion in water or an aqueous solvent.

12. The assembly according to any one of claims 8 and 9, or the assembly prepared by the method according to claim 10, for use in therapeutic, preventive, or veterinary applications by dispersion in water or an aqueous solvent.

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