COMPLEX BETWEEN LACTOFERRIN AND SILICA, PROCESS FOR THE PRODUCTION THEREOF, AND COMPOSITION THEREOF FOR ORAL HYGIENE

Abstract

Disclosed is a complex including at least one lactoferrin and at least one silica having a specific surface area included between 100 m2/g and 1000 m2/g (BET area, measured according to standard ISO 9277-2010), the complex being obtainable by adding the lactoferrin to the silica in the form of a sol-gel with an aqueous solvent, and subsequent drying of the sol-gel. The complex is particularly suitable for use in oral care products, in particular toothpastes, mouthwashes, gels, chewing gum, tablets for use in oral care, gingival dyes, and the like. The complex features a high level of stability and bio-availability, such that it has an effective, long-lasting antibacterial and antioxidant action within the oral cavity. The complex is particularly suitable for the prevention or treatment of diseases of dental apparatus, in particular for the prevention or treatment of periodontitis or peri-implantitis, preferably in patients suffering from diabetes.

Claims

1. A complex of at least one lactoferrin and at least one silica having a specific surface area comprised between 100 m.sup.2/g and 1000 m.sup.2/g (BET area, measured according to standard ISO 9277-2010), said complex being obtainable by adding said lactoferrin to said silica in the form of a sol-gel with an aqueous solvent, and subsequent drying of the sol-gel.

2. The complex according to claim 1, wherein said at least one silica is a synthetic silica, selected from fumed silicas and precipitated silicas.

3. The complex according to claim 1, wherein said at least one silica is in the form of an aerogel.

4. The complex according to claim 3, wherein said at least one silica in the form of an aerogel has a porosity greater than 85% and a density ranging from 0.003 g/cm.sup.3 to 0.8 g/cm.sup.3.

5. The complex according to claim 3, wherein said at least one silica is obtained from vegetable waste, in particular from husk deriving from the processing of grains.

6. The complex according to claim 1, wherein said at least one lactoferrin and said at least one silica are present in a weight ratio of 0.05:1 to 10:1.

7. The complex according to claim 1, in the form of particles surface treated with at least one polyol.

8. A process for preparing a complex according to claim 1, which comprises: preparing a silica having a specific surface area ranging from 100 m.sup.2/g to 1,000 m.sup.2/g, preferably from 150 m.sup.2/g to 700 m.sup.2/g (BET area, measured according to the standard ISO 9277-2010) in a sol-gel form with an aqueous solvent; adding at least one lactoferrin to the silica in a sol-gel form; subjecting the mixture thus obtained to a drying step.

9. The process according to claim 8, which further comprises treating the dried mixture by adding at least one polyol.

10. The process according to claim 8, wherein the drying step is carried out by treatment with supercritical CO2.

11. The process according to claim 8, wherein the drying step is carried out by lyophilization.

12. The process according to claim 10, wherein the drying step with supercritical CO2 is carried out in an autoclave, at a temperature ranging from 15 C. to 60 C. and a pressure ranging from 80 bars to 150 bars.

13. The process according to claim 11, wherein the drying step by means of lyophilization is carried out by cooling under vacuum at a temperature between 80 C. and 30 C. and subsequent heating, again under vacuum, at a temperature not greater than 60 C.

14. The process according to claim 13, wherein the lyophilization is carried out at a pressure between 0.1 bar and 0.8 bar.

15. A composition for oral hygiene, which comprises at least one complex according to claim 1, and at least one physiologically acceptable excipient.

16. The composition according to claim 15, wherein said at least one complex is in admixture with at least one hydrophilic silica not complexed with lactoferrin.

17. The composition according to claim 16, comprising: from 10% to 70% by weight of at least one silica; from 0.05% to 15% by weight of at least one lactoferrin; from 25% to 80% by weight of an aqueous phase.

18. The composition according to claim 15, wherein the aqueous phase comprises a mixture of water and a polyol.

19. A method for the prevention or treatment of diseases of dental apparatus, comprising locally applying an effective amount of the complex of claim 1 within an oral cavity.

20. The complex according to claim 1, wherein the at least one silica has a specific surface area comprised between 150 m.sup.2/g and 700 m.sup.2/g.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 is a plot of antioxidant effect in the form of % ROS reduction, over time, for Examples 1-3;

[0052] FIG. 2 is a plot of antibacterial effect in the form of % bacterial load reduction, over time, for Examples 1-3;

[0053] FIG. 3 is a plot of antioxidant effect in the form of % ROS reduction, over time, for Examples 4-6; and

[0054] FIG. 4 is a plot of antibacterial effect in the form of % bacterial load reduction, over time, for Examples 4-6.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] The following embodiment examples are provided for the sole purpose of illustrating the present invention and are not to be intended to limit the scope of protection defined by the appended claims.

Examples 1-3

[0056] The compositions shown in Table 1 were prepared (% by weight of the total composition):

TABLE-US-00001 TABLE 1 EXAMPLE 1 2 3 (*) glycerol 51.6 51.0 74.0 water 7.0 15.0 25.0 hydrophilic 40.0 30.0 silica rice husk 0.4 3.0 silica lactoferrin 1.0 1.0 1.0 (*) comparative [0057] Hydrophilic silica: hydrophilic fumed silica having a specific surface area of about 200 m.sup.2/g (BET area, measured according to standard ISO 9277-2010) (commercial product Aerosil 200 by Evonik); [0058] Rice husk silica: silica in the form of an aerogel having a specific surface area of about 550 m.sup.2/g (BET area, measured according to standard ISO 9277-2010) and a density of about 0.050 g/cm.sup.3.

[0059] The rice husk silica and lactoferrin were previously complexed through the following process. The rice husk silica was prepared as described in Example 1 of WO 2016/193877, with the difference that, after obtaining the silica sol-gel, this was added to the lactoferrin and then subjected to drying with supercritical CO.sub.2 as described in the same Example 1, with the exception that the silica sol-gel added with lactoferrin was treated in the autoclave at a temperature of 50 C. and a pressure of 100 bar, for a time equal to 6 hours, in order to obtain the drying of the sol-gel without causing degradation of lactoferrin.

[0060] The complex thus obtained and the other ingredients of the composition were mixed in a flask with the aid of a magnetic bar, until a homogeneous formulation was obtained. It should be noted that hydrophilic silica has been added as such, i.e. not complexed with lactoferrin.

[0061] Antioxidant and antibacterial properties of these compositions were verified, evaluating variation of efficacy over time.

[0062] Antioxidant Activity (Scavenger).

[0063] Antioxidant activity (scavenger for free radicals) of the above compositions was evaluated by measuring the ability to counteract the formation of free radicals (ROS) in cell cultures of human keratinocytes. For this purpose, keratinocytes were treated with scalar concentrations of the product (1:2 dilutions starting from 1.0 mg/ml) and then exposed to UVA rays at room temperature to stimulate ROS production. The untreated cells represent the negative control. At the same time, a neutral red uptake (NRU) assay was carried out before and after irradiation in order to verify that cell viability did not significantly decrease under the experimental conditions. The results of the NRU test before exposure to UVA rays demonstrated the absence of cell viability inhibition at all concentrations used. Therefore, all the concentrations were taken into consideration for the ROS dosing.

[0064] The results obtained by the ROS dosing demonstrated the antioxidant action, expressed as % reduction of ROS with respect to the negative control.

[0065] More in detail, the materials and operating conditions used are reported hereinbelow.

[0066] Human keratinocytes (Huker) were used, grown in DMEM (Dulbecco's Modified Eagle medium) containing 10% of foetal bovine serum (FBS) and 1% of antibiotics (penicillin and streptomycin), incubated under standard culture conditions (37 C., 5% CO.sub.2). Good cell cultivation practices were applied.

[0067] In order to simulate a condition of environmental stress capable of inducing formation of ROS, the cells were subjected to irradiation with UVA rays. The lamp used in the experiments was a sunlight simulator that reproduces the solar spectrum with a constant UVA emission range comprised between 315 nm and 400 nm and with a radiance of 1.7 mW/cm.sup.2. The emission of UVB is appropriately screened to avoid cytotoxic damage directed to cell cultures.

[0068] The effects on ROS production at different irradiation times were evaluated: 4, 8, 12, 16 and 20 min.

[0069] Each sample of the tested compositions was dissolved in water and then diluted in a growth medium to the desired final concentrations comprised between 0.0156 and 1.0 mg/ml (1:2 dilutions). 2,7-dichlorofluorescine diacetate (DCFDA) was used as a fluorimetric tracer, dissolved in dimethylsulfoxide (DMSO) at a concentration of 50 mM and then diluted in the appropriate buffer up to a concentration of use of 250 M.

[0070] Each cell sample (human keratinocytes) was seeded in 96-well plates. Once a semi-confluent monolayer was reached, the cells were washed with phosphate buffer (PBS) and then incubated with the DCFDA solution for 20 min under standard culture conditions. DCFDA was then removed, the cells were washed in PBS, treated with the different concentrations of the tested composition and with the positive control, incubated for 20 min at standard conditions and then irradiated with UVA for 4, 8, 12, 16 and 20 min. At the end of each irradiation period a measurement with a fluorometer was performed at the excitation wavelength of 485 nm and emission wavelength of 530 nm.

[0071] In order to verify that the cell viability did not vary significantly in the presence of the composition under examination and in the experimental conditions applied, the neutral red uptake (NRU) assay was performed before and after irradiation of the cells. Neutral red (NR) is a weakly cationic probe that passes through the cell membrane by nonionic passive diffusion and is concentrated in lysosomes, where it binds by electrostatic interactions to anionic sites of the matrix. The uptake of NR depends on the ability of the cells to create pH gradients, determined in turn by ATP production. The NR thus only accumulates in living cells, from which it is extracted with an acidic ethanol solution: the absorbance of the solution, measured at the spectrophotometer, is proportional to cell viability.

[0072] After gently removing the medium, the cells were treated with neutral red (NR) (50 g/ml) and incubated for 3 hours under standard conditions. The cells were subsequently washed with PBS to remove the dye residues and the NR was extracted. The plates were gently agitated for at least 10 min (to facilitate the dissolution of NR) and the absorbance (optical density, OD) was determined through spectrophotometric measurement at 540 nm. The absorbance measured at 540 nm is proportional to the cell viability. The percentages were calculated based on the absorbance values at 540 nm and taking the absorbance of the negative control (untreated cells) as 100%. In the absence of UVA irradiation, the cell viability of the keratinocytes treated with the product was comparable to that of the untreated keratinocytes for all the concentrations tested, therefore all the concentrations were taken into account in the ROS dosages.

[0073] Antimicrobial Activity.

[0074] Each composition was tested to verify the antimicrobial activity with respect to E. coli. An amount equal to 9 g of the composition was added to 1 ml of inoculum of E. coli (ATCC 8739) so as to obtain a final charge of about 10.sup.5 CFU. After increasing contact times (1, 4, 8 and 24 hours), 0.5 ml of the suspension was taken to carry out the plate count (surface spatulation method). The % variation of the bacterial count was therefore determined with respect to the initial one.

[0075] The results are shown in the following tables, where the left column shows the times (in minutes for Table 2 and in hours for Table 3). The same results are reported in the graphs attached as FIG. 1 and FIG. 2.

TABLE-US-00002 TABLE 2 (antioxidant effect: % ROS reduction) EXAMPLE 1 2 3 (*) t = 8 50 40 60 t = 12 65 65 30 t = 16 40 50 20 t = 20 25 30 10 (*) comparative

TABLE-US-00003 TABLE 3 (antibacterial effect: % bacterial load reduction) EXAMPLE 1 2 3 (*) t = 1 30 25 45 t = 4 45 35 55 t = 8 70 75 58 t = 24 70 77 60 (*) comparative

[0076] From the results obtained, it appears evident that the complex comprising lactoferrin and silica makes it possible to increase efficacy both in terms of antioxidant and antibacterial effect in a more gradual way and maintain them over time, compared to lactoferrin used alone.

Examples 4-6

[0077] Examples 2 and 3 were repeated under the same conditions and with the same ingredients shown above (Examples 4 and 5). A further comparative example (Example 6) was also carried out, again with the same procedure and the same ingredients, wherein the lactoferrin was simply mixed with the hydrophilic silica, i.e. without preparing a complex according to the present invention, wherein silica is prepared in a sol-gel form, lactoferrin is added and then dried. The compositions are reported in Table 4 (% by weight of the total composition):

TABLE-US-00004 TABLE 4 EXAMPLE 4 5 (*) 6 (*) glycerol 51.0 74.0 51.0 water 15.0 25.0 15.0 hydrophilic 30.0 33.0 silica rice husk 3.0 silica lactoferrin 1.0 1.0 1.0 (*) comparative

[0078] Antioxidant and antibacterial properties of these compositions were verified, evaluating the variation of the efficacy over time, as described in Examples 1-3.

[0079] The results are reported in the following tables, where the left column shows the times (in minutes for Table 5 and in hours for Table 6). The same results are reported in the graphs attached as FIG. 3 and FIG. 4.

TABLE-US-00005 TABLE 5 (antioxidant effect: % ROS reduction) EXAMPLE 4 5 (*) 6 (*) t = 4 35 60 55 t = 8 38 58 50 t = 12 60 35 30 t = 16 55 25 20 t = 20 48 15 10 (*) comparative

TABLE-US-00006 TABLE 6 (antibacterial effect: % bacterial load reduction) EXAMPLE 4 5 (*) 6 (*) t = 1 26 45 50 t = 4 42 55 55 t = 8 60 58 60 t = 24 77 60 63 (*) comparative

[0080] From the results obtained, it appears evident that the complex comprising lactoferrin and silica makes it possible to increase efficacy both in terms of antioxidant and antibacterial effect in a more gradual way and to maintain them over time, compared to lactoferrin used alone, as well as compared to a simple mixture of lactoferrin with silica, without forming a complex according to the present invention.