BR102021002053A2 - PROCESS FOR OBTAINING THE NANOCELLULOSE MEMBRANE (NANOVIT) AS A VITAMIN D3 RELEASE SYSTEM, MEMBRANE (NANOVIT) AND ITS USE AS A VITAMIN D3 RELEASE SYSTEM - Google Patents

Abstract

The present patent application refers to a membrane (1) of crystalline nanocellulose coupled to an adhesive material, (2) the process of obtaining said membrane and (3) its use as a controlled release system of substances, such as vitamin D. Epidemiological studies show that a significant portion of the world's population, regardless of age, ethnicity and geographic location, has low serum levels of vitamin D3. In this context, the incorporation of vitamin D3 in new drug delivery systems, such as transdermal systems, have been pointed out as an alternative for the administration of this drug. The membranes were characterized by high resolution electron microscopy (SEM-FEG), Fourier transform infrared spectroscopy (FT-IR), dynamometer tensile test and drug release kinetics by ultraviolet visible molecular spectroscopy (UV- vis). The membrane showed reproducibility in the synthesis, excellent physical properties such as flexibility, transparency and elasticity, as well as adequate strength for use in the tensile test for biomedical application. The release of vitamin D3 in alcoholic solution occurred in an increasing and fast way, with values of 3029 IU mL-1 of vitamin D3 in 60 minutes. In this way, this study revealed the possibility of using the membrane in the sustained release of vitamin D3, obtaining a new bionanomaterial with conditions of application in the release of this drug, which may in the future become a new model of supplementation for people with vitamin D3 deficiency.

Description

PROCESS FOR OBTAINING THE NANOCELLULOSE MEMBRANE (NANOVIT) AS A VITAMIN D3 RELEASE SYSTEM, MEMBRANE (NANOVIT) AND ITS USE AS A VITAMIN D3 RELEASE SYSTEM

[001] The present patent application refers to the process of obtaining the membrane (NANOVIT) as a vitamin D3 release system, as well as the crystalline nanocellulose membrane coupled to an adhesive material and its use as a controlled release system substances, such as vitamin D3.

APPLICATION FIELD

[002] The present invention patent application demonstrates the innovation in the technical field related to the pharmaceutical industry, where the main application is in the release of vitamin D3 in individuals with vitamin D deficiency.

STATUS OF THE TECHNIQUE

[003] In recent years, vitamin D3 has received increased attention due to the resurgence of vitamin D3 deficiency and rickets as a global health issue. Epidemiological studies show low levels of vitamin D3 in the entire world population, regardless of age, education or race. Numerous formulations, such as liposomes, lipid emulsions and mixed micelles, have been created for the administration of vitamin D3. However, these formulations are unstable and degradation and oxidation easily occur during storage. Thus, the incorporation of vitamin D3 in new drug delivery systems, such as the bionanomaterial that is proposed in this patent application, may become an alternative for the administration of this drug.

[004] A study was conducted with the aim of exploring the assessment of whether transdermal administration of vitamin D is feasible. On 50 female medical students, this study was carried out. Age, weight and height were taken, a detailed history and clinical examination were performed. Blood was collected for the level of 25 hydroxy vitamin D3 (25OHD). Two women had >30 ng/mL 25OHD and were excluded from the study. Participants were divided into two groups of 24 in each arm. All participants mistakenly agreed not to change their eating habits and lifestyle until the end of the study. The women's study group was invited to apply; Top-D (Aloe Vera-based Vitamin D3) (Patency Pending) was developed at King Fahd University Hospital, AlKhobar, with each gram of Top-D cream delivering 5,000 IU of Vitamin D3. The second group used 1 gram of Aloe vera gel. Participants did not know which group they belong to. A second blood sample was collected at the end of 3 months and the data were analyzed. Data from 48 women were available for analysis. The mean age was 22.58 ± 1.95 years. The mean pre-treatment 25OHD in the study group was 12.05 ng/ml ± 6.54 and post-treatment was 37.95 ng/ml ± 6.43 (P = 0.001, CI <28.582). In the control group, pre-treatment 25OHD was 11.4 ng/mL ± 3.97 and post-treatment was 10.58ng/mL ± 3.03 (Mir Sadat-Ali et al, 2014). Said study is a study on topical application in cream, unlike the present invention which comprises a sustained-release patch, with gradual release.

[005] The biocompatibility of nanofibrillated cellulose (NFC) hydrogels has been evaluated by in vitro cell studies keeping in mind the potential use as a novel wound dressing. NFC underwent TEMPO-mediated oxidation and was cross-linked by Ca2+ to create an autonomous hydrogel that was tested with human dermal fibroblasts and keratinocytes. NFC hydrogels and compounds with collagen or kaolin were included in the study, as well as the commercial Aquacel dressing as reference material. The effect of materials on cell viability and migration was studied by measuring cell metabolic activity, investigating cell morphologies and performing a migration assay. Cells on NFC hydrogels had increased proliferation and loudness morphologies than cells grown on Aquacel, while cell migration was similar to that of cells on Aquacel. NFC hydrogels show potential to become new wound dressing materials and further studies will investigate their biocompatibilities further (Gunta Celma, 2017). The present study presents similarities, but differences in the composition of the membrane, which is nanofibrillated nanocellulose, while the present invention consists of crystalline nanocellulose with another obtaining process. Furthermore, the membrane is cross-linked with Calcium, while in the present invention the membrane is not cross-linked, but has improved elasticity with PVA, which is biocompatible, in addition to the use of particle-carrying surfactant.

[006] The present study is about wound cauterization and not about the release of vitamin D via topical administration. In the present invention, vitamin D would be used as a supplement in case of low content, especially for elderly people who forget to take their medication, or who have other medications in their routine. Due to the compatibility of nanocellulose with the skin, it could also be used for healing. Nanocellulose is cellulose in the form of nanostructures, that is, characteristics not exceeding 100 nm, at least in one dimension. These nanostructures include nanofibrils, found in bacterial cellulose; nanofibers, present particularly in electrospun matrices; and nanowhiskers, nanocrystals, nanorods and nanoballs. These structures can be further assembled into larger two-dimensional (2D) and three-dimensional (3D) nano, micro and macrostructures, such as nanoplates, membranes, films, microparticles and macroscopic porous matrices. There are four main sources of nanocellulose: bacteria (Gluconacetobacter), plants (trees, shrubs, herbs), algae (Cladophora) and animals (Tunicata). Nanocellulose has emerged for a wide range of industrial, technological and biomedical applications, namely for adsorption, ultrafiltration, packaging, conservation of historical artifacts, thermal insulation and fire retardation, energy extraction and storage, acoustics, sensory, controlled drug delivery and , particularly for tissue engineering. Nanocellulose holds promise for use in scaffolding to engineer blood vessels, neural tissue, bone, cartilage, liver, adipose tissue, urethra and dura mater, to repair connective tissue and congenital heart defects, and to build contact lenses and protective barriers. . This review is focused on the applications of nanocellulose in skin tissue engineering and wound healing as a scaffold for cell growth, for wound cell delivery and as a material for advanced wound dressings along with drug delivery, transparency and sensory. The potential cytotoxicity and immunogenicity of nanocellulose are also discussed (Lucie Bacakova et al, 2019).

OBJECTIVES OF THE INVENTION

[007] Thus, the objective of the present patent application is to develop a bioproduct as a new tool for the release of vitamin D3, or even the incorporation of new drugs in this system. The membrane can be used in cosmetology as a new method of treating injuries caused by skin aging and as it has characteristics of a biocompatible material, it can be used as an alternative for skin replacement in patients with second and third degree burns.

DESCRIPTION OF THE INVENTION AND DRAWINGS

[008] The present invention patent application will be better explained by the presentation of the methodology, tables and figures. The invention has been divided into steps which will be further explained below.

[009] The method of obtaining the nanocellulose membrane with vitamin D3 includes the following steps.
- Obtaining the materials being: Nanocellulose, material where at least one dimension is smaller than 100 nm, whose source material is from a natural and biodegradable source, such as cotton lint or eucalyptus wood. It is important to note that the nanocellulose used in the production of this membrane was obtained by a chemical process through acid hydrolysis of cotton lint, but nothing prevents the process of obtaining it from being mechanical. The objective here is not the process of obtaining nanocellulose, but the bioproduct obtained.

[010] The other reagents used were purchased commercially: Vitamin D3 (Fagron® (Fabr/Orig: Zhejiang Garden Biochemical High-Tech, lot C20170603A-5): crystalline powder, whitish, with an initial concentration of 40,000,000 International Units (IU ) per gram; liquid glycerin (Synth®), used to solubilize vitamin D3; tween 80 (Dinâmica®): Surfactant used to promote the solubility of hydrophobic substances in water, such as vitamin D3; Polyvinyl alcohol-PVAl (Dinâmica®, Lot 94332): water-soluble synthetic polymer used to increase the viscosity of solutions Used as a skin permeation agent Ultrapure water (resistivity < 18.3 MΩ/cm) was used to prepare the solutions.

tracionado, Δ ℓ - alongamento sofrido, ℓ comprimento final do corpo de prova após ser tracionado.[011] The characteristics of the nanovit membrane are in the fact that the nanovit membrane has a diameter of 93.0 ± 1.4 mm, 3.75 ± 0.35 mm in thickness and 12.10 ± 1.27 g of mass . Mechanical tests of traction and elongation suffered by the control membranes and nanovit membrane, the Young's modulus, the specific deformation and the force necessary for the rupture of the membranes were carried out. Table 1 presents the results.

tension, Δ ℓ - elongation suffered, ℓ final length of the specimen after being tensioned.

[012] The analysis of the tensile test data suggest that the chemical composition of the membranes influences the elongation and in particular the presence of VITD3 leads to an increase in the elasticity of the material. It is noted in Table 4 that the nanovit membrane showed elongation values of 40.8 mm with a standard deviation of 2.76, higher than the control membrane with 27.1 mm with a standard deviation of 1.87.

[013] The Young's modulus, indicates the intrinsic stiffness of the material. The nanovit membrane had an average value of 0.46 MPa, with a standard deviation of 0.02, while the Young's modulus of the control membrane averaged 0.40 MPa, with a standard deviation of 0.05. The higher the Young's modulus, the greater the deformation resistance of the material. The percentage of deformation of the material containing vitamin D3 was 64.7%. Once a tension was applied to the material, it was plastically deformed, not returning to its original shape. Polymers, such as cellulose membranes, present an intermediate behavior between materials with elastic characteristics and liquids with high viscosity (HERCULANO, 2014).

[014] The mechanical tests to which the samples were submitted revealed a hypothesis that the drug added to the polymeric matrix of the nanovit membrane can positively influence the resistance and specific deformation of the material in relation to the control membrane. The drug can theoretically provide an increase in cross-linking, an increase in the interaction between the chains by van der Walls forces and hydrogen bonds, forming a three-dimensional network within the material, strengthening the membrane. The formation of crosslinks has a great effect on the mechanical properties of polymeric materials. In controlled release technology, it is very important that the mechanical characteristics of the materials are well known, as the strength of the bonds between the components present in the membranes influences the local drug delivery systems. Matrices with cross-links between the polymer chains allow the membranes to absorb more water without dissolving and thereby adhering to application surfaces, thus allowing for longer drug release periods.

[015] In compressive strength tests, the force, at a constant strain rate, required to compress or break a material placed between two controlled parallel plates is determined. It is observed in Table 2 that the nanovit membranes presented higher values of compressive strength than the control membranes. In the tear strength test, where the force required to break the membrane is determined, it is clear that membranes rich in VITD3 have greater strength.

[016] Analyzing the mechanical characteristics found in the membranes, it can be said that the addition of the active component provided an improvement in the mechanical strength of the nanovit membrane.

[017] Preparation and obtaining of the nanovit Membrane: According to Table 1, two types of membranes were produced in this study. Being a membrane containing VITD3 and a control membrane without the addition of the active ingredient. A membrane with a concentration of 40,000 IU mL-1 of VITD3, called nanovit membrane, was produced. In the membrane called control, glycerin was added without the addition of the drug.

[018] Liquid glycerin was used as a solubilizing agent for VITD3 and as a plasticizing agent in the mixture to enhance membrane formation. For the synthesis of membranes with the active ingredient, a solution containing liquid glycerin and vitamin was prepared. 5.0 g of a 1:50 diluted solution of VITD3 in absolute ethyl alcohol was weighed and added to 30 ml of liquid glycerin (v/v). So having 4,000,000 IU of vitamin in this solution. To weigh the compounds, a Marte/Shimadzu® analytical balance, with a precision of 0.0001g, model AW 220, was used.

[019] The components were processed according to the "casting" technique, which consists of preparing a solution capable of forming membranes, according to the methodology of Fakhouri et al. (2007).

[020] Figure 1 shows a flowchart of the technique used to produce the nanocellulose membrane plus the active component vitamin D3. 15 ml of nanocellulose (NC) (1) was measured and added to 50 ml of 5% PVAL solution (2). This solution was subjected to sonification for 60 min (3) for a complete dissolution of the nanocellulose, in an ultrasonic sonicator, Ultronique®, Ecosonics, with a power of 500 W, a frequency of 20 khz and a titanium macrotip with a diameter of 13 mm. After these procedures, the PVAL and NC solutions were mixed with glycerin solutions with different concentrations of VITD3 (4). After these steps, the emulsifier tween 80 (5) was added to all solutions as a surface-active agent. Then, the sonication of this solution was performed for 60 minutes (3). The solutions (6A and 6B) were placed in petri dishes (7) and left at room temperature for evaporation for 48 hours (8). Then they were left in an oven for another 24 hours at 60°C (9).

[020] To assess whether the nanovit membrane can be applied in drug delivery, the receiver liquid was used whose optical response was ideal, due to the identification procedure used, the UV-Vis molecular absorption spectrometry. The recipient liquid chosen was absolute ethyl alcohol. Release into the receptor fluid indicates that the drug has been incorporated into the membrane and that it can be released as a function of time. Indicating the possibility of application of the bionanomaterial developed in prolonged/sustained release of the drug.

[021] The nanovit membrane showed 3 distinct phases of drug release, initially the drug was released on the surface of the material, in 5 min where it reached a concentration of 1066 IU mL-1; then, between 5 min and 30 min, the drug is released, incorporated into the internal region of the membrane (“bulk”), with values between 1066 IU ml-1 and 2965 IU ml-1 ; and finally, the membrane reached a plateau of VITD3 release with constant values with a maximum peak of 3063 IU mL-1, indicating that the membrane showed prolonged/sustained release characteristics.

[022] Figures 2A, 2B and 2C present the general aspects and visual characterization of the nanocellulose membrane.
Figure 3 presents the mechanical tensile and elongation test of nanocellulose membranes.
Figure 4 shows the kinetics of Vitamin D3 release in ethyl alcohol using the nanovit membrane.
Figure 5 shows the electron microscopy (FEG-SEM) analysis of the nanovit membrane with a 25,000-fold magnification before release into receptor systems and 50,000-fold magnification after drug release into the receptor system.

[023] It is important to emphasize that the laboratory experiments are carried out in receiver liquids with fixed volume, where there is saturation of the drug in the solution, being used only as a method of verification of the release. There is no saturation effect on the skin, considering that vitamin D3, when in contact with the skin, is metabolized by the living organism.

Claims (4)

"PROCESS FOR OBTAINING THE NANOCELLULOSE MEMBRANE (NANOVIT) AS A VITAMIN D3 RELEASE SYSTEM", said process for obtaining the membrane, characterized by comprising the following steps:

• - liquid glycerin as a solubilizing agent for VITD3 and as a plasticizing agent in the mixture to enhance membrane formation (membrane without the active ingredient);
• - liquid glycerin and vitamin (active ingredient): in which 5.0 g of a 1:50 diluted solution of VITD3 in absolute ethyl alcohol was weighed and added to 30 ml of liquid glycerin (v/v); wherein thus having 4,000,000 IU of vitamin in this solution;
• - in which the composition of the membranes (with and without active ingredient) is composed of PVAL 5% (50)/Nanocellulose (NC) (15) (v/v) in mL;
• - in which the processing of components by “casting”;
• - in which the solution was subjected to sonication for 60 min (3) for a complete dissolution of the nanocellulose, with a power of 500 W, a frequency of 20 khz and a titanium macrotip with a diameter of 13 mm;
• - in which the glycerin solutions with different concentrations of VITD3 (4) were mixed with the 5% PVAL and NC solutions;
• - in which 5mL of the tween 80 emulsifier (5) was added to all solutions as a surface-active agent;
• - in which the sonication of this solution was carried out for 60 minutes (3);
• - in which the solutions (6A and 6B) were placed in petri dishes (7) and left at room temperature for evaporation for 48 hours (8);
• - in which the solutions (6A and 6B) were left in an oven for another 24 hours at 60°C (9).

"NANOCELLULOSE MEMBRANE (NANOVIT) AS A VITAMIN D3 RELEASE SYSTEM", said membrane characterized by being prepared by the process presented in claim 1. "NANOCELLULOSE MEMBRANE (NANOVIT) AS A VITAMIN D3 RELEASE SYSTEM", according to claim 2, characterized in that it can be applied in drug delivery, using the receiver liquid, absolute ethyl alcohol. "USE OF NANOCELLULOSE MEMBRANE (NANOVIT) AS A VITAMIN D3 RELEASE SYSTEM", according to claims 2 and 3, characterized by the fact that the drug release on the surface of the material, in 5 min, reached a concentration of 1066 IU mL-1; then, between 5 min and 30 min, the drug is released, incorporated into the internal region of the membrane (“bulk”), with values between 1066 IU ml-1 and 2965 IU ml-1 ; and finally, the membrane reached a plateau of VITD3 release with constant values with a maximum peak of 3063 IU mL-1, indicating that the membrane showed prolonged/sustained release characteristics.

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