WO2018102895A1

WO2018102895A1 - Process for producing polymer membranes by electrospinning, polymer membranes containing pterodon pubescens benth and arrabidaea chica verlot extracts and uses thereof

Info

Publication number: WO2018102895A1
Application number: PCT/BR2017/000134
Authority: WO
Inventors: Tais Helena Costa SALLES; Mary Ann FOGLIO; Fabiana Volpe ZANUTTO; Ilza Maria Oliveira SOUZA; Marcos Akira D'AVLLA
Original assignee: Universidade Estadual De Campinas - Unicamp
Priority date: 2016-12-05
Filing date: 2017-11-17
Publication date: 2018-06-14
Also published as: BR102016028506B1; BR102016028506A2

Abstract

The present invention relates to a process for producing polymer membranes by electrospinning by combining Pterodon pubescens Benth and Arrabidaea chica Verlot extracts for use in guided bone and tissue regeneration, wherein the pharmacological healing and antimicrobial activities are combined with anti-inflammatory and analgesic activity, attributed to Arrabidaea chica and Pterodon pubescens, respectively.

Description

PROCESS TO. OBTAINING POLYMERIC MEMBRANES BY

ELECXROSPINNING, POLYMERIC MEMBRANES containing Pterodon pubescens Benth and Arrabidaea chica extracts. Verlot And Its Uses

[001] The present invention is in the field of engineering and pharmacy, and describes a process for obtaining polymeric membranes by electrospinning by combining Pterodoji pubescens Benth and ArraJbidaea chica Verlot extracts for application in bone and tissue regeneration guided.

BACKGROUND OF THE INVENTION

Electrospinning is a simple and convenient method for producing ultrafine fiber with micro or nanometer scale diameters. Electrophilic scaffolds have a reticular structure with high surface area to volume, high porosity and interconnected pores, which are similar to the natural extracellular matrix (ECM), features that can increase cell adhesion, proliferation and growth (Meng et al. . 2010).

The scaffolds developed by the electrospinning process can be employed as physical barriers in Guided Tissue Regeneration (RTG). The principles of RTG have been applied not only in dentistry or orthopedics, as in their more traditional form, but also in other medical specializations. Understanding the mechanisms involved is as important as checking the types of physical barriers employed at RTG, as the material adopted is related to the results obtained. The application of these membranes aims at bone tissue regeneration, promoting the repair of the region through the faithful synthesis of the original tissue and may be spontaneous or induced (associated with one or more plant extracts: Arrabiadaea chica and / or Pterodon pubescens). Currently-, this technique is extrapolated to various tissues, such as nervous, cartilaginous and connective tissue (Iamaguti et al, 2008).

[004] ArraJbiadaea chica (Humb. & Bonpl.) Verlot (Bignoniaceae), found mainly in the Amazon Region, popularly known as crajiru, carajiru, pariri and chica, is a woody liana, which has leaves that provide red pigments, carajurina and carajurone, used by the Brazilian Indians as a dye and healing agent. Traditional medicine gives the species a broad spectrum of properties, such as anti-inflammatory, astringent, and therapeutic properties, as well as its use in treating skin conditions (psoriasis, wounds, ulcers, pyoderma), leukemia, oral and uterine cancer. , as well as being used for caries prevention and as a cosmetic (Gentry, 1992; Kalil, 2000).

The genus Pterodon comprises four species native to Brazil: Pterodon abruptus Benth. , Pterodon apparucuri Pedersoli, Pterodon pubescens Benth., [Pterodon Emarginatus Vogel) and Pterodon polygalaeflorus Benth. The plant species Pterodon pubescens Benth. (Botanical synonym: Pterodon emarginatus Vog.) Is a tree of the family Leguminosae-Papillonoideae, found in the Brazilian Cerrado, mainly in the states of ... Minas Gerais, São Paulo, Goiás and Mato Grosso do Sul and popularly known as faveiro, sucupira -white, sucupira, sucupira or sucupira-lisa ^' (Lorenzi, 1998). Some authors have suggested that the vaiacapanic skeleton of furan diterperms is involved with the anti-inflammatory, anti-edematogenic, analgesic properties of Pterodon pubescens seed oil (Nunan et al., 1982; Carvalho et al., 1999; Silva et al., 2004).

Thus, three different polymeric membrane compositions obtained by the elecrospinninr process were developed, containing plant extracts Pterodon pubescens Benth (Sucupira) and / or Arrabidaea chica Verlot for applications in guided tissue regeneration, as these extracts have anti-inflammatory and healing activity. respectively. It is noteworthy that physical barriers should promote cellular response (Arrabidaea chica Verlot: stimulation of healing) and not produce an inflammatory reaction (Pterodon pubescens Benth: helps to combat the anti-inflammatory process and has analgesic properties due to the presence of Vouacapans). Therefore, it was possible to develop a membrane with the capacity to release plant active principles, with these biological activities, being these extracts used for the first time in association.

TECHNICAL STATE

EP1558444B1 describes a process and composition for producing silk biomaterials, for example fibers, films, foams. In this process at least one biocompatible polymer such as (ethylene oxide) poly (PEO) and polyethylene glycol (PEG) has been mixed with the silk protein, wherein the protein is obtained from Bombyx morl before processing, for example, electrospinning. However, this document is limited to the use of 2 possible polymers for obtaining the membranes. The use of natural polymers, specific in this case to silk fibroin, hinders their reproducibility, as it is a polymer that depends on the regional climatic conditions where the silk cocoon is manufactured and collected. Synthetic polymers used in our work, are polymers already recognized by the literature and approved by the FDA. The present invention, on the other hand, is associated with two plant extracts that have never been presented together in the literature. With the electrospinning technique employed, it becomes possible to associate plant extracts with other polymers such as poly (lactic-co-glycolic acid) (PLGA), cellulose, gelain, polyvinylpyrrolidone (PVP), poly lactic acid (PLLA) , etc., which increases the possibilities. of applications in the biomedical field.

EP2254608B1 relates to biologically active cell-free scaffolds composed of cell extract and / or extracellular compartments for use in tissue regeneration. The cells are seeded on top of scaffolds that are capable of receiving mechanical forces and have a standardized surface. These cells can be from animals, humans or plants, with application for repair and regeneration of tissues or organs. These scaffolds have been associated with extracellular and / or intracellular extracts from said cells or tissues. Scaffolds associated with cells present the difficulty of storage and stability, and difficult control of cell growth, thus obtaining scaffolds with different parameters and characteristics. The present invention has differently developed a membrane capable of releasing plant active principles with biological activities, being these extracts used for the first time in association, and which have analgesic, anti-inflammatory and healing properties, which favors tissue regeneration.

US7172765B3 describes fibrous and / or bioabsorbable biodegradable articles and methods for use in medical applications. Such articles which are formed by electrospinning fibers of biodegradable and / or bioabsorbable material, comprise a compound (or asymmetric compound) of different biodegradable and / or bioabsorbable fibers. Such articles having specific medical uses include a adhesion reducing barrier and a controlled release system. This paper presents an overview of the electrospinning method, involving the parameters for obtaining membranes for tissue regeneration. They suggest a possible association with active ingredients such as drugs, but tests were not performed with them, but only the use of polymers to manufacture the membranes. Already the present invention shows the association of Food and Drug Administration (FDA) approved synthetic polymers associated with plant extracts that have biological principles that assist in tissue regeneration, making this membrane promising for biomedical and dental applications. According to the in vitro results, there was a significant cell proliferation.

Thus, with the process described in the present invention, it was possible to develop a polymeric membrane for the purpose of regenerating bone tissue, promoting the repair of the region through the faithful synthesis of the original tissue. being induced by the association of plant extracts: Arrabiadaea chica and / or Pterodon pubescens. It is noteworthy that physical barriers should promote cellular response, since Arrabidaea chica Verlot stimulates healing) and not produce an inflammatory reaction, as Pterodon pubescens Benth helps fight the anti-inflammatory process and has analgesic properties due to the presence of I'm going to cover it. Therefore, the membrane has the ability to release plant active principles, with these biological activities, being these extracts used for the first time in association.

SUMMARY OF THE INVENTION

[011] The present invention aims to propose a process for obtaining electrospininic polymeric membranes by combining the extracts of Pterodon pubescens Benth and -Arrabidaea chica Verlot for application in guided bone and tissue regeneration.

It is an additional object polymeric membranes comprising the combination of 0.25 mg to 1.0 g Pterodon pubescens and 0.10 to 1.0 g Arrabidaea chica and at least one guided tissue regeneration polymer (RTG) .

BRIEF DESCRIPTION OF THE FIGURES

[013] FIG. 1 shows micrographs of the fibers, where A represents PCL-S; B represents PCL-C and C represents PCL-S;

[014] FIG. 2 shows absorbance x time graphs showing the evaluation of cytotoxicity and cell growth of polymeric membranes with BALB / c 3T3 plant extracts. BALB / c 3T3 were treated with different membranes for 24 h, 3 and 7 days. The results represent the means and standard deviations of the experiment. Anova I test. * P <0.05.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for obtaining electrospinning polymeric membranes comprising the following steps:

a} Collection of plant material;

a.l) from Pterodon pubescens Benth;

a.2) Arrabidaea chica Verlot;

b) Preparation of Pterodon pubescens plant extract

Benth;

B. l) crush 1kg of fruits on dry ice;

b.2) add with mechanical stirring in a 3x stainless steel tank the volume of dichloromethane;

b.3) leave in contact with the extracting solvent for 1.5 hours under agitation;

b.4) repeat steps b.1), b.2) and b.3) three times, totaling 4.5 hours of extraction;

b.5) collecting, filtering and transferring the extraction liquid to a round bottom flask and concentrated under vacuum in a rotary evaporator at 40 ° C;

(c) drying and milling of Arrabidaea chica Verlot plant material;

(d) preparation of the vegetable extract of Arrabidaea chica Verlot;

d.l) - transfer with mechanical agitation 3kg of leaves to a stainless steel tank;

d.2) use 70% ethanol, acidified with 0.3% citric acid PA, in the ratio of 1 (plant): 5 (solvent) w / v. d.3) repeat steps dl) and d.2) for three times of 90 minutes each;

d.4) filtering the resulting solution under light-protected vacuum;

d.5) concentrate the crude extract under vacuum to 80% reduction of the final volume;

d.6) neutralizing the crude extract with ammonium hydroxide to pH 6.5-7.00;

e) Preparation of electrospinning solutions:

and. l) mixing 20% mass by weight of 80000 g / mol molar polymer, 80% mass of acetone and the plant extracts obtained in (b) and (c) by stirring for at least 3 hours at 23 and 28 ° C;

e.2) electrophyte at a temperature ranging from 23 to 28 ° C, a humidity ranging from 52 to 75%, a flow rate ranging from 0.5 to 5 mL / h and a voltage range from 10 to 30 kV and a variable needle tip distance to the collecting plate between 10 and 20 cm.

It is an additional object polymeric membranes comprising the combination of 0.25 mg to 1.0 g Pterodon pubescens and 0.10 to 1.0 g Arrabidaea chica and at least one polymer for guided tissue regeneration (RTG). ).

[017] Polymeric membranes optionally comprise:

humectant is selected from the group consisting of propylene glycol and glycerin, preferably glycerin;

nonionic surfactant is selected from the group consisting of polysorbate 80 and triton X-100, preferably triton X-100; - permeation promoter is selected from the group consisting of ethoxydiglycol and 1-methyl-2-pyrrolidone, preferably 1-methyl-2-pyrrolidone; and

The solvent mixture is selected from the group consisting of dichloromethane, DMF, ethyl alcohol, acetic acid or formic acid water, sodium chloride solution, acetone and chloroform, preferably acetone and chloroform.

Vegetable Material Collection

From Pterodon pubescens Benth. (Sucupira)

[018] The fruits of Pterodon pubescens were collected and identified by Prof. Dr. Jorge Yoshio Tamashiro from the Botanical Department of IB-UNICAMP. The exsiccates were deposited in the Herbarium of the Institute of Biology of the State University of Campinas under the voucher 179739. Thus, regardless of the region where the fruit is collected or the species of it, it is possible to incorporate the plant extract to the polymeric membrane via the process. electrospinning.

From Arrabidaea chica Verlot

[019] The leaves of the A. chica creeper were collected and identified by Dr. Glyn Mara Figueira. An exsicata was deposited in the Herbarium of CPQBA / Unicamp under No. 1865. Thus, regardless of the region from which the fruit is collected or the species thereof, it is possible to incorporate the plant extract into the polymeric membrane via the electrospinning process.

Preparation of Plant Extracts

From Pterodon pubescens Benth. (Sucupira) [020] To obtain the sucupira extract, the fruits (lkg) were first crushed with dry ice to increase the contact surface and consequently to obtain a higher extraction yield. Then the ground fruits were transferred to a stainless steel tank (5 liters capacity) with mechanical stirring and under them was added 3 times the volume of dichloromethane (3 liters). The plant material was left in contact with the extracting solvent for 1.5 hours while stirring. This procedure was repeated 3 times, totaling a period of 4.5 hours of extraction. After this period, the extracting liquid was collected, filtered, transferred to a round bottom flask and concentrated under vacuum in a rotary evaporator (Buchi® RE 120) at 40 ° C. At the end of the procedure, a total of 450g of sucupira extract was obtained and stored in glass bottles and stored at room temperature.

From Arrabidaea chica Verlot

[021] Prior to the preparation of the Arrabideae chica plant extract, it is first necessary to dry the leaves for 48 hours in an oven at 40 ° C with forced ventilation and then grind in a 40 mesh sieve hammer mill. The dry and molten material was packaged in laminated bags made of polypropylene inner film, coated with an outer opaque aluminum layer to protect the plant material from oxidative action from light, oxygen and moisture.

After drying and milling, 3 kg of leaves were weighed and transferred to a 50 liter stainless steel tank with mechanical stirring. The crude extract was obtained using 70% ethanol (technical grade), acidified with 0.3%. of citric acid PA at a ratio of 1 (plant): 5 (solvent) w / v. The process was repeated 3 times of 90 minutes each. The resulting solution was filtered under vacuum protected from light. The crude extract was concentrated under vacuum to 80% reduction of the final volume, this extract was neutralized with ammonium hydroxide to pH 6.5-7.00.

Preparation of electrospinning solutions

All solutions were stirred for three hours at 23 ° C subjected to the electrospinning process on the same day of preparation. Table 1 shows the composition of the electrophobic membranes (mass%).

Table 1:

S: Sucupira; C: Arrabidaea chica

Electrospinning

The electrospinning system used in the present invention consisted of a 10 mL syringe and a 0.55 mm diameter needle, an infusion pump, a high voltage source and a grounded collection plate.

[025] The solutions were spun at room temperature at 25 ° C and 52% humidity, with a flow rate of 2 mL / h and voltages of 12 to 13 kV. The distance from the needle tip to the collecting plate was 12 cm.

Characterization of polymeric membranes obtained

[026] Membrane morphology was assessed by scanning electron microscopy (SEM). For observation by SEM, the samples were coated in gold. The diameters The mean fibers were determined by SEM image analysis using the image analysis program, where the diameters of 50 fibers of each randomly chosen sample were measured.

The SEM micrographs reveal, for the adopted conditions, the formation of a dense network of fibers with a cylindrical morphology, without the presence of defects like beads structure that are commonly observed in electrospinning nanofibers. The average fiber diameter of PCL-S, PCL-C and PCL-SC was 0.45 (± 0.12) one, 1.25

(± 0.53) one and 1.91 (± 0.7) one respectively.

Cell Culture

Non-carcinogenic established strain BALB / c 3T3 (murine fibroblast) was used. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (SFB) and 1% antibiotic (penicillin and streptomycin). Cells were plated in 75 cm ² bottles at a density of 2 x 10 cells / ml, ^and incubated at 37 C under humid atmosphere with 5% CO2.

Cytotoxicity Assays

Cell Viability Assay Through Reduction of MTT ((4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-tetrazolium 3-Jaromide).

In that assay, cell viability was assessed by measuring the ability of cells to reduce MTT ((4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-tetrazolium) 3-bromide to formazan. Formazan is an insoluble pigment that is extracted from cells and spectrophotornetrically quantified at λ = 570 nm. MTT reduction is mainly catalyzed by mitochondrial dehydrogenases and also cytoplasm. Therefore, alteration of mitochondrial function may be detected by varying the MTT reduction capacity.

Cells were plated at a density of 6 x 10 ³⁴ cells / ml in 24-well plates that already contained laminates with or without biomaterial being studied. Subsequently, 5% CO ₂ was incubated at 37 ° C for 24h. After 24h incubation, the medium was removed, the wells were washed with phosphate buffered saline (PBS) and added to the serum free medium containing MTT dye (0.5 mg / mL). After incubation for 3h at 37 ° C, the medium was carefully removed and 100 µl of ethanol added for solubilization of formazan. The plates were shaken for 5 minutes and the absorbance corresponding to each well was read in the plate reader at λ = 570 nm. Values were expressed as percentages of MTT reduction relative to control, where cells were not exposed to test agents (Mosmann, 1983).

[006] FIG. 2 shows the results of cytotoxicity and cell growth, which represents the progressive growth of 3T3 cells grown on the PCL scaffolds associated with plant extracts over time 24 hours, 3 days and 7 days. It can be observed from the cell viability results that the membranes present biocompatibility in all compounds when exposed to the 3T3 lineage and there was significant difference in cell proliferation, evaluated by the MTT method, between the extracts analyzed. All membranes were analyzed for the control group and the control vs. control group was observed. PCL-S showed significant difference only in the 7 day time interval (p <0.01). For the PCL-C group. control group there was a significant difference (p <0.001) in the time intervals of 3 and 7 days. The PCL-SC group presented better results in all time intervals analyzed (p <0.01) for 24 hours and p <0.001 for 3 and 7 days.

[008] In vitro and in vivo studies have demonstrated the healing capacity of Arrabidaea chica crude extract with increased fibroblast production, trypanocidal action, in vitro antifungal action and antimicrobial activity (Barbosa et al., 2008; Jorge et al, 2010 ; Hofling et al., 2010). In the present invention, significant growth of fibroblasts in the Arrabidaea chica extract membrane was observed (FIG. 2). The induction of fibroblast growth and, consequently, the healing action of the extracts may be related to the presence of anthocyanins (Taffarello et al., 2013), which proves that the extract was released into the environment stimulating the growth of lineage fibroblasts. 3Q3. The association of the two plant extracts favored the stimulation of fibroblast proliferation by 148% higher than the positive control FIG. 2.

[009] Studies in the literature confirm the antinoceptive and anti-inflammatory activities of Pterodon pubescens extract, which are attributed to the presence of geranilgeraniol and voaucapanes alone (Spindola et al., 2010; Spindola et al., 2011). [010] In the study by Vieira et al, 2008 it was shown that activities in the cell viability assay with alcoholic extract from Pterodon pubescens seeds at a concentration of 30 mg / mL exerted an antitumor activity and could kill cells. Note that in the present study, the concentration of sucupira extract used in the polymeric membranes (Table 1) did not show cltotoxicity and stimulated significant cell growth over 7 days (p <0.01) (FIG. 2).

Statistical test

[011] After data collection and variable categorization, the values were transferred to the GraphPad Prism 5 program. Statistical analyzes of the results were analyzed by applying the statistical tests. Variance Analysis (ANOVA) + Multiple Comparison with the Tukey test. A significance level of 5% was considered, ie, the data were statistically significant for p <0.05.

Thus, according to the process described in the present invention under the conditions adopted in the process, membranes containing plant extracts were successfully obtained, showing no cytotoxic effects. This demonstrated that this membrane has potential application for Guided Tissue Regeneration (RTG) and may be a new alternative in the market with lower cost. The association of the extracts is being applied for the first time, where it combines the pharmacological activities of healing action and antimicrobial with anti-inflammatory and analgesic action, attributed respectively to Arrabidaea chica and Pterodon pubescens.

Claims

Process for obtaining polymeric poly (ε-caprolactone) (PCL) membranes characterized by being electrospinning and comprising the following steps:

(a) collection of plant material;

b) Preparation of Pterodon pubescens plant extract

Benth;

(c) drying and milling of Arrabidaea chica Verlot plant material;

(d) preparation of the vegetable extract of Arrabidaea chica Verlot; and

e) Preparation of electrospinning solutions.

Process according to Claim 1, characterized in that the plant material collected from Pterodon pubescens Benth is the fruit and Arrabidaea chica Verlot is the leaf.

Process according to Claim 1, characterized in that step b) further comprises: b.l) crushing 1kg of the fruits on dry ice;

b.4) repeat steps b.l), b.2) and b.3) three times, totaling 4.5 hours of extraction;

b.5) collect, filter and transfer the extraction liquid to a round bottom flask and concentrate under vacuum in a rotary evaporator at 40 ° C.

Process according to Claim 1, characterized in that step c) comprises drying the leaves for 48 hours in an oven at 40 ° C with forced ventilation and then grinding in a 40 mesh sieve hammer mill.

Process according to Claim 1, characterized in that step d) further comprises: d.l) transferring with mechanical stirring 3kg of sheets to a stainless steel tank;

d.2) use 70% ethanol, acidified with 0.3% P.A citric acid, in the ratio of 1 (plant): 5 (solvent) w / v.

d.3) repeat steps d.l) and d.2) for three times of 90 minutes each;

d.4) filtering the resulting solution under light-protected vacuum;

d.6) neutralize the crude extract with ammonium hydroxide to pH 6.5-7.00.

Process according to Claim 1, characterized in that step e) further comprises: e.1) mixing 20% mass of 80000 g / mol molar mass polymer, 80% acetone mass and extracts vegetables obtained in (b) and (c) by stirring for at least 3 hours at 23 and 28 ° C;

e.2) electrophyte at a variable temperature between 23 and 28ºC, a variable humidity between 52 and 75%, with a variable flow rate between 0.5 and 5 mL / h and variable voltages between 10 and 30 kV and with a variable distance from the tip of the needle to the plate. collector between 10 and 20 cm.

7. Polymeric membranes characterized by the fact obtainable by the process as defined in any one of claims 1 to 5, comprise the combination of 0.25 mg to 1.0 g Pterodon pubescens and 0.10 to 1.0 g Arrabidaea chica and at least one polymer.

Polymeric membranes according to claim 7, characterized in that the polymer is selected from the group consisting of poly (lactic acid glycolic acid), poly (glycolic acid), polybenzimidazole, polycarbonate, polyurethane, polystyrene, oxide. polyethylene, polyvinyl alcohol, collagen, fibroin, hyaluronic acid, gelatin, poly (L-lactic acid), polyvinylpyrrolidone, polyhydroxybutyrate, chitin, chitosan and polycaprolactone (PCL), preferably PCL.

Polymeric membranes according to claim 7, characterized in that the average fiber diameter is 0.45 (± 0.12) µm.

Membranes according to claims 7 to 9, characterized in that it optionally comprises:

nonionic surfactant is selected from the group consisting of polysorbate 80 and triton X-100, preferably triton X-100;

- permeation promoter is selected from the group consisting of ethoxydiglycol and 1-methyl-2-pyrrolidone, preferably 1-methyl-2-pyrrolidone; and

- solvent mixture is selected from the group consisting of dichloromethane, DMF, ethyl alcohol, acetic acid or formic acid water, sodium chloride solution, acetone and chloroform, preferably acetone. and chloroform.

Use of the polymeric membranes as defined in any one of claims 7 to 10, characterized in that they are for guided tissue regeneration (RTG).