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    • 专利摘要:
    Abstract. The present invention provides hybrid nanofibers comprising a mixture of the components of an aloe vera gel and a synthetic polymer selected from poly-3-hydroxybutyrate-co-3-hydroxyvalerate (phbv), poly-l-lactic acid (plla) and polydioxanone (pds), and where the average diameter of the hybrid nanofibers is between 0.3 and 1.5 microns. Also, the present invention provides a method of manufacturing said hybrid nanofibers. The hybrid nanofibers of the invention can be used in tubular prostheses of nerve reconnection after axotomy of peripheral nerves, in dressings and sutures to improve the reinnervation, and therefore the sensory recovery, of lesions in sensory organs such as the skin (eg burns, ulcers, surgical incisions, etc.) among others. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2579161A2
    申请号:ES201600173
    申请日:2016-03-08
    公开日:2016-08-05
    发明作者:Maximina MONZÓN MAYOR；María Del Mar ROMERO ALEMÁN
    申请人:Universidad de las Palmas de Gran Canaria；
    IPC主号:
    专利说明:

    Aloe Vera hybrid nanofibers.
    Field of the Invention
    The present invention belongs to the field of tissue engineering. In particular, it refers to the application of aloe vera hybrid nanofibers for nerve regeneration.
    Background of the invention
    After traumatic injuries, the success of axonal regrowth in both the Central Nervous System (CNS) and in wide spaces between peripheral nerve lines, and functional reinnervation in the target tissues as a result of CNS lesions, peripheral nerves or local wounds (eg wounds cutaneous) is currently a challenge in the field of regenerative biomedicine. The peripheral nerves have a capacity for spontaneous regrowth provided that contact between the nerve ends is restored as the Schwann cells of the distal nerve end offer a favorable microenvironment. However, the structure and function of the regenerated nerves differs from normal health conditions. In addition, the re-preservation of the target organs is usually clinically disappointing with significant and persistent functional deficits.
    Until now, the star procedure for the regeneration of peripheral nerves consists in replacing the damaged area with autologous and heterologous tissue transplants. However, these have important limitations such as the reduced availability of the former and the possibility of immunological rejection of the latter. As an alternative, the use of natural and synthetic materials that reproduce the natural micrometric and natural nanometric organization of the extracellular matrix of healthy tissues and provide an optimal microenvironment for cell adhesion, growth, proliferation and differentiation has been proposed.
    The literature recognizes the usefulness of synthetic polymers such as poly3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) and poly-L-Ictic acid (PLLA) in axonal regrowth [PLLA (Corey el al., J. Biomed. Mater Res. A 2007, 83 (3) 636-645; Wang el al., J. Neural Eng. 2009, 6 (1), 016001), PHBV (Masaeli el al., 2013, PLoS One 8 (2) e57157 ), Prabhakaran al. , 2013, Biotechnol. Bioeng. 11 0 (1 0) 2775-84)]. In addition, I Uslu el al (Hacettepe J. Biol. & Chem., 2010, 38 (1)) discloses the use of aloe vera hybrid nanofibers with synthetic polymers of polyvinyl alcohol / polyvinylpyrrolidone! polyethylene glycol for wound dressing. Gupta al. (J. Biomater. Tissue Eng., 2013, 3 (5) 503-11) disclose hybrid nanofibers of aloe vera, polyvinyl alcohol, polyethylene oxide and carboxymethyl cellulose. Jithendra the al. (ACS Appl. Malter. Interfaces, 2013. 5, 7291-8) disclose nanofibers of aloe vera, collagen and chitosan for tissue engineering. Shanmugavel al. (J. Biomalter. Appl., 2013, 29 (1) 46-58) disclose nanofibers of aloe vera, silk fibroin and caprolactone for bone tissue engineering. Sungaya al, 2014 disclose nanofibers of aloe vera, silk fibroin and hydroxyapatite for osteogenesis. None of these documents mention structures that are efficient in the growth and regeneration of nerve tissue.
    Therefore, there is a need in the state of the art to achieve new structures that are more efficient in nerve regeneration, in particular in the functional reinnervation in target tissues, peripheral nerves or local wounds.
    Description of the invention
    The object of the present invention is to provide structures that allow nerve reconnection after axotomy of peripheral nerves and to improve the sensory recovery of lesions in sensory organs such as the skin (eg burns, ulcers, surgical incisions, etc.) among others, the Hybrid nanofibers provided in the present invention promote nerve regeneration by additionally serving as a structural support for nerve tissue growth. In addition, the biocompatible nature of the hybrid nanofibers of the invention prevents rejection in the body.
    In a first aspect the invention relates to hybrid nanofibers comprising a mixture of the components of an aloe vera gel and a synthetic polymer, wherein the synthetic polymer is selected from poly-3-hydroxybutyrate-co-3-hydroxivalerate (PHBV ), poly-L-Ictic acid (PLLA), polydioxanone (PDS) and derived mixtures, and where the average diameter of hybrid nanofibers is between 0.3 and 1.5 microns.
    In a second aspect, the invention relates to a process for the manufacture of the aloe vera hybrid nanofibers comprising:
    a) preparing a solution of an aloe vera gel or derivatives of an aloe vera gel in a solvent selected from hexafluoride-2-propanol, polyvinyl alcohol (PVA), chloroform solution: methanol or derived mixtures,
    b) mixing the solution of aloe vera gel or derivatives of aloe vera gel of step a), with a synthetic polymer selected from poly-3-hydroxybutyrate-co-3-hydroxyivalerate (PHBV), poly-L-Ictic acid ( PLLA), polydioxanone (PDS) and derivative mixtures, for the formation of a hybrid polymer solution, and
    c) injecting the mixture of step b) into an electrowinning equipment for the manufacture of the hybrid nanofibers of aloe vera by electrowinning.
    Likewise, the invention also relates to hybrid nanofibers comprising a mixture of the components of an aloe vera gel and a synthetic polymer selected from poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), poly-L-Ictic acid (PLLA), polydioxanone (PDS) and derivative mixtures, where the hybrid nanofibers have an average diameter between 0.3 and 1.5 microns, obtained by the above-mentioned procedure.
    In one aspect, the invention relates to the aloe vera hybrid nanofibers for use as a medicament. In a further aspect, the invention relates to the use of aloe vera hybrid nanofibers for the manufacture of a medicament for the regeneration of nerve tissue of any vertebrate or to promote the growth, proliferation or differentiation of any cell type located in any tissue. , organ, or organ system where nerve tissue is present. In particular, the regeneration or growth of nerve tissue in the presence of the hybrid nanofibers of the invention occurs without requiring the presence of other additives or growth factors that promote the growth of nerve tissue.
    In addition, the invention also relates to the use of hybrid nanofibers for the manufacture of tubular prostheses, dressings or sutures, as well as tubular prostheses, dressings or sutures comprising the aloe vera hybrid nanofibers.
    Description of the figures
    The figures included in the description illustrate particular embodiments of the present invention. Furthermore, in combination with the text of the description they serve to explain the principles on which the invention is based.
    Figure 1: Table showing the particular conditions used for electro-spinning formation of synthetic polymer nanofibers, and aloe vera hybrid nanofibers in example 1 of the invention.
    Figure 2: Micrograph obtained by a scanning electron microscope of electro-spun polymers: A. PLLA, B. PLLA + aloe, C. POS, O. POS + aloe; E. PHBV; F. PHBV + aloe. Scales (A-F): 60 ~ m. Scales in the box (A-F): 6 ~ m.
    Figure 3 Immunofluorescence images of dorsal root ganglion explants (GRD) neurons cultured in the presence of PHBV synthetic polymer aligned nanofibers (A), and in the presence of PHBV and aloe vera (B) hybrid aligned nanofibers belonging to the invention. Figure 3C is a representative scheme of Figures 3A and 38, showing the neuronal bodies (spherical structures) and their nerve extensions following the path of the aligned nanofibers. The arrows in Figures 3A, 38 and 3C indicate the location of the growth cones. Scale (3A and 3B): 200 ~ m.
    Detailed statement of the invention
    Aloe Vera hybrid fibers and synthetic polymer
    The present invention relates to hybrid nanofibers comprising a mixture of the components of an aloe vera gel and a synthetic polymer, wherein the synthetic polymer is selected from poly-3-hydroxybutyrate-co-3-hydroxivalerate (PHBV), poly acid -Lactic (PLLA), polydioxanone (PDS) and derivative mixtures, and where the average diameter of hybrid nanofibers is between 0.3 and 1.5 microns.
    In the context of the present invention, the hybrid nanofibers of aloe vera, refer to the hybrid nanofibers comprising a mixture of the components of the aloe vera gel and a synthetic polymer selected from poly-3-hydroxybutyrate-co-3hydroxivalerate (PHBV ), poly-L-Ictic acid (PLLA), polydioxanone (POS) and derived mixtures.
    Aloe vera gel is extracted from the Aloe Vera plant of the liliaceae family. The Aloe vera plant is a succulent plant that contains more than 75 potentially bioactive components such as vitamins, enzymes, minerals, sugars, saponins, salicylic acids and amino acids, among these the essential amino acids usine, threonine, valine, leucine, phenylalanine and methionine . In addition, it contains antrones (aloe-emodin, aloin A, aloin B. 8-0-methyl-7-hydroxyaloin A, 8-0-methyl-7-hydroxyaloin BY 1 O-hydroxyaloin A), phenyl pyranas (aloin A and aloin B) and chromones (aloesin, 8-C-glucosyl7-Q-methy- (S) -aloesol, isoaloeresin D and aloeresin E). The plant has spear-shaped leaves that contain the Aloe Vera gel responsible for providing stiffness to the leaves. The Aloe Vera plant is a tropical or subtropical plant of the Aloe genus, which usually requires temperatures above 10 ° C for its cultivation. In a preferred embodiment, the aloe vera gel of the aloe vera hybrid nanofibers of the
    The invention comes from the Aloe Vera plants of the Canary Islands. In another particular embodiment, the aloe vera gel of the hybrid nanofibers of the invention is derived from the Aloe Barbadensis Miller plant.
    Aloe vera gel is usually separated from the plant by procedures known in the state of the art. Thus, for example, US 2016/0015041 discloses a procedure that is based on slicing the Aloe Vera leaves and grinding them for gel extraction. Other procedures for gel extraction are disclosed in US 3,878,197 or in US4959214.
    In the context of the invention, the term "the components of an aloe vera gel" refers to the components present in any aloe vera gel extracted from the Aloe Vera plant and, where appropriate, sterilized and stabilized, between those found in sugar-linked mucilaginous polysaccharides such as glucose, acemannan, mannose, glucomanose, rhamnose, xylose, arabinose, galactose, aldopentose and cellulose; Carbohydrates, acids, organic salts, enzymes, sterols, triacylglycerides, amino acids, RNA, traces of alkaloids, vitamins and various minerals. Some state-of-the-art documents that disclose the analysis of the composition of aloe vera gels are Reynolds al. (Journal of Ethnopharmacology 68 (1999) 3-37) and J. H. Hamman (Molecules, 13 (2008) 1599-1616).
    The aloe vera gel extracted from the plant oxidizes rapidly in the air, decomposing and in many cases losing its properties. Aloe vera gel to be used away from its origin must be sterilized and stabilized once separated from the plant. Various new technologies have been described for the stabilization of the aloe vera gel, such as conservation by high hydrostatic pressures, ohmic heating, electrical pulses, microwaves, gamma radiation and ultrasound. R.N. Domínguez-Fernández (Mexican Journal of Chemical Engineering 11 (2012) 23-43) mentions some of the procedures known in the state of the art for stabilizing aloe vera gel.
    The authors have observed that the aloe vera hybrid nanofibers of the invention are also biodegradable and biocompatible. It is believed that these properties are due to the presence of aloe vera gel components in hybrid nanofibers.
    The hybrid nanofibers of the invention comprising a mixture of the components of the aloe vera gel and a synthetic polymer selected from poly-3-hydroxybutyrate-co3-hydroxivalerate (PHBV), poly-L-Ictic acid (PLLA), polydioxanone (PDS ) and derived mixtures, have an average diameter between 0.3 and 1.5 microns, preferably between 0.5 and 1.5 microns, more preferably between 0.8 and 1.5 microns. In a preferred embodiment, the hybrid nanofibers have an average diameter between 0.8 and 1.3 microns, preferably between 0.9 and 1.2 microns, more preferably between 1 and 1.1 microns. The inventors have observed that the aloe vera hybrid nanofibers of the invention presenting the ranges of indicated diameters serve as support for the adhesion and guidance of nerve cells in axonal growth during a regenerative process, and of healing of any tissue or organ that Present nervous structures. In this sense, the hybrid nanofibers of the
    The present invention are particularly useful as a support for axonal regeneration in the peripheral nervous system (SNP) and in the central nervous system (CNS). In addition, the inventors of the present invention have observed that the aloe vera hybrid nanofibers of the invention achieve the growth of regenerated neurites of greater
    5 length than in the presence of synthetic nanofibers of synthetic polymers, at equal time of action, and without the need for growth factor supplements.
    On the other hand, the inventors have proven that the presence of the components of an aloe vera gel in hybrid nanofibers can in certain cases reduce the diameter of synthetic polymer nanofibers prepared under the same conditions. Thus, the aloe vera / POS hybrid nanofibers have a smaller diameter than the POS nanofibers prepared under the same conditions, as shown in Figures 2e and 20. This same effect has also been observed in the aloe vera / PLLA hybrid nanofibers. (Figures 2A and 2B). In contrast, the diameter of aloe nanofibers
    15 Vera / PHBV hardly varies with respect to PHBV nanofibers. Wang the al. (Acta Biomater., 2010, 6 (8) 2970-2978) reports that the diameter of PLLA synthetic nanofibers influences nerve growth. The inventors of the present invention have observed that PHBV / aloe vera hybrid nanofibers improve nerve growth with respect to pure PHBV having the same diameter. Figure 3 shows the growth of nerve endings in the presence of Aloe Vera / PHBV aligned hybrid nanofibers of the invention (Figure 3A) and in the presence of PHBV synthetic polymer aligned nanofibers (Figure 3B). The images show that the growth of nerve endings is surprisingly superior in the presence of the hybrid nanofibers of the invention.
    In a particular embodiment, the hybrid nanofibers of the invention contain between 3 and 5% of aloe vera gel and between 10 and 12% of synthetic polymer, wherein the synthetic polymer is selected from poly-3-hydroxybutyrate-co-3-hydroxivalerate. (PHBV), polylactic acid (PLLA), polydioxanone (POS) and derived mixtures, and have an average diameter of hybrid nanofibers between 0.3 and 1.5 microns. Preferably, the nanofibers h1 flanges contain 4% by weight of components of an aloe vera gel and 11% by weight of synthetic polymer.
    In a particular embodiment, the hybrid nanofibers of the invention contain between 3 and
    35 5% by weight of the components of an aloe vera gel and between 10 and 12% of a synthetic polymer, where the synthetic polymer is selected from poly-3-hydroxybutyrate-co3-hydroxivalerate (PHBV), poly-L- acid Lactic (PLLA), polydioxanone (PDS) and derivative mixtures, and where hybrid nanofibers have an average diameter between 0.8 and 1.5 microns. Preferably, hybrid nanofibers contain 4% by weight of components of an aloe vera gel and 11% by weight of synthetic polymer.
    In a particular embodiment, the weight ratio of the components of an aloe vera gel and synthetic polymer in the hybrid nanofibers of the invention is between 17:83 and 33:67. Preferably, the weight ratio of the components of a gel
    45 aloe vera / synthetic polymer is between 20:80 and 30:70, more preferably between 23:77 and 27:73. In a preferred embodiment, the weight ratio of the components of a synthetic polymer aloe veral gel is between 24:76 and 26:74.
    In another embodiment, the hybrid nanofibers of the invention can be aligned, that is to say oriented in the same direction, or disordered, that is to say oriented in different directions with respect to each other. Preferably, the hybrid nanofibers of the invention are aligned. The authors of the present invention have observed that when the hybrid nanofibers of the invention are aligned, greater nerve growth occurs.
    In a particular embodiment, the hybrid nanofibers of the invention comprise a mixture of the components of an aloe vera gel obtained from the Aloe Barbadensis Miller plant, and poly-3-hydroxybutyrate-co-3-hydroxivalerate (PHBV), wherein said nanofibers Hybrids have an average diameter between 0.8 and 1.5 microns, and where the nanofibers are aligned in a certain direction.
    In another particular embodiment, the hybrid nanofibers comprise a mixture of the components of an aloe vera gel obtained from the Aloe Barbadensis Miller plant, and poly-L-Ictic acid (PLLA), where the hybrid nanofibers have an average diameter between 0, 8 and 1.5 microns and are aligned in a certain direction.
    In another particular embodiment, the hybrid nanofibers comprise a mixture of the components of an aloe vera gel obtained from the Aloe Barbadensis Miller plant, and polydioxanone (POS), where the hybrid nanofibers have an average diameter between 0.8 and 1.5 microns and are aligned in a certain direction.
    Manufacturing procedure of the hybrid nanofibers of aloe vera and synthetic polymer
    In one aspect, the invention relates to the process for the manufacture of hybrid nanofibers comprising:
    a) Prepare a solution of an aloe vera gel or derivatives of an aloe vera gel in a solvent selected from hexafluoride-2-propanol, polyvinyl alcohol (PVA), chloroform solution: methanol or derived mixtures.
    b) mixing the solution of aloe vera gel or derivatives of aloe vera gel of step a), with a synthetic polymer selected from poly-3-hydroxybutyrate-co3-hydroxyvalerate (PHBV), poly-L-Ictic acid ( PLLA), polydioxanone (PDS) and derivative mixtures, for the formation of a hybrid polymer solution, and
    c) injecting the mixture of step b) into an electrowinning equipment for the manufacture of the hybrid nanofibers of aloe vera by electrowinning.
    According to the procedure described above, in step a) a solution of an aloe vera gel or derivatives of an aloe vera gel is prepared, in hexafluoride-2-propanol (HFIP), polyvinyl alcohol (PVA), chloroform solution: methanol or derived mixtures. Said solution usually has a concentration of 25 to 50 mg / ml of aloe vera gel. In a particular embodiment the solvent of the aloe vera solution is a chloroform: methanol solution with a 3: 1 volume ratio.
    The aloe vera gel used in the present invention can have different origins. However, the use of aloe vera gel from the Canary Islands is preferred. In a particular embodiment, the aloe vera gel of the hybrid nanofibers of the invention is derived from the Aloe Barbadensis Miller plant.
    In the context of the present invention, the term "aloe vera gel derivatives" refers to the different ways in which aloe vera gel can be presented, such as lyophilisate of the gel, gel juice, plant extract, gel solution, or in solution without aloin and / or with high concentration in acemannan. In a particular embodiment, the aloe vera gel derivatives in step a) are selected from lyophilisate of the gel, gel juice, plant extract, gel solution, in solution without aloin and / or with high concentration in acemannan.
    So-called "components of an aloe vera gel" are present in all derivatives of aloe vera gel.
    In step b) of the procedure described above, the solution of the aloe vera gel or derivatives of the aloe vera gel of step a) is mixed with a synthetic polymer selected from poly-3-hydroxybutyrate-co-3-hydroxivalerate (PHBV), poly-L-Ictic acid (PLLA), polydioxanone (PDS) and derivative mixtures, for the formation of a hybrid polymer solution.
    In a particular embodiment, the hybrid polymer solution contains aloe vera gel obtained from the Aloe Barbadens; s MilJer plant, and the synthetic polymer is poly3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV).
    In a preferred embodiment, the weight ratio of the aloe vera gel and synthetic polymer in the hybrid polymer solution of step b) is between 17:83 and 33:67. The weight ratio of aloe vera gel and synthetic polymer influences the average diameter of hybrid nanofibers. Generally, the diameter of the nanofibers is greater when the concentration of the synthetic polymer in the solution increases. Preferably, the weight ratio of aloe vera gel / synthetic polymer in the hybrid polymer solution of step b) is between 20:80 and 30:70, more preferably between 23:77 and
    27:73 In a preferred embodiment, the weight ratio of aloe vera gel / synthetic polymer is between 24:76 and 26:74.
    In step c) of the process of the invention, the mixture of step b) is injected into an electrowinning equipment for the manufacture of the hybrid nanofibers of aloe vera by electrowinning. In general, the electrowinning process is affected by system parameters such as polymer molecular weight, molecular weight distribution and dissolution properties such as viscosity, and surface tension. In addition, the electrowinning process may be affected by process parameters such as flow rate, electrical potential, distance between capillary and collector, etc. These parameters are optimized to control the characteristics of the nanofibers obtained.
    In the electro-spinning equipment the mixture is loaded into a syringe pump connected to an electrode. The solution is propelled at a flow rate between 0.7 and 1.2 ml / h, preferably at a flow rate between 0.8 and 1 ml / h, more preferably between 0.9 and 1 ml / h; and applying a potential of 10 to 15 kV, preferably 11 to 14 kV, more preferably 12 to 13 kV, from the syringe to a rotating collecting wheel. The rotating collector wheel can rotate at a speed between 2000 and 4000 rpm. In a particular embodiment, the speed of rotation of the collector wheel is between 3000 and 3500 rpm. In particular, when the rolling speed is between 3000 and 3500 rpm, the hybrid nanofibers obtained are aligned. The rotating collecting wheel is located at a distance of 8 to 12 cm from the syringe pump, preferably at a distance of 9 to 11 cm from the syringe pump, more preferably at a distance of 10 cm from the syringe pump . The conditions used in the electro-spinning are environmental conditions of 60-70% humidity and 20-30 ° C temperature. Figure 1 of the present application shows by way of example the conditions used for the manufacture of synthetic polymer nanofibers and for the manufacture of the hybrid nanofibers of the invention by electrowinning according to step e) of the process of the invention under ambient conditions of 25 ± 1 ° C and with a relative constant humidity of 65 ± 5%.
    In a further aspect, the invention relates to hybrid nanofibers comprising a mixture of the components of the aloe vera gel and a synthetic polymer selected from poly-3-hydroxybutyrate-co-3-hydroxivalerate (PHBV), poly-L acid -Ictic (PLLA), polydioxanone (PDS) and derivative mixtures, where the hybrid nanofibers have an average diameter between 0.3 and 1.5 microns, obtained by the method of the invention described above. Figures 28, 20 and 2F show the SEM micrographs of hybrid nanofibers belonging to the invention obtained by the described procedure.
    The aloe vera hybrid nanofibers of the present invention can be used for tissue engineering applications, in particular for the regeneration of nerve tissue. Thus, one aspect of the invention relates to the use of hybrid nanofibers for the manufacture of a medicament for the regeneration of nerve tissue of any vertebrate or to promote the growth, proliferation or differentiation of any cell type located in any tissue, organ, or organ system where nerve tissue is present.
    In one aspect, the invention relates to the aloe vera hybrid nanofibers of the invention for use as a medicament.
    In another aspect, the invention relates to the use of the aloe vera hybrid nanofibers for the manufacture of a medicament for the regeneration of the nervous tissue of any vertebrate or to promote the growth, proliferation or differentiation of any cell type located in any tissue, organ, or system of organs where nerve tissue is present.
    In another aspect, the invention relates to the aloe vera hybrid nanofibers of the invention for use in the treatment of nerve tissue regeneration of any vertebrate, or of growth, proliferation or differentiation of any cell type located in any tissue, organ , or organ system where nerve tissue is present.
    In a particular embodiment, the aloe vera hybrid nanofibers of the invention are used for the manufacture of a medicament for the regeneration of nerve tissue of any vertebrate or to promote the growth, proliferation or differentiation of any cell type located in any tissue, organ, or organ system where nerve tissue is present, in the absence of any other additive or growth factor that promotes the regeneration of nerve tissue.
    In particular, the aloe vera hybrid nanofibers of the invention can be used for the regeneration of nerve tissue in nerve connection prostheses, dressings and sutures in skin wounds. In this regard, one aspect of the invention relates to a tubular prosthesis comprising the aloe vera hybrid nanofibers of the invention. Another aspect of the present invention relates to dressings comprising the aloe vera hybrid nanofibers of the invention. Another additional aspect refers to the sutures comprising the hybrid nanofibers of the invention.
    Also, the aloe vera hybrid nanofibers of the invention can also be used for the regeneration of other tissues, such as tendon, ligament and bone tissues.
    Honey and synthetic polymer hybrid nanofibers
    Other hybrid nanofibers comprise a mixture of components of a honey bee and a synthetic polymer, where the synthetic polymer is selected from poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), poly-L-Ictic acid (PLLA), polydioxanone ( PDS), biodegradable thermoplastic polyurethanes for medical use, polycaprolactone (pel), polyvinyl alcohol (PVA), polyoelide eo-glycolide (PlGA), polyhydroxyalkanoalos (PHAs), polypropylene carbonate (PPC) and derived mixtures. In a particular embodiment, the hybrid nanofibers of honey may comprise a mixture of components of honey and biodegradable thermoplastic polyurethanes for medical use such as Z3A1 or Z9A1 (Biomer Technology L TD), DegraPol® (urethane polyester formed by two dialing polyester joined by a isocyanate group) or Desmopan® 9370A (ether of 4 carbon atoms) from Bayer.
    In a preferred embodiment, the hybrid nanofibers of honey and synthetic polymer comprise a mixture of the components of a honey bee and a synthetic polymer selected from poly-3-hydroxybutyrate-co-3-hydroxivalerate (PHBV), poly-Lactic acid ( PLLA), polydioxanone (PDS) and derived mixtures.
    Honey is produced by bees from the nectar of flowers or secretions of living parts of plants or excretions of plant-sucking insects. Honey from bees is extracted directly from the honeycomb and is stored in a sterile container at 8 ° C before the formation of hybrid nanofibers.
    For the preparation of hybrid nanofibers of honey, the honey used can be any type of honey. In particular, the honey used may be flower honey such as monofloral, multifloral, mountain, mountain or desert honey. Honey from hybrid nanofibers can also be honeydew honey, honey roela, dew honey or forest honey.
    In general, the composition of honey known in the state of the art comprises the following components:
    14-22% by weight of water,
    28-44% by weight of fructose,
    22-40% by weight glucose,
    0.2-7% by weight sucrose,
    2-16% by weight of maltose, 0.1-8% by weight of other sugars,
    0.2-2% by weight of proteins and amino acids,
    0.5-1% by weight of vitamins, enzymes, organic lying hormones,
    0.5-1% by weight of minerals, and
    0.2-1% by weight of ashes.
    In a particular embodiment the honey composition comprises:
    18% by weight of water,
    38% by weight of fructose,
    31% by weight glucose,
    1% by weight sucrose,
    7.5% by weight of maltose, and
    5% by weight of other sugars.
    The honey used by the inventors comes from a local beekeeper from Gran Canaria registered in the General Sanitary Registry of Food and Food Companies (RGSEAA): María del Rosario Cazarla López, N ° RGSEAA: 23.03229 / GC.
    In a particular embodiment, the hybrid honey nanofibers comprising a mixture of the components of a honey and a synthetic polymer, contain between 5 and 10% by weight of the components of a honey and 10% by weight of a synthetic polymer selected from poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), poly-L-Ictic acid (PLLA), polydioxanone (PDS), polyeaprolaetone (pel), polyvinyl alcohol (PVA), eoglycolide polyaetide (PLGA), polyhydroxyalkanoates (PHAs ), polypropylene carbonate (PPC) and biodegradable thermoplastic polyurethanes for medical use such as Z3A1 or Z9A1 (Biomer Technology L TD), DegraPol® (urethane polyester formed by two polyester dials joined by an isocyanate group, Desmopan® 9370A (ether of 4 carbon ties) from Bayer or derived mixtures Preferably, the hybrid nanofibers contain between 6 and 8% by weight of a honey and 10% by weight of a synthetic polymer.
    In a particular embodiment, the weight ratio of the honey and synthetic polymer components in the hybrid nanofibers of the invention is between 33:67 and 50:50. Preferably, the weight ratio of the honey and synthetic polymer components is between 35:65 and 48:52, preferably between 37:62 and 46:54, more preferably between 40:80 and 45:55. In a preferred embodiment, the weight ratio of synthetic polymer is between 42:58 and 44:58.
    In a particular embodiment, the hybrid honey and synthetic polymer nanofibers of the invention have an approximate diameter of between 0.3 and 1.5 microns, preferably between 0.5 and 1.3, more preferably between 0.7 and 1.2 . In another embodiment, the hybrid honey and synthetic polymer nanofibers of the invention have an approximate diameter of between 0.8 and 1.1 microns, preferably between 0.9 and 1 microns. Hybrid honey nanofibers, as well as aloe vera hybrid nanofibers, serve as support for the adhesion and guidance of nerve cells in axonal growth in a regenerative and healing process of any tissue or organ that presents structures
    5 nervous In particular, the hybrid honey nanofibers of the present invention serve as support for axonal regeneration in the peripheral nervous system (SNP) and central nervous system (CNS).
    In another embodiment, the hybrid honey nanofibers of the invention can be aligned, that is to say oriented in the same direction, or disordered, that is to say oriented in different directions with respect to each other. Preferably, the hybrid honey nanofibers of the invention are aligned.
    In a particular embodiment, the hybrid honey nanofibers comprising a mixture
    15 components of a honey, and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), have an average diameter between 0.8 and 1.5 microns and are aligned in a certain direction.
    In another particular embodiment, the hybrid honey nanofibers comprising a mixture of components of a honey, and poly-L-Ictic acid (PLLA), have an average diameter between 0.8 and 1.5 microns and are aligned in a given address.
    In another particular embodiment, the hybrid honey nanofibers comprising a mixture of components of a honey, and polydioxanone (POS), have a diameter
    25 average between 0.8 and 1.5 microns and are aligned in a certain direction.
    Manufacturing procedure of hybrid nanofibers of honey and synthetic polymer
    The hybrid honey and synthetic polymer nanofibers of the invention can be obtained by a manufacturing process similar to that used for the manufacture of the hybrid fibers of aloe vera and synthetic polymer. The process for manufacturing hybrid honey nanofibers comprises:
    A) Prepare a solution of a honey in a solvent selected from hexafluoride-2propanol, polyvinyl alcohol (PVA), 1% acetic acid and trifluoroacetic acid (TFA).
    b) mixing the honey solution from step a), with a synthetic polymer selected from poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), poly-L-Ictic acid (PLLA), polydioxanone (POS), polyurethanes Biodegradable thermoplastics for medical use, polyeaprolaelone (pel), polyoelide eo-glieólido (PlGA), polyhydroxyalkanoalos (PHAs), polypropylene carbonate (PPC), biodegradable thermoplastic polyurethanes for medical use such as Z3A1 or Z9A 1, OemoPol® or O70 Grap 93® and derived mixtures, for the formation of a hybrid polymer solution, and
    c) injecting the mixture of step b) into an electro-spinning equipment for the manufacture of the hybrid nanofibers of honey by electro-spinning.
    According to the procedure described above, in step a) a solution of honey is prepared, in hexafluoride-2-propanol (HFIP), polyvinyl alcohol (PVA), 1% acetic acid and trifluoroacetic acid (TFA). This solution usually has a concentration of 50 to 100 mg / ml of honey.
    In step b) of the procedure described above, the honey solution of step a) is mixed with a synthetic polymer selected from poly-3-hydroxybutyrate-co-3-hydroxyivalerate (PHBV), poly-L-Ictic acid (PLLA) , polydioxanone (POS), polycaprolactone (PCL), co-glycolide polyactide (PLGA), polyhydroxyalkanoates (PHAs), polypropylene carbonate (PPC), biodegradable thermoplastic polyurethanes for medical use such as Z3A 1 or Z9A 1, OegraPol® or Oesmopan® 9370A and derived mixtures, for the formation of a hybrid polymer solution.
    In a particular embodiment, the weight ratio of honey and synthetic polymer in the hybrid polymer solution is between 33:67 and 50:50 (honey: synthetic polymer). Preferably, the honey / synthetic polymer weight ratio in the hybrid polymer solution is between 35:65 and 48:52, preferably between 37:62 and 46:54, more preferably between 40:60 and 45:55. In a preferred embodiment, the honey / synthetic polymer weight ratio in the hybrid polymer solution is comprised between
    42:58 and 44:56.
    In step c) of the process of the invention, the mixture of step b) is injected into an electrowinning equipment for the manufacture of hybrid nanofibers of honey by electrowinning. In particular, the mixture is loaded into a syringe pump connected to an electrode. The solution is propelled at a flow rate between 2 and 3 ml / h, preferably at a flow rate between 2.5 and 3 ml / h, more preferably between 2.5 and 2.8 ml / h; and applying a potential of 7 to 11 kV, preferably 8 to 10 kV, more preferably 8 to 9 kV, from the syringe to a rotating collecting wheel. The rotating collector wheel can rotate at a speed between 2000 and 4000 rpm, preferably 3000 rpm. The rotating collecting wheel is located at a distance of 8 to 12 cm from the syringe pump, preferably at a distance of 9 to 11 cm from the syringe pump, more preferably at a distance of 10 cm from the syringe pump . The conditions used in the electro-spinning are environmental conditions of 60-70% humidity and 20-25 ° C temperature.
    The hybrid nanofibers obtained by the process described above comprise a mixture of honey components and a synthetic polymer selected from poly-3-hydroxybutyrate-co-3-hydroxivalerate (PHBV), poly-L-Ictic acid (PLLA) and polydioxanone (PDS ), polyeaprolaetone (pel), polyether eo-glycolide (PlGA), polyhydroxyalkanoates (PHAs), polypropylene carbonate (PPC), biodegradable thermoplastic polyurethanes for medical use such as Z3A 1 or Z9A 1, OegraPol® or Oesmopan® 9370A and derivative mixtures , where hybrid nanofibers have an average diameter between 0.3 and 1.5 microns. In a preferred embodiment, the hybrid nanofibers obtained by the process described above comprise a mixture of honey and a synthetic polymer selected from poly-3-hydroxybutyrate-co-3-hydroxyivalerate (PHBV), poly-L-Ictic acid (PLLA), polydioxanone (POS) and derived mixtures.
    Hybrid honey nanofibers can be used for tissue engineering applications, in particular for the regeneration of nerve tissue. Also, hybrid honey fibers can be used in bandages and dressings to cover wounds.
    The invention relates to the hybrid honey nanofibers of the invention for use as a medicament. Also, the invention relates to hybrid nanofibers of honey for use in the treatment of nerve tissue regeneration of any vertebrate, or growth, proliferation or differentiation of any cell type located in any
    5 tissue, organ, or organ system where nerve tissue is present.
    Examples
    1. Preparation of aligned hybrid fibers of aloe vera and synthetic polymer
    Initially, a solution of 25-50 mg / ml of aloe vera (Prod. # 001, Luciano Reverón e Niños S.L., Tenerife) laboratories in Hexafluoro-2-propanol (HFIP) was prepared. To obtain the hybrid solution (AV / PHBV), 10-12% (w / w) of PHBV (SigmaAldrich, Prod. No. 403121) was added to the above solution. To get nanofibers aligned
    15 using the electrospining technique, the hybrid polymer solution was loaded into a syringe pump (Hardvard Apparatus PhO Ultra) with a needle (G20, 0.9 mm diameter) whose tip was connected to an electrode (spinnerette). 12-15 Kv was applied with a high voltage source (Spellman 60N300) while the syringe pump fired the solution (flow rate of 0.9 ml / h) under ambient conditions of 60-65% humidity and 22-25 ° C temperature towards a target wheel (90 mm diameter / 12 mm thickness) located 12 cm from the electrode. The aligned nanofiber beams were collected on the target wheel rotating at 3000 rpm. Figure 2 shows the SEM micrographs of the nanofibers obtained.
    In addition, Figures 1 A-E show the SEM micrographs of the aligned fibers obtained by the method of the invention of the synthetic polymers PLLA (Fig. 2A), PDS (Fig. 2C) and PHBV (Figure 2E); as well as the hybrid fibers of aloe vera of the invention of PLLA + Aloe vera (Fig. 2B); POS + Aloe Vera (Figure 20).
    2. Comparative trials of neuritic growth
    Comparative tests were performed from the explant culture of the dorsal root ganglion (GRO) of neonatal rat (Sprague Oawley) in a) standard culture medium (OMEM / F12 (1: 1)] to establish control conditions and b) OMEM / F12 (1: 1)
    35 containing the invented hybrid nanometric matrix (AV / PHBV) and other pure reference (PHBV and PLLA) as experimental substrate of neuritic growth. Rat GRO explants were incubated in an oven at 37 ° C and 5% CO2 atmosphere for 6 days and culture medium renewal at 3 days.
    After the incubation period, the rat GRO explants were fixed with a solution of 4% paraformaldehyde in phosphate buffered saline and specific antibodies were immunolabelled for the identification of neurons. Subsequently, digital images were taken in a fluorescence microscope equipped with an image capture system. The images were processed for statistical analysis.
    45 A statistical analysis was performed on each of the polymers summarizing the positive area relationships by medium and interquartile ranges in each of the treatment groups. Statistical analyzes showed that the highest neuritic growth rate occurred with the AV / PHBV aligned hybrid nanofibers compared to the absence of aloe in pure PHBV aligned nanofibers (p <0.001) and pure PLLA (p = O.049).
    权利要求:
    Claims (17)
    [1]
    one. Hybrid nanofibers comprising a mixture of the components of an aloe vera gel and a synthetic polymer, wherein the synthetic polymer is selected from poly3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), poly-L-Ictic acid (PLlA), polydioxanone (PDS) and derivative mixtures, and where the average diameter of hybrid nanofibers is between 0.3 and 1.5 microns.
    [2]
    2. The hybrid nanofibers according to claim 1, wherein the hybrid nanofibers have an average diameter between 0.8 and 1.5 microns.
    [3]
    3. The hybrid nanofibers according to any one of claims 1 to 2, wherein the hybrid nanofibers comprise between 3 and 5% by weight of the components of aloe vera, and between 10 and 12% of synthetic polymer.
    [4]
    Four. The hybrid nanofibers according to any one of claims 1 to 3, wherein the aloe vera gel is obtained from the Aloe Barbadensis Miller plant.
    [5]
    5. The hybrid nanofibers according to any one of claims 1 to 4, wherein said nanofibers are aligned in a certain direction.
    [6]
    6. The hybrid nanofibers according to claim 1, wherein said nanofibers comprise a mixture of the components of an aloe vera gel obtained from the Aloe Barbadensis Miller plant and poly-3-hydroxybutyrate-co-3-hydroxivalerate (PHBV), wherein said hybrid nanofibers have an average diameter between 0.8 and 1.5 microns and where the nanofibers are aligned in a certain direction.
    [7]
    7. Process for manufacturing hybrid nanofibers according to claims 1 to 6, comprising:
    a) preparing a solution of an aloe vera gel or derivatives of an aloe vera gel in a solvent selected from hexafluoride-2-propanol, polyvinyl alcohol (PVA), chloroform solution: methanol or derived mixtures,
    b) mixing the solution of the aloe vera gel or derivatives of the aloe vera gel of step a), with a synthetic polymer selected from poly-3-hydroxybutyrate-co3-hydroxyvalerate (PHBV), poly-L-Ictic acid ( PLLA), polydioxanone (PDS) and derivative mixtures, for the formation of a hybrid polymer solution, and
    c) injecting the mixture of step b) into an electrowinning equipment for the manufacture of the hybrid nanofibers of aloe vera by electrowinning.
    [8]
    8. The process according to claim 7, wherein the aloe vera gel derivatives in step a) are selected from lyophilisate of the gel, gel juice, plant extract, gel solution, or in solution without aloin and / or canker sore. concentration in acemannan.
    [9]
    9. The process according to any of claims 7 or 8, wherein in step b) the weight ratio of the aloe vera gel and the synthetic polymer is comprised between
    17:83 and 33:67.
    [10]
    10. The process according to any of claims 7 to 9 wherein the aloe vera gel is obtained from the Aloe Barbadensis Miller plant and the synthetic polymer is poly3-hydroxybutyrate-co-3-hydroxyvalerate (P HBV).
    [11 ]
    eleven . Hybrid nanofibers comprising a mixture of the components of a gel
    aloe vera and a synthetic polymer selected from poly-3-hydroxybutyrate-co-3-hydroxyivalerate (PHBV), poly-L-Ictic acid (PLLA), polydioxanone (PDS) and derived mixtures, where hybrid nanofibers have an average diameter between 0 , 3 and 1.5 microns, obtained by the process according to claims 7 to 10.
    [12]
    12. Hybrid nanofibers according to any of claims 1 to 6 and 11 for use as a medicament.
    [13]
    13. Use of hybrid nanofibers according to any of claims 1 to 6 and 11, for the manufacture of a medicament for the regeneration of nerve tissue of any vertebrate or to promote the growth, proliferation or differentiation of any cell type located in any tissue, organ , or organ system where nerve tissue is present.
    [14]
    14. Use of the hybrid nanofibers according to any of claims 1 to 6 and 11 for the manufacture of tubular prostheses, dressings and sutures.
    [15]
    fifteen. Tubular prosthesis comprising the hybrid nanofibers according to any one of claims 1 to 6 and 11.
    [16]
    16. Dressing comprising the hybrid nanofibers according to claims 1 to 6 and 11.
    [17]
    17. Sutures comprising the hybrid nanofibers according to claims 1 to 6 and 11.
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    同族专利:
    公开号 | 公开日
    EP3461788A1|2019-04-03|
    EP3428117A1|2019-01-16|
    EP3428117A4|2019-11-13|
    ES2579161R1|2016-09-30|
    WO2017153619A1|2017-09-14|
    ES2579161B2|2017-03-28|
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    WO2009133059A2|2008-05-02|2009-11-05|Evonik Degussa Gmbh|Nanofiber matrices formed from electrospun hyperbranched polymers|
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    WO2014027965A1|2012-08-15|2014-02-20|National University Of Singapore|Wound dressing nanomesh impregnated with human umbilical cord wharton's jelly stem cells|
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    WO2015183228A1|2014-05-27|2015-12-03|Duymuş Ethem|Nano fiber cover for wounds and ambustions with an additive containing natural antiseptic|WO2020049339A1|2018-09-06|2020-03-12|Bio-On S.P.A.|Membrane to re-establish continuity of injured biological tissues|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    ES201600173A|ES2579161B2|2016-03-08|2016-03-08|Aloe Vera hybrid nanofibers|ES201600173A| ES2579161B2|2016-03-08|2016-03-08|Aloe Vera hybrid nanofibers|
    EP17762582.9A| EP3428117A4|2016-03-08|2017-03-02|Hybrid aloe vera nanofibres|
    PCT/ES2017/000029| WO2017153619A1|2016-03-08|2017-03-02|Hybrid aloe vera nanofibres|
    EP18173213.2A| EP3461788A1|2016-03-08|2017-03-02|Hybrid honey nanofibers|
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