WO2014044894A1 - Controlled-relase formulation - Google Patents

Description

 Controlled Release Formulation

FIELD OF THE INVENTION

 The present invention provides a controlled release composition, a drug delivery system for the controlled release composition and a method of preparing the controlled release composition. In particular, the present invention provides a controlled release formulation loaded with bone morphogenic proteins for the treatment of brain tumors, more particularly, for the treatment of Multiform Glioblastoma. The formulation of the present invention is also useful for tissue regeneration, more particularly cartilage regeneration.

BACKGROUND OF THE INVENTION

 The following discussion of the background of the invention is provided only to help the reader understand the invention, and is not admitted to describe or constitute information prior to the present invention.

Controlled release of macromolecules has been a primary objective for pharmaceutical technology in recent decades (R. Langer, Nature 263, 1976, pp 797). This is particularly relevant for the controlled release of many chemokines, growth factors, morphogens or other physiologically active proteins that have a heparin binding domain and are normally active at very low levels, but have a high turnover rate in the organism (M. Biondi, Adv Drug Deliv Ap 60, 2008, pp 229). Therefore, to produce biological effects that require maintenance of signaling, a controlled release device must maintain low but constant levels of the proteins at the site of action. For these proteins, not only temporal regulation is important, but also spatial regulation. Many therapies are based on increasing the concentration of therapeutic proteins at the site of action or a chemotactic gradient. The implantation of drug delivery systems in the region of interest generally allows to obtain sustained and effective levels of protein at the site of action, thus avoiding side effects at remote sites in the body. In this sense, a controlled release device, effective and composed primarily of pharmaceutically acceptable biodegradable polymers, should be able to effectively encapsulate therapeutic proteins and provide sustained release for several days or weeks. A technical challenge associated with this objective is the lack of affinity between proteins and most of the biodegradable polymers used for the formation of implants for controlled and sustained release, which are mostly hydrophobic.

 A relevant application to this type of systems is the development of implantable systems that allow the regeneration of cartilage "in situ". From a technological point of view the regeneration of functional cartilage is still a clinical challenge that requires new solutions (Huey, Science 338, 2012, pp. 917). In this sense, the development of formulations capable of releasing one or more growth factors in a controlled manner and from an adequate support for cell infiltration and tissue formation is required (Holland, Osteoarthritis and Cartilage 15 (2), 2007, pp. 187). The morphogen TGF-P3 is the active substance most used for the regeneration of cartilage, since its ability to differentiate mesenchymal stem cells from chondrocytes has been demonstrated. BMP-7 has also demonstrated pro-regenerative capacity of cartilage, both as a solo treatment (Chubinskaya, International Orthopedics 31, 2007, pp. 773), or in combination with TGF-β (Kim, Tissue Engineering Part A, 15 ( 7), 2009, pp. 1543).

The sustained release of therapeutic proteins in the brain could become another important application for such technology, since virtually all macromolecules are unable to cross the blood-brain barrier. Therefore, the surgical implantation of a therapeutic protein in a controlled release device is an appropriate technological strategy to achieve exposure to the drug in the brain. While this invasive strategy may not be the first choice for minor diseases, it could be considered for the most important conditions, such as brain cancer, where surgical procedures are required in any case.

High-grade malignant astrocytic gliomas are the most common and aggressive primary tumors of the Central Nervous System, representing 52% of all cases of primary brain tumors. Glioblastoma multiforme (GBM) is the most common glioma in adults and the typical survival time of the patient is less than one year. A fraction of the cells in the GBM share biological properties with normal neural stem cells such as: resistance to radiation and chemotherapy, infiltrative nature and proliferative behavior. These cells are capable of starting new tumors from isolated cells, indicating that GBMs contain tumor-initiating cells, also called stem cells. cancer (CSC). Currently, it is thought that these cells are located at the apex of the functional hierarchy and that they are responsible for tumor recurrence, which makes them a very attractive target for cancer therapy. Unfortunately, these types of cells have active mechanisms that make them resistant to conventional therapies such as: high level of expression of drug resistance proteins, increased quiescence period, higher levels of anti-apoptotic signaling and a greater capacity to repair the DNA To solve this problem and eliminate the CSC stem cell-like properties, some strategies have been explored such as treating these cells with different types of growth factors, for example bone morphogenic proteins (BMPs). In 2006, Piccirillo et al. "Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells", Nature, Vol. 444, pg 761-765, showed that glioblastoma CSCs can be differentiated into astroglia-like cells by treatment with BMP-4 both in vitro as in vivo, and that this process suppresses the tumorigenic capacity of CSCs. Another BMP with very important functions in the Central Nervous System is BMP-7. This BMP has the ability to regulate the survival, migration and differentiation of cells, after an injury or stroke of the Central Nervous System, and demonstrates neuroprotective activity. It has recently been shown that BMP-7 is secreted by endogenous neural stem cells as a paracrine tumor suppressor due to its ability to induce CSC differentiation.

 However, from a pharmaceutical point of view, the use of BMPs as tumor suppressors in GBM therapy is limited due to the short half-life of this growth factor in the body (few minutes), and its inability to pass through the blood-brain barrier. Therefore, it is still necessary to provide adequate treatment for GBM.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a new controlled release formulation (hereinafter, the controlled release formulation of the invention) capable of encapsulating a hydrophilic protein, which has a heparin binding domain, in a hydrophobic matrix. The formulation is intended to avoid the usual lack of affinity between hydrophobic controlled release peptides and polymers. This lack of affinity was addressed in this invention by integrating proteins into colloidal intermediates coated with polyoxyethylene for direct encapsulation in biodegradable matrices. The polyoxyethylene coating provides the colloidal system with a polymeric coating with intermediate solubility parameters that should improve the interaction between the protein and the biodegradable matrix.

 The general scheme of the process for preparing the controlled release formulation of the invention is illustrated in Figure 9, in which (i) a hydrophilic protein, which has a heparin binding domain specifically binds to a polysulphated polymer, (ii) the resulting anionic complex is then joined through electrostatic interaction with a cationic polyoxyethylene derivative in a process similar to a layer-by-layer ionic interaction process, and (iii) because the polyoxyethylene coating guarantees high compatibility With the biodegradable polymer, the complex can be efficiently encapsulated in the biodegradable polymer matrix thereby constituting the controlled release formulation of the invention.

 Therefore, a first aspect of the invention relates to a composition (hereinafter composition of the invention) suitable for the controlled release of hydrophilic proteins having a heparin binding domain, comprising:

 to. A surface layer of a biodegradable hydrophobic polymer; and b. A nucleus of a cationic polyoxyethylene derivative physically bonded to a polysulphated polymer which in turn is physically bonded to a hydrophilic protein with a heparin binding domain.

In the context of the present invention, hydrophilic proteins that have a heparin binding domain include all proteins that have any of the following three consensus sequences XBBXBX, XBBBXXBX or XBBBXXBBBXXBBX, where B is a base and X an amino acid residue Hydropathic (neutral and hydrophobic). However, it has been determined that other proteins have heparin binding sequences even without these consensus sequences. These proteins, sometimes, contain basic amino acids that are far apart in the primary structure but that in the tertiary structure are nearby forming cationic regions capable of interacting very closely with heparin. The 3D conformation analysis of these proteins has suggested that a spatial separation between the basic amino acids of approximately 20 Amgstrons or less is Important for interactions. These cationic pockets are not normally in the same region of the pharmacologically active site of the protein.

In particular, hydrophilic proteins with a heparin binding domain include, but are not limited to, growth factors such as: endothelial vascular growth factor (VEGF), interleukins (IL), transforming growth factor (TGF), factor of epidermal growth (EGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), stromal cell derived factor (SDF), growth and differentiation factor (GDF), platelet factor (PF), ligand of the family of chemokines CC (CCL), ligand of the family of chemokines CXC, ligand of the family of chemokines C, chemokines CX ₃ C, antithrombins, neurotrophins, ligands of the family of neurotrophic factor derived glial cell line (GFL ), bone morphogenic proteins (BMPs), chymotrypsinogen or any combination thereof. More particularly, hydrophilic proteins that have a heparin binding domain include TGF and / or BMPs. In a particular embodiment, bone morphogenic proteins (BMPs) are selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8b and BMP-14, or any combination thereof. In a particular embodiment, transforming growth factors (TGF) are selected from the group consisting of TGF-βΙ, TGF-P2, TGF-P3, or any combination thereof. In a more particular embodiment of the invention, hydrophilic proteins with a heparin binding domain are BMP-7 and / or TGF-P3.

In the context of the present invention, biodegradable hydrophobic polymers include all those polymers that do not form a homogeneous 5% solution (w / w) in water and that when implanted in the body are reabsorbed in less than 2 years. In particular, biodegradable hydrophobic polymers include polyesters, polyanhydrides, poly (ortho esters), polyamides, poly (alkyl cyanoacrylates), polyimides, polyester-poly (ethylene glycol) copolymers, polyphosphazenes, poly (phosphoesters) or any combination of the same. In a more particular embodiment, the hydrophobic biodegradable polymers include polyesters of the group consisting of poly (hydroxivalerate), polycaprolactone, poly (lactic acid), poly (lactic-co-glycolic) (PLGA), poly (hydroxybutyrate), poly (hydroxybutyrate) -co-valerate), polydioxanone, poly (8-caprolactone), and poly (glycolic acid). In a particular embodiment, the polyanhydride is poly (methyl vinyl ether-alt-maleic anhydride) (pMVEMA). In a further embodiment, the hydrophobic biodegradable polymers they include polyphosphoesters such as polyphosphoester urethane. In a more particular embodiment, hydrophobic biodegradable polymers include polyamides of the group consisting of poly (amino acids), in which the amino acids are hydrophobic amino acids such as alanine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan or tyrosine; polyamide; a linear polymer of γ-aminobutyric acid (GABA) or nylon 6,6, polycaprolactam. In a more particular embodiment, the hydrophobic biodegradable polymer is polyurethane. In an even more particular embodiment, the hydrophobic biodegradable polymer is PLGA.

 In the context of the present invention, the cationic polyoxyethylene derivative is a grafted, block or random copolymer of polyoxyethylene and optionally polyoxypropylene having at least one potentially cationizable chemical group and where the percentage of polyoxyethylene and polyoxypropylene groups in the copolymer is at least 30% by weight. In a preferred form, the cationizable group is a primary, secondary, tertiary or quaternary amine. In a more preferred form, the cationic polyoxyethylene derivative can be selected from any of the following groups of compounds: polyethylene glycol amines (ie methoxypolyethylene glycol amine), poloxamines, chitosan polyoxyethylene derivatives (ie, healthy quito-g-PEG), cationic polyamino acid-polyoxyethylene derivatives (ie, polylysine-g-PEG, polyarginine-g-PEG), polyoxyethylene derivatives of cationic or cationic cationic proteins (ie, cationic albumin-PEG, cationic polyethylene-PEG gelatin) , polyaminoesters derived from polyoxyethylene or any combination thereof. In a preferred embodiment of the invention, the cationic polyoxyethylene derivative is methoxypolyethylene glycol amine or poloxamine. In the context of the present invention, the polysulfated polymer is a biodegradable, non-cyclic polymer having at least 1 KDa and having at least three sulfate groups. The polymer can be any of the following compounds: polysulphated silk fibroin, dendritic polyglycerol sulfate or a sulfated proteoglycan. Preferably, the polysulfated polymer is a polysaccharide such as polysulphated hyaluronic acid, polysulphated dextran, polysulphated proteoglycans, heparan sulfate and / or heparin, or any combination thereof. In a more preferred embodiment, the polysulphated polymer is heparan sulfate, polysulfated dextran or heparin.

Always respecting the structure and composition of the invention, said formulations may also comprise other pharmaceutical excipients to provide additional technical characteristics. Examples of such excipients may be surfactants (lecithin, polyvinyl alcohol, poloxamer, Span, Tween, bile salts, etc.), surface modifying agents (DOTAP, benzalkonium, cetylpyridinium), insoluble basic salts that co-encapsulate in the matrix ( calcium and magnesium hydroxide), porogenic agents (sodium chloride granules) and / or cryoprotectants (glucose, trehalose).

 In a more particular embodiment, the composition of the invention comprises as a biodegradable hydrophobic polymer the PLGA, the pMVEMA or the poly (8-caprolactone), as a cationic polyoxyethylene derivative a poloxamine or methoxypolyethylene glycol amine, as a hydrophilic protein having a binding domain to heparin a BMP and / or a TGF, in particular BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8b, BMP-14, TGF-βΙ, TGF-p2 and / or TGF-p3 and as polysulfated polymer of heparan sulfate, polysulfated dextran or heparin. In an even more particular embodiment of the invention, the composition of the invention comprises PLGA, poloxamine, lecithin, BMP-7 and heparin. In another even more particular embodiment of the invention, the composition of the invention comprises PLGA, poloxamine, BMP-7, TGF-P3 and heparin.

 In a further embodiment, the composition of the invention may be a microsphere (hereinafter, microspheres of the invention), a film, a nanoparticle or a porous matrix. In a particular embodiment, the microspheres of the invention have an average size larger than 10 in order to avoid phagocytosis. More particularly, the microsphere has an average size between 10 and 90um, more particularly between 10 and 30 um.

 In a particular embodiment, the composition of the invention may be a nanoparticle (hereinafter nanoparticles of the invention) with an average size between 80 and 500 nm. More particularly the nanoparticle has a size between 100 and 300 nm.

 In another particular embodiment, the composition of the invention may be a film (hereinafter films of the invention).

In another additional embodiment, the composition of the invention may be a porous matrix (hereinafter porous matrix of the invention). The porous matrix can have different pore sizes and porosities that can be controlled during the preparation process. The average pore size of the porous matrices is between CU20 and 800. More particularly, it is between 100 and 500 um. A second aspect of the present invention relates to a composition suitable for the controlled release of hydrophilic proteins with a heparin binding domain, comprising:

 to. A hydrophobic biodegradable polymer;

 b. A cationic polyoxyethylene derivative as a biocompatible emulsifier;

 C. A hydrophilic protein with a heparin binding domain; and d. A polysulphated polymer physically bound to the hydrophilic protein with a heparin binding domain,

wherein the composition is obtainable by mixing an aqueous solution or a solid composition, preferably in the form of a lyophilized powder, comprising a cationic polyoxyethylene derivative, a hydrophilic protein with a heparin binding domain and a polysulfated polymer bonded together. with an organic medium comprising a hydrophobic biodegradable polymer.

 A third aspect of the invention relates to a method for producing microspheres, nanoparticles, porous matrices or film of any of the above aspects of the invention, comprising:

 to. Dissolve the cationic polyoxyethylene derivative in a solution having a hydrophilic protein with a heparin binding domain and a polysulphated polymer;

 b. Dry by lyophilization of the mixture resulting from step (a); C. Resuspend the dry powder of step (b) in an organic solvent containing a biodegradable hydrophobic polymer, and d. Collect microspheres, nanoparticles, porous matrices or films. In a more particular embodiment, the microsphere obtained by the above procedure comprises as a biodegradable hydrophobic polymer the PLGA, as a cationic polyoxyethylene derivative a poloxamine, as a hydrophilic protein having a heparin binding domain a BMP, and as a heparin polysulphated polymer. In an even more particular embodiment of the invention, the microsphere comprises PLGA, poloxamine, BMP-7 and heparin.

In another more particular embodiment, the nanoparticle obtained by the above process comprises as a biodegradable hydrophobic polymer the PLGA, as a cationic polyoxyethylene derivative a poloxamine, and as a heparin polysulphated polymer. In another more particular embodiment, the m atri z poro sa obtained by the above procedure comprises as a biodegradable hydrophobic polymer the PLGA, as a cationic polyoxyethylene derivative a poloxamine, as a hydrophilic protein having a heparin binding domain a TGF or a BMP, and as heparin polysulfated polymer. In an even more particular embodiment of the invention, the porous matrix comprises PLGA, poloxamine, BMP-7, TGF-P3 and heparin.

 In another more particular embodiment, the film obtained by the above process comprises as a biodegradable hydrophobic polymer PLGA, pMVEMA or poly (8-caprolactone); as a cationic polyoxyethylene derivative a poloxamine or methoxypolyethylene glycol amine; as a hydrophilic protein having a heparin binding domain a BMP; and as polysulfated polymer heparin or dextran polysulfate. In an even more particular embodiment of the invention, the film comprises PLGA, pMVEMA or poly (8-caprol actone); pol oxamine or methoxypolyethylene glycol amine; BMP-7; and heparin or dextran sulfate.

 A fourth aspect of the invention relates to a drug delivery system (hereinafter drug delivery system of the invention) for controlled release comprising the composition or the microsphere, nanoparticle, film or porous matrix of any of the aspects of the invention. In a particular embodiment of this aspect of the invention, the drug delivery system is a stereotactic device or apparatus capable of introducing the composition of any of the above aspects of the invention into the brain. In another particular embodiment, the drug delivery system is an atroscopic surgery device capable of introducing the invention of any of the previous aspects of the invention into the joint.

 A fifth aspect of the invention relates to the composition according to any of the previous aspects of the invention or the drug delivery system of the invention, for use in therapy, particularly, for use in the treatment of brain tumors; more particularly, for use in the treatment of glioblastoma multiforme (GBM), particularly when the composition comprises a BMP selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 BMP - 8b.

A further aspect of the invention relates to the composition according to any of the previous aspects of the invention or the drug delivery system of the invention, for use in therapy, particularly, for use in the treatment of cartilage regeneration; particularly when the composition comprises a BMP selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 or BMP-8b and BMP-14 and / or a TGF selected from the TGF group -βΙ, TGF-p2 or TGF-p3.

 A further aspect of the invention relates to the composition according to any of the previous aspects of the invention or the drug delivery system of the invention, for the preparation of a medicament, particularly, for use in the treatment of brain tumors, in particular glioblastoma multiforme (GBM), and in the regeneration of cartilage.

 A sixth aspect of the invention relates to a pharmaceutically acceptable composition having as its active ingredient or as one of its active ingredients the composition of the microspheres, films, porous matrix or nanoparticles or any of the above aspects of the invention. In an embodiment of the present invention, the pharmaceutically acceptable composition for use in therapy, particularly, in the treatment of brain tumors or in the regeneration of cartilage; more particularly, in the treatment of glioblastoma multiforme (GBM), preferably when the composition or microsphere of the invention comprises a BMP selected from the group consisting of BMP-2, BMP-5, BMP-6, BMP-7, BMP-8b and BMP-14 and / or a TGF selected from the group of TGF-β 1, TGF-p2 or TGF-p3.

BRIEF DESCRIPTION OF THE FIGURES

 Figure 1: Effect of soluble BMP-7 on the ability of U-87MG cells cultured as neurospheres to form new neurospheres and on the size of the formed neurospheres. A) Neurospheres formed de novo were cultured in the presence of BMP-7, B) Neurospheres formed when previously exposed to soluble BMP-7.

Figure 2: The cell cycle profile of U-87MG cells cultured as neurospheres treated with soluble BMP-7. After six days in culture, the neurospheres were pulsed with BrdU and the incorporation of BrdU into the newly synthesized DNA of replicating cells was analyzed by flow cytometry. Left: Neurospheres cultured in the absence of soluble BMP-7 in the culture medium (control). Right: Neurospheres cultured in the presence of soluble BMP-7 (50 ng / mi) in the culture medium. Figure 3: BMP-7 causes a pro- f feren ci ad or effect on cells grown as neurospheres. Cytofluorimetric quantification of the nestin, GFAP or doublecortin markers in neurospheres cultured in the absence (control, "A") or presence of soluble BMP-7 (50 ng / mi, "B") in culture medium for six days. As another control, the profiles of the same cells are also presented, whether or not treated with BMP-7, but marked with a non-specific IgG ("C" and "D", respectively). Figure 4: Microscopy images of microspheres obtained by nanocomplex encapsulation technique. A) White microspheres (heparin concentration of 0.2% w / w); B) Microspheres loaded with 0.2% of chemotrypsinogen; C) white microspheres corresponding to a load of 0.01%; D) BMP-7 charged microspheres at a load of 0.01%. The white bar (lower right corner) corresponds to 50 μιη (A, CD), less in B where it corresponds to 10 μτη.

Figure 5: In vitro release profile of BMP-7 encapsulated in microspheres composed of PLGA: TI 107 and heparin.

Figure 6: Effect of BMP-7 and the different components of the microspheres on the ability of U-87MG cells to form new neurospheres (A) and on the size of deformed neurospheres (B). The size of the neurospheres was classified as small (area between 1450 and 5918 um ² ), medium (area between 5919 and 11340 um ² ) and large (area between 11341 and 85296 um ² ). The groups tested were: neurosphere (control) culture medium, heparin in control medium, Tetronic (1107) in control medium, BMP-7 in control medium and BMP-7, heparin and Tetronic in culture medium (Mixture ). The data represent the mean ± standard deviation of six experiments.

Figure 7: Effect of BMP-7 released (50 ng / mi) from the microspheres in 300 and 90 days (30dR and 90dR groups, respectively) on the ability of U-87MG cells to form new neurospheres (A) and on size of neurospheres (B). As controls, the culture medium of neurospheres (control), BMP-7 (50 ng / mi) in control medium (BMP7), medium where the white microparticles are incubated during the same time intervals (30dC and 90dC), were also analyzed. , and the white microparticle incubation medium with 50 ng / mL BMP-7 (90dC + BMP7). The size of the neurospheres was classified into small (area between 1450 and 5918 um ² ), medium (area between 5919 and 11340 um ² ) and large (area between 11341 and 85296 um ² ). The data represent the mean ± standard deviation of six experiments. Figure 8: Optical microscope images of the neurospheres resulting in the culture of U-87MG cells in the neurosphere for 7 days (control) or cultured in: control medium with 50 ng / mi BMP-7 (BMP-7) , control medium incubated for 37 ⁰ C for 15 days after exposure of the cells, white microsphere incubation medium (90 day time point, 90dC), white microsphere incubation medium at 90 days with 50 ng / mL of BMP-7 (90dC + BMP7), microsphere release medium (90 day time point, 90dR). Arrows show uninitiated tumors.

 Figure 9: General scheme of the steps of the procedure used to integrate the proteins into the devices of the present invention. Legend: Protein with a heparin-binding region (A), poly-sulfated polymer (B), polysulfated protein-polymer complex (C), cationic polyoxyethylene (D) cationic, polyoxyethylene-coated polysulfated protein-polymer complex (E), encapsulation in the biodegradable polymer.

Figure 10: (A) Preparation of films with different combination of polymers for the controlled release of growth factors. The photo shows a real movie prepared by this method next to a coin. (B) Study design: plate distribution of the different polymers.

 Figure 11: Release of BMP-7 from films prepared with the different compositions. (A) Films composed of poly (8-caprolactone) (PCL); (B) films composed of poly (lactic-co-glycolic) (PLGA); (C) films composed of pMVEMA. The compositions have poloxamine or methoxypolyethylene glycol amine, and dextran sulfate or heparin as indicated in the figure. BMP-7 concentrations were detected by ELISA. Data represent the mean ± standard deviation of three experiments.

 Figure 12: SEM images of PLGA-poloxamine-heparin microparticles at different incubation times in 1% PSA BSA solution at time 0 (a), 24 h (b), 1 week (c), 2 weeks (d), 4 weeks (e) and 8 weeks (f).

Figure 13: SEM images of PLGA microspheres in degradation process that reveal the hollow interior. The white bar in the lower right corner equals 50 μιη.

Figure 14: TEM images of heparin / poloxamine nanoparticles in a PLGA matrix. The white bar in the lower right corner equals 100 nm. Figure 15: Stab ilid ad of the nanoparticles of PLGA in aqueous medium at room temperature and 4 ° C for 28 days. Data represent the mean ± standard deviation of three experiments.

 Figure 16: Preparation of porous matrices by the "solvent casting / salt leaching" method. The polymer mixture is added dissolved in a known amount of salt, homogenized and introduced into the molds (A). Once the solvent is evaporated, a polymer matrix is left with the homogeneously distributed salt (B), which is subsequently removed by dissolution in water, leaving the porous matrix ready for use (C). Photo (C) shows a porous matrix prepared by this method next to a coin, as a size reference.

 Figure 17: SEM surface images (A and B) and vertical sections (C and D) of porous heparin-poloxamine-PLGA matrices. It can be seen how in A and C, whose manufacturing process used a higher concentration of polymer (20% with respect to the solvent), the number of pores is smaller than in B and D (10% of polymer with respect to the solvent) , which presents a more open structure.

 Figure 18: Mice after 3 months of tumor growth (A). To the control group, the tumor cells were injected with white microparticles and to the BMP-7 group, microparticles loaded with this factor. Size of the tumors of both groups of mice at the end of the study (B). Western Blot that reveals the phosphorylation of the Smad-1/5/8 pathway in mice administered with loaded microparticles (C).

 Figure 19: Appearance of pellets of hMSCs grown for 21 days in chondrogenic medium and supplemented with microparticles loaded with BMP-7 (A), or without BMP-7 (B). Relative expression of Sox9 in "pellets" with BMP-7 ("Test") and targets ("Control") (C). Histology of the control pellets (D) and those treated with BMP-7 (E).

 Figure 20: Appearance of porous matrices loaded with BMP-7 / TGF-P3 at the beginning of the experiment (A) and after 21 days (B). Section of control porous matrices in which no staining of cartilage is observed by safranine (A) and porous matrix with BMP-7 / TGF-P3 in which clearly stained areas indicative of cartilaginous tissue (B) appear.

Figure 21: The relative expression of the Sox9, aggrecan and type II collagen genes in hMSCs grown in basal medium and in porous matrices loaded with BMP-7 / TGF-P3 ("Test") with respect to cells grown in white porous matrices ("Control"). In the 21-day studies (A) and (B) significant increases (p <0.05) were observed in the expression of chondrogenic ores SOX9 and aggrecano. In the 28-day study, a significant increase in the expression of the aggrecan and type II collagen genes was observed. DETAILED DESCRIPTION OF THE INVENTION

 The present invention provides a new way to encapsulate a hydrophilic protein that has a heparin binding domain in a hydrophobic matrix and achieve controlled release thereof. More particularly, the present invention solves the problem of the treatment of brain tumors, particularly GBM, by providing a new controlled release formulation having a hydrophilic protein with a heparin binding domain, in particular a BMP, more particularly, BMP- 2, BMP-4, BMP -5, BMP-6, BMP-7, BMP-8b and / or BMP-14, which can be implanted in the brain and improves the protein's ability to reduce CSC tumorogenicity, particularly GBM CSC. In order to arrive at the present invention, the potential of a certain BMP, BMP-7, to suppress GBM CSC was studied, and for which the U-87MG glioma cell line was cultured as neurospheres, planting them as a unicellular suspension in serum-free culture medium supplemented with mitogens (EGF and basic FGF), and a decrease in the ability to form new neurospheres (tumorogenicity) of the U-87MG glioma cell line treated with BMP-7 soluble in two different concentrations was observed (50 and 100 ng / mL) (Figure 1A).

Next, the ability of the U-87MG glioma cell line to recover its ability to form neurospheres when previously exposed to BMP-7 was analyzed and later the culture medium factor was removed. We have observed that pre-exposure to BMP-7 does not affect the ability of cells to form neurospheres, however, surprisingly, a certain increase in the formation of neurospheres was observed in cultures treated with 50 and 100ng / ml BMP-7 soluble and a smaller increase for those treated with 150ng / mL soluble BMP-7 (Figure IB). This surprising result indicates that BMP-7 has to be supplied to these cells in a sustained manner in order to totally decrease their tumorogenicity.

In this sense, a polymeric device is designed to encapsulate BMP-7 and protect it from degradation due to the short half-life of BMPs in the body, which limits its use as a therapeutic drug. This system of Release could be used in the treatment of GBM to release this growth factor in a controlled manner in the Central Nervous System.

By incorporating the protein in the form of a nanocomplex dispersion, we were able to synthesize PLGA-based polymer particles with a very high yield for the various compositions (Table I and II below).

Tables I. Characteristics of the PLGA microspheres, obtained by means of the nanocomplex encapsulation method, whether or not loaded with achymotrypsinogen-heparin

 * Control corresponding to the theoretical load of 0.1%

** Control corresponding to the theoretical load of 0.2%

Table II Characteristics of PLGA microspheres, obtained by the nanocomplex encapsulation method, loaded or not with BMP-7

 Average size Efficiency of

performance

 Composition of encapsulation particles

 (%)

 (μιη) ± SD (%)

PLGA: T1107 87.7 ± 6.2 26.0 ± 12.3 - (8: l) / heparin *

PLGA: T1107 (8: l) / BMP-7- heparin 90.5 ± 3.6 21.6 ± 11, 1 93.5 ± 4.8

0.01% theoretical load

 * Control corresponding to the theoretical load c e 0.01%

Although the particles obtained were very heterogeneous in size, they can be classified as microspheres (Figure 4), with an average size of 10 to 30 microns. The results obtained here suggest that the incorporation of Tetronic 1107, heparin and BMP-7 into PLGA did not affect the performance of the microsphere synthesis process, neither its morphology nor its size (Table I, Figure 4).

Triphasic release profiles in vitro of BMP-7 obtained in this invention, in PBS (pH 7.4) containing 1% (w / v) BSA and free culture medium neurospheres mitogen at 37 ⁰ C (Figure 5); they were surprisingly longer and more constant than the typical release profiles of other macromolecules encapsulated in PLGA microspheres. Polymeric microspheres preserve the antigenic activity of the encapsulated growth factor for a long period, as determined by ELISA.

Mechanistic studies on the processes that govern the release of microspheres have indicated that there is a process of biodegradation of the polymer during this process (Figure 12). In addition, the morphological study carried out during degradation has shown that at least a significant fraction of the larger microparticles are hollow inside (Figure 13), which may be relevant to the observed release kinetics.

 Due to the results obtained with soluble BMP-7 on the ability of U-87MG cells, grown as neurospheres, to form new neurospheres from a single-cell suspension of these cells at a clonal dilution, we decided to test whether the protected BMP-7 within polymer microspheres, it retains this effect when released.

In this sense, we have been able to determine that BMP-7 released at 30 and 90 days of incubation induced a significant decrease in the number of newly formed neurospheres compared to the control neurosphere culture and with BMP-treated cells. 7 soluble, poloxamine, heparin and their combinations Additionally, a significant reduction in the size of the neurospheres was obtained when treated with BMP-7 released after 90 days of incubation.

 Figures 6, 7 and 8 show the results obtained with BMP-7 released from microspheres on the tumorigenic capacity of U-87MG cells grown as neurospheres. Additionally, we have tested the effects of soluble BMP-7, poloxamine, heparin and their combinations on the tumorigenic capacity of U-87MG cells grown as neurospheres, in order to determine their contribution to the results obtained.

The significant decrease in tumorigenicity of U-87MG cells cultured as neurospheres, when treated with BMP-7 released from particles composed of PLGA, heparin and Tetronic 1107, confirms that encapsulation of BMP-7 in polymeric microspheres is a strategy effective to protect this factor and preserve its biological activity. Likewise, the released BMP-7 not only affected the ability of these cells to form neurospheres, but this growth factor influenced the proliferation capacity of these cells also causing the reduction of the size of the formed neurospheres; This confirms the capacity of the growth factor to reduce the recurrence of GBM. These results indicate that the encapsulation of BMP-7 in the microsphere of the present invention improves its activity, resulting in an anti-tumorigenic and anti-proliferative effect.

 In an in vivo study, tumor formation was compared for 3 months from primary glioma lines (12012) grafted on the flank of the naked mouse and in the presence of BMP-7 loaded microspheres or white (control) microspheres. The study revealed a much lower growth of BMP-7 microparticle implanted tumors compared to the control (Figure 18), and that said biological effect correlated with a measured activation in the BMP-7 signaling pathway tumor (p- Smad 1/5/8).

The success of this peptide administration technology has led us to consider other pharmaceutical forms, always based on the concept of preformation of a sulphated-protein polymer complex with cationic heparin-derived polyoxyethylene-derived region, and its integration into a biodegradable polymer Thus, we have developed in addition to microspheres, polymeric films (Figures 10 and 11), nanoparticles (Figure 14), and porous matrices (Figure 16 and 17). We consider that nanoparticles could be an interesting alternative means for the release of BMP in antitumor therapies, since the system can allow its injection by various routes, and is stable to storage for at least one month (Figure 15).

Controlled release systems of growth factors and other similar proteins also have a notable interest in functional regeneration therapies of organs and tissues, since they allow the formation of pro-regenerative signals at the space-time level. The spatial control allows a regional delivery of the drug in the area of implantation of the device, thus avoiding side effects in other tissues. Temporary control allows to sustain the effect of the growth factor or morphogen, which, as previously mentioned, usually has a very reduced half-life. As a test of this application, we have studied the use of the technologies of the invention for the regeneration of cartilage, a current challenge of orthopedic medicine.

In a first study, we compared cartilage formation from human mesenchymal stem cells cultured for 21 days in the form of a pellet, in chondrogenic medium and supplemented with BMP-7 microspheres or with white microspheres (control). The macroscopic and histological observation led us to conclude that the supplementation of the chondrogenic medium with a BMP-7 controlled release system allows cartilage quality to improve. In addition, quantitative PCR studies demonstrated an overexpression of the SOX9 early chondrogenic marker for pellets cultured with BMP-7 with respect to the controls (Figure 19). Based on this result, we decided to develop devices for cartilage regeneration based on porous films or matrices. In a systematic study, we lyophilize BMP-7 complexes and integrate them into films using up to 12 different compositions resulting from changing: (1) the biodegradable polymer controlled release, which could be PLGA, poly (8-caprolactone) (PCL) or pMVEMA;

(2) the complexed sulfated polymer, which could be heparin or dextran sulfate; Y

(3) the polyoxyethylene derivative cation which could be Tetronic ^® or amine methoxypolyethylene (Figure 10). Studies showed a release of

BMP-7 later for the PCL, and a more pronounced "burst" effect for the pMVEMA. The effect of the other components was modest (Figure 11). Due to its intermediate release profile and previous experience with microspheres we decided to use the PLGA system: heparin: tetronic to form porous matrices for regeneration of cartilage. In this case, two growth factors, BMP-7 and TGF-P3 were co-encapsulated as heparin and Tetronic® complexes in the PLGA matrices, which were precipitated on a salt mask of granulometry known to form porous structures. , porous matrices (Figure 16). The bioactivity of these porous matrices loaded with growth factors was studied in another chondrogenesis trial. In this trial, mesenchymal stem cells were seeded on the porous matrices, with or without factors, and grown in chondrogenic medium without factors for 21 and 28 days. After 21 days, the formation of tissue in the porous matrix with factors was evident even at the macroscopic level. Histological studies showed a much higher cartilage formation for porous matrices with factors than in control porous matrices (Figure 20). Gene expression studies confirmed this more pronounced chondrogenic process of porous matrices loaded with BMP-7 and TGF-P3 than controls. Specifically, overexpersion of the early chondrogenic SOX9 marker was observed at 21 days, and overexpression of the aggregate cartilage marker. At 28 days of culture, porous matrices with factors overexpressed markers of extracellular matrix of aggrecan cartilage and type II collagen (Figure 21).

 In summary, this new controlled release formulation can be used to encapsulate any hydrophilic protein that has a heparin binding domain in a hydrophobic matrix and achieve controlled release of the protein with this type of encapsulation, which is much longer and more constant than the typical release profiles of other similar biodegradable polymer devices.

Therefore, a first aspect of the invention relates to a composition suitable for the controlled release of hydrophilic proteins with a heparin binding domain, comprising:

 to. A surface layer of a biodegradable hydrophobic polymer, and b. A nucleus of a cationic polyoxyethylene derivative physically bonded to a polysulphated polymer which in turn is physically bonded to a hydrophilic protein with a heparin binding domain.

In the context of the present invention, hydrophilic proteins that have a heparin binding domain include all proteins that have any of the following three consensus sequences XBBXBX, XBBBXXBX or XBBBXXBBBXXBBX, where B is a base and X is a hydropathic amino acid residue (neutral and hydrophobic). However, it has been determined that other proteins have heparin binding sequences even without these consensus sequences. These sometimes contain basic amino acids that in their tertiary structure are close forming cationic regions, capable of interacting very closely with heparin. Likewise, the 3D conformation analysis of these proteins has suggested that a spatial separation between the basic amino acids of 20 Amgstrons or less is important for interactions. These cationic pockets are not normally in the same region of the pharmacologically active site of the protein.

In particular, hydrophilic proteins that have a heparin binding domain include, but are not limited to, growth factors such as: endothelial vascular growth factor (VEGF), interleukins (IL), transforming growth factor (TGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), stromal cell derived factor (SDF), growth and differentiation factor (GDF), platelet factor (PF) , ligand of the family of chemokines CC (CCL), ligand of the family of chemokines CXC, ligand of the family of chemokines C, chemokines CX ₃ C, antithrombins, neurotrophins, ligands of the family of neurotrophic factor derived glial cell line ( GFL), BMPs, chymotrypsinogen or any combination thereof. More particularly, hydrophilic proteins that have a heparin binding domain include BMPs selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8b, BMP- 14 or any combination thereof. In a more particular embodiment of the invention, it is the BMP-7 bone morphogenic protein.

Here, bone morphogenic protein 7 or BMP7 (also known as osteogenic protein-1 or OP-1) is used which is a protein encoded in humans by the BMP7 gene whose protein structure can be found in the bank of genes with the accession number AAG43508. The transforming growth factor-beta 3, or TGF-P3, which is a protein encoded in humans by the TGFB3 gene, is also used.

In the context of the present invention, biodegradable hydrophobic polymers include all those polymers that do not form a homogeneous 5% solution (w / w) in water and that when implanted in the body are reabsorbed in less than 2 years. In particular, biodegradable hydrophobic polymers include polyesters, polyanhydrides, poly (ortho esters), polyamides, poly (alkyl cyanoacrylates), polyimides, polyester-poly (ethylene glycol) copolymers, polyphosphazenes, poly (phosphoesters) or any combination thereof. In a more particular embodiment, the hydrophobic biodegradable polymers include polyesters of the group consisting of poly (hydroxyvalerate), polycaprolactone (PCL), poly (lactic-co-glycolic) (PLGA), poly (hydroxybutyrate), poly (hydroxybutyrate-co- valerate), polydioxanone or poly (glycolic acid). In another more particular embodiment the hydrophobic polymers include polyanhydrides of the pMVEMA group. In a further embodiment, hydrophobic biodegradable polymers include polyphosphoesters such as urethane polyphosphorester. In a more particular embodiment, hydrophobic biodegradable polymers include polyamides of the group consisting of poly (amino acids), in which the amino acids are hydrophobic amino acids such as alanine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan or tyrosine; polyamide; a linear polymer of γ-aminobutyric acid (GABA) or nylon 6,6, polycaprolactam. In a more particular embodiment, the hydrophobic biodegradable polymer is polyurethane. In an even more particular embodiment, the biodegradable hydrophobic polymer is PLGA, PCL or pMVEMA, which can be implanted in the Central Nervous System, either by open surgery or by a stereotactic technique, or implanted in the joints by arthroscopic surgery. .

 A biodegradable hydrophobic polymer having an average molecular weight (Mw average) of 5,000-300,000 (five thousand to three hundred thousand), more preferably from 20,000 to 100,000 (twenty thousand to one hundred thousand), is preferred herein because the yield of the production of microspheres can be reduced and the stability of the polysaccharide reduced due to the difficulty in molecular formation if the Mw is outside the aforementioned range.

In the context of the present invention, the cationic polyoxyethylene derivative is a grafted, block or random graft copolymer of polyoxyethylene and polyoxypropylene having at least one potentially cationizable chemical group and where the percentage of polyoxyethylene and polyoxypropylene groups in the copolymer is at least 30% by weight. In a preferred form, the cationizable group is a primary, secondary, tertiary or quaternary amine. In a more preferred form, the cationic polyoxyethylene derivative can be selected from any of the following groups of compounds: methoxypolyethylene glycol amine, poloxamines, chitosan polyoxyethylene derivatives (Le., Chitosan-g-PEG), cationic derivatives of polyamino acids-polyoxyethylene (i. E., Poly sina-g-PEG, polyarginine-g-PEG), polyoxyethylene derivatives of cationic or cationized proteins (ie, albumin- Cationic PEG, cationic JEG-PEG), polyethyleneimine poly oxy ethylene derivatives, polyoxyethylene derived polyamino esters or any combination thereof. In a preferred embodiment of the invention, the cationic polyoxyethylene derivative is methoxypolyethylene glycol amine or poloxamine. The name 'Poloxamine' denotes symmetric polyalkoxylated block polymers of ethylenediamine according to the general type:

 I. [(PEG) X- (PPG) Y] 2-NCH2CH2N - [(PPG) Y- (PEG) X] 2, in which PEG is "polyethylene glycol" and PPG is "polypropylene glycol".

 Each name of Poloxamine is followed by an arbitrary code number, according to the numerical average values of the respective monomer units indicated by X and Y in the previous general type.

 For Poloxamine Tetronic 1107, Y is 20 and X is 60. For Poloxamine Tetronic 90R4, Y and X is 18 is 16.

 Methoxypolyethylene glycol amine denotes a synthetic polymer of the structure:

(PEG) Z- H2

where PEG is polyethylene glycol, and Z a numerical value indicating the amount of ethylene glycol monomers in the polymer. Z has a value between 5 and 200.

In the context of the present invention, the polysulfated polymer is a biodegradable, non-cyclic polymer having at least 1 KDa and having at least three sulfate groups. The polymer can be any of the following compounds: polysulphated silk fibroin, dendritic polyglycerol sulfate or a sulfated proteoglycan. Preferably, the polysulphated polymer is a polysaccharide such as polysulphated hyaluronic acid, polysulfated dextran or polysulfated proteoglycans. In a more preferred embodiment, the polysulphated polymer is heparan sulfate, polysulfated dextran or heparin.

Always respecting the structure and composition of the invention, said formulations may also comprise other pharmaceutical excipients to provide additional technical characteristics. Examples of such excipients may be surfactants (lecithin, polyvinyl alcohol, poloxamer, Span, Tween, bile salts, etc.), surface charge modifying agents (DOTAP, benzalkonium, cetylpyridinium), insoluble basic salts that co-encapsulate in the matrix (calcium and magnesium hydroxide), porogenic agents (granules of sodium chloride) and / or cryoprotectants (glucose, trehalose).

 In a more particular embodiment, the composition of the invention comprises as a biodegradable hydrophobic polymer the PLGA, the PCL or the pMVEMA; as a cationic polyoxyethylene derivative, methoxypolyethylene glycol amine or a poloxamine; as a hydrophilic protein having a heparin binding domain a BMP and / or a TGF, in particular BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8b, BMP-14, TGF-βΙ, TGF-p2 and / or TGF-p3; and as polysulphated polymer of heparan sulfate, polysulfated dextran or heparin. In an even more particular embodiment of the invention, the composition of the invention comprises PLGA, PCL or pMVEMA; methoxypolyethylene glycol amine or a poloxamine; BMP-7, BMP-8b, BMP-14, TGF-βΙ, TGF-P2, TGF-P3; and as polysulphated polymer of heparan sulfate, polysulfated dextran or heparin. The encapsulation of BMP-7 in polymer microspheres composed of PLGA and poloxamine in association with heparin is a very attractive strategy for GBM therapy. This controlled release system can be implanted during tumor resection and improves the ability of BMP-7 to reduce the tumorigenicity of GBM CSC. In addition, this polymeric device could be introduced through the use of a stereotactic technique in the brain in cases where the patient needs successive applications of BMP-7.

 On the other hand, the use of BMP-7 and / or TGF-P3 encapsulated in microspheres or porous matrices of PLGA, poloxamine in association with heparin is a promising strategy for cartilage regeneration as it has been verified in primary cell lines Human mesenchymal mother. It should be noted that the cartilage generated from porous matrices capable of releasing the two factors was performed under conditions in which there was no supplementation of any other external growth factor. Therefore, this result suggests an adequate capacity of the device to promote the generation of cartilage "in situ" once the implantation is carried out.

In a further embodiment, the composition of the invention can be a microsphere with an average size larger than 10 in order to avoid phagocytosis. More particularly, the microsphere has an average size between 10 and 90um. In another real left further, the composition of the invention may be a nanoparticle with an average size between 80 and 500 nm. More particularly the nanoparticle has a size between 100 and 300 nm.

 In another additional embodiment, the composition of the invention may be a film. In another additional embodiment, the composition of the invention may be porous matrix. The porous matrix can have diverse pore sizes and porosities that can be controlled by the characteristics and proportion of porogenic agent used. The pore size of the porous matrices is between 20 and 800 um. More particularly, it is between 100 and 500 um.

In the present invention, the polysulphated polymer forms a specific bond with the hydrophilic protein with a heparin binding domain, thus being able to stabilize the protein drug and significantly favor controlled or sustained release by decreasing the initial release of the drug. This notable improvement in sustained release can be seen in Figure 5 for BMP-7.

As used herein, the terms "physically linked" or the like refer to any type of physical bonds induced by the physical process without any chemical reaction, and examples of the physical bonds include without limitation, an adsorption, a cohesion, crosslinking and entrapment. The compositions described herein are biocompatible. In this sense, the microsphere and the drug delivery system according to the present invention are advantageous in terms of biocompatibility.

In a preferred aspect of the invention, the composition of the invention is a microsphere that preferably has an average size larger than 10 in order to avoid phagocytosis, in particular, it is preferred that the microsphere have an average particle size between 10 and 90 microns

 In a more preferred embodiment the composition of the invention comprises PLGA (50:50), PCL, or pMVEMA; TI 107 poloxamine or methoxypolyethylene glycol amine; polysulphated heparin or dextran.

 A second aspect of the invention relates to a composition suitable for the controlled release of hydrophilic proteins with a heparin binding domain, comprising:

 to. A hydrophobic biodegradable polymer;

b. A cationic polyoxyethylene derivative as a biocompatible emulsifier; C. A hydrophilic protein with a heparin binding domain, and

 d. A polysulphated polymer physically bound to the hydrophilic protein with a heparin binding domain,

wherein the composition is obtainable by mixing an aqueous solution or a solid composition, preferably in the form of a lyophilized powder, comprising elements b), c) and d) linked together with an organic medium comprising element a).

 A third aspect of the present invention relates to a method for producing the composition of microspheres, nanoparticles, films or porous matrix of any of the above aspects of the invention, comprising:

 to. Dissolve the cationic polyoxyethylene derivative in a solution having a hydrophilic protein with a heparin binding domain and a polysulphated polymer;

 b. Dry by lyophilization of the mixture resulting from step (a); C. Resuspend the dry powder of step (b) in an organic solvent containing a biodegradable hydrophobic polymer, and d. Collect microspheres, nanoparticles, porous matrices or films.

It is widely known by the person skilled in the art how to proceed to obtain different final forms of a formulation from an organic phase. So you can select, depending on your needs, the method of collecting microspheres, nanoparticles, films or porous matrices. For example, to prepare nanoparticles, the person skilled in the art could do so by following the following references: (1) Csaba, N., Caamaño, P., Sánchez, A., Domínguez, F., & Alonso, M. J. (2005). PLGA: poloxamer and PLGA: poloxamine blend nanoparticles: new carriers for gene delivery. Biomacromolecules, 6 (1), 271-278; (2) d'Angelo, L, García-Fuentes, M., Parajó, Y., Welle, A, Vántus, T., Horváth, A, ... & Alonso, M. J. (2010). Nanoparticles based on PLGA: poloxamer blends for the delivery of proangiogenic growth factors. Molecular pharmaceutics, 7 (5), 1724-1733.

The preparation of microspheres from an organic phase is described in Tobío, M., Nolley, J., Guo, Y., Mclver, J., & Alonso, MJ (1999). A novel system based on a poloxamer / PLGA blend as a tetanus toxoid delivery vehicle. Pharmaceutical research, 16 (5), 682-688. Film preparation is described for example in García-Fuentes, M., Giger, E., Meinel, L., & Merkle, HP (2008). The effect of hyaluronic acid on silk fibroin conformation. Biomaterials, 29 (6), 633-642. Jeong, JH, Lim, DW, Han, DK, & Park, TG (2000). Synthesis, characterization and protein adsorption behaviors of PLGA / PEG di-block co-polymer blend films. Colloids and Surfaces B: Biointerfaces, 18 (3), 371-379.

 The preparation of porous matrix is widely known and for example is described in García-Fuentes, M., Meinel, A. J., Hilbe, M., Meinel, L., & Merkle, H. P. (2009). Silk fibroin / hyaluronan scaffolds for human mesenchymal stem cell culture in tissue engineering. Biomaterials, 30 (28), 5068-5076. Barbanti, S. H., Zavaglia, C. A. C, & Duek, E. A. D. R. (2008). Effect of salt leaching on PCL and PLGA (50/50) resorbable scaffolds. Materials Research, 11 (1), 75-80.

 The product produced by the above-mentioned synthesis method provides high encapsulation efficiency. In addition, this method protects the proteins from denaturation caused by the organic solvent at the water-in-oil interface and from inactivation in the presence of water. In addition, the cationic polyoxyethylene derivative and the polysulfated polymer of the composition protect the hydrophilic protein, especially BMP-7 and / or TGF-P3, during the synthesis process. The cationic polyoxyethylene derivative, particularly methoxypolyethylene glycol amine or poloxamine as a surfactant, is capable of forming micelles and the polysulphated polymer, especially heparan sulfate, polysulphated dextran or heparin, stabilizes the drug during the lyophilization process. In this case, the polysulfated polymer can also stabilize the hydrophilic protein, especially BMP-7 and / or TGF-P3, by specific binding sites. The absence of an external aqueous phase in the present microsphere formation method prevents the diffusion of the protein from the internal phase favoring its entrapment.

In addition, through the use of this method, the rapid initial release (burst effect) of the devices was reduced, and that of the PLGA, Tetronic and heparin microspheres was less than 3% of the encapsulated BMP-7, indicating that the Amount of protein located on the surface of the particle is very small. This is due to the presence of the cationic polyoxyethylene derivative in the formulation, in particular due to the presence of poloxamine, which in fact favors the entrapment of the hydrophilic protein during microsphere synthesis and with its incorporation into the protein solution (previous to freeze drying). It has been observed that when The surfactant added in this phase reduces the burst effect compared to its incorporation into the organic phase together with PLGA. In addition, when poloxamine is added in this phase, the encapsulation efficiency increases.

In a preferred embodiment of this aspect of the invention, the composition of the invention is a microsphere that is preferred to have an average size larger than 10 in order to avoid phagocytosis, and it is particularly preferred that the microsphere have an average particle size. between 10 and 90 um.

In another additional embodiment, the composition of the invention can be a nanoparticle with an average size between 80 and 500 nm. More particularly the nanoparticle has a size between 100 and 300 nm.

 In another additional embodiment, the composition of the invention may be a film. In another additional embodiment, the composition of the invention may be porous matrix. The porous matrix can have diverse pore sizes and porosities that can be controlled by the characteristics and proportion of porogenic agent used. The pore size of the porous matrices is between 20 and 800 um. More particularly, it is between 100 and 500 um.

 A fourth aspect of the invention relates to a drug delivery system for controlled release comprising the composition according to any of the above aspects.

As used herein, a drug delivery system includes those that involve drug preparation, route of administration, site orientation, metabolism and toxicity.

In a preferred embodiment of the fourth aspect of the invention, the medicament delivery system is a stereotactic device or apparatus capable of introducing the microspheres of the invention into the brain. In another particular embodiment of this aspect of the invention, the drug delivery system is an atroscopic surgery device capable of introducing the invention of any of the previous aspects of the invention into the joint.

 In a fifth aspect of the invention, the microsphere, film, porous matrix or nanoparticle of any of the above aspects of the invention or the drug delivery system of the invention is employed in the therapy.

In a sixth aspect of the invention, the composition or microsphere of any aspect of the foregoing of the invention or the drug delivery system of the invention is employed in the treatment of brain tumors, particularly the glioblastoma multiforme, preferably when the composition comprises any of the following hydrophilic proteins: BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8b or BMP-14, particularly BMP-7.

 A further aspect of the invention relates to the composition of any of the above aspects of the invention or the drug delivery system of the invention, for use in therapy, particularly, for use in the treatment of cartilage regeneration. ; particularly when the composition comprises a BMP selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 or BMP-8b and BMP-14 and / or a TGF selected from the TGF group -βΙ, TGF-p2 or TGF-β3.

 In a seventh aspect of the invention, the composition or microsphere of any aspect of the foregoing of the invention or the drug delivery system of the invention is used for the manufacture of a medicament for the treatment of brain tumors, in particular for the treatment of glioblastoma multiforme, preferably when the composition comprises any of the following hydrophilic proteins: BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8b, BMP-14, particularly BMP- 7. In a further embodiment of this aspect of the invention the composition of any previous aspect of the invention or the drug delivery system of the invention is employed for the regeneration of tissues or organs, specifically for the regeneration of cartilage, preferably when the composition it comprises a BMP selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 or BMP-8b and BMP-14 and / or a TGF selected from the group of TGF-βΙ, TGF -p2 or TGF-p3. The following examples serve to illustrate the present invention, these examples are not intended in any way to limit the scope of the invention.

EXAMPLES

 Example 1. MATERIALS AND METHODS

1.1 Materials

For cell studies, the immortalized human glioblastoma U-87MG cell line (ATCC HTB-14TM) was used. For cell culture, Dulbecco medium modified by Eagle (DMEM), DMEM-F12 1: 1 medium, serum free B-27 supplement, L-glutamine, penicillin-streptomycin, trypsin and Fetal bovine serum, acquired from Invitrogen (Spain). Recombinant human epidermal growth factor and recombinant human basic fibroblast growth factor were purchased from PeproTech (United Kingdom) and Accumax de Millipore (Spain) was obtained. Recombinant human bone morphogenic protein 7 (rhBMP-7) (pl 8, 1 MW = 28.8 kDa) was purchased from PeproTech (UK).

 For flow cytometry studies, bromodeoxyuridine (BrdU), anti-BrdU antibody conjugated with fluorescein isothiocyanate (FITC), ribonuclease A (RNAse) of bovine pancreas and Triton X-100 were used, all acquired in Sigma Aldrich (Spain) ; propidium iodide acquired from Calbiochem (Spain); BD Cytofix ™ fixation buffer, BD Phosflow ™ III permeabilization buffer, human anti-nestin antibody labeled with the pyridine-chlorophyll A (PerCP) and cyanine 5.5 (CyTM5.5) (PerCP-CyTM5.5) protein complex, antibody anti-doublecortin and glia anti-fibrillar acidic protein antibody (GFAP), both antibodies labeled with phycoerythrin (PE), all acquired from BD Biosciences (Spain).

 Poly (D, L-lactide-co-glycolide) 50:50 Resomer ® RG 503 (PLGA) polymer (PM = 34 kDa) was purchased from Boehringer Ingelheim (Germany). Polymers pMVEMA (poly (methyl vinyl ether-alt-maleic anhydride), poly (8-caprolactone), methoxypolyethylene glycol amine, polysulphated dextran, Tetronic ® 90R4 (T90R4 HBL = 1-7 PM = 7.2 kDa), Tetronic ® 1107 (TI 107 HBL = 24 PM = 15 kDa), bovine pancreas a-chymotrypsinogen (pl 9.5 PM = 25.7 kDa), grade IA sodium heparin salt of the porcine intestinal mucosa, cottonseed oil and soy lecithin, all acquired from Sigma Aldrich (Spain): ELISA assays used: human anti-BMP-7 polyclonal antibody obtained in rabbit and human anti-BMP-7 antibody obtained in biotinylated rabbit, both acquired from PeproTech (United Kingdom); avidin-peroxidase conjugate, azino-bis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS), BSA, Tween 20 and sodium phosphate buffer (PBS) (pH 7.4), which were purchased from Sigma Aldrich ( Spain) The other solvents and reagents used were of a high degree of purity.

1.2 U-87MG neurosphere culture

U-87MG cells were grown in neurosphere culture medium (DMEM / F-12 (1: 1), L-glutamine, serum free B-27 supplement, 1% penicillin-streptomycin, 20 ng / mi EGF , 20 ng / mi of bFGF). After 6 days, the neurospheres will subcultured by centrifugation, disintegrated with Accumax and plated at 40,000 cells / ml in fresh neurosphere culture medium.

 1.3 Neurosphere proliferation studies

 The U-87MG cell neurospheres were disintegrated and cultured at high dilution (500 cells / well, clonal conditions) in 96-well culture plate with 200μL of neurosphere medium. The effect of BMP-7 on proliferation was tested by adding 50, 100 or 150 ng / ml of BMP-7 per well. The cells were cultured under these conditions for 6 days and the culture was characterized in terms of the number and size of the microspheres formed by microscopy using the Cell A version 2.6 computer program (Olympus Soft Imaging Solutions GMBH, Germany).

 In addition, the ability to form new neurospheres of the cells resulting from the previous experiment was checked (control vs BMP-7 treated cells). For this experiment, the neurospheres are formed from isolated U-87MG cells (1600 cells, 100 mm of plate) by culturing in 10 ml of neurosphere medium with either (50, 100 or 150 ng / mi) or without BMP-7 After 6 days, the neurospheres were disintegrated with Accumax and seeded at a low density in a 96-well plate (500 cells / well). All samples were cultured with neurosphere medium (without BMP-7). The effect of previous exposure to BMP-7 was tested by counting and measuring the resulting neurospheres by microscopy using the Cell A version 2.6 computer program (Olympus Soft Imaging Solutions GmbH).

 1.4 Cell cycle studies

For cell cycle analysis, the neurospheres were cultured in the middle of neurospheres with (50 ng / mi) or without BMP-7 for 6 days. Then, the neurospheres were pulsed with 100 mM BrdU for 8 hours. After this time, the neurospheres disintegrated and the cells were washed with PBS. Then frozen ethanol was added to 1 million cells at a final concentration of 75%. The cells were centrifuged for 5 minutes and washed twice. The cells were centrifuged and the pellet was resuspended with 2 N hydrochloric acid. The cells were incubated for 20 minutes at room temperature and washed. The cells were centrifuged and the pellet was resuspended with 0.1 M sodium borate solution, pH 8.5. The cells were incubated for 2 hours at room temperature and washed again. The cells were centrifuged and 10 μΕ was added to them of FITC-conjugated anti-BrdU antibody diluted in DPBS containing 0.5% (w / v) BSA and 0.5% Tween 20. The cells were incubated for 1 hour, washed and centrifuged. The cells were incubated overnight in the dark with PBS containing propidium iodide (10 mg / mL), RNase (500 ug / mi) and 0.1%) of Triton X-100 and analyzed for FITC against fluorescence by flow cytometry (Becton Dickinson flow cytometer, FACScanTM model, Spain).

 1.5 Immunocytochemistry

 To determine the percentage of U-87MG cells with specific molecular markers of neuronal stem cells, neurons and / or astrocytes in a population of neurospheres, the disintegrated U-87MG neurospheres were washed with PBS and centrifuged for 5 minutes. The pellet was resuspended with BD Cytofix ™ fixation buffer and incubated for 20 minutes in the dark. The cells were washed twice with PBS and centrifuged. Ice-cold Permeation Buffer III BD PhosFlow ™ was added to the cells and then incubated on ice for 30 minutes. The cells were washed twice with PBS and centrifuged. The cells were incubated on ice for 30 minutes in the dark with 100 ul of PBS containing 0.5%> (w / v) of BSA and 5 ul of each conjugate antibody to be analyzed (human anti-nestin antibody labeled with PerCP-CyTM5.5; PE-labeled anti-doublecortin antibody, and PE-labeled anti-GFAP antibody). The cells were washed twice with PBS and centrifuged. The cells were then resuspended in 400 ul of PBS to be analyzed in a flow cytometer (Becton Dickinson, FACScanTM model, Spain).

Example 2. Effect of soluble BMP-7 on the ability of U-87MG cells cultured as neurospheres to form new neurospheres and on the size of the formed neurospheres.

To analyze the effect of BMP-7 on the ability of U-87MG to form de novo neurospheres, the disintegrated cells of neurospheres were cultured as a unicellular suspension at a clonal dilution and cultured for 6 days in a neurosphere culture medium supplemented with different doses of BMP-7 (doses 50, 100 and 150 ng / mi). Then, to analyze the pre-exposure effect of U-87MG cells cultured as neurospheres at different doses of BMP-7, as described above, on the sixth day of culture the neurospheres were disintegrated and seeded as a suspension. single cell at clonal dilution in culture medium of Neurospheres for another 6 days. Neurospheres were counted and measured by microscopy (Figure 1).

 Example 3. BMP-7 causes a pro-differentiation effect in neurospheres

 Based on the results obtained with soluble BMP-7 on the ability to initiate tumors of the U-87MG cells detailed in Example 2 above, it was decided to continue working on 50 ng / mL of this growth factor and analyzed whether the observed effect Neurosphere culture is due to the fact that this factor acts on the regulation of the cell cycle, or because it induces the differentiation of tumor-initiating cells isolated from neurospheres. Similar cell cycle profiles were obtained from U-87MG cells cultured as neurospheres in neurosphere medium supplemented with 50 ng / mL soluble BMP-7 and those grown as control (Figure 2).

 The effect of soluble BMP-7 (50 ng / mi) on the regulation of differentiation of cancer stem cells was studied. For this purpose, some specific molecular markers representative of neural stem cells (nestin), astrocytes (GFAP) and neurons (doublecortin) in U-87MG cells cultured as BMP-7 treated neurospheres as well as in untreated ones are analyzed. This growth factor induced a moderate increase in the percentage of cells expressing GFAP and the percentage of cells expressing doublecortin was reduced, without affecting those expressing nestin (Figure 3). This indicates that soluble BMP-7 (at 50ng / ml) induces a slight differentiation of these cells with CSC-like properties to more differentiated cells with astrocytic characteristics, and decreases the population of cells with specific molecular marker of neurons. These results are represented in the table below herein. This table shows the results obtained from the molecular markers analyzed in a unique way for the cell population. Soluble BMP-7 increases the percentage of cells in the population with specific astrocyte molecular marker (GFAP) and reduces the percentage of cells with specific molecular marker of neurons.

Sample / Molecular Marker (%) Nestin GFAP Doublecortin

U-87MG cells cultured as 94.2 11.7 24, 1 neurospheres

U-87MG cells cultured as

 Neurospheres in the presence of BMP-7 96.8 21.6 11.2 (50ng / mL)

The results obtained with soluble BMP-7 on the population of neurospheres show that BMP-7 acts as a suppressor of the tumorigenic capacity of GBM CSC, reducing recurrence and metastasis and improving patient survival.

 Example 4. Release of BMP-7 from polymer microspheres

 4.1 Preparation of PLGA microspheres: Tetronic ® 1107 loaded with BMP-

7

An "oil-in-oil" (O / O) solvent extraction / evaporation technique was used, where the protein to be encapsulated in the oily internal phase was incorporated in the form of a nanocomplex. Briefly, 2.5 mg of TI 107 was dissolved in 300 ul of a protein solution in a 1: 1 mass ratio with heparin before microencapsulation. The resulting mixture was frozen and then lyophilized (primary drying at -35 ° C, secondary drying at 25 ⁰ C; Liophylizer VirTis Genesis 25GB, USA) after incubation for 30 min at room temperature to ensure interaction of the components. Subsequently, the dry powder was resuspended with 400 ul of acetonitrile containing 20 mg of PLGA. Under vigorous stirring, this suspension was added dropwise to 4 ml of cottonseed oil containing 0.5% (w / v) soybean lecithin. After that, the suspension was subjected to ultrasound for 20 seconds using a Branson 250 sonicator (40W outlet, Danbury, CT, USA) and stirred for 45 min in an extraction hood. Two milliliters of petroleum ether was then added to the suspension to harden the immature microspheres and the preparation is stirred for another 10 minutes in the extraction hood. Finally, the microspheres formed were collected by vacuum filtration using a nitrocellulose membrane (25 mm, 0.22 m), washed with petroleum ether and dried by freezing. The obtained microspheres were stored at 4 ⁰ C until use.

Two proteins were encapsulated. First a pro-enzyme, chymotrypsinogen A (NCBI taxonomic identifier = 9913), which has high molecular weight and a Highly cationic pocket comprising 6 Usina residues between positions 78 and 93 of its sequence and non-ammonium amino acids. Two theoretical loads of chymotrypsinogen A, 0.1% and 0.2% (w / w) were tested, which requires 20 mg or 40 mg of the protein in the first aqueous solution of 300 ul. For BMP-7, 2 ug of the protein was added to the 300 ul aqueous solution.

 4.2 Morphology and size of PLGA microspheres: Tetronic

 The morphology and particle size parameters were analyzed by scanning electron microscopy (SEM) (scanning electron microscope LEO-435vp, UK). The samples were placed on a metal sample holder and covered in vacuo with a thin layer of gold-palladium. The size of the microspheres was calculated from micrographs using the DigitalMicrograph (TM) 3.7.0 computer program (Gatan Software, USA).

 4.3 Determination of the encapsulation efficiency of a-chymotrypsinogen

The efficiency of chymotrypsinogen encapsulation was evaluated by a protein extraction method. Briefly, 5 mg of my crop joints was dissolved in 100 ul of dimethyl sulfoxide under continuous agitation (350 rpm, Heidolf, Promax 2020, Germany) for 1 hour. Then, 400 ul of 0.05 M NaOH solution containing 0.5% (w / v) SDS was added to the microsphere solution and the mixture was incubated for another hour under the same conditions. Finally, the sample was centrifuged at 7000 g for 5 minutes at 25 ⁰ C. Protein free microspheres were subjected to the same procedure as a control of the experiment. Chymotrypsinogen encapsulated in the particles was quantified from the supernatant by the Lowry protein assay (Micro BCA Protein Assay Kit Pierce Biotechnology Inc., USA) performed according to the manufacturer's instructions. The results are illustrated in Table I.

 4.4 Determination of encapsulation efficiency for BMP-7

For this determination, a protein extraction method was designed. Briefly, 1 mg of the microspheres was dissolved with 1 ml of dichloromethane under stirring by means of a horizontal stirrer (Heidolph, Promax 2020, Germany) at 350 rpm. The solution was filtered under vacuum using a nitrocellulose membrane (25 mm, 0.22 mm). Next, 3 ml of PBS (pH 7.4) containing 0.1% (w / v) of BSA and 0.05%) Tween 20 were added to the membrane and the mixture was incubated with stirring at 250 rpm for at least 6 hours BMP-7 free microspheres were subjected to the same procedure as a control of this experiment. The BMP-7 encapsulation was quantified by an ELISA as described below. The results are illustrated in table II.

 4.5 In vitro release studies

 Samples comprising 1 mg of PLGA: Tetronic microspheres loaded with BMP-7 and heparin were incubated with 500 ul of PBS (pH 7.4) containing 1% (w / v) of BSA or free neurosphere culture medium of mitogens with stirring (100 rpm) at 37 ° C. At the scheduled time points of 12 hours to 60 days, the microspheres were centrifuged at 7000 g for 10 min at 4 ° C. The BMP-7 released from the antigenically active microspheres was determined by ELISA as described below. At various release points, microspheres of the release medium were also collected, isolated and dried at room temperature, and analyzed by scanning electron microscopy to determine their morphology and structure.

4.6 Quantification of BMP-7 by ELISA

The 96-well microtiter plate was coated with 100 ul of rabbit anti-rhBMP-7 polyclonal antibody at 1 mg / ml in Dulbecco PBS and incubated overnight at 4 ° C in a wet container. The plate was washed three times with PBS containing 0.05% Tween 20 (PBST). To minimize non - specific interactions, 300ÍL PB S containing 1% (w / v) BSA to each well and the plate was incubated for 1 hour at 37 ⁰ C in a humid container were added. After this time, the plate was washed three times with PBST and 100 ul of standard BMP-7 solution and test samples diluted in PBS containing 0.1% (w / v) of BSA and were added to the wells 0.05% Tween 20 (PBST-BSA). The plate was incubated for 4 hours at 37 ⁰ C in a humid container and washed three times again. Then, 100 ul of biotinylated anti-rhBMP-7 antibody (0.5 mg / mL) in PBST-BSA was added to each well. The plate was incubated for 2 hours at 37 ⁰ C in a humid container and washed three times. Then, to each well they were added 100 ul of conjugate avidin-peroxidase diluted 1: 2000 in PBST-BSA and the plate was incubated for 1 hour at 37 ⁰ C in a humid container. The plate was washed three times and 100 ul of substrate (ABTS) was added to the wells. After color development, the plate was read at 405 nm in a microplate reader (Biorad, Microplate reader model 680, Japan).

 4.7. In vivo study of the effect of microspheres with BMP-7.

Tumor cells from xenografts of the primary glioma line 12012 were isolated, centrifuged, resuspended in complete medium and counted. For another On the other hand, 20 mg of microspheres loaded with 10 ug of BMP-7 were resuspended in 75 uL of complete medium. To this suspension approximately 2-3 million cells were added in 75 uL of medium. At the time prior to injection in the side of the naked mouse, an equivalent volume of Matrigel ™ (150 uL) was added. Eight mice were used to inject the two flanks. Four of these mice were implanted with BMP-7 microspheres as indicated, and other mice were injected with white microspheres. The time of the experiment was 3 months, after which the tumors were removed for macroscopic analysis. In addition, an activation analysis of the BMP pathway in tumors was performed, by western blot analysis for the phosphorylated protein Smad 1/5/8.

 4.8. In vitro release profile of BMP-7 encapsulated in microspheres composed of PLGA: TI 107 and heparin and mechanistic studies.

The microspheres were obtained by the nanocomplex encapsulation technique, as described above. The BMP-7 retained its antigenic activity for more than 2 months under both conditions: PBS containing 1% BSA (w / v) and mitogen-free neurosphere culture medium, under agitation (100 rpm) at 37 ⁰ C (Fig . 5). The morphological analysis carried out by scanning electron microscopy of microspheres at different release points showed a progressive degradation of the structures during the middle incubation process (Fig. 12). This degradation of the polymer of the device seems to be the dominant process that controls drug release. During this degradation study we could also observe that a significant fraction of the microspheres had a hollow internal structure (Fig. 13).

 4.9. Effect of the different components of the microspheres on the ability of U-87MG cells grown as neurospheres to form new neurospheres and on the size of de novo-formed neurospheres.

The number of neurospheres of the U-87MG cells cultured in neurosphere culture medium (Control) or the different components of the formulation was measured: heparin, Tetronic (1107), BMP7 (50 ng / mi) or a mixture of the three components (mixture) (Fig. 6). As can be seen, treatments with only BMP-7 (ie BMP-7 or the mixture) produced a detectable reduction in the number of neurospheres (Fig. 6A). The analysis of the size of the neurospheres resulting from these experiments suggests that Tetronic and heparin reduce the fraction of large neurospheres BMP-7 either alone or in admixture with the microsphere components does not influence the size of the neurospheres (Fig. 6B).

4.10. Effect of BMP-7 released from microspheres on the ability of U-87MG cells grown as neurospheres to form new neurospheres and on the size of de novo-formed neurospheres.

 The effect of BMP-7 released in 30, 60 and 90 days on the number of neurospheres can be seen in Figure 7A (RM30d, RM60d, RM90d). As can be seen, BMP-7 released from the microspheres almost completely inhibits the formation of neurospheres, with values below 2% of the formation of neurospheres compared to cells grown in the medium of neurospheres (Control). Interestingly, BMP-7 at 50 ng / mi in control medium induced only a 40% decrease in neurosphere formation. To explain the effect of BMP-7 released from the formulation, the effect of the release medium was also tested from a blank microsphere formulation (ie, without BMP-7). The results showed that the formulation release medium itself generates an inhibitory effect on the formation of neurospheres (RJVBOd-Control, RM60d-Control, RM90d-Control), probably due to the consumption of some nutrients from the culture medium during the weeks of incubation. However, the experiments also showed that a physical mixture of the blank release medium and BMP-7 resulted in a further decrease in neurosphere formation (see Fig. 7A, RM90d-Control + BMP7). Therefore, these results explain the drastic effect observed with microsphere release media as a combined effect between the consumption of cell culture medium used for release studies and the conserved bioactivity of BMP-7. It is even possible that the experiments point to an improvement in the bioactivity of BMP-7 released from the formulation, which in this case should be explained as a result of the inclusion of this protein in a nanocomplex structured by heparin-Tetronic.

The analysis of the size of the resulting neurospheres is shown in Figure 7B. As can be seen, the blank microparticle release medium produces a smaller fraction of large neurospheres. Although this could be partially due to the release of heparin and Tetronic as noted earlier, our experiments indicate that this is related to the partial consumption of the medium during the release experiment. Also, cultured neurospheres in control medium that had been pre-incubated at 37 ° C for 15 days (control 15 incubated), they were also smaller. As noted earlier, BMP-7 in the control medium does not appear to drastically change the size of the neurospheres. The physical mixtures of BMP-7 and microparticle release medium (RM90d-Control + BMP-7) also showed smaller neurospheres similar to those observed only with the release medium. Interestingly, BMP-7 released from loaded microparticles showed almost no large or medium neurospheres, which can be explained as a combined effect of the consumption of the release medium and the effect of BMP-7 bioactivity. Typical neurospheres produced by these treatments are shown in the figure. 8, which very explicitly present the drastic effect of BMP-7 released from microparticles in the formation of neurospheres. 4.11 Effect of the microspheres with BMP-7 on tuntoral grafts of the primary culture of glioma 12012 implanted in a naked mouse.

The in vivo study showed a larger size of the tumors developed on the flanks of the mice to which the white microspheres had been implanted with respect to which microspheres were implanted with BMP-7. This effect could be observed both on the mice before the tumor was removed, and on the tumors removed. Analysis of westem blot performed on tumors showed a correlation between their size and the presence of p-Smad 1/5/8, indicator of the activation of the BMP pathway in these tumors (Figure 18).

Example 5. Preparation of polymeric nanoparticles

 5.1. Methods of preparation and characterization of PLGA nanoparticles: Tetronic: heparin

The nanoparticles were prepared by a modified solvent diffusion technique. First, an aqueous solution was prepared with 15 μ% Üeparin and 3.75 mg poloxamine. The solution was left for 30 min at room temperature, frozen at -20 ° C and lyophilized (primary drying at -35 ° C for 24 h, secondary drying at 0 ° C for 24 h and at room temperature for 14 h; Freeze Dryer Labconco Corp.). The lyophilized solid was dissolved in 2 mL of acetone to which 30 mg of PLGA was added. The mixture was stirred at 200 rpm until total dissolution of the PLGA. At that time, 25 mL of Milli-Q water was added at one time and left under stirring, at 700 rpm, for 20 minutes. The solvent mixture was removed in the rotary evaporator at 30 ° C until the volume was reduced by approximately half. Finally, make up to 10 mL with Milli-Q water. The nanoparticles obtained were characterized with respect to their average size and polydispersion index by photonic correlation spectroscopy (Zetasizer®, Malvern Instruments, UK). For this, the samples were diluted 1: 20 (v / v) in Milli-Q water. Each analysis was performed in triplicate and with a detection angle of 173 °. The zeta potential was determined using the Doppler laser anemometry technique from the average electrophoretic mobility data of the particles, after 1: 20 dilution (v / v) in a lmM aqueous solution of KC1 (Zetasizer®). The stability studies of the nanoparticles were carried out through the analysis of the average size over time both at 4 ° C and at room temperature.

 The morphological analysis of the nanostructures obtained was carried out by means of transmission electron microscopy (Libra 200 FE OMEGA). To this end, aliquots of the nanoparticle formulations were deposited on a copper grid and treated with a 2% phosphotungstic acid solution (p / v).

5.2. Characteristics of PLGA nanoparticles: Tetronic: heparin and storage stability study

 Through this method, PLGA nanoparticles were obtained that encapsulated heparin / poloxamine complexes, with the purpose of a possible future incorporation of growth factors.

The average size obtained from the nanoparticles obtained was 135 ± 5 nm (n = 8), with a polydispersion of 0.1, indicative of a single mode population. As for the potential Z, parameter indicating the surface charge of the nanosystem, the average value obtained was -35 ± 7 mV. From the analysis of the TEM microscopy images it is observed that the nanoparticles have an irregular morphology, with some deviation from the sphericity (Figure 14).

 To check the stability of the nanoparticles, measurements of size and potential Z were made at different times (Figure 15). For this, the nanoparticles were kept in suspension, both at room temperature and at 4 ° C, for 1 month. At room temperature the nanoparticles maintain their stability, in regard to their size and potential Z, for at least 24 hours. Although the size remains unchanged during the 4 weeks of the experiment, the potential decreases very slowly over time.

In the case of particles stored at 4 ° C, they are stable in terms of their size and potential Z for at least 2 weeks, maintaining again the stable size throughout the entire time of the experiment, and increasing polydispersion slightly from 0.1 to 0.2.

 Overall, these data indicate adequate chemical and colloidal stability characteristics of these nanometric formulations during prolonged storage processes.

 Example 6. Preparation and characterization of BMP-7 loaded films and study of different compositions.

 6.1. Methods of preparation and characterization of films loaded with BMP-7 from various compositions.

In this study films loaded with BMP-7 were prepared exploring all possible combinations within the following chemical space: (1) as biodegradable polymers, PLGA, PCL (Sigma-Aldrich, Ref. 440744) and poly (methyl vinyl ether-alt- maleic anhydride) (pMVEMA; Sigma-Aldrich, Ref. 416339); (2) Tetronic 1107 and methoxypolyethylene glycol amine were studied as cationic derivatives of polyoxyethylene; (3) as polysulphated polymers, polidextran sulfate (Sigma-Aldrich, Ref. D8906) and heparin.

For the preparation of the films, first 4 eppendorf tubes were incubated with 4 ug of BMP-7, 4 ug of polysulphated polymer and completed with sterile Milli-Q water up to 300 uL. The components were allowed to incubate for 30 min. at room temperature. Then 1.25 mg of cationic polyoxyethylene derivative was added and the mixture was allowed to incubate another 30 min. at room temperature. At this time, to each of the 4 eppendorf tubes a single combination of sulfated polymer, two possible, and cationic derivative of polyoxyethylene, two other alternatives had been added. These mixtures were then lyophilized, by primary drying at -35 ° C for 24 h, secondary drying at 0 ° C for 24 h and at room temperature for 14 h (Labconco Corp. lyophilizer). The lyophilisate of each eppendorf was resuspended in 400 uL of acetonitrile. In a 96-well plate an amount of 10 uL (0.1 ug of BMP-7 per well) was added, following the scheme of Figure 10B for combinations of sulfated polymer and cationic polyoxyethylene derivative. On the other hand, 500 mg of each biodegradable polymer was dissolved in 5 mL of an organic solvent (PLGA and pMVEMA in acetonitrile and PCL in dichloromethane). 100 uL per well was added following the scheme of Figure 10B. The result is plates with the 12 possible combinations of components and three replicated per prototype. The organic solvents were left evaporate, 5 hours at room temperature and 19 hours in desiccator, for a total of 24 h. Figure 10 illustrates the process of film formation and the arrangement of samples of different compositions tested on the multiwell plate.

The prepared films were analyzed morphologically at the macroscopic level, and the release profile of BMP-7 was studied as follows: 200 uL of PBS medium with 1% bovine serum albumin (w / v) per well was added, the plate and kept at 37 ° C with gentle agitation. Release samples were obtained at 24 h, 1 week, 3 weeks and 6 weeks, which were analyzed by ELISA as described in example 4.6.

6.2. Characterization of films loaded with BMP-7 from various compositions.

 The preparation method resulted in films with a homogeneous structure and without fractures (see example in Fig. 10A). The different films showed different release pulses, the selected biodegradable polymer being the fundamental factor that controls the process (Figure 11). Thus, the most important pulses occurred in PCL films at 3 weeks, and accounted for 20-30% of the BMP-7 loaded. An important disintegration was observed in pMVEMA films, so it is possible that an important part of BMP-7 was already released in the interval between the first and second release point, and was already degraded.

Example 7. Porous microspheres and matrices for cartilage regeneration. 7.1. Methods of preparation and characterization of porous matrices.

The procedure for obtaining porous matrices is based on the "solvent casting / salt leaching" method, already described in the literature, adapted for encapsulation of complex factors according to the techniques of the invention. First, 20 μg of BMP-7, 600 ng of TGF-P3 and 20.6 μg of heparin were dissolved in 300 μΕ of Milli-Q water (growth factor ratio: heparin (w / w) 1: 1) . The solution was incubated for 30 minutes at room temperature. After this time, 10 mg of Tetronic 1 107 was added and incubated for another 30 minutes at room temperature to allow interaction of the components. The sample was then frozen at -20 ° C and lyophilized (primary drying at -35 ° C for 24 h, secondary drying at 0 ° C for 24 h and at room temperature for 14 h; Labconco Corp. freeze dryer). Subsequently, the lyophilisate was dissolved in 400 μΕ of acetone to which 40 or 80 mg of PLGA was added, resulting in a percentage of polymer in relation to the solvent of 10 or 20% (w / v), respectively. Mix stirred until complete dissolution of the PLGA. At this time 480 mg of screened NaCl (particle diameter between 180-250 μιη) was added such that the solvent: porogen ratio was 1: 1, 2. Immediately, the mixture was transferred to 4 cylindrical molds of 0.9 mm diameter and a thickness between 1-1.5 mm. The molds were left in an extraction hood until complete removal of the solvent (4 hours). After this time the porous matrix was demolished and its surface was equalized with the help of a scalpel. To remove the porogen, the matrix was immersed in distilled water for 1 week, during which time the water was renewed every 24 hours. Figure 16 shows the mold and demoulding device used for the generation of this example. The method described here yielded 4 porous matrices of approximately 4 μg BMP-7 and 120 ng TGF-P3 per porous matrix.

7.2. Formation of cartilage in pellets from microspheres with BMP-7.

All steps of the experiment were performed under sterile conditions. First, human mesenchymal stem cells (hMSCs) were collected from a culture plate, by trypsinization. The cells were washed plates twice with PBS to remove the expansion medium. 1 mL of cell suspension - half a million hMSCs close to the confluence - was distributed in each 2 mL eppendorf tube. 1 mL with 20 mg of white PLGA microspheres (control) or loaded with BMP-7 (made according to the procedures of Example 4.1) was added to the cell suspension. The cell and microsphere suspension was centrifuged (300 g, 10 min, 4 ° C) and the supernatant was removed 2 cells of chondrogenic medium (ITS with 10 ng / mL TGF-P3) were added to the cell pellets and kept in an incubator at 37 ° C and 5% C02. mL of medium every 3 days and the pellets were kept in culture for 21 days.

7.3. Formation of cartilage from porous matrices with BMP-7 / TGFP-3 pellets from microspheres with BMP-7.

All steps of the experiment were performed under sterile conditions. First, after removing the porogen for 1 week in distilled water, the porous matrices were anchored to a culture plate by means of a needle (3 porous matrices per well in 6-well culture plates). It was allowed to dry for 4 hours prior to planting the cells. To collect the hMSCs from the culture plate, the plates were washed twice with PBS to remove the expansion medium and trypsinized. 40 μΕ of cell suspension (1 million hMSCs near the confluence) were distributed in each porous matrix. Cells were incubated on the porous matrix for 3 hours without adding culture medium but moistening the porous matrix so that it does not dry out. After this time, 5 mL of culture medium was added in each well. 12 growth replicas were made in porous matrices loaded with BMP-7 / TGF-P3 and 12 replicas in porous matrices without growth factors. 2.5 ml of medium was changed every 3 days and the porous matrices were kept in culture for 2 different times: group (a) 8 porous matrices for 3 weeks, group (b) 4 porous matrices for 4 weeks.

 7.4. Chondrogenic process evaluation

The differentiation of hMSCs to cartilage cells, both in pellet and in porous matrix, was verified by studies of mRNA gene expression by means of real-time quantitative PCR (Taqman) and histology. For gene expression studies, the relative expression levels of the mRNAs of Sox9, aggrecan, and type II collagen genes, involved in the chondrogenesis process, were estimated, and in addition, the levels of 2 housekeeping genes were estimated genes "), Actin B and GAPDH. For histology studies, the presence of cartilage was determined by staining Safranina-O.

 7.5. Characterization of porous matrices.

 The most used vehicles for the administration of growth factors in tissue engineering are porous matrices. These systems contain inside an intricate network of pores that allow the three-dimensional growth of the cells inside. At the same time, they can incorporate in their structure growth factors necessary to achieve cell differentiation. In this case, we apply the composition of PLGA, Tetronic and heparin of the invention to the co-encapsulation of two growth factors, BMP-7 and TGF-P3, in porous matrix type structures.

The method used in the manufacture of porous matrix is based on the use of a porogen (in this case NaCl) together with the mixture of polymers dissolved in an organic solvent. The removal of the organic solvent by evaporation and that of the porogen by dissolution in water, lead to the obtaining of a porous matrix with a homogeneous porous structure. In Figure 16 you can see the molds used in the manufacturing process and the size and macroscopic morphology of the porous matrices. With regard to the internal structure, the preparation of two types of porous matrices was carried out in which the proportion of polymer with respect to the solvent was 10 and 20% (w / v) respectively, which in the end it also results in a greater or lesser concentration of polymer with respect to the porogen. From the SEM microscopy images (Figure 17) it is observed that both are porous materials, although the structure of the matrix with a smaller proportion of polymer is much more open, as one would expect. The purpose of the porous matrix is to serve as an anchor and support to the cells, but at the same time said structure must provide adequate mechanical properties.

7.6. Chondrogenesis studies performed with microspheres with BMP-7.

 The TGF-P3 growth factor is a fundamental component in the chondrogenic standard medium. To evaluate the possible additional effect of the addition of controlled release microspheres of BMP-7 in the cartilage differentiation process, it was decided to conduct a differentiation study with hMSCs grown in pellets. Cell pellets were cultured in chondrogenic medium (STI with 10 ng / mL of TGF-P3) and with BMP-7 loaded microspheres or with white microspheres. As all pellets were grown 21 days in chondrogenic medium, the possible differences found could only be due to the controlled release of BMP-7 by the microspheres.

The macroscopic appearance of the pellets with microspheres with BMP-7 and the controls was slightly different (Figure 19). The microsphere pellets with BMP-7 were slightly larger, their appearance more viscous and their color slightly more intense, which suggested a more intense differentiation.

 Confirmation that the differentiation process actually took place was done by PCR in which the expression of SOX9, an early marker of chondrogenesis, was determined. A greater relative expression of Sox9 was observed in the pellets with BMP-7 loaded microspheres than in the controls. On the other hand, histology images also reveal that there is differentiation to cartilage, although it had not occurred throughout the entire length of the pellet, and that it was more intense and homogeneous than in the control pellets (Figure 19).

 7.7. Chondrogenesis studies performed in porous matrices with BMP-7 and TGF-P3.

Taking into account the good results obtained with the BMP-7 loaded microparticles, the joint administration of both factors of growth, TGF-P3 and BMP-7, in a controlled way in the same vehicle suitable for tissue engineering. In this co-encapsulation example, porous matrices were chosen as a release vehicle, systems widely used in tissue engineering since they condition the final shape of the mass of differentiated cells, which would be of great interest in the regeneration of cartilage in orthopedics

 The porous matrices described in example 7.5 were used, and specifically, those prepared from 20% (w / v) solutions of PLGA. Porous matrices with a theoretical load of 4 μg of BMP-7 and 120 ng of TGF-P3 per porous matrix were used for this study, and as a control white porous matrices. HMSCs were seeded in the porous matrices and cultured in control medium (STI) without TGF-P3 for 21 and 28 days. Thus, in this experimental design, all chondrogenic factors can come only from the porous matrix itself.

At 21 days of the experiment, the porous matrices that did not carry growth factors inside, presented a similar appearance to the initial one, with a substantial reduction in size probably due to the settlement of the cells inside which caused a contraction effect. On the other hand, the appearance of the porous matrices loaded with BMP-7 and TGF-P3 was substantially different from that of the beginning of the experiment and that of the control porous matrices (Figure 20). The initial porous translucent structure resulted in a more compact structure, a slight increase in size and a slightly viscous mass that could be due to the extracellular matrix components.

 Histology tests by staining with safranin-0 confirmed the presence of cartilage tissue in porous matrices with growth factors. The generation of cartilage in this stain can be seen as a red color resulting from the binding of safranine dye to peptidoglycan. On the other hand, the porous control matrices did not show positive staining with said dye (Fig. 20).

The results of quantitative PCR showed that the Sox9 gene had a relative expression 9 times greater in porous matrices loaded with growth factors than in the controls, and an overexpression of aggrecan close to 10 times greater (Figure 21). Aggrecan is a fundamental marker of the extracellular matrix of cartilage and Sox9 a transcription factor associated with the early stages of chondrogenesis. In this case, it was not possible to detect type II collagen, probably because we were in an initial stage of chondrogenesis. At 28 days, the presence of aggrecan continued to be observed, and the presence of type II collagen began to be observed, overexpressed in the porous matrices loaded with control factors. In this case, it was not possible to detect Sox9, which may indicate that we are at a later stage of the differentiation process (Figure 21).

 One skilled in the art readily appreciates that the present invention is well adapted to accomplish the objectives and obtain the aforementioned purposes and advantages, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are illustrative, and are not intended as limitations on the scope of the invention.

 It will be apparent to one skilled in the art that variable substitutions and modifications to the invention described herein can be made without departing from the scope and spirit of the invention.

 All publications mentioned in the specification are indicative of the levels of ordinary skill in the art to which the invention pertains. All publications are incorporated here by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.

 The invention described here by way of illustration may be suitably practiced in the absence of any element or elements, limitation or limitations, which is not specifically described herein. Thus, for example, in each case herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be substituted with any of the other two terms. The terms and expressions that have been used are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the characteristics shown and described or parts thereof, but it is recognized that several modifications are possible within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been specifically described by preferred embodiments and optional features, for the modification and variation of the concepts described herein, those skilled in the art can be used, and that such modifications and variations are within the scope of the present invention as defined by the appended claims.

Claims

 1. A composition suitable for the controlled release of hydrophilic proteins with a heparin binding domain, comprising:  to. A surface layer of a biodegradable hydrophobic polymer, and b. A nucleus of a cationic polyoxyethylene derivative physically bonded to a polysulphated polymer which in turn is physically bonded to a hydrophilic protein with a heparin binding domain.  2. A composition suitable for the controlled release of hydrophilic proteins with a heparin binding domain, comprising:  to. A hydrophobic biodegradable polymer;  b. A cationic polyoxyethylene derivative as a biocompatible emulsifier;  C. A hydrophilic protein with a heparin binding domain; and d. A polysulphated polymer physically bound to the hydrophilic protein with a heparin binding domain, wherein the composition is obtainable by mixing an aqueous solution or a solid composition, preferably in the form of a lyophilized powder, comprising elements b), c) and d) linked together with an organic medium comprising element a). 3. The composition according to any one of claims 1 to 2, wherein the hydrophilic protein with a heparin binding domain is selected from the group consisting of: endothelial vascular growth factor (VEGF), interleukins (IL), growth factor transformant (TGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), stromal cell derived factor (SDF), growth and differentiation factor (GDF), platelet factor (PF), ligand of the family of chemokines CC (CCL), ligand of the family of chemokines CXC, ligand of the family of chemokines C, chemokines CX ₃ C, antithrombins, neurotrophins, ligands of the family of derived neurotrophic factor the glial cell line (GFL), bone morphogenic proteins (BMPs), chymotrypsinogen or any combination thereof. 4. The composition according to claim 3, wherein the hydrophilic protein with a heparin binding domain is a bone morphogenic protein and / or a transforming growth factor.  5. The composition according to claim 4, wherein the bone morphogenic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8b, and BMP-14 or any combination thereof.  6. The composition according to claim 4, wherein the transforming growth factor is selected from the group consisting of TGF-βΙ, TGF-P2, TGF-P3 or any combination thereof. 7. The composition according to any of the preceding claims, wherein:  to. The biodegradable hydrophobic polymer is selected from the group consisting of polyesters, polyanhydrides, poly (orthoesters), polyamides, poly (alkyl cyanoacrylates), polyimides, polyester-poly (ethylene glycol) copolymers, polyphosphazenes, poly (phosphoesters) or any combination thereof;  b. The cationic polyoxyethylene derivative is selected from the group consisting of poloxamines, polyethylene glycol amines, chitosan-polyoxyethylene derivatives, cationic polyamino acid-polyoxyethylene derivatives, polyoxyethylene derivatives of cationic or cationized proteins, polyoxyethylene polyethylene ethylene derivatives, polyoxy ethylene ethylene derivatives any of its combinations; Y  C. The polysulphated polymer is selected from the group consisting of polysulphated hyaluronic acid, polysulphated dextran, polysulphated proteoglycans, heparan sulfate, heparin, or any combination thereof.  8. The composition of claim 7 wherein the polysulphated polymer is heparan sulfate, polysulfated dextran or heparin.  9. The composition according to any of claims 7 or 8, wherein the hydrophobic biodegradable polymer is poly (lactic-co-glycolic) (PLGA), poly (s-caprolactone) (PCL) or poly (methyl vinyl ether-alt -maleic anhydride) (pMVEMA). 10. The composition of any one of claims 7 to 9, wherein the cationic polyoxyethylene derivative is poloxamine or methoxypolyethylene glycol amine. 11. The composition according to claim 1 wherein:  to. The biodegradable hydrophobic polymer is poly (lactic-co-glycolic) (PLGA), poly (s-caprolactone) (PCL) or poly (methyl vinyl ether-alt-maleic anhydride) (pMVEMA);  b. The cationic polyoxyethylene derivative is poloxamine or methoxypolyethylene glycol amine;  C. The polysulphated polymer is heparan sulfate, polysulfated dextran or heparin; Y  d. Hydrophilic proteins with a heparin binding domain are selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8b, BMP-14, TGF-βΙ, TGF-p2 and / or TGF-p3. 12. The composition according to claim 11 wherein the hydrophilic proteins with a heparin binding domain are BMP-7 and / or TGF-P3.  13. The composition according to any one of the preceding claims wherein the composition is a microsphere, a film, a nanoparticle or a porous matrix.  14. The composition according to claim 13 wherein the microsphere has an average size larger than 10.  15. The composition according to claim 14 wherein the microsphere has an average size between 10 and 90um.  16. The composition according to claim 13 wherein the nanoparticle has a size between 80 and 500 nm.  17. The composition according to claim 13 wherein the porous matrix has a pore size between 20 and 800 um. 18. A system for the controlled release of drugs comprising the composition according to any of claims 1 to 12, or the microspheres according to any of claims 13 to 15, or the nanoparticles according to claim 13 or 16, or the porous matrix according to claim 13 or 17, or the films according to claim 13. 19. The drug delivery system according to claim 18, wherein the system is a stereotactic device or apparatus capable of introducing the composition according to any of claims 1 to 12, or the microspheres according to any of claims 13 to 15, or the nanoparticles according to the claim 13 or 16, or the porous matrix according to claim 13 or 17, or the films according to claim 13, in the brain.  20. The system for the controlled release of drugs according to claim 18, wherein the system is an atroscopic surgery device capable of introducing the composition according to any of claims 1 to 12, or the microspheres according to any of claims 13 to 15, or the nanoparticles according to claim 13 or 16, or the porous matrix according to claim 13 or 17, or the films according to claim 13, in the joints.  21. A method of producing microspheres according to any of claims 13 to 15, or the nanoparticles according to claim 13 or 16, or the porous matrix according to claim 13 or 17, or the films according to claim 13, comprising: a. Dissolve the cationic polyoxyethylene derivative in a solution having a hydrophilic protein with a heparin binding domain and a polysulphated polymer;  b. Dry by lyophilization of the mixture resulting from step (a); C. Resuspend the dry powder of step (b) in an organic solvent containing a biodegradable hydrophobic polymer, and  d. Collect microspheres, nanoparticles, porous matrices or films. 22. Use of the composition according to any of claims 1 to 12, or the microspheres according to any of claims 13 to 15, or the nanoparticles according to claim 13 or 16, or the porous matrix according to claim 13 or 17, or films according to claim 13, or the drug delivery system according to claims 18 to 20 for the manufacture of a medicament. 23. Use according to claim 22, wherein the medicament is for the treatment of brain tumors or for the regeneration of cartilage.  24. Pharmaceutical composition comprising the composition according to any of claims 1 to 12, or the microspheres according to any of claims 13 to 15, or the nanoparticles according to claim 13 or 16, or the porous matrix according to claim 13 or 17, or the films according to claim 13. 25. The pharmaceutical composition according to claim 24 further comprising pharmaceutically acceptable excipients.