WO2013106944A2 - Method for selective targeting of nanoencapsulated cells on target tissue or organs - Google Patents

Method for selective targeting of nanoencapsulated cells on target tissue or organs Download PDF

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WO2013106944A2
WO2013106944A2 PCT/CL2013/000003 CL2013000003W WO2013106944A2 WO 2013106944 A2 WO2013106944 A2 WO 2013106944A2 CL 2013000003 W CL2013000003 W CL 2013000003W WO 2013106944 A2 WO2013106944 A2 WO 2013106944A2
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cells
nanoencapsulated
antibody
cell
nanoencapsulation
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WO2013106944A3 (en
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Roberto Alejandro EBENSPERGER GONZÁLEZ
Daniel Jordi HACHIM DÍAZ
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Pontificia Universidad Católica De Chile
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes

Definitions

  • the present invention describes a method and system of cell targeting with the purpose of directing nanoencapsulated mesenchymal stem cells derived from human or animal adipose tissue (ADSC) to specific tissues or organs by using antibodies attached to cell therapy that seeks resolve physiological and pathophysiological deficiencies, through the administration and / or implantation of living cells and / or tissues to the organism.
  • ADSC adipose tissue
  • the present invention comprises the use of nanoencapsulated mesenchymal stem cells derived from human adipose tissue (usually lipoaspirated) whose minimum phenotype is: positive for CD105, CD90, CD73 and negative for CD14, CD19, CD45 and HLA-DR.
  • Nanoencapsulated mesenchymal cells have the ability to differentiate into fat cells, osteoblasts and chondrocytes.
  • Cell therapy seeks to resolve physiological and pathophysiological deficiencies, through the administration and / or implantation of living cells and / or tissues to the body. These are classified according to the source of obtaining, with autologous (from the same patient), allogeneic (from other donors) and xenogenic (from other animal species) implants. Ideally, the most recommended source for these therapies are those of autologous origin, since the rejection of the implant in the organism is avoided, since it is not recognized as a foreign agent 1 . Allogeneic and xenogenic sources have a potential use within the pharmaceutical and clinical market, due to the greater feasibility of obtaining, cultivating and standardizing all processes, before reaching the patient. However, xenogenic implants have a lower biosecurity profile, for this reason the use of allogeneic implants is preferred. The appropriate conditions and technologies are currently being developed so that these treatments reach as many patients as possible (1) .
  • cell therapy shows some limitations, mainly in the decrease of cell viability due to the inflammatory environment at the implantation site and the generation of a possible immune response, which can lead to rejection of transplanted cells and tissues. and its subsequent destruction (2) .
  • stem cells represents one of the most promising alternatives for cell therapy and tissue engineering, since they have the potential to differentiate into multiple cell types and to secrete factors that stimulate tissue growth and regeneration (4) .
  • Particularly novel is the use of mesenchymal stem cells of allogeneic origin, thanks to their low immunogenicity potential and immunomodulatory capacity (5, 6) .
  • There are different types of stem cells being classified according to their proliferative abilities, differentiation, tissue extraction and embryonic germ lines.
  • Totipotential Stem Cell a cell that has the potential to give rise to all body tissues including extraembryonic tissues and replicate itself. In this case there is only one cell type with these characteristics, the Embryonic Stem Cell (ESC), which originates immediately after fertilization.
  • ESC Embryonic Stem Cell
  • Pluripotential Stem Cell they are native to the three embryonic layers, the endoderm, mesoderm and ectoderm. These cells give rise to different types of tissues corresponding to their own cell line, which have the ability to regenerate themselves.
  • Multipotential Stem Cell they are cells of adult origin, present in a small proportion in each tissue of the organism, and have a certain degree of differentiation, since they come from the different germ lines. They are able to differentiate both in-vivo and in-vivo to the respective cellular subtype of the tissue of origin.
  • HSC Hematopoietic Stem Cells
  • MSC Mesenchymal Stem Cells
  • MSC Mesenchymal Stem Cells
  • ADSC adipose tissue
  • MSCs as multipotential cells, are capable of giving rise to different cell subtypes by cell differentiation, in-vitro conditions those described are adipocytes, osteocytes, chondrocytes, myocytes, skin, neurons, hematopoietic cells and other types of connective tissues (7) .
  • the support chosen for the present invention corresponds to the nanoencapsulation.
  • the ADSC are initially nanoencapsulated and subsequently bound to a specific reagent or molecule that grants a specific destination for its function in a chosen tissue or organ.
  • nanoencapsulation support was chosen by the inventors, because it has several advantages over other support means described in the state of the art.
  • the macrocapsules are porous polymeric supports (ideally biodegradable), which allow the irrigation of nutrients and some would allow the neovascularization of the implant.
  • macrocapsules there are several types of macrocapsules, microporous ones, fiber compounds and those with selective permeability membranes stand out.
  • One of the notable limitations in obtaining these Macrocapsules is the difficulty in manufacturing methods, since they must maintain an optimal pore size and that each area of the macrocapsule and pores are interconnected. This allows cells to enter and proliferate within them, without interfering with the exchange of nutrients, molecules and metabolic wastes, preventing the formation of cellular necrotic centers in areas of high cell density within the macrocapsule, making it more feasible the use of microporous beds.
  • Vascular Perfusion Devices (2) In relation to Vascular Perfusion Devices (2) , they are defined as such from the point of view of implantation. Looking at it from the point of view of its physicochemical properties, they can be defined as an intravascular implantation macrocapsule. This macrocapsule has the advantage that its intravascular implantation allows better irrigation and access to nutrients, elimination of metabolic wastes and an exchange of bioactive substances.
  • Microencapsulation is by far the most studied and used support. Some of the best known are polyelectrolyte complexation, interfacial inversion, in-situ polymerization and spray drying. On an industrial scale, spray drying is an easy process to perform, since only the parameters in the device must be controlled, however, it is expensive and does not allow the encapsulation of living material. Depending on the technique used, experimental conditions and polymers used, microcapsules of different sizes and attributes are obtained, such as pore size, number of cells encapsulated by microcapsule, among others.
  • PEGylation (3) Another support used is by means of PEGylation (3) , a process used with the aim of seeking greater immunoisolation.
  • PEGylation shows interesting results, giving greater protection against the immune system by steric impairment.
  • this technique is not viable by itself, and must be complemented with other encapsulation techniques, since it does not provide adequate mechanical protection. Its main limitations are the alteration of solute diffusion and the decrease in cell viability, due to the binding of PEG to surface proteins, which interfere with cellular functions.
  • the characteristics of the nanocapsules constructed, using the layer by layer method can be handled by factors such as; the type of polyelectrolyte, concentration, molecular mass, number of layers, final layer charge, pH, salts present and incubation time in the solution in which deposition occurs. These factors modify characteristics such as: pore size, surface charge density, elasticity, degree of hydratation of the nanocapsule, determining permeability, release kinetics, mechanical resistance of the nanocapsule, among others (11 '12) .
  • the nanoencapsulation therefore has potential capabilities to encapsulate all types of material (charged nanoparticles, proteins, DNA / RNA, cells), forming part of the tempering or coating (deposited by electrostatic interactions to form part of the nanocapsule). It is even possible to introduce substances into the hollow nanocapsules, such as active ingredients, using temperates that are then degraded (eg melamine formaldehyde) and when the active ingredients are introduced, the open and closed pore state is manipulated of the nanocapsules. With this we can use them as a vehicle of administration in numerous therapies, for delivery on a specific target (using for example nanocarriers deposited on the surface of the nanocapsules) or for the controlled release of active ingredients and other applications, according to the required clinical needs.
  • temperates eg melamine formaldehyde
  • the cell as a temperate (of net negative charge), to use nanoencapsulation as a vehicle that allows its application and overcomes some limitations associated with cell therapy and other types of encapsulation used (13 ) .
  • Pioneers in the field of cellular nanoencapsulation, using this method, are those carried out by Diaspro, Krol and collaborators ' 10 ' 12, H 15) initially with prokaryotic cells and subsequently, with mammalian eukaryotic cells.
  • the nanocapsule would function as a support that provides protection and mechanical resistance, since the high degree of hydration transforms these coatings into thin layers of gel.
  • This in addition to the small cutting size in the pores of the nanocapsule, allows an even more effective immuno-isolation than other types of cell delivery devices used and substantially improves cell viability.
  • these results are inconclusive, being rather controversial, especially in relation to cell viability and the fragility of mammalian cells. For this reason, it is unknown if this method is completely harmless to cells, transforming a process, which for other applications is simple, into something much more complex (15) .
  • polyelectrolytes that will be part of the nanocapsule.
  • the polyelectrolytes can be both bioactive substances, which retain their activity in the nanocapsules, therefore, they can interact with the cells as a substrate or a factor of the cellular microenvironment that stimulates growth, some specific cellular function, or an emulation of the extracellular matrix (ECM) of the cells, seeking to mimic the medium in which the cells in the tissue they originally come from are originally. Therefore, the choice of polyelectrolytes is critical for obtaining additional benefits, which is why a brief review of the polyelectrolytes used in this invention is given below.
  • the polyelectrolytes used correspond to polycations and polyanions.
  • Poly (allylamine) Chloride (PAH) (16) Poly (diallyldimethylammonium) chloride (PDADMAC) (17 ' 8) , Chitosan (Ch) and Poly-L-lysine (PLL) are described.
  • the polyanions can be: sodium poly (styrene) sulfonate (PSS) (16) , Hyaluronic Acid (HA) and Chondroitin Sulfate (CS).
  • Poly Chloride (allylamine) is a weak cationic polyelectrolyte, with positively charged amino groups at physiological pH, with a pKa of 9.3 and exhibiting different mechanical properties and conformations, depending on the pH and ionic strength of the solution. The greatest interactions it generates are electrostatic, ionic and hydrophobic. It is prepared from the polymerization of allylamine.
  • this polycation is closely related to colloidal applications and encapsulations. These applications vary in a wide spectrum, from the properties to stabilize or flocculate suspensions, stabilize emulsions (useful in the food, pharmaceutical and cosmetic industry), to the purification of water, product of the attractive interactions of the polymer, when added in small quantities .
  • it is one of the most used polymers, together with Poly (styrene sulfonate) by the layer by layer method, to obtain nanocapsules of potential use, as a delivery system for cell therapy drugs (10 , 12) .
  • Polychloride (diallyldimethylammonium) is a polyelectrolyte of synthetic origin, consisting of diallyldimethylammonium monomers that has a quaternary amino group, classifying it as a strong polycation and electrolyte, so its properties are not dependent on either ionic strength or pH of the solutions. It is characterized by a high solubility in aqueous solvents and a dense electrical charge. Therefore, it is a polyelectrolyte of more twisted conformations and with numerous ionic interactions against counterions (18) .
  • PDADMAC has wide uses, both in the industrial and scientific areas. Many of its load stabilizing properties are used as additives and emulsion stabilizers, thus determining the final properties of cosmetic products. Its flocculant properties are used as water purifying agents, in the preparation of suspensions, in the manufacture of paper and also in the mining and oil industry 07) .
  • R H or COCH 3
  • This polycation is a natural polysaccharide, derived from one of the most abundant organic substances in nature, chitin. Chitin is found in the shell of shrimp, crabs and other crustaceans, in the thin mantle of plankton, in the exoskeleton of insects, in squid cartilage and in the cell membranes of some fungi (20) .
  • Chitosan a polysaccharide derivative with positively charged amino groups, units of p- (1-4) -D-glucosamine, with a pKa of 6.5.
  • the degree of deacetylation classifies it as low, medium and high molecular weight.
  • the reaction generates acetic acid, thereby explaining that the solubility of this polysaccharide occurs in acidified solutions and depends on the pH of the solution (20) .
  • this polysaccharide makes it possible to use it in a wide range of applications, such as cosmetic products, water purification, waste recovery, also in the food industry, agriculture and medicine.
  • Chitosan is a molecule that absorbs large amounts of water, making it an important ingredient in some moisturizers (20) .
  • Chitosan is soluble in acidified water. This solubility, added to its viscosity, can make it thicker or lighter, as required, for use, for example, in the food industry.
  • Chitin and its derivatives are totally biodegradable and therefore are highly recommended to preserve the environment. It is one of the most promising substances in the field of biodegradable plastics, as an alternative to traditional plastic.
  • the antifungal properties, together with their biodegradability, are ideal conditions for use in agriculture (20) .
  • PLL Poly-L-lysine
  • the source of obtaining Poly-L-lysine comes from the fermentation of Streptomices, a process used to produce it industrially (24) .
  • Poly-L-lysine is a small polypeptide, with 25 to 30 residues of the amino acid L-lysine, so its molecular weight is variable. Due to its amino groups, it has a positive charge at physiological pH, since they have a pKa of 9.0. As described above, the electrical and charging properties classify it as a polycation. Structurally it corresponds to the ⁇ -poly-L-lysine, named for the union that occurs between the amino group of the ⁇ position and the carboxyl group of the amino acid. It is soluble in water, slightly soluble in ethanol and insoluble in organic solvents such as ethyl acetate. The activity of poly-L-lysine is not pH dependent and is stable at high temperatures.
  • Antimicrobial properties for this polypeptide are described, because the positively charged surfaces they form inhibit the growth of microorganisms. Its mechanism of action would be related to the ability to electrostatically adsorb to the bacterial cell surface, followed by an imbalance and an abnormal distribution of the cytoplasm, causing damage to the bacteria (25) . This quality is widely used as a preservative in the food industry.
  • Sodium poly (styrene) sulfonate is an anionic polyelectrolyte, of synthetic origin, obtained either by the polymerization of styrene sulfonate monomers or by the sulfonation of poly (styrene) (26) .
  • this polyelectrolyte is known as kayexalate ( ', useful for treating hyperkalemia, for its known selectivity to potassium ions (which are trapped in the intestine), administered orally or enema.
  • kayexalate useful for treating hyperkalemia, for its known selectivity to potassium ions (which are trapped in the intestine), administered orally or enema.
  • Hyaluronic acid is a linear polysaccharide, consisting of alternating disaccharide units of (1, 4) -aD-glucuronic acid and (1, 3) -pN-acetyl-D-glucosamine.
  • the properties of this polyanion are given by the carboxylate substituents of glucuronic acid, a functional group that confers a negative charge with a pKa of 3.4 (29) .
  • Hyaluronic acid also known as a glycosaminoglycan, is found in all tissues and body fluids of vertebrates. It is an important component in the extracellular matrix (ECM) of connective tissue, being more abundant in umbilical cord, synovial fluid, skin and vitreous humor. It is also found in bacteria. Hyaluronic acid is synthesized in the plasma membrane and is catabolized by receptor-mediated endocytosis, followed by lysosomal degradation. Its half-life in humans and animals fluctuates from less than 1 day to several days. (30) . The carboxylic groups of hyaluronic acid are fully ionized at extracellular pH. Its osmotic activity plays an important role in the movement and homeostasis of water.
  • ECM extracellular matrix
  • this compound is obtained from numerous sources. The most common are the product of bioengineering techniques, using bacterial fermentation. The sources most commonly used to obtain them are the vitreous humor and synovial fluid of cattle (restricted due to mad cow disease), umbilical cord, shark fin and the crests of roosters (30) .
  • Chondroitin sulfate is a sulfated glucosaminoglycan, related to Heparan, Dermatan and Keratan Sulfates. Its fixed composition consists of a linear chain with alternating disaccharides of N-acetylgalactosamine and N-glucuronic acid, of which the total product is a mixture of sulfates, substituted in different positions, where the majority are Chondroitin 6- sulfate and Chondroitin 4-sulfate As a result of the numerous and varied substitutions, Chondroitin Sulfate has a charge density and variable molecular weights.
  • Chondroitin sulfate has the ability to interact with extracellular matrix proteins, thanks to the negative charges of the molecule, important to regulate the broad spectrum of cellular mechanisms.
  • Coindritin sulfate has an anti-inflammatory activity, because it stimulates synthesis and inhibits the degradation of proteoglycans, acting as a necessary substrate to stabilize cell membranes and regenerate damage to cartilage. It promotes the growth of glycosoaminoglycans in the extracellular matrix, thus it is possible to cause synergism of these agents in co-administration of Non-Spheroidal Anti-Inflammatory Analgesics (NSAIDs). It can also stimulate the synthesis and inhibition of collagen degradation. For this reason it is used for arthritis and osteoarthritis, since in addition to its anti-inflammatory effects, glycosoaminoglycan levels in the extracellular matrix of human articular chondrocytes and cartilage increase. Some studies suggest that this activity is mainly due to sulfate groups, which replace these glucosamine derivatives (38) .
  • chondroitin sulfate is well known, it is used for osteoarthritis, co-administered with N-acetyl-glucosamine, so in some countries they consider it as a drug (non-steroidal anti-rheumatic anti-inflammatory), but not in the United States where it is regulated as a food supplement.
  • the sources of Chondroitin sulfate extraction, and from which mainly the different proportions of sulfate varieties are obtained, are bovine, porcine or marine cartilage, by means of proteolysis and purification.
  • the one obtained from bovine trachea (with 95% purity) is the most used and studied, therefore its efficacy and safety are the best known (39) .
  • Figure 1 Layer-to-layer deposition cycle on a loaded temper. Stage 1: polycation solution; stage 2: washing; stage 3: polyanion solution; stage 4: washing.
  • Figure 2 Fluorescence microscopy of cells with the antibody or conjugate: Fluorescence Microscopy images at a magnification of 200x to determine the presence of the conjugation of the IgG-1 FITC antibody on the surface of nanoencapsulated ADSCs. Images (a) and (b) show nanoencapsulated ADSCs in the presence of antibody but without the addition of the chemical reagents NHSS and EDAC, which does not emit green fluorescence of the FITC antibody. Images (c) and (d) correspond to nanoencapsulated ADSCa in the presence of antibody and the addition of the chemical reagents NHSS and EDAC, which shows green fluorescence corresponding to the conjugated lgG1-FITC antibody on the surface of nanoencapsulated ADSCs.
  • Figure 3 Cell flow cytometry histogram with the conjugated antibody: Flow cytometry histograms for the determination of the conjugation of the lgG-1 FITC antibody, using the chemical reagents [N-hydroxy-sulfo-succinimide (NHSS) and 1 -ethil-3- [3-dimethylaminopropyl] carbodiimide (EDAC)], for nanoencapsulated ADSCs in the presence of crosslinkers (gray line) and in the absence of chemical reagents (black line). Shift to the right of the fluorescence intensity of cells with conjugated antibody (gray) with respect to the control (black) indicates the conjugation of the antibody to the surface of the nanoencapsules.
  • NHSS N-hydroxy-sulfo-succinimide
  • EDAC 1 -ethil-3- [3-dimethylaminopropyl] carbodiimide
  • Figure 4 Cell viability of cells with the conjugated antibody.
  • Figure 5 Cellular visibility images of cells with the conjugated antibody.
  • ADSCs correspond to nanoencapsulated and conjugated ADSCs to a lgG-1 antibody using NHSS and EDAC as chemical reagents.
  • Image C corresponds to the overlay of previous images in the same field. 200x magnification
  • Figure 6 Cell growth curves with the conjugated antibody.
  • ADSC ADSC Growth curve of the ADSCs and ADSCs naoencapsulated and conjugated to the lgG1 antibody. Each point on the curve represents the average of 3 samples +/- SEM.
  • Figure 8 Microscopy images at 200x magnification corresponding to nanoencapsulated ADSC and conjugated to the lgG1 antibody, subjected to differentiation stimuli for osteoblast (b) and its control (a). Osteoblast differentiation is revealed by the presence of calcium deposits colored red by red Alizarin.
  • FIG. 9 Adhesion and specific retention of ADSC cells conjugated to CD31 antibody on human endothelial cells (HUVEC) -
  • the images show the specificity with which nanoencapsulated cells bound to a specific antibody directed to a particular tissue, in this case human endothelium, remarkably increases the ability of the cells to fix on it and be retained.
  • the figure shows nanoencapsulated fat stem cells to which an anti-CD31 antibody was attached, which specifically recognizes the PECAM-1 molecule on the surface of endothelial cells derived from human umbilical cord.
  • the images correspond to photographs of phase contrast (upper panel) and fluorescence (lower panel).
  • nanoencapsulated cells As controls, cells without nanoencapsular (images a and b) were used; nanoencapsulated cells (images c and d); nanoencapsulated cells bound to an isotype antibody (images e and f) and nanoencapsulated cells bound to a specific anti-CD31 antibody (images g and h).
  • nanoenGapsulaeión-elegiar deliversHa-present invention describes a design of a method of selective direction of stem cells mesenchymal from nanoencapsulated adipose tissue destined to white organs or tissues, as a strategy to make possible its administration from an allogeneic source and its accumulation or selective retention in a specific tissue.
  • this technology provides new tools for tissue engineering, as well as for novel pharmaceutical and biomedical applications.
  • the objective of the present invention is the covalent binding of a specific antibody to the polymer used in the last layer of nanoencapsulated mesenchymal stem cells, with the purpose of directing the systemically administered cells (such as an intravenous injection of a cell suspension) to a specific organ or tissue such that they accumulate or are selectively retained in said organ or tissue.
  • the organs or tissues to which the mesenchymal stem cells can be directed are selected from among others, liver, kidney, intestine, pancreas, prostate, lung, heart, spleen, brain, joints.
  • the selection of the antibody to be bound to the nanoencapsulated cells is selected depending on the organ in which it is desired that they accumulate and / or also on the particular structure of an organ or tissue to which it is desired to be preferentially fixed, for example hepatic parenchyma, tubule. proximal or distal in the case of the nephron, beta islets of the pancreas, etc.
  • the preferential or selective accumulation will depend on the specificity of the bound antibody and the level of expression of the antigen or protein in said organ or tissue.
  • stages 2 and 3 can be performed simultaneously or sequentially.
  • the intravenous systemic administration of mesenchymal stem cells with therapeutic objectives has a wide distribution in the organism '40, 4) , being preferably located in peripheral organs such as lung, spleen, liver and kidney; and being trapped in greater proportion or quantity in the pulmonary microvasculature (42) . Additionally, of the cells that are trapped in the microvasculature of the various organs after an intravenous administration, an even smaller fraction manages to transfer or escape or migrate from the vasculature to graft the organ '43, 4 ) and for which there are also reports that they remain in the organ for more than 4 weeks (3) . It is for these reasons that alternatives to intravenous systemic administration have been studied in order to increase the proportion or amount of stem cells administered that reach the target organ.
  • intramyocardial (5) or intraosseous administration ⁇ 43 has been attempted for this purpose, observing a greater accumulation of the cells administered in the white organ, but without altering or affecting the distribution to other organs peripherals such as liver, spleen and lung (45) .
  • the independence of the route of administration in the distribution of stem cells to peripheral organs makes it possible to state that the non-covalent or covalent binding of antibodies directed against specific structures present in a white organ will allow a greater accumulation of the modified cells. in said organ, as well as a greater graft ("engraftment") of them in it after a simple intravenous systemic administration, and thus avoid a route of direct administration in the white organ or in an artery that irrigates it. Then, once in the organ, the cells accumulated there would begin with their repair actions of the damaged tissue.
  • anti-asialoglycoprotein arylhydrocarbon antireceptor, anti-cMet for liver
  • anti-von Willebrand endothelin antireceptor
  • anti-angiopoietin anti-ICAM1
  • Type 2 anti-collagen anti-coindritin-4-sulfate, anti-keratan sulfate for cartilage
  • anti-VCAM1 for colon and mesenteric lymph nodes
  • specific tumor antigens etc.
  • the list of examples is not exhaustive and represents only a few examples.
  • the method of selective targeting of mesenchymal stem cells from nanoencapsulated adipose tissue destined to white organs or tissues is described in detail according to the 3 main stages of: nanoencapsulation, binding of a bifunctional reagent to nanoencapsulated cells, and binding of specific antibody to the bifunctional reagent bound to the nanoencapsulated cells.
  • HBSS Hank's balancee! Salt Solution
  • nanoencapsulated cells bound to the specific antibody are considered according to the procedures described above and are contacted with the target cells chosen from those that express the specific antigen for which the antibody is directed.
  • This method of directionality of nanoencapsulated cells with specific antibody to white cells can be extrapolated to organs or tissues of patients for therapeutic purposes.
  • HBSS Hank's balanced Salt Solution
  • Hyaluronic Acid Chondroitin Sulfate 1: 1 of 1 mg / mL using 2- (N-morpholino) ethanesulfonic acid (MES) buffer pH 7.4 as solvent at 4 ° C, add an amount of N-hydroxysulfosuccinimide ( NHSS) dissolved in MES buffer pH 7.4, sufficient to maintain a final concentration of 1.1 mg / mL. Incubate for 15 minutes at 4 ° C.
  • MES 2- (N-morpholino) ethanesulfonic acid
  • NHSS N-hydroxysulfosuccinimide
  • ANTI-CD31 antibody SPECIFIC EXAMPLE THEN DESCRIBED IN EXAMPLE 3
  • concentration 100 pg / mL and a sufficient amount of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide solution (EDAC) dissolved in MES buffer pH 7.4, so that the concentration of EDAC in the mixture is 2 mM or 400 pg / mL.
  • EDAC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide solution
  • an isotype antibody of the IgG1 conjugated lgG1 type (Fluorescein isothiocyanacyte) was used.
  • Hyaluronic Acid Chondroitin Sulfate 1: 1 of 1 mg / mL using 2- (N-morpholino) ethanesulfonic acid (MES) buffer pH 7.4 as solvent at 4 ° C, add an amount of N- Hydroxysulfosuccinimide (NHSS) dissolved in MES buffer pH 7.4, sufficient to maintain a final concentration of 1.1 mg / mL.
  • MES 2- (N-morpholino) ethanesulfonic acid
  • NHSS N- Hydroxysulfosuccinimide
  • the cells obtained at this stage are analyzed by two methods: Fluorescence microscopy and flow cytometry.
  • HUVECs human endothelial cells derived from umbilical cord
  • ADSC nanoencapsulation and anti-CD31 antibody conjugation was performed, according to conditions. Before performing the cell coating procedure, the antibody was conjugated to the components of the outer layer and the second layer. For it:
  • Hyaluronic Acid Chondroitin sulfate 1: 1 of 1 mg / mL dissolved in MES buffer at pH 7.4; Sufficient amount of an NHSS solution (dissolved in MES buffer at pH 7.4) was added to maintain a final concentration of 1.1 mg / mL. It was incubated for 15 minutes at 4 ° C.
  • Adhered HUVECs were observed by red and light field fluorescence filter microscopy for ADSC, under their different conditions.
  • MSC mesenchymal stem cells
  • MSCs have alternative mechanisms of action to exert their reparative effect on damaged tissue.
  • pro-regenerative factors such as, for example, anti-inflammatory, angiogenic, neurotrophic, immunomodulatory and antifibrotic factors (6) .
  • MSCs have already demonstrated clear therapeutic potential, there are currently some aspects related to their use that need to be optimized.
  • clinical studies involving MSC use one of two routes of administration: a) intravenous injection of a large bolus of cells, or b) a direct injection into the affected tissue.
  • the first involves exposing the patient to large doses of cells (between 1 and 5 million cells per kg of weight) circulating throughout the body, with the inconvenience of high production costs involved and potential risks of side effects; while the second method involves invasive and expensive infusion procedures.
  • new studies have been developed that have tried to optimize MSC-based therapy either by increasing its potency or increasing its delivery or disposition in white tissues.
  • the one related to the present invention is that of "targeting" to a specific tissue.
  • various approaches have been described, among which we find, for example, the enzymatic modification of the CD44 surface receptor of the MSCs in an E-selectin binding domain (48) and the coupling of specific tissue antibodies on the cell surface (49 ) .
  • tissue-specific antibodies we find two alternatives described.
  • the first uses palmitoylated G protein, which allows its attachment to the cell membrane and subsequently the coupling of an antibody, by an affinity mechanism, to the Q protein (5O, 51) £
  • the method describes a mixed coupling that includes covalent and affinity type junctions, which uses biotin and streptavidin and biotinylated ligands (52) .
  • a new and different method of coupling tissue-specific antibodies on the cell surface by means of covalent binding is described ( Figures 2 and 3).
  • a strategy is used that uses the nanoencapsulation of the MSC by means of biocompatible polymers (chitosan, hyaluronic acid and chondroitin sulfate) to which a tissue-specific antibody is covalently attached to the cell surface of the MSC, by the use of chemical reagents that allow or facilitate this binding by serving as catalysts by activating the carboxylic residues present in the polyanion.
  • biocompatible polymers chitosan, hyaluronic acid and chondroitin sulfate
  • the main utilities of this methodology consist in being able to selectively and specifically direct cells to a tumor with the objective of attacking and selectively destroying it by means of some strategy known by the state of the art.
  • stem cells we find the possibility of selectively and specifically directing these cells to damaged tissues or organs allowing greater accumulation or "engraftment" of the cells in the tissue so that they can exert their reparative effect.
  • COPD chronic obstructive disease
  • heart failure and acute myocardial infarction
  • inflammatory bowel disease inflammatory vascular diseases
  • joint diseases such as osteoarthritis or arthritis
  • neurodegenerative diseases such as Parkinson's disease, Alzheimer's , etc.
  • the present invention can be applied to any type of cell that wants to target a specific target, be it a damaged tissue or a tumor.
  • the cells to be used can be selected from mesenchymal stem cells, hematopoietic stem cells, endothelial progenitor cells, induced pluripotential stem cells or iPS, embryonic stem cells, CD4 T lymphocytes, CD8 T lymphocytes, regulatory T lymphocytes, Natural Killer or NK cells, monocytes / macrophages; and white organs include liver, heart, lung, kidney, pancreas, brain, intestine and colon, vascular endothelium, joints and blood vessels in general and tumors.
  • Diaspro A Nanocapsules: a European Community interdisciplinary network in the nanobiosciences. IEEE Trans Nanobioscience 2004; 3: 1-2.
  • Margolis RU Lalley K, Kiang WL, Crockett C, Margolis RK. Isolation and properties of a soluble chondroitin sulfate proteoglycan from brain. Biochem Biophys Res Commun 1976; 73: 1018-24.

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Abstract

Cell-targeting method and system for targeting nanoencapsulated cells derived from human or animal tissue on specific organs or tissue by means of the use of conjugated antibodies, for cell therapy, which seeks to resolve physiological and pysiopathological deficiencies through the administration and/or implantation of live cells in target tissue or organs of the human or animal body.

Description

METODO DE DIRECCIÓN SELECTIVA DE CÉLULAS NANOENCAPSULADAS A ÓRGANOS O TEJIDOS BLANCO.  SELECTIVE ADDRESS METHOD OF NANOENCAPSULATED CELLS TO ORGANS OR WHITE FABRICS.
La presente invención describe un método y sistema de direccionamiento celular (cell targeting) con el propósito de dirigir células madre mesenquimales nanoencapsuladas derivadas de tejido adiposo humano o animal (ADSC) a tejidos u órganos específicos mediante el uso de anticuerpos adheridos para terapia celular que busca resolver deficiencias fisiológicas y fisiopatológicas, mediante la administración y/o implantación de células vivas y/o tejidos al organismo. The present invention describes a method and system of cell targeting with the purpose of directing nanoencapsulated mesenchymal stem cells derived from human or animal adipose tissue (ADSC) to specific tissues or organs by using antibodies attached to cell therapy that seeks resolve physiological and pathophysiological deficiencies, through the administration and / or implantation of living cells and / or tissues to the organism.
La presente invención comprende el uso de células madre mesenquimales nanoencapsuladas derivadas de tejido adiposo humano (usualmente lipoaspirados) cuyo fenotipo mínimo es: positivo a CD105, CD90, CD73 y negativo para CD14, CD19, CD45 y HLA-DR. The present invention comprises the use of nanoencapsulated mesenchymal stem cells derived from human adipose tissue (usually lipoaspirated) whose minimum phenotype is: positive for CD105, CD90, CD73 and negative for CD14, CD19, CD45 and HLA-DR.
Las células mesenquimales nanoencapsuladas presentan la capacidad de diferenciarse en células adiposas, osteoblastos y condrocitos. Nanoencapsulated mesenchymal cells have the ability to differentiate into fat cells, osteoblasts and chondrocytes.
ESTADO DE LA TÉCNICA STATE OF THE TECHNIQUE
La terapia celular busca resolver deficiencias fisiológicas y fisiopatológicas, mediante la administración y/o implantación de células vivas y/o tejidos al organismo. Éstas se clasifican según la fuente de obtención, existiendo implantes autólogos (provenientes del mismo paciente), alogénicos (provenientes de otros donantes) y xenogénicos (provenientes de otras especies animales). En condiciones ideales la fuente más recomendada para estas terapias son las de origen autólogo, ya que al provenir del mismo paciente se evita el rechazo del implante en el organismo, al no reconocerlo como un agente extraño1. Fuentes de obtención alogénicos y xenogénicos tienen un potencial uso dentro del mercado farmacéutico y clínico, debido a la mayor factibilidad de obtención, cultivo y estandarización de todos los procesos, antes de llegar al paciente. Sin embargo, los implantes xenogénicos tienen un perfil de bioseguridad más bajo, por esta razón se prefiere la utilización de implantes alogénicos. Actualmente se están desarrollando las condiciones y tecnologías adecuadas para que estos tratamientos alcancen el mayor número de pacientes posibles(1). Cell therapy seeks to resolve physiological and pathophysiological deficiencies, through the administration and / or implantation of living cells and / or tissues to the body. These are classified according to the source of obtaining, with autologous (from the same patient), allogeneic (from other donors) and xenogenic (from other animal species) implants. Ideally, the most recommended source for these therapies are those of autologous origin, since the rejection of the implant in the organism is avoided, since it is not recognized as a foreign agent 1 . Allogeneic and xenogenic sources have a potential use within the pharmaceutical and clinical market, due to the greater feasibility of obtaining, cultivating and standardizing all processes, before reaching the patient. However, xenogenic implants have a lower biosecurity profile, for this reason the use of allogeneic implants is preferred. The appropriate conditions and technologies are currently being developed so that these treatments reach as many patients as possible (1) .
Para implantes alogénicos y xenogénicos, la terapia celular muestra algunas limitaciones, principalmente en la disminución de la viabilidad celular debido al ambiente inflamatorio en el sitio de implantación y la generación de una posible respuesta inmune, que puede llevar al rechazo de las células y tejidos trasplantados y su posterior destrucción(2). For allogeneic and xenogenic implants, cell therapy shows some limitations, mainly in the decrease of cell viability due to the inflammatory environment at the implantation site and the generation of a possible immune response, which can lead to rejection of transplanted cells and tissues. and its subsequent destruction (2) .
Además, se presentan limitaciones en torno a la inherente fragilidad de las células mamíferas, producto de estrés celular al que se somete antes de ser administrado al paciente, incluyendo el manejo, el transporte y el almacenamiento. Se suman a estas condiciones adversas los requerimientos metabólicos celulares, necesarios para asegurar la viabilidad celular(2, 3). In addition, there are limitations around the inherent fragility of mammalian cells, a product of cellular stress to which it is subjected before being administered to the patient, including handling, transport and storage. Add to these adverse conditions the cellular metabolic requirements, necessary to ensure cell viability (2, 3) .
Para sortear estas limitaciones, investigaciones multidisciplinarias han utilizado herramientas inspiradas en ingeniería en tejidos, biotecnología, y nanotecnología. Aplicado a la biomedicina, esta ciencia se engloba bajo el concepto que los investigadores llaman bionanotecnología. To overcome these limitations, multidisciplinary research has used tools inspired by tissue engineering, biotechnology, and nanotechnology. Applied to biomedicine, this science is encompassed under the concept that researchers call bionanotechnology.
Células Madre. Mother cells.
La utilización de células madre, representa una de las alternativas más prometedoras para la terapia celular y la ingeniería en tejidos, ya que tienen la potencialidad de diferenciarse en múltiples tipos celulares y la de secretar factores que estimulan el crecimiento y regeneración tisular(4). Lo particularmente novedoso, es la utilización de células madre mesenquimales de origen alogénico, gracias a su bajo potencial de inmunogenicidad y su capacidad inmunomoduladora(5, 6). Existen diferentes tipos de células madre, clasificándose según sus capacidades proliferativas, de diferenciación, del tejido de extracción y de líneas germinales embrionarias. The use of stem cells represents one of the most promising alternatives for cell therapy and tissue engineering, since they have the potential to differentiate into multiple cell types and to secrete factors that stimulate tissue growth and regeneration (4) . Particularly novel is the use of mesenchymal stem cells of allogeneic origin, thanks to their low immunogenicity potential and immunomodulatory capacity (5, 6) . There are different types of stem cells, being classified according to their proliferative abilities, differentiation, tissue extraction and embryonic germ lines.
• Célula Madre Totipotencial: célula que tiene el potencial de dar origen a la totalidad de los tejidos del organismo incluidos los tejidos extraembrionarios y replicarse a sí misma. En este caso existe un solo tipo celular con estas características, la Célula Madre Embrionaria (ESC), que se origina inmediatamente después de la fecundación. • Totipotential Stem Cell: a cell that has the potential to give rise to all body tissues including extraembryonic tissues and replicate itself. In this case there is only one cell type with these characteristics, the Embryonic Stem Cell (ESC), which originates immediately after fertilization.
• Célula Madre Pluripotencial: son originarias de las tres capas embrionarias, el endodermo, mesodermo y ectodermo. Estas células dan origen a los diferentes tipos de tejidos correspondientes a su propia línea celular, que tienen la capacidad de autoregenerarse. • Pluripotential Stem Cell: they are native to the three embryonic layers, the endoderm, mesoderm and ectoderm. These cells give rise to different types of tissues corresponding to their own cell line, which have the ability to regenerate themselves.
• Célula Madre Multipotencial: son células de origen adulto, presentes en una pequeña proporción en cada tejido del organismo, y poseen cierto grado de diferenciación, ya que provienen de las diferentes líneas germinales. Son capaces de diferenciarse tanto in-vítro como in-vivo al respectivo subtipo celular del tejido de origen. • Multipotential Stem Cell: they are cells of adult origin, present in a small proportion in each tissue of the organism, and have a certain degree of differentiation, since they come from the different germ lines. They are able to differentiate both in-vivo and in-vivo to the respective cellular subtype of the tissue of origin.
La mayoría de los estudios en células madre, incluso en ensayos clínicos humanos han utilizado Células Madre Hematopoyéticas (HSC) y también Células Madre Mesenquimales (MSC), ambas células multipotenciales provenientes de la Médula Ósea(7). Most studies in stem cells, even in human clinical trials have used Hematopoietic Stem Cells (HSC) and also Mesenchymal Stem Cells (MSC), both multipotential cells from the Bone Marrow (7) .
Las Células Madre Mesenquimales (MSC), también se pueden obtener de tejido adiposo (ADSC)(8). Éstas, a diferencia de otras fuentes, permiten su obtención de forma menos invasiva y sin transgresión ni conflictosmorales, ya que el tejido adiposo del que se obtiene, como liposucciones y abdominoplastías, normalmente se consideran desechos. Mesenchymal Stem Cells (MSC) can also be obtained from adipose tissue (ADSC) (8) . These, unlike other sources, allow them to be obtained less invasively and without transgression or moral conflicts, since the adipose tissue from which it is obtained, such as liposuctions and tummy tucks, is usually considered waste.
Estas MSC, como células multipotenciales son capaces de dar origen mediante diferenciación celular, en condiciones in-vitro, a diferentes subtipos celulares de las que se describen adipocitos, osteocitos, condrocitos, miocitos, piel, neuronas, células hematopoyéticas y otros tipos de tejidos conectivos(7). These MSCs, as multipotential cells, are capable of giving rise to different cell subtypes by cell differentiation, in-vitro conditions those described are adipocytes, osteocytes, chondrocytes, myocytes, skin, neurons, hematopoietic cells and other types of connective tissues (7) .
Administración de ADSC mediante Soportes o Scaffolds. ADSC Administration through Stands or Scaffolds.
En el área de ingeniería en tejidos, se han diseñado diferentes dispositivos que posibilitan la administración de células, conocidos como scaffolds, con el objetivo de buscar el soporte adecuado para las células y el implante. También se buscan algunas ventajas, con el objetivo de superar ciertas limitaciones inherentes a la terapia celular, como ofrecer inmunoaislamiento celular junto con entregar una protección mecánica y fisicoquímica, prolongando la viabilidad celular. Se han diseñado diferentes tipos de dispositivos, desde los más simples hasta los más complejos, pudiendo ser combinados entre sí. En relación a estos soportes podemos encontrar, macroencapsulación, dispositivos de perfusión vascular, microencapsulación, PEGilación y nanoencapsulación(2). In the area of tissue engineering, different devices have been designed that allow the administration of cells, known as scaffolds, in order to find the right support for the cells and the implant. Some advantages are also sought, with the aim of overcoming certain limitations inherent in cell therapy, such as offering cellular immunoisolation along with delivering mechanical and physicochemical protection, prolonging cell viability. Different types of devices have been designed, from the simplest to the most complex, and can be combined with each other. In relation to these supports we can find, macroencapsulation, vascular perfusion devices, microencapsulation, PEGylation and nanoencapsulation (2) .
El soporte elegido para la presente invención, corresponde a la nanoencapsulación. De esta manera la ADSC son inicialmente nanoencapsuladas y posteriormente ligadas a un reactivo o molécula específica que otorga una destinación específica para su función en un tejido u órgano elegido. The support chosen for the present invention corresponds to the nanoencapsulation. In this way the ADSC are initially nanoencapsulated and subsequently bound to a specific reagent or molecule that grants a specific destination for its function in a chosen tissue or organ.
El soporte de nanoencapsulación fue elegido por los inventores, debido a que presenta diversas ventajas con respecto a otros medios de soporte descritos en el estado del arte. The nanoencapsulation support was chosen by the inventors, because it has several advantages over other support means described in the state of the art.
En relación a la Macroencapsulación'2, 9), las macrocápsulas son soportes poliméricos porosos (idealmente biodegradables), que permiten la irrigación de nutrientes y algunos permitirían la neovascularización del implante. In relation to the Macroencapsulation ' 2, 9) , the macrocapsules are porous polymeric supports (ideally biodegradable), which allow the irrigation of nutrients and some would allow the neovascularization of the implant.
Existen variados tipos de macrocápsulas, se destacan los microporosos, los compuestos por fibras y los que poseen membranas de permeabilidad selectiva. Una de las limitaciones destacables en la obtención de estas macrocápsulas es la dificultad en los métodos de fabricación, ya que deben mantener un tamaño óptimo de poro y que cada zona de la macrocápsula y los poros estén interconectados. Esto permite que las células se introduzcan y proliferen dentro de ellas, sin interferir con el intercambio de nutrientes, moléculas y desechos metabólicos, evitando la formación de centros necróticos celulares en zonas de alta densidad celular dentro de la macrocápsula, por lo que es más factible el uso de lechos microporosos. Sin embargo, no es posible implantar estas macrocápsulas en lugares de poca irrigación sanguínea, pues la tasa de intercambio de nutrientes y desechos debe ser más dinámica. Desde el punto de vista del inmunoaislamiento, es necesario el uso de tratamiento inmunosupresor previo a la implantación (aunque leve), ya que el tamaño de poro permite la infiltración de componentes del sistema inmune. Además, estas macrocápsulas no han mostrado resultados satisfactorios, cuando son utilizadas en aplicaciones que necesitan una importante masa celular para cumplir con los objetivos, debido a su elevada razón macrocápsula:masa celular, es necesario que el implante sea de un elevado tamaño para que contenga la densidad celular adecuada y que cumpla con las necesidades terapéuticas deseadas. Además, el elevado volumen de la macrocápsula no permitiría la difusión adecuada de nutrientes, llevando a núcleos de necrosis y posteriores reacciones inmunológicas. There are several types of macrocapsules, microporous ones, fiber compounds and those with selective permeability membranes stand out. One of the notable limitations in obtaining these Macrocapsules is the difficulty in manufacturing methods, since they must maintain an optimal pore size and that each area of the macrocapsule and pores are interconnected. This allows cells to enter and proliferate within them, without interfering with the exchange of nutrients, molecules and metabolic wastes, preventing the formation of cellular necrotic centers in areas of high cell density within the macrocapsule, making it more feasible the use of microporous beds. However, it is not possible to implant these macrocapsules in places with low blood supply, as the exchange rate of nutrients and wastes must be more dynamic. From the point of view of immunoisolation, the use of immunosuppressive treatment prior to implantation (although mild) is necessary, since the pore size allows infiltration of components of the immune system. In addition, these macrocapsules have not shown satisfactory results, when they are used in applications that need an important cell mass to meet the objectives, due to their high macrocapsule ratio: cell mass, it is necessary that the implant be of a large size to contain adequate cell density and that meets the desired therapeutic needs. In addition, the high volume of the macrocapsule would not allow adequate diffusion of nutrients, leading to necrosis nuclei and subsequent immunological reactions.
En relación a los Dispositivos de Perfusión Vascular(2), se definen como tales desde el punto de vista de la implantación. Mirándolo desde el punto de vista de sus propiedades físico-químicas, se pueden definir como una macrocápsula de implantación intravascular. Esta macrocápsula posee la ventaja de que su implantación intravascular posibilita una mejor irrigación y acceso a nutrientes, eliminación de desechos metabólicos y un intercambio de sustancias bioactivas. In relation to Vascular Perfusion Devices (2) , they are defined as such from the point of view of implantation. Looking at it from the point of view of its physicochemical properties, they can be defined as an intravascular implantation macrocapsule. This macrocapsule has the advantage that its intravascular implantation allows better irrigation and access to nutrients, elimination of metabolic wastes and an exchange of bioactive substances.
En relación a la Microencapsulación(1 , 3), ésta corresponde a una técnica que experimentalmente ha mostrado mejores resultados, al prolongar la viabilidad celular, entregar un soporte mecánico y resistencia a los factores ambientales. Sin embargo, presenta limitaciones en la difusión de solutos y nutrientes. Por su elevada razón de volumen microcápsula:masa celular, permite la implantación sólo en lugares de gran perfusión. Así también, el tamaño de corte en los poros de la membrana es lo suficientemente grande como para permitir la infiltración de componentes, que generan respuesta inmunológica. In relation to Microencapsulation (1, 3) , this corresponds to a technique that has experimentally shown better results, by prolonging cell viability, delivering mechanical support and resistance to environmental factors. However, it has limitations in the diffusion of solutes and nutrients. By its high microcapsule volume ratio: cell mass, allows implantation only in places of great perfusion. Also, the cutting size in the pores of the membrane is large enough to allow infiltration of components, which generate an immune response.
Sus métodos de fabricación resultan en microcápsulas de características muy variables, las más adecuadas son complejas y tienen un elevado costo. Its manufacturing methods result in microcapsules of very variable characteristics, the most suitable are complex and have a high cost.
La microencapsulación es lejos el soporte más estudiado y utilizado. Algunos de los más conocidos son complejación de polielectrolitos, inversión interfacial, polimerización in-situ y spray drying. A escala industrial, el spray drying es un proceso fácil de realizar, ya que solo deben controlarse los parámetros en el aparato, sin embargo, es de elevado costo y no permite la encapsulacion de material viviente. Dependiendo de la técnica utilizada, condiciones experimentales y polímeros utilizados, se obtienen microcápsulas de diferentes tamaños y atributos, como tamaño de poro, número de células encapsuladas por microcápsula, entre otros. Microencapsulation is by far the most studied and used support. Some of the best known are polyelectrolyte complexation, interfacial inversion, in-situ polymerization and spray drying. On an industrial scale, spray drying is an easy process to perform, since only the parameters in the device must be controlled, however, it is expensive and does not allow the encapsulation of living material. Depending on the technique used, experimental conditions and polymers used, microcapsules of different sizes and attributes are obtained, such as pore size, number of cells encapsulated by microcapsule, among others.
Otro soporte utilizado es mediante PEGilación(3), proceso utilizado con el objetivo de buscar un mayor inmunoaislamiento. La PEGilación muestra interesantes resultados, otorgando una mayor protección frente al sistema inmune por impedimento estérico. Sin embargo, esta técnica no es viable por sí sola, debiendo ser complementada con otras técnicas de encapsulacion, ya que no entrega protección mecánica adecuada. Sus principales limitaciones son la alteración de la difusión de solutos y la disminución de la viabilidad celular, por la unión del PEG a proteínas de superficie, que interfieren con las funciones celulares. Another support used is by means of PEGylation (3) , a process used with the aim of seeking greater immunoisolation. PEGylation shows interesting results, giving greater protection against the immune system by steric impairment. However, this technique is not viable by itself, and must be complemented with other encapsulation techniques, since it does not provide adequate mechanical protection. Its main limitations are the alteration of solute diffusion and the decrease in cell viability, due to the binding of PEG to surface proteins, which interfere with cellular functions.
En relación a la nanoencapsulación, ésta se logra mediante una técnica conocida como layer by layer (capa por capa), que consta en la deposición alternada de finas capas de polielectrolitos (de grosor nanométrico) con cargas superficiales contrarias sobre un templado cargado, tratándose por lo tanto de un proceso de autoensamblaje, mediado por interacciones electrostáticas (figura 1). Este método es utilizado en un amplio campo de aplicaciones en la industria, ciencias y tecnología, por ser un método simple y de bajo costo(10) In relation to nanoencapsulation, this is achieved by a technique known as layer by layer, which consists in the alternate deposition of thin layers of polyelectrolytes (of nanometric thickness) with opposite surface charges on a charged temper, being treated by therefore of a self-assembly process, mediated by electrostatic interactions (Figure 1). This method is used in a wide range of applications in industry, science and technology, as it is a simple and low-cost method (10)
Las características de las nanocápsulas construidas, utilizando el método capa por capa, pueden ser manejadas por factores como; el tipo de polielectrolito, concentración, masa molecular, número de capas, carga de la capa final, el pH, sales presentes y tiempo de incubación en la solución en que ocurre la deposición. Estos factores modifican características como: tamaño del poro, densidad de carga superficial, elasticidad, grado de hídratación de la nanocápsula, determinando la permeabilidad, cinética de liberación, resistencia mecánica de la nanocápsula, entre otras(11' 12). The characteristics of the nanocapsules constructed, using the layer by layer method, can be handled by factors such as; the type of polyelectrolyte, concentration, molecular mass, number of layers, final layer charge, pH, salts present and incubation time in the solution in which deposition occurs. These factors modify characteristics such as: pore size, surface charge density, elasticity, degree of hydratation of the nanocapsule, determining permeability, release kinetics, mechanical resistance of the nanocapsule, among others (11 '12) .
La nanoencapsulación tiene por lo tanto, potenciales capacidades para encapsular todo tipo de material (nanopartículas cargadas, proteínas, ADN/ARN, células), formando parte del templado o del recubrimiento (depositándose mediante interacciones electrostáticas para formar parte de la nanocápsula). Se puede, inclusive introducir sustancias en el interior de las nanocápsulas huecas, como por ejemplo, principios activos, utilizando templados que luego son degradados (ej. Melamina formaldehído) y al momento de introducir los principios activos se manipula el estado de poro abierto y cerrado de las nanocápsulas. Con esto podemos utilizarlas como vehículo de administración en numerosas terapias, para la entrega en un blanco específico (utilizando por ejemplo nanocarriers depositados en la superficie de las nanocápsulas) o para la liberación controlada de principios activos y otras aplicaciones, según las necesidades clínicas requeridas. The nanoencapsulation therefore has potential capabilities to encapsulate all types of material (charged nanoparticles, proteins, DNA / RNA, cells), forming part of the tempering or coating (deposited by electrostatic interactions to form part of the nanocapsule). It is even possible to introduce substances into the hollow nanocapsules, such as active ingredients, using temperates that are then degraded (eg melamine formaldehyde) and when the active ingredients are introduced, the open and closed pore state is manipulated of the nanocapsules. With this we can use them as a vehicle of administration in numerous therapies, for delivery on a specific target (using for example nanocarriers deposited on the surface of the nanocapsules) or for the controlled release of active ingredients and other applications, according to the required clinical needs.
En la presente invención, es de interés la utilización de la célula como un templado (de carga neta negativa), para utilizar la nanoencapsulación como vehículo que posibilite su aplicación y supere algunas limitaciones asociadas a la terapia celular y a otros tipos de encapsulación utilizados(13). Pioneros en el campo de la nanoencapsulación celular, utilizando este método, son los realizados por Diaspro, Krol y colaboradores'10' 12, H 15) inicialmente con células procariontes y posteriormente, con células eucariontes mamíferas. In the present invention, it is of interest to use the cell as a temperate (of net negative charge), to use nanoencapsulation as a vehicle that allows its application and overcomes some limitations associated with cell therapy and other types of encapsulation used (13 ) . Pioneers in the field of cellular nanoencapsulation, using this method, are those carried out by Diaspro, Krol and collaborators ' 10 ' 12, H 15) initially with prokaryotic cells and subsequently, with mammalian eukaryotic cells.
Algunos estudios provenientes de diversos autores, intentan demostrar indicios de mejoras sustanciales en la superación de las limitaciones presentes en terapia celular, debido a que la nanoencapsulación consta de un recubrimiento conformacional, eliminando así el volumen ocupado por la interfase de la membrana de la cápsula y la célula (encapsulada individualmente), por lo tanto, el volumen del implante se reduce drásticamente, permitiendo implantaciones menos invasivas y con una mayor densidad celular. A la vez, esta delgada capa permite una difusión adecuada de solutos y nutrientes'10, 12). Some studies from various authors, try to show signs of substantial improvements in overcoming the limitations present in cell therapy, because the nanoencapsulation consists of a conformational coating, thus eliminating the volume occupied by the interface of the capsule membrane and the cell (individually encapsulated), therefore, the implant volume is drastically reduced, allowing for less invasive implants and with a higher cell density. At the same time, this thin layer allows adequate diffusion of solutes and nutrients' 10, 12) .
Al tratarse de un recubrimiento que solo se deposita mediante interacciones no covalentes, no altera las estructuras celulares, preservando el metabolismo celular y la proliferación. Por lo tanto, la nanocápsula funcionaría como un soporte que entrega protección y resistencia mecánica, pues el alto grado de hidratación transforma estos recubrimientos en delgadas capas de gel. Esto, sumado al pequeño tamaño de corte en los poros de la nanocápsula, permite un inmunoaislamiento aún más eficaz que otros tipos de dispositivos de administración celular utilizados y mejora sustancialmente la viabilidad celular. Sin embargo, estos resultados no son concluyentes, siendo más bien controversiales, sobretodo en relación a viabilidad celular y a la fragilidad de las células mamíferas. Por esta razón, se desconoce si este método es del todo inocuo para las células, transformando un proceso, que para otras aplicaciones es simple, en algo mucho más complejo (15). As it is a coating that is only deposited through non-covalent interactions, it does not alter cell structures, preserving cell metabolism and proliferation. Therefore, the nanocapsule would function as a support that provides protection and mechanical resistance, since the high degree of hydration transforms these coatings into thin layers of gel. This, in addition to the small cutting size in the pores of the nanocapsule, allows an even more effective immuno-isolation than other types of cell delivery devices used and substantially improves cell viability. However, these results are inconclusive, being rather controversial, especially in relation to cell viability and the fragility of mammalian cells. For this reason, it is unknown if this method is completely harmless to cells, transforming a process, which for other applications is simple, into something much more complex (15) .
Otro interesante efecto está relacionado con la elección de los polielectrolitos que formarán parte de la nanocápsula. Los polielectrolitos pueden ser a la vez sustancias bioactivas, las cuales conservan su actividad en las nanocápsulas, por lo tanto, pueden interactuar con las células como un sustrato o un factor del microambiente celular que estimule el crecimiento, alguna función celular específica, o una emulación de la matriz extracelular (ECM) de las células, buscando imitar el medio en el cual originalmente están las células en el tejido del cual provienen. Por tanto, la elección de los polielectrolitos es crítica para la obtención de beneficios adicionales, por esto es que a continuación, se entrega una breve revisión de los polielectrolitos utilizados en esta invención. Another interesting effect is related to the choice of polyelectrolytes that will be part of the nanocapsule. The polyelectrolytes can be both bioactive substances, which retain their activity in the nanocapsules, therefore, they can interact with the cells as a substrate or a factor of the cellular microenvironment that stimulates growth, some specific cellular function, or an emulation of the extracellular matrix (ECM) of the cells, seeking to mimic the medium in which the cells in the tissue they originally come from are originally. Therefore, the choice of polyelectrolytes is critical for obtaining additional benefits, which is why a brief review of the polyelectrolytes used in this invention is given below.
Los polielectrolitos utilizados corresponden a policationes y polianiones. Dentro de los policationes se describe el Cloruro de Poli(alilamina) (PAH)(16), Cloruro de Poli(dialildimetilamonio) (PDADMAC)(17' 8), Chitosan (Ch) y Poli-L-lisina (PLL). Los polianiones pueden ser: poli(estireno) sulfonato de sodio (PSS) (16),Ácido Hialurónico (HA) y Condroitin sulfato (CS). The polyelectrolytes used correspond to polycations and polyanions. Within the polycations, Poly (allylamine) Chloride (PAH) (16) , Poly (diallyldimethylammonium) chloride (PDADMAC) (17 ' 8) , Chitosan (Ch) and Poly-L-lysine (PLL) are described. The polyanions can be: sodium poly (styrene) sulfonate (PSS) (16) , Hyaluronic Acid (HA) and Chondroitin Sulfate (CS).
Cloruro de Poli(alilamina) (PAH). Poly (allylamine) chloride (PAH).
Figure imgf000010_0001
Figure imgf000010_0001
Cloruro de Poli(alilamina) es un polielectrolito catiónico débil, con grupos amino cargados positivamente a pH fisiológico, con un pKa de 9,3 y que exhibe diferentes propiedades mecánicas y conformaciones, dependiendo del pH y de la fuerza iónica de la solución. Las mayores interacciones que genera son electrostáticas, iónicas e hidrofóbicas. Se prepara a partir de la polimerización de alilamina. Poly Chloride (allylamine) is a weak cationic polyelectrolyte, with positively charged amino groups at physiological pH, with a pKa of 9.3 and exhibiting different mechanical properties and conformations, depending on the pH and ionic strength of the solution. The greatest interactions it generates are electrostatic, ionic and hydrophobic. It is prepared from the polymerization of allylamine.
La utilización de este policatión está estrechamente relacionada con las aplicaciones coloidales y las encapsulaciones. Estas aplicaciones varían en un amplio espectro, desde las propiedades para estabilizar o flocular suspensiones, estabilizar emulsiones (útiles en industria alimenticia, farmacéutica y cosmética), hasta la purificación de agua, producto de las interacciones atractivas del polímero, cuando se adiciona en pequeñas cantidades. En el campo de la encapsulación, es uno de los polímeros más utilizados, conjuntamente con el Poli(estireno sulfonato) por el método layer by layer, para la obtención de nanocápsulas de potencial uso, como sistema de entrega de drogas de terapia celular(10, 12). The use of this polycation is closely related to colloidal applications and encapsulations. These applications vary in a wide spectrum, from the properties to stabilize or flocculate suspensions, stabilize emulsions (useful in the food, pharmaceutical and cosmetic industry), to the purification of water, product of the attractive interactions of the polymer, when added in small quantities . In the field of encapsulation, it is one of the most used polymers, together with Poly (styrene sulfonate) by the layer by layer method, to obtain nanocapsules of potential use, as a delivery system for cell therapy drugs (10 , 12) .
Cloruro de Poli(dialildimetilamonio) (PDADMAC) Poly (diallyldimethylammonium) chloride (PDADMAC)
Figure imgf000011_0001
Figure imgf000011_0001
El Cloruro de Poli(dialildimetilamonio) es un polielectrolito de origen sintético, conformado por monómeros de dialildimetilamonio que posee un grupo amino cuaternario, clasificándolo como un policatión y electrolito fuerte, por lo que sus propiedades no son dependientes ni de la fuerza iónica ni del pH de las soluciones. Se caracteriza por una gran solubilidad en solventes acuosos y una densa carga eléctrica. Por lo tanto, es un polielectrolito de conformaciones más torsionadas y con numerosas interacciones iónicas frente a contraiones(18). Polychloride (diallyldimethylammonium) is a polyelectrolyte of synthetic origin, consisting of diallyldimethylammonium monomers that has a quaternary amino group, classifying it as a strong polycation and electrolyte, so its properties are not dependent on either ionic strength or pH of the solutions. It is characterized by a high solubility in aqueous solvents and a dense electrical charge. Therefore, it is a polyelectrolyte of more twisted conformations and with numerous ionic interactions against counterions (18) .
A pesar de su naturaleza sintética, PDADMAC posee amplios usos, tanto en el área industrial como en la científica. Muchas de sus propiedades estabilizadores de carga son utilizadas como aditivos y estabilizadores de emulsiones, determinando así las propiedades finales de productos cosméticos. Sus propiedades floculantes son utilizadas como agentes purificadores de agua, en la preparación de suspensiones, en la fabricación de papel y también en la industria minera y petrolera07). Despite its synthetic nature, PDADMAC has wide uses, both in the industrial and scientific areas. Many of its load stabilizing properties are used as additives and emulsion stabilizers, thus determining the final properties of cosmetic products. Its flocculant properties are used as water purifying agents, in the preparation of suspensions, in the manufacture of paper and also in the mining and oil industry 07) .
Es utilizado dentro del campo científico principalmente por su función como agente gelificante y quelante, por sus propiedades hidrofílicas, interacciones iónicas y electrostáticas. En lo que concierne a encapsulaciones existen numerosas aplicaciones. Investigaciones de Fournier y colaboradores(19), destacan su potencial uso en nanoencapsulación, debido a que posibilita una mayor viabilidad celular en comparación con otros policationes estudiados, tanto naturales como sintéticos. It is used within the scientific field mainly for its function as a gelling and chelating agent, for its hydrophilic properties, ionic and electrostatic interactions. As regards encapsulations, there are numerous applications. Research by Fournier et al. (19) , highlights its potential use in nanoencapsulation, because it makes possible a greater cell viability compared to other policationes studied, both natural and synthetic.
Chitosan (Ch). Chitosan (Ch).
Figure imgf000012_0001
Figure imgf000012_0001
R = H o COCH3 R = H or COCH 3
Polímero de unidades fi~(l-4)-D*glttcosa?nma  Unit polymer fi ~ (l-4) -D * glttcosa? Nma
Este policatión es un polisacárido natural, derivado de una de las sustancias orgánicas más abundantes en la naturaleza, la quitina. La quitina se encuentra en la coraza de camarones, cangrejos y otros crustáceos, en el fino manto del plancton, en el exoesqueleto de los insectos, en el cartílago del calamar y en las membranas celulares de algunos hongos(20). This polycation is a natural polysaccharide, derived from one of the most abundant organic substances in nature, chitin. Chitin is found in the shell of shrimp, crabs and other crustaceans, in the thin mantle of plankton, in the exoskeleton of insects, in squid cartilage and in the cell membranes of some fungi (20) .
Al someter la quitina a una N-desacetilación genera Chitosan (Esquema de Obtención), un derivado polisacárido con grupos amino cargados positivamente, unidades de p-(1-4)-D-glucosamina, con un pKa de 6,5. El grado de desacetilación lo clasifica en bajo, medio y alto peso molecular. Además, la reacción genera ácido acético, explicando con esto que la solubilidad de este polisacárido ocurre en soluciones acidificadas y depende del pH de la solución(20). By subjecting chitin to an N-deacetylation generates Chitosan (Obtaining Scheme), a polysaccharide derivative with positively charged amino groups, units of p- (1-4) -D-glucosamine, with a pKa of 6.5. The degree of deacetylation classifies it as low, medium and high molecular weight. In addition, the reaction generates acetic acid, thereby explaining that the solubility of this polysaccharide occurs in acidified solutions and depends on the pH of the solution (20) .
Esquema de obtención de Chitosan a partir de Quitina
Figure imgf000013_0001
Scheme of obtaining Chitosan from Chitin
Figure imgf000013_0001
Figure imgf000013_0002
Figure imgf000013_0002
Muchas de las propiedades de este polisacárido, hacen posible su utilización en una amplia gama de aplicaciones, como productos cosméticos, purificación de agua, recuperación de desechos, también en industria alimenticia, agricultura y medicina. Many of the properties of this polysaccharide make it possible to use it in a wide range of applications, such as cosmetic products, water purification, waste recovery, also in the food industry, agriculture and medicine.
Su naturaleza catiónica lo hace capaz de adherirse a superficies cargadas negativamente como piel y cabello, por lo tanto útil en productos cosméticos. A la vez, permite la neutralización de cargas contrarias, causando la precipitación de partículas y/o coloides cargados negativamente, útil en la purificación de soluciones. Por lo anterior, se infiere su capacidad floculante de suspensiones(20). Its cationic nature makes it capable of adhering to negatively charged surfaces such as skin and hair, therefore useful in cosmetic products. At the same time, it allows the neutralization of opposite charges, causing the precipitation of negatively charged particles and / or colloids, useful in the purification of solutions. Therefore, its flocculant capacity of suspensions is inferred (20) .
El chitosan es una molécula que absorbe grandes cantidades de agua, por lo que es un ingrediente importante en algunas cremas humectantes(20). Chitosan is a molecule that absorbs large amounts of water, making it an important ingredient in some moisturizers (20) .
Sus propiedades antibacteriales y antivirales están muy indicadas para aplicaciones médicas(21), curar heridas, en suturas y como ayuda en operaciones de cataratas y tratamiento de enfermedades ambulatorias. Mientras que en frutas y verduras frescas sirve como protector antimicrobiano, o agente preservante(22). Chitosan, así como quitina y sus derivados, han demostrado no ser tóxicos ni producir alergias, por lo que el organismo humano no produce ningún rechazo hacia estos componentes(20, 23). Its antibacterial and antiviral properties are very suitable for medical applications (21) , to heal wounds, in sutures and as an aid in cataract operations and treatment of outpatient diseases. While in fresh fruits and vegetables it serves as an antimicrobial protector, or preservative agent (22) . Chitosan, as well as chitin and its derivatives, have proven not to be toxic or produce allergies, so the human organism does not produce any rejection of these components (20, 23) .
El chitosan es soluble en agua acidificada. Esta solubilidad, sumada a su viscosidad, puede hacerlo más espeso o más ligero, según se requiera, para uso, por ejemplo, en la industria alimenticia. Chitosan is soluble in acidified water. This solubility, added to its viscosity, can make it thicker or lighter, as required, for use, for example, in the food industry.
La quitina y sus derivados son totalmente biodegradables y por eso están muy recomendados para preservar el medio ambiente. Es una de las sustancias más prometedoras en el campo de los plásticos biodegradables, como alternativa al plástico tradicional. Las propiedades antihongos, en conjunto con su biodegradabilidad son condiciones idóneas para el uso en agricultura(20). Chitin and its derivatives are totally biodegradable and therefore are highly recommended to preserve the environment. It is one of the most promising substances in the field of biodegradable plastics, as an alternative to traditional plastic. The antifungal properties, together with their biodegradability, are ideal conditions for use in agriculture (20) .
Poli-L-lisina (PLL). Poly-L-lysine (PLL).
Figure imgf000014_0001
Figure imgf000014_0001
La fuente de obtención de Poli-L-lisina proviene de la fermentación de Streptomices, proceso utilizado para producirla industrialmente(24). The source of obtaining Poly-L-lysine comes from the fermentation of Streptomices, a process used to produce it industrially (24) .
Poli-L-lisina es un polipéptido de pequeño tamaño, con 25 a 30 residuos del aminoácido L-lisina, por lo que su peso molecular es variable. Debido a sus grupos amino, tiene carga positiva a pH fisiológico, pues poseen un pKa de 9,0. Por lo anteriormente descrito, las propiedades eléctricas y de carga lo clasifican como un policatión. Estructuralmente corresponde a la ε-poli-L-lisina, denominada así por la unión que ocurre entre el grupo amino de la posición ε y el grupo carboxilo del aminoácido. Es soluble en agua, ligeramente soluble en etanol e insoluble en solventes orgánicos como acetato de etilo. La actividad de la poli-L-lisina no es dependiente del pH y es estable a altas temperaturas. Se describen propiedades antimicrobianas para este polipéptido, debido a que las superficies con carga positiva que forman, inhiben el crecimiento de microorganismos. Su mecanismo de acción estaría relacionado con la capacidad de adsorberse electrostáticamente a la superficie celular de la bacteria, seguido de un desequilibrio y una distribución anormal del citoplasma, causando daño en la bacteria(25). Esta cualidad es utilizada ampliamente como preservante en la industria alimenticia. Poly-L-lysine is a small polypeptide, with 25 to 30 residues of the amino acid L-lysine, so its molecular weight is variable. Due to its amino groups, it has a positive charge at physiological pH, since they have a pKa of 9.0. As described above, the electrical and charging properties classify it as a polycation. Structurally it corresponds to the ε-poly-L-lysine, named for the union that occurs between the amino group of the ε position and the carboxyl group of the amino acid. It is soluble in water, slightly soluble in ethanol and insoluble in organic solvents such as ethyl acetate. The activity of poly-L-lysine is not pH dependent and is stable at high temperatures. Antimicrobial properties for this polypeptide are described, because the positively charged surfaces they form inhibit the growth of microorganisms. Its mechanism of action would be related to the ability to electrostatically adsorb to the bacterial cell surface, followed by an imbalance and an abnormal distribution of the cytoplasm, causing damage to the bacteria (25) . This quality is widely used as a preservative in the food industry.
Su amplia utilización, se debe principalmente a su perfil de biocompatibilidad con el organismo, por su seguridad como preservante, por promover la adherencia en cultivos celulares y por sus propiedades de gelificación utilizadas en recubrimientos y lechos poliméricos, dada su capacidad de captar grandes cantidades de agua, así como por sus interacciones electrostáticas con otros iones. Sin embargo, numerosos autores describen un efecto tóxico e inmunogénico, sobre todo cuando es inyectado intravenosamente*2, 3). Its wide use is mainly due to its biocompatibility profile with the organism, for its safety as a preservative, for promoting adhesion in cell cultures and for its gelation properties used in coatings and polymer beds, given its ability to capture large amounts of water, as well as for its electrostatic interactions with other ions. However, numerous authors describe a toxic and immunogenic effect, especially when injected intravenously * 2, 3) .
Poli(estireno) sulfonato de sodio (PSS)(16). Sodium poly (styrene) sulphonate (PSS) (16).
Figure imgf000015_0001
Figure imgf000015_0001
El poli(estireno) sulfonato de sodio es un polielectrolito aniónico, de origen sintético, obtenido ya sea por la polimerización de monómeros de estireno sulfonato o por la sulfonación de poli(estireno)(26). Sodium poly (styrene) sulfonate is an anionic polyelectrolyte, of synthetic origin, obtained either by the polymerization of styrene sulfonate monomers or by the sulfonation of poly (styrene) (26) .
Estructuralmente, a diferencia del poli(estireno) (molécula principalmente hidrocarbonada y de gran lipofilicidad), sus propiedades cambian totalmente cuando se introduce un sustituyente sulfonilo (un grupo ácido de pKa 2,5) en posición 4 del anillo bencénico, que le confiere una gran solubilidad en agua. A pH fisiológico, casi la totalidad de la molécula está cargada negativamente, generando interacciones iónicas, por lo que se le conoce como una resina de intercambio iónico(26, 27). Structurally, unlike poly (styrene) (mainly hydrocarbon and high lipophilicity molecule), its properties change completely when a sulfonyl substituent (an acidic group of pKa 2.5) is introduced in position 4 of the benzene ring, which gives it a high water solubility TO Physiological pH, almost the entire molecule is negatively charged, generating ionic interactions, which is why it is known as an ion exchange resin (26, 27) .
En el área médica, este polielectrolito es conocido como kayexalato( ', útil para tratar hiperpotasemias, por su conocida selectividad ante los iones potasio (los cuales son atrapados en el intestino), administrándose por vía oral o enema. Así es capaz de absorber 1 mEq de potasio por cada gramo de poli(estireno) sulfonato, liberando 1 mEq de Sodio. Se describen propiedades como espermicida y antimicrobiano, cuando es utilizado tópicamente. In the medical area, this polyelectrolyte is known as kayexalate ( ', useful for treating hyperkalemia, for its known selectivity to potassium ions (which are trapped in the intestine), administered orally or enema. Thus it is able to absorb 1 mEq of potassium for each gram of poly (styrene) sulphonate, releasing 1 mEq of Sodium Properties described as spermicide and antimicrobial, when used topically.
También es utilizado como un plastificador en cementos. En nanoencapsulación, numerosos trabajos lo utilizan conjuntamente con cloruro de poli(alilamina)(10' 12). It is also used as a plasticizer in cements. In nanoencapsulation, numerous works use it in conjunction with poly (allylamine) chloride (10 '12) .
Ácido Hialurónico (HA). Hyaluronic Acid (HA).
Figure imgf000016_0001
Figure imgf000016_0001
El ácido hialurónico es un polisacárido lineal, que consta de unidades disacáridas alternantes de ácido (1 ,4)-a-D-glucurónico y (1 ,3)-p-N-acetil-D- glucosamina. Las propiedades de este polianión están dadas por los sustituyentes de carboxilato del ácido glucurónico, un grupo funcional que le confiere carga negativa con un pKa de 3,4(29). Hyaluronic acid is a linear polysaccharide, consisting of alternating disaccharide units of (1, 4) -aD-glucuronic acid and (1, 3) -pN-acetyl-D-glucosamine. The properties of this polyanion are given by the carboxylate substituents of glucuronic acid, a functional group that confers a negative charge with a pKa of 3.4 (29) .
El ácido hialurónico, conocido también como un glicosaminoglicano, se encuentra en todos los tejidos y fluidos corporales de los vertebrados. Es un componente importante en la matriz extracelular (ECM) del tejido conectivo, siendo más abundante en cordón umbilical, fluido sinovial, piel y humor vitreo. También se encuentra en bacterias. El ácido hialurónico es sintetizado en la membrana plasmática y es catabolizado por endocitosis mediada por receptores, seguido por degradación lisosomal. Su vida media en humanos y animales fluctúa de menos de 1 día a varios días.(30). Los grupos carboxílicos del ácido hialurónico están totalmente ionizados a pH extracelular. Su actividad osmótica juega un rol importante en el movimiento y homeostasis del agua. Enlaces de hidrógeno secundarios a lo largo del polisacárido crean torsiones, generando zonas hidrofóbicas, que permiten la asociación entre cadenas de ácido hialurónico, a pesar de sus cargas negativas. El alto grado de hidratación, es capaz de retener un porcentaje de vmiles de veces mayor a su peso, en conjunto con su estructura rígida, promueve una configuración extendida, entregándole distintas propiedades de viscoelasticidad que no son constantes, sino variables según sus movimientos oscilatorios. Hyaluronic acid, also known as a glycosaminoglycan, is found in all tissues and body fluids of vertebrates. It is an important component in the extracellular matrix (ECM) of connective tissue, being more abundant in umbilical cord, synovial fluid, skin and vitreous humor. It is also found in bacteria. Hyaluronic acid is synthesized in the plasma membrane and is catabolized by receptor-mediated endocytosis, followed by lysosomal degradation. Its half-life in humans and animals fluctuates from less than 1 day to several days. (30) . The carboxylic groups of hyaluronic acid are fully ionized at extracellular pH. Its osmotic activity plays an important role in the movement and homeostasis of water. Secondary hydrogen bonds along the polysaccharide create torsions, generating hydrophobic zones, which allow the association between hyaluronic acid chains, despite their negative charges. The high degree of hydration, is able to retain a percentage of vmiles of times greater than its weight, together with its rigid structure, promotes an extended configuration, giving it different properties of viscoelasticity that are not constant, but variable according to their oscillatory movements.
Sus múltiples propiedades, tanto físico-químicas, como biológicas, le dan una amplia variedad de usos. Its multiple properties, both physical-chemical and biological, give it a wide variety of uses.
El uso en cosmética es muy conocido y demandado, se emplea para hidratación de la epidermis ya que reconstituye las fibras que sostienen los tejidos de la piel, por ello, en cremas previene la acentuación de arrugas por deshidratación y ayuda a retener agua(3 ). The use in cosmetics is well known and demanded, it is used for hydration of the epidermis since it reconstitutes the fibers that support the skin tissues, therefore, in creams it prevents the accentuation of wrinkles by dehydration and helps retain water (3) .
En las cirugías estéticas, su conocida participación en matriz extracelular como soporte en tejidos, además de ser biodegradable y de gran biocompatibilidad, se avala su utilización como material de relleno en implantes, que se inyecta subcutáneamente. Además de alisar los pliegues subcutáneos, estimula la producción de colágeno, haciendo estos resultados más evidentes y duraderos en el tiempo. Se utiliza por lo tanto, para reestructurar arrugas, así como para reducir marcas y cicatrices, producto del acné severo y otros problemas cutáneos que ocasionen pérdida de piel(32). In aesthetic surgeries, its known participation in extracellular matrix as a support in tissues, in addition to being biodegradable and of great biocompatibility, its use as a filler material in implants, which is injected subcutaneously, is endorsed. In addition to smoothing the subcutaneous folds, it stimulates the production of collagen, making these results more evident and lasting over time. It is therefore used to restructure wrinkles, as well as to reduce marks and scars, severe acne and other skin problems that cause skin loss (32) .
En el área médica se utiliza en viscosuplementación, técnica para sustituir el líquido sinovial perdido durante las artroscopías. También es factible su utilización en mesoterapia. Inclusive, numerosos países tienen incluido en la farmacopea al ácido hialurónico, ya que es utilizado como cicatrizante de heridas y para úlceras de decúbito, por medio de aplicación tópica. Se emplea como suplemento nutricional para tratamientos por dolores articulares, osteoartritis y tejido conjuntivo(33). In the medical area it is used in viscosupplementation, a technique to replace the synovial fluid lost during arthroscopy. Its use in mesotherapy is also feasible. Even numerous countries have included in the Pharmacopoeia to hyaluronic acid, as it is used as wound healing and for pressure sores, by topical application. It is used as a nutritional supplement for treatments for joint pain, osteoarthritis and connective tissue (33) .
Industrialmente, este compuesto se obtiene a partir de numerosas fuentes. Las más comunes son producto de técnicas de bioingeniería, utilizando fermentación bacteriana. Las fuentes más utilizadas para su obtención son el humor vitreo y líquido sinovial de ganado vacuno (restringido debido a enfermedad de las vacas locas), cordón umbilical, aleta de tiburón y las crestas de los gallos(30). Industrially, this compound is obtained from numerous sources. The most common are the product of bioengineering techniques, using bacterial fermentation. The sources most commonly used to obtain them are the vitreous humor and synovial fluid of cattle (restricted due to mad cow disease), umbilical cord, shark fin and the crests of roosters (30) .
Condroitin Sulfato (CS). Chondroitin Sulfate (CS).
Figure imgf000018_0001
Figure imgf000018_0001
Condroitin 6sulfato Condroitin 4-tsulfato  Chondroitin 6 Sulfate Chondroitin 4-Tsulfate
El Condroitin sulfato es un glucosaminoglicano sulfatado, emparentado con Heparán, Dermatán y Keratán Sulfatos. Su composición fija consta de una cadena lineal con disacáridos alternados de N-acetilgalactosamina y N-ácido glucurónico, de los cuales el producto total es una mezcla de sulfatos, sustituidos en diferentes posiciones, donde los mayoritarios son el Condroitin 6- sulfato y el Condroitin 4-sulfato. Producto de las numerosas y variadas sustituciones, el Condroitin sulfato posee una densidad de carga y pesos moleculares variables. Sus grupos funcionales ácidos, tanto carboxilatos como sulfonatos, con pKas de 3,5 y 3,0 respectivamente, están totalmente ionizados a pH fisiológico, siendo estas cargas negativas las que confieren la característica de un polianión(35). El Condroitin sulfato es un componente importante en la mantención e integridad de la matriz extracelular de tejidos conectivos del cuerpo, cartílago, piel, vasos sanguíneos, tendones y ligamentos. Esta función la cumple formando parte de proteoglicanos, en el cual se encuentra unido por ejemplo, ácido hialurónico, aminoácidos, entre otros. En el cartílago, sus fuertes cargas le confieren un alto grado de hidratación, otorgando así su característica resistencia a la compresión*36'. Chondroitin sulfate is a sulfated glucosaminoglycan, related to Heparan, Dermatan and Keratan Sulfates. Its fixed composition consists of a linear chain with alternating disaccharides of N-acetylgalactosamine and N-glucuronic acid, of which the total product is a mixture of sulfates, substituted in different positions, where the majority are Chondroitin 6- sulfate and Chondroitin 4-sulfate As a result of the numerous and varied substitutions, Chondroitin Sulfate has a charge density and variable molecular weights. Its acid functional groups, both carboxylates and sulphonates, with pKas of 3.5 and 3.0 respectively, are fully ionized at physiological pH, these negative charges confer the characteristic of a polyanion (35) . Chondroitin sulfate is an important component in the maintenance and integrity of the extracellular matrix of connective tissues of the body, cartilage, skin, blood vessels, tendons and ligaments. This function is fulfilled by forming part of proteoglycans, in which it is linked, for example, hyaluronic acid, amino acids, among others. In cartilage, its strong loads give it a high degree of hydration, thus giving it its characteristic compressive strength * 36 '.
El Condroitin sulfato tiene la capacidad de interactuar con las proteínas de la matriz extracelular, gracias a las cargas negativas de la molécula, importantes para regular el amplio espectro de mecanismos celulares. Chondroitin sulfate has the ability to interact with extracellular matrix proteins, thanks to the negative charges of the molecule, important to regulate the broad spectrum of cellular mechanisms.
Todas estas funciones no están tan estudiadas como en otro de sus análogos, el Heparán sulfato. Un ejemplo de ello, es el aumento de los niveles de proteoglicanos provenientes del Condroitin sulfato, cuando existe un daño en el sistema nervioso central, que impide la regeneración de terminales nerviosas dañadas(37). All these functions are not as studied as in another of its analogues, heparan sulfate. An example of this is the increase in proteoglycan levels from Chondroitin sulfate, when there is damage to the central nervous system, which prevents the regeneration of damaged nerve terminals (37) .
El Coindritin sulfato posee una actividad antiinflamatoria, porque estimula la síntesis e inhibe la degradación de proteoglicanos, actuando como un sustrato necesario para estabilizar las membranas celulares y regenerar el daño al cartílago. Promueve el crecimiento de glucosoaminoglicanos en la matriz extracelular, así es posible provocar sinergismo de estos agentes en coadministración de Analgésicos Antiinflamatorios No Esferoidales (AINEs). Igualmente, puede estimular la síntesis e inhibición de la degradación de colágeno. Por esto mismo es utilizado para artritis y artrosis, ya que además de sus efectos antiinflamatorios, aumentan los niveles de glucosoaminoglicanos en la matriz extracelular de condrocitos articulares humanos y cartílago. Algunos estudios sugieren que esta actividad se debe principalmente a los grupos sulfato, que sustituyen a estos derivados de glucosaminas(38). Coindritin sulfate has an anti-inflammatory activity, because it stimulates synthesis and inhibits the degradation of proteoglycans, acting as a necessary substrate to stabilize cell membranes and regenerate damage to cartilage. It promotes the growth of glycosoaminoglycans in the extracellular matrix, thus it is possible to cause synergism of these agents in co-administration of Non-Spheroidal Anti-Inflammatory Analgesics (NSAIDs). It can also stimulate the synthesis and inhibition of collagen degradation. For this reason it is used for arthritis and osteoarthritis, since in addition to its anti-inflammatory effects, glycosoaminoglycan levels in the extracellular matrix of human articular chondrocytes and cartilage increase. Some studies suggest that this activity is mainly due to sulfate groups, which replace these glucosamine derivatives (38) .
Producto de las propiedades mencionadas anteriormente, el uso médico del Condroitin sulfato es bastante conocido, se utiliza para artrosis, coadministrada con N-acetil-glucosam¡na, por lo que en algunos países lo consideran como un fármaco (antiinflamatorio antirreumático no esteroidal), no así en Estados Unidos donde es regulado como suplemento alimenticio. Due to the properties mentioned above, the medical use of chondroitin sulfate is well known, it is used for osteoarthritis, co-administered with N-acetyl-glucosamine, so in some countries they consider it as a drug (non-steroidal anti-rheumatic anti-inflammatory), but not in the United States where it is regulated as a food supplement.
Las fuentes de extracción del Condroitín sulfato, y de las cuales principalmente se obtienen las diferentes proporciones de las variedades de sulfatos, son el cartílago bovino, porcino o marino, por medio de proteólisis y purificación. El obtenido de tráquea bovina (con un 95% de pureza) es el más utilizado y estudiado, por lo tanto su eficacia y su seguridad son las más conocidas(39). The sources of Chondroitin sulfate extraction, and from which mainly the different proportions of sulfate varieties are obtained, are bovine, porcine or marine cartilage, by means of proteolysis and purification. The one obtained from bovine trachea (with 95% purity) is the most used and studied, therefore its efficacy and safety are the best known (39) .
DESCRIPCIÓN DE LAS FIGURAS DESCRIPTION OF THE FIGURES
Figura 1 : Ciclo de deposición capa a capa sobre un templado cargado. Etapa 1 : solución de policatión; etapa 2: lavado; etapa 3: solución de polianión; etapa 4: lavado. Figure 1: Layer-to-layer deposition cycle on a loaded temper. Stage 1: polycation solution; stage 2: washing; stage 3: polyanion solution; stage 4: washing.
Figura 2: Microscopía de fluorescencia de células con el anticuerpoo conjugado: Imágenes de Microscopía de Fluorescencia a un aumento de 200x para determinar la presencia de la conjugación del anticuerpo lgG-1 FITC en la superficie de ADSCs nanoencapsuladas. Las imágenes (a) y (b) muestran ADSCs nanoencapsuladas en presencia de anticuerpo pero sin la adición de los reactivos químicos NHSS y EDAC, la que no emite fluorescencia verde del anticuerpo FITC. Las imágenes (c) y (d) corresponden a ADSCa nanoencapsuladas en presencia de anticuerpo y la adición de los reactivos químicos NHSS y EDAC, la cual muestra fluorescencia verde que corresponde al anticuerpo lgG1-FITC conjugado en la superficie de las ADSCs nanoencapsuladas. Figure 2: Fluorescence microscopy of cells with the antibody or conjugate: Fluorescence Microscopy images at a magnification of 200x to determine the presence of the conjugation of the IgG-1 FITC antibody on the surface of nanoencapsulated ADSCs. Images (a) and (b) show nanoencapsulated ADSCs in the presence of antibody but without the addition of the chemical reagents NHSS and EDAC, which does not emit green fluorescence of the FITC antibody. Images (c) and (d) correspond to nanoencapsulated ADSCa in the presence of antibody and the addition of the chemical reagents NHSS and EDAC, which shows green fluorescence corresponding to the conjugated lgG1-FITC antibody on the surface of nanoencapsulated ADSCs.
Figura 3: Histograma de Citometría de flujo de células con el anticuerpo conjugado: Histogramas de Citometría de Flujo para la determinación de la conjugación del anticuerpo lgG-1 FITC, mediante los reactivos químicos [N- hidroxi-sulfo-succinimida (NHSS) y 1-ethil-3-[3-dimetilaminopropil]carbodiimida (EDAC)], para ADSCs nanoencapsuladas en presencia de crosslinkers (línea gris) y en ausencia de reactivos químicos (línea negra). Desplazamiento a la derecha de la intensidad de fluorescencia de células con anticuerpo conjugado (gris) respecto del control (negro) indica la conjugación del anticuerpo a la superficie de las nanoencapsuladas. Figure 3: Cell flow cytometry histogram with the conjugated antibody: Flow cytometry histograms for the determination of the conjugation of the lgG-1 FITC antibody, using the chemical reagents [N-hydroxy-sulfo-succinimide (NHSS) and 1 -ethil-3- [3-dimethylaminopropyl] carbodiimide (EDAC)], for nanoencapsulated ADSCs in the presence of crosslinkers (gray line) and in the absence of chemical reagents (black line). Shift to the right of the fluorescence intensity of cells with conjugated antibody (gray) with respect to the control (black) indicates the conjugation of the antibody to the surface of the nanoencapsules.
Figura 4: Viabilidad celular de células con el anticuerpo conjugado. Figure 4: Cell viability of cells with the conjugated antibody.
Viabilidad cellular después del proceso de conjugación del anticuerpo en ADSCs nanoencapsuladas, utilizando los reactivos químicos NHSS y EDAC. Control (columna A) corresponde a ADSCs en presencia del anticuerpo pero sin la adición de los reactivos químicos. ADSC con anticuerpo conjugado a lgG1 (columna B). Las columnas representan el promedio de 3 muestras +/- SEM. (ns) no hay diferencia estadísticamente significativa. Cellular viability after the antibody conjugation process in nanoencapsulated ADSCs, using the chemical reagents NHSS and EDAC. Control (column A) corresponds to ADSCs in the presence of the antibody but without the addition of chemical reagents. ADSC with antibody conjugated to lgG1 (column B). The columns represent the average of 3 samples +/- SEM. (ns) there is no statistically significant difference.
Figura 5: Imágenes de vibilidad celular de células con el anticuerpo conjugado. Figure 5: Cellular visibility images of cells with the conjugated antibody.
Las imágenes de viabilidad celular bajo el microscopio de fluorescencia muestran en A a las ADSC viables y que tienen conjugado el anticuerpo (en verde) y en B a las ADSC muertas y que tienen conjugado el anticuerpo (en rojo). Las ADSC corresponden a ADSCs nanoencapsuladas y conjugadas a un anticuerpo lgG-1 utilizando NHSS y EDAC como reactivos químicos. La imagen C corresponde a la superposición de las imágenes previas en el mismo campo. Magnificación 200x. The cell viability images under the fluorescence microscope show in A the viable ADSCs and that they have conjugated the antibody (in green) and in B the dead ADSCs and that they have conjugated the antibody (in red). ADSCs correspond to nanoencapsulated and conjugated ADSCs to a lgG-1 antibody using NHSS and EDAC as chemical reagents. Image C corresponds to the overlay of previous images in the same field. 200x magnification
Figura 6: Curvas de crecimiento de células con el anticuerpo conjugado. Figure 6: Cell growth curves with the conjugated antibody.
Curva de crecimiento de las ADSCs y ADSCs naoencapsuladas y conjugadas al anticuerpo lgG1. Cada punto en la curva representa el promedio de 3 muestras +/- SEM. ADSC: y = 37,6 e0,2x, k = 0,2, Tau= 5,2, Tiempo de duplicación = 3, r2 = 0,929. ADSC Conjugadas al anticuerpo; y = 34,7 e0,2x, k = 0,2, Tau= 5, Tiempo de duplicación = 3, r2 = 0,96. Growth curve of the ADSCs and ADSCs naoencapsulated and conjugated to the lgG1 antibody. Each point on the curve represents the average of 3 samples +/- SEM. ADSC: y = 37.6 e0.2x, k = 0.2, Tau = 5.2, Duplication time = 3, r2 = 0.929. ADSC Conjugated to the antibody; y = 34.7 e0.2x, k = 0.2, Tau = 5, Duplication time = 3, r2 = 0.96.
-Figura^rlnTageTieTde Microscopía a un aumento de 200x correspondientes a ADSC nanoencapsuladas y conjugadas al anticuerpo lgG1 , sometidas a estímulos de diferenciación para adipocito (b) y su control (a). La diferenciación a adipocitos es revelada por la presencia de gotitas de lípidos coloreadas de rojo mediante Oil red. -Figure ^ rlnTageTieTde Microscopy at 200x magnification corresponding to nanoencapsulated ADSC and conjugated to the lgG1 antibody, subjected to differentiation stimuli for adipocyte (b) and its control (a). Adipocyte differentiation is revealed by the presence of droplets of lipids colored red by Oil red.
Figura 8: Imágenes de Microscopía a un aumento de 200x correspondientes a ADSC nanoencapsuladas y conjugadas al anticuerpo lgG1 , sometidas a estímulos de diferenciación para osteoblasto (b) y su control (a). La diferenciación a osteoblastos es revelada por la presencia de depósitos de calcio coloreados de rojo mediante Alizarina roja. Figure 8: Microscopy images at 200x magnification corresponding to nanoencapsulated ADSC and conjugated to the lgG1 antibody, subjected to differentiation stimuli for osteoblast (b) and its control (a). Osteoblast differentiation is revealed by the presence of calcium deposits colored red by red Alizarin.
Figura 9: adhesión y retención específica de células ADSC conjugadsas a anticuerpo CD31 sobre células endoteliales humanas (HUVEC)— Figure 9: Adhesion and specific retention of ADSC cells conjugated to CD31 antibody on human endothelial cells (HUVEC) -
Las imágenes muestran la especificidad con que las células nanoencapsuladas unidas a un anticuerpo específico dirigido a un tejido particular, en este caso endotelio humano, aumenta notablemente la capacidad de las células de fijarse en él y quedar retenidas. En la figura se muestra células madre adiposas nanoencapsuladas a las cuales se les unió un anticuerpo anti-CD31 , el cual reconoce específicamente la molécula PECAM-1 en la superficie de células endoteliales derivadas de cordón umbilical humano. Las imágenes corresponden a fotografías de contraste de fase (panel superior) y fluorescencia (panel inferior). Como controles se usaron células sin nanoencapsular (imágenes a y b); células nanoencapsuladas (imágenes c y d); células nanoencapsuladas unidas a un anticuerpo isotipo (imágenes e y f) y células nanoencapsuladas unidas a un anticuerpo específico anti-CD31 (imágenes g y h). The images show the specificity with which nanoencapsulated cells bound to a specific antibody directed to a particular tissue, in this case human endothelium, remarkably increases the ability of the cells to fix on it and be retained. The figure shows nanoencapsulated fat stem cells to which an anti-CD31 antibody was attached, which specifically recognizes the PECAM-1 molecule on the surface of endothelial cells derived from human umbilical cord. The images correspond to photographs of phase contrast (upper panel) and fluorescence (lower panel). As controls, cells without nanoencapsular (images a and b) were used; nanoencapsulated cells (images c and d); nanoencapsulated cells bound to an isotype antibody (images e and f) and nanoencapsulated cells bound to a specific anti-CD31 antibody (images g and h).
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
Conocidos los antecedentes mencionados previamente y considerando los beneficios que entrega la nanoenGapsulaeión-eelularHa-presente invención describe un diseño de un método de dirección selectiva de células madre mesenquimales provenientes de tejido adiposo nanoencapsuladas destinadas a órganos o tejidos blanco, como una estrategia para hacer posible su administración desde una fuente alogénica y su acumulación o retención selectiva en un tejido específico. De esta manera, el desarrollo de esta tecnología proporciona nuevas herramientas para la ingeniería en tejidos, así como también para novedosas aplicaciones farmacéuticas y biomédicas. Known the previously mentioned background and considering the benefits that nanoenGapsulaeión-eelular deliversHa-present invention describes a design of a method of selective direction of stem cells mesenchymal from nanoencapsulated adipose tissue destined to white organs or tissues, as a strategy to make possible its administration from an allogeneic source and its accumulation or selective retention in a specific tissue. In this way, the development of this technology provides new tools for tissue engineering, as well as for novel pharmaceutical and biomedical applications.
El objetivo de la presente invención consiste en la unión covalente de un anticuerpo específico al polímero utilizado en la última capa de las células madre mesenquimales nanoencapsuladas, con el propósito de dirigir las células administradas en forma sistémica (como una inyección intravenosa de una suspensión celular) a un órgano o tejido específico de tal forma que éstas se acumulen o bien sean retenidas de manera selectiva en dicho órgano o tejido. Los órganos o tejidos a los que se pueden dirigir las células madre mesenquimales se seleccionan de entre otros, hígado, riñon, intestino, páncreas, próstata, pulmón, corazón, bazo, cerebro, articulaciones. La selección del anticuerpo a unir a las células nanoencapsuladas se selecciona dependiendo del órgano en el cual se desea que se acumulen y/o también de la estructura particular de un órgano o tejido al que se desea se fijen preferentemente, por ejemplo parénquima hepático, túbulo proximal o distal en el caso del nefrón, islotes beta del páncreas, etc. En cada caso, la acumulación preferente o selectiva dependerá de la especificidad del anticuerpo unido y del nivel de expresión del antigeno o proteina en dicho órgano o tejido. The objective of the present invention is the covalent binding of a specific antibody to the polymer used in the last layer of nanoencapsulated mesenchymal stem cells, with the purpose of directing the systemically administered cells (such as an intravenous injection of a cell suspension) to a specific organ or tissue such that they accumulate or are selectively retained in said organ or tissue. The organs or tissues to which the mesenchymal stem cells can be directed are selected from among others, liver, kidney, intestine, pancreas, prostate, lung, heart, spleen, brain, joints. The selection of the antibody to be bound to the nanoencapsulated cells is selected depending on the organ in which it is desired that they accumulate and / or also on the particular structure of an organ or tissue to which it is desired to be preferentially fixed, for example hepatic parenchyma, tubule. proximal or distal in the case of the nephron, beta islets of the pancreas, etc. In each case, the preferential or selective accumulation will depend on the specificity of the bound antibody and the level of expression of the antigen or protein in said organ or tissue.
Una descripción breve de las etapas del método de la presente invención, corresponden a: A brief description of the steps of the method of the present invention correspond to:
1. - Nanoencapsulación: las células madre mesenquimales humanas se nanoencapsulan utilizando primero una capa del polielectrolito consistente en polímero de chitosan (policatión). 1. - Nanoencapsulation: human mesenchymal stem cells are nanoencapsulated using first a layer of the polyelectrolyte consisting of chitosan polymer (polycation).
2. Activación de los grupos carboxilos tanto del ácido hialurónico como del condroitin sulfato mediante reactivos químicos para facilitar o permitir la unión o conjugación con el anticuerpo específico. Por lo tanto, la unión entre los grupos funcionales de ácido carboxílico pertenecientes al ácido hialurónico y condroitín sulfato y los grupos funcionales amino de los residuos lisina de la fracción Fe del anticuerpo (Ab) se hace efectiva por la activación de los ácidos carboxílicos mediada por la N-hidroxisulfosuccinimida (NHSS). Esto acelera y mejora el rendimiento de la reacción catalizada posteriormente por 1-etil-3-(3- dimetilaminopropil)carbodiimida (EDAC), la cual es responsable de la unión entre el ácido carboxílico y el grupo amino y que transcurre mediante la formación de un intermediario inestable, la o-acilisourea, que es fácilmente atacada por un grupo amino para formar finalmente la unión entre el anticuerpo y la superficie de la nanocápsula. 2. Activation of the carboxyl groups of both hyaluronic acid and chondroitin sulfate by chemical reagents to facilitate or allow binding or conjugation with the specific antibody. Therefore, the binding between the carboxylic acid functional groups belonging to hyaluronic acid and chondroitin sulfate and the amino functional groups of the lysine residues of the Fe fraction of the antibody (Ab) is made effective by the activation of the carboxylic acids mediated by N-hydroxysulfosuccinimide (NHSS). This accelerates and improves the yield of the reaction subsequently catalyzed by 1-ethyl-3- (3- dimethylaminopropyl) carbodiimide (EDAC), which is responsible for the union between the carboxylic acid and the amino group and which takes place through the formation of an unstable intermediate, o-acylisourea, which is easily attacked by an amino group to finally form the junction between the antibody and the nanocapsule surface.
3. Unión de anticuerpo específico a través de residuos amino libres de las lisinas de la fracción Fe del anticuerpo a los residuos carboxílicos activados tanto del ácido hialurónico como del condroitín sulfato de la etapa anterior. Sobre el residuo carboxilo activado se une un anticuerpo específico, dirigido contra una molécula que se exprese específicamente en el tejido blanco u objetivo, ya sea en condiciones fisiológicas normales o en situaciones patológicas o anormales. 3. Binding of specific antibody through free amino residues of the lysines of the Fe fraction of the antibody to activated carboxylic residues of both hyaluronic acid and chondroitin sulfate from the previous step. On the activated carboxyl residue a specific antibody is bound, directed against a molecule that is specifically expressed in the target or target tissue, either in normal physiological conditions or in pathological or abnormal situations.
En otras palabras lo que sucede es lo siguiente: In other words, what happens is the following:
HA/CS-COOH + NHSS > intermediario 1 + EDAC > intermediario 2HA / CS-COOH + NHSS> intermediary 1 + EDAC> intermediary 2
(o-acilisourea) + -NH2-Ab > HA/CS-CO-NH-Ab (o-acylisourea) + -NH2-Ab> HA / CS-CO-NH-Ab
Alternativamente, las etapas 2 y 3 se pueden realizar de manera simultanea o secuencial. Alternatively, stages 2 and 3 can be performed simultaneously or sequentially.
4. Nanoencapsulación con la capa final de ácido hialurónico/coindritin sulfato que contiene el anticuerpo unido. 5. Unión de las células nanoencapsuladas con anticuerpo específico a las células blanco ya sea en condiciones fisiológicas normales o en situaciones patológicas o anormales. 4. Nanoencapsulation with the final hyaluronic acid / coindritin sulfate layer containing the bound antibody. 5. Union of nanoencapsulated cells with specific antibody to white cells either in normal physiological conditions or in pathological or abnormal situations.
La administración sistémica por vía intravenosa de las células madre mesenquimales con objetivos terapéuticos presenta una amplia distribución en el organismo'40, 4 ), localizándose preferentemente en órganos periféricos como pulmón, bazo, hígado y riñon; y quedando atrapadas en mayor proporción o cantidad en la microvasculatura pulmonar (42). Adicionalmente, de las células que quedan atrapadas en la microvasculatura de los diversos órganos tras una administración intravenosa, una fracción aún más pequeña logra traspasar o escapar o emigrar de la vasculatura para injertar el órgano'43, 4) y para las cuales además existen reportes de que permanecen en el órgano por más de 4 semanas( 3). Es por estas razones que se han estudiado alternativas a la administración sistémica por vía intravenosa con el objetivo de aumentar la proporción o cantidad de células madre administradas que alcancen el órgano blanco. Es así, por ejemplo, que con este propósito se ha intentado la administración intramiocárdica( 5) o intraósea<43), observándose una mayor acumulación de las células administradas en el órgano blanco, pero sin que se altere o afecte la distribución hacia otros órganos periféricos como hígado, bazo y pulmón(45). The intravenous systemic administration of mesenchymal stem cells with therapeutic objectives has a wide distribution in the organism '40, 4) , being preferably located in peripheral organs such as lung, spleen, liver and kidney; and being trapped in greater proportion or quantity in the pulmonary microvasculature (42) . Additionally, of the cells that are trapped in the microvasculature of the various organs after an intravenous administration, an even smaller fraction manages to transfer or escape or migrate from the vasculature to graft the organ '43, 4 ) and for which there are also reports that they remain in the organ for more than 4 weeks (3) . It is for these reasons that alternatives to intravenous systemic administration have been studied in order to increase the proportion or amount of stem cells administered that reach the target organ. Thus, for example, that intramyocardial (5) or intraosseous administration <43) has been attempted for this purpose, observing a greater accumulation of the cells administered in the white organ, but without altering or affecting the distribution to other organs peripherals such as liver, spleen and lung (45) .
De esta manera, la independencia de la ruta de administración en la distribución de las células madre a órganos periféricos permite plantear que la unión no-covalente o covalente de anticuerpos dirigidos contra estructuras específicas presentes en un órgano blanco permitirá una mayor acumulación de las células modificadas en dicho órgano, así como también un mayor injerto ("engraftment") de las mismas en él tras una simple administración sistémica intravenosa, y evitar así una ruta de administración directa en el órgano blanco o en una arteria que lo irriga. Entonces, una vez en el órgano, las células allí acumuladas comenzarían con sus acciones reparadoras del tejido dañado. Es así entonces, que entre los anticuerpos que pueden ser usados con tal propósito podemos mencionar por ejemplo: anti-asialoglicoproteína, antireceptor de arilhidrocarburos, anti-cMet para hígado; anti-von Willebrand, antireceptor de endotelina, anti-angiopoietina, anti-ICAM1 , para endotelio; anticolágeno tipo 2, anti-coindritín-4-sulfato, anti-queratán sulfato para cartílago; anti-VCAM1 para colon y nodulos linfáticos mesentéricos; anti-antígenos tumorales específicos; etc. La lista de ejemplos no es exhaustiva y representa sólo algunos ejemplos. In this way, the independence of the route of administration in the distribution of stem cells to peripheral organs makes it possible to state that the non-covalent or covalent binding of antibodies directed against specific structures present in a white organ will allow a greater accumulation of the modified cells. in said organ, as well as a greater graft ("engraftment") of them in it after a simple intravenous systemic administration, and thus avoid a route of direct administration in the white organ or in an artery that irrigates it. Then, once in the organ, the cells accumulated there would begin with their repair actions of the damaged tissue. Thus, among the antibodies that can be used for this purpose, we can mention for example: anti-asialoglycoprotein, arylhydrocarbon antireceptor, anti-cMet for liver; anti-von Willebrand, endothelin antireceptor, anti-angiopoietin, anti-ICAM1, for endothelium; Type 2 anti-collagen, anti-coindritin-4-sulfate, anti-keratan sulfate for cartilage; anti-VCAM1 for colon and mesenteric lymph nodes; specific tumor antigens; etc. The list of examples is not exhaustive and represents only a few examples.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DETAILED DESCRIPTION OF THE INVENTION
El método de dirección selectiva de células madre mesenquimales provenientes de tejido adiposo nanoencapsuladas destinadas a órganos o tejidos blanco, se describe detalladamente de acuerdo a las 3 etapas principales de: nanoencapsulación, unión de un reactivo bifuncional a las células nanoencapsuladas, y unión de anticuerpo específico al reactivo bifuncional unido a las células nanoencapsuladas. The method of selective targeting of mesenchymal stem cells from nanoencapsulated adipose tissue destined to white organs or tissues, is described in detail according to the 3 main stages of: nanoencapsulation, binding of a bifunctional reagent to nanoencapsulated cells, and binding of specific antibody to the bifunctional reagent bound to the nanoencapsulated cells.
1.- Primera capa de nanoencapsulación Protocolo: 1.- First layer of nanoencapsulation Protocol:
1. Obtener una suspensión de células a partir de una placa o botella de cultivo. 1. Obtain a cell suspension from a plate or culture bottle.
2. Realizar 2 lavados con Hank's balancee! Salt Solution (HBSS) para eliminar la presencia de medio de cultivo, utilizando centrifugación a 200 g por 5 minutos, retirar sobrenadante y resuspender pellet de células en HBSS. Preparación para 1 Litro de HBSS (llevar a volumen final utilizando agua grado Milli Q): 2. Perform 2 washes with Hank's balancee! Salt Solution (HBSS) to eliminate the presence of culture medium, using centrifugation at 200 g for 5 minutes, remove supernatant and resuspend pellet cells in HBSS. Preparation for 1 Liter of HBSS (bring to final volume using Milli Q grade water):
Figure imgf000027_0001
Figure imgf000027_0001
3. Resuspender pellet proveniente del segundo lavado en una solución de Policatión (Chitosan 500 g/mL,bajo peso molecular 448869 Sigma- Aldrich), considerando una relación de volumen de pelletpolicatión de 1 :10. Incubar por 10 minutos a temperatura ambiente. 3. Resuspend pellet from the second wash in a Polycation solution (Chitosan 500 g / mL, low molecular weight 448869 Sigma-Aldrich), considering a pellet volume polycation ratio of 1: 10. Incubate for 10 minutes at room temperature.
4. Centrifugar a 200 g por 5 minutos y retirar el sobrenadante para eliminar el polielectrolito en exceso. 4. Centrifuge at 200 g for 5 minutes and remove the supernatant to remove excess polyelectrolyte.
5. Realizar un lavado con HBSS con un volumen equivalente a 10 veces el volúmen de pellet. - — - - n del anticuerpo y nanoencapsulación final: 1 mL de solución Acido Hialurónico.Condroitín Sulfato 1 :1 de 1 mg/mL utilizando el ácido 2-(N-morfolino)etanolsulfonico (MES) buffer pH 7.4 como solvente a 4°C, adicionar una cantidad de N- hidroxisulfosuccinimida (NHSS) disuelta en MES buffer pH 7.4, suficiente para mantener una concentración final de 1.1 mg/mL. Incubar durante 15 minutos a 4°C. gregar posteriormente a la mezcla 40 uL de anticuerpo de concentración 100 g/mL y una cantidad suficiente de solución 1-etil-3- (3-dimetilaminopropil)carbodiimida (EDAC) disuelta en MES buffer pH 7.4, para que la concentración de EDAC en la mezcla sea de 2 mM o 400 pg/mL Incubar durante 2 horas a 4°C Lavar las células nanoencapsuladas con chitosan con buffer MES pH 7.4, centrifugando a 200 g durante 5 minutos. Eliminar sobrenadante y resuspender el pellet de céluas nanoencapsuladas con chitosan en la solución que contiene Acido Hialurónico, Condroitin Sulfato y el anticuerpo unido. Incubar durante 10 minutos a temperatura ambiente. Realizar dos lavados con HBSS y centrifugar a 200 g durante 5 minutos cada vez. Resuspender las células en un buffer apropiado para ser administradas. 3. Direccionalidad de las células nanoencapsuladas con anticuerpo espécifico a células blanco. 5. Perform a wash with HBSS with a volume equivalent to 10 times the volume of pellet. - - - - n of antibody and final nanoencapsulation: 1 mL of Hyaluronic Acid solution. 1: 1 Chondroitin Sulfate of 1 mg / mL using 2- (N-morpholino) ethanesulfonic acid (MES) buffer pH 7.4 as solvent at 4 ° C, add a amount of N-hydroxysulfosuccinimide (NHSS) dissolved in MES buffer pH 7.4, sufficient to maintain a final concentration of 1.1 mg / mL. Incubate for 15 minutes at 4 ° C. subsequently add to the mixture 40 uL of antibody of concentration 100 g / mL and a sufficient amount of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide solution (EDAC) dissolved in MES buffer pH 7.4, so that the EDAC concentration in the mixture is 2 mM or 400 pg / mL Incubate for 2 hours at 4 ° C Wash the nanoencapsulated cells with chitosan with MES buffer pH 7.4, centrifuging at 200 g for 5 minutes. Remove supernatant and resuspend the pellet of nanoencapsulated cells with chitosan in the solution containing Hyaluronic Acid, Chondroitin Sulfate and the bound antibody. Incubate for 10 minutes at room temperature. Perform two washes with HBSS and centrifuge at 200 g for 5 minutes at a time. Resuspend the cells in an appropriate buffer to be administered. 3. Directionality of nanoencapsulated cells with specific antibody to white cells.
Se consideran las células nanoencapsuladas y unidas al anticuerpo específico según los procedimentos descritos anteriormente y se ponen en contacto con las células blanco elegidas entre aquellas que expresen el antigeno específico para el cual el anticuerpo está dirigido. The nanoencapsulated cells bound to the specific antibody are considered according to the procedures described above and are contacted with the target cells chosen from those that express the specific antigen for which the antibody is directed.
1 . Preparar una suspensión de células que expresan el antigeno. one . Prepare a suspension of cells that express the antigen.
2. Poner en contacto las células del punto 1 , en contacto con las células nanoencapsuladas unidas al anticuerpo específico. 2. Contact the cells of point 1, in contact with the nanoencapsulated cells bound to the specific antibody.
Este método de direccionalidad de las células nanoencapsuladas con anticuerpo espécifico a células blanco, puede ser extrapolado a órganos o tejidos de pacientes con fines terapéuticos This method of directionality of nanoencapsulated cells with specific antibody to white cells can be extrapolated to organs or tissues of patients for therapeutic purposes.
EJEMPLOS Ejemplo 1 : EXAMPLES Example 1:
CONJUGACIÓN DE ANTICUERPO ANTI-CD31 A CÉLULAS ADSC NANOENCAPSULADAS CONJUGATION OF ANTI-CD31 ANTIBODY TO NANOENCAPSULATED ADSC CELLS
Etapas: Stages:
1. Obtener una suspensión de células a partir de una placa o botella de cultivo. 1. Obtain a cell suspension from a plate or culture bottle.
2. Realizar 2 lavados con Hank's balanceó Salt Solution (HBSS) para eliminar la presencia de medio de cultivo, utilizando centrifugación a 200 g por 5 minutos, retirar sobrenadante y resuspender pellet de células en HBSS. Resuspender pellet proveniente del segundo lavado en una solución de Policatión (Chitosan 500 pg/mL,bajo peso molecular 448869 Sigma-Aldrich), considerando una relación de volumen de pelletpolicatión de 1 :10. Incubar por 10 minutos a temperatura ambiente. Centrifugar a 200 g por 5 minutos y retirar el sobrenadante para eliminar el polielectrolito en exceso. Realizar un lavado con HBSS con un volúmen equivalente a 10 veces el volúmen de pellet. A 1 ml_ de solución Acido Hialurónico:Condroitín Sulfato 1 :1 de 1 mg/mL utilizando el ácido 2-(N-morfolino)etanolsulfonico (MES) buffer pH 7.4 como solvente a 4°C, adicionar una cantidad de N- hidroxisulfosuccinimida (NHSS) disuelta en MES buffer pH 7.4, suficiente para mantener una concentración final de 1.1 mg/mL. Incubar durante 15 minutos a 4°C. Agregar posteriormente a la mezcla 40 uL de anticuerpo ANTI-CD31 (EJEMPLO ESPECIFICO QUE LUEGO SE DESCRIBIRA EN EJEMPLO 3) de concentración 100 pg/mL y una cantidad suficiente de solución 1-etil-3-(3-dimetilaminopropil)carbodiimida (EDAC) disuelta en MES buffer pH 7.4, para que la concentración de EDAC en la mezcla sea de 2 mM o 400 pg/mL. Incubar durante 2 horas a 4°C Lavar las células nanoencapsuladas con chitosan con buffer MES pH 7.4, centrifugando a 200 g durante 5 minutos. Eliminar sobrenadante y resuspender el pellet de céluas nanoencapsuladas con chitosan en . la solución que contiene Acido Hialurónico, Condroitin Sulfato y el anticuerpo unido. 12. Incubar durante 10 minutos a temperatura ambiente. 2. Perform 2 washes with Hank's balanced Salt Solution (HBSS) to eliminate the presence of culture medium, using centrifugation at 200 g for 5 minutes, remove supernatant and resuspend pellet cells in HBSS. Resuspend pellet from the second wash in a Polycation solution (Chitosan 500 pg / mL, low molecular weight 448869 Sigma-Aldrich), considering a pellet volume polycation ratio of 1: 10. Incubate for 10 minutes at room temperature. Centrifuge at 200 g for 5 minutes and remove the supernatant to remove excess polyelectrolyte. Perform a wash with HBSS with a volume equivalent to 10 times the volume of pellet. To 1 ml_ of solution Hyaluronic Acid: Chondroitin Sulfate 1: 1 of 1 mg / mL using 2- (N-morpholino) ethanesulfonic acid (MES) buffer pH 7.4 as solvent at 4 ° C, add an amount of N-hydroxysulfosuccinimide ( NHSS) dissolved in MES buffer pH 7.4, sufficient to maintain a final concentration of 1.1 mg / mL. Incubate for 15 minutes at 4 ° C. Then add to the mixture 40 uL of ANTI-CD31 antibody (SPECIFIC EXAMPLE THEN DESCRIBED IN EXAMPLE 3) of concentration 100 pg / mL and a sufficient amount of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide solution (EDAC) dissolved in MES buffer pH 7.4, so that the concentration of EDAC in the mixture is 2 mM or 400 pg / mL. Incubate for 2 hours at 4 ° C. Wash the nanoencapsulated cells with chitosan with MES buffer pH 7.4, centrifuging at 200 g for 5 minutes. Remove supernatant and resuspend the pellet of nanoencapsulated cells with chitosan in. the solution containing Hyaluronic Acid, Chondroitin Sulfate and the bound antibody. 12. Incubate for 10 minutes at room temperature.
13. Realizar dos lavados con HBSS y centrifugar a 200 g durante 5 minutos cada vez. 13. Perform two washes with HBSS and centrifuge at 200 g for 5 minutes at a time.
14. Resuspender las células en un buffer apropiado para ser administradas. 14. Resuspend the cells in an appropriate buffer to be administered.
Ejemplo 2: Example 2:
Determinación de la conjugación del anticuerpo a las células mediante microscopía de fluorescencia y citometrá de flujo. Determination of antibody conjugation to cells by fluorescence microscopy and flow cytometry.
Para comprobar la unión específica de un anticuerpo a la superficie de una célula nanoencapsulada se utilizó un anticuerpo isotipo del tipo lgG1 conjugado a FITC (Isotiociacianato de fluoresceina). To verify the specific binding of an antibody to the surface of a nanoencapsulated cell, an isotype antibody of the IgG1 conjugated lgG1 type (Fluorescein isothiocyanacyte) was used.
1. A 1 ml_ de solución Acido Hialurónico:Condroitín Sulfato 1 :1 de 1 mg/mL utilizando el ácido 2-(N-morfolino)etanolsulfonico (MES) buffer pH 7.4 como solvente a 4°C, adicionar una cantidad de N- hidroxisulfosuccinimida (NHSS) disuelta en MES buffer pH 7.4, suficiente para mantener una concentración final de 1.1 mg/mL. 1. At 1 ml_ of solution Hyaluronic Acid: Chondroitin Sulfate 1: 1 of 1 mg / mL using 2- (N-morpholino) ethanesulfonic acid (MES) buffer pH 7.4 as solvent at 4 ° C, add an amount of N- Hydroxysulfosuccinimide (NHSS) dissolved in MES buffer pH 7.4, sufficient to maintain a final concentration of 1.1 mg / mL.
2. Incubar durante 15 minutos a 4°C. 2. Incubate for 15 minutes at 4 ° C.
3. Agregar posteriormente a la mezcla 40 uL del anticuerpo, lgG1-FITC (fluoesceína isotioscianato), a una concentración 100 pg/mL y una cantidad suficiente de solución 1-etil-3-(3- dimetilaminopropil)carbodiimida (EDAC) disuelta en MES buffer pH 7.4, para que la concentración de EDAC en la mezcla sea de 2 mM o 400 pg/mL. 3. Subsequently add to the mixture 40 uL of the antibody, lgG1-FITC (fluoscein isothioscyanate), at a concentration of 100 pg / mL and a sufficient amount of 1-ethyl-3- (3- dimethylaminopropyl) carbodiimide solution (EDAC) dissolved in MES buffer pH 7.4, so that the EDAC concentration in the mixture is 2 mM or 400 pg / mL.
4. Incubar durante 2 horas a-4°C . _ - - . 5. Lavar las células nanoencapsuladas con chitosan con buffer MES pH 7.4, centrifugando a 200 g durante 5 minutos, de acuerdo a como se obtuvieron en la etapa 3 del Ejemplo 1.. 4. Incubate for 2 hours at 4 ° C-. _ - - . 5. Wash the nanoencapsulated cells with chitosan with MES buffer pH 7.4, centrifuging at 200 g for 5 minutes, according to how they were obtained in step 3 of Example 1 ..
6. Eliminar sobrenadante y resuspender el pellet de céluas nanoencapsuladas con chitosan en la solución que contiene Acido Hialurónico, Condroitin Sulfato y el anticuerpo unido. 6. Remove supernatant and resuspend the pellet of nanoencapsulated cells with chitosan in the solution containing Hyaluronic Acid, Chondroitin Sulfate and the bound antibody.
7. Incubar durante 10 minutos a temperatura ambiente. 7. Incubate for 10 minutes at room temperature.
8. Realizar dos lavados con HBSS y centrifugar a 200 g durante 5 minutos cada vez. 8. Perform two washes with HBSS and centrifuge at 200 g for 5 minutes at a time.
9. Resuspender las células en un buffer apropiado. 9. Resuspend the cells in an appropriate buffer.
Las células obtenidas en esta etapa son analizadas por dos métodos: Microscopía de fluorescencia y citometría de flujo. The cells obtained at this stage are analyzed by two methods: Fluorescence microscopy and flow cytometry.
Ejemplo 3 Example 3
Ejemplo de utilización de células nanoencapsuladas unidas a anticuerpo anti- CD31 dirigidas a tejido endotelial humano y observación de una mayor adhesión o "engraftment" específico de las células Example of use of nanoencapsulated cells bound to anti-CD31 antibody directed to human endothelial tissue and observation of increased adhesion or specific "engraftment" of the cells
1. Se sembraron 2 x 105 ADSCs en placas de 24 pocilios un día previo al estudio, para formar una alfombra de células en cada pocilio. Se crearon las siguientes 4 condiciones: 1. 2 x 10 5 ADSCs were seeded in 24-well plates one day prior to the study, to form a carpet of cells in each well. The following 4 conditions were created:
Condición 1 : ADSCs Condition 1: ADSCs
Condición 2: ADSCs Nanoencapsuladas Condition 2: Nanoencapsulated ADSCs
Condición 3: ADSCs Nanoencapsuladas y conjugadas al anticuerpo IgG-Condition 3: Nanoencapsulated and conjugated to IgG- antibody ADSCs
1 Condición 4: ADSCs Nanoencapsuladas y conjugadas al anticuerpoone Condition 4: Nanoencapsulated and antibody conjugated ADSCs
CD31 CD31
2. Al día siguiente, se preparó una suspensión de 1 x 105 HUVECs marcadas (células endoteliales humanas derivadas de cordón umbilical) con TAMRA para cada condición. Para ello se tripsinizó una botella de cultivo con 1 x 106 HUVECs y se lavó 2 veces con PBS. A continuación se realizó el mareaje de estas células en 200 uL de una solución de 200 uM de TAMRA en PBS, incubándolas a 37°C durante 10 minutos. Se lavó 2 veces con PBS. Finalmente, se resuspendió el pellet celular en una solución de PBS + 0,5% BSA + 1mM EDTA. 2. The next day, a suspension of 1 x 10 5 labeled HUVECs (human endothelial cells derived from umbilical cord) was prepared with TAMRA for each condition. For this, a culture bottle was trypsinized with 1 x 10 6 HUVECs and washed twice with PBS. Subsequently, the marking of these cells was carried out in 200 uL of a solution of 200 uM of TAMRA in PBS, incubating them at 37 ° C for 10 minutes. It was washed 2 times with PBS. Finally, the cell pellet was resuspended in a solution of PBS + 0.5% BSA + 1mM EDTA.
3. Se realizó la nanoencapsulación de ADSC y conjugación de anticuerpo anti-CD31 , según condiciones. Antes de realizar el procedimiento de recubrimiento celular, se realizó la conjugación de los anticuerpos a los componentes de la capa exterior y de la segunda capa. Para ello: 3. ADSC nanoencapsulation and anti-CD31 antibody conjugation was performed, according to conditions. Before performing the cell coating procedure, the antibody was conjugated to the components of the outer layer and the second layer. For it:
A 1 mL de una solución de Acido Hialurónico: Condroitin sulfato 1 :1 de 1 mg/mL disuelto en buffer MES a pH 7,4; se agregó la cantidad suficiente de una solución de NHSS (disuelto en buffer MES a pH 7,4) para mantener una concentración final de 1.1 mg/mL. Se incubó durante 15 minutos a 4°C. At 1 mL of a solution of Hyaluronic Acid: Chondroitin sulfate 1: 1 of 1 mg / mL dissolved in MES buffer at pH 7.4; Sufficient amount of an NHSS solution (dissolved in MES buffer at pH 7.4) was added to maintain a final concentration of 1.1 mg / mL. It was incubated for 15 minutes at 4 ° C.
A continuación, se agregó 40 uL del anticuerpo IgG a la condición 3, y 40 uL del anticuerpo CD31 para la condición 4 e inmediatamente a ambas se agregó la cantidad suficiente de una solución de EDAC (disuelta en buffer MES a pH 7,4) para mantener una concentración final de 2 mM (400 pg/mL). Se Incubó durante 2 horas a 4°C. Next, 40 uL of the IgG antibody was added to condition 3, and 40 uL of the CD31 antibody for condition 4, and immediately enough of an EDAC solution (dissolved in MES buffer at pH 7.4) was added to both. to maintain a final concentration of 2 mM (400 pg / mL). It was incubated for 2 hours at 4 ° C.
4. Paralelamente, se realizaron dos lavados de las células de cada pocilio, con HBSS. Posteriormente a las condiciones 2, 3 y 4 se agregó 200 uL de solución de Chitosan 500 pg/mL disuelto en HBSS pH 6,5 y se incubó durante 10 minutos a temperatura ambiente. Se realizaron dos lavados con HBSS. 5. Sólo para la condición 2, se agregó 200 uL de una solución de Acido Hialurónico: Condroitin sulfato 1 :1 de 1 mg/mL disuelto en HBSS pH 7,4. Se incubó durante 10 minutos a temperatura ambiente. 4. In parallel, two washes of the cells of each well were performed, with HBSS. Subsequently to conditions 2, 3 and 4, 200 uL of Chitosan solution 500 pg / mL dissolved in HBSS pH 6.5 was added and incubated for 10 minutes at room temperature. Two washes were performed with HBSS. 5. Only for condition 2, 200 uL of a solution of Hyaluronic Acid: Chondroitin sulfate 1: 1 of 1 mg / mL dissolved in HBSS pH 7.4 was added. It was incubated for 10 minutes at room temperature.
6. Después de 2 horas de incubación, se agregó 200 uL de las soluciones de polianiones conjugados a anticuerpos IgG y CD31 a las condiciones 3 y 4, respectivamente. Se incubó durante 10 minutos a temperatura ambiente. 6. After 2 hours of incubation, 200 uL of the conjugated polyanion solutions to IgG and CD31 antibodies were added to conditions 3 and 4, respectively. It was incubated for 10 minutes at room temperature.
7. Se lavaron todos los posillos dos veces con HBSS. Posteriormente, se agregó 200 uL de una solución de PBS + 0,5% BSA + 1 mM EDTA. Se incubó durante 10 minutos a temperatura ambiente. 7. All the posts were washed twice with HBSS. Subsequently, 200 uL of a solution of PBS + 0.5% BSA + 1 mM EDTA was added. It was incubated for 10 minutes at room temperature.
8. Se retiró el sobrenadante a todos los pocilios y se agregó a cada uno, una suspensión de 1 x 105 HUVECs en 200 uL. Se incubó durante 30 minutos a 4°C. Se realizaron 3 lavados con una solución de PBS + 0,5% BSA + 1 mM EDTA. 8. The supernatant was removed from all wells and a suspension of 1 x 10 5 HUVECs in 200 uL was added to each. It was incubated for 30 minutes at 4 ° C. 3 washes were performed with a solution of PBS + 0.5% BSA + 1 mM EDTA.
9. Se observaron HUVECs adheridas mediante microscopía de fluorescencia filtro rojo y a campo claro para ADSC, en sus diferentes condiciones. 9. Adhered HUVECs were observed by red and light field fluorescence filter microscopy for ADSC, under their different conditions.
Discusión Discussion
El gran desarrollo de conocimiento acerca de la biología de las células madre se ha aplicado a la reparación de diversos tejidos. Sin embrago, la entrega precisa de estas células reparadoras en el tejido dañado o en el sitio de la lesión representa un aspecto fundamental de cualquier estrategia de terapia celular o ingeniería de tejidos basada en las células madre. Particularmente, hoy las células madre mesenquimales (MSC) representan una alternativa muy prometedora para la terapia celular. Tal como su nombre lo sugiere, originalmente las MSC se caracterizaron en función de su capacidad para diferenciarse en grasa, hueso y cartílago, linajes celulares derivados del mesénquima; y por lo tanto la idea inicial para un uso terapéutico se dirigió hacia la reconstrucción o reparación de estos tejidos. Sin embargo, debido a los resultados obtenidos en diversos estudios preclínicos, que demostraron que una recuperación funcional del tejido sucedía en una gran variedad de tejidos después de inyectar las MSC, sin que se observara una mayor diferenciación de las MSC inyectadas en células propias del tejido reparado, condujo a proponer que, in vivo, las MSC poseen mecanismos de acción alternativos para ejercer su efecto reparador en el tejido dañado. Es así, que hoy día se acepta que las MSC estimulan la recuperación y la regeneración a través de la producción y secreción de numerosos factores pro-regenerativos como por ejemplo, factores anti-inflamatorios, angiogénicos, neurotróficos, inmunomoduladores y antifibróticos ( 6). De esta manera, resulta evidente que desarrollar un sistema de disposición sistémica efectivo de estas células en el sitio específico a ser reparado o donde se ubica la lesión, o cualquier órgano o tejido de interés terapéutico, tendrá un enorme impacto en la utilidad terapéutica de las células madre mesenquimales, o bien de cualquier tipo celular que ejerza un efecto reparador o terapéutico (ejemplo, células del sistema inmune capaces de ser dirigidas contra un tumor). The great development of knowledge about the biology of stem cells has been applied to the repair of various tissues. However, the precise delivery of these repair cells in the damaged tissue or at the site of the lesion represents a fundamental aspect of any cell therapy or tissue engineering strategy based on the stem cells. Particularly, today mesenchymal stem cells (MSC) represent a very promising alternative for cell therapy. As the name suggests, the MSCs were originally characterized by their ability to differentiate into fat, bone and cartilage, cell lineages derived from the mesenchyme; and therefore the initial idea for therapeutic use was addressed towards the reconstruction or repair of these tissues. However, due to the results obtained in various preclinical studies, which demonstrated that a functional recovery of the tissue occurred in a wide variety of tissues after injecting the MSCs, without observing a greater differentiation of the MSCs injected into the tissue's own cells. repaired, it led to propose that, in vivo, MSCs have alternative mechanisms of action to exert their reparative effect on damaged tissue. Thus, it is now accepted that MSCs stimulate recovery and regeneration through the production and secretion of numerous pro-regenerative factors such as, for example, anti-inflammatory, angiogenic, neurotrophic, immunomodulatory and antifibrotic factors (6) . In this way, it is evident that developing an effective systemic arrangement of these cells at the specific site to be repaired or where the lesion is located, or any organ or tissue of therapeutic interest, will have an enormous impact on the therapeutic utility of mesenchymal stem cells, or of any cell type that exerts a reparative or therapeutic effect (eg, immune system cells capable of being directed against a tumor).
Aunque las MSC ya han demostrado un claro potencial terapéutico, actualmente existen algunos aspectos relativos a su uso que requieren ser optimizados. Actualmente, los estudios clínicos que involucran MSC utilizan una de dos vía de administración: a) inyección intravenosa de un bolus grande de células, o b) una inyección directa en el tejido afectado. Al respecto, ninguna de estas aproximaciones es óptima. La primera implica exponer al paciente a grandes dosis de células (entre 1 y 5 millones de células por Kg de peso) circulando por todo el cuerpo, con el inconveniente de altos costos de producción implicados y potenciales riesgos de efectos secundarios; mientras que el segundo método implica procedimientos de infusión invasivos y costosos. En este sentido, se han desarrollado nuevos estudios que han intentado optimizar la terapia basada en MSC ya sea aumentando su potencia o aumentando su entrega o disposición en los tejidos blanco. Entre las estrategias de optimización de la terapia con MSC, o células en general, encontramos: i) aumento de la secreción de factores de crecimiento mediante transgénesis, ii) predisposición o "priming" de las células para mejorar la supervivencia o función postinoculación, iii) aumentar la destinación o "homing" utilizando mecanismos endógenos y iv) apuntar o "targeting" a un tejido específico usando señales o pistas tejido-específicas ( 7). Although MSCs have already demonstrated clear therapeutic potential, there are currently some aspects related to their use that need to be optimized. Currently, clinical studies involving MSC use one of two routes of administration: a) intravenous injection of a large bolus of cells, or b) a direct injection into the affected tissue. In this regard, none of these approaches is optimal. The first involves exposing the patient to large doses of cells (between 1 and 5 million cells per kg of weight) circulating throughout the body, with the inconvenience of high production costs involved and potential risks of side effects; while the second method involves invasive and expensive infusion procedures. In this sense, new studies have been developed that have tried to optimize MSC-based therapy either by increasing its potency or increasing its delivery or disposition in white tissues. Among the strategies for optimizing therapy with MSC, or cells in general, we find: i) increased secretion of growth factors through transgenesis, ii) predisposition or "priming" of cells to improve survival or post-inoculation function, iii ) increase the destination or "homing" using endogenous mechanisms and iv) point or "targeting" to a specific tissue using tissue-specific signals or clues (7) .
De estas cuatro estrategias, la que tiene relación con la presente invención es la de "targeting" a un tejido específico. Actualmente, se han descrito diversas aproximaciones entre las cuales encontramos por ejemplo, la modificación enzimática del receptor de superficie CD44 de las MSC en un dominio de unión de E-selectina(48) y el acoplamiento de anticuerpos tejido específicos sobre la superficie celular(49). Para el acoplamiento de anticuerpos tejido-específicos encontramos dos alternativas descritas. La primera usa proteína G palmitoilada, lo que permite su fijación a la membrana celular y posteriormente el acoplamiento de un anticuerpo, por un mecanismo de afinidad, a la proteína Q(5O, 51) £| segunc|0 método describe un acoplamiento mixto que incluye uniones de tipo covalente y por afinidad, que utiliza biotina y streptavidina y ligandos biotinilados(52) . Of these four strategies, the one related to the present invention is that of "targeting" to a specific tissue. Currently, various approaches have been described, among which we find, for example, the enzymatic modification of the CD44 surface receptor of the MSCs in an E-selectin binding domain (48) and the coupling of specific tissue antibodies on the cell surface (49 ) . For the coupling of tissue-specific antibodies we find two alternatives described. The first uses palmitoylated G protein, which allows its attachment to the cell membrane and subsequently the coupling of an antibody, by an affinity mechanism, to the Q protein (5O, 51) £ | is unc | The method describes a mixed coupling that includes covalent and affinity type junctions, which uses biotin and streptavidin and biotinylated ligands (52) .
En la presente invención se describe un método nuevo y distinto de acoplamiento de anticuerpos tejido-específicos sobre la superficie celular por medio de unión covalente (figuras 2 y 3). Para ello se hace uso de una estrategia que utiliza la nanoencapsulación de la MSC mediante polímeros biocompatibles (quitosano, ácido hialurónico y condroitin sulfato) a los cuales se les une de manera covalente un anticuerpo tejido-específico a la superficie celular de la MSC, mediante el uso de reactivos químicos que permiten o facilitan esta unión sirviendo como catalizadores al activar los residuos carboxílicos presentes en el polianión. In the present invention a new and different method of coupling tissue-specific antibodies on the cell surface by means of covalent binding is described (Figures 2 and 3). For this, a strategy is used that uses the nanoencapsulation of the MSC by means of biocompatible polymers (chitosan, hyaluronic acid and chondroitin sulfate) to which a tissue-specific antibody is covalently attached to the cell surface of the MSC, by the use of chemical reagents that allow or facilitate this binding by serving as catalysts by activating the carboxylic residues present in the polyanion.
Entre las características y ventajas que representa el método descrito en la presente invención tenemos que utiliza- polímeros- biocompatibles-no tóxicos que no alteran la viabilidad (figuras 4 y 5) y función celular, dado que nuestros resultados demuestran que sus características de proliferación y de multipotencialidad de las MSC no se alteran (figuras 6 y 7, respectivamente). Esto quiere decir, que las MSC son capaces de diferenciarse a adipocito y osteoblasto cuando se encuentran nanoencapsuladas y con el anticuerpo conjugado. Adicionalmente es un método sencillo de desarrollar y compatible con una producción masiva o a mayor escala, dado que su viabilidad se mantiene elevada durante el proceso de nanoencapsulación y posterior conjugación del anticuerpo (figuras 4 y 5, respectivamente). Among the characteristics and advantages of the method described in the present invention, we have to use biocompatible-non-toxic polymers that do not alter viability (Figures 4 and 5) and cellular function, since our Results show that their proliferation and multipotentiality characteristics of MSCs are not altered (Figures 6 and 7, respectively). This means that MSCs are able to differentiate into adipocyte and osteoblast when they are nanoencapsulated and with the conjugated antibody. Additionally, it is a simple method of developing and compatible with a massive or large-scale production, given that its viability remains high during the nanoencapsulation process and subsequent conjugation of the antibody (Figures 4 and 5, respectively).
Entonces, considerando la independencia de la ruta de administración en la distribución de las células madre a órganos periféricos, nos permite plantear que la presente invención (unión covalente de anticuerpos dirigidos contra estructuras específicas presentes en un órgano blanco) permitirá una mayor acumulación de las células modificadas en dicho órgano, así como también un mayor injerto ("engraftment") de las mismas en él, tras una simple administración sistémica intravenosa, y evitar así una ruta de administración directa en el órgano blanco o en una arteria que lo irriga. Then, considering the independence of the route of administration in the distribution of stem cells to peripheral organs, it allows us to state that the present invention (covalent binding of antibodies directed against specific structures present in a white organ) will allow a greater accumulation of cells modified in said organ, as well as a greater graft ("engraftment") of them in it, after a simple intravenous systemic administration, and thus avoid a route of direct administration in the white organ or in an artery that irrigates it.
Esto se demuestra claramente en los resultados obtenidos en la Figura 9 donde se observa una mayor adhesión y retención (f) específica de células ADSC conjugadas a anticuerpo CD31 sobre células endoteliales humanas (HUVEC) con respecto al control de células no nanoencapsuladas (b). This is clearly demonstrated in the results obtained in Figure 9 where there is greater adhesion and specific retention (f) of ADSC cells conjugated to CD31 antibody on human endothelial cells (HUVEC) with respect to the control of non-nanoencapsulated cells (b).
Las principales utilidades de esta metodología, consisten en poder dirigir selectivamente y específicamente células a un tumor con el objetivo de atacarlo y destruirlo selectivamente mediante alguna estrategia conocida por el estado del arte. The main utilities of this methodology consist in being able to selectively and specifically direct cells to a tumor with the objective of attacking and selectively destroying it by means of some strategy known by the state of the art.
En el caso de las células madre, encontramos la posibilidad de dirigir selectivamente y específicamente estas células a tejidos u órganos dañados permitiendo una mayor acumulación o "engraftment" de las células en el tejido para que puedan ejercer su efecto reparador. A modo de ejemplo, podemos considerar la insuficiencia renal crónica, el daño hepático por cualquier causa, el daño pulmonar debido a enfermedad obstructiva crónica (EPOC), la insuficiencia cardíaca e infarto agudo de miocardio, enfermedad inflamatoria intestinal, enfermedades inflamatorias vasculares, enfermedades de las articulaciones como por ejemplo artrosis o artritis, enfermedades neurodegenerativas como por ejemplo enfermedad de Parkinson, Alzheimer, etc. In the case of stem cells, we find the possibility of selectively and specifically directing these cells to damaged tissues or organs allowing greater accumulation or "engraftment" of the cells in the tissue so that they can exert their reparative effect. As an example, we can consider chronic renal failure, liver damage from any cause, lung damage due to chronic obstructive disease (COPD), heart failure and acute myocardial infarction, inflammatory bowel disease, inflammatory vascular diseases, joint diseases such as osteoarthritis or arthritis, neurodegenerative diseases such as Parkinson's disease, Alzheimer's , etc.
La presente invención puede ser aplicada a cualquier tipo de célula que se quiera dirigir a un blanco específico, sea un tejido dañado o un tumor. Las células a utilizar pueden ser seleccionadas entre células madre mesenquimales, células madre hematopoyéticas, células progenitoras endoteliales, células madre pluripotenciales inducidas o iPS, células madre embrionarias, linfocitos T CD4, linfocitos T CD8, linfocitos T regulatorios, células Natural Killer o NK, monocitos/macrófagos; y los órganos blanco incluyen hígado, corazón, pulmón, riñon, páncreas, cerebro, intestino y colon, endotelio vascular, articulaciones y vasos sanguíneos en general y tumores. The present invention can be applied to any type of cell that wants to target a specific target, be it a damaged tissue or a tumor. The cells to be used can be selected from mesenchymal stem cells, hematopoietic stem cells, endothelial progenitor cells, induced pluripotential stem cells or iPS, embryonic stem cells, CD4 T lymphocytes, CD8 T lymphocytes, regulatory T lymphocytes, Natural Killer or NK cells, monocytes / macrophages; and white organs include liver, heart, lung, kidney, pancreas, brain, intestine and colon, vascular endothelium, joints and blood vessels in general and tumors.
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Claims

REIVINDICACIONES
1. Método de direccionamiento celular de células a tejidos u órganos específicos, CARACTERIZADO porque comprende las etapas de: 1. Method of cell targeting of cells to specific tissues or organs, CHARACTERIZED because it comprises the steps of:
i) Nanoencapsulación de células, con una primera capa que comprende un policatión;  i) Nanoencapsulation of cells, with a first layer comprising a polycation;
ii) Activación de los grupos carboxilos del polianión, mediante reactivos químicos que facilitan o permiten la unión o conjugación del anticuerpo específico; y en que el polianión está comprendido dentro de la capa final de nanoencapsulación;  ii) Activation of the carboxylic groups of the polyanion, by chemical reagents that facilitate or allow the binding or conjugation of the specific antibody; and in which the polyanion is comprised within the final nanoencapsulation layer;
iii) Unión de anticuerpo específico a los residuos carboxílicos activados del polianión;  iii) Specific antibody binding to the activated carboxylic residues of the polyanion;
iv) Nanoencapsulación de las células nanoencapsuladas de la etapa i) con la capa final que comprende el anticuerpo específico y el polianión resultante de la etapa iii).  iv) Nanoencapsulation of the nanoencapsulated cells of step i) with the final layer comprising the specific antibody and the polyanion resulting from step iii).
v) Unión de la célula nanoencapsulada de la etapa iv) a una célula blanco;  v) Union of the nanoencapsulated cell of step iv) to a white cell;
en que las etapas ii) y iii) pueden ser simultáneas o secuenciales in which stages ii) and iii) can be simultaneous or sequential
2. Método de acuerdo a la reivindicación 1 , CARACTERIZADO porque las células a ser nanoencapsuladas se seleccionan de células madres mesenquimales humanas o de animales. 2. Method according to claim 1, CHARACTERIZED in that the cells to be nanoencapsulated are selected from human or animal mesenchymal stem cells.
3. Método de acuerdo a la reivindicación 2, CARACTERIZADO porque las células tienen un fenotipo mínimo, que es: positivo a CD105, CD90, CD73 y negativo para CD14, CD19, CD45 y HLA-DR. 3. Method according to claim 2, CHARACTERIZED because the cells have a minimum phenotype, which is: positive for CD105, CD90, CD73 and negative for CD14, CD19, CD45 and HLA-DR.
4. Método de acuerdo a la reivindicación 1 , CARACTERIZADO porque la etapa i) de unión del policatión para la nanoencapsulación comprende: 4. Method according to claim 1, CHARACTERIZED in that step i) of polycation bonding for nanoencapsulation comprises:
obtener una suspensión de células seleccionadas; obtain a suspension of selected cells;
lavar y centrifugar las células para eliminar la presencia de residuos; wash and centrifuge the cells to eliminate the presence of residues;
resuspender e incubar el pellet proveniente de la etapa anterior con una solución de un policatión; resuspend and incubate the pellet from the previous stage with a solution of a polycation;
centrifugar la suspensión de la etapa anterior y eliminar el sobrenadante; lavar las células nanoencapsuladas con el policatión. centrifuge the suspension from the previous stage and remove the supernatant; wash the nanoencapsulated cells with the polycation.
5. Método de acuerdo a la reivindicación 4, CARACTERIZADO porque el policatión utilizado para la primera capa de nanoencapsulacion se selecciona de cloruro de poli(alilamina) (PAH) y/o cloruro de poli(dialildimetilamonio) (PDADMAC) y/o Chitosan (Ch) y/o poli-L-lisina (PLL). 5. Method according to claim 4, CHARACTERIZED in that the polycation used for the first nanoencapsulation layer is selected from poly (allylamine) chloride (PAH) and / or poly (diallyldimethylammonium) chloride (PDADMAC) and / or Chitosan ( Ch) and / or poly-L-lysine (PLL).
6. Método de acuerdo a la reivindicación 5, CARACTERIZADO porque el policatión es Chitosan (Ch). 6. Method according to claim 5, CHARACTERIZED because the polycation is Chitosan (Ch).
7. Método de acuerdo a la reivindicación 1 , CARACTERIZADO porque el polianión utilizado para la capa final de nanoencapsulacion se selecciona de poli(estireno) sulfonato de sodio (PSS) y/o ácido hialurónico (HA) y/o condroitin sulfato (CS). 7. Method according to claim 1, CHARACTERIZED in that the polyanion used for the final nanoencapsulation layer is selected from sodium poly (styrene) sulphonate (PSS) and / or hyaluronic acid (HA) and / or chondroitin sulfate (CS) .
8. Método de acuerdo a la reivindicación 7, CARACTERIZADO porque el polianión utilizado para la capa final de nanoencapsulacion se selecciona de ácido hialurónico (HA) y/o condroitin sulfato (CS). 8. Method according to claim 7, CHARACTERIZED in that the polyanion used for the final nanoencapsulation layer is selected from hyaluronic acid (HA) and / or chondroitin sulfate (CS).
9. Método de acuerdo a la reivindicación 1 , CARACTERIZADO porque los reactivo químicos se seleccionan de N-hidroxisulfosuccinimida (NHSS) y 1-etil- 3-(3-dimetilaminopropil)carbodiimida (EDAC). 9. Method according to claim 1, CHARACTERIZED in that the chemical reagents are selected from N-hydroxysulfosuccinimide (NHSS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC).
10. Método de acuerdo a la reivindicación 1 , CARACTERIZADO porque las etapas ii) y iii) de unión del reactivo bifuncional al polianión, y unión a un anticuerpo comprende: 10. Method according to claim 1, CHARACTERIZED in that steps ii) and iii) of binding the bifunctional reagent to the polyanion, and binding to an antibody comprises:
i) mezclar una solución de polianiones, que comprende ácido hialurónico:condroitín sulfato 1 :1 de 1 mg/mL en buffer MES (ácido 2-(N- morfolino)etanolsulfonico), pH 7,4 a 4°C, con el reactivo N- hidroxisulfosuccinimida (NHSS); i) mixing a solution of polyanions, comprising hyaluronic acid: 1: 1 chondroitin sulfate of 1 mg / mL in MES buffer (2- (N-morpholino) ethanesulfonic acid), pH 7.4 at 4 ° C, with the reagent N-hydroxysulfosuccinimide (NHSS);
ii) Incubar durante 15 minutos a 4°C; iii) Agregar a la mezcla anterior 40 uL de anticuerpo seleccionado (100 pg/mL) en presencia de una solución 1-etil-3-(3-dimetilaminopropil)carbodiimida (EDAC) disuelta en buffer MES pH 7.4; ii) Incubate for 15 minutes at 4 ° C; iii) Add to the previous mixture 40 uL of selected antibody (100 pg / mL) in the presence of a 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC) solution dissolved in MES buffer pH 7.4;
iv) incubar durante 2 horas a 4°C. iv) incubate for 2 hours at 4 ° C.
11. Método de acuerdo a la reivindicación 1 , CARACTERIZADO porque la etapa final de nanoencapsulación y unión a un anticuerpo iv) y v) comprende: proveer las células nanoencapsuladas con chitosan de acuerdo a la reivindicación 6 y mezclar con la solución que contiene el ácido hialurónico, el condroitín sulfato y el anticuerpo unido, obtenido de acuerdo a la reivindicación 10. 11. Method according to claim 1, CHARACTERIZED in that the final stage of nanoencapsulation and binding to an antibody iv) and v) comprises: providing the nanoencapsulated cells with chitosan according to claim 6 and mixing with the solution containing the hyaluronic acid , chondroitin sulfate and bound antibody, obtained according to claim 10.
12. Método de direccionamiento celular de células a tejidos u órganos específicos, CARACTERIZADO porque comprende dirigir células nanoencapsuladas unidas a anticuerpos específicos con capacidad de unión a antígenos de células de tejidos u órganos, que presentan deficiencias fisiológicas y/o fisiopatológicas. 12. Method of cell targeting of cells to specific tissues or organs, CHARACTERIZED because it comprises directing nanoencapsulated cells bound to specific antibodies capable of binding to tissue or organ cell antigens, which have physiological and / or pathophysiological deficiencies.
PCT/CL2013/000003 2012-01-18 2013-01-17 Method for selective targeting of nanoencapsulated cells on target tissue or organs WO2013106944A2 (en)

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CL2012000145A CL2012000145A1 (en) 2012-01-18 2012-01-18 Method of cell targeting of cells to specific tissues or organs comprising the steps of nanoencapsulation of cells with a first layer comprising a polycation and a final layer comprising a polyanion with activated carboxy groups to which a specific antibody against a cell binds White.

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
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