WO2022058809A1 - Matrice polymère multicouche - Google Patents

Matrice polymère multicouche Download PDF

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Publication number
WO2022058809A1
WO2022058809A1 PCT/IB2021/057367 IB2021057367W WO2022058809A1 WO 2022058809 A1 WO2022058809 A1 WO 2022058809A1 IB 2021057367 W IB2021057367 W IB 2021057367W WO 2022058809 A1 WO2022058809 A1 WO 2022058809A1
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matrix
cellulose
polymer
layer
alkaline solution
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Spanish (es)
Inventor
Juana María Alejandra AYALA
Juan Carlos Cruz Jimenez
Carolina MUÑOZ CAMARGO
Juan Sebastián ORTIZ TINOCO
Daniela RUBIO OLAYA
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Universidad de los Andes Colombia
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Universidad de los Andes Colombia
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B23/00—Layered products comprising a layer of cellulosic plastic substances, i.e. substances obtained by chemical modification of cellulose, e.g. cellulose ethers, cellulose esters, viscose
    • B32B23/14—Layered products comprising a layer of cellulosic plastic substances, i.e. substances obtained by chemical modification of cellulose, e.g. cellulose ethers, cellulose esters, viscose characterised by containing special compounding ingredients
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B9/00—Layered products comprising a layer of a particular substance not covered by groups B32B11/00 - B32B29/00
    • B32B9/02—Layered products comprising a layer of a particular substance not covered by groups B32B11/00 - B32B29/00 comprising animal or vegetable substances, e.g. cork, bamboo, starch
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B9/00—Layered products comprising a layer of a particular substance not covered by groups B32B11/00 - B32B29/00
    • B32B9/04—Layered products comprising a layer of a particular substance not covered by groups B32B11/00 - B32B29/00 comprising such particular substance as the main or only constituent of a layer, which is next to another layer of the same or of a different material
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0084—Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L1/00—Compositions of cellulose, modified cellulose or cellulose derivatives
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/04—Alginic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof

Definitions

  • the present invention refers to the general field of biomaterials, particularly to a porous, adherent and biocompatible multilayer polymeric matrix that allows the growth of adherent and suspended cells and the control of the mobilization and fixation of active compounds.
  • the polymeric matrix of the invention can be used in various applications, such as detection systems, cell growth systems and experimental organ models, among others. Additionally, the invention relates to the method of manufacturing the polymeric matrix by layer-by-layer deposition.
  • biomaterials correspond to those used to evaluate, cure, correct or replace any tissue, organ or function of the human body.
  • This field has had a vertiginous advance in recent times, due to the great scientific advances in materials for medical application, in particular due to the development of new biomaterials whose structure and morphology try to imitate biological tissues and which are increasingly approaching more to have the characteristic mechanical and biological properties required to achieve the desired biological action.
  • biopolymers which are versatile materials, with multiple chemical configurations and that allow the generation of composite materials with synergistic properties.
  • Biopolymers in addition to having biocompatible characteristics and high affinity for environments that simulate in vivo conditions, can be modified to improve desired characteristics such as resistance, swelling capacity, adherence to surfaces, among others.
  • the modifications of the biopolymers can be associated with the formation of composite polymers as combinations of two of them or their crosslinking.
  • Cross-linked biopolymers have new properties attributed to different interchain interactions. This is directly related to one of the most sought-after properties for applications in encapsulation and biomaterials, which is the swelling capacity, since it allows the absorption of large amounts of solvent that facilitate the migration of molecules into the interior of the materials and therefore, allow the encapsulation of compounds.
  • characteristics associated with resistance and adhesion to the surface are mainly associated with the composition and molecular conformation of the biopolymers.
  • One of the most versatile families of polymers due to their composition are the polyelectrolytes, which are of great interest due to their biocompatibility and wide variety of applications, since the difference between ionic charges improves the adherent capacities on other materials and even cells.
  • These polymeric biomaterials are an area of maximum interest, as reflected in the number of scientific articles and related patents.
  • patent US2009/0047517 describes a multilayer polymeric assembly comprising polymeric layers covalently linked by crosslinking.
  • the manufacturing process of the multilayer polymeric material is carried out by layer-by-layer (LbL) assembly, which allows obtaining a wide variety of multilayer assemblies with different compositions and controlled physical properties.
  • the process also comprises modifying the multilayer polymeric assembly, by reacting at least one functional group of one of the layers with a compound selected from antifouling agents, antimicrobials, chelating compounds, fluorescent compounds, antibodies, scavenging substances and physiologically active compounds.
  • the use of different polymeric materials in the different layers of the assembly allows the properties of the assembly to be adjusted for specific applications, such as for controlled or sustained drug release applications.
  • patent US2005/0287111 refers to devices comprised of an LbL film that covers a surface of a substrate, where said film comprises binding agents that interact with cells.
  • LbL films are assembled by serial application of individual layers, that are associated with each other by non-covalent bonds. This technology is applicable to a variety of possible polymers, in particular polyelectrolytes. Films come in various architectures (number of layers, thickness of individual layers, chance of layers melting, total film thickness, etc.).
  • agents that can be incorporated on the surface or inside of said films and their molecular structure allows visualizing, measuring or monitoring an agent in vivo or in vitro through detection techniques such as spectroscopic, photochemical, biochemical, immunochemical , electrical, optical, chemical and others.
  • patent GB2553074 describes a device for cell culture that comprises a three-dimensional scaffold, which can be composed of natural or synthetic or hybrid polymers.
  • the scaffold has modified porosity and may allow controlled diffusion of agents from the scaffold into the cell population growing on the outer surface of the scaffold.
  • Such devices can be used in applications involving cell isolation. Among some of their advantages, they allow the isolation and release of cells (by layer degradation), under mild conditions.
  • the present invention relates to a porous, adherent and biocompatible multilayer polymeric matrix, comprising an upper layer and a lower layer both of an adherent porous polymer and an intermediate layer of a swellable porous polymer.
  • the adherent porous polymer (APP) is a natural polymer cross-linked with an alkaline solution.
  • the swellable porous polymer (PPH) is a modified or unmodified natural polymer cross-linked with an acidic polyelectrolyte.
  • the matrix comprises active compounds, therapeutic compounds or mixtures thereof immobilized in one or more of the polymeric layers.
  • the invention refers to the application of the porous multilayer polymeric matrix in detection systems, cell growth systems and experimental organ models, among others.
  • the invention relates to the method for making the porous multilayer polymeric matrix by layer-by-layer deposition.
  • FIG. 1 Diagram of the conformation by layers of the polymeric matrix. The composition of each of the layers and how they are distributed on the surface is evidenced.
  • FIG. 2 Properties of the upper and lower layers of natural polymer cross-linked with alkaline solution.
  • FIG. 3 Properties of the intermediate layer of modified or unmodified polymer crosslinked with polyelectrolyte.
  • FIG. 4 Diagram of the conformation by layers of the polymeric matrix according to Example 5. The composition of each of the layers and how they are distributed on the surface are shown.
  • FIG. 5 Biocompatibility of the matrix.
  • the present invention is directed to a porous multilayer polymeric matrix comprising an upper layer and a lower layer, both of an adherent porous polymer and an intermediate layer of a swellable porous polymer.
  • multi-layer polymeric matrix refers to a biomaterial comprising 3 layers from adherent and swellable porous polymers.
  • the upper and lower layers of the matrix are made of a porous adherent polymer (PPA).
  • PPA porous adherent polymer
  • adhere reme porous polymer refers to a thermosetting natural polymer cross-linked with an alkaline solution.
  • the upper and lower layers of the polymeric matrix allow it to be adherent and biocompatible.
  • thermalostable natural polymer refers to a polymer of biological origin with the ability to form non-covalent interchain bonds when subjected to crosslinking processes due to changes in pH that stabilize the internal interactions between polymer chains.
  • natural thermosetting polymer can be selected from cross-linkable, sticky, pH-sensitive natural polymers including, but not limited to polysaccharides (eg, cellulose, pectin, gellan gum, methylcellulose, xyloglucan, curdlan, konjac glucomannan, hyaluronic acid, chitosan, alginate, chitin , dextran and starch); proteins (for example, collagen, ovalbumin, lactalbumin, and gelatin) and native or modified extracellular matrices from organs of any vertebrate and mixtures thereof (intestinal submucosa, placenta, myocardium, bladder, etc.).
  • polysaccharides eg, cellulose, pectin, gellan gum, methylcellulose, xyloglucan, curdlan, konjac glucomannan, hyaluronic acid, chitosan, alginate, chitin , dextran and starch
  • proteins for example,
  • the natural polymer is selected from pectin, hyaluronic acid, chitosan, alginate, chitin, dextran, collagen, ovalbumin, gelatin, extracellular matrices of intestinal submucosa, placenta, myocardium, bladder, and mixtures thereof.
  • the natural polymer is found in solution or suspension in concentrations between 10 to 50 mg/mL, 15 to 30 mg/mL or 30 to 50 mg/mL.
  • the natural polymer is egg white.
  • thermosetting natural polymer is cross-linked with an alkaline solution, which makes the PPA formed have an adjustable pH according to the desired application.
  • the adjustable pH range is between 9 and 10.5, which allows PPA to reduce its swelling capacity and therefore increase its stability in aqueous media and at temperatures above 30°C, common conditions for applications that seek to resemble the physiological environment such as organ models and cell culture production.
  • the crosslinking alkaline solution is of the heterobifunctional type, which allows multiple conjugations between polymeric chains of different nature.
  • the alkaline solution corresponds to high reactivity crosslinkers and is selected from, without being limited to, the following: - strong bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide and calcium hydroxide; carbodiimides such as N,N-dicyclohexylcarbodiimide, l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, and diisopropylcarbodiimide; imidoesters such as dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate;
  • maleimides such as sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate, N-[4-(p-maleimidophenyl)-butyryl] (MPB) or 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC );
  • haloacetyls such as acetyl chloride, ethanoyl chloride, propanoyl chloride, butanoyl chloride and malonyl bromide chloride;
  • - hydrazides such as formic hydrazide, carbohydrazide, acetidrazide, methyl hydrazinecarboxylate, oxalyldihydrazide, 1,2-diacetylhydrazine; alkoxyamines such as 2,2,5-trimethyll-4-phenyl-3-azahexane-3-nitroxide, N-tert-butyl-O-[l-[4-(chloromethyl)phenyl]ethyl]-N-(2- methyl-l-phenylpropyl)hydroxylamine; and diazirines such as 3-(4-bromophenyl)-3-(trifluoromethyl)-3H-diazirine, succinimidyl 6-(4,4'-azipentanamido)hexanoate, succinimidyl 4,4'-azipentanoate, sulfosuccinimidyl 4,4'-azipentano
  • the strong base is sodium hydroxide
  • the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)
  • the hydrazide is carbohydrazide.
  • the crosslinking alkaline solution is sodium hydroxide.
  • the natural thermosetting polymer and the crosslinking alkaline solution are mixed in a ratio between 20: 1 and 200: 1, between 20: 1 and 50: 1, between 50: 1 and 200: 1, until forming the PPA that makes up the upper layers. and bottom of the array.
  • the ratio is 50:1.
  • the concentration of the alkaline solution is between 0.5 and 5 M, between 0.5 and 2.5 M, between 0.5 and 1 M or between 1 M and 5 M.
  • the alkaline solution is at a concentration of 1 M.
  • the PPA that makes up the upper and lower layers of the matrix is characterized by the following parameters:
  • PPA is characterized by a porosity that ranges between 70% and 75%, which allows the encapsulation or trapping of molecules or compounds inside it. Additionally, the porosity gives it the swelling capacity that is of great importance to resemble physiological environments, as well as a unique surface morphology, which allows the anchoring of protein structures present in adherent cells.
  • PPA is characterized by a water retention capacity of no more than 300%, between 100% and 300%, between 150% and 200%, and between 200% and 300%. In one embodiment, the water retention of the PPA is 150%. These water retention values allow the PAA to remain hydrated with less chance of hydrolytic degradation. Additionally, regarding the encapsulation of molecules, these levels of swelling allow the particles to migrate through the matrix and control their sustained release into the medium.
  • PPA - Resistance
  • the time in which the material remains stable must range between 12 and 48 hours, with 24 hours being the optimum, with a percentage of weight loss between 5% and 10%.
  • a 24-hour stability enables its use in multiple short-term applications associated with compound encapsulation, cell culture growth, and organ model manufacturing.
  • the percentage of associated weight loss during this period is acceptable since it does not induce significant changes to the PPA and, consequently, its capacity in the applications of interest.
  • PPA is characterized by a viscosity between 0.1 to 1 Pa*s, between 0.1 to 0.5 Pa*s and between 0.5 to 1 Pa*s, which guarantees the correct deposition of the layers. Due to the method proposed for the elaboration of the matrix and the deposition of the layers explained later, the values of The viscosity obtained are optimal for carrying out the spin coating process with the PPA and a uniform layer is formed. Viscosities greater than 2 Pa*s make the process difficult, causing the formation of irregularities on the surface of the layers.
  • PPA is characterized by a firmness between 2.5 N and 5.15 N, which varies depending on the thickness of the layers. These values ensure that the PPA has firmness characteristics that give it stability over time, especially after the drying process, since it prevents the formation of cracks and the breaking of the layer formed. These processes are associated with high rates of hydrolytic degradation due to the premature breakdown of polymeric networks.
  • PPA favors the formation of three-dimensional polymeric networks, which gives it uniformity and hardness, for this reason the interaction with molecules such as water is based on repelling the effects of hydrolysis that can damage and collapse the network.
  • the decrease in hydrogen bonds due to the presence of the alkaline solution as a crosslinker gives the PPA surface hydrophobic characteristics that favor stability in aqueous media. This allows PPA to remain stable for prolonged periods of time in interaction with aqueous media. This makes it ideal for applications where permanent contact with aqueous media is sought.
  • PPA has the ability to actively interact with other structures due to its high content of proteins and molecules with highly reactive radicals along with amino, hydroxyl, sulfhydryl, phenolic and carboxyl groups at the ends of the chains, which can be easily modified by interacting with other inorganic structures or organic proteins, forming stable bonds. This ability makes the PPA add versatility to the matrix and it can adhere to multiple surfaces in different conditions.
  • the PPA of the polymeric matrix is suitable for molecular interaction with the adherent proteins of the cells, improving their viability. Therefore, it is biocompatible. Additionally, PPA is characterized by a cytotoxicity of less than 5%, indicating which is viable for cells in suspension. Additionally, the interaction between the material and the cells favors the formation of adherent protein structures essential for the survival of cells that require anchoring to the surface. On the other hand, PPA gives stability to the polymeric matrix, since it allows the control of the conditions conducive to cell growth in in vitro and in vivo conditions, such as high humidity and temperature between 28°C and 40°C. , ensuring that the multilayer matrix does not degrade before the time required for cell growth.
  • the intermediate layer consists of a swellable porous polymer (PPH).
  • PPH swellable porous polymer
  • the term "swellable porous polymer” refers to a polymer derived from modified or unmodified polysaccharides or cellulose crosslinked with a solution of an acidic polyelectrolyte.
  • the intermediate layer of the polymeric matrix allows the mobilization and fixation of molecular compounds.
  • modified or unmodified polymer derived from polysaccharides or cellulose is a substance that has a great swelling capacity, absorbing large amounts of polar liquids such as water, as well as high versatility to adsorb and retain compounds. This allows the encapsulation and mobilization of molecules inside the intermediate layer, allowing the passage of water without the immobilized components being quickly released.
  • unmodified polymer refers to a polymer of natural or synthetic origin without additional chemical modifications to those initially required for its synthesis and condensation. That is, these polymers do not have functional groups or physical modifications at the molecular level that change the base form of the original polymer chains.
  • modified polymer refers to a polymer of natural or synthetic origin with physical or chemical modifications that alter the base structure of the chains polymeric and, ultimately, that give additional properties or characteristics to those of the base material.
  • the polymer when the polymer is a polysaccharide derivative, it is selected from, but not limited to, chitosan, alginate, xanthan gum, guar gum, hyaluronic acid, starch, gum acacia and carrageenan.
  • the polymer when the polymer is a cellulose derivative it is selected from, but not limited to, alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester and nitrocellulose.
  • the polymer when it is a cellulose derivative, it is selected from methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxybutylmethylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethylcellulose (CMC) and cellulose triacetate.
  • the polymer is CMC.
  • polyelectrolyte that has highly reactive groups at the ends of the chains.
  • polyelectrolyl refers to a polymer with ionizable groups such as polyanions and polycations that give it the ability to change its spatial conformation according to the surrounding conditions.
  • the polyelectrolyte is selected from, but not limited to, polyglycolic acid (PGA), polylactic acid (PLA), polyacrylic acid (PAA), polyamides, poly-2-hydroxy butyrate (PHB), gelatin, (A, B)- polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), poly(L-lysine) (PLL), poly(L-glutamic acid) (PGA), and mixtures thereof.
  • the polyelectrolyte is PAA.
  • the polymer derived from polysaccharides or modified or unmodified cellulose and the polyelectrolyte are mixed in a ratio between 10:4 and 2:0.5, between 10:4 and 3: 1, between 10: 1 and 3: 1, between 10: 1 and 2:0.5, until forming the PPH that forms the intermediate layer.
  • the ratio is 5:1.
  • the polymer is found in a concentration between 0.6 and 1.5% w/v, between 0.6 and 1% w/v or between 1 and 1.5% w/v, and in a particular embodiment, it is found in a concentration of 1% w/v. Additionally, the polyelectrolyte is found in a concentration between 0.1% and 0.4% p/v, between 0.2% and 0.4% p/v, between 0.3% and 0.4% p/v , and in a particular embodiment, in a concentration of 0.3% w/v.
  • the PPH is characterized by different conformations when it is swollen and when it is dry. Particularly, when it dries, it forms aggregates with the immobilized molecules, and once it swells, it allows these aggregates to dissolve and migrate through the matrix, preventing the molecules from migrating to the medium and eventually detaching from the matrix. Additionally, the PPH allows interaction with polar molecules such as water, facilitating its entry and exit from the layer with minimal damage, which allows for considerable hydration in one of the layers of the matrix. This allows the aggregates containing the immobilized molecules to migrate through the matrix, without losing their ability to absorb water and remain stable in liquid media.
  • the PPH is characterized by the following parameters:
  • Adsorption capacity PPH is characterized by its ability to interact with carboxyl, carboxylate and nitrile groups with substrates in polar liquid media, which gives it an adsorption capacity between 850-1000 mg/g. This facilitates the effective immobilization of active compounds within the intermediate layer.
  • PPH is characterized by having a swelling capacity of over 500% and remaining stable for a period between 12 and 24 hours. This capacity allows the polymeric matrix to be kept hydrated to prevent the other layers from fracturing and losing their properties.
  • PPH is characterized by large and widely separated pores.
  • the porosity of PPH ranges from 50% to 70% with pore sizes between 2 pm and 6 pm. This facilitates the effective immobilization of active compounds within the intermediate layer, disappearing the pores and showing a uniform surface.
  • the multilayer polymeric matrix according to the present invention has the structure that is outlined in the Figure. 1, and is made by methods known in the art such as those encompassed by surface deposition methods for the formation of thin coatings. These include but are not limited to interfacial deposition processes such as Sol-gel, spin-coating or dip-coating.
  • the multilayer matrix is obtained by layer-by-layer deposition according to the following steps:
  • the adequate volume refers to the amount of PPA or PPH solution necessary to achieve that each layer has a thickness between 10 pm and 500 pm, depending on the application of interest and the area of the surface where are deposited.
  • the multilayer polymeric matrix of the present invention has the structure outlined in Figure 1.
  • the upper, intermediate and lower layers of the polymeric matrix are characterized by a thickness between 10 pm and 500 pm, between 10 pm and 50 pm, between 50 pm and 150 pm, between 150 pm and 300 pm, or between 300 and 500 pm, depending on the application of interest and the surface area where they are deposited.
  • the polymeric matrix of the present invention is characterized by greater stability (longer duration over time), and this is due, without wanting to be bound by a definitive explanation, to the formation of polymeric bonds at the interfaces of the layers that allow improving their resistance.
  • the nature of the PPA and PPH polymers guarantees the biocompatibility that allows the adequate growth of adherent and suspended cells.
  • the polymeric matrix of the present invention is characterized by a porosity that allows a controlled diffusion of active compounds of interest, since by combining the porosities of the three individual layers, an active control of mobilization and fixation of compounds is achieved according to with its size, which allows the molecules to be separated and differentiated within the matrix.
  • the multilayer matrix is characterized by a pH between 7 and 8.
  • the matrix has a pH of 7.4.
  • the three layers keep the pH stable and at adequate values for cells, which allows for adequate cell viability.
  • basic pH favors the interaction of the matrix with the water, thanks to the polarization of anionic functional groups, allowing the matrix to swell to desired levels without being affected in a period of approximately 72 hours.
  • the multilayer polymeric matrix according to the present invention may have therapeutic compounds of natural or synthetic origin immobilized in one or more of the layers.
  • Therapeutic compounds of natural origin have biological activity and can be used to modulate deregulated cellular processes that cause undesirable health conditions or control the proliferation of pathogenic agents.
  • Examples of the biological activity of said compounds include, but are not limited to antiviral, antifungal, antibacterial, anticancer, immunomodulatory properties or modulation of metabolic process regulation cascades. Additionally, they can be used to fulfill detection, biocatalysis and in vitro identification functions.
  • the active compounds serve to obtain complementary functionalities in the matrix.
  • These compounds include, but are not limited to, nucleic acids, enzymes that metabolize compounds to remove them or generate desired products, antibodies that specifically detect a molecule or that specifically bind to and immobilize a molecule or compound, and hormones and metabolites that support cellular processes.
  • These compounds incorporated in the matrix serve to promote cell growth due to their growth-promoting effect; have antibiotic or antifungal activity; they serve to evaluate the effect of specific compounds on cells and in the case of molecules such as DNA, RNA, nucleotides, they allow the evaluation of changes in metabolism or cell expression, among other possible applications of the matrix.
  • the active compounds are selected from enzymes and antibodies.
  • Therapeutic compounds include, but are not limited to antimicrobial agents, anesthetics, analgesics, anticancer agents, angiogenic agents, antiseptics, antibiotics, fibrotic agents, antimitotics, chelating agents, peptides, proteins, DNA, RNA, nucleotides, liposomes, blood products, hormones, water soluble silver salts, growth factors, and combinations thereof.
  • the therapeutic compounds are selected from growth factors and antibiotics.
  • Therapeutic compounds are added to the matrix in amounts ranging from 1 mg/mL to 1 g/mL.
  • the multilayer polymeric matrix has multiple applications that include, without being limited to, rapid detection systems for microorganisms in different matrices ranging from food to pharmaceutical products, contaminants in water and food, cell cultures, systems for cell growth , experimental organ models, controlled release, biocatalysis systems for environmental, industrial and pharmaceutical applications, toxin and contaminant agent trapping systems, among others.
  • an Al composition was prepared with egg white, which was obtained from fresh chicken eggs. Carefully, after cleaning the outside of an egg with 70% ethanol, a hole was opened to remove its internal contents, the egg white was slowly poured into a container, avoiding mixing with the yolk. Once the egg white was obtained, 50 mL of it were taken and its pH was evaluated. Subsequently, a 1M sodium hydroxide (NaOH) solution was added dropwise until a pH of 10.5 was reached.
  • NaOH sodium hydroxide
  • compositions A2-A4 are prepared as indicated in Table 1.
  • the swelling capacity of the layer was determined from continuous measurements of the weight of the layer after immersion for a total time of 24 hours in distilled water. The initial weight of the totally dry layer was taken to obtain a reference value. The PPA film was removed from the water at different time intervals, the excess water was removed with the help of a 0.22 pm filter paper, the weight was taken and it was submerged again until the total time was completed. To calculate the percentage of swelling over time, the following formula was used:
  • Swelling (%) Ps Pm x 100, Ps where Ps is dry weight and Pm is the weight after immersing the PPA layer in water.
  • the layer was immersed in distilled water for 72 hours and additionally kept under incubation at 37°C.
  • the initial weight of the totally dry layer was taken and once submerged, it was removed from the water at different time intervals and the excesses were removed with 0.22 pm filter paper to take the weight again.
  • P is the initial dry weight
  • Pf is the weight after immersing the PPA layer in the water.
  • the proposed formulation has a high resistance to the proposed humidity and temperature conditions with a loss of less than 10% of its initial mass in the total evaluation time, which is beneficial for applications such as cell cultures, since the multilayer system is maintained under these conditions and requires a minimum time of 72 hours to be successful (FIG. 2B).
  • composition B1 was prepared with carboxymethyl cellulose (CMC 263.2 g/mol) and polyacrylic acid (PAA 72.06 g/mol). Ultrapure water (Milli Q-plus system, Millipore) was used to dilute both compounds.
  • a deposition method was used to ensure a uniform distribution.
  • 10 pL of composition B 1 were deposited on the surface of a 24mm x 24mm coverslip.
  • the intermediate layer film was formed with a spin coater, positioning the coverslip on the support with the help of a vacuum system.
  • the computer was then programmed to spin at 3,000 rpm with an acceleration of 250 rpm/s for 30 seconds.
  • compositions B2-B4 are prepared as indicated in Table 2.
  • an absorption assay was performed, dissolving different molecules in phosphate buffered saline (PBS).
  • Three versions of the proposed PPH formulation were synthesized and each was immersed in one of the solutions for 24 hours after initial dry weight measurement. Next, the PPH was removed at different time intervals, the excess water was removed with a 0.22 pm filter paper and weighed. According to the initial concentration of the solutions and the absorbed water, it was possible to calculate the concentration of adsorbed and immobilized molecules in each layer (FIG. 3C).
  • the swelling capacity of the layer was determined from constant measurements of its weight after submerging it for a total time of 24 hours in distilled water. The initial weight of the totally dry layer was taken to obtain a reference value. Then, at different time intervals, the film was removed from the PPH, the excess water was removed with the help of a 0.22 pm filter paper, the weight was taken and finally it was submerged again in the water until the total time was completed. . To calculate the percentage of swelling over time, the following formula was used: 100, where Ps is dry weight and Pm is the weight after submerging the PPH layer in water.
  • Example 5 Obtaining a multilayer polymeric matrix
  • the layer of egg white cross-linked with NaOH was deposited through the spin coating method as described in composition Al of Example 1. Subsequently, this layer was subjected to UV radiation for 5 minutes to ensure its sterility, sealed and exposed. radicals that allow the formation of bonds. Next, the second layer was deposited on its surface and formed with the composition B1 described in Example 3. Next, this second layer was subjected again UV light for 10 minutes and allowed to stand at room temperature for an additional 10 minutes, before depositing the surface layer in the same way as for the first one and thus complete the formation of the matrix in its entirety. (FIG. 4).
  • Example 6 Characterization of a multilayer polymeric matrix
  • MTT cell viability assay
  • Vero cells ATCC, CCL-81 were grown in a cell culture dish in the presence of DMEM supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin (growth medium). complete) until approximately 80% confluency is complete.
  • the cells were detached from the culture flask with hot trypsin at a concentration IX, then cells stained with trypan blue were counted in a Neubauer chamber to dilute them again in medium without serum and obtain a final concentration of 200,000 cells/ mL.
  • the multilayer polymeric matrix with the cell culture medium was established as a target, in order to rule out effects of the polymer on the test reagents.
  • DMSO dimethyl sulfoxide
  • Blank, positive control and assay were incubated for 24 hours at 37°C and 5-6.5% CO2.
  • 10 pL of the MTT staining reagent were added and incubated for 4 hours under the previously mentioned conditions.
  • 100 pL of the solubilization solution were added and 100 pL of the medium were taken to measure its absorbance in a spectrophotometer at a wavelength of 650nm. The percentage of viability was calculated with the following formula:
  • the multilayer matrix preserves cell integrity, maintaining its viability at values greater than 95%, validating that the components of the matrix and its conformation are biocompatible and safe for use in cell systems for research and clinical applications (FIG. 5A ).
  • Vero cells ATCC, CCL-81 were initially grown in a cell culture dish with DMEM supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin (complete growth medium). ) until approximately 80% confluence was completed. The cells were detached from the culture flask with hot trypsin at a concentration IX, then cells stained with trypan blue were counted in a Neubauer chamber to dilute them again in medium without serum and obtain a final concentration of 200,000 cells/ mL.
  • the cells were cultured on the surface of the matrix that was synthesized on the surface of a coverslip to place it in the bottom of the wells of a 24-well microplate for cell cultures.
  • the cells were allowed to incubate for 24 hours at 37°C and 5-6.5% CO2.
  • the culture medium was removed and the cells were washed with PBS to remove residues, then ImL of 4% (v/v) formaldehyde was added to fix the cells for 10 minutes.
  • triton was added at a concentration of 4X and allowed to react for 5 minutes.
  • the nucleus was stained by adding 1 mL of DAPI for 10 minutes. Excess DAPI was removed to stain the structures. cell membrane proteins that adhere to the surface of the multilayer polymeric matrix by adding 1 mL of Alexa Fluor 488.
  • each well was washed again and the cells were observed in an Olympus FV1000 confocal microscope.
  • multilayer polymeric matrix examples include, but are not limited to two general groups comprising encapsulation of compounds and/or molecules and tissue engineering. Examples of possible applications within these two categories will be given below.
  • microorganisms within the category of encapsulation are applications such as the detection of microorganisms, substances and metabolites.
  • detection systems based on the use of antibodies and reporter enzymes that bind to specific proteins of the organism and report their presence through an increase in fluorescence, a change in color or the increase in concentration of a specific substance that can be measured with the help of specialized instruments.
  • Possible applications framed in the detection of microorganisms include, but are not limited to, microbiological evaluation of food, water, drugs, cell cultures and diagnosis of diseases caused by microorganisms.
  • reporter molecules or microsensors that, when reacting with the substance of interest, generate a detectable reaction or allow the evaluation of its concentration in the medium.
  • Possible applications in this context include, but are not limited to, evaluation of the quality of water, air, food, drugs and the diagnosis of diseases associated with imbalances in metabolites or the presence of detectable toxic substances.
  • tissue engineering Within the category of tissue engineering are applications in suspension and adherent cell cultures in two and three dimensions and organ models with simple morphology.
  • Possible applications for two-dimensional adherent cell cultures include the evaluation of cell lines for clinical or research purposes. Within the evaluation, the effect of components previously immobilized in the multilayer polymeric matrix, the migration and subsequent evaluation of metabolites generated by the cells as a reaction to a treatment within the multilayer polymeric matrix, the differentiation of stem cells in different tissues by the effect of growth factors and metabolites previously immobilized in the matrix.
  • the applications associated with 3D cell cultures include, but are not limited to, the encapsulation of cells for the formation of spheroids that resemble a physicochemical environment closer to in vivo, since they facilitate cell-cell and cell-matrix interaction, their use It can be extended to tumor and cancer research, therapeutic transplants, evaluation of the effect of drugs and clinical studies. Additionally, within this category are organ models with simple morphology as a barrier model for the evaluation of the effect of substances on cellular structures more complex than those of two dimensions, skin model, intestine lumen model, among others.

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Abstract

La présente invention concerne une matrice polymère multicouche poreuse, adhérente et biocompatible qui permet la croissance de cellules adhérentes et en suspension et un contrôle actif de mobilisation et de fixation de composés actifs. La matrice polymère de l'invention peut être utilisée dans des applications diverses, telles que des systèmes de détection, des systèmes de croissance cellulaire et des modèles d'organes expérimentaux, entre autres. L'invention concerne en outre le procédé d'élaboration de la matrice par dépôt couche par couche.
PCT/IB2021/057367 2020-09-21 2021-08-10 Matrice polymère multicouche Ceased WO2022058809A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007215519A (ja) * 2006-02-20 2007-08-30 Fujifilm Corp 細胞培養担体
US20150209188A1 (en) * 2010-08-16 2015-07-30 Fasttrack Medical Solutions Llc Peripheral Hydrogel Wound Dressing
US20170166884A1 (en) * 2015-12-15 2017-06-15 Worcester Polytechnic Institute Coated Cell Culture Apparatus and Methods of Use
CA2740597C (fr) * 2008-10-17 2017-11-21 Sofradim Production Timbres chirurgicaux multicouches renfermant un premier precurseur hydrogel et un deuxieme precurseur hydrogel

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007215519A (ja) * 2006-02-20 2007-08-30 Fujifilm Corp 細胞培養担体
CA2740597C (fr) * 2008-10-17 2017-11-21 Sofradim Production Timbres chirurgicaux multicouches renfermant un premier precurseur hydrogel et un deuxieme precurseur hydrogel
US20150209188A1 (en) * 2010-08-16 2015-07-30 Fasttrack Medical Solutions Llc Peripheral Hydrogel Wound Dressing
US20170166884A1 (en) * 2015-12-15 2017-06-15 Worcester Polytechnic Institute Coated Cell Culture Apparatus and Methods of Use

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BORGES J.: "Layer-by-Layer Assembly of Light-Responsive Polymeric Multilayer Systems", ADV. FUNCT. MATER, vol. 24, 2014, pages 5624 - 5648, XP001591636, DOI: 10.1002/adfm. 20140105 0 *
BOUDOU T.: "Multiple Functionalities of Polyelectrolyte Multilayer Films: New Biomedical Applications", ADV. MATER., vol. 22, 2010, pages 441 - 467, XP055917140, DOI: 10.1002/adma.200901327 *
WITTMER C. ET AL.: "Multilayer Nanofilms as Substrates for Hepatocellular Applications", BIOMATERIALS, vol. 29, no. 30, October 2008 (2008-10-01), pages 4082 - 4090, XP025479387, DOI: 10.1016/j.biomaterials. 2008.06.027 *

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