WO2021119864A1 - Multi-polymer hydrogel as a device for administering pharmaceutical and cellular therapeutic components - Google Patents

Multi-polymer hydrogel as a device for administering pharmaceutical and cellular therapeutic components Download PDF

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Publication number
WO2021119864A1
WO2021119864A1 PCT/CL2020/050120 CL2020050120W WO2021119864A1 WO 2021119864 A1 WO2021119864 A1 WO 2021119864A1 CL 2020050120 W CL2020050120 W CL 2020050120W WO 2021119864 A1 WO2021119864 A1 WO 2021119864A1
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hydrogel
multipolymeric
therapeutic
hydrogels
allows
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Spanish (es)
French (fr)
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Jorge Toledo Alonso
Brian RIVAS TIZNADO
Seidy PEDROSO SANTANA
Carolina GOMEZ GAETE
Maria Daniela MERINO FUENTES
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Universidad de Concepción
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/718—Starch or degraded starch, e.g. amylose, amylopectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/34—Copper; Compounds thereof
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61K47/56—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/20—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing organic materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/24—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/26—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/28—Polysaccharides or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/62—Compostable, hydrosoluble or hydrodegradable materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L29/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an alcohol, ether, aldehydo, ketonic, acetal or ketal radical; Compositions of hydrolysed polymers of esters of unsaturated alcohols with saturated carboxylic acids; Compositions of derivatives of such polymers
    • C08L29/02—Homopolymers or copolymers of unsaturated alcohols
    • C08L29/04—Polyvinyl alcohol; Partially hydrolysed homopolymers or copolymers of esters of unsaturated alcohols with saturated carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L3/00—Compositions of starch, amylose or amylopectin or of their derivatives or degradation products
    • C08L3/02—Starch; Degradation products thereof, e.g. dextrin
    • 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
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L71/00—Compositions of polyethers obtained by reactions forming an ether link in the main chain; Compositions of derivatives of such polymers
    • C08L71/02—Polyalkylene oxides

Definitions

  • Multipolymeric hydrogel as a device for the administration of pharmaceutical and cellular therapeutic components.
  • This technology is related to the pharmaceutical and medical industry, in particular a medical device is presented, which allows the application of pharmaceutical and cellular therapies, in a controlled manner over time, and centralized at the application site.
  • This type of wound is especially vulnerable to being infected by pathogens such as S. aureus and E co / i. These infections slow down the epithelial regeneration process and facilitate the incorrect deposition of collagen, worsening the patient's diagnosis 1 .
  • the most used form of prevention is the use of dressings that act as barriers against microbes, however, many times the dressing may not be 100% sterile.
  • the dressing only covers the outer layer and in order to heal, it is necessary to remove the dressing from time to time, increasing the chances of suffering an infection 2 .
  • Tissue engineering is a field that mixes the areas of cell biology, biomaterial engineering and medicine, in order to develop strategies that allow the regeneration of damaged tissue, through grafts that replace damaged tissue, or through localized release. of compounds that facilitate epithelial regeneration 3 .
  • materials are needed that can be a support for the graft cells or the therapeutic compound, that possess antimicrobial properties to prevent infection, and that are biocompatible.
  • hydrogels have great potential to be applied in the regeneration of epithelial tissue ⁇ 5 .
  • Hydrogels are three-dimensional polymeric structures that retain large amounts of liquid without losing their structure and are formed from the physical or chemical crosslinking of one or more constituent polymers.
  • tissue engineering hydrogels have been used to encapsulate cells, proteins, and and other bioactive compounds, to later release them locally and systematically in damaged tissue 6 ' 7 ' 8 .
  • Natural biopolymers such as chitosan and starch have been used to synthesize biocompatible hydrogels that are useful for use in tissue engineering 9,10 .
  • synthetic polymers such as polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG), which are biocompatible and have been used to generate hydrogels with biological applications 11 .
  • Table 1 describes some hydrogels used as support matrix, which have been patented and are for sale. Table 1: List of commercially available hydrogels used as support matrix. 12 '13
  • Calamine-zinc gelatin Gelatin used as a wound dressing to decrease pain and itching.
  • Partial thickness carboxymethylcellulose granugel and full propylene glycol Partial thickness carboxymethylcellulose granugel and full propylene glycol.
  • Intrasite Gel for superficial wounds and deep propylene glycol Intrasite Gel for superficial wounds and deep propylene glycol.
  • Woundtab Carboxymethylcellulose and capable of absorbing glycerol bacteria and retaining them in its structure.
  • none of the commercially available hydrogels uses a combination of polymers that includes compounds that, when degrading, enhance re-epithelialization 1,4 , favoring the improvement of the patient.
  • Figure 1 Photographs of the macroscopic and microscopic structure of the multipolymeric hydrogels (A-C) and chitosan-starch (D-F), obtained by chemical synthesis.
  • Figure 2 Graph of the cell viability percentages obtained by the MTT assay in the HEP2 cell line, incubated with fragments of the hydrogels. Each sample was studied in triplicate and the results indicate that none of the hydrogels had significant negative effects on the viability of the cell line.
  • Figure 3 Characterization of the hydrogels in terms of swelling capacity. The samples were incubated in alkaline phosphate buffer (PBS) and analyzed at different times for 24 hours.
  • Figure 4 Microfotog raffles of the hydrogels before and after the incorporation of copper nanoparticles and elemental analysis by energy dispersive (EDX) for the detection of copper in the hydrogels. Chitosan-starch (AB), multipolymeric (CD). The EDX analysis allowed to observe the presence of copper in the hydrogels after being incubated with the nanoparticles.
  • EDX energy dispersive
  • Figure 5 Infrared spectra of hydrogels obtained by Fourier transform infrared spectroscopy (FTIR).
  • A tension of the N-H groups, visible in the chitosan-starch hydrogel;
  • C O-H and C-O-C tension, from starch, visible in both hydrogels.
  • Figure 6 Time course of wound healing in mice treated with different hydrogels. 1 + Cu: chitosan-starch hydrogel loaded with copper nanoparticles; 1-Cu: chitosan-starch hydrogel; 2 + Cu: multipolymeric hydrogel loaded with copper nanoparticles, 2-Cu: multipolymeric hydrogel, negative control: mice with wounds without hydrogel treatment. The averaged results and their standard deviation are plotted for days 0, 3, 6, 9, and 13 after wound development.
  • the present technology consists of a medical device, which allows the application of pharmaceutical and cellular therapies, in a controlled manner over time, and centralized in the application site, by serving as support and protection for the therapeutic component.
  • This device consists of a single piece: a multipolymeric hydrogel matrix, with the ability to swell and absorb biocomposites with therapeutic properties, which is biocompatible and biodegradable under physiological conditions.
  • the support has the ability to absorb exudates in wounds and favors their healing, reducing inflammation.
  • the therapeutic principle to be administered must be added to the device, before its application, being absorbed and retained by the polymeric network and subsequently released at the site of interest.
  • the active component to be integrated can be a nanoparticle, an antibiotic, a drug, a growth stimulator or cell regenerator, or directly cells from the patient, such as stem cells, which are They incorporate the hydrogel and use it as a support for their multiplication and formation of new tissues.
  • the polymeric matrix that forms the device has characteristics that make it ideal for the absorption, retention and release of cells and drugs.
  • the hydrogel comprises the inclusion of the following compounds: a) 2-4% Chitosan; b) 0.01-4% Poly ivi ni l-alcohol; c) 2-4% Starch; d) 0.01-4% Polyethylene Glycol; e) 250-500 pL of crosslinking agent; and f) 80-92% slightly acidic aqueous solution.
  • the crosslinking agent used is preferably glutaraldehyde or genipin, and the aqueous solution is used at pH 2.0.
  • the presence of these polymers guarantees electrostatic interaction with the bioactive components, while allowing the passage of substances and cells throughout the structure, through the formation of channels with a diameter greater than 100 pm.
  • the lattice that forms the matrix establishes a high level of retention, while mechanisms associated with the polymers that comprise it, such as sensitivity to pH, hydrolysis and enzymatic action, guarantee the biodegradability and subsequent release of the retained material.
  • the different polymers also provide chemical and structural characteristics that in some cases are reinforced and in others they complement each other, providing properties to the matrix such as: flexibility, rigidity, antimicrobial activity, and high swelling capacity, without losing its three-dimensional structure, and high strength and resistance to pressure / impact, without losing its biodegradability properties.
  • this technology enables a more efficient therapeutic effect of the drugs or cellular components to be administered, by functioning as an absorption and protection device for the agent. therapeutic.
  • the active component is released in a controlled way over time, which guarantees a greater availability of it, and a persistent or sustained activity, while it can reduce the occurrence of side / adverse effects such as infections (due to its nature or antimicrobial property ).
  • the therapeutic agent will be protected against the action of external elements such as proteolytic enzymes and cells of the immune system that contribute to its degradation and clearance, it will be contained in a support with a certain degree of structural rigidity, which guarantees its permanence in the site of application, and its activity will be enhanced by the antimicrobial effect of the hydrogel, which inhibits the occurrence of infections at the site of application.
  • the biodegradable and biocompatible characteristics of the device allow its gradual metabolic elimination, without any type of toxic reaction associated with it.
  • the innovation is in the advanced research and development stage.
  • the prototype hydrogels have been synthesized, they have been characterized in terms of morphology, mechanical resistance and chemical composition, absorption capacity and retention of chemical compounds. Specifically, studies were carried out for the loading of copper nanoparticles, the presence of which has been verified by X-ray scattering analysis. Similarly, the effect of hydrogels on the wound healing process was studied in a mouse model, with Obvious results in accelerating the healing process and reducing inflammation.
  • composition and methodology for obtaining the hydrogel are carried out by step dissolution in a unique combination of polymers: starch, polyethylene glycol, polyvinyl alcohol and chitosan, dissolved in a 2% aqueous solution of acetic acid. Subsequently, a known amount of a crosslinking agent is added to it to cause crosslinking of the polymers in the hydrogel.
  • This chemical method of obtaining the hydrogel is combined with a physical method, since the mixture undergoes several freezing and thawing cycles, and it is subsequently lyophilized under vacuum.
  • we develop two hydrogels one rnultipolymeric that includes the four polymers and only one with chitosan-starch (Table 2).
  • Table 2 Composition of hydrogels.
  • the macro and microscopic structure of the hydrogel matrix was evaluated by means of Scanning Electron Microscopy JEOL JSM-6380 LV (USA), in a device that, in addition, coupled an energy dispersive X-ray fluorescence detector (EDX) to study their morphology and polymeric matrix.
  • EDX energy dispersive X-ray fluorescence detector
  • the chitosan-starch hydrogel presented a swelling capacity close to 10 times the increase of its initial dry weight, while the multipolymeric hydrogel doubled its dry weight when incorporating the aqueous solution (Figure 3).
  • the EDX analyzes showed that after 12 hours in the PBS solution with copper nanoparticles (CuNps), the hydrogels retained these nanoparticles, since a copper emission is observed (Figure 4B and D).
  • FTIR Fourier transform infrared spectroscopy
  • Infrared spectra were obtained using a Nicolet, Nexus FTIR with ATR detector (USA) and Nicolet Lexus 470 with PAS detector (USA), using a KBr pellet as a target.
  • the samples were analyzed at room temperature in a range of 4000-500 cm-1.
  • the amino groups from the different individual components of both hydrogels were identified, as well as the formation of triple bonds, which shows that the devices are made up of their individual chemically cross-linked components ( Figure 5).
  • Example 3 Evaluation of the effect of hydrogels on wound healing.
  • Group 1-Cu Chitosan-starch hydrogel.
  • Group 2-Cu Multipolymeric hydrogel.
  • Negative control group Control of animals without hydrogel treatment.
  • Hydrogels were observed to decrease healing time compared to the negative control without hydrogel, based on two factors: 1) the groups treated with hydrogel decreased the size of the wounds on all the days analyzed, while the untreated control group presented an increase in the size of the wound and greater inflammation ( Figure 6, days 3 and 6). 2) 77.8% of the animals treated with hydrogel completely healed before day 13, while of the mice in the control group, only 40% healed before day 13.

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Abstract

Disclosed is a multi-polymer hydrogel used as a medical device for administering pharmaceutical and cellular therapeutic components, which comprises at least the following components: 2-4% chitosan; 0.01-4% polyvinyl alcohol; 2-4% starch; 0.01-4% polyethylene glycol; 250-500 µL crosslinker; and 80-92% mildly acidic aqueous solution. The hydrogel allows pharmaceutical and cellular therapies to be applied in a time-controlled manner and centrally on the application site, acting as a support and protective element for the therapeutic component.

Description

Hidrogel multipolimérico como dispositivo para la administración de componentes terapéuticos farmacéuticos y celulares. Multipolymeric hydrogel as a device for the administration of pharmaceutical and cellular therapeutic components.
Sector técnico Technical sector
Esta tecnología está relacionada con la industria farmacéutica y medica, en particular se presenta un dispositivo médico, que permite la aplicación de terapias farmacéuticas y celulares, de manera controlada en el tiempo, y centralizada en el sitio de aplicación. This technology is related to the pharmaceutical and medical industry, in particular a medical device is presented, which allows the application of pharmaceutical and cellular therapies, in a controlled manner over time, and centralized at the application site.
Estado del arte State of the art
Las quemaduras, golpes o ciertas enfermedades congénitas, pueden provocar heridas graves en la piel. Este tipo de heridas es especialmente vulnerable a ser infectada por agentes patógenos como S.aureus y E co/i. Estas infecciones ralentizan el proceso de regeneración epitelial y facilitan la deposición incorrecta de colágeno, empeorando el diagnóstico del paciente1. La forma de prevención más usada, es el uso de apósitos que actúan como barreras frente a los microbios, sin embargo, muchas veces el apósito puede no ser 100% estéril. Además, en caso de heridas con muchas capas de profundidad, el apósito solo cubre la capa externa y para poder hacer las curaciones, se requiere quitar el apósito cada cierto tiempo, incrementándose las probabilidades de sufrir una infección2. Burns, blows or certain congenital diseases can cause serious skin wounds. This type of wound is especially vulnerable to being infected by pathogens such as S. aureus and E co / i. These infections slow down the epithelial regeneration process and facilitate the incorrect deposition of collagen, worsening the patient's diagnosis 1 . The most used form of prevention is the use of dressings that act as barriers against microbes, however, many times the dressing may not be 100% sterile. In addition, in the case of wounds with many layers of depth, the dressing only covers the outer layer and in order to heal, it is necessary to remove the dressing from time to time, increasing the chances of suffering an infection 2 .
La ingeniería de tejidos es un campo que mezcla las áreas de biología celular, ingeniería de biomateriales y la medicina, con el fin de desarrollar estrategias que permitan la regeneración del tejido dañado, mediante injertos que reemplacen el tejido dañado, o a través de la liberación localizada de compuestos que faciliten la regeneración epitelial3. Para lograr esto, se necesitan materiales que puedan ser un soporte para las células del injerto o el compuesto terapéutico, que posean propiedades antimicrobianas para evitar infecciones y que sean biocompatibles. Dentro de este contexto, es que los hidrogeles poseen un gran potencial para ser aplicados en la regeneración de tejido epitelial ^5. Tissue engineering is a field that mixes the areas of cell biology, biomaterial engineering and medicine, in order to develop strategies that allow the regeneration of damaged tissue, through grafts that replace damaged tissue, or through localized release. of compounds that facilitate epithelial regeneration 3 . To achieve this, materials are needed that can be a support for the graft cells or the therapeutic compound, that possess antimicrobial properties to prevent infection, and that are biocompatible. Within this context, it is that hydrogels have great potential to be applied in the regeneration of epithelial tissue ^ 5 .
Los hidrogeles son estructuras poliméricas tridimensionales, que retiene grandes cantidades de líquido sin perder su estructura y se forman a partir del entrecruzamiento físico o químico de uno o más polímeros constituyentes. En la ingeniería de tejidos, los hidrogeles se han utilizado para encapsular células, proteínas i y otros compuestos bioactivos, para posteriormente liberarlos de forma localizada y de manera sistemática en el tejido dañado6'7'8. Biopolímeros naturales como el quitosano y almidón, han sido usados para sintetizar hidrogeles biocompatibles y útiles para ser utilizados en ingeniería de tejidos9,10. También existen algunos polímeros sintéticos, como el polivinil alcohol (PVA), polivinil pirrolidona (PVP) y polietilenglicol (PEG), que son biocompatibles y han sido usados para generar hidrogeles con aplicaciones biológicas11. Hydrogels are three-dimensional polymeric structures that retain large amounts of liquid without losing their structure and are formed from the physical or chemical crosslinking of one or more constituent polymers. In tissue engineering, hydrogels have been used to encapsulate cells, proteins, and and other bioactive compounds, to later release them locally and systematically in damaged tissue 6 ' 7 ' 8 . Natural biopolymers such as chitosan and starch have been used to synthesize biocompatible hydrogels that are useful for use in tissue engineering 9,10 . There are also some synthetic polymers, such as polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG), which are biocompatible and have been used to generate hydrogels with biological applications 11 .
Invenciones similares han sido propuestas y algunas de ellas patentadas, para su uso en la fabricación de lentes de contacto, de productos tipo apósitos para cubrir y tratar heridas, como soportes estructurales en la ingeniería de tejidos, como dispositivos para la liberación de fármacos, y como componentes ecológicos en productos de higiene. Similar inventions have been proposed and some of them patented, for use in the manufacture of contact lenses, dressing-type products to cover and treat wounds, as structural supports in tissue engineering, as drug delivery devices, and as ecological components in hygiene products.
En la tabla 1 se describen algunos hidrogeles usados como matriz de soporte, que han sido patentados y se encuentran a la venta. Tabla 1: Lista de hidrogeles usados como matriz de soporte disponibles comercialmente.12'13 Table 1 describes some hydrogels used as support matrix, which have been patented and are for sale. Table 1: List of commercially available hydrogels used as support matrix. 12 '13
Producto Material del hidrogel DescripciónProduct Hydrogel Material Description
Hidrogel con óxido de zinc y calamina incorporado,Hydrogel with incorporated zinc oxide and calamine,
Calamine-zinc gelatin Gelatina usado como apósito para heridas para disminuir dolor y picazón.Calamine-zinc gelatin Gelatin used as a wound dressing to decrease pain and itching.
Hidrogel con plataHydrogel with silver
ALGICELL Ag. Suprasorb incorporada, usada comoALGICELL Ag. Suprasorb incorporated, used as
Alginato A+Ag apósito de heridas con propiedad antimicrobiana.Alginate A + Ag wound dressing with antimicrobial property.
Hidrogel con polihexanida Prontosan Hidroetilcelulosa usada como apósito para heridas antiséptico.Hydrogel with polyhexanide Prontosan Hydroethylcellulose used as an antiseptic wound dressing.
Gel tópico con PGF recombinante humanoTopical gel with recombinant human PGF
REGRAN EX Ca rboxi m eti I cel u I osa incorporado para el tratamiento de úlceras diabéticas. REGRAN EX Carboxi m eti I cell u I ose incorporated for the treatment of ulcers diabetic
Hidrogel para elHydrogel for him
Pectina, tratamiento de heridas dePectin, wound treatment of
Granugel carboximetilcelulosa y espesor parcial y propilen glicol completas. Partial thickness carboxymethylcellulose granugel and full propylene glycol.
Hidrogel usado paraHydrogel used for
Carboximetilcelulosa y Carboxymethylcellulose and
Intrasite Gel heridas superficiales y propilen glicol profundas. Intrasite Gel for superficial wounds and deep propylene glycol.
Hidrogel indicado enHydrogel indicated in
Carboximetilcelulosa de conjunto con un apósitoCarboxymethylcellulose in conjunction with a dressing
Purilon Gel sodio secundario para heridas necróticas y quemaduras.Purilon Secondary sodium gel for necrotic wounds and burns.
Polietilenglicol y Hidrogel usado para llenarPolyethylene Glycol and Hydrogel used to fill
Aquaflo propilenglicol cavidades poco profundas. Hidrogel superabsorbenteAquaflo propylene glycol shallow cavities. Super absorbent hydrogel
Woundtab Carboximetilcelulosa y capaz de absorber glicerol bacterias y retenerlas en su estructura. Woundtab Carboxymethylcellulose and capable of absorbing glycerol bacteria and retaining them in its structure.
Como se puede observar en la tabla, ninguno de los hidrogeles comercialmente disponibles utiliza una combinación de polímeros que incluya compuestos que al degradarse potencie la re-epitelización1,4, favoreciendo la mejora del paciente. As can be seen in the table, none of the commercially available hydrogels uses a combination of polymers that includes compounds that, when degrading, enhance re-epithelialization 1,4 , favoring the improvement of the patient.
Referencias: References:
1. Jayakumar, R., Prabaharan, M., Kumar, P. S., Nair, S. V., & Tamura, H. (2011). Biomaterials based on chitin and chitosan ¡n wound dressing applications. Biotechnology advances, 25(3), 322-337. 1. Jayakumar, R., Prabaharan, M., Kumar, P. S., Nair, S. V., & Tamura, H. (2011). Biomaterials based on chitin and chitosan ¡n wound dressing applications. Biotechnology advances, 25 (3), 322-337.
2. Aoyagi, S., Onishi, H., & Machida, Y. (2007). Novel chitosan wound dressing loaded with minocydine for the treatment of severe burn wounds. International Journal of pharmaceutics, 330 12), 138-145. 2. Aoyagi, S., Onishi, H., & Machida, Y. (2007). Novel chitosan wound dressing loaded with minocydine for the treatment of severe burn wounds. International Journal of pharmaceutics, 330 12), 138-145.
3. Nicodemus, G. D., & Bryant, S. 1 (2008). Cell encapsulation in biodegradable hydrogels for tissue engineering applications. Tissue Engineering Part B: Reviews, 14(2), 149-165. 4. 5. Hoffman, A. S. (2012). Hydrogels for biomedical applications. Advanced drug delivery reviews, 64,3. Nicodemus, G. D., & Bryant, S. 1 (2008). Cell encapsulation in biodegradable hydrogels for tissue engineering applications. Tissue Engineering Part B: Reviews, 14 (2), 149-165. 4. 5. Hoffman, A. S. (2012). Hydrogels for biomedical applications. Advanced drug delivery reviews, 64,
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5. Lee, 1 H., 8i Kim, H. W. (2018). Emerging properties of hydrogels in tissue engineering. Journal of tissue engineering, 9, 2041731418768285. 6. Wu, X., Black, L, Santacana-Laffitte, G., & Patrick Jr, C. W. (2007). Preparation and assessment of glutaraldehyde-crosslinked collagen-chitosan hydrogels for adipose tissue engineering. Journal of biomedicaí meteríais research Part A, 81(1), 59-65. 5. Lee, 1 H., 8i Kim, HW (2018). Emerging properties of hydrogels in tissue engineering. Journal of tissue engineering, 9, 2041731418768285. 6. Wu, X., Black, L, Santacana-Laffitte, G., & Patrick Jr, CW (2007). Preparation and assessment of glutaraldehyde-crosslinked collagen-chitosan hydrogels for adipose tissue engineering. Journal of biomedical research Part A, 81 (1), 59-65.
7. Sudheesh Kumar, P. T., Lakshmanan, V. K., Anilkumar, T. V., Ramya, C, Reshmi, P., Unnikrishnan, A. G., & Jayakumar, R. (2012). Flexible and mlcroporous chltosan hydrogel/nano ZnO composlte bandages for wound dresslng: In vltro and ¡n vivo evaluatlon. ACS apptied materials & interfaces, 5), 2618-2629. 7. Sudheesh Kumar, P. T., Lakshmanan, V. K., Anilkumar, T. V., Ramya, C, Reshmi, P., Unnikrishnan, A. G., & Jayakumar, R. (2012). Flexible and mlcroporous chltosan hydrogel / nano ZnO composlte bandages for wound dresslng: In vltro and ¡n vivo evaluatlon. ACS apptied materials & interfaces, 5), 2618-2629.
8. Huang, N., Un, J., Ll, S., Deng, Y., Kong, S., Hong, P. 8i Hu, Z. (2018). Preparation and evaluatlon of squld ¡nk polysaccharlde-chltosan as a wound-heallng sponge. Materials Science and Engineering: C, 82, 354-362. 8. Huang, N., Un, J., Ll, S., Deng, Y., Kong, S., Hong, P. 8i Hu, Z. (2018). Preparation and evaluatlon of squld ¡nk polysaccharlde-chltosan as a wound-heallng sponge. Materials Science and Engineering: C, 82, 354-362.
9. Ismall, H., Iraní, M., 8i Ahmad, Z. (2013). Starch-based hydrogels: present status and appllcatlons. International Journal ofPoiymeric Materials and Poiymeric Biomateriais, 62(7), 411420. 9. Ismall, H., Iranian, M., 8i Ahmad, Z. (2013). Starch-based hydrogels: present status and appllcatlons. International Journal of Poiymeric Materials and Poiymeric Biomateriais, 62 (7), 411420.
10. Anltha, A., Rejinold, N. S.,Bumgardner, J. D., Nair, S. V., 8i Jayakumar, R. (2012). Approaches for functlonal modlflcatlon or cross-llnklng of chltosan. Chitosan-based systems for biopharmaceuticais: deiivery, targeting and poiymer therapeutics, 1, 108-124. 10. Anltha, A., Rejinold, N. S., Bumgardner, J. D., Nair, S. V., 8i Jayakumar, R. (2012). Approaches for functlonal modlflcatlon or cross-llnklng of chltosan. Chitosan-based systems for biopharmaceuticais: deiivery, targeting and poiymer therapeutics, 1, 108-124.
11. Ool, H. W., Hafeez, S., Van Blltterswljk, C. A., Moronl, L., 8i Baker, M. B. (2017). Hydrogels that listen to cells: a revlew of cell-responslve strategles ¡n blomaterlal deslgn for tissue regeneratlon. Materials Horizons, 4(6), 1020-1040. 11. Ool, H. W., Hafeez, S., Van Blltterswljk, C. A., Moronl, L., 8i Baker, M. B. (2017). Hydrogels that listen to cells: a revlew of cell-responslve strategles ¡n blomaterlal delgn for tissue regeneratlon. Materials Horizons, 4 (6), 1020-1040.
12. Caló, E., 8i Khutoryanskly, V. V. (2015). Biomedicaí appllcatlons of hydrogels: A revlew of patents and commerdal producís. European Poiymer Journal, 65, 252-267. 12. Caló, E., 8i Khutoryanskly, V. V. (2015). Biomedicaí appllcatlons of hydrogels: A revlew of patents and commerdal producís. European Poiymer Journal, 65, 252-267.
13. Ll, J., 8i Mooney, D. J. (2016). Deslgnlng hydrogels for controlled drug deiivery. Nature Reviews Materials, 1(12), 16071. 13. Ll, J., 8i Mooney, D. J. (2016). Deslgnlng hydrogels for controlled drug deiivery. Nature Reviews Materials, 1 (12), 16071.
14. Tsao, C. T., Chang, C. H., Li, Y. D., Wu, M. F., Un, C. P., Han, J. L, ... & Hsieh, K. H. (2011). Development of chltosan/dlcarboxyllc acid hydrogels as wound dresslng materlals. Journal of Bloactlve and Compatible Polymers, 26(5), 519-536. 14. Tsao, C. T., Chang, C. H., Li, Y. D., Wu, M. F., Un, C. P., Han, J. L, ... & Hsieh, K. H. (2011). Development of chltosan / dlcarboxyllc acid hydrogels as wound dresslng materlals. Journal of Bloactlve and Compatible Polymers, 26 (5), 519-536.
Breve Descripción De Las Figuras Brief Description Of The Figures
Figura 1: Fotografías de la estructura macroscópica y microscópica de los hidrogeles multipolimérico (A-C) y quitosano-almidón (D-F), obtenidos por síntesis química. Figure 1: Photographs of the macroscopic and microscopic structure of the multipolymeric hydrogels (A-C) and chitosan-starch (D-F), obtained by chemical synthesis.
Figura 2: Gráfica de los porcentajes de viabilidad celular obtenidos mediante el ensayo de MTT en la línea celular HEP2, incubada con fragmentos de los hidrogeles. Cada muestra se estudió por triplicado y los resultados indican que ninguno de los hidrogeles tuvo efectos negativos significativos en la viabilidad de la línea celular. Figure 2: Graph of the cell viability percentages obtained by the MTT assay in the HEP2 cell line, incubated with fragments of the hydrogels. Each sample was studied in triplicate and the results indicate that none of the hydrogels had significant negative effects on the viability of the cell line.
Figura 3: Caracterización de los hidrogeles en cuanto a capacidad de hinchamiento. Las muestras fueron incubadas en tampón fosfato alcalino (PBS) y analizadas a diferentes tiempos durante 24 horas. Figura 4: Microfotog rafias de los hidrogeles antes y después de la incorporación de nanopartículas de cobre y análisis elemental mediante energía dispersiva (EDX) para la detección del cobre en los hidrogeles. Quitosano-almidón (A-B), multipolimérico (C-D). El análisis por EDX permitió observar la presencia de cobre en los hidrogeles después de ser incubados con las nanopartículas. Figure 3: Characterization of the hydrogels in terms of swelling capacity. The samples were incubated in alkaline phosphate buffer (PBS) and analyzed at different times for 24 hours. Figure 4: Microfotog raffles of the hydrogels before and after the incorporation of copper nanoparticles and elemental analysis by energy dispersive (EDX) for the detection of copper in the hydrogels. Chitosan-starch (AB), multipolymeric (CD). The EDX analysis allowed to observe the presence of copper in the hydrogels after being incubated with the nanoparticles.
Figura 5: Espectros infrarrojos de los hidrogeles obtenidos por espectroscopia infrarroja por transformada de Fourier (FTIR). A: tensión de los grupos N-H, visible en el hidrogel quitosano-almidón; (B) formación del enlace C=C, por posible entrecruzamiento, visible en el hidrogel multipolimérico; (C) O-H y tensión C-O-C, proveniente del almidón, visible en ambos hidrogeles. Figure 5: Infrared spectra of hydrogels obtained by Fourier transform infrared spectroscopy (FTIR). A: tension of the N-H groups, visible in the chitosan-starch hydrogel; (B) formation of the C = C bond, by possible crosslinking, visible in the multipolymeric hydrogel; (C) O-H and C-O-C tension, from starch, visible in both hydrogels.
Figura 6: Curso temporal de la cicatrización de la herida en ratones tratados con diferentes hidrogeles. 1+Cu: hidrogel quitosano-almidón cargado con nanopartículas de cobre; 1-Cu: hidrogel quitosano-almidón; 2+Cu: hidrogel multipolimérico cargado con nanopartículas de cobre, 2-Cu: hidrogel multipolimérico, control negativo: ratones con heridas sin tratamiento con hidrogel. Se grafican los resultados promediados y su desviación estándar para los días 0, 3, 6, 9 y 13 posteriores al desarrollo de la herida. Figure 6: Time course of wound healing in mice treated with different hydrogels. 1 + Cu: chitosan-starch hydrogel loaded with copper nanoparticles; 1-Cu: chitosan-starch hydrogel; 2 + Cu: multipolymeric hydrogel loaded with copper nanoparticles, 2-Cu: multipolymeric hydrogel, negative control: mice with wounds without hydrogel treatment. The averaged results and their standard deviation are plotted for days 0, 3, 6, 9, and 13 after wound development.
Divulgación De La Invención Disclosure of Invention
La presente tecnología consiste en un dispositivo médico, que permite la aplicación de terapias farmacéuticas y celulares, de manera controlada en el tiempo, y centralizada en el sitio de aplicación, al servir como soporte y protección al componente terapéutico. Este dispositivo está formado por una única pieza: una matriz de hidrogel multipolimérica, con capacidad de hinchamiento y absorción de biocompuestos con propiedades terapéuticas, que resulta biocompatible y biodegradable en condiciones fisiológicas. Además, el soporte tiene la capacidad de absorber exudados en heridas y favorece su cicatrización, reduciendo la inflamación. The present technology consists of a medical device, which allows the application of pharmaceutical and cellular therapies, in a controlled manner over time, and centralized in the application site, by serving as support and protection for the therapeutic component. This device consists of a single piece: a multipolymeric hydrogel matrix, with the ability to swell and absorb biocomposites with therapeutic properties, which is biocompatible and biodegradable under physiological conditions. In addition, the support has the ability to absorb exudates in wounds and favors their healing, reducing inflammation.
El principio terapéutico que se quiera administrar debe ser adicionado al dispositivo, antes de su aplicación, siendo absorbido y retenido por la red polimérica y posteriormente liberado en el sitio de interés. El componente activo a integrar puede ser una nanopartícula, un antibiótico, un fármaco, un estimulador del crecimiento o regenerador celular, o directamente células del paciente, como células madre, que se incorporan al hidrogel y que lo usan como soporte para su multiplicación y formación de nuevos tejidos. The therapeutic principle to be administered must be added to the device, before its application, being absorbed and retained by the polymeric network and subsequently released at the site of interest. The active component to be integrated can be a nanoparticle, an antibiotic, a drug, a growth stimulator or cell regenerator, or directly cells from the patient, such as stem cells, which are They incorporate the hydrogel and use it as a support for their multiplication and formation of new tissues.
La matriz polimérica que forma el dispositivo posee características que lo hacen idóneo para la absorción, retención y liberación de células y fármacos. The polymeric matrix that forms the device has characteristics that make it ideal for the absorption, retention and release of cells and drugs.
En particular el hidrogel comprende la inclusión de los siguientes compuestos: a) 2 - 4 % de Quitosano; b) 0,01 - 4 % de Pol ivi ni l-alcohol ; c) 2 - 4 % de Almidón; d) 0,01 - 4% de Polietilenglicol; e) 250 - 500 pL de agente entrecruzante; y f) 80 — 92% de solución acuosa ligeramente ácida. In particular, the hydrogel comprises the inclusion of the following compounds: a) 2-4% Chitosan; b) 0.01-4% Poly ivi ni l-alcohol; c) 2-4% Starch; d) 0.01-4% Polyethylene Glycol; e) 250-500 pL of crosslinking agent; and f) 80-92% slightly acidic aqueous solution.
El agente entrecruzante utilizado es, de forma preferencial, glutaraldehido o genipina, y la solución acuosa se utiliza a pH 2,0. The crosslinking agent used is preferably glutaraldehyde or genipin, and the aqueous solution is used at pH 2.0.
La presencia de estos polímeros garantiza la interacción electrostática con los componentes bioactivos, a la vez que permite el paso de sustancias y células por toda la estructura, mediante la formación de canales de diámetro superior a 100 pm. El enrejado que forma la matriz establece un alto nivel de retención, mientras que mecanismos asociados a los polímeros que la integran, como la sensibilidad a pH, a hidrólisis y a la acción enzimática, garantizan la biodegradabilidad y posterior liberación del material retenido. Los diferentes polímeros aportan también características químicas y estructurales que en algunos casos se refuerzan y en otros se complementan, proporcionando propiedades a la matriz tales como: flexibilidad, rigidez, actividad antimicrobiana, y elevada capacidad de hinchamiento, sin que pierda su estructura tridimensional, y una alta fortaleza y resistencia a presión/impacto, sin que se pierdan sus propiedades de biodegradabilidad. The presence of these polymers guarantees electrostatic interaction with the bioactive components, while allowing the passage of substances and cells throughout the structure, through the formation of channels with a diameter greater than 100 pm. The lattice that forms the matrix establishes a high level of retention, while mechanisms associated with the polymers that comprise it, such as sensitivity to pH, hydrolysis and enzymatic action, guarantee the biodegradability and subsequent release of the retained material. The different polymers also provide chemical and structural characteristics that in some cases are reinforced and in others they complement each other, providing properties to the matrix such as: flexibility, rigidity, antimicrobial activity, and high swelling capacity, without losing its three-dimensional structure, and high strength and resistance to pressure / impact, without losing its biodegradability properties.
En la industria farmacéutica es necesario contar con formulaciones o dispositivos que permitan una entrega controlada del agente activo, esta tecnología posibilita un efecto terapéutico más eficiente de los fármacos o componentes celulares a administrar, al funcionar como un dispositivo de absorción y protección del agente terapéutico. El componente activo se libera de manera controlada en el tiempo, lo que garantiza una mayor disponibilidad del mismo, y una actividad persistente o sostenida, a la vez que puede reducir la ocurrencia de efectos secundarios/adversos como infecciones (por su naturaleza o propiedad antimicrobiana). De igual manera, el agente terapéutico estará protegido ante la acción de elementos externos como enzimas proteolíticas y células del sistema inmunitario que contribuyan a su degradación y aclaramiento, estará contenido en un soporte con determinado grado de rigidez estructural, lo que garantiza su permanencia en el sitio de aplicación, y su actividad se reforzará por el efecto antimicrobiano del hidrogel, que inhibe la ocurrencia de infecciones en el sitio de aplicación. Las características biodegradables y biocompatibles del dispositivo, posibilitan su eliminación metabólica gradual, sin que surja ningún tipo de reacción tóxica asociada al mismo In the pharmaceutical industry, it is necessary to have formulations or devices that allow a controlled delivery of the active agent, this technology enables a more efficient therapeutic effect of the drugs or cellular components to be administered, by functioning as an absorption and protection device for the agent. therapeutic. The active component is released in a controlled way over time, which guarantees a greater availability of it, and a persistent or sustained activity, while it can reduce the occurrence of side / adverse effects such as infections (due to its nature or antimicrobial property ). In the same way, the therapeutic agent will be protected against the action of external elements such as proteolytic enzymes and cells of the immune system that contribute to its degradation and clearance, it will be contained in a support with a certain degree of structural rigidity, which guarantees its permanence in the site of application, and its activity will be enhanced by the antimicrobial effect of the hydrogel, which inhibits the occurrence of infections at the site of application. The biodegradable and biocompatible characteristics of the device allow its gradual metabolic elimination, without any type of toxic reaction associated with it.
La innovación se encuentra en etapa de investigación y desarrollo avanzada. Se han sintetizado los hidrogeles prototipo, se han caracterizado en cuanto a morfología, resistencia mecánica y composición química, capacidad de absorción y retención de compuestos químicos. Específicamente, se realizaron estudios para la carga de nanopartículas de cobre, cuya presencia ha sido comprobada mediante análisis de dispersión de rayos X. De igual manera se estudió el efecto de los hidrogeles en el proceso de cicatrización de heridas en un modelo de ratón, con resultados evidentes en la aceleración del proceso de cicatrización y la reducción de la inflamación. The innovation is in the advanced research and development stage. The prototype hydrogels have been synthesized, they have been characterized in terms of morphology, mechanical resistance and chemical composition, absorption capacity and retention of chemical compounds. Specifically, studies were carried out for the loading of copper nanoparticles, the presence of which has been verified by X-ray scattering analysis. Similarly, the effect of hydrogels on the wound healing process was studied in a mouse model, with Obvious results in accelerating the healing process and reducing inflammation.
Ejemplos De Aplicación Application Examples
Ejemplo 1: Elaboración del hidrogel. Example 1: Preparation of the hydrogel.
La composición y la metodología de obtención del hidrogel se realizan mediante disolución escalonada en una combinación única de los polímeros: almidón, polietilen- glicol, polivinil-alcohol y quitosano, en disueltos en una solución acuosa al 2% de ácido acético. Posteriormente se le agrega una cantidad conocida de un agente entrecruzante para producir el entrecruzamiento de los polímeros en el hidrogel. Este método químico de obtención del hidrogel es combinado con un método físico, pues a la mezcla se le realizan varios ciclos de congelación y descongelación, y es posteriormente es liofilizada al vacío. Como ejemplo desarrollamos dos hidrogeles, uno rnultipolimérico que incluye los cuatro polímeros y uno solo con quitosano-almidón (Tabla 2). The composition and methodology for obtaining the hydrogel are carried out by step dissolution in a unique combination of polymers: starch, polyethylene glycol, polyvinyl alcohol and chitosan, dissolved in a 2% aqueous solution of acetic acid. Subsequently, a known amount of a crosslinking agent is added to it to cause crosslinking of the polymers in the hydrogel. This chemical method of obtaining the hydrogel is combined with a physical method, since the mixture undergoes several freezing and thawing cycles, and it is subsequently lyophilized under vacuum. As an example we develop two hydrogels, one rnultipolymeric that includes the four polymers and only one with chitosan-starch (Table 2).
Tabla 2: Composición de hidrogeles.
Figure imgf000009_0001
Table 2: Composition of hydrogels.
Figure imgf000009_0001
Ejemplo 2: Caracterización de los hidrogeles. Example 2: Characterization of hydrogels.
Se evaluó la estructura macro y microscópica de la matriz de los hidrogeles mediante Microscopía Electrónica de Barrido JEOL JSM-6380 LV (USA), en un equipo que, además, acopla un detector de fluorescencia de rayos X por energía dispersiva (EDX) para estudiar la morfología y matriz polimérica de los mismos. Se observó una estructura uniforme para ambas alternativas, con una morfología de redes bien definidas y homogéneas (Figura 1) The macro and microscopic structure of the hydrogel matrix was evaluated by means of Scanning Electron Microscopy JEOL JSM-6380 LV (USA), in a device that, in addition, coupled an energy dispersive X-ray fluorescence detector (EDX) to study their morphology and polymeric matrix. A uniform structure was observed for both alternatives, with a well-defined and homogeneous network morphology (Figure 1)
Ambos hidrogeles resultaron inocuos para la línea celular humana (Hep 2). Lo que confirma la biocompatibilidad de los productos que lo conforman, aún en las condiciones de asociación en que generaron ambos dispositivos (Figura 2). Both hydrogels were innocuous for the human cell line (Hep 2). This confirms the biocompatibility of the products that comprise it, even under the association conditions in which both devices were generated (Figure 2).
El hidrogel de quitosano-almidón presentó una capacidad de hinchamiento cercana a 10 veces el incremento de su peso seco inicial, mientras que el hidrogel rnultipolimérico duplicó su peso seco al incorporar la solución acuosa (Figura 3). Además, se evaluó la capacidad de ambos hidrogeles para incorporar nanopartículas de cobre como un componente activo, que contribuye a la cicatrización de heridas. Los análisis EDX demostraron que después de 12 horas en la solución de PBS con nanopartículas de cobre (CuNps), los hidrogeles retuvieron estas nanopartículas, ya que se observa una emisión de cobre (Figura 4B y D). The chitosan-starch hydrogel presented a swelling capacity close to 10 times the increase of its initial dry weight, while the multipolymeric hydrogel doubled its dry weight when incorporating the aqueous solution (Figure 3). In addition, the ability of both hydrogels to incorporate copper nanoparticles as an active component, which contributes to wound healing, was evaluated. The EDX analyzes showed that after 12 hours in the PBS solution with copper nanoparticles (CuNps), the hydrogels retained these nanoparticles, since a copper emission is observed (Figure 4B and D).
La caracterización de grupos funcionales se realizó mediante espectroscopia infrarroja con transformación de Fourier (FTIR). Los espectros infrarrojos fueron obtenidos utilizando un equipo Nicolet, Nexus FTIR con detector ATR (EEUU) y Nicolet Lexus 470 con detector PAS (EEUU), usando como blanco una pastilla de KBr. Las muestras fueron analizadas a temperatura ambiente en un rango de 4000-500 cm-1. Se identificaron los grupos aminos provenientes de los distintos componentes individuales de ambos hidrogeles, así como la formación de triples enlaces lo que demuestra que los dispositivos están conformados por sus componentes individuales entrecruzados químicamente (Figura 5). The characterization of functional groups was carried out using Fourier transform infrared spectroscopy (FTIR). Infrared spectra were obtained using a Nicolet, Nexus FTIR with ATR detector (USA) and Nicolet Lexus 470 with PAS detector (USA), using a KBr pellet as a target. The samples were analyzed at room temperature in a range of 4000-500 cm-1. The amino groups from the different individual components of both hydrogels were identified, as well as the formation of triple bonds, which shows that the devices are made up of their individual chemically cross-linked components (Figure 5).
Ejemplo 3: Evaluación del efecto de los hidrogeles en la cicatrización de heridas. Example 3: Evaluation of the effect of hydrogels on wound healing.
Se realizaron ensayos de cicatrización de heridas en modelos murinos, usando ratones hembras CF1 de 6 semanas de vida, con heridas dérmicas de 6 mm de diámetro. Se generaron grupos de animales con n=5, a los que se les aplicaron hidrogeles de 6 mm de diámetro por 1,5 mm de altura. Se realizó una aplicación única de cada hidrogel.Wound healing assays were performed in murine models, using 6-week-old female CF1 mice with 6-mm diameter dermal wounds. Groups of animals with n = 5 were generated, to which hydrogels of 6 mm in diameter by 1.5 mm in height were applied. A single application of each hydrogel was made.
Grupo 1+Cu: Hidrogel quitosano-almidón cargado con nanopartículas de cobre. Group 1 + Cu: Chitosan-starch hydrogel loaded with copper nanoparticles.
Grupo 1-Cu: Hidrogel quitosano-almidón. Group 1-Cu: Chitosan-starch hydrogel.
Grupo 2+Cu: Hidrogel multipolimérico cargado con nanopartículas de cobre. Group 2 + Cu: Multipolymeric hydrogel loaded with copper nanoparticles.
Grupo 2-Cu: Hidrogel multipolimérico. Group 2-Cu: Multipolymeric hydrogel.
Grupo control neg.: Control de animales sin tratamiento con hidrogel. Negative control group: Control of animals without hydrogel treatment.
Se tomaron fotografías individuales de las heridas a los tiempos: día 0, 3, 6, 9, 13 y se midió el diámetro de las heridas en milímetros. Los resultados obtenidos fueron promediados para realizar la comparación entre los grupos de ratones (Figura 6). Individual photographs of the wounds were taken at times: day 0, 3, 6, 9, 13 and the diameter of the wounds was measured in millimeters. The results obtained were averaged to make the comparison between the groups of mice (Figure 6).
Se observó que los hidrogeles disminuyen el tiempo de cicatrización en comparación con el control negativo sin hidrogel, basado en dos factores: 1) los grupos tratados con hidrogel disminuyeron el tamaño de las heridas en todos los días analizados, mientras que el grupo control no tratado presentó un incremento en el tamaño de la herida y una mayor inflamación (Figura 6, días 3 y 6). 2) El 77,8 % de los animales tratados con hidrogel cicatrizó totalmente antes del día 13, mientras que de los ratones del grupo control, sólo el 40 % cicatrizó antes del día 13. Hydrogels were observed to decrease healing time compared to the negative control without hydrogel, based on two factors: 1) the groups treated with hydrogel decreased the size of the wounds on all the days analyzed, while the untreated control group presented an increase in the size of the wound and greater inflammation (Figure 6, days 3 and 6). 2) 77.8% of the animals treated with hydrogel completely healed before day 13, while of the mice in the control group, only 40% healed before day 13.
Se observó una disminución de los efectos de inflamación y enrojecimiento de las heridas, así como la no aparición de exudados, cuando estaban presentes los hidrogeles. Esto contribuyó a la formación de una costra protectora en las heridas, que disminuye infecciones e incrementa la cicatrización. A decrease in the effects of inflammation and redness of the wounds, as well as the non-appearance of exudates, was observed when the hydrogels. This contributed to the formation of a protective scab on the wounds, which reduces infections and increases healing.
El cobre no parece generar por sí solo una respuesta tóxica en los ratones, ya que no hubo rascado en los ratones del grupo 1+Cu. El hidrogel de quitosano-almidón con y sin cobre, mostró los mejores resultados conductuales y de regeneración celular de los ratones. Copper does not appear to generate a toxic response by itself in the mice, as there was no scratching in the 1 + Cu mice. The chitosan-starch hydrogel with and without copper showed the best behavioral and cell regeneration results in the mice.

Claims

REIVINDICACIONES
1. Un hidrogel multipolimérico como dispositivo médico para la administración de componentes terapéuticos farmacéuticos y celulares CARACTERIZADO porque comprende al menos los siguientes componentes: a. 2-4 % de Quitosano; b. 0,01-4% de Pol ivi ni l-alcohol ; c. 2-4% de Almidón; d. 0,01-4% de Polietilenglicol; e. 250 - 500 pL de agente entrecruzante; y f. 80 - 92% de solución acuosa ligeramente ácida. 1. A multipolymeric hydrogel as a medical device for the administration of pharmaceutical and cellular therapeutic components CHARACTERIZED because it comprises at least the following components: a. 2-4% Chitosan; b. 0.01-4% Pol ivi ni l-alcohol; c. 2-4% Starch; d. 0.01-4% Polyethylene Glycol; and. 250-500 pL of crosslinking agent; and f. 80-92% slightly acidic aqueous solution.
2. Un hidrogel multipolimérico, según reivindicación 1, CARACTERIZADO porque el agente entrecruzante utilizado es preferentemente glutaraldehido o genipina; y la solución acuosa se utiliza a pH 2,0. 2. A multipolymeric hydrogel, according to claim 1, CHARACTERIZED in that the crosslinking agent used is preferably glutaraldehyde or genipin; and the aqueous solution is used at pH 2.0.
3. Un hidrogel multipolimérico, según reivindicación 1, CARACTERIZADO porque está formado por una única pieza con capacidad de hinchamiento y absorción de biocompuestos con propiedades terapéuticas. 3. A multipolymeric hydrogel, according to claim 1, CHARACTERIZED in that it is formed by a single piece with the ability to swell and absorb biocomposites with therapeutic properties.
4. Un hidrogel multipolimérico, según reivindicación 1, CARACTERIZADO porque presenta una forma del tipo enrejado con canales de diámetro superior a 100 pm, que permiten la interacción electrostática con los componentes bioactivos, y permite el paso de sustancias y células por toda la estructura. 4. A multipolymeric hydrogel, according to claim 1, CHARACTERIZED in that it has a lattice-type shape with channels of diameter greater than 100 pm, which allow electrostatic interaction with the bioactive components, and allows the passage of substances and cells throughout the structure.
5. Un hidrogel multipolimérico, según reivindicación 1, CARACTERIZADO porque posee propiedades como: flexibilidad, rigidez, actividad antimicrobiana, y elevada capacidad de hinchamiento, sin que pierda su estructura tridimensional, y una alta fortaleza y resistencia a presión/impacto, sin que se pierdan sus propiedades de biodegradabilidad. 5. A multipolymeric hydrogel, according to claim 1, CHARACTERIZED because it has properties such as: flexibility, rigidity, antimicrobial activity, and high swelling capacity, without losing its three-dimensional structure, and high strength and resistance to pressure / impact, without being lose their biodegradability properties.
6. Un hidrogel multipolimérico, según reivindicación 1, CARACTERIZADO porque el principio terapéutico que se quiera administrar es adicionado al dispositivo, antes de su aplicación, siendo absorbido y retenido por la red polimérica y posteriormente liberado en el sitio de interés 6. A multipolymeric hydrogel, according to claim 1, CHARACTERIZED in that the therapeutic principle to be administered is added to the device, before its application, being absorbed and retained by the polymeric network and subsequently released in the site of interest
7. Un hidrogel multipolimérico, según reivindicación 1, CARACTERIZADO porque el principio terapéutico puede ser una nanopartícula, un antibiótico, un fármaco, un estimulador del crecimiento o regenerador celular, o directamente células del paciente, como células madre. 7. A multipolymeric hydrogel, according to claim 1, CHARACTERIZED in that the therapeutic principle can be a nanoparticle, an antibiotic, a drug, a growth stimulator or cell regenerator, or directly cells from the patient, such as stem cells.
8. Un hidrogel multipolimérico, según reivindicación 1, CARACTERIZADO porque biocompatible y biodegradable en condiciones fisiológicas. 8. A multipolymeric hydrogel, according to claim 1, CHARACTERIZED because it is biocompatible and biodegradable under physiological conditions.
9. Uso del hidrogel multipolimérico, según reivindicación 1, CARACTERIZADO porque su utilización permite la aplicación de terapias farmacéuticas y celulares, de manera controlada en el tiempo, y centralizada en el sitio de aplicación, al servir como soporte y protección al componente terapéutico. 9. Use of the multipolymeric hydrogel, according to claim 1, CHARACTERIZED in that its use allows the application of pharmaceutical and cellular therapies, in a controlled manner over time, and centralized in the application site, by serving as support and protection for the therapeutic component.
10. Uso del hidrogel multipolimérico, según reivindicación 9, CARACTERIZADO porque su utilización permite absorber exudados en heridas y favorece su cicatrización, reduciendo la inflamación. 10. Use of the multipolymeric hydrogel, according to claim 9, CHARACTERIZED in that its use allows the absorption of exudates in wounds and favors their healing, reducing inflammation.
11. Uso del hidrogel multipolimérico, según reivindicación 9, CARACTERIZADO porque su utilización permite que el componente activo se libera de manera controlada en el tiempo, y permite reducir la ocurrencia de efectos secundarios/adversos como infecciones, por su naturaleza o propiedad antimicrobianas. 11. Use of the multipolymeric hydrogel, according to claim 9, CHARACTERIZED in that its use allows the active component to be released in a controlled manner over time, and allows reducing the occurrence of secondary / adverse effects such as infections, due to its antimicrobial nature or property.
12. Uso del hidrogel multipolimérico, según reivindicación 9, CARACTERIZADO porque su utilización permite proteger al agente terapéutico de la acción de elementos externos como enzimas proteolíticas y células del sistema inmunitario que contribuyan a su degradación y aclaramiento. 12. Use of the multipolymeric hydrogel, according to claim 9, CHARACTERIZED in that its use allows the therapeutic agent to be protected from the action of external elements such as proteolytic enzymes and cells of the immune system that contribute to its degradation and clearance.
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