WO2023084140A1 - Hydrogels ayant des domaines dendritiques de type carbosilane, leur préparation et leurs utilisations - Google Patents

Hydrogels ayant des domaines dendritiques de type carbosilane, leur préparation et leurs utilisations Download PDF

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WO2023084140A1
WO2023084140A1 PCT/ES2022/070728 ES2022070728W WO2023084140A1 WO 2023084140 A1 WO2023084140 A1 WO 2023084140A1 ES 2022070728 W ES2022070728 W ES 2022070728W WO 2023084140 A1 WO2023084140 A1 WO 2023084140A1
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hydrogel
dendritic
hydrogels
molecule
biologically active
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Sandra GARCÍA GALLEGO
Francisco Javier De La Mata De La Mata
Andrea BARRIOS GUMIEL
Judith RECIO RUIZ
Silvia MUÑOZ SÁNCHEZ
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Universidad De Alcalá
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • 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
    • 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
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/003—Dendrimers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/003—Dendrimers
    • C08G83/004—After treatment of dendrimers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels

Definitions

  • the present invention refers to hydrogels that have crosslinking points formed by dendritic macromolecules that contain a skeleton of carbosilane nature.
  • the materials of the invention are versatile agents for the transport and release of molecules with biological activity, as well as antimicrobial agents per se, and are useful for their application in medicine and cosmetics.
  • Hydrogels are hydrophilic polymeric networks with numerous applications in the field of biomedicine, due to their great biocompatibility, porosity and high water content (Caló E., et al., Eur. Polym. J. 2015, 65, 252-267 ). Hydrogels can be reversible, whose networks are formed by molecular entanglement and/or secondary forces, or chemically stable, where network formation is accomplished by covalent bonding. Traditionally, hydrogels are prepared by three-dimensional polymerization of monomers or crosslinking of already formed polymers. The polymerization technique often results in materials containing significant levels of residual monomers, requiring long purification processes to avoid the possible sustained release of toxic compounds.
  • hydrogels are excellent platforms for sustained and controlled drug release.
  • hydrophilic drugs examples include amphiphilic hydrogels, with hydrophobic and hydrophilic domains in the same structure, capable of transporting molecules of both natures, are few in the bibliography (Larra ⁇ eta E., et al. J. Funct. Biomater. 2018, 9, 13).
  • endowing the hydrogels with a capacity to respond to a stimulus makes it possible to successfully control the release of the drug.
  • thermosensitive hydrogels undergo reversible swelling or collapse in response to local temperature alteration, which can be exploited to modulate drug release (Zhang K., et al., Ge/s, 2021, 7, 3, 77).
  • these hydrogels include those based on polymers such as poloxamer (Pluronic®), poly(N-isopropylacrylamide) (polyNIPAAm), polycaprolactone (PCL), polyglycolate (PGA) and polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the main problem with these "responsive" polymers is their poor mechanical properties, such as low tensile strength and low Young's modulus, which limit their practical applications.
  • Increasing the density of the crosslink, copolymerization with other monomers, and incorporation into other composite materials are several methods that improve the mechanical strength of the polymer, although they may negatively reduce the stimulus-response properties.
  • Nanotechnology has been integrated into the development process of hydrogels in order to improve their properties, in relation to the characteristics of the material and its formulation.
  • These nanosystems include micelles, metal nanoparticles, liposomes, carbon nanotubes, and dendritic systems, among others.
  • dendrimers are highly branched structures of the polymeric type with a monodisperse and globular structure, whose controlled synthesis allows establishing a unique structure-reactivity relationship between nanoscopic systems.
  • the multivalent, multifunctional and monodisperse nature of dendrimers and dendrons as defined building units in hydrogels offers important advantages (Sanyal A., et al., Molecules, 2016, 21, 497).
  • the dendrimers used mainly comprise backbones of the polyamide (PAMAM, polylysine), polyamine (PPI), polyether (PBzE) or polyester (bisMPA, glycerol) type.
  • PAMAM polyamide
  • PPI polyamine
  • PBzE polyether
  • bisMPA bisMPA, glycerol
  • carbosilane-type dendritic systems have proven to be very versatile with promising applications as transport agents for nucleic material and/or drugs, as antibacterial, anticancer or antiviral agents, among others (Serramia MJ, et al., Journal of Controlled Release 2015, 200, 60-70, Milowska, K. et al., International Journal of Pharmaceutics 2015, 484, 268-275; Sanchez-Rodriguez J., et al., Journal of Biomedical Nanotechnology 2015, 11, 1783-1798; Fuentes-Paniagua E., et al., Rsc Advances
  • the present invention includes covalent hydrogels with dendritic domains of carbosilane structure, whose skeleton is made up of silicon-carbon and carbon-carbon bonds.
  • Dendritic systems act as multivalent crosslinking sites that generate networks by reacting with hydrophilic or amphiphilic polymers.
  • the family of dendritic polymers significantly increases the control of the properties of the networks formed.
  • the invention also provides a procedure for obtaining the hydrogels of the present invention and their uses in the medical and cosmetic area, since the amphiphilic hydrogels, which comprise dendritic domains of carbosilane nature, of the present invention represent an interesting alternative for the use in the pharmaceutical and cosmetic industry.
  • the hydrogels of the present invention refer to a type of polymer-based material characterized by its ability to absorb water or different fluids and that are useful in the pharmaceutical industry, and can be used as new functional materials, for example, for the treatment of microbial diseases or for the transport and release of drugs or bioactive molecules.
  • a first aspect of the present invention refers to a hydrogel comprising a dendritic molecule of carbosilane nature with terminal alkene groups and a functionalized hydrophilic or amphiphilic polymer with two terminal thiol groups directly cross-linked with each other covalently through a thioether bond.
  • the dendritic molecule of carbosilane nature is selected from among a dendrimer and a dendron with a core or polyfunctional focal point, respectively, and further comprising an outer layer consisting of the same or different units of the group of formula (I): where: n is an integer ranging from 0 to 4, preferably n is 0 or 1;
  • Ri is a (C1-C4)alkyl group, preferably R1 is CH3 ; and m is an integer ranging from 1 to 3, preferably m is 2.
  • dendritic molecule in the present invention a highly branched macromolecule where the growth units, branches or ramifications have a carbosilane skeleton.
  • This dendritic molecule of carbosilane nature can be selected from a dendrimer or a dendron, the latter also known as a dendritic wedge.
  • Carbosilane dendrimer it refers to a macromolecule of the branched polymeric type with a spherical shape, where the growth nucleus of the dendrimer is polyfunctional, the growth units, branches or ramifications have a carbosilane skeleton and the external layer, surface or periphery of the dendrimer that consists of the same or different units of the group of formula (I). This surface or periphery would be that corresponding to the extremities of the ramifications.
  • nucleus in the present invention is meant a covalently bonded polyvalent element or compound with at least two branches, that is, it should at least be divalent.
  • the nucleus is tetravalent and more preferably, the nucleus is a silicon atom.
  • nucleus is divalent and more preferably, the nucleus is /V,/V-(b ⁇ sh ⁇ drox ⁇ et ⁇ l)et ⁇ lend ⁇ am ⁇ na.
  • the nucleus can be any polyfunctional derivative from which it is possible to grow a dendrimer of carbosilane nature, of those known to a person skilled in the art, such as, for example, and without limitation, a polyphenolic nucleus, or an amino or polyamino.
  • alkyl refers in the present invention to aliphatic, linear or
  • the dendrimer can be first, second, or third generation.
  • the core of the hydrogel precursor dendrimer is silicon or the divalent group -((CH 2 )2OH))N-CH2-CH2-(OH(CH 2 )2)N-.
  • the dendrimer is first, second or third generation.
  • the dendrimer is a homofunctional dendrimer and is selected from among Si-GiV 8 (1), Si-G 2 V 16 (2), N 2 O 2 -G 2 V 8 (3) and N 2 O 2 -G 3 V 16 (4):
  • the hydrogels of the invention use, as crosslinking points, a carbosilane dendron with terminal groups of the alkene type.
  • “carbosilane dendron” it refers in the present invention to a highly branched cone-shaped macromolecule that is defined by a focal point, the growth units, branches or ramifications, which start from said focal point, have a carbosilane skeleton and the external layer, surface or periphery of said ramifications incorporates functional groups consisting of units the same or different from the group of formula (I), already described.
  • the focal point can be selected from the group -(CH 2 )ZR 2 , where: z is an integer ranging from 1 to 10, preferably from 1 to 5 and more preferably is 4; and
  • R 2 is a group selected from -OH, -N 3 , -Br, -COOH, -NH 2 , -N(CH 3 ) 2 , thioacetate (MeCOS), phthalimide (Pht).
  • the hydrogel precursor dendron is a heterofunctional dendritic structure with peripheral alkene groups and other additional groups at the focal point that, once the hydrogel is formed, can be used to modify the hydrogel later.
  • the dendron can be first, second, or third generation.
  • generation (G n ) refers to the number of iterative steps that are necessary for the preparation of the dendrimer, where n is an integer, preferably 1-3.
  • the hydrogel precursor dendritic molecule is a dendron where the focal point is selected from -OH or -N 3 , more preferably second or third generation.
  • the dendron is selected from among HO-G 3 V 8 (5) and N 3 -G 3 V 8 (6):
  • the functionalized polymer with two terminal thiol groups (P(SH) 2 ) precursor of the hydrogel of the invention must be hydrophilic in nature or amphiphilic in nature.
  • Hydrophilic polymers are well known in the state of the art and can preferably be a polyethylene glycol (or PEG).
  • This functionalized polymer with the terminal thiol groups can be represented in an abbreviated form as SH-PEG-SH, in which -PEG- is understood to represent the following structural unit:
  • a typically ranges from about 2 (0.2 kDa) to 450 (20 kDa).
  • PEG polymers can include other groups such as acyl groups, represented by the following unit:
  • the functionalized PEG polymers of the present invention have a molecular weight between 0.35 and 20 kDa. Preferably between 1 and 10 kDa.
  • the PEG polymer precursor of the hydrogel of the present invention can be selected from among the following structures (L1) and (L2):
  • the precursor polymer of the present invention may be amphiphilic in nature.
  • PLU poloxamer
  • This PLU polymer is a generic block copolymer made up of two PEG units, separated by a central polypropylene glycol (PPG) unit.
  • Pluronic® are commercially available (Pluronic® from BASF or Poloxamer from ICI) and in a wide range of molecular weights and lengths of the constituent blocks.
  • the functionalized PLU polymer is selected from among the following structures (L3) and (L4): where a typically ranges from about 2 to 136 and b from about 6 to 69.
  • the functionalized PLU polymers of the present invention have a molecular weight between 0.35 and 20 kDa. Preferably between 1 and 10 kDa.
  • the stoichiometry between the alkenes of the dendritic molecule and the thiol groups of the polymer, alkene:thiol is 1:1 or 1:k, where k is a number less than 1.
  • RECTIFIED SHEET (RULE 91) Another aspect of the present invention refers to the use of the hydrogel of the invention as a vehicle for the transport and release of a biologically active molecule.
  • the hydrogel of the present invention further comprises a biologically active molecule covalently bound or encapsulated within the pores.
  • biologically active molecule is understood in the present invention any substance that can influence the physical and biochemical properties of a biological organism, including, but not limited to, viruses, bacteria, fungi, animals and humans.
  • biologically active molecules include any substance intended for the diagnosis, cure, alleviation, treatment, or prevention of disease in humans or other animals, or to enhance physical well-being. or mental health of humans or animals, including for physical well-being substances with cosmetic applications, such as antioxidant, moisturizing, firming or regenerating.
  • biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, dyes, nucleosides, oligonucleotides, oligosaccharides, polysaccharides, vaccines, cells, or viruses.
  • Classes of biologically active agents include, but are not limited to, antiparasites, antibiotics, fungicides, antiviral agents, anti-inflammatory agents, antitumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, antioxidant agents, and the like.
  • another aspect of the present invention relates to the hydrogel of the invention for use as a medicament.
  • Another aspect of the invention refers to the use of the hydrogel for the preparation of a medicine or a cosmetic composition.
  • the therapeutic or diagnostic use of the hydrogel will depend on the type of biologically active molecule contained in the hydrogel of the present invention.
  • the biologically active molecule is an antitumor agent
  • another aspect of the invention will refer to the hydrogel for its use in the treatment of cancer or to the use of the hydrogel for the manufacture of a medicament for the treatment of cancer.
  • the biologically active molecule is an antibiotic or a cationic group, such as cysteamine or 2-(dimethylamino)ethanethiol hydrochloride, which provide the hydrogel with antibiotic properties, so another aspect of the invention would be the hydrogel for use in the treatment of infectious diseases caused by bacteria or the use of the hydrogel for the preparation of a medicine for the treatment of infectious diseases caused by bacteria.
  • an antibiotic or a cationic group such as cysteamine or 2-(dimethylamino)ethanethiol hydrochloride
  • the biologically active molecule is an antioxidant agent, such as caffeic acid, for which reason another aspect of the invention could be the use of the hydrogel in cosmetic applications.
  • compositions comprising the hydrogel of the invention.
  • This composition can be a cosmetic or pharmaceutical composition.
  • the composition when the composition is a pharmaceutical composition, the composition may further comprise a pharmaceutically acceptable carrier or excipient and when the composition is a cosmetic composition, the composition may contain cosmetically acceptable carriers or excipients.
  • the composition is in a form suitable for topical, oral or parenteral administration.
  • Another aspect of the present invention refers to a process for obtaining the hydrogels described in the present invention that comprises: the thiol-ene reaction initiated by UV light between the dendritic molecule with terminal alkene groups and the hydrophilic or amphiphilic polymer functionalized with two terminal thiol groups, in the presence of a photoinitiator and an organic solvent.
  • Any photoinitiator known to any person skilled in the art for this type of reaction can be used, such as dimethoxyphenylacetophenone (DMPA), 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) or Phenyl-2.
  • DMPA dimethoxyphenylacetophenone
  • Irgacure 2959 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone
  • Phenyl-2 Phenyl-2.
  • the organic solvent used in the reaction can be a solvent or a mixture of solvents, such as tetrahydrofuran (THF), methanol (MeOH), dichloromethane (DCM) or acetone, or a mixture thereof, preferably THF/MeOH (tetrahydrofuran/methanol).
  • solvents such as tetrahydrofuran (THF), methanol (MeOH), dichloromethane (DCM) or acetone, or a mixture thereof, preferably THF/MeOH (tetrahydrofuran/methanol).
  • the dithiol polymer P(SH) 2 preferably based on polyethylene glycol PEG X (SH) 2 or poloxamer PLU X (SH) 2 , is obtained commercially (eg Sigma-Aldrich) or by substituting the terminal hydroxyl groups of the commercial precursors with thiol groups, using degradable (preferably ester or amide) or non-degradable (preferably ether or thioether) bonds using protocols described in the literature (Macdougall LJ et al., Biomacromolecules, 2018, 19, 1378-1388; Stenstróm P. et al, Molecules , 2021 , 26, 2364).
  • the synthesis of hydrogels is performed from dendrimers or dendrons with vinyl or allyl groups, for example Si-GiV 8 (1), Si-G 2 Vi 6 (2), N 2 O 2 -G 2 V 8 (3), N 2 O 2 -G 3 V 16 (4), HO-G 3 V 8 (5), N 3 -G 3 V 8 (6), and a dithiol polymer P(SH) 2 by thiol-ene type reactions initiated by ultraviolet light. Control of the alkene:thiol stoichiometry makes it possible to obtain networks with different numbers of functional groups.
  • the reaction between the dendritic precursor and the dithiol polymer is carried out in the presence of a photoinitiator, preferably DMPA, and an organic solvent or a mixture, preferably THF/MeOH, in a single step and initiated by irradiation with ultraviolet light, using a 1:1 alkene:thiol stoichiometry (Scheme 1).
  • a photoinitiator preferably DMPA
  • an organic solvent or a mixture preferably THF/MeOH
  • the reaction between the dendritic precursor and the dithiol polymer P(SH) 2 is carried out in the presence of a photoinitiator and an organic solvent or a mixture, preferably THF/MeOH, in a single step and initiated by irradiation with ultraviolet light, using a stoichiometry
  • the process of the invention also comprises the reaction between the dendritic hydrogel with free alkene groups described in scheme 2 and a biologically active molecule with a thiol group (F-SH).
  • This molecule can contain this thiol group or it can be functionalized prior to the synthesis described, for example the molecule (F-SH) can be cysteamine hydrochloride or 2-(dimethylam ⁇ no)ethaneth ⁇ ol hydrochloride.
  • the thiol-ene reaction is carried out in the presence of a photoinitiator, preferably DMPA, and an organic solvent or a mixture, preferably THF/MeOH/H 2 O, in a single step and initiated by irradiation with ultraviolet light, using an alkene stoichiometry. :thiol 1:1 (Scheme 2.B).
  • a photoinitiator preferably DMPA
  • an organic solvent or a mixture preferably THF/MeOH/H 2 O
  • the method of the invention also comprises the reaction between the dendritic hydrogel with heterofunctional precursor dendritic molecules, such as dendrons whose focal points are free hydroxyl groups (OH) and a biologically active molecule with a carboxylic acid group (COOH).
  • This molecule can contain this carboxylic group or it can be functionalized prior to the synthesis described, for example, the molecule can be ibuprofen, which could be modified with 2-mercaptoethanol prior to anchoring to the hydrogel, or caffeic acid.
  • the reaction is carried out using standard esterification reagents, preferably 1,1'-carbonyldiimzazole (CDI)/cesium fluoride (CsF) or N-(3-
  • the method of the invention also comprises the reaction between the dendritic hydrogel with heterofunctional precursor dendritic molecules, such as dendrons whose focal points are free azide groups (N 3 ) and a biologically active molecule with an alkyne group.
  • This molecule can contain that alkyne group or it can be functionalized prior to the synthesis described, for example, the molecule can be rhodamine previously modified with propargylamine.
  • the reaction can be carried out in the presence of copper sulfate and sodium ascorbate in water, using a 1:1.2 azide:alkyne stoichiometry. With this synthesis, the biologically active molecules are covalently linked, through triazole bonds.
  • the method further comprises adding a biologically active molecule to the hydrogel obtained in step (a) by encapsulation or covalent linkage via thioethers, esters or triazole bonds.
  • the obtained hydrogel is covalently functionalized with a biologically active molecule preferably by: i. thiol-ene reaction of unreacted terminal alkene groups and thiol groups of biologically active molecules with thiol groups;
  • the biologically active molecule is encapsulated in the hydrogel such that it is
  • the encapsulation process is known to any person skilled in the art and particularly comprises: preparing a solution in an organic solvent that is easy to evaporate, such as ethanol, with a saturated concentration of the biologically active molecule and immersing the hydrogel of the invention in the above solution at room temperature (approx. 25 °C) or a higher temperature (for example 50 °C, to increase solubility) with slight agitation (up to approx. 150 rpm).
  • Fig. 1 Examples of functional dendritic hydrogels, before and after their modification with a fluorescent agent, linked by ester (A) and triazole (B) bond.
  • Fig. 2 Examples of antibacterial activity of neutral (H3b) and cationic (H4b) dendritic hydrogels, compared to the commercial Urgo® Resistant Antiseptic dressing containing the antibiotic benzalkonium chloride at 960 mg/m 2 (bottom). Dendritic hydrogels perse (A) and loaded with the antibiotic ciprofloxacin (B).
  • Fig. 3 Example of release curves of caffeic acid (solid line, H5 hydrogel) and ciprofloxacin (dashed line, H3b hydrogel) in water at room temperature.
  • the Si-GiV s (1) dendrimer 50 mg, 1 eq.), described in Strasák T., et al., Organometallics 2012, 31, 19, 6779-6786, and the polymer PEG 1k (SH) 2 (L1 , 1 kDa) (268 mg, 4 eq.), described in Macdougall, L. et al., Biomacromolecules 2018, 19, 5, 1378-1388, are dissolved in a THF:MeOH mixture and DMPA (8 mg, 5 mol% alkene). The mixture is exposed to UV light (At 365 nm). After 6 h, the hydrogel is purified by successive washings in acetone. The H1 hydrogel is isolated as a yellowish solid (% cross-linking - 90%).
  • the dendrimer N 2 O 2 -G 2 V 8 (3) (100 mg, 1 eq.) and the polymer PEG 1k (SH) 2 (L1 , 1 kDa) (536 mg, 4 eq.) are dissolved in a mixture THF:MeOH and DMPA (11 mg, 5 mol% alkene) is added.
  • the mixture is exposed to UV light (At 365 nm).
  • the hydrogel is purified with acetone washes.
  • the dendrimer N 2 O 2 -G 2 V 8 (3) (100 mg, 1 eq.) and the polymer PEG 1k (SH) 2 (L1 , 1kDa) (402 mg, 3 eq. for H3a and 268 mg, 2 eq for H3b) are dissolved in a THF:MeOH mixture and DMPA (8 mg and 5 mg respectively, 5 mol% alkene) is added.
  • the mixture is exposed to UV light (At 365 nm).
  • H3 hydrogels are exposed to a THF:MeOH:H 2 O mixture and DMPA (11 and 7 mg for H3a and H3b respectively, 5 mol% alkene) is added together with 2-(dimethylamino)ethanethiol hydrochloride (33 mg and 40 mg for H3a and for H3b respectively, 1,2
  • thermoresponsive dendritic hydrogel H-[(Si-GiV 8 )x( PLU L35 (SH) 2 )] H5
  • thermoresponsive dendritic hydrogel H-[(N3-G 3 V 8 )X(PLU L35 (SH) 2 )] H7)
  • the dendron HO-G 3 V 8 (5) (47.6 mg, 0.056 mmol) and the polymer PLU L6 I(SH) 2 (L3, L61) (500 mg, 0.226 mmol), described in Example 11, were dissolve in a mixture
  • thermoresponsive dendritic hydrogel H-[(N3-G3V 8 )x(PLU L61 (SH)2)] H9
  • the dendron N 3 -G 3 V 8 (6) 49.1 mg, 0.056 mmol
  • the polymer PLUL6I(SH) 2 L3, L61
  • DMPA 3 mg, 5 mol% alkene
  • the mixture is exposed to UV light ( ⁇ 365 nm).
  • the hydrogel is purified with acetone washes.
  • thermoresponsive polymers PLU X (SH) 2 (type L3)
  • the activity as a drug transport system of selected hydrogels has been evaluated by two approaches: encapsulation within the pores or covalent bonding in the available terminal functional groups.
  • Encapsulation For the encapsulation assay, a solution of the drug in ethanol is prepared at a concentration of 50 mg/mL and the hydrogel (65 mg) is immersed in 1 mL of said solution for 20 h at 25 °C with slight agitation (150 rpm). Subsequently, the hydrogel is dried under vacuum and the amount of drug retained is quantified.
  • Anchoring can also be accomplished by the formation of degradable, such as ester, or nondegradable, such as triazole, covalent bonds.
  • degradable such as ester
  • nondegradable such as triazole
  • covalent bonds The hydrogels have been modified with drugs (ibuprofen) or fluorescent agents (rhodamine), visually and analytically checking the anchorage.
  • drugs ibuprofen
  • fluorescent agents rhodamine
  • RECTIFIED SHEET (RULE 91) It is carried out by means of usual reactions assisted by CDI or EDCI, while the anchoring with triazole is produced by CuAAC click type reaction.
  • the charge of the hydrogel depends on both the nature of the hydrogel and the agent to be attached. Examples:
  • Rhodamine B (1.2 eq.) dissolved in dichloromethane (4 mL) is reacted with EDCI HCI (1.2 eq.). The mixture is stirred for 30 min and DMAP (0.2 eq.) is added. Subsequently, the H8 hydrogel (70 mg) is immersed and maintained with slight agitation (150 rpm). After 20 h, the hydrogel is purified with dichloromethane washes and dried, obtaining a pink colored hydrogel confirming rhodamine anchoring (Fig. 1A).
  • Rhodamine B isothiocyanate 50 mg, 0.093 mmol was previously modified by reaction with propargylamine (6 ⁇ L, 0.093 mmol) to generate the alkyne derivative of Rhodamine B. Subsequently, an aqueous solution of the alkyne derivative of Rhodamine (1.2 eq .), copper sulfate (1.2 eq., 2.8 mg, 0.011 mmol) and sodium ascorbate (2.4 eq., 4.5 mg, 0.0226 mmol). The H9 hydrogel is immersed and kept at 50 °C, with slight agitation (150 rpm) for 20 h. The hydrogel is purified with water and acetone washes and dried, obtaining a pink colored hydrogel confirming rhodamine anchorage (Fig. 1B).
  • Ibuprofen (7.5 mg, 1.5 eq/OH gel) was previously activated by reaction with CDI (8.44 mg, 1.5 eq/OH gel) in ethyl acetate for 30 minutes at 50 °C, to generate the imidazolide derivative of ibuprofen. Subsequently, CsF catalyst (0.74 mg, 0.2 eq/OH gel) is added. The H3a hydrogel (47.5 mg) is immersed and kept at 50 °C, with slight agitation (150 rpm) for 20 h. The hydrogel is purified by washing with water and acetone and dried in vacuo, obtaining a yellow colored hydrogel that confirms the anchorage of ibuprofen.
  • Caffeic acid acetonide (5.76 mg, 1.5 eq/OH gel) was previously activated by reaction with CDI (5.70 mg, 1.5 eq/OH gel) in ethyl acetate for 30 minutes at 50 °C, to generate the caffeic imidazolide derivative. Subsequently, CsF catalyst (0.5 mg, 0.2 eq/OH gel) is added.
  • the H3a hydrogel (32.0 mg) is immersed and kept at 50 °C, with slight agitation (150 rpm) for 20 h. The hydrogel is purified by washing with water and acetone and dried in vacuo, obtaining a brown colored hydrogel that confirms the anchorage of the caffeic acid.
  • RECTIFIED SHEET (RULE 91) Release test The hydrogels are immersed in an aqueous solution (2 mL), in a static mode or with slight agitation, at the desired temperature and the evolution in the amount of drug released is quantified over time by HPLC. Depending on the nature of the hydrogel and the encapsulated drug, different results are obtained, ranging from rapid release (93% in 2 h for caffeic acid in H5; 90% in 3 h for ciprofloxacin in H3b, Figure 3) to longer release. sustained, which could be modulated by temperature (for example, using the H5 thermoresponsive hydrogel). Covalently anchored hydrogels are stable in water and require more aggressive pH conditions for drug release.
  • the antibacterial activity of selected hydrogels was evaluated in Staphylococcus aureus (Gram+) by zone of inhibition assay, using a commercial dressing impregnated with an antibacterial drug as a positive control.
  • a solution of said bacteria is spread on a PCA plate and both hydrogels and the dressing with the same size are placed on these to favor their comparison.
  • the PCA plate is incubated 24 h at 37 °C.
  • the results showed that both neutral hydrogels (for example, H3b) and cationically charged hydrogels (for example, H4b), inhibited bacterial growth in the contact zone, without generating an inhibition halo, Fig. 2A.
  • the test was repeated using the same ciprofloxacin-loaded hydrogels.
  • a saturated solution of ciprofloxacin in chloroform is prepared at 50 °C and the hydrogel (65 mg) is immersed in 1 mL of said solution for 20 h at 50 °C with gentle shaking (150 rpm). Subsequently, the hydrogel is dried under vacuum and the amount of drug retained is quantified, finding values of 1 mg/100 mg. In this case, the hydrogels were shown to inhibit bacterial growth, generating an inhibition halo, Figure 2.B.
  • the antibiotic-loaded H4b hydrogel produced superior inhibition (average 5 mm halo) than the commercial Urgo Resistant Antiseptic dressing (average 3 mm halo) containing the antibiotic benzalkonium chloride at 960 mg/m 2 , used as
  • thermoresponsive capacity of the hydrogels of the invention As an example of the thermoresponsive capacity of the hydrogels of the invention, the determination method used and the results obtained for selected compounds are detailed.
  • the swelling capacity of the hydrogels was evaluated over time, immersing the hydrogel in 2 ml_ of water at the desired temperature (25, 37 and 45 °C) and measuring its weight and size at different times.
  • the H1 hydrogel which contains the PEGi k (SH) 2 polymer in its composition, did not show significant differences at the different temperatures, Fig. 4.
  • the H5 hydrogel which contains the PLU L35 (SH) 2 polymer, showed a clear decrease in swelling capacity with increasing temperature. This behavior can be exploited to modulate the encapsulation and release of a bioactive molecule from the hydrogel.

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Abstract

La présente utilisation se rapporte à des hydrogels ayant des domaines dendritiques de type carbosilane. L'invention concerne également le procédé pour l'obtention et les utilisations de ces hydrogels en tant que matières antimicrobiennes et pour la libération de molécules bioactives en vue de leur utilisation dans l'industrie pharmaceutique et cosmétique.
PCT/ES2022/070728 2021-11-12 2022-11-11 Hydrogels ayant des domaines dendritiques de type carbosilane, leur préparation et leurs utilisations WO2023084140A1 (fr)

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Citations (3)

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