Nanogels comprising bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules

Abstract

Novel nanogels comprising one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules and methods of making the same. Biomaterial implants, medical devices or bioprostheses coated with nanogels and methods of producing the same.

Description

Technical Field

[0001] This invention relates to nanogels comprising bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, particularly vitamin E derivatives. The invention also relates to medical devices, biomaterial implants or bioprosthetics coated with nanogels, methods for preparing such nanogels, and methods for coating medical devices, biomaterial implants or bioprosthetics, particularly catheters. Background Technology

[0002] Most medical devices, biomaterial implants, or bioprosthetics can raise biocompatibility issues. Importantly, the implantation of foreign substances into the vascular system activates the clotting pathway, which can lead to thrombotic complications. For example, the surface roughness of medical devices, biomaterial implants, or bioprosthetics is a significant factor influencing thrombosis.

[0003] Medical devices, biomaterial implants, or bioprosthetics can also become infected, and treatment for such infections usually requires the administration of antibiotics targeting the causative bacteria.

[0004] Furthermore, increasing resistance to antibiotics has increased the demand for antibiotics that exhibit both antibacterial efficacy and resistance to antibiotic resistance. Since traditional one-target-one-molecule approaches cannot meet this demand, alternative methods are needed for multi-target antibiotics.

[0005] Nanogels are known in the prior art. WO2018 / 122318A1 describes a nanogel made of a first hydrophilic polymer or copolymer having catechol groups and crosslinked with a second hydrophilic polymer having one or more reactive groups; the nanogel also includes bioactive molecules, therapeutic molecules or drugs.

[0006] These nanogels can be directly anchored or attached to the surface of medical devices, biomaterial implants, or bioprosthetics, and can release bioactive molecules, therapeutic molecules, or drugs. However, the challenge in using these nanogels lies in the loading of water-insoluble or hydrophobic bioactive molecules, therapeutic molecules, or drugs, leading to irregular release kinetics and lower pharmacological efficiency.

[0007] Furthermore, only a small amount of hydrophobic bioactive molecules, therapeutic molecules, or drugs are released from this nanogel after the release begins. Therefore, direct pharmacological effects cannot be achieved rapidly, and bacterial adhesion to medical devices, biomaterial implants, or bioprosthetics cannot be completely prevented. Summary of the Invention

[0008] We have now discovered improved nanogels containing one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives or most preferably amphiphilic cyclodextrins; said nanogels exhibit improved antibacterial efficacy and resistance to antibiotics.

[0009] This invention provides a surprisingly effective loading of hydrophobic bioactive molecules, therapeutic molecules, or drugs in nanogels, along with stable, gradual, and continuous release over several weeks. Hydrophobic bioactive molecules, therapeutic molecules, or drugs can bind to and be released simultaneously with hydrophilic bioactive molecules, therapeutic molecules, or drugs within such nanogels. Furthermore, nanogels containing bioactive molecules, therapeutic molecules, or drugs advantageously maintain long-term stability in liquid suspensions, particularly in aqueous media, by preserving their structural integrity and uniform distribution in the dispersed state.

[0010] Therefore, the nanogel of the present invention is used to treat or prevent bacterial infections, particularly for topical application to host mammals.

[0011] This invention provides the use of this novel nanogel in coating medical devices, biomaterial implants or bioprosthetics, particularly catheters; it also provides improvements to medical devices, biomaterial implants or bioprosthetics.

[0012] Medical devices, biomaterial implants, or bioprosthetics coated with the nanogel of the present invention advantageously provide a more uniform, hydrophilic, and smooth surface and content uniformity, resulting in more reproducible kinetic release of hydrophobic bioactive molecules, therapeutic molecules, or drugs from the coated medical device, biomaterial implant, or bioprosthetic, and subsequently improving pharmacological efficiency.

[0013] Meanwhile, medical devices, biomaterial implants or bioprosthetics coated with the nanogel of the present invention advantageously reduce bacterial adhesion to the surface of the medical device, biomaterial implant or bioprosthetic, especially when the nanogel contains a combination of antibacterial agents and antiviral agents or bactericides. Detailed Implementation

[0014] This invention relates to novel nanogels, which are three-dimensional cross-linked particles with submicron particle sizes that provide a larger carrying space and can be used to incorporate bioactive molecules, therapeutic molecules or drugs encapsulated by amphiphilic molecules, particularly vitamin E derivatives or amphiphilic cyclodextrins.

[0015] This nanogel, used for the treatment and prevention of bacterial infections, particularly bacterial virulence, is especially suitable for topical application to host mammals requiring this treatment.

[0016] This nanogel can also anchor or adhere to the surface of any biomaterial or medical device, whether metallic or polymeric, or on bioprosthetics, thereby reducing or preventing infection and improving the biocompatibility and blood compatibility of temporary or permanent implant materials to help maintain their function and increase their durability.

[0017] According to a first aspect, the present invention provides a nanogel made of poly(methacrylamide) of formula (1) with a quinone group. (1) Where x is an integer greater than 1, preferably x is between 1 and 100; The poly(methacrylamide) is crosslinked with polymers containing primary or secondary amine groups; and The nanogel contains one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules. The amphiphilic molecules are preferably vitamin E derivatives, more preferably D-α-tocopherol polyethylene glycol succinate. The amphiphilic molecules can also be amphiphilic cyclodextrins, preferably hydroxypropyl-β-cyclodextrin.

[0019] Alternatively, the present invention also provides nanogels made of poly(vinylquinone) represented by formula (6). (6) Where n is an integer greater than 1; preferably, n is between 1 and 100; The poly(vinylquinone) is crosslinked with polymers containing primary or secondary amine groups; and The nanogel contains one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules. The amphiphilic molecules are preferably vitamin E derivatives, more preferably D-α-tocopherol polyethylene glycol succinate. The amphiphilic molecules can also be amphiphilic cyclodextrins, preferably hydroxypropyl-β-cyclodextrin.

[0021] Alternatively, the present invention also provides nanogels made from quinone polymers or combinations of copolymers made from poly(methacrylamide) and poly(vinylquinone). The invention is also extended to all polymers or copolymers containing quinone groups.

[0022] The poly(methacrylamide) of formula (1) with a quinone group is obtained by oxidation of the catechol group (also known as the benzene 1,2-diol group) of P(mDOPA) of formula (2). The oxidation is preferably carried out in an aqueous medium under alkaline conditions at a pH above 10 (preferably between pH 10 and 12).

[0023] (2) Similarly, poly(vinylquinone) of formula (6) is obtained by oxidizing the catechol group (also known as the benzene 1,2-diol group) of poly(vinylcatechol) of formula (7). (7) Where n is an integer greater than 1, preferably between 1 and 100.

[0026] Oxidation is preferably carried out in an aqueous medium under alkaline conditions with a pH above 10 (preferably between pH 10 and 12).

[0027] Amphiphilic molecules are molecules that are both hydrophilic and lipophilic. When dispersed in water, amphiphilic molecules have the property of self-assembling into, for example, micelles. They can be surfactants, such as sodium lauryl sulfate, 1-octanol, cocamidopropyl betaine, benzalkonium chloride, phospholipids, cholesterol, glycolipids, fatty acids, vitamin-polyether copolymers, such as vitamin E-polyoxyethylene copolymers, especially D-α-tocopherol polyethylene glycol succinate (TPGS). Amphiphilic molecules also refer to amphiphilic cyclodextrins obtained by grafting hydrocarbon chains onto the hydroxyl groups of cyclodextrins, such as hydroxypropyl β-cyclodextrin or sulfobutyl ether β-cyclodextrin. Amphiphilic cyclodextrins can incorporate hydrophobic biomolecules, therapeutic molecules, or drugs into their hydrophobic cavities.

[0028] For clarity, the term "amphiphilic molecule" does not extend to molecules containing both organic and hydrophilic groups, or to those that fall under the definition of bioactive molecules, therapeutic molecules, or drugs in this invention. For example, vancomycin, minocycline, or ticagrelor are not considered amphiphilic molecules.

[0029] Polymers with primary or secondary amine groups can be polyallylamine, polyethyleneamine, polyvinylamide, polyvinyl alcohol, poly(meth)acrylate, poly(meth)acrylamide, polyurethane, polyethylene glycol (PEG), and polyelectrolytes (cationic, anionic, or amphoteric) with primary or secondary amine active groups. Polymers with primary or secondary amine groups can be natural or synthetic polymers with primary or secondary amine functional groups, such as polyvinylamine, chitosan, or proteins.

[0030] In a preferred embodiment, the polymer with primary amine groups may include polyallylamine, such as poly(allylamine hydrochloride), also known as PAH, as shown below, where p is an integer greater than 1, preferably between 10 and 300: .

[0031] Bioactive molecules, therapeutic molecules, or drugs can be antibiotics, anti-biofilm formation agents, antiplatelet agents, anticoagulants, antithrombotic agents, and anticalcification agents.

[0032] Bioactive agents (also known as bioactive molecules) are molecules derived from plants, seeds, fungi, animals, humans, or microorganisms, or that can be synthesized. They can include any agent intended to be delivered to molecules, cells, tissues, or organs to modulate or alter molecular or cellular function, including therapeutic effects. Bioactive agents include, but are not limited to, pharmaceutically active compounds or diagnostic compounds. Bioactive molecules or bioactive compounds include, but are not limited to, nucleotides (aptamers, RNAi, antisense oligonucleotides), peptides, oligopeptides, proteins, apoproteins, glycoproteins, antigens and antibodies or their antibody fragments, receptors and other membrane proteins, and retro-inversonucleotides. Oligopeptides, protein analogs of which at least one peptide bond is not substituted with a peptide bond, enzymes, coenzymes, enzyme inhibitors, amino acids and their derivatives, hormones, lipids, phospholipids, liposomes, ricin or fragments of ricin; toxins (e.g., aflatoxin, digoxin, xanthotoxin, red yeast rice toxin); analgesics (e.g., aspirin, ibuprofen, and acetaminophen); bronchodilators (e.g., theophylline and salbutamol); beta-blockers (e.g., propranolol, metoprolol, atenolol, labetalol, timolol, pentbuprofen, and indrolol); antimicrobial agents (e.g., those mentioned above, as well as ciprofloxacin, sinofloxacin, and norfloxacin); antihypertensive agents (e.g., clonidine, methyldopa, prazosin, verapamil, nifedipine, atadepril, and enalapril); cardiovascular drugs, including antiarrhythmic drugs. Cardiac glycosides, antianginal drugs, and vasodilators; central nervous system drugs, including stimulants, psychotropic drugs, antimanic drugs, and sedatives; antiviral agents; antihistamines (e.g., chlorpheniramine and brompheniramine); cancer drugs, including chemotherapeutic agents (e.g., chlorambucil, carboplatin, busulfan derivatives, doxorubicin, etoposide, topotecan (TPT)); tranquilizers (e.g., diazepam, chlordiazepoxide, oxazepam, alprazolam, and triazolam); antidepressants (e.g., ... Examples of anticonvulsants include: fluoxetine, amitriptyline, nortriptyline, and imipramine; H-2 antagonists (e.g., nizatidine, cimetidine, famotidine, and ranitidine); anticonvulsants; antinausea drugs; prostaglandins; muscle relaxants; anti-inflammatory substances; stimulants; decongestants; antiemetics; diuretics; anticonvulsants; bronchodilators; anti-Parkinson's drugs; expectorants; antitussives; mucolytics; vitamins; minerals and nutritional supplements.Other molecules include nucleotides; oligonucleotides; polynucleotides; and their well-known and biologically functional analogs and derivatives, including, for example, methylated polynucleotides and nucleotide analogs having thiophosphate bonds; plasmids, granules, artificial chromosomes, other nucleic acid vectors; antisense polynucleotides, including those substantially complementary to at least one endogenous nucleic acid or those having a sequence opposite to at least a portion of the genome of a selected virus or retrovirus; promoters; enhancers; inhibitors; and other ligands that regulate gene transcription and translation.

[0033] Bioactive agents can be anti-infective agents. Anti-infective agents include, but are not limited to, antibiotics such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, gerdemycin, herbimycin, rifaximin, loracarbose, ertapenem, doripenem, imipenem / cilastatin, and meropenem. Penem, Cefadroxil, Cefazolin, Cefathiophene, Cefadroxil, Cefaclor, Cefamandole, Cefoxitin, Cefproxil, Cefuroxime, Cefixime, Cefdinir, Ceffitoren, Cefoperazone, Cefotaxime, Cefpodoxime, Cefozidine, Cefbutan, Cefazolin, Rothiazide, Cefpiram, Ceftropine fosamil), cefepime, teicoplanin, vancomycin, travancin, dabavancin, orivincin, clindamycin, lincomycin, dapoxetine, azithromycin, clarithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, ranezoli, amoxicillin, ampicillin, piperacillin, ticarcillin, bacillus subtilisin, colistin, polymyxin B, propofol, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, fleroxacin, mesylate (maf) (enide), sulfacetyl, sulfadimethoxine, silver sulfadimethoxine, sulfadimethoxine, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfonamidochrysoidine, demeclocyline, doxycycline, minocycline, tetracycline hydrochloride, tetracycline, chlorpheniramine, dapsone, rifampin, rifabutin, arspehnamine, chloramphenicol, fosfomycin, metronidazole, thiamphenicol, tigecycline, tebuconazole, and trimethoprim.

[0034] Antibiotics can be bacteriostatic, inhibiting growth without killing bacteria, or bactericidal, killing bacteria. The following antibiotics in the list above are generally considered bacteriostatic: minocycline (especially against Staphylococcus aureus), tetracyclines, macrolides, clindamycin, ranezoli, chloramphenicol, chlorhexidine, or alexidine (when used at low levels) or combinations thereof, while the other antibiotics in the list above are generally bactericidal.

[0035] Antibiotics can also be antiviral agents when provided at low levels.

[0036] Virulence factors are molecules produced by pathogens that allow them to colonize, evade the immune system, and damage host cells. Antiviral agents target the virulence factors of pathogens, rather than killing or stopping their growth, thereby eliminating infectious pathogens. Unlike bactericidal antibiotics that induce resistance, antiviral agents do not create selective pressure to develop resistance. Antiviral agents interfere with the interaction between pathogens, particularly bacteria, and their host mammals, thereby reducing damage to the host and weakening the bacteria's pathogenicity. Antiviral agents can inhibit the production of bacterial toxins or prevent them from adhering to tissues.

[0037] Antibiofilm formation agents include, but are not limited to, naturally occurring peptides such as human cathelicidin LL-37 or bovine indole, or synthetic peptides such as 1018, natural compounds having a 2-aminoimidazole group, 2-aminoimidazole-based inhibitors, benzimidazole analogs, indole-triazole-amide analogs, and plant-derived biofilm inhibitors (e.g., emodin, phlorizin, casbane diterpenes). Diterpene, resveratrol and its oligomers, sulfur derivatives, brominated furanone analogs, brominated pyrrole alkaloids, skyllamycins and (-)-ageloxime D-structures, cephalosporins, N-acylhomoserine lactone analogs, carolacton, molecules that interfere with amyloid formation, fatty acids, nitric oxide donors, ionic liquids (such as 1-alkyl-3-methylimidazolium chloride, 1-alkylquinolineium bromide), all of these agents can be used in combination with conventional antibiotics.

[0038] Antiplatelet agents include, but are not limited to, irreversible cyclooxygenase inhibitors (e.g., aspirin and Disgren), adenosine diphosphate (ADP) receptor inhibitors (e.g., clopidogrel (Plavix), prasugrel (Effient), ticagrelor (Brilique and Brilinta), ticlopidine (Ticlid)), phosphodiesterase inhibitors (e.g., cilostazol), protease-activated receptor-1 (PAR-1) antagonists (e.g., vorapaxar (Zontivity), glycoprotein IIB / IIIA inhibitors (for intravenous use only) (e.g., abciximab (ReoPro), eptifibatide (Integrilin), tirofiban (Aggrastat)), adenosine reuptake inhibitors (e.g., dipyridamole (Persantine)), and thromboxane inhibitors. Inhibitors, thromboxane synthase inhibitors and thromboxane receptor antagonists (e.g., terutroban), glycoprotein VI inhibitors (e.g., Revacept), glycoprotein Ib inhibitors, and von Willebrand factor inhibitors.

[0039] Anticoagulants include, but are not limited to, acenitrocoumarin, romaine, dicumarol, ethylbiscoumacetate, phenylpropanol, warfarin, chlorobenzylindion, dipjenadione, phenylindion, ticlomarol, bemiparin, certoparin, ardeparin, dalteparin, enoxaparin, nalteparin calcium, parecene, heparin sodium, dabigatran, apixaban, betrixabaan, darexaban, edoxaban, omexaban, rivaroxaban, alteplase, danaladiparin, tinzaparin, and fondaparin.

[0040] Thrombolytic agents include, but are not limited to, alteplase, reteplase, tenecteplase, teruloplasm, urokinase, aniplasm, montelpeplase, streptokinase, anclobetaase, fibrinolytic enzyme, and fibrinolytic enzyme.

[0041] Anti-calcification agents include, but are not limited to, bisphosphonates, aluminum salts, glutaraldehyde, aminooleic acid, and metalloproteinase inhibitors.

[0042] In a preferred embodiment, the nanogel according to the invention comprises antibiotics and / or antiplatelet agents.

[0043] In another preferred embodiment, the nanogel according to the invention comprises a combination of antibacterial agents, more preferably a combination of bacteriostatic agents and antiviral agents, wherein the antiviral agents inhibit bacterial growth and bacterial adhesion when the nanogel is coated on a surface.

[0044] Advantageously, when nanogels are coated onto medical devices, biomaterial implants, or bioprosthetics, nanogels containing this combination of antibacterial and antiviral agents delay bacterial growth (measured by metabolic rate) and increase anti-adhesion. Preferably, the molecular ratio of the antibacterial agent to the antiviral agent is 1:1 to 1:10, more preferably 1:2.

[0045] In a more preferred embodiment, the antibacterial agent is minocycline.

[0046] In another preferred embodiment, the antibacterial agent is chlorhexidine.

[0047] In a preferred embodiment, the bioactive molecule, therapeutic molecule, or drug is a triazolo(4,4-d)-pyrimidine derivative of formula (3), or a pharmaceutically acceptable salt or solvation thereof, or a solvation thereof or a solvation of such salt, provided that R is CH2 or a bond. 1 Not propyl; when X is CH2 and R 1 When it is CH2CH2CF3, butyl, or pentyl, R 2 The phenyl group on X must be replaced by fluorine; when X is OCH2CH2 and R 1 When it is propyl, R 2 The phenyl group on the surface must be replaced by fluorine. (3) Where R 1 It is a C3-5 alkyl group optionally substituted with one or more halogen atoms; R 2 It is a phenyl group, optionally substituted with one or more halogen atoms; R 3 and R 4 All are hydroxyl groups; R is OH or XOH, where X is CH2, OCH2CH2 or a bond.

[0049] The triazolo(4,4-d)-pyrimidine derivatives of formula (3) advantageously possess antiplatelet activity and also have antibacterial activity. They are particularly useful for reducing or preventing infection from blood-contaminated medical devices, biomaterial implants, or bioprosthetics, and for preventing thrombosis, when inserted into or implanted in a mammalian host. Thrombosis can indeed promote infection from blood-contaminated medical devices, biomaterial implants, or bioprosthetics. The mammalian host can be a human patient or an animal.

[0050] In the most preferred embodiment, the triazolo(4,4-d)-pyrimidine derivative is (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol, also known as triafluocyl.

[0051] In another preferred embodiment, the triazolo(4,4-d)-pyrimidine derivative is (1S,2R,3S,4R)-4-[7-[(1R,2S)-2-(3,4-difluorophenyl)-cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol, also known as Fluometacyl or Fluometacyl ® As shown in equation (5).

[0052] (5) In another preferred embodiment, the bioactive molecule, therapeutic molecule or drug is a pyrimidine derivative represented by formula (4), or an optical isomer thereof, a racemic mixture thereof, a pharmaceutically acceptable acid addition salt, a pharmaceutically acceptable metal salt or alkylated ammonium salt or a prodrug; (4) in: X 1 and X 2 Independently, N, CH, CR 8 , where R 8 It is C 1-6 Alkyl, C 2-6 alkenyl or C 2-6 Alkyne group; except if X 1 or X 2 If one of them equals N, then the remaining X 1 or X 2 Selected from CH, CR 8 .

[0055] -Y- is either -O- or -S-; R 11 and R 12 C is independent 1-6 -alkyl, C 2-6 -Alkenyl, C 2-6 -Alynyl group, C 3-6 -cycloalkyl, aryl, aryl-C 1-6-alkyl, wherein the alkyl or cycloalkyl group is optionally mono- or poly-substituted with OH or halogen, and the aryl group is optionally substituted with halogen, -C 1-6 Alkyl, -C 1-6 Alkoxy, -OH, -NO2, -CN, -NH2, -NHR 8 -N(R) 8 )2-COOH, -COOR 8 -CONH2, -CONHR 8 -CON(R) 8 )2、-SO2NH2、-SO2NHR 8 or -SO2N(R) 8 )2. Mono- or poly-substituted; R 13 R 14 R 15 R 16 and R 17 Independently, it consists of H, halogens, and C. 1-6 Alkyl, C 1-6 Alkoxy, -OH, -NO2, -CN, -NH2, -NHR 8 -N(R) 8 )2-COOH, -COOR 8 -CONH2, -CONHR 8 -CON(R) 8 )2、-SO2NH2、-SO2NHR 8 or -SO2N(R) 8 )2.

[0056] The pyrimidine derivatives of formula (4) have advantageous antibacterial activity. They are particularly useful for reducing or preventing infections from medical devices, biomaterial implants, or bioprosthetics when inserted into or implanted in a mammalian host. The mammalian host can be a human patient or an animal.

[0057] In another preferred embodiment, the bioactive molecule, therapeutic molecule, or drug is biguanide, preferably chlorhexidine, hereinafter also referred to as chlorhexidine chloride.

[0058] The biguanide or HN(C(NH)NH2)2 used in this article refers to formula (7). (7) Biguanides can be chlorhexidine, alexiidine, and polyhexylbiguanide. The chlorhexidine used in this article refers to chlorhexidine base (8), also known as chlorhexidine chloride. (8) However, it can also refer to chlorhexidine salts, such as chlorhexidine diphosphanilate, chlorhexidine digluconate, chlorhexidine diacetate, chlorhexidine dinitrate, chlorhexidine dihydrochloride, chlorhexidine dichloride, chlorhexidine acetate, chlorhexidine dipropionate, chlorhexidine maleate, chlorhexidine succinate, chlorhexidine thiosulfate, chlorhexidine diphosphate, chlorhexidine malate, chlorhexidine dibenzoate, chlorhexidine diisophthalate, chlorhexidine dilaurate, and chlorhexidine distearate.

[0061] The term "alexidin" as used in this article refers to alexidin base, but it can also refer to alexidin hydrochloride, alexidin dihydrochloride, alexidin monoacetate, alexidin diacetate, alexidin gluconate, alexidin digluconate, and mixtures thereof.

[0062] The bioactive agent, therapeutic molecule or drug is dispersed in a solvent together with an amphiphilic molecule, preferably a vitamin E derivative or an amphiphilic cyclodextrin, more preferably hydroxypropyl-β-cyclodextrin, and then added to the resulting dispersion of poly(methacrylamide) or poly(vinylquinone) with a quinone group.

[0063] The solvent can be any solvent containing an OH group. The solvent should not contain NH and / or -SH bonds to avoid interaction with the catechol groups. Solvents can be, for example, water, alcohols (e.g., methanol, ethanol, butanol, propanol, etc.) or combinations thereof.

[0064] Vitamin E derivatives can be any copolymer obtained by esterification of vitamin E (also known as α-tocopherol succinate) with an ester (such as vitamin E acetate) or with a linear or branched polyether (such as polyoxyethylene, polyoxypropylene, polyoxypropylene-polyoxyethylene copolymer, polyethylene glycol, polypropylene glycol, etc.).

[0065] The polyalkylene glycol has a molecular weight of 500 to 2000, preferably 750 to 1000, and most preferably 1000.

[0066] In a preferred embodiment, the vitamin E derivative is D-α-tocopherol polyethylene glycol succinate (TPGS) as shown in Formula A.

[0067] TPGS is a copolymer obtained by esterification of vitamin E (also known as α-tocopherol succinate) and polyethylene glycol (PEG 750 or PEG1000).

[0068]

[0069] Amphiphilic molecules, particularly vitamin E derivatives, and even more so TPGS copolymers, efficiently form micelles in solvents such as water or aqueous solutions containing 0-60% alcohol, such as ethanol. TPGS encapsulates hydrophobic bioactive agents, therapeutic molecules, or drugs, and through sustained release from the nanogel over several weeks, preferably more than 10 days, increases the loading capacity of such hydrophobic bioactive agents, therapeutic molecules, or drugs in the nanogel, as well as their efficiency and bioavailability.

[0070] When TPGS (bioactive agent, therapeutic molecule or drug) is mixed in a ratio of 1:1 w / w to 5:1 w / w, preferably 2:1 by weight, the TPGS copolymer can effectively form micelles with hydrophobic bioactive molecules, therapeutic molecules or drugs in aqueous solution.

[0071] The hydrophilic groups of TPGS copolymers, polyethylene glycol (PEG), form the crown of the micelles, while the hydrophobic groups, tocopheryl succinate, form their core. The hydrophobic core of the micelles can dissolve poorly soluble or insoluble drugs and partially protect bioactive agents, therapeutic molecules, or drugs from the effects of the aquatic environment. Encapsulating hydrophobic bioactive agents, therapeutic molecules, or drugs with TPGS molecules, when inserted into nanogels, contributes to their improved stability.

[0072] Micelles obtained by such encapsulation of bioactive agents, therapeutic molecules or drugs have an average particle size of 10 nm to 100 nm, preferably 10 nm.

[0073] Bioactive agents, therapeutic molecules or drugs are encapsulated in micelles along with amphiphilic molecules (preferably TPGS) and retained in nanogels.

[0074] The nanogel comprises micelles of amphiphilic molecules, preferably TPGS, and a bioactive agent, therapeutic molecule, or drug. The nanogel has a diameter less than 1000 nm, for example, about 100 nm to 300 nm. For example, the diameter of the nanogel can be less than about 500 nm, less than about 300 nm, less than about 200 nm, or less than about 150 nm. In a specific embodiment, the nanogel of the present invention has a diameter of about 150 nm to about 250 nm. In a specific embodiment, the diameter of the nanogel of the present invention is about 100 nm to about 250 nm.

[0075] Nanogels can also contain amphiphilic molecules, such as amphiphilic cyclodextrin (Cy) structures or vesicles, incorporating hydrophobic bioactive agents, therapeutic molecules or drugs into the hydrophobic cavities of Cy.

[0076] When micelles or vesicles are loaded into nanogels, the amphiphilic molecules, preferably TGPS, and more preferably hydroxypropyl-β-cyclodextrin, surprisingly reduce bacterial adhesion to the coated medical devices, biomaterial implants, or bioprosthetics, and do not inhibit the pharmacological effects of bioactive agents, therapeutic molecules, or drugs as they would in solvent mixtures.

[0077] The nanogels according to the invention can advantageously load higher levels of bioactive molecules, therapeutic molecules, or drugs. Therefore, when coated onto medical devices, biomaterial implants, or bioprosthetics, the nanogels according to the invention also advantageously allow for longer release upon contact with cells, tissues, or organs.

[0078] The nanogels according to the invention can advantageously load hydrophilic and hydrophobic bioactive agents, therapeutic molecules, or drugs together. Preferably, the molecular ratio of the hydrophilic to hydrophobic bioactive agents, therapeutic molecules, or drugs is 1:0 to 1:1; more preferably 1:0.5.

[0079] The nanogels according to the present invention maintain their structural integrity and prevent aggregation or degradation over time. The stable nanogels maintain their dispersed state, ensuring uniform distribution and optimal performance. The stable nanogel structure enhances the controlled release of drugs.

[0080] According to a second aspect, the present invention provides a method for preparing a nanogel comprising one or more bioactive molecules, therapeutic molecules or drugs, and an amphiphilic molecule, wherein the nanogel is obtained by one of two methods, depending on whether each bioactive molecule, therapeutic molecule or drug in the nanogel is loaded alone or simultaneously with the amphiphilic molecule: a) When each bioactive molecule, therapeutic molecule, or drug is individually loaded with an amphiphilic molecule, the method comprises the following sequential steps: i) Mix the poly(methacrylamide) of formula (1) with only one bioactive molecule, therapeutic molecule or drug and an amphiphilic molecule, preferably a vitamin E derivative or an amphiphilic cyclodextrin; (1) Where x is an integer greater than 1, preferably x is between 1 and 100; ii) Add a polymer solution with primary or secondary amine groups to the resulting mixture obtained in step i) to produce a cross-linked nanogel containing a bioactive molecule, therapeutic molecule or drug and an amphiphilic molecule, preferably a vitamin E derivative or an amphiphilic cyclodextrin. iii) Repeat steps i) and ii) for each additional bioactive molecule, therapeutic molecule, or drug. iv) Mix each cross-linked nanogel obtained in step iii) to obtain a resulting nanogel containing one or more biomolecules, therapeutic molecules or drugs.

[0082] b) When a bioactive molecule, therapeutic molecule, or drug is simultaneously loaded with an amphiphilic molecule, the method comprises the following sequential steps: i) Mix the poly(methacrylamide) of formula (1) with a quinone group with one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins; (1) Where x is an integer greater than 1, preferably x is between 1 and 100; ii) Add a polymer solution with primary or secondary amine groups to the resulting mixture obtained in step i) to produce a cross-linked nanogel containing one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins. In the first step i), micelles or vesicles are directly formed after a solution of poly(methacrylamide) of formula (1) with a quinone group is added to a mixture of one or more bioactive molecules, therapeutic molecules, or drugs with an amphiphilic molecule, preferably a vitamin E derivative or an amphiphilic cyclodextrin, in a solvent. The solvent can be water, an alcohol, or a combination thereof. Ethanol is preferred. The solution of poly(methacrylamide) with a quinone group can be water, an alcohol, or a combination thereof, but water is preferred.

[0084] The addition was carried out with stirring at room temperature.

[0085] In the second step ii), the polymer with primary or secondary amine groups reacts with poly(methacrylamide) of formula (1) via a quinone-amine reaction to generate a nanogel containing one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins, in solution.

[0086] Alternatively, the present invention provides a method for preparing nanogels comprising a bioactive molecule, a therapeutic molecule or a drug and an amphiphilic molecule, wherein the nanogel is obtained by one of two methods, depending on whether each bioactive molecule, therapeutic molecule or drug is loaded separately or simultaneously with the amphiphilic molecule in the nanogel: a) When each bioactive molecule, therapeutic molecule, or drug is individually loaded with an amphiphilic molecule, the method comprises the following sequential steps: i) Mix the poly(vinyl)quinone group of formula (6) with only one bioactive molecule, therapeutic molecule or drug and an amphiphilic molecule, preferably a vitamin E derivative or an amphiphilic cyclodextrin; ii) Add a polymer solution with primary or secondary amine groups to the resulting mixture obtained in step i) to produce a cross-linked nanogel containing a bioactive molecule, therapeutic molecule or drug and an amphiphilic molecule, preferably a vitamin E derivative or an amphiphilic cyclodextrin. iii) Repeat steps i) and ii) for each additional bioactive molecule, therapeutic molecule, or drug. iv) Mix each cross-linked nanogel obtained in step iii) to obtain a resulting nanogel containing two or more biomolecules, therapeutic molecules or drugs.

[0087] b) When a bioactive molecule, therapeutic molecule, or drug is simultaneously loaded with an amphiphilic molecule, the method comprises the following sequential steps: i) Mixing the poly(vinyl)quinone of formula (6) with one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins; (6) Where n is an integer greater than 1, preferably between 1 and 100; ii) Add a polymer solution with primary or secondary amine groups to the resulting mixture obtained in step i) to produce a cross-linked nanogel containing one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins.

[0089] In the first step i), after the poly(vinyl)quinone solution of formula (6) is added to a mixture of one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins in a solvent, micelles or vesicles are formed and encapsulated with amphiphilic molecules.

[0090] The solvent can be water or alcohol or a combination thereof, but ethanol is preferred.

[0091] The solution of poly(vinylquinone) can be water or alcohol or a combination thereof, but water is preferred.

[0092] The addition was carried out with stirring at room temperature.

[0093] In the second step ii), the polymer with primary or secondary amine groups reacts with poly(vinylquinone) of formula (6) through quinone-amine interaction to generate a nanogel containing one or more bioactive molecules, therapeutic molecules or drugs encapsulated with amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins.

[0094] In a preferred embodiment, the vitamin E derivative is D-α-tocopherol polyethylene glycol succinate.

[0095] In another preferred embodiment, the amphiphilic cyclodextrin is hydroxypropyl β-cyclodextrin.

[0096] In another preferred embodiment, the polymer with primary or secondary amine groups includes polyallylamine, preferably poly(allylamine hydrochloride), also known as PAH, as shown below, where p is an integer greater than 1, preferably p is between 10 and 300, and most preferably p is 160:

[0097] The solvent can be any solvent containing an OH group, so that at least one hydrogen atom readily interacts in hydrogen bonds. Examples include water, alcohols such as methanol, ethanol, butanol, propanol, etc., or combinations thereof.

[0098] In a preferred embodiment, the solvent is an alcohol, preferably ethanol.

[0099] In a preferred embodiment, the bioactive molecule, therapeutic molecule, or drug is a triazolo(4,4-d)-pyrimidine derivative of formula (3), most preferably a (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidine-3- [7-[(1R,2S)-2-(3,4-difluorophenyl)-cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazol[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol (also known as fluometacyl) ® ).

[0100] In another preferred embodiment, the bioactive molecule, therapeutic molecule, or drug is biguanide, with chlorhexidine being the most preferred.

[0101] In another preferred embodiment, a triazolo(4,4-d)-pyrimidine derivative is combined with an antibacterial antibiotic, preferably minocycline. The triazolo(4,4-d)-pyrimidine derivative is preferably tereflux, flumetrexed, or Fluometacyl. ® .

[0102] In another preferred embodiment, a triazolo(4,4-d)-pyrimidine derivative is combined with biguanide (most preferably chlorhexidine).

[0103] According to a third aspect, the present invention provides biomaterial implants, medical devices, or bioprosthetics, wherein their surfaces or portions are coated with a quinone-based poly(methacrylamide) nanogel of formula (1). (1) Where x is an integer greater than 1, preferably in the range of 1 to 100; The poly(methacrylamide) is crosslinked with polymers containing primary or secondary amine groups; and The nanogel contains one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives, and more preferably D-α-tocopherol polyethylene glycol succinate.

[0105] Alternatively, the present invention provides biomaterial implants, medical devices, or bioprosthetics wherein their surfaces or portions are coated with a nanogel made of poly(vinyl)quinone of formula (6). (6) Where n is an integer greater than 1, preferably in the range of 1 to 100; The poly(vinyl)quinone is crosslinked with a polymer containing primary or secondary amine groups; and The nanogel contains one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives, and more preferably D-α-tocopherol polyethylene glycol succinate.

[0107] Alternatively, the present invention provides biomaterial implants, medical devices or bioprosthetics wherein their surfaces or portions are coated with a nanogel made of poly(methacrylamide) and polyvinylquinone or copolymers thereof containing quinone.

[0108] The present invention is also extended to all biomaterial implants, medical devices or bioprosthetics, wherein the surface or part thereof is coated with a nanogel made of a polymer or copolymer with a quinone group.

[0109] Biomaterial implants, medical devices, or bioprosthetics coated with the nanogel according to the invention advantageously provide a more uniform, hydrophilic, and smooth surface.

[0110] The more uniform, hydrophilic, and smooth surface of biomaterial implants, medical devices, or bioprosthetics coated with nanogels advantageously reduces damage during insertion into human patients or animals and reduces thrombosis.

[0111] Biomaterial implants, medical devices, or bioprosthetics coated with nanogels containing bioactive molecules, therapeutic molecules, or drugs, as well as amphiphilic molecules, also advantageously provide more reproducible kinetic release of hydrophobic bioactive molecules, therapeutic molecules, or drugs from the coated nanogels and from the medical devices, biomaterial implants, or bioprosthetics, resulting in better pharmacological efficiency of the bioactive molecules, therapeutic molecules, or drugs, particularly in preventing complications of infection and thrombosis.

[0112] Furthermore, biomaterial implants, medical devices, or bioprosthetics coated with the nanogel according to the present invention also advantageously reduce bacterial adhesion to their surfaces.

[0113] When micelles containing one or more bioactive molecules, therapeutic molecules, or drugs encapsulated with amphiphilic molecules, preferably vitamin E derivatives, and more preferably D-α-tocopherol polyethylene glycol succinate, are loaded into nanogels coated on the surface of biomaterial implants, medical devices, or bioprosthetics, bacterial adhesion to the surface of the biomaterial implants, medical devices, or bioprosthetics is reduced. This anti-adhesion effect is synergistically enhanced when an antibacterial agent is present in the nanogel.

[0114] Furthermore, amphiphilic molecules, particularly TPGS in nanogel coatings, do not inhibit the pharmacological effects of bioactive agents, therapeutic molecules, or drugs as they would when used in solution.

[0115] Biomaterial implants can be any implantable foreign material used clinically in a host mammal, such as prosthetic joints, pacemakers, implantable cardioverter defibrillators, catheters including intravascular or urinary catheters or materials, stents including coronary stents, mechanical and biological prosthetic heart valves, intraocular lenses, dental implants, etc.

[0116] Medical devices can be, but are not limited to, any instrument, tool, apparatus, implant, etc., related to the medical field or human or veterinary practice, or intended for the prevention or treatment of disease. Medical devices can include all natural and synthetic materials, as well as fibrous and non-fibrous materials. For example, these materials can include metals, plastics, paper, glass, ceramics, textiles, rubber, polymers, composite materials, or any other material or combination of materials. Exemplary medical devices include, but are not limited to, catheters of any kind; cannulas; needles; stents of any size, shape, or location; coils of any size, shape, or location; contact lenses; intrauterine devices (IUDs); peristaltic pump chambers; endotracheal tubes; gastrointestinal feeding tubes; arteriovenous shunts; condoms; oxygenators and renal membranes; gloves; pacemaker leads; wound dressings; metal pins, plates, and screws; metal artificial hip joints; artificial knees; and gels. In embodiments, the nanogel of the present invention can be used to coat catheters to prevent bacterial infection.

[0117] Biological prostheses can be, but are not limited to, prostheses made of biological materials. Examples include heart valves, pericardial grafts, vascular grafts, bladder prostheses, tendon prostheses, hernia patches, surgical meshes, and skin substitutes.

[0118] In one embodiment, the nanogel of the present invention can be used to coat bioartificial heart valves, such as decellularized porcine heart valves or bovine pericardium, to prevent bacterial infection and thrombosis.

[0119] Coated biomaterial implants, medical devices, or bioprosthetics can be used in human or animal hosts for the diagnosis, prevention, or treatment of diseases or for medical practice.

[0120] In a preferred embodiment, the polymer with primary or secondary amine groups is poly(allylamine hydrochloride) of formula (2), wherein p is an integer greater than 1, preferably between 10 and 300.

[0121] In another preferred embodiment, the bioactive molecule, therapeutic molecule, or drug is a triazolo(4,4-d)-pyrimidine derivative of formula (3) having antibacterial and antiplatelet properties; preferably (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5 -(2-hydroxyethoxy)-1,2-cyclopentanediol, also known as terefrol or ticagrelor; or (1S,2R,3S,4R)-4-[7-[(1R,2S)-2-(3,4-difluorophenyl)-cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol, also known as fluometacyl. ® .

[0122] In another preferred embodiment, the bioactive molecule, therapeutic molecule, or drug is biguanide, more preferably chlorhexidine.

[0123] In another preferred embodiment, a triazolo(4,4-d)-pyrimidine derivative is combined with an antibacterial antibiotic, preferably minocycline. Most preferably, the triazolo(4,4-d)-pyrimidine derivative is tereflux, flumetrexed, or Fluometacyl. ® .

[0124] In another preferred embodiment, a triazolo(4,4-d)-pyrimidine derivative is combined with biguanide, more preferably chlorhexidine.

[0125] The nanogels according to the invention can be anchored or attached to the surface of biomaterial implants, medical devices, or bioprosthetics using various physical or chemical methods known in the art. For example, the surface of the biomaterial implant, medical device, or bioprosthetic can be electrically grafted, deposited layer by layer, spin-coated, sprayed, or simply immersed in a mixed solution of a quinone-based poly(methacrylamide) of formula (1) with bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules (preferably vitamin E derivatives).

[0126] Alternatively, it is possible to immerse the surface of a biomaterial implant, medical device, or bioprosthetic in a mixed solution of poly(vinylquinone) and one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules (preferably vitamin E derivatives).

[0127] Bioactive molecules, therapeutic molecules, or drugs are encapsulated together with amphiphilic molecules (preferably TPGS) and loaded into nanogels, and gradually and continuously released over time from biomaterial implants, medical devices, or bioprosthetics into mammalian hosts. The mammalian host can be a human patient or an animal.

[0128] When micelles are loaded into nanogels, amphiphilic molecules, particularly TGPS, surprisingly reduce bacterial adhesion on the surface without hindering the pharmacological effects of bioactive agents, therapeutic molecules, or drugs.

[0129] When the antibacterial agent is present in the nanogel, this anti-adhesion effect is synergistically enhanced.

[0130] In a fourth aspect, the present invention provides a method for producing a medical device, biomaterial implant, or bioprosthetic having a nanogel-coated surface, said surface comprising one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins; the method comprises the following sequential steps: ci) Optionally, the surface to be coated is immersed in a dopamine buffer solution; cii) Immerse the surface coated in step ci) into a polymer solution containing primary or secondary amine groups; then ciii) Immerse the coated surface obtained in step cii) in a liquid suspension containing one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins; said cross-linked nanogel is obtained in a liquid suspension, preferably in water, by one of the two methods of the present invention; then civ) Drying the cross-linked coated surface obtained in ciii) to obtain a coated cross-linked nanogel surface containing one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins. cv) Optionally repeat steps cii) to civ) to obtain a surface coated with a multilayer cross-linked nanogel containing one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins; Alternatively, the present invention also provides a method for producing a medical device, biomaterial implant, or bioprosthetic having a cross-linked monolayer or optionally multilayer coated surface, said surface comprising one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins; said method comprising the following sequential steps: di) Optionally, the surface to be coated is immersed in a dopamine buffer solution; dii) Immerse the surface coated in step di) into a polymer solution containing primary or secondary amine groups; then diii) Immerse the resulting coated surface obtained in step dii) into a mixture of poly(methacrylamide) of formula (1) containing one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins. (1) Where x is an integer greater than 1, preferably in the range of 1 to 100; div) Drying step diii) The cross-linked coated surface obtained to obtain a coated cross-linked monolayer surface containing one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins. dv) Optionally repeat steps dii) to div) to obtain a surface coated with a cross-linked multilayer containing one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins.

[0132] In optional steps c1) and di), the medical device, biomaterial implant, or bioprosthetic is first immersed in a buffer solution, particularly a Tris buffer solution containing dopamine, to firmly anchor the first polymer layer to the surface of the medical device, biomaterial implant, or bioprosthetic. Then, a primer coating of polydopamine PDA is formed on the surface of the medical device by polymerization of dopamine molecules having a 4-(2-aminoethyl)phenyl-1,2-diol motif.

[0133] In steps cii) and dii), the polymer with primary or secondary amine groups is preferably PAH, and the covalent grafting of PAH onto the primer coating occurs through an amine / quinone reaction and / or Schiff base formation.

[0134] In step diii), the pre-coated surface obtained in step dii) is immersed in a solution containing poly(methacrylamide) of formula (1) with quinone groups, mixed with one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins. Covalent grafting of the polymer layer is carried out through the same reaction between primary or secondary amines, preferably from the PAH monolayer and the quinone groups of the poly(methacrylamide) of formula (1), and / or Schiff base formation. (1) Where x is an integer greater than 1, preferably x is between 1 and 100.

[0136] In step ciii), the pre-coated surface obtained in step cii) is immersed in a liquid suspension containing one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins; the liquid suspension is preferably water.

[0137] In steps cii) to cv), covalent grafting with a polymer (preferably PAH) containing primary or secondary amine groups occurs between quinone-containing layers coated on a medical device, biomaterial implant, or bioprosthetic, resulting in a cross-linked nanogel coating on the medical device, biomaterial implant, or bioprosthetic. In steps dii) to dv), covalent grafting with a polymer (preferably PAH) containing primary or secondary amine groups occurs between monolayers coated on a medical device, biomaterial implant, or bioprosthetic, resulting in a cross-linked multilayer coating on the medical device, biomaterial implant, or bioprosthetic.

[0138] Similar to poly(vinylquinone), the present invention also provides a method for producing medical devices, biomaterial implants, or bioprosthetics with a surface coated with a nanogel, said surface comprising one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins; said method comprising the following sequential steps: (ei) Optionally, the surface to be coated is immersed in a dopamine buffer solution; eii) Immerse the surface coated in step ei) into a polymer solution containing primary or secondary amine groups; then eiii) Immerse the coated surface obtained in step eii) into a liquid suspension containing one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins, wherein the cross-linked nanogels are obtained in a liquid suspension by one of the two methods of the present invention, preferably in water. eiv) Drying the cross-linked coated surface obtained in step eiii) to obtain a coated cross-linked nanogel surface containing one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins. ev) Optionally repeat steps eii) to eiv) to obtain a surface coated with a multilayer cross-linked nanogel containing one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins.

[0139] Alternatively, the present invention also provides a method for producing a medical device, biomaterial implant, or bioprosthetic having an optionally multi-layered coated surface, comprising the following steps: fi) Optionally, the surface to be coated is immersed in a dopamine buffer solution; fii) Immerse the surface coated in step fi) into a polymer solution containing primary or secondary amine groups; then fiii) Immerse the resulting coating surface obtained in step fii) into poly(vinylquinone) of formula (6) and one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or a mixture of amphiphilic cyclodextrins; (6) Where n is an integer greater than 1, preferably between 1 and 100; fiv) The cross-linked coated surface obtained in drying step fiii) is used to obtain a cross-linked monolayer coated surface containing one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins. fv) Optionally repeat steps fii) to fiv) to obtain a cross-linked multilayer coated surface containing one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives.

[0141] In steps ei) and fi), the medical device, biomaterial implant, or bioprosthetic is first immersed in a buffer solution, particularly a Tris buffer solution containing dopamine (DOPA), to firmly anchor the first layer of coating to the surface of the medical device, biomaterial implant, or bioprosthetic. Then, a primer coating of PDA is generated on the surface of the medical device through the polymerization of dopamine molecules having a 4-(2-aminoethyl)phenyl-1,2-diol motif.

[0142] In steps eii) and fii), the polymer with primary or secondary amine groups is PAH, and the covalent grafting of PAH onto the primer coating occurs through an amine / quinone reaction and / or Schiff base formation at room temperature.

[0143] In step fiii), the pre-coated surface obtained in step fii) is immersed in a poly(vinylquinone) of formula (6) mixed with one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules (preferably vitamin E derivatives or amphiphilic cyclodextrins). The covalent grafting of the polymer layer is carried out through the same reaction between the primary amine of the PAH monolayer and the quinone group of the poly(vinylquinone) of formula (6) and / or Schiff base formation.

[0144] In steps eii) to ev), covalent grafting with a polymer (preferably PAH) containing primary or secondary amine groups occurs between nanogel layers coated on a medical device, biomaterial implant, or bioprosthetic, resulting in a cross-linked nanogel coating on the medical device, biomaterial implant, or bioprosthetic.

[0145] In steps fii) to fv), covalent grafting with a polymer (preferably PAH) containing primary or secondary amine groups occurs between monolayers coated on a medical device, biomaterial implant, or bioprosthetic, resulting in a cross-linked multilayer coating on the medical device, biomaterial implant, or bioprosthetic.

[0146] Alternatively, the present invention also provides a method for producing a medical device, biomaterial implant, or bioprosthetic having a coated surface comprising a nanogel made of poly(methacrylamide)quinone of formula (1) and polyvinylquinone of formula (6) or copolymers thereof.

[0147] The present invention is also extended to methods for producing biomaterial implants, medical devices or bioprosthetics whose surfaces are coated with nanogels made of polymers or copolymers with quinone groups.

[0148] The obtained nanogel may contain one or more, preferably two or more, bioactive molecules, therapeutic molecules, and / or drugs. Bioactive molecules may include antibiotics and / or antiplatelet agents.

[0149] The method of the present invention can be used without a primer coating step. In this case, the coating adhesion is based on the adhesive properties of free quinone groups present on the surface of the nanogel, which is sufficient to coat and fix the nanogel according to the invention onto the surface of medical devices, biomaterial implants, or bioprosthetics.

[0150] By repeating steps cii) and ciii) or eii) and eiii) of the above method, medical devices, biomaterial implants, or bioprosthetics with two or more layers of cross-linked nanogels coated on their surfaces can be produced. Medical devices, biomaterial implants, or bioprosthetics containing 2, 3, 4, 5, or more layers of nanogels can be produced.

[0151] In a preferred embodiment, the bioactive molecule, therapeutic molecule, or drug is a triazolo(4,4-d)-pyrimidine derivative of formula (3) having antibacterial and antiplatelet properties; preferably (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidine-3-yl]-5- (2-Hydroxyethoxy)-1,2-cyclopentanediol, also known as terefrol or ticagrelor; or (1S,2R,3S,4R)-4-[7-[(1R,2S)-2-(3,4-difluorophenyl)-cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol, also known as fluometacyl. ® .

[0152] In another preferred embodiment, a triazolo(4,4-d)-pyrimidine derivative is combined with an antibacterial antibiotic, preferably minocycline. The triazolo(4,4-d)-pyrimidine derivative is preferably tereflux, flumetrexed, or Fluometacyl. ® .

[0153] In another preferred embodiment, the bioactive molecule, therapeutic molecule, or drug is biguanide, with chlorhexidine being the most preferred.

[0154] The method of the present invention can be used to coat only a portion of the surface of a medical device, biomaterial implant, or bioprosthetic, or substantially all or the entire surface of a medical device, biomaterial implant, or bioprosthetic.

[0155] The present invention also provides coated medical devices, biomaterial implants or bioprosthetics produced according to the present invention or by the methods of the present invention, for preventing or reducing infection when the medical device, biomaterial implant or bioprosthetic is implanted in a mammal, which may be a human patient or an animal.

[0156] In a fifth aspect, the present invention also provides pharmaceutical compositions comprising a nanogel according to the invention or a nanogel produced by the method of the invention, for the prevention or reduction of infection when applied topically to a host mammal. The host mammal may be a human patient or an animal.

[0157] In a preferred embodiment, the pharmaceutical composition comprising the nanogel of the present invention is administered to an animal, preferably a dog, sheep or a cow, for the treatment of a skin disease caused by a bacterial infection, such as mastitis or pyoderma.

[0158] The pharmaceutical composition containing the nanogel according to the invention is preferably a gel, but may also be a physiologically compatible liquid composition.

[0159] In addition to bioactive molecules, pharmaceutical compositions may include therapeutic molecules or drugs and amphiphilic molecules; excipients, preservatives, solvents, and / or viscosity modifiers. Solvents are, for example, water, saline or any other physiological solution, ethanol, glycerol, oils (e.g., vegetable oils), or mixtures thereof. Viscosity modifiers include, for example, carboxymethyl cellulose.

[0160] In a preferred embodiment, the bioactive molecule, therapeutic molecule, or drug is an anti-infective agent, bactericide, antiviral agent, or bacteriostatic agent, preferably minocycline or chlorhexidine.

[0161] In a preferred embodiment, the bioactive molecule, therapeutic molecule, or drug is torefluzole.

[0162] In a sixth aspect, the present invention also provides the use of nanogels produced according to or by the methods of the present invention for inhibiting bacterial adhesion to the surface of medical devices, particularly the surface of catheters.

[0163] Methods for inhibiting bacterial adhesion to surfaces may include the following steps: i) Optionally, the surface to be inhibited from bacterial adhesion is immersed in a dopamine buffer solution; ii) Immerse the surface optionally coated in step i) in a polymer solution containing primary or secondary amine groups; iii) Immerse the surface obtained in step ii) in a solution of poly(methacrylamide) of formula (1) containing a quinone group, which is mixed with one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins; iv) Dry the cross-linked coated surface obtained in step iii) to obtain a coated cross-linked monolayer surface containing one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins.

[0164] Alternatively, methods for inhibiting bacterial adhesion to a surface may include the following steps: i) Immerse the surface from which bacterial adhesion is to be inhibited in a dopamine buffer solution; ii) Immerse the surface optionally coated in step i) in a polymer solution containing primary or secondary amine groups; iii) Immerse the surface obtained in step ii) in a solution of poly(vinylquinone) of formula (6) mixed with one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins; iv) Dry the cross-linked coated surface obtained in step iii) to obtain a coated cross-linked monolayer surface containing one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins.

[0165] Alternatively, methods for inhibiting bacterial adhesion to a surface may include the following steps: i) Optionally, the surface to be inhibited from bacterial adhesion is immersed in a dopamine buffer solution; ii) Immerse the surface coated in step i) in a polymer solution containing primary or secondary amine groups; then iii) Immerse the coated surface obtained in step ii) into a liquid suspension containing one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins; the cross-linked nanogel is obtained in a liquid suspension, preferably in water, by one of the two methods of the present invention; then iv) Dry the cross-linked coated surface obtained in step iii) to obtain a coated cross-linked nanogel surface containing one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins. v) Optionally repeat steps cii) to civ) to obtain a surface coated with a multilayer cross-linked nanogel containing one or more bioactive molecules, therapeutic molecules or drugs and amphiphilic molecules, preferably vitamin E derivatives or amphiphilic cyclodextrins.

[0166] The invention is further described by way of non-limiting embodiments only, with reference to the accompanying drawings and experimental examples below.

[0167] Figure 1 Dynamic size analysis (dynamic light scattering, DLS) of the nanogel diameter in the aqueous suspension NTT containing terefluphene and TPGS according to the present invention is shown compared with that of the nanogel NT containing only terefluphene according to WO2018 / 122318.

[0168] Figure 2The diameters of nanogels in aqueous suspensions containing only tereflux (A) or TPGS (B) according to the present invention are shown. Solid line: DLS analysis achieved 1 hour after nanogel formation. Dashed line: DLS analysis achieved 24 hours later.

[0169] Figure 3 The surface morphology of a polyurethane (PU) catheter coated with nanogel according to the present invention (NTT) is shown by SEM analysis, compared with the surface morphology of a PU catheter coated with nanogel (NT) according to WO2018 / 122318.

[0170] Figure 4 The kinetic release of terefothide from nanogel (NTT) according to the present invention is shown compared with the kinetic release of terefothide from nanogel (NT) according to WO2018 / 122318.

[0171] Figure 5 The diameters of the nanogels in an aqueous suspension containing tereflux, TPGS, and minocycline are shown. Solid line: DLS analysis achieved 1 hour after nanogel formation. Dashed line: DLS analysis achieved 24 hours later.

[0172] Figure 6 Staphylococcus aureus (Staphylococcus aureus) S. aureus Real-time microcalorimetric measurements of BAA-1556 metabolic activity, expressed as heat flux. Solid lines represent control bacteria with a carrier (0.6% ethanol); thick dashed lines represent bacteria treated with 10 μg / mL tereflux; thick streaked lines represent bacteria treated with 20 μg / mL tereflux; thin dashed lines represent bacteria treated with tereflux and TPGS (10 μg / mL: 10 μg / mL); thin streaked lines represent bacteria treated with tereflux and TPGS (20 μg / mL: 20 μg / mL).

[0173] Figure 7 Real-time microcalorimetric measurements of the metabolic activity of Staphylococcus aureus BAA-1556 adhered to the catheter are shown, expressed as heat flow. Solid line - uncoated PU catheter; dashed line - PU catheter coated with nanogel loaded with toreflux; dotted line - PU catheter coated with nanogel loaded with toreflux-TPGS.

[0174] Figure 8 Real-time microcalorimetry measurements of the metabolic activity of bacteria adhering to the duct are shown, expressed as heat flux. Staphylococcus aureus BAA-1556 (MRSA) at a dilution of 10-1 -6Baseline growth at time of day (no catheter fragment); B - Control, uncoated catheter fragment; C - NGM - Catheter fragment coated with 5 layers of nanogel, with minocycline 0.5 mg / mL added to the last nanogel layer and grafted with PEG (t=0 day); D - NTM - Catheter fragment coated with 5 layers of nanogel loaded with 0.05 mg / mL ticagrelor, then minocycline (0.5 mg / mL) added to the last nanogel layer and grafted with PEG (t=0 day); E - NTTM - Catheter fragment coated with 5 layers of nanogel loaded with a combination of Triafluocyl® / TPGS (0.1 / 0.2 mg / mL, w / w), then minocycline (0.5 mg / mL) added to the last nanogel layer (t=0 day); F - NGM - Catheter fragment coated with 5 layers of nanogel, with minocycline 0.5 mg / mL added to the last nanogel layer and grafted with PEG (t=20 day); G - NTM - Catheter fragment coated with 0.05 mg / mL ticagrelor... Five layers of Triafluocyl® nanogels were used, followed by the addition of minocycline (0.5 mg / mL) to the final nanogel layer and grafting with PEG (t=20 days); H-NTTM-catheter fragments were coated with a five-layer nanogel loaded with a combination of Triafluocyl® / TPGS (0.1 / 0.2 mg / mL, w / w), followed by the addition of minocycline (0.5 mg / mL) to the final nanogel layer (t=20 days).

[0175] Figure 9 The DLS analysis of the diameter of nanogels in the aqueous suspension is shown. The solid line indicates the presence of chlorhexidine (Chc) and TPGS, and the dashed line indicates the absence of TPGS.

[0176] Figure 10 The DLS analysis of the diameter of nanogels in the aqueous suspension is shown. Solid lines indicate the presence of tereflux, chlorhexidine (Chc), and TPGS, while dashed lines indicate the absence of TPGS.

[0177] Figure 11 It shows the presence of Fluometacyl ® Photographs of nanogels with and without Chc and TPGS (NFCcT) and without TPGS (NFCc), showing complete precipitation of NFCc after 48 hours.

[0178] Figure 12 Real-time microcalorimetric measurements of bacterial metabolic activity adhering to the duct are shown, expressed as heat flux. Curve A - Control Pseudomonas aeruginosa ( P. aeruginosa 10 -7 Bacterial growth; Curve B - bacteria in a PU catheter coated with nanogel, the catheter being loaded with Fluometacyl ®And chlorhexidine chloride (NFCc); Curve C - bacteria with PU conduits coated with nanogel, which were loaded with Fluometacyl in the presence of TPGS (NFCcT). ® And chlorhexidine chloride.

[0179] Figure 13 Real-time microcalorimetric measurements of the metabolic activity of *Pseudomonas aeruginosa* adhering to a catheter are shown, expressed as heat flux. Curve A – Bacteria with a PU catheter coated with a nanogel and loaded with Fluometacyl. ® And chlorhexidine chloride (NFCc); Curve B - bacteria with PU conduits coated with nanogel, which are loaded with Fluometacyl ® And immersed in chlorhexidine chloride solution (1 mg / mL) (NF / Cc); Curve C - bacteria with PU catheters coated with nanogel, which were loaded with Fluometacyl in the presence of TPGS (NFCcT). ® And chlorhexidine chloride.

[0180] Figure 14 DLS analysis of the diameter of a nanogel containing a mixture of flumetrexed, chlorhexidine, and polymyxin B (NFCccP) is shown.

[0181] Figure 15 DLS analysis of the diameter of nanogels in aqueous suspension is shown before (solid line) and after (dashed line) the addition of PAH. The nanogels contain fluorometholone and cyclodextrin (NFCy).

[0182] Figure 16 The stability study of the diameter of nanogels of NF, NFT, and NFCy in aqueous suspensions with different concentrations of fluorometholone and stabilizers (TPGS or cyclodextrin) is shown using DLS analysis.

[0183] Figure 17 Real-time microcalorimetric measurements of bacterial metabolic activity are shown, expressed as heat flux. Curve A - Control: Staphylococcus aureus MRSA 10 -6 Bacterial growth; curve B-containing Fluometacyl ® (10 μg / mL) bacteria; curve C - containing Fluometacyl ® Bacteria containing 10 μg / mL and hydroxypropyl-β-cyclodextrin (42 μg / mL); Curve D - bacteria containing fluometacyl (20 μg / mL); Curve E - bacteria containing fluometacyl ® Bacteria containing 20 μg / mL of hydroxypropyl-β-cyclodextrin and 42 μg / mL of hydroxypropyl-β-cyclodextrin.

[0184] Figure 18 Real-time microcalorimetric measurements of bacterial metabolic activity adhering to the duct are shown, expressed as heat flux. Curve A - Control: Staphylococcus aureus MRSA 10 -6 Bacterial growth; Curve B - bacteria in a PU catheter coated with nanogel, the catheter being loaded with Fluometacyl ® And TPGS (0.2 / 0.4 mg / mL, w / w); Curve C - bacteria with PU catheters coated with nanogel, which are loaded with Fluometacyl ® And hydroxypropyl-β-cyclodextrin (0.2 / 15 mg / mL, w / w).

[0185] The following table of references summarizes the different nanogel compositions obtained according to the present invention in the following embodiments:

[0186] Example 1: Preparation of nanogels according to the present invention using P(mDOPA)tereflux and TPGS (also known as NTT). The nanogel preparation is similar to that disclosed in WO2018 / 122318A1, except for the drug loading in the presence of additional amphiphilic molecules.

[0187] The nanogels are prepared in a liquid solution. After cross-linking, the nanogels remain suspended in the liquid solution.

[0188] 1.1 Oxidation of PmDOPA → Pox(mDOPA) Homopolymers of methacrylamide with 3,4-dihydroxy-L-phenylalanine (P(mDOPA)) were synthesized according to Faure et al in Adv Funct. Mater. 2012; 22:5271-5282 and oxidized in an aqueous medium under alkaline conditions for 12 hours to form water-soluble Pox(mDOPA). The oxidized catechol groups of Pox(mDOPA) are essential for the covalent interaction of PAHs at room temperature via amine / quinone reactions and / or Schiff base formation, and are therefore necessary for the preparation of stable crosslinked nanogels in aqueous suspensions.

[0189] P(mDOPA) (2.5 mg) was dissolved in distilled water (5 mL), and NaOH (0.1 M) was slowly added to raise the pH to above 10 and promote the oxidation of the catechol groups of P(mDOPA).

[0190] 1.2 Preparation of Terifolene / TPGS Mixture in Ethanol Terifolium (also known as ticagrelor, supplied by Polpharma) and TPGS supplied by MedChemExpress LLC were dissolved separately in ethanol to prepare stock solutions of 3.33 mg / mL. 160 μL of the terifolium solution and 160 μL of the TPGS solution were mixed with a magnetic stirrer (300 rpm) and then concentrated under vacuum to approximately 160 μL at room temperature.

[0191] 1.3 Preparation of nanogels using Terifolene / TPGS and PAH Pox(mDOPA) (5 mL, 0.5 mg / mL) was added to the concentrated mixture obtained at point 1.2 while stirring with a magnetic stirrer (300 rpm). After homogenization at room temperature for one hour, an aqueous PAH solution (0.5 mL, 0.5 mg / mL) provided by Sigma-Aldrich at pH 10 was slowly added to the mixture. The solution was reacted at room temperature with vigorous stirring with a magnetic stirrer (500 rpm) for 1 hour.

[0192] Nanogels with diameters ranging from 100 nm to 350 nm were observed in aqueous suspensions using dynamic light scattering (Zetasizer Advance Pro, Malvern).

[0193] Example 2: Preparation of nanogels according to the present invention using polyvinyl(quinone), tereflux and TPGS 2.1 Oxidation of polyvinylcatechol → polyvinylquinone.

[0194] Similar to P(mDOPA), poly(vinylcatechol) supplied by Polykey was oxidized in an aqueous medium under alkaline conditions for 12 hours to form water-soluble polyvinylquinone. The oxidized catechol groups of poly(vinylcatechol) are essential for the covalent interactions of PAHs at room temperature via amine / quinone reactions and / or Schiff base formation, and are therefore necessary for the preparation of stable cross-linked nanogels in aqueous suspensions.

[0195] Poly(vinylcatechol) (10 mg) was dissolved in distilled water (20 mL), and NaOH (0.1 M) was slowly added to raise the pH to above 10 over 12 hours and promote the oxidation of the catechin groups of poly(vinylcatechol).

[0196] 2.2 Preparation of Terifolene / TPGS Mixture in Ethanol Terifolol (also known as ticagrelor) and TPGS were dissolved separately in ethanol to prepare stock solutions of 3.33 mg / mL for Terifolol and TPGS, respectively. 160 μL of Terifolol solution and 160 μL of TPGS solution were mixed with a magnetic stirrer (300 rpm) and then concentrated under vacuum to approximately 160 μL at room temperature.

[0197] 2.3 Preparation of nanogels using Terifolene / TPGS and PAH Poly(diethylquinone) (5 mL, 0.5 mg / mL) was added to the concentrated mixture obtained at point 2.2 while stirring with a magnetic stirrer (300 rpm). After homogenization for one hour, an aqueous PAH solution (0.5 mL, 0.5 mg / mL) at pH 10 was slowly added to the mixture. The solution was reacted at room temperature with vigorous stirring with a magnetic stirrer (500 rpm) for 1 hour.

[0198] Nanogels with diameters ranging from 100 nm to 350 nm were observed in aqueous suspensions using dynamic light scattering (Zetasizer Advance Pro, Malvern).

[0199] Example 3: Preparation of a medical device having the nanogel according to the present invention The method according to the present invention is used to coat a polyurethane catheter provided by Carfill (intravascular grade polyurethane tube 5Fr).

[0200] Step 1: Immerse the catheter (4.5 cm long) in a dopamine Tris buffer solution (pH = 7.4) from Aldrich (0.2 g·L⁻¹). -1 ) for 5 hours.

[0201] Step 2: After rinsing twice with 5 mL of water, immerse the modified conduit substrate in a PAH aqueous solution (pH>10) for 1 hour, and rinse twice with 5 mL of water.

[0202] Step 3: Immerse the modified catheter substrate obtained in Step 2 in an aqueous solution of the nanogel loaded with bioactive molecules prepared according to Example 1 or Example 2 for 18 hours, and rinse twice with 5 mL of water.

[0203] Repeat steps 2 to 3 to build a multilayer assembly of nanogels on the surface of the coating device (five times to obtain a five-layer assembly of cross-linked nanogels).

[0204] Example 4: Comparison of the nanogel NTT according to the present invention with the nanogel NT of WO2018 / 122318A1 To compare with the nanogels of the present invention, nanogels containing and without tereflux were prepared according to WO2018 / 122318A1.

[0205] Preparation of nanogels without tereflux (NG): P(mDOPA) (2.5 mg) was dissolved in distilled water (5 mL), and NaOH (0.1 M) was slowly added to raise the pH to above 10 and promote the oxidation of the catechol groups of P(mDOPA). After standing overnight at room temperature, an aqueous solution of PAH at pH 10 (0.55 mL, 0.5 g / L) was slowly added to the Pox(mDOPA) solution. The solution was reacted for 1 hour at room temperature with vigorous stirring (500 rpm using a magnetic stirrer). Nanogels with diameters ranging from 100 nm to 250 nm were observed in the aqueous suspension by Zetasizer Advance Pto (Malvern).

[0206] Preparation of nanogels containing tereflux (NT): P(mDOPA) (2.5 mg) was dissolved in distilled water (5 mL), and NaOH (0.1 M) was slowly added to raise the pH to above 10 and promote the oxidation of the catechol groups of P(mDOPA). After standing overnight at room temperature, 0.16 mL of tereflux solution (3.33 mg / mL, in DMSO) was added dropwise to the Pox(mDOPA) solution while stirring at room temperature (using a magnetic stirrer at 300 rpm). After homogenization for one hour, PAH aqueous solution (0.5 mL, 0.5 g / L) was slowly added to the mixture. The solution was reacted for 1 hour at room temperature under vigorous stirring (using a magnetic stirrer at 500 rpm). Nanogels with diameters ranging from 120 nm to 750 nm were observed in the aqueous suspension by dynamic light scattering (Zetasizer Advance Pro, Malvern).

[0207] 4.1. Particle size and stability The diameter of the nanogel according to the present invention is compared with the diameter of the nanogel (NT) obtained in WO2018 / 122318A1 with and without tereflux.

[0208] Compared to nanogels without tereflux and TPGS (ranging from 100 nm to 250 nm) and nanogels containing only tereflux (ranging from 120 nm to 750 nm), the diameter of the nanogels of this invention (ranging from 100 nm to 350 nm) is significantly between the diameter of the nanogel particles themselves and the diameter of nanogel particles containing tereflux. For example... Figure 1As shown, the nanogel of the present invention also exhibits a low size distribution.

[0209] Figure 2 The increase in NT size of the nanogel after 24 hours is shown (dashed line), which describes the formation or precipitation of nanogel aggregates, not observed in the NTT nanogel that remained stable after 24 hours.

[0210] Therefore, the nanogel according to the present invention is more stable than the nanogel disclosed in WO2018 / 122318A1.

[0211] 4.2 Surface morphology from Figure 3 As can be seen, SEM analysis of the coated conduit revealed a significant difference between the NT coating and the NTT coating. A more uniform and smooth surface was observed with the NTT coating, while large particle aggregation was observed in the NT coating, which increased the surface roughness.

[0212] 4.3 Surface hydrophilicity Contact angle measurement is a useful method for determining surface hydrophilicity. Table 1 reports the contact angle analysis of glass coverslips (CS) provided by WTR. CS were coated with polydopamine (CS-PDA). Single-layer, triple-layer, and five-layer NTT (CS-NTT 1-layer, 3-layer, and 5-layer) were compared with a five-layer nanogel according to WO2018 / 122318 A1 NT (CS-NT 5-layer PEG). As in WO2018 / 122318A1, PEG (MW 2000 g / mol) was covalently linked to the top of the nanogel coating. Contact angles were measured after 30 seconds.

[0213] Compared to PDA (primer polydopamine), the static contact angle was significantly reduced in the presence of a nanogel layer (NT or NTT). A coated surface with five NTT layers (nanogel with TPGS) exhibited a much lower water contact angle (18.3°) than a coated surface with the same number of NT layers (35.5°), indicating that the presence of PEG chains (1000 g / mol) in the TPGS molecule significantly improved surface hydrophilicity, even after the deposition of only one NTT layer (25.3°).

[0214] Table 1

[0215] 4.3 Kinetic Release The in vitro drug release from the coated catheter surface was quantified at specified time points over a period of up to 10 days. The effect of the presence of TPGS on drug release kinetics was analyzed using UV spectrophotometry, and drug release curves were established for each catheter based on the experimental data. Figure 4 ).

[0216] The presence of deposits in the NT coating was found to significantly affect the drug release kinetics of the coated catheter. Drug release data indicated that both coatings exhibited gradual and continuous drug release. However, significant variability was observed in the NT coating, suggesting a lack of uniformity. Furthermore, the kinetics of toripalopexy release with NT was much faster than with NTT, resulting in a shorter release duration, which will negatively impact the duration of the coating's pharmacological activity.

[0217] 4.4 Terifolene content in coated catheters According to the method of the present invention (NTT) or WO2018 / 122318A1 (NT), six 4.5 cm long polyurethane catheters (intravascular grade polyurethane tubes 5Fr) provided by Carfill are coated with NTT or NT.

[0218] The content of terefrolactone in the coated tubing (4.5 cm) was determined by HPLC on a Waters Acquity UPLC system, which consists of a quaternary solvent delivery system, a syringe with adjustable injection volume, a temperature-controlled autosampler, a column thermostat, and a photodiode array detector. The assay method was performed according to the monograph on ticagrelor (European Pharmacopoeia 10.4). Briefly, the analytical column was an XBridge phenyl column, 150 x 4.6 mm, 3 μm (Waters), with a Security Guard phenyl column, 3 x 4 mm (Phenomenex). A 50 μL injection volume was used at a flow rate of 1.0 mL / min at 40°C (column temperature). Mobile phase A: phosphate buffer pH: 3.0 - water - acetonitrile (1:89:10 v / v / v). Mobile phase B: phosphate buffer pH: 3.0 - water - acetonitrile (1:29:70 v / v / v). The detection wavelength was 300 nm.

[0219] The data shown in Table 2 indicate that NTT-coated catheters achieved twice the amount of terefthol compared to catheters coated with NT.

[0220] Table 2

[0221] Example 5: Preparation of nanogels according to the present invention using P(mDOPA)tereflux, minocycline and TPGS (also known as NTTM) 5.1 Oxidation of PmDOPA → Pox(mDOPA).

[0222] A homopolymer of methacrylamide containing 3,4-dihydroxy-L-phenylalanine (P(mDOPA)) was oxidized in an aqueous medium under alkaline conditions for 12 hours to form water-soluble Pox(mDOPA). The oxidized catechol groups of Pox(mDOPA) are essential for the covalent interaction of PAHs at room temperature via amine / quinone reactions and / or Schiff base formation, and are therefore necessary for the preparation of stable cross-linked nanogels in aqueous suspensions.

[0223] P(mDOPA) (2.5 mg) was dissolved in distilled water (5 mL), and NaOH (0.1 M) was slowly added to raise the pH to above 10 and promote the oxidation of the catechol groups of P(mDOPA) to Pox(mDOPA).

[0224] 5.2 Preparation of Terifolene / Minocycline / TPGS Mixture in Ethanol Terifolium (also known as ticagrelor), minocycline, and TPGS were dissolved separately in ethanol to prepare a stock solution of 3.33 mg / mL. 160 μL of terifolium solution, 320 μL of TPGS solution, and 320 μL of minocycline solution were mixed with stirring (300 rpm) and then concentrated under vacuum at room temperature to approximately 160 μL.

[0225] 5.3 Preparation of nanogels using tereflux / minocycline / TPGS and PAH Pox(mDOPA) (5 mL, 0.5 mg / mL) was added to the concentrated mixture obtained at point 5.2 with stirring. After homogenization at room temperature for 1 hour, an aqueous PAH solution (0.5 mL, 0.5 mg / mL) at pH 10 was slowly added to the mixture. The solution was reacted with vigorous stirring (500 rpm) at room temperature for 1 hour.

[0226] Stable nanogels with diameters ranging from 150 nm to 350 nm were observed in aqueous suspensions using dynamic light scattering (Malvern). Figure 5 ) Similar to NTT nanogels, NTTM nanogels are stable within 24 hours of formation. In contrast, NT nanogels according to WO2018 / 122318A1 are unstable (Table 3).

[0227] Table 3

[0228] On the other hand, when tereflux is released from the nanogel prepared according to the method of the present invention and thus encapsulated by TPGS, as shown in the following examples, surprisingly little inhibitory effect of TPGS is observed.

[0229] Example 6: Effect of TPGS on the inhibitory activity of tereflux on bacterial growth Staphylococcus aureus (ATCC BAA-1556, MRSA) was grown overnight in TSB (trypsin-soybean broth) medium, and then diluted 1×10⁻⁶ in fresh TSB. 4 The bacterial suspension was then diluted 300 μL and aliquots were supplemented with 10 μg / mL and 20 μg / mL of Polpharma alone or a mixture of Polpharma and TPGS (MedChemExpress LLC (Europe)) (1:1 w / w). As described in the examples above, both Polpharma and TPGS were prepared in anhydrous ethanol and refrigerated in anhydrous ethanol as master stock. Polpharma, TPGS, or ethanol (final concentration 0.6%) was added to the bacterial suspension as a carrier to obtain concentrations (10 μg / mL and 20 μg / mL), followed by brief vortexing. The bacterial aliquots were then distributed into dedicated inactive inserts in 48-well plates and grown statically at 37°C for 24 hours. Bacterial growth and metabolic activity were measured in real-time using Calscreener technology, as described at https: / / cordis.europa.eu / project / id / 784514.

[0230] exist Figure 6 In this study, real-time microcalorimetric measurements of bacterial metabolic activity were expressed as heat flow and represented bacterial growth in TSB medium. Solid lines represent control bacteria with a carrier (0.6% ethanol); thick dashed lines represent bacteria treated with 10 μg / mL tereflux; thick streaked lines represent bacteria treated with 20 μg / mL tereflux; thin dashed lines represent bacteria treated with tereflux and TPGS (10 μg / mL: 10 μg / mL); and thin streaked lines represent bacteria treated with tereflux and TPGS (20 μg / mL: 20 μg / mL).

[0231] At a concentration of 10 μg / mL, tereflux (also known as ticagrelor) significantly reduced the growth of Staphylococcus aureus, while at higher doses of 20 μg / mL, bacterial growth was completely inhibited. Notably, the addition of an equal volume (w / w) of TPGS to tereflux significantly reduced its inhibitory effect on bacterial growth.

[0232] Example 7: Testing the anti-adhesion properties of nanogel coatings against Staphylococcus aureus using Terifolium alone and Terifolium in combination with TPGS.

[0233] Staphylococcus aureus (ATCC BAA-1556, MRSA) was grown overnight in TSB (trypsin-soybean broth) medium, and then diluted 1×10⁻⁶ in fresh TSB. 6 The nanogel coating was then added to aliquots of 1000 μL diluted bacterial suspension, consisting of two PU catheter sheets (0.5 cm long) coated with either terefraxetine (0.5 mg / mL) or terefraxetine-TPGS (0.5 mg / mL w / w). Uncoated PU catheters served as a control. The nanogel coating was prepared according to the protocols described in Examples 1 and 4. The bacterial solution with the catheters was incubated at 37°C and 220 rpm for 30 minutes. Subsequently, the catheters were washed twice with saline (0.9% NaCl) and placed in dedicated inactive inserts in 48-well plates containing 300 μL of fresh TSB medium, and grown statically at 37°C for 24 hours using Calscreener.

[0234] exist Figure 7 In the figure, real-time microcalorimetric measurements of bacterial metabolic activity adhering to the catheter are expressed as heat flow. Solid line - control uncoated PU catheter; dashed line - PU catheter coated with nanogel loaded with toreflux; dotted line - PU catheter coated with nanogel loaded with toreflux-TPGS.

[0235] The nanogel coating containing tereflux can increase the anti-adhesion of PU catheters, which is seen as a delay in the peak metabolic rate. The presence of TPGS in the coating can further shift the peak of bacterial growth, indicating less bacterial adhesion on the catheter surface.

[0236] Example 8: The combination of Terifolide-TPGS and the antimicrobial agent minocycline endows the nanogel (NTTM) coating with long-term anti-adhesion properties against Staphylococcus aureus.

[0237] Staphylococcus aureus (ATCC BAA-1556, MRSA) was grown overnight in TSB (trypsin-soybean broth) medium, and then diluted 1×10⁻⁶ in fresh TSB. 6 Subsequently, two catheter fragments (0.5 cm long) were added to an aliquot of the 1000 μL diluted bacterial suspension. The catheter fragments were coated with nanogel loaded with tereflux (0.05 mg / mL, NTM) or tereflux-TPGS (0.1 / 0.2 mg / mL w / w, NTTM) and supplemented with minocycline as an antibacterial agent.

[0238] Nanogel solutions prepared using Pox (mDOPA) and PAH (NG) A PAH aqueous solution (pH 10, 0.5 mL, 0.5 mg / mL) was slowly added to Pox(mDOPA) (5 mL, 0.5 mg / mL) while stirring with a magnetic stirrer (300 rpm). The solution was reacted at room temperature with vigorous stirring (500 rpm) for 1 hour.

[0239] Nanogels (NG) with diameters ranging from 100 nm to 300 nm were observed in aqueous suspensions using dynamic light scattering (Zetasizer Advance Pro, Malvern).

[0240] The catheter nanogel coating was performed according to the protocol described in Example 3. Minocycline (0.5 mg / mL) was added to the final layer of the nanogel, either to NG, NT, or NTT, resulting in the formation of the following variants, NGM, NTM, or NTTM, respectively. Uncoated catheter fragments served as controls. Two groups of coated catheter fragments were tested: “washed” (long-term drug release assay) after incubation in PBS / DMSO 2% for 20 days and “unwashed” (no incubation in buffer, time = 0 days) to observe the length of time the anti-adhesion activity of the coating was tested.

[0241] To test the anti-adhesion properties of the nanogel coating, the bacterial solution containing the aforementioned vessels was incubated at 37°C and 220 rpm for 30 minutes. Subsequently, the vessels were washed twice with saline (0.9% NaCl) and placed in a dedicated inactive insert in a 48-well plate containing 300 μL of fresh TSB medium, and grown using Calscreener under static conditions at 37°C for 24 hours. Microcalorimetric readings were obtained.

[0242] exist Figure 8 In this study, real-time microcalorimetric measurements of the metabolic activity of bacteria adhering to the duct were expressed as heat flux. Staphylococcus aureus (MRSA) at a dilution of 10-1... -6Baseline growth at time of day (no catheter fragment); B - Control, uncoated catheter fragment; C - NGM - Catheter fragment coated with 5 layers of nanogel, with minocycline 0.5 mg / mL added to the last nanogel layer and grafted with PEG (t=0 day); D - NTM - Catheter fragment coated with 5 layers of nanogel loaded with 0.05 mg / mL tereflux, then minocycline (0.5 mg / mL) added to the last nanogel layer and grafted with PEG (t=0 day); E - NTTM - Catheter fragment coated with 5 layers of nanogel loaded with a combination of tereflux / TPGS (0.1 / 0.2 mg / mL, w / w), then minocycline (0.5 mg / mL) added to the last nanogel layer (t=0 day); F - NGM - Catheter fragment coated with 5 layers of nanogel, with minocycline 0.5 mg / mL added to the last nanogel layer and grafted with PEG (t=20 day); G - NTM - Catheter fragment coated with 0.05 mg / mL tereflux / TPGS (0.1 / 0.2 mg / mL, w / w) and loaded with PEG (t=20 day); Five-layer nanogels of tereflux (0.1 / 0.2 mg / mL) were coated with minocycline (0.5 mg / mL) and then grafted with PEG (t=20 days); H-NTTM-catheter fragments were coated with five-layer nanogels loaded with a combination of tereflux / TPGS (0.1 / 0.2 mg / mL, w / w), followed by the addition of minocycline (0.5 mg / mL) to the final nanogel layer (t=20 days).

[0243] Introducing minocycline into the coating resulted in a complete reduction of bacterial adhesion to the catheter NGM, NTM, and NTTM, as shown by the background levels at the start of the drug release test (at day 0, curves C, D, and E).

[0244] The anti-adhesion effects of the coated catheters, particularly NTM and NTTM (curves G and H), persisted for at least 20 days (duration of drug release in this experimental setting). Terifolide and minocycline (NTM, NTTM) exhibited a significant synergistic effect compared to unloaded terifolide coated NGM. This effect was enhanced in the presence of TPGS (NTTM), which was attributed to the movement and reduction of trace thermal signals.

[0245] Using a combination of Tereflex and TPGS offers the possibility of increasing the Tereflex loading, which leads to a stronger synergistic effect between Tereflex and minocycline (NTTM vs. NTM). This clearly demonstrates that the NTTM coating is superior to the NTM coating, exhibiting a more significant and sustained anti-adhesion effect in the long term (≥ 20 days).

[0246] Example 9: Preparation of the nanogel (Nanogel NCT) according to the present invention using P(mDOPA), chlorhexidine chloride and TPGS The nanogel preparation was similar to that disclosed in Example 1, except for the drug loading. We used chlorhexidine chloride as the active pharmaceutical ingredient and TPGS as the amphiphilic molecule.

[0247] 9.1 Oxidation of PmDOPA → Pox(mDOPA).

[0248] A homopolymer of methacrylamide containing 3,4-dihydroxy-L-phenylalanine (P(mDOPA)) was oxidized in an aqueous medium under alkaline conditions for 12 hours to form water-soluble Pox(mDOPA). The oxidized catechol groups of Pox(mDOPA) are essential for the covalent interaction of PAHs at room temperature via amine / quinone reactions and / or Schiff base formation, and are therefore necessary for the preparation of stable cross-linked nanogels.

[0249] P(mDOPA) (2.5 mg) was dissolved in distilled water (5 mL), and NaOH (0.1 M) was slowly added to raise the pH to above 10 and promote the oxidation of the catechol groups of P(mDOPA).

[0250] 9.2 Preparation of chlorhexidine chloride / TPGS mixture in DMSO Chlorhexidine chloride (Chc) and TPGS were dissolved separately in DMSO to prepare stock solutions of 10 mg / mL Chc and TPGS. 115 μL of the Chc solution and 234 μL of the TPGS solution were mixed at room temperature with stirring (300 rpm) for 10 minutes.

[0251] 9.3 Preparation of Nanogels Using Chlorhexidine Chloride / TPGS and PAH Pox(mDOPA) (5 mL, 0.5 mg / mL) was added to the mixture obtained at point x.2 with stirring (300 rpm). After homogenization at room temperature for one hour, an aqueous PAH solution (0.5 mL, 0.5 mg / mL) at pH 10 was slowly added to the mixture. The solution was reacted with vigorous stirring (500 rpm) at room temperature for 1 hour.

[0252] Nanogels (NCcT) with diameters ranging from 100 nm to 350 nm were observed in liquid suspensions using dynamic light scattering (Zetasizer Advance Pro, Malvern).

[0253] Comparison of the nanogel NCCT according to the present invention with nanogel NCC without TPGS: particle size and stability The nanogel particle size of Example 9 of the present invention is compared with the nanogel diameter obtained without TPGS (NCc).

[0254] Preparation of nanogels containing chlorhexidine chloride (NCc): P(mDOPA) (2.5 mg) was dissolved in distilled water (5 mL), and NaOH (0.1 M) was slowly added to raise the pH to above 10 and promote the oxidation of the catechol groups of P(mDOPA). After standing overnight at room temperature, 0.110 mL of Chc solution (10 mg / mL, in DMSO) was added dropwise to the Pox(mDOPA) solution with stirring (300 rpm) at room temperature. After homogenization for one hour, PAH aqueous solution (0.5 mL; 0.5 g / L) at pH 10 was slowly added to the mixture. The solution was reacted with vigorous stirring (500 rpm) at room temperature for 1 hour. Large-diameter nanogels (NCs) with diameters greater than 500 nm were observed in the mixture by dynamic light scattering (Zetasizer Advance Pro, Malvern).

[0255] like Figure 9 As shown, compared with the diameter (NCc) of nanogels without TPGS, the nanogels loaded with Chc exhibit a lower diameter of 100 nm to 350 nm in the presence of TPGS (NCcT), and have a lower size distribution.

[0256] Example 10: Using P(mDOPA), Fluometacyl ® Nanogels (NFCcT nanogels) according to the present invention were prepared using chlorhexidine chloride and TPGS. The nanogel preparation was similar to that disclosed in Example 1, except for the drug loading. We used Fluometacyl... ® (Fluo) and chlorhexidine chloride and TPGS are amphiphilic molecules.

[0257] 10.1 Oxidation of PmDOPA → Pox(mDOPA) A homopolymer of methacrylamide containing 3,4-dihydroxy-L-phenylalanine (P(mDOPA)) was oxidized in an aqueous medium under alkaline conditions for 12 hours to form water-soluble Pox(mDOPA). The oxidized catechol groups of Pox(mDOPA) are essential for the covalent interaction of PAHs at room temperature via amine / quinone reactions and / or Schiff base formation, and are therefore necessary for the preparation of stable cross-linked nanogels in an aqueous medium.

[0258] P(mDOPA) (2.5 mg) was dissolved in distilled water (5 mL), and NaOH (0.1 M) was slowly added to raise the pH to above 10 and promote the oxidation of the catechol groups of P(mDOPA).

[0259] 10.2 Preparation of Fluometacyl in DMSO ® Chlorhexidine chloride / TPGS mixture Chlorhexidine chloride (Chc) and TPGS were dissolved separately in DMSO to prepare stock solutions of 10 mg / mL Fluo, Chc, and TPGS. The solutions were stirred at room temperature (300 rpm) for 10 minutes, and then 115 μL of Fluometacyl was added. ® The solution, 115 μL of Chc solution and 234 μL of TPGS solution were mixed.

[0260] 10.3 Preparation of Nanogels using Fluo, Chc / TPGS and PAH Pox(mDOPA) (5 mL, 0.5 mg / mL) was added to the mixture obtained at point 12.2 with stirring (300 rpm). After homogenization at room temperature for 1 hour, an aqueous PAH solution (0.5 mL, 0.5 mg / mL) at pH 10 was slowly added to the mixture. The solution was reacted with vigorous stirring (500 rpm) at room temperature for 1 hour.

[0261] Nanogels (NFCcT) with diameters ranging from 100 nm to 350 nm were observed in a mixed solution using dynamic light scattering (Zetasizer Advance Pro, Malvern).

[0262] A comparison of the nanogel NFCc according to the present invention with the nanogel NFCc without TPGS.

[0263] The diameter of the nanogel according to Example 10 of the present invention is compared with that of a nanogel loaded with Fluo and Chc but without TPGS (NFCc).

[0264] Preparation of chlorhexidine chloride-containing nanogels (NFCc): P(mDOPA) (2.5 mg) was dissolved in distilled water (5 mL), and NaOH (0.1 M) was slowly added to raise the pH to above 10 and promote the oxidation of the catechol groups of P(mDOPA). After standing overnight at room temperature, 0.11 mL of Chc solution (10 mg / mL, in DMSO) and 0.11 mL of Fluo solution (10 mg / mL, in DMSO) were added to the Pox(mDOPA) solution, respectively, with stirring (300 rpm) at room temperature. After homogenization for one hour, PAH aqueous solution (0.5 mL, 0.5 g / L) at pH 10 was slowly added to the mixture. The solution was reacted with vigorous stirring (500 rpm) at room temperature for 1 hour. Large-diameter nanogels (NFCc) with diameters greater than 500 nm were observed in the mixture by dynamic light scattering (Zetasizer Advance Pro, Malvern).

[0265] like Figure 10 As shown, the diameter (NFCc) of the nanogel loaded with Fluometacyl in the presence of TPGS (NFCcT) is significantly larger than that of the nanogel without TPGS. ® The nanogels of Chc exhibit low particle sizes ranging from 100 nm to 350 nm and have a low size distribution.

[0266] After 24 hours, an increase in the size of NFCc was observed, indicating the formation and precipitation of nanogel aggregates, a phenomenon not observed in NFCc that remained stable after 24 hours. Figure 11 ) Example 11: With or without TPGS, the loading of Fluometacyl ® The anti-adhesion properties of catheters coated with chlorhexidine nanogel were tested against Pseudomonas aeruginosa.

[0267] 11.1 Preparation of Samples for Microcalorimetry Pseudomonas aeruginosa (ATCC 15442) was grown overnight in TSB (trypsin-soy broth) medium, and then diluted 1×10⁻⁶ in fresh TSB. 7 The concentration was increased by 1000 times. Subsequently, two PU catheter fragments (0.5 cm long) were added to aliquots of the diluted bacterial suspension. These catheter fragments were coated with five layers of nanogel loaded with Fluometacyl. ® (0.2 mg / mL) and chlorhexidine chloride (0.2 mg / mL), with or without TPGS (0.4 mg / mL). Nanogel coatings were prepared according to the following protocol, to which the respective bioactive molecules were added.

[0268] The bacterial solution with the tubes was incubated at 37°C and 220 rpm for 30 minutes. Subsequently, the tubes were washed twice with saline (0.9% NaCl) and placed in a dedicated non-activated insert in a 48-well plate with 300 μL of fresh TSB medium, and grown under static conditions at 37°C for 24 hours using Calscreener.

[0269] 11.2 Preparation and drug loading of nanogels (NFCc and NFCcT) containing Pox (mDOPA) and PAH ® (and chlorhexidine) While stirring with a magnetic stirrer (300 rpm), a PAH aqueous solution (pH 10) of 0.5 mL (0.5 mg / mL) was slowly added to Pox(mDOPA) (5 mL, 0.5 mg / mL), which had been pre-mixed with Fluometacyl. ® Incubate with chlorhexidine chloride (CHLXc, 0.2 mg / mL) and TPGS (0.4 mg / mL) with or without for 10 minutes. React the solution at room temperature with vigorous stirring (500 rpm) for 1 hour.

[0270] Nanogels with diameters ranging from 100 nm to 300 nm were observed in aqueous suspensions using dynamic light scattering (Zetasizer Advance Pro, Malvern).

[0271] The test drug is kept frozen in the form of the following stock solution: Fluometacyl ® (3.3 mg / mL stock solution in DMSO, prepared according to Eur J Med Chem 2020 Dec 15:208:112767), chlorhexidine chloride (3.3 mg / mL stock solution in DMSO, Merck), TPGS (3.3 mg / mL stock solution in DMSO, Merck).

[0272] Figure 12 Real-time microcalorimetry measurements of bacterial metabolic activity adhered to the ducts were displayed, expressed as heat flux. Curve A - Control: Pseudomonas aeruginosa 10 -7 Bacterial growth; Curve B - bacteria in a PU catheter coated with nanogel, the catheter being loaded with Fluometacyl ® And chlorhexidine chloride (NFCc); Curve C - bacteria with PU conduits coated with nanogel, which were loaded with Fluometacyl in the presence of TPGS (NFCcT). ® And chlorhexidine chloride.

[0273] Compared to coatings without TPGS, coatings containing Fluometacyl in the presence of TPGS have higher viscosity. ® The nanogel coating with chlorhexidine chloride imparts higher anti-adhesion properties to the PU catheter. This is viewed as a variation in the time it takes for the metabolic activity of bacteria growing from the population adhering to the catheter to peak during the initial incubation period. In other words, the fewer bacteria attached to the catheter fragment, the later the metabolic activity occurs.

[0274] TPGS, as an amphiphilic molecule, enhances both hydrophobic and hydrophilic antibacterial molecules such as Fluometacyl. ® Retention of chlorhexidine in nanogel coatings.

[0275] Example 12: Fluometacyl loaded in the presence of TPGS ® The catheters coated with chlorhexidine nanogel showed stronger anti-adhesion properties against Pseudomonas aeruginosa.

[0276] 12.1 Preparation of Samples for Microcalorimetry Pseudomonas aeruginosa (ATCC 15442) was grown overnight in TSB (trypsin-soy broth) medium, and then diluted 1×10⁻⁶ in fresh TSB. 7 The concentration was increased by 1000 times. Subsequently, two PU catheter fragments (0.5 cm long) were added to aliquots of the 1000 mL diluted bacterial suspension. The two PU catheter fragments were coated with five layers of Fluometacyl-loaded fluid. ® (0.2 mg / mL) nanogel, and soaked (1 hour) in an aqueous solution of chlorhexidine chloride (1 mg / mL), or supplemented with 2 PU catheter fragments (0.5 cm long), the 2 PU catheter fragments being coated with 5 layers of Fluometacyl loaded in the presence or absence of TPGS (0.4 mg / mL). ® Nanogels containing chlorhexidine chloride (0.2 mg / mL) and chlorhexidine chloride (0.2 mg / mL) were prepared. The nanogel coatings were prepared according to the following protocol, to which the respective bioactive molecules were added.

[0277] The bacterial solution with the tubes was incubated at 37°C and 220 rpm for 30 minutes. Subsequently, the tubes were washed twice with saline (0.9% NaCl) and placed in a dedicated non-activated insert in a 48-well plate with 300 mL of fresh TSB medium, and grown under static conditions at 37°C for 24 hours using Calscreener.

[0278] 12.2 Preparation and drug loading of nanogels containing Pox (mDOPA) and PAH (NFCcT) While stirring with a magnetic stirrer (300 rpm), a PAH aqueous solution (pH 10) of 0.5 mL (0.5 mg / mL) was slowly added to Pox(mDOPA) (5 mL, 0.5 mg / mL), which had been pre-mixed with Fluometacyl. ® Incubate for 10 minutes with Fluometacyl® (0.2 mg / mL) and chlorhexidine chloride (Cc, 0.2 mg / mL) and with or without TPGS (0.4 mg / mL). React the solution at room temperature with vigorous stirring (500 rpm) for 1 hour. Alternatively, instead of loading the nanogel with both Fluometacyl® and chlorhexidine chloride, the nanogel can be loaded with Fluometacyl® under stirring (orbital oscillator). ® The nanogel-coated catheter was immersed in an aqueous solution of Chc (1 mg / mL) for 1 hour to allow chlorhexidine to be mixed with Fluometacyl. ® Individual load.

[0279] Nanogels with diameters ranging from 100 nm to 300 nm were observed in aqueous suspensions using dynamic light scattering (Zetasizer Advance Pro, Malvern).

[0280] The test drug is kept frozen in the form of the following stock solution: Fluometacyl ® (3.3 mg / mL stock solution in DMSO, prepared according to Eur J Med Chem 2020 Dec 15:208:112767), chlorhexidine chloride (3.3 mg / mL stock solution in DMSO, Merck), TPGS (3.3 mg / mL stock solution in DMSO, Merck). For the immersion protocol, a fresh aqueous solution of 1 mg / mL was prepared from the powder.

[0281] Figure 13 Real-time microcalorimetric measurements of bacterial metabolic activity adhering to the catheter are shown, expressed as heat flux. Curve A - bacteria with a PU catheter coated with nanogel, the catheter being loaded with Fluometacyl® and chlorhexidine chloride (NFCc); Curve B - bacteria with a PU catheter coated with nanogel, the catheter being loaded with Fluometacyl® and immersed in a chlorhexidine chloride solution (1 mg / mL) (NF / Cc); Curve C - bacteria with a PU catheter coated with nanogel, the catheter being loaded with Fluometacyl® and chlorhexidine chloride in the presence of TPGS (NFCcT).

[0282] In the presence of TPGS, nanogel coatings carrying Fluometacyl® and chlorhexidine exhibit the strongest anti-adhesion properties, which is considered a shift in peak metabolic signaling. Immersion in chlorhexidine yields moderate performance compared to methods simultaneously loading Fluometacyl® and chlorhexidine. This supports the positive effects of TPGS on loading bioactive molecules into nanogel coatings.

[0283] Example 13: Stability of mixtures of Fluometacyl®, Chlorhexidine, and Polymyxin B with and without TPGS.

[0284] The nanogel preparation was similar to the preparation methods disclosed above for NF and NFT, except that we used Fluometacyl at the same concentration (0.2 mg / mL). ® A mixture of chlorhexidine and polymyxin B.

[0285] The stability of different active pharmaceutical ingredients or drugs can be problematic due to potential intermolecular interactions. In this example, we clearly demonstrate that the presence of TPGS increases the stability of the nanogel in liquid suspension. NFCcP nanogels containing three different drugs or APIs without TPGS produced large aggregates with very low stability. However, in the presence of TPGS, the NFCcP nanogel exhibited better stability, and DLS analysis showed that the nanogel diameter in the liquid suspension was approximately on the micrometer scale. Figure 14 In the study, a mixture of nanogels NFT, NCT, and NPT showed that the nanogel diameters ranged from 100 nm to 350 nm and exhibited higher stability in liquid suspensions.

[0286] Example 14: Using P(mDOPA)Fluometacyl ® Nanogels (NFCy) according to the present invention were prepared by reacting hydroxypropyl-β-cyclodextrin with hydroxypropyl-β-cyclodextrin.

[0287] The preparation of nanogels is similar to that disclosed above, except that we use hydroxypropyl-β-cyclodextrin (hereinafter also referred to as cyclodextrin) instead of TPGS.

[0288] 14.1 Oxidation of PmDOPA → Pox(mDOPA).

[0289] A homopolymer of methacrylamide containing 3,4-dihydroxy-L-phenylalanine (P(mDOPA)) was oxidized in an aqueous medium under alkaline conditions for 12 hours to form water-soluble Pox(mDOPA). The oxidized catechol groups of Pox(mDOPA) are essential for the covalent interaction of PAHs at room temperature via amine / quinone reactions and / or Schiff base formation, and are therefore necessary for the preparation of stable crosslinked nanogels in liquid dispersions.

[0290] P(mDOPA) (2.5 mg) was dissolved in distilled water (5 mL), and NaOH (0.1 M) was slowly added to raise the pH to above 10 and promote the oxidation of the catechol groups of P(mDOPA).

[0291] 14.2 Preparation of Fluometacyl in DMSO ® Hydroxypropyl-β-cyclodextrin mixture 75 μL of 40% hydroxypropyl-β-cyclodextrin solution (400 mg / mL, in H2O) provided by Merck and 60 μL of flumetrexed solution (3.3 mg / mL, in DMSO) were mixed with a magnetic stirrer (300 rpm).

[0292] 14.3 Using Fluometacyl ® Preparation of nanogels from hydroxypropyl-β-cyclodextrin and PAH Pox(mDOPA) (0.87 mL, 0.5 mg / mL) was added to the mixture obtained at point 14.2 while stirring with a magnetic stirrer (300 rpm). DLS analysis was performed. Figure 15 The presence of substances smaller than 10 nm, characterizing cyclodextrin molecules, was observed. After homogenization at room temperature for 1 hour, an aqueous PAH solution (0.087 mL, 0.5 mg / mL) at pH 10 was slowly added to the mixture. The solution was reacted at room temperature with vigorous stirring (500 rpm) for 1 hour. Figure 15 The diagram shows the nanogels NFCy (dashed lines) in liquid suspension with diameters ranging from 100 nm to 350 nm, as observed by dynamic light scattering (Zetasizer Advance Pro, Malvern) compared to the solution before the addition of PAH (without nanogels) (solid lines).

[0293] Example 15: Stability comparison of the nanogels NF, NFT, and NFCy according to the present invention, wherein different concentrations of Fluometacyl ® TPGS or hydroxypropyl-β-cyclodextrin are amphiphilic molecules.

[0294] The preparation of nanogels is similar to the preparation methods disclosed above for NF, NFT, and NFCy, except that we used different concentrations of Fluometacyl. ® (0.2 mg / mL, 0.4 mg / mL and 0.8 mg / mL) and we maintained the same ratios as the amphiphilic molecules (Fluo:TPGS 1:2 and Fluo:cyclodextrin 1:75).

[0295] The size and polydispersity of different nanogels were tracked by DLS on days 0, 1, and 4. We can clearly see that with increasing fluorometholone concentration, the size and polydispersity of nanogel NF increased, leading to precipitation. However, in the presence of the amphiphilic molecules TPGS and hydroxypropyl-β-cyclodextrin as stabilizers, nanogels NFT and NFCy exhibited higher stability.

[0296] Example 16: In tests against Staphylococcus aureus, hydroxypropyl-β-cyclodextrin did not affect Fluometacyl compared to TPGS. ® Antibacterial effect in liquid media.

[0297] 16.1 Preparation of Samples for Microcalorimetry Staphylococcus aureus (MRSA, ATCC 6538) was grown overnight in TSB (trypsin-soy broth) medium, and then diluted 1×10⁻⁶ in fresh TSB. 7 Fluometacyl was then added to aliquots of the diluted bacterial suspension in 1000 mL. ® (10 μg / mL or 20 μg / mL) or Fluometacyl ® A mixture of (10 μg / mL or 20 μg / mL) and hydroxypropyl-β-cyclodextrin (42 μg / mL) was then placed in a dedicated non-activated insert in a 48-well plate and grown at 37 °C for 24 hours using Calscreener.

[0298] 16.2 Preparation of Stock Solutions Fluometacyl ® (Prepared according to Eur J Med Chem 2020 Dec 15:208:112767) Keep frozen as a 3.3 mg / mL EtOH stock solution. Hydroxypropyl-β-cyclodextrin (Merck) is stored in powder form at room temperature and a suitable stock solution in H2O is prepared fresh before use.

[0299] Figure 17Real-time microcalorimetric measurements of bacterial metabolic activity are shown, expressed as heat flux. Curve A - Control: Staphylococcus aureus MRSA 10 -6 Bacterial growth; curve B-containing Fluometacyl ® (10 μg / mL) bacteria; curve C - containing Fluometacyl ® Bacteria containing 10 μg / mL and hydroxypropyl-β-cyclodextrin (42 μg / mL); curve D-containing Fluometacyl ® (20 μg / mL) bacteria; curve E - containing Fluometacyl ® Bacteria containing 20 μg / mL of hydroxypropyl-β-cyclodextrin and 42 μg / mL of hydroxypropyl-β-cyclodextrin.

[0300] In solution, hydroxypropyl-β-cyclodextrin did not attenuate Fluometacyl compared to TPGS (in the previous examples). ® Its antibacterial activity.

[0301] Example 17: Fluometacyl loaded in the presence of TPGS or hydroxypropyl-β-cyclodextrin ® The anti-adhesion properties of the nanogel-coated catheter were tested against Staphylococcus aureus.

[0302] 17.1 Preparation of Samples for Microcalorimetry Staphylococcus aureus (MRSA, ATCC 6538) was grown overnight in TSB (trypsin-soy broth) medium, and then diluted 1×10⁻⁶ in fresh TSB. 7 The concentration was increased by 1000 times. Subsequently, two PU catheter fragments (0.5 cm long) were added to each aliquot of the 1000 mL diluted bacterial suspension. These catheter fragments were coated with five layers of Fluometacyl-loaded fluid. ® (0.2 mg / mL) and TPGS (0.4 mg / mL) or Fluometacyl ® Nanogels containing 0.2 mg / mL of hydroxypropyl-β-cyclodextrin and 15 mg / mL of hydroxypropyl-β-cyclodextrin were prepared. The nanogel coatings were prepared according to the following protocol, to which the respective bioactive molecules were added.

[0303] The bacterial suspension was incubated with the tubes at 37°C and 220 rpm for 30 minutes. Subsequently, the tubes were washed twice with saline (0.9% NaCl) and placed in a dedicated non-activated insert in a 48-well plate containing 300 mL of fresh TSB medium, and grown under static conditions at 37°C for 24 hours using Calscreener.

[0304] 17.2 Preparation and drug loading of nanogels containing Pox (mDOPA) and PAH (NG) While stirring with a magnetic stirrer (300 rpm), a PAH aqueous solution (pH 10) of 0.5 mL (0.5 mg / mL) was slowly added to Pox(mDOPA) (5 mL, 0.5 mg / mL), which had been pre-mixed with Fluometacyl. ® (0.2 mg / mL) and TPGS (0.4 mg / mL) or Fluometacyl ® Pre-incubate with 0.2 mg / mL of hydroxypropyl-β-cyclodextrin (15 mg / mL) for 10 minutes. React the solution at room temperature with vigorous stirring (500 rpm) for 1 hour.

[0305] Nanogels with diameters ranging from 100 nm to 300 nm were observed using dynamic light scattering (Zetasizer Advance Pro, Malvern).

[0306] The test drug was kept frozen in the form of the following stock solution: Fluometacyl ® (3.3 mg / mL stock solution in DMSO, prepared according to Eur J Med Chem 2020 Dec 15:208:112767), TPGS (3.3 mg / mL stock solution in DMSO, Merck). Hydroxypropyl-β-cyclodextrin (Merck) is stored in powder form at room temperature and a suitable stock solution in H2O is prepared fresh before use.

[0307] Figure 18 Real-time microcalorimetric measurements of bacterial metabolic activity adhering to the duct are shown, expressed as heat flux. Curve A - Control: Staphylococcus aureus MRSA 10 -6 Bacterial growth; Curve B - bacteria in a PU catheter coated with nanogel, the catheter being loaded with Fluometacyl ® And TPGS (0.2 / 0.4, w / w); Curve C - bacteria with PU catheters coated with nanogel, which are loaded with Fluometacyl ® And hydroxypropyl-β-cyclodextrin (0.2 / 15, w / w).

[0308] Compared to coatings containing TPGS, coatings containing Fluometacyl ®The nanogel coating with hydroxypropyl-β-cyclodextrin imparts higher anti-adhesion properties to the PU catheter. This is viewed as a reduction in signal and a shift in the peak time of metabolic activity from the bacteria growing in the population that adheres to the catheter during initial incubation. In other words, the fewer bacteria attached to the catheter fragment, the later metabolic activity occurs.

[0309] Hydroxypropyl-β-cyclodextrin is more effective than TPGS in increasing Fluometacyl levels. ® Retention rate in nanogel coatings.

Claims

1. A nanogel made of poly(methacrylamide) of formula (1) with quinone groups, said poly(methacrylamide) being crosslinked with a polymer having primary or secondary amine groups; （1） Where x is an integer greater than 1, preferably x is between 1 and 100; and / or The nanogel is made of poly(vinylquinone) represented by formula (6), which is crosslinked with a polymer having primary or secondary amine groups; （6） Where n is an integer greater than 1, preferably between 1 and 100; The nanogel contains one or more bioactive molecules, therapeutic molecules or drugs, and amphiphilic molecules.

2. The nanogel according to claim 1, wherein the amphiphilic molecule is a vitamin E derivative, preferably D-α-tocopherol polyethylene glycol succinate.

3. The nanogel according to claim 1, wherein the amphiphilic molecule is an amphiphilic cyclodextrin, preferably hydroxypropyl-β-cyclodextrin.

4. The nanogel according to any one of claims 1 to 3, wherein the polymer with primary or secondary amine groups is poly(allylamine hydrochloride) of formula (2). （2） Where p is an integer greater than 1; preferably, p is between 10 and 300.

5. The nanogel according to any one of claims 1 to 4, wherein the bioactive molecule, therapeutic molecule or drug is a triazolo(4,4-d)-pyrimidine derivative of formula (3). （3） wherein R 1 is C3-5alkyl optionally substituted with one or more halogen atoms; R 2 is phenyl optionally substituted with one or more halogen atoms; R 3 and R 4 are both hydroxyl; R is OH or XOH, wherein X is CH2, OCH2CH2or a bond; or a pharmaceutically acceptable salt or solvate thereof, or a solvate of it or of such a salt, provided that when X is CH2or a bond, R 1 is not propyl; when X is CH2and R 1 is CH2CH2CF3, butyl or pentyl, the phenyl group on R 2 must be substituted with fluorine; when X is OCH2CH2and R 1 is propyl, the phenyl group on R 2 must be substituted with fluorine; Preferably, the triazolo(4,4-d)-pyrimidine derivative is (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]- triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol, also known as Tresflotrexed; or is (1S,2R,3S,4R)-4-[7-[(1R,2S)-2-(3,4-difluorophenyl)-cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol, also known as Fluometacyl or Fluometacyl ® or a combination thereof.

6. The nanogel according to any one of claims 1 to 4, wherein the bioactive molecule, therapeutic molecule or drug is a pyrimidine derivative or optical isomer thereof represented by formula (4), a racemic mixture, a pharmaceutically acceptable acid addition salt, a pharmaceutically acceptable metal salt or alkylated ammonium salt or a prodrug; （4） in: X 1 and X 2 are independently N, CH, CR 8 wherein R 8 is C 1-6 alkyl, C 2-6 alkenyl or C 2-6 alkynyl; with the proviso that if one of X 1 or X 2 equals N, the remaining X 1 or X 2 is selected from CH, CR 8 ; -Y- is either -O- or -S-; R 11 and R 12 are independently C 1-6 -alkyl, C 2-6 -alkenyl, C 2-6 -alkynyl, C 3-6 -cycloalkyl, aryl, aryl-C 1-6 -alkyl, wherein alkyl or cycloalkyl is optionally mono- or poly- substituted by OH or halogen, and aryl is optionally mono- or poly- substituted by halogen, -C 1-6 -alkyl, -C 1-6 -alkoxy, -OH, -NO2, -CN, -NH2, -NHR 8 , -N(R 8 )2-COOH, -COOR 8 , -CONH2, -CONHR 8 , -CON(R 8 )2, -SO2NH2, -SO2NHR 8 or -SO2N(R 8 )2; R 13 , R 14 , R 15 , R 16 and R 17 are independently H, halogen, C 1-6 1-6 alkyl, C 1-6 1-6 alkoxy, -OH, -NO2, -CN, -NH2, -NHR 8 , -N(R 8 )2, -COOH, -COOR 8 , -CONH2, -CONHR 8 , -CON(R 8 )2, -SO2NH2, -SO2NHR 8 or -SO2N(R 8 )2.

7. The nanogel according to claim 2, wherein the ratio of the bioactive molecule, therapeutic molecule or drug and D-α-tocopherol polyethylene glycol succinate is from 1:1 w / w to 1:5 w / w.

8. The nanogel according to claim 3, wherein the ratio of the bioactive molecule, therapeutic molecule or drug and hydroxypropyl-β-cyclodextrin is from 1:1 w / w to 1:200 w / w.

9. The nanogel according to any one of claims 1 to 8, wherein the one or more bioactive molecules, therapeutic molecules or drugs are antiviral agents or bactericides and bacteriostatic agents, preferably minocycline or chlorhexidine.

10. The nanogel according to any one of claims 1 to 9, wherein the biologically active molecule, therapeutic molecule or drug is fluometacyl or Fluometacyl in combination with chlorhexidine. ® in combination with chlorhexidine.

11. The nanogel according to any one of claims 1 to 10, wherein the biologically active molecule, therapeutic molecule or drug is fluometacyl or Fluometacyl ® in combination with chlorhexidine and polymyxin B.

12. The nanogel according to any one of claims 1 to 11, for treating or preventing bacterial infections.

13. The nanogel according to any one of claims 1 to 12, for topical application.

14. A biomaterial implant, medical device or bioprosthetic, wherein its surface or part is coated with a nanogel according to any one of claims 1 to 11.

15. The biomaterial implant or medical device according to claim 14, wherein the medical device is a catheter.

16. A method of preparing a nanogel comprising one or more biologically active molecules, therapeutic molecules or drugs and an amphiphilic molecule, wherein the nanogel is obtained by one of the following two methods depending on whether each biologically active molecule, therapeutic molecule or drug is loaded separately or simultaneously with the amphiphilic molecule in the nanogel: a) when each biologically active molecule, therapeutic molecule or drug is loaded separately with the amphiphilic molecule, the method comprises the following sequential steps: i) mixing a poly(methacrylamide) bearing a quinone group of formula (1) with only one biologically active molecule, therapeutic molecule or drug and an amphiphilic molecule; （1） wherein x is an integer greater than 1, preferably x is between 1 and 100; and / or mixing a poly(vinylquinone) represented by formula (6) with only one biologically active molecule, therapeutic molecule or drug and an amphiphilic molecule; （6） wherein n is an integer greater than 1, preferably n is between 1 and 100; ii) adding a solution of a polymer bearing a primary or secondary amine group to the resulting mixture obtained in step i) to produce a cross-linked nanogel comprising one biologically active molecule, therapeutic molecule or drug and an amphiphilic molecule; iii) repeating steps i) and ii) for each additional biologically active molecule, therapeutic molecule or drug; iv) mixing each cross-linked nanogel obtained in step iii) to obtain a resulting nanogel comprising two or more biologically molecules, therapeutic molecules or drugs and an amphiphilic molecule; b) when the biologically active molecule, therapeutic molecule or drug is loaded simultaneously with the amphiphilic molecule; the method comprises the following sequential steps: i) mixing a poly(methacrylamide) bearing a quinone group of formula (1) with one or more biologically active molecules, therapeutic molecules or drugs and an amphiphilic molecule; （1） wherein x is an integer greater than 1, preferably x is between 1 and 100; and / or mixing a poly(vinylquinone) represented by formula (6) with one or more biologically active molecules, therapeutic molecules or drugs and an amphiphilic molecule; （6） wherein n is an integer greater than 1, preferably n is between 1 and 100; ii) adding a solution of a polymer bearing a primary or secondary amine group to the resulting mixture of step i) to produce a nanogel comprising one or more biologically active molecules, therapeutic molecules or drugs and an amphiphilic molecule.

17. The method according to claim 16, wherein the amphiphilic molecule is a vitamin E derivative, preferably D-a-tocopheryl polyethylene glycol succinate.

18. The method according to claim 16, wherein the amphiphilic molecule is an amphiphilic cyclodextrin, preferably hydroxypropyl- -cyclodextrin.

19. The method according to claim 16, wherein the ratio of biologically active molecule, therapeutic molecule or drug to D-a-tocopheryl polyethylene glycol succinate is between 1 : 1 w / w and 1 :5 w / w.

20. The method according to claim 16, wherein the ratio of biologically active molecule, therapeutic molecule or drug to hydroxypropyl- -cyclodextrin is between 1 : 1 and 1 :

200.

21. The method of any one of claims 16 to 20, wherein the one or more biologically active molecules, therapeutic molecules, or drugs is a triazolo(4,4-d)-pyrimidine derivative of Formula (3); preferably, the triazolo(4,4-d)-pyrimidine derivative is (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H- [1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol), also known as Trelagliptin; or (1S,2R,3S,4R)-4-[7-[(1R,2S)-2-(3,4-difluorophenyl)-cyclopropyl]amino]-5-(propylthio)-3H- 1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol, also known as Fluometacyl ® or a combination thereof.

22. The method according to any one of claims 16 to 20, wherein the biologically active molecule, therapeutic molecule or drug is a pyrimidine derivative represented by formula (4) or an optical isomer, a racemic mixture, a pharmaceutically acceptable acid addition salt, a pharmaceutically acceptable metal salt or an alkylated ammonium salt or a prodrug thereof; （4） wherein: X 1 and X 2 independently are N, CH, CR 8 wherein R 8 is C 1-6 alkyl, C 2-6 alkenyl or C 2-6 alkynyl; with the proviso that if one of X 1 or X 2 equals N, the remaining X 1 or X 2 is selected from CH, CR 8 , - Y- is -O- or -S-; R 11 and R 12 are independently C 1-6 -alkyl, C 2-6 -alkenyl, C 2-6 -alkynyl, C 3-6 -cycloalkyl, aryl, aryl-C 1-6 -alkyl, wherein alkyl or cycloalkyl is optionally mono- or poly- substituted by OH or halogen and aryl is optionally mono- or poly- substituted by halogen, -C 1-6 -alkyl, -C 1-6 -alkoxy, -OH, -NO2, -CN, -NH2, -NHR 8 , -N(R 8 )2-COOH, -COOR 8 , -CONH2, -CONHR 8 , -CON(R 8 )2, -SO2NH2, -SO2NHR 8 or -SO2N(R 8 )2; R 13 , R 14 , R 15 , R 16 and R 17 are independently H, halogen, C 1-6 alkyl, C 1-6 alkoxy, -OH, -NO2, -CN, -NH2, -NHR 8 , -N(R 8 )2-COOH, -COOR 8 , -CONH2, -CONHR 8 , -CON(R 8 )2, -SO2NH2, -SO2NHR 8 or -SO2N(R 8 )2.

23. The method according to any one of claims 16 to 20, wherein the one or more biologically active molecule, therapeutic molecule or drug is an antiviral agent or a bactericide and a bacteriostat, preferably minocycline or chlorhexidine.

24. A method for producing a medical device, a biomaterial implant or a bioprosthesis having a nanogel-coated surface comprising one or more biologically active molecule, therapeutic molecule or drug and an amphiphilic molecule; said method comprising the following successive steps: ci) optionally immersing the surface to be coated in a buffered solution of dopamine; cii) immersing the surface coated in step ci) in a solution of a polymer bearing a primary or secondary amine group; then ciii) immersing the resulting coated surface obtained in step cii) in a liquid suspension of a cross-linked nanogel comprising one or more biologically active molecule, therapeutic molecule or drug and an amphiphilic molecule; then civ) drying the cross-linked coated surface obtained in step ciii) to obtain a coated cross-linked nanogel surface comprising one or more biologically active molecule, therapeutic molecule or drug and an amphiphilic molecule; cv) optionally repeating steps cii) to civ) to obtain a surface coated with a plurality of layers of cross-linked nanogel comprising one or more biologically active molecule, therapeutic molecule or drug and an amphiphilic molecule.

25. A method for producing a medical device, a biomaterial implant or a bioprosthesis having a coated surface, wherein the coating is obtained by the following successive steps: i) optionally immersing the surface to be coated in a buffered solution of dopamine; ii) immersing the surface coated with dopamine in step i) in a solution of a polymer bearing a primary or secondary amine group; then iii) immersing the resulting coated surface obtained in step ii) in a mixture of poly(methacrylamide) bearing quinone groups of formula (1) with one or more biologically active molecule, therapeutic molecule or drug and an amphiphilic molecule （1） wherein x is an integer greater than 1, preferably x is between 1 and 100; and / or in a poly(vinylquinone) of formula (6) with one or more biologically active molecule, therapeutic molecule or drug and an amphiphilic molecule （6） wherein n is an integer greater than 1, preferably n is between 1 and 100; iv) drying the cross-linked coated surface obtained in step iii) to obtain a coated cross-linked monolayer surface comprising one or more biologically active molecule, therapeutic molecule or drug and an amphiphilic molecule; v) optionally repeating steps ii) to iv) to obtain a surface coated with a plurality of layers of cross-linked multilayers comprising one or more biologically active molecule, therapeutic molecule or drug and an amphiphilic molecule, preferably vitamin E derivatives.

26. The method according to claim 24 or 25, wherein the amphiphilic molecule is a vitamin E derivative, preferably D-a-tocopheryl polyethylene glycol succinate.

27. The method according to claim 24 or 25, wherein the amphiphilic molecule is an amphiphilic cyclodextrin, preferably hydroxypropyl-beta-cyclodextrin.

28. The method according to any one of claims 224 to 27, wherein the one or more biologically active molecule, therapeutic molecule or drug is a triazolo(4,4-d)-pyrimidine derivative or a pyrimidine derivative and a bacteriostatic agent, preferably minocycline or chlorhexidine.

29. The method of any one of claims 24 to 27, wherein the one or more biologically active molecules, therapeutic molecules, or drugs is (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4- difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2- hydroxyethoxy)-1,2-cyclopentanediol, also known as Tetrifluotecine; or (1S,2R,3S,4R)-4-[7-[(1R,2S)-2-(3,4-difluorophenyl)-cyclopropyl]amino]-5-(propylthio)-3H-1,2,3- triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol, also known as Fluometacyl or Fluometacyl ® or combinations thereof.

30. A pharmaceutical composition comprising the nanogel according to any one of claims 1 to 11 for use in the treatment or prevention of bacterial virulence in a host mammal by topical administration.

31. Use of the nanogel according to any one of claims 1 to 11 as a bacterial adhesion inhibitor on the surface of a biomaterial implant, a medical device or a bioprosthesis.

32. The use according to claim 31, wherein the biomaterial implant, medical device or bioprosthesis is a catheter.

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