WO2023279594A1 - Vésicule composite d'électrolyte homopolymère, son procédé de préparation, vésicule antibactérienne, vésicule contenant des substances hydrophobes et vésicule anti-adhésion - Google Patents

Vésicule composite d'électrolyte homopolymère, son procédé de préparation, vésicule antibactérienne, vésicule contenant des substances hydrophobes et vésicule anti-adhésion Download PDF

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WO2023279594A1
WO2023279594A1 PCT/CN2021/127452 CN2021127452W WO2023279594A1 WO 2023279594 A1 WO2023279594 A1 WO 2023279594A1 CN 2021127452 W CN2021127452 W CN 2021127452W WO 2023279594 A1 WO2023279594 A1 WO 2023279594A1
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vesicle
vesicles
homopolyelectrolyte
formula
complex
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Chinese (zh)
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孙静
王美瑶
林敏�
赵英杰
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青岛科技大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/04—Nitro compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10—Alpha-amino-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/48—Polymers modified by chemical after-treatment
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/07—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media from polymer solutions
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2377/00—Characterised by the use of polyamides obtained by reactions forming a carboxylic amide link in the main chain; Derivatives of such polymers
    • C08J2377/04—Polyamides derived from alpha-amino carboxylic acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the assembly method of polyelectrolyte complexes has many unique properties, such as higher efficiency, better controllability, and ease of large-scale production.
  • bionanoparticles with good stability and solubility can also be constructed by simple complexation to achieve efficient drug delivery.
  • Kataoka's research group prepared two oppositely charged block copolymers by introducing PEG molecules, and mixed them in equal proportions in water to form nanoscale PIC micelles, in which the charged block part formed a hydrophobic
  • the core of the polyelectrolyte complex, and the uncharged hydrophilic block as the shell form a stable nano-micelle.
  • the purpose of the present invention is to provide a homopolyelectrolyte complex vesicle and its preparation method, antibacterial vesicle, hydrophobic substance-loaded vesicle and anti-adhesion vesicle.
  • the homopolyelectrolyte composite vesicles provided by the invention are assembled from two oppositely charged homopolymers, the raw materials are easy to obtain, and the cost is low, and the obtained vesicles have good membrane permeability characteristics, and can contain hydrophobic substances. It has bactericidal properties in an acidic environment, the surface of the vesicles has the characteristics of zwitterions, and has good anti-protein adhesion properties.
  • the mixed solution is neutralized with hydrochloric acid, and the obtained neutralized solution is dialyzed to obtain an aqueous solution of homopolyelectrolyte complex vesicles.
  • the preparation method of the amine group-containing clustered peptide in the side chain comprises the following steps:
  • Glyoxylic acid and R- NH2 are subjected to aldehyde-amine condensation reaction, and the resulting reaction solution is mixed with hydrochloric acid to perform a salt-forming reaction to obtain a compound having a structure shown in formula a;
  • the temperature of the salt-forming reaction is 100-110° C., and the time is 20-24 hours;
  • the acidic buffer is acetate buffer.
  • the present invention also provides a method for preparing antibacterial vesicles described in the above scheme, comprising the following steps:
  • the homopolyelectrolyte complex vesicle powder is dissolved in acid buffer solution to obtain antibacterial vesicles.
  • the present invention also provides a vesicle containing hydrophobic substances, comprising the homopolyelectrolyte composite vesicles described in the above scheme or the homopolyelectrolyte composite vesicles prepared by the preparation method described in the above scheme and entrapped in the homopolyelectrolyte composite vesicles
  • the hydrophobic substance inside the polyelectrolyte complex vesicle; the hydrophobic substance is a hydrophobic dye or a hydrophobic drug.
  • the hydrophobic dye is Nile Red; the hydrophobic drug is paclitaxel and/or doxorubicin.
  • the concentrated product is centrifuged to obtain an aqueous solution of hydrophobic substance-loaded vesicles.
  • the present invention also provides an anti-adhesion vesicle prepared by a method comprising the following steps:
  • aqueous solution of the homopolyelectrolyte complex vesicles or the aqueous solution of the vesicles carrying hydrophobic substances is mixed with a condensing agent to carry out cross-linking reaction to obtain anti-adhesion vesicles.
  • the degree of cross-linking on the surface of the anti-adhesion vesicles is 17.9%-35.1%.
  • the invention provides a homopolyelectrolyte composite vesicle, which is obtained by self-assembly of a clustering peptide containing an amino group in a side chain and a clustering peptide containing a carboxyl group in a side chain.
  • Polypeptide poly(N-substituted glycine), polypeptoid
  • Polypeptide is a biopolymer with a structure similar to that of a polypeptide. Since the hydrogen on the N is replaced, there are no hydrogen bond donors and chiral centers on its main chain. Dadi simplifies the interaction of the main chain, has better solubility in some common solvents, and has better biocompatibility.
  • the invention introduces groups with opposite charges (amine group and carboxyl group) on the side chain of the clustering peptide, and the two clustering peptides with opposite charges self-assemble into a stable nanoscale vesicle structure through electrostatic interaction.
  • the polar amide groups in the polypeptide backbone are water-soluble, and despite the absence of hydrophilic blocks, the amide groups present on the polypeptide backbone The group can still undergo hydrogen bond interaction with water molecules, thereby effectively stabilizing the assembly; in addition, there are unpaired free charges on the surface of the clustered peptide, and because the positive and negative charges are evenly dispersed on the surface of the vesicle, it is not easy to interact with other charged peptides.
  • the homopolyelectrolyte composite vesicles provided by the present invention have good membrane permeability properties, and have good bactericidal effects under acidic conditions; can contain hydrophobic substances; and the surface of the vesicles has the characteristics of zwitterions, and has good
  • the property of anti-protein adhesion has broad application prospects in the antifouling physiological protein solution system.
  • the present invention also provides a vesicle containing a hydrophobic substance, comprising the homopolyelectrolyte composite vesicle described in the above scheme and the hydrophobic substance contained inside the homopolyelectrolyte composite vesicle.
  • the hydrophobic substance-loaded vesicles provided by the present invention can contain various hydrophobic dyes or drug molecules, and because the hydrophobic substance-loaded vesicles contain carboxyl and amino groups, they are pH-responsive and can be used in acidic ( pH value is 5.0-7.4) or alkaline environment (pH value is 8-11) can release hydrophobic substances.
  • the vesicles When the vesicles are placed in an environment with a different pH than their own, the vesicles are destroyed to a certain extent, thereby The encapsulated hydrophobic substances are released to different degrees, so as to achieve efficient drug delivery; in addition, the release behavior of the encapsulated molecules in the inner cavity can be effectively regulated by lightly cross-linking the surface of the vesicles.
  • Fig. 2 is the NMR spectrum of the N-allyl N-carboxylic acid anhydride prepared by embodiment 1;
  • Fig. 3 is the nuclear magnetic hydrogen spectrogram of the clustering peptide PNAG that embodiment 1 prepares;
  • Fig. 4 is the polymkeric substance PNAG-NH that embodiment 1 prepares The nuclear magnetic proton spectrum figure
  • Figure 6 is a transmission electron micrograph of PNAG 47 -NH 2 +PNAG 46 -COOH vesicles prepared in Example 6;
  • the side chain carboxyl group-containing clustering peptide has a structure shown in formula III or formula IV:
  • m, n, p, and q represent degrees of polymerization, and the m, n, p, and q are independently 10-90, preferably 32-82, and more preferably 45-75.
  • the dispersity of the clustered peptides containing amine groups in the side chains and the clustered peptides containing carboxyl groups in the side chains is preferably 1-1.3, more preferably 1.01-1.25; the dispersity specifically refers to polymer The ratio of weight average molecular weight to number average molecular weight (Mw/Mn).
  • the preferred molar ratio of the clustered peptides containing amine groups in the side chains to the clustered peptides containing carboxyl groups in the side chains is (0.25-4):1, more preferably (1-3):1, most preferably Preferably 1:1.
  • the particle size of the homopolyelectrolyte complex vesicles is preferably 100-400 nm, more preferably 200-300 nm.
  • the present invention also provides a method for preparing homopolyelectrolyte complex vesicles described in the above scheme, comprising the following steps:
  • the preparation method of the amine group-containing clustered peptide in the side chain preferably includes the following steps:
  • poly(N-substituted glycine), mercaptoethylamine and photoinitiator are mixed to carry out click chemical reaction to obtain a side chain-containing amine-containing clustered peptide;
  • the poly(N-substituted glycine) is poly( N-allylglycine) (PNAG) or poly(N-propargylglycine) (PNPG).
  • Glyoxylic acid and R- NH2 are subjected to aldehyde-amine condensation reaction, and the resulting reaction solution is mixed with hydrochloric acid to perform a salt-forming reaction to obtain a compound having a structure shown in formula a;
  • glyoxylic acid and R- NH2 are subjected to aldehyde-amine condensation reaction, and the obtained reaction solution is mixed with hydrochloric acid to perform a salt-forming reaction to obtain a compound having a structure shown in formula a.
  • R- NH2 is allylamine or propargylamine;
  • the glyoxylic acid is preferably monohydrate glyoxylic acid;
  • the molar ratio of the glyoxylic acid and R- NH2 is preferably 1:(2.5 ⁇ 3.5), more preferably 1:(2.8 ⁇ 3.2), more preferably 1:3;
  • the glyoxylic acid is preferably used in the form of glyoxylic acid aqueous solution, the quality of the glyoxylic acid aqueous solution
  • the fraction is preferably 40 to 60%, more preferably 50%;
  • the solvent used for the aldehyde-amine condensation reaction is preferably dichloromethane; the present invention has no special requirements on the amount of dichloromethane, which can make the aldehyde-amine condensation reaction smooth Just proceed.
  • the temperature of the aldolamine condensation reaction is preferably room temperature, and the time is preferably 20-24 hours, more preferably 22-23 hours.
  • the present invention preferably removes the dichloromethane in the reaction solution, and then adds hydrochloric acid to carry out the salt-forming reaction.
  • the method for removing dichloromethane is preferably rotary evaporation; the concentration of the hydrochloric acid is preferably 1-2mol/L; the temperature of the salt-forming reaction is preferably 100-110°C, more preferably 103- 105°C, the salt-forming reaction is preferably carried out under reflux conditions; the time for the salt-forming reaction is preferably 20-24 hours, more preferably 22-23 hours.
  • the method for removing moisture is preferably rotary steaming; the temperature of the recrystallization is preferably -20°C; in a specific embodiment of the present invention, the recrystallization is preferably carried out in a refrigerator; the recrystallization is preferably carried out in a refrigerator; The number of times of recrystallization is preferably 3 times.
  • the solid product obtained by the first recrystallization is dissolved in methanol again, and the resulting solution is added to tetrahydrofuran for the second recrystallization, and the method for the third recrystallization is analogous ;
  • the volume ratio of methanol and tetrahydrofuran used in the single recrystallization process is preferably 1:(5-10), more preferably 1:(6-8).
  • the present invention mixes the compound with the structure shown in formula a, di-tert-butyl dicarbonate (Boc 2 O) and triethylamine for substitution reaction to obtain the compound with the structure shown in formula b.
  • R in formula b is allyl
  • the compound having the structure shown in formula b is N-tert-butoxycarbonyl-N-allyl substituted glycine hydrochloride
  • R in formula b is propargyl
  • the compound having the structure shown in formula b is N-tert-butoxycarbonyl-N-propargyl substituted glycine hydrochloride.
  • the temperature of the substitution reaction is preferably room temperature, specifically 25°C, and the time of the substitution reaction is preferably 20-24 hours, more preferably 22-23 hours;
  • the compound having the structure represented by formula a is dissolved in deionized water, and then di-tert-butyl dicarbonate (Boc 2 O) and triethylamine are added for substitution reaction.
  • the obtained reaction solution is preferably post-treated to obtain a compound having a structure represented by formula b.
  • the post-treatment preferably includes the following steps: extracting the obtained reaction liquid with n-hexane to obtain an aqueous phase; adjusting the pH value of the lower liquid to 2 to obtain an acidic liquid; extraction with an acidic liquid to obtain an ethyl acetate phase; extract the ethyl acetate phase with saturated brine to obtain an ethyl acetate phase; dry the ethyl acetate phase with anhydrous sodium sulfate and filter, and extract the ethyl acetate phase from the filtrate The ethyl acetate is removed to obtain a compound having the structure shown in formula b.
  • the number of times of the normal hexane extraction is preferably 3 times;
  • the pH regulator for adjusting the pH value of the lower layer liquid is preferably hydrochloric acid, and the concentration of the hydrochloric acid is preferably 2mol/L;
  • the saturated saline The number of times of extraction is preferably 3 times;
  • the drying time of the anhydrous sodium sulfate is preferably 24h;
  • the method for removing ethyl acetate in the filtrate is preferably rotary evaporation.
  • the solvent for the ring-forming reaction is preferably anhydrous dichloromethane;
  • the molar ratio of the compound having the structure shown in formula b to phosphorus oxychloride is preferably 1:(2 ⁇ 3), more preferably is 1:(2.2 ⁇ 2.5), the phosphorus oxychloride plays a role in promoting the ring formation of the compound with the structure shown in formula b;
  • the temperature of the ring formation reaction is preferably 0 ⁇ 25°C, more preferably 5 ⁇ 20°C, the time is preferably 3-4 hours;
  • the ring-forming reaction is preferably carried out under conditions of ice bath, anhydrous and nitrogen protection.
  • the present invention mixes the compound with the structure shown in formula c and benzylamine for polymerization reaction to obtain poly(N-substituted glycine), specifically poly(N-allyl glycine) (PNAG) or poly(N-propargylglycine) (PNPG).
  • poly(N-substituted glycine) specifically poly(N-allyl glycine) (PNAG) or poly(N-propargylglycine) (PNPG).
  • the present invention performs post-treatment on the obtained reaction liquid to obtain the compound having the structure shown in formula c.
  • the post-treatment preferably includes the following steps: sedimentation of the obtained reaction solution in cold ether, followed by centrifugation and drying to obtain a compound having a structure shown in formula c; the temperature of the cold ether is preferably 0 ⁇ 25°C, more preferably 5 ⁇ 20°C; the number of times of said sedimentation is preferably 3 times.
  • the present invention mixes poly(N-substituted glycine), mercaptoethylamine and a photoinitiator under ultraviolet light conditions to perform a click chemical reaction to obtain a side chain-containing amine group-containing peptide,
  • the poly(N-substituted glycine) is PNAG
  • the obtained product is specifically a clustered peptide (PNAG-NH 2 ) having the structure shown in formula I
  • the poly(N-substituted glycine) is PNPG
  • the obtained product is specifically a clustered peptide (PNPG-(NH 2 ) 2 ) having the structure shown in formula II.
  • the photoinitiator is preferably benzoin dimethyl ether.
  • benzoin dimethyl ether absorbs energy and generates free radicals to catalyze the reaction of thiols and double bonds;
  • the poly(N- The molar ratio of carbon-carbon double bond (or carbon-carbon triple bond) in substituted glycine) to benzoin dimethyl ether and mercaptoethylamine is preferably (20 ⁇ 25):1:(90 ⁇ 100), more preferably It is (22 ⁇ 23):1:(93 ⁇ 95);
  • the solvent of the click chemical reaction is preferably N,N-dimethylformamide; the present invention has no special requirements on the amount of the solvent, which can ensure the click chemical reaction The reaction can proceed smoothly.
  • the temperature of the click chemical reaction is preferably room temperature, and the time is preferably 3 h; the click chemical reaction is preferably carried out under a protective atmosphere; the protective atmosphere is preferably nitrogen.
  • the obtained reaction solution is preferably dialyzed and freeze-dried in sequence to obtain a clustered peptide with an amine group in the side chain.
  • the molecular weight of the dialysis bag for dialysis is preferably 100-3500; the dialysis time is preferably 3 days; the freeze-drying temperature is preferably -20°C, and the time is preferably 24 hours.
  • the preparation method of the carboxyl group-containing clustered peptide in the side chain comprises the following steps:
  • the obtained product when the poly(N-substituted glycine) is PNAG, the obtained product is specifically a clustered peptide (PNAG-COOH) having a structure shown in formula III, and when the poly(N-substituted glycine) is In the case of PNPG, the obtained product is specifically a clustering peptide (PNPG-(COOH) 2 ) having a structure shown in formula IV;
  • the conditions for peptides are the same, only mercaptoethylamine is replaced with mercaptopropionic acid, and will not be repeated here.
  • the present invention self-assembles the clustering peptides containing amino groups in the side chains and the clustering peptides containing carboxyl groups in the side chains in water to obtain the An aqueous solution of the homopolyelectrolyte complex vesicles.
  • the present invention when the sample concentration is higher, the present invention is preferably prepared by the following method:
  • the mixed solution is neutralized with hydrochloric acid, and the obtained neutralized solution is dialyzed to obtain an aqueous solution of homopolyelectrolyte complex vesicles.
  • the present invention mixes the side chain amino group-containing polymer peptide aqueous solution with the side chain carboxyl group-containing alkaline aqueous solution to obtain a mixed solution.
  • concentration of the aqueous solution of the amine-containing peptide in the side chain is preferably 2 mg/mL; the molar ratio of the amine-containing peptide in the side chain to the carboxyl-containing peptide in the side chain is preferably (0.25-4):1, more preferably (1-3):1, most preferably 1:1.
  • the present invention uses hydrochloric acid to neutralize the mixed solution, and dialyzes the obtained neutralized solution to obtain an aqueous solution of homopolyelectrolyte complex vesicles.
  • the concentration of the hydrochloric acid is preferably 1mol/L; the molar ratio of the hydrochloric acid to the sodium hydroxide is preferably (0.5 ⁇ 1.5):1, most preferably 1:1; And make the sodium ion in the system form sodium chloride, and then remove sodium chloride by dialysis.
  • the present invention has no special requirements on the specific conditions of the dialysis, as long as the sodium chloride can be removed.
  • an aqueous solution of homopolyelectrolyte complex vesicles is obtained, and the concentration of the aqueous solution of homopolyelectrolyte complex vesicles obtained by this method is preferably 1-2 mg/mL.
  • the present invention After obtaining the aqueous solution of homopolyelectrolyte complex vesicles, the present invention also preferably freeze-dries the aqueous solution of homopolyelectrolyte complex vesicles to obtain homopolyelectrolyte complex vesicle powder.
  • the freeze-drying temperature is preferably -20°C, and the time is preferably 24 hours.
  • the present invention also provides an antibacterial vesicle, comprising the homopolyelectrolyte complex vesicle described in any one of the above schemes and an acidic buffer, the pH of the acidic buffer is preferably 4-6, more preferably 5.
  • the acidic buffer is preferably an acetate buffer; the acetate buffer is preferably a sodium acetate-acetic acid buffer solution; the present invention has no special preparation method for the sodium acetate-acetic acid buffer Requirements, the method well known to those skilled in the art can be adopted; in a specific embodiment of the present invention, the sodium acetate-acetic acid buffer solution is preferably prepared by acetic acid, sodium acetate and sodium chloride, the acetic acid and sodium acetate The total concentration is preferably 10mmol/L, and the concentration of the sodium chloride is preferably 142mmol/L.
  • the present invention also provides a method for preparing antibacterial vesicles described in the above scheme, comprising the following steps:
  • the polyelectrolyte composite vesicle powder is dissolved in an acid buffer to obtain the antibacterial vesicle.
  • the present invention prepares the homopolyelectrolyte composite vesicle powder according to the preparation method described in the above scheme, and the specific preparation conditions are not repeated.
  • DLC represents the loading capacity of hydrophobic substances
  • m 1 is the mass of hydrophobic substances packed
  • m is the mass of vesicles.
  • the present invention also provides a method for preparing a vesicle loaded with a hydrophobic substance described in the above scheme, comprising the following steps:
  • the concentrated product is centrifuged to obtain vesicles loaded with hydrophobic substances.
  • the present invention first prepares the aqueous solution of homopolyelectrolyte composite vesicles according to the preparation method described in the above scheme, and the specific preparation conditions are not repeated.
  • the present invention mixes the aqueous solution of the homopolyelectrolyte composite vesicle and the hydrophobic substance solution for vortex balance, and then removes the organic solvent in the vortex mixing system , to obtain the concentrated product.
  • the time for the vortex balance is preferably 2 hours; in the present invention, the organic solvent in the system is preferably removed under nitrogen flow.
  • the present invention centrifuges the concentrated product to obtain vesicles carrying hydrophobic substances.
  • the present invention has no special requirements on the centrifugation method, as long as the hydrophobic substances not encapsulated in the vesicles can be removed.
  • the aqueous solution of the homopolyelectrolyte composite vesicle or the aqueous solution of the vesicle loaded with hydrophobic substance is mixed with a condensing agent to carry out a cross-linking reaction to obtain an anti-adhesive vesicle.
  • an aqueous solution of homopolyelectrolyte complex vesicles or an aqueous solution of vesicles loaded with hydrophobic substances is firstly prepared according to the method described in the above scheme, and the specific preparation conditions are not repeated here.
  • aqueous solution is prepared from the carboxyl group-containing cluster peptide in the side chain.
  • the specific preparation method is the same as the above-mentioned scheme, only the cluster peptide containing amine groups in the side chain is replaced with the cluster peptide containing amine groups grafted with fluorescent materials.
  • the preparation method of the amino group-containing clustering peptide grafted with a fluorescent material preferably includes the following steps:
  • the fluorescent material, the amino group-containing clustering peptide in the side chain and the solvent are mixed and reacted to obtain the amino group-containing clustering peptide grafted with the fluorescent material.
  • the fluorescent material is preferably fluorescein isothiocyanate (FITC) or rose bengal isothiocyanate; the mass ratio of the fluorescent material to the side chain-containing amine group-containing cluster peptide is preferably 1 :(10 ⁇ 90), more preferably 1:(20 ⁇ 80); the solvent is preferably N,N-dimethylformamide; the temperature of the reaction is preferably room temperature, and the time is preferably 20 ⁇ 24h.
  • the condensing agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and the condensing agent is preferably used in the form of a condensing agent solution , the concentration of the condensing agent solution is preferably 20mg/mL, the solvent of the condensing agent solution is preferably water; ) surface COO - group molar ratio is preferably (0.5 ⁇ 10): 1, specifically preferably 0.5: 1, 1: 1, 5: 1 or 10: 1; in the present invention, by controlling the amount of condensing agent can Indirectly control the degree of cross-linking of amine and carboxyl groups on the surface of the vesicles, and then effectively regulate the anti-adhesion properties of the vesicles. When the vesicles are encapsulated with hydrophobic substances, the degree of cross-linking can also control the hydrophobic substances The release behavior can be effectively regulated
  • the present invention preferably removes the unreacted condensing agent by dialysis.
  • the present invention has no special requirements on the conditions of the dialysis, and the conditions well known to those skilled in the art can be used.
  • the degree of cross-linking on the surface of the anti-adhesion vesicles is preferably 17.9%-35.1%, more preferably 23.6-27.1%.
  • the purity of NCA is determined by a hydrogen nuclear magnetic resonance spectrometer, and the test conditions are: Bruker 500MHz, and the solvent is CDCl 3 ; the number-average molecular weight of the polymer is determined by gel permeation chromatography, and the GPC condition: SSI pump connected to Wyatt Optilab
  • the solvent is DMF, the flow rate is 1 mL ⁇ min -1 , and the test temperature is 50°C.
  • N-tert-butoxycarbonyl-N-allyl substituted glycine hydrochloride (15g, 0.067mol) was dissolved in 200mL of anhydrous dichloromethane, and phosphorus trichloride ( 14.54mL, 0.168mol), reacted for 3h under the protection of nitrogen, removed the solvent by rotary evaporation, introduced into the glove box and settled three times with tetrahydrofuran and n-hexane (volume ratio: 1:10), and obtained a colorless transparent liquid after pumping dry, namely N - Allyl N-carboxylic acid anhydride.
  • N-allyl N-carboxylic acid anhydride monomer (1.4g, 0.1mol) is dissolved in anhydrous tetrahydrofuran (100mg/mL), adds the benzylamine initiator ( 2.1mmol), reacted at 55°C for 24h, and then settled in cold ether to obtain a white solid poly(N-allylglycine) (PNAG), with a degree of polymerization of 47 and a number average molecular weight of 4.5kg/mol.
  • PNAG white solid poly(N-allylglycine)
  • the molecular weight distribution is 1.08.
  • Fig. 2 is the proton nuclear magnetic spectrum figure of gained N-allyl N-carboxylic acid anhydride
  • Fig. 3 is the proton nuclear magnetic spectrum figure of gained poly (N-allyl glycine)
  • Fig. 4 is gained PNAG 47 -NH 2 hydrogen nuclear magnetic spectrum Spectrum: As can be seen from Figures 2 to 4, the present invention has indeed obtained a product with a corresponding structure.
  • Step (5) is the same as in Example 1 to obtain a polymer having the structure shown in formula I-2, which is denoted as PNAG 32 -NH 2 .
  • N-tert-butoxycarbonyl-N-propargyl substituted glycine hydrochloride (15g, 0.067mol) was dissolved in 200mL of anhydrous dichloromethane, and phosphorus trichloride was added while stirring in an ice bath ( 14.54mL, 0.168mol), reacted for 3h under the protection of nitrogen, removed the solvent by rotary evaporation, introduced into the glove box and recrystallized three times with tetrahydrofuran and n-hexane (1:10), and obtained N-propargyl N-carboxylic acid anhydride after pumping dry.
  • step (5) is replaced with mercaptopropionic acid to obtain a polymer having the structure shown in formula III-1, which is denoted as PNAG 47 -COOH.
  • step (5) is replaced with mercaptopropionic acid to obtain a polymer with the structure shown in formula IV-1, which is denoted as PNPG 46 -(COOH) 2 .
  • HCl the volume ratio of hydrochloric acid and sodium hydroxide aqueous solution is 1:1
  • Figure 6 is the transmission electron microscope image of the obtained PNAG 47 -NH 2 +PNAG 47 -COOH vesicles; it can be seen from Figure 6 that PNAG 47 -NH 2 and PNAG 47 -COOH formed a uniform vesicle structure through self-assembly, and the vesicles The particle size of the bubbles is 30 to 300 nm.

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Abstract

La présente invention concerne une vésicule composite d'électrolyte homopolymère, son procédé de préparation, une vésicule antibactérienne, une vésicule contenant des substances hydrophobes et une vésicule anti-adhésion. Dans la présente invention, deux polypeptides présentant des charges opposées sont autoassemblés en une vésicule composite d'électrolyte homopolymère à l'échelle nanométrique stable au moyen d'une action électrostatique. La vésicule présente des caractéristiques de perméation de membrane relativement bonnes et des effets de stérilisation relativement bons dans des conditions acides et peut encapsuler des colorants ou des médicaments hydrophobes pour obtenir une administration efficace des médicaments. De surcroît, la surface de la vésicule présente des caractéristiques zwittérioniques et présente une performance de résistance à l'adhésion des protéines relativement bonne. La vésicule est plus stable après une réticulation légère à sa surface, elle présente une meilleure résistance à l'adhésion et peut réguler et contrôler efficacement la vitesse de libération des médicaments internes.
PCT/CN2021/127452 2021-07-07 2021-10-29 Vésicule composite d'électrolyte homopolymère, son procédé de préparation, vésicule antibactérienne, vésicule contenant des substances hydrophobes et vésicule anti-adhésion WO2023279594A1 (fr)

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