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WO2011106993A1 - Nanoparticules d'or modifiées par un aminopyrimide, leur procédé de préparation et leur utilisation - Google Patents

Nanoparticules d'or modifiées par un aminopyrimide, leur procédé de préparation et leur utilisation Download PDF

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Publication number
WO2011106993A1
WO2011106993A1 PCT/CN2011/000341 CN2011000341W WO2011106993A1 WO 2011106993 A1 WO2011106993 A1 WO 2011106993A1 CN 2011000341 W CN2011000341 W CN 2011000341W WO 2011106993 A1 WO2011106993 A1 WO 2011106993A1
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aminopyrimidine
gold nanoparticles
modified
solution
mercapto
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PCT/CN2011/000341
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Chinese (zh)
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蒋兴宇
赵玉云
田月
刘文文
张伟
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国家纳米科学中心
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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
    • A61K47/02—Inorganic 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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 present invention relates to a series of aminopyrimidine modified gold nanoparticles, a process for the preparation of the nanoparticles and their antibacterial use. Background technique
  • nano-silver is unstable to light and harmful to human body.
  • the antibacterial effect of nano-titanium dioxide depends on the light source, and the dosage of nano-oxidation is too high.
  • the development of nano gold as an antibacterial agent is not much.
  • Sabo-Attwood TL et al. Normal, RS; Stone, JW; Gole, A.; Murphy, CJ; Sabo-Attwood, TL, Targeted photothermallysis of the pathogenic bacteria, pseudomonas aeruginosa, with gold nanorods.
  • 2-Mercaptopyrimidine or 4-amino-2-mercaptopyrimidine is considered to have antibacterial properties by inhibiting the synthesis of bacterial t-RNA.
  • Mostafa S. L et al (mostafa, SI; Hadjiliadis, N" Biologically active 2-thione-4, 6-diamino-5-hydroxypyrimidine transition metal complexes. Transition Metal Chemistry 2008, 1-6) tested 2-mercapto-4, The antibacterial activity of 6-diamino-5-hydroxypyrimidine showed a slight antibacterial effect when its concentration was above 20 mg/mL; when the pyrimidine molecule was coordinated with metallic silver, the antibacterial concentration was reduced to 10 mg/mL.
  • This series of gold nanoparticles can be used as an antibacterial drug.
  • Another object of the present invention is to provide a process for the preparation of the aminopyrimidine modified gold nanoparticles.
  • the invention provides a gold nanoparticle wherein the surface of the gold nanoparticle is modified by an aminopyrimidine molecule.
  • the aminopyrimidine molecule modified on the surface of the gold nanoparticle is one or more of 2-mercapto-4,6-diaminopyrimidine, 2-mercapto-4-aminopyrimidine and/or 2,4-diamino-6. - Mercaptopyrimidine.
  • the aminopyrimidine molecularly modified gold nanoparticle provided by the invention has a particle size ranging from 1.0 to 10.0 nm; the molar ratio of the aminopyrimidine molecule to the gold element in the nanoparticle is 0.1:1 to 0 ⁇ 9:1 .
  • the above aminopyrimidine modified gold nanoparticle aqueous solution may be stored at 4 ° C for at least one year at a concentration of 0.1 to 1 mg / ml, and may be stored at 4 ° C or -20 ° C for at least one year after lyophilization, freeze-dried particles Still soluble In the water.
  • the present invention provides a method for preparing the above gold nanoparticles, the steps of which include:
  • reducing agent for example, sodium borohydride or sodium ascorbate
  • the molar ratio of the chloroauric acid to the aminopyrimidine molecule in the mixed solution in the step (1) is 1: 1 to 1 : 10; the molar ratio of the chloroauric acid to the reducing agent is 1: 1 ⁇ 1 : 10 .
  • the chloroauric acid, the aminopyrimidine and the reducing agent are dissolved in a polar solvent or a non-polar solvent, and further preferably, the polar solvent is selected from the group consisting of water, disulfoxide, decyl alcohol, ethanol, tetrahydrofuran, The polar solvent is selected from the group consisting of toluene and diphenylbenzene.
  • the mixed solution further comprises an organic acid or a mineral acid, preferably, acetic acid, propionic acid or hydrochloric acid; the volume content of the acid in the mixed solution is 0.5% to 10%;
  • the non-ionic surfactant is further included in the mixed solution, and the nonionic surfactant is preferably Triton X-100, Tween and polyethylene glycol, and the volume content of the nonionic surfactant in the mixed solution is 1 % ⁇ 10%.
  • the non-polar solvent further comprises a phase transfer catalyst
  • the phase transfer catalyst is preferably tetra-n-octyl ammonium bromide, tetrabutylammonium bromide, tetrabutyl hydrogen sulfate.
  • the reaction time of the above preparation method is 0.5 hours or more, and the reaction temperature range is below room temperature, preferably between room temperature and zero degrees Celsius, including room temperature and zero degrees Celsius; further preferably, the reaction time is 0.5 to 3 hours.
  • the present invention provides an antibacterial agent comprising aminopyrimidine modified gold nanoparticles.
  • the antibacterial agent is selected from the group consisting of one or more anti-Gram-negative bacteria drugs, anti-Gram-positive bacteria drugs, and/or anti-multidrug resistant clinical isolate drugs.
  • the Gram-negative bacteria comprise Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteria, Salmonella typhi, Proteus and Cholera;
  • the Gram-positive bacteria comprise Staphylococcus epidermidis, Pneumococci, Diphtheria Bacillus and tetanus;
  • the multi-drug resistant clinical isolate comprises multi-drug resistant E. coli and Pseudomonas aeruginosa.
  • the present invention provides the use of gold nanoparticles for the preparation of antibacterial agents.
  • the antibacterial agent is selected from the group consisting of one or more anti-Gram-negative bacteria drugs, anti-Gram-positive bacteria drugs, and/or anti-multidrug resistant clinical isolate drugs.
  • the Gram-negative bacteria comprise Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteria, Salmonella typhi, Proteus and Cholera;
  • the Gram-positive bacteria comprise Staphylococcus epidermidis, Pneumococci, Diphtheria Bacillus and tetanus;
  • the multi-drug resistant clinical isolate comprises multi-drug resistant E. coli and Pseudomonas aeruginosa.
  • the present invention provides a method of treating a disease caused by a bacterium and a disease, disorder or condition associated therewith, the method comprising administering a therapeutically effective amount of the above aminopyrimidine modified gold nanoparticle after bacterial infection in a mammal Granules or the above antibacterial drugs.
  • the method of treatment comprises administering a therapeutically effective amount of aminopyrimidine modified gold nanoparticles to a patient by intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, or oral administration, and may be administered in a single administration.
  • the drug can be administered twice a day, once a day, three times in a row, or the like, and the gold nanoparticles can be dissolved in an aqueous solution or a 5% dextrose solution.
  • the present invention has at least the following advantages:
  • the aminopyrimidine-modified gold nanoparticle of the present invention is a 2-mercapto-4,6-diaminopyrimidine, 2-mercapto-4-aminopyrimidine which has no antibacterial activity per se and is often used as a pyrimidine prodrug. / or 2,4-diamino-6-mercaptopyrimidine modified to gold nanoparticles prepared, has excellent antibacterial activity.
  • the aminopyrimidine modified gold nanoparticle of the invention has the advantages of small single particle size distribution, good particle dispersion in water (see Figures 2 ⁇ 5), and can be at 4 ° C or -20 ° C. After at least one year of storage, the lyophilized particles are still soluble in water.
  • the aminopyrimidine modified gold nanoparticles consist of gold nanoparticles and aminopyrimidine molecules bound thereto.
  • the aminopyrimidine is selected from one or more of 2-mercapto-4,6-diaminopyrimidines (called DAPT), 2-mercapto-4-aminopyrimidine (called APT) and/or 2,4-diamino-6.
  • DAPT 2-mercapto-4,6-diaminopyrimidine
  • APT 2-mercapto-4-aminopyrimidine
  • iDAPT 2-mercapto-4-amino-6
  • Figure 1 the chemical structure of the three aminopyrimidine molecules is shown in Figure 1.
  • Single aminopyrimidine molecularly modified gold nanoparticles comprise 2-mercapto-4,6-diaminopyrimidine modified gold nanoparticles (called Au-DAPT), 2-mercapto-4-aminopyrimidine modified gold nanoparticles Au-APT) and 2,4-diamino-6-mercaptopyrimidine-modified gold nanoparticles (called Au-iDAPT); mixed aminopyrimidine-modified gold nanoparticles containing 2-mercapto-4,6-diaminopyrimidine Gold nanoparticles co-modified with 2-mercapto-4-aminopyrimidine (called Au DAPT/APT), 2-mercapto-4,6-diaminopyrimidine and 2,4-diamino-6-mercaptopyrimine Gold nanoparticles (called Au-DAPT/iDAPT), 2-mercapto-4-aminopyrimidine and 2,4-diamino-6-mercaptopyrim-modified gold nanoparticles (called Au-AP
  • the method for preparing the above nanoparticles may comprise the following steps: (1) mixing the raw material chloroauric acid with an aminopyrimidine molecule and a surfactant to form a uniform solution; (2) reducing the above solution by using a reducing agent such as sodium borohydride or sodium ascorbate; The chloroauric acid forms aminopyrimidine-modified gold nanoparticles; (3) removing by-products such as salts and salts in the reaction system, and purifying the aminopyrimidine-modified gold nanometers from the exemplary embodiments of the embodiments include:
  • Formulate tetra-n-octyl ammonium bromide, tetrabutylammonium bromide, tetrabutylammonium hydrogen sulfate, benzyltriethylammonium chloride, dodecyltrimethylammonium chloride or 1 ⁇ 10 mM
  • the solvent is removed under reduced pressure in the dark red solution formed in the above polar solvent, or a large amount of ethanol is added to the deep red solution formed in the non-polar solvent, and the solvent is removed after separation, and pure water is added.
  • Dialysis was carried out in pure water for 48 hours with a dialysis bag having a molecular weight of 3 KDa or more, and water was changed every 2 hours; the solution after dialysis was filtered and sterilized by a 0.22 ⁇ microporous filter to obtain an aminopyrimidine-modified aqueous solution of gold nanoparticles, which was placed. Store at 4°C or store at -20 °C after lyophilization.
  • the chloroauric acid comes from Shanghai Jiushan Chemical Co., Ltd., sodium borohydride, sodium ascorbate, polyethylene glycol (molecular weight 2000 ⁇ 5000), ethanol, toluene, diphenylbenzene, tetra-n-octyl ammonium bromide, tetrabutyl Ammonium bromide, tetrabutylammonium hydrogen sulfate, benzyltriethylammonium chloride, dodecyltrimethylammonium chloride and tetradecyltrimethylammonium chloride from Sinopharm Group Beijing Chemical Reagent Company, 2-Mercapto-4,6-diaminopyrimidine from Merck, 2-mercapto-4-aminopyrimidine and 2,4-diamino-6-mercaptopyrimidine, disulfoxide, decyl alcohol, tetrahydrofuran, anhydrous acetic acid Triton X-100 and Tween (
  • the particle size of the aminopyrimidine-modified gold nanoparticles was determined by a transmission electron microscope (Tecnai G2 20 S-TWIN transmission electron microscope, FEI, USA), and the ratio of gold to aminopyrimidine in the nanoparticles was determined by X-ray photoelectron energy.
  • the instrument (ESCALab220I-XL, VG Scentific, UK) was used to determine the concentration of the aqueous nanoparticle solution (gold concentration) as determined by inductively coupled plasma atomic emission spectroscopy (ICP-OES, American PE).
  • the present invention performs cytotoxicity test on the aminopyrimidine-modified gold nanoparticles provided, and proves that the aminopyrimidine-modified gold nanoparticles have no effect on the cell proliferation rate, that is, no cytotoxicity.
  • the invention detects the antibacterial activity of the provided aminopyrimidine modified gold nanoparticles, and the specific specific detection methods include:
  • E Escherichia coli
  • CGMCC1.2389 Gram-negative bacteria
  • dysentery bacillus Salmonella typhi
  • Proteus or Vibrio cholerae in other Gram-negative bacteria Can be
  • Pseudomonas aeruginosa CGMCC1.2387, Gram-negative bacteria
  • MDR E multi-drug resistant Escherichia coli
  • epidermidis needs to be cultured for 16 hours, others need to be cultured) 8 to 10 hours), dilute the bacterial solution with fresh broth medium to make the OD value (600 nm) of the bacterial solution 0.1.
  • Each group is added with different concentrations of aqueous drug solution (the drug can be aminopyrimidine modified gold nanoparticles, amino group).
  • coli bacteria solution was added to all 12 portions, cultured at 37 V, shaking at 200 rpm, and the turbidity of the bacterial solution was measured after 24 hours.
  • the lowest concentration of the drug in the 4 groups was not turbid (OD value ⁇ 0.05 at 600 nm), which is the minimum inhibitory concentration (MIC) of the drug against Escherichia coli.
  • the present invention provides the use of the above aminopyrimidine-modified gold nanoparticles as a nano antibacterial agent, which is a drug resistant to Gram-negative bacteria, a drug resistant to Gram-positive bacteria, and/or resistant Drug-resistant clinical isolates of drugs.
  • the Gram-negative bacteria comprises Escherichia coli, Pseudomonas aeruginosa, Shigella, Salmonella typhi, Proteus or Vibrio cholerae
  • said Gram-positive bacteria include Staphylococcus epidermidis, pneumonia Diplococcus, diphtheria or tetanus
  • the drug-resistant clinical isolates include multi-drug resistant Gram-negative bacteria, such as multidrug-resistant E. coli and multidrug resistant patina Monocytogenes, etc.
  • the present invention provides a method for treating bacterial infection in vivo using the above aminopyrimidine modified gold nanoparticles as a nano antibacterial agent.
  • exemplary specific treatments include: intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection or oral aminopyrimidine modified gold nanoparticles, E. coli or P. aeruginosa infection, gold nanoparticles, gold
  • the solvent of the nanoparticles is water or 5% glucose, which is administered in a single administration, twice a day, once a day, three times in a row.
  • the invention provides a method for synthesizing an aminopyrimidine which has a small particle size distribution, a small particle size distribution, good water dispersibility, can be stored at 4 ° C or -20 ° C for more than one year, and is non-toxic to human primary cells.
  • Modified gold nanoparticles, synthetic methods and antibacterial uses, ie, 2-mercapto-4,6-diaminopyrimidine, 2-mercapto-4-aminopyrimidine and/or 2,4-Diamino-6-mercaptopyrimidine was modified (bound) onto gold nanoparticles to prepare aminopyrimidine-modified gold nanoparticles, and the aminopyrimidine-modified gold nanoparticles exhibited excellent antibacterial activity.
  • the beneficial effects of the invention are:
  • 2-Mercapto-4,6-diaminopyrimidine, 2-mercapto-4-aminopyrimidine and/or 2,4-diamino-6-mercaptopyrimidine have no antibacterial activity per se, and have antibacterial properties after modification with gold nanoparticles.
  • the effect, not the old drug modification, but belongs to the class I new drug, can be widely used in the preparation of antibacterial drugs, such as anti-gram-negative bacteria drugs, anti-gram-positive bacteria drugs or / and anti-multidrug resistance Clinical isolate drug;
  • the aminopyrimidine-modified gold nanoparticle of the present invention has a small particle size distribution, and the preparation method is simple and easy to preserve.
  • Figure 1 is 2-mercapto-4,6-diaminopyrimidine (called DAPT), 2-mercapto-4-aminopyrimidine (called APT) and 2,4-diamino-6-mercaptopyrimidine (iDAPT) Chemical structure.
  • DAPT 2-mercapto-4,6-diaminopyrimidine
  • APT 2-mercapto-4-aminopyrimidine
  • iDAPT 2,4-diamino-6-mercaptopyrimidine
  • Figure 2 is a TEM image of 2-mercapto-4,6-diaminopyrimidine modified gold nanoparticles prepared in a polar solvent.
  • Figure 3 is a 2-nonyl-4,6-diaminopyrimidine modified gold nanoparticle prepared in a non-polar solvent.
  • Figure 4 is a TEM image of 2-mercapto-4-aminopyrimidine modified gold nanoparticles.
  • Figure 5 is a TEM image of 2,4-diamino-6-mercaptopyrimidine modified gold nanoparticles.
  • Figure 6 shows the effects of different concentrations of 2-indolyl-4,6-diaminopyrimidine modified gold nanoparticles on the proliferation of human umbilical vein endothelial cells.
  • FIG. 7 shows 4-aminothiophenol-modified gold nanoparticles (A) and 6-mercaptopurine-modified gold nanoparticles (B). The best way to implement the invention
  • the 2-mercapto-4,6-diaminopyrimidine modified gold nanoparticles are prepared in a polar solvent by the steps of: (1) preparing a reaction solution:
  • Dioxin was removed under reduced pressure from the above dark red solution, pure water was added, and dialyzed in pure water for 48 hours with a dialysis bag having a molecular weight cut off of 3 kDa, and water was changed every 2 hours; the solution after dialysis was passed through a 0.22 ⁇ microporous filter. Filtration and sterilization to obtain a 2-mercapto-4,6-diaminopyrimidine modified gold nanoparticle aqueous solution, stored at 4 ° C, or lyophilized - : C storage: , , , , ⁇ , , , , , . Inductively coupled plasma atomic emission spectroscopy (ICP-OES).
  • ICP-OES Inductively coupled plasma atomic emission spectroscopy
  • a 10 ⁇ nanoparticle aqueous solution was dropped on a 400 mesh carbon support membrane copper mesh, and after natural drying, the size and shape of the nanoparticles were observed by a transmission electron microscope ( ⁇ ).
  • the gold nanoparticles were washed three times with pure water, and after X-ray photoelectron spectroscopy (XPS), the molar ratio of the ligand molecule aminopyrimidine to the elemental gold in the nanoparticles was obtained.
  • the aqueous solution containing the 2-mercapto-4,6-diaminopyrimidine-modified gold nanoparticles synthesized in this Example 1 is dark red, and it can be seen from Fig. 2 ( ⁇ ) that 2-mercapto-4,6-diaminopyrimidine can be seen.
  • the modified gold nanoparticles have a particle size of 2.2 to 5.6 nm; the molar ratio of the 2-mercapto-4,6-diaminopyrimidine molecule to the gold element is 0.4:1.
  • the 2-mercapto-4,6-diaminopyrimidine modified gold nanoparticles are prepared in a non-polar solvent, the steps of which are:
  • a solution of 4 mM tetra-n-octyl ammonium bromide in toluene was prepared by adding 21.7 mg of tetra-n-octyl ammonium bromide solid to 20 ml of toluene and sonicating.
  • a 10 ⁇ nanoparticle aqueous solution was dropped on a 400 mesh carbon support membrane copper mesh, and after natural drying, the size and shape of the nanoparticles were observed by a transmission electron microscope ( ⁇ ).
  • the gold nanoparticles were washed three times with pure water, and after X-ray photoelectron spectroscopy (XPS), the molar ratio of the ligand molecule aminopyrimidine to the elemental gold in the nanoparticles was obtained.
  • the aqueous solution containing the 2-mercapto-4,6-diaminopyrimidine-modified gold nanoparticles synthesized in this Example 2 is dark red, and it can be seen from Fig. 3 ( ⁇ ) that 2-mercapto-4,6-diaminopyrimidine can be seen.
  • the modified gold nanoparticles have a particle size of 3.4 to 10.0 nm.
  • a gold nanoparticle containing 2-mercapto-4-aminopyrimidine is prepared by the steps of:
  • the nano-particles were dissolved in a 400-mesh carbon support film copper mesh. After drying, the size and shape of the nanoparticles were observed by transmission electron microscopy (TEM). The gold nanoparticles are washed three times with pure water, and after X-ray photoelectron spectroscopy (XPS), the molar ratio of the ligand molecules to the elemental gold in the nanoparticles is obtained.
  • TEM transmission electron microscopy
  • XPS X-ray photoelectron spectroscopy
  • the aqueous solution containing the 2-mercapto-4-aminopyrimidine-modified gold nanoparticles synthesized in this example is dark red, and the particles of 2-mercapto-4-aminopyrimidine-modified gold nanoparticles can be seen from FIG. 4 (TEM image).
  • the diameter is 1.5 to 6.0 nm; the molar ratio of 2-nonyl-4-aminopyrimidine to gold is 0.2:1.
  • Table 1 The above analysis and measurement results are shown in Table 1.
  • a solution of 2,4-diamino-6-mercaptopyrimidine tetrahydrofuran at a concentration of 100 mM was prepared by adding 142 mg of 2,4-diamino-6-mercaptopyrimidine solid to 10 ml of tetrahydrofuran and adding 400 ⁇ M of anhydrous acetic acid. And 80 ⁇ M Tween 80 to obtain a mixed solution containing 2,4-diamino-6-mercaptopyrimidine;
  • the above dark red solution was decompressed to remove tetrahydrofuran, and pure water was added thereto, and dialyzed in pure water for 48 hours with a dialysis bag having a molecular weight cut off of 3 KDa, and water was changed every 2 hours; the dialyzed solution was filtered and sterilized by a 0.22 ⁇ microporous filter.
  • An aqueous solution of 2,4-diamino-6-mercaptopyrimidine-modified gold nanoparticles was obtained, stored at 4 ° C, or stored at -20 ° C after lyophilization.
  • the concentration of the 2,4-diamino-6-mercaptopyrimidine-modified gold nanoparticle aqueous solution can be measured by an inductively coupled plasma atomic emission spectroscopy (ICP-OES).
  • ICP-OES inductively coupled plasma atomic emission spectroscopy
  • a 10 ⁇ nanoparticle aqueous solution was dropped on a 400 mesh carbon support membrane copper mesh, and after natural drying, the size and shape of the nanoparticles were observed by a transmission electron microscope ( ⁇ ).
  • the gold nanoparticles are washed three times with pure water, and after X-ray photoelectron spectroscopy (XPS), the molar ratio of the ligand molecules to the elemental gold in the nanoparticles is obtained.
  • XPS X-ray photoelectron spectroscopy
  • the aqueous solution containing the 2,4-diamino-6-mercaptopyrimidine-modified gold nanoparticles synthesized in this Example 3 is dark red, and 2,4-diamino-6-mercaptopyrimidine can be seen from Fig. 5 ( ⁇ ).
  • the modified gold nanoparticles have a particle size of 3.5 to 10.0 nm, and the molar ratio of the 2,4-diamino-6-mercaptopyrimidine molecule to the gold element is 0.3:1.
  • Table 1 The above analysis and measurement results are shown in Table 1.
  • the above dark red solution was decompressed to remove tetrahydrofuran, and pure water was added thereto, and dialyzed in pure water for 48 hours with a dialysis bag having a molecular weight cut off of 3 KDa, and water was changed every 2 hours; the dialyzed solution was filtered and sterilized by a 0.22 ⁇ microporous filter.
  • An aqueous solution of gold nanoparticles modified with 2-mercapto-4,6-diaminopyrimidine/2-mercapto-4-aminopyrimidine is obtained, stored at 4 ° C, or stored at -20 ° C after lyophilization.
  • the concentration of 2-indolyl-4,6-diaminopyrimidine/2-mercapto-4-aminopyrimidine-modified aqueous gold nanoparticles can be measured by inductively coupled plasma atomic emission spectroscopy (ICP-OES).
  • ICP-OES inductively coupled plasma atomic emission spectroscopy
  • a 10 ⁇ L aqueous solution of the nanoparticles was dropped on a 400-mesh carbon support film copper mesh, and after natural drying, the size and shape of the nanoparticles were observed by a transmission electron microscope ( ⁇ ).
  • the gold nanoparticles are washed three times with pure water, and after X-ray photoelectron spectroscopy (XPS), the molar ratio of the ligand molecules to the elemental gold in the nanoparticles is obtained.
  • XPS X-ray photoelectron spectroscopy
  • Example 5 An aqueous solution containing 2-mercapto-4,6-diamino-pyrimidin-1 3 ⁇ 4 / 2- mercapto-4-amino pyrimidine synthesis of Example 5 of the present embodiment is modified gold particles is deep red, a particle size of 2.0 ⁇ 6.0 nm.
  • Example 6
  • Cytotoxicity assay of 2-mercapto-4,6-diaminopyrimidine modified gold nanoparticles of the present invention The second generation of human umbilical vein endothelial cells with good growth state was inoculated into 96-well plates according to ⁇ ⁇ ⁇ 4 cells/well. After adhering to the wall, 200 ⁇ M of M199 medium containing 100 g/ml gold nanoparticles was added in the second row, and M199 medium 200 containing 10 and 50 g/ml gold nanoparticles was sequentially added to the third and fourth rows. ⁇ , add six holes per line. In the fifth row, 200 ⁇ l of M199 medium containing no nanoparticles was added.
  • FIG. 6 shows that the aqueous solution of gold nanoparticles containing 2-mercapto-4,6-diaminopyrimidine modified has no effect on cell proliferation rate, ie no cytotoxicity, at concentrations up to 100 g/ml.
  • Example 7 The above cytotoxicity test uses the 2-mercapto-4,6-diaminopyrimidine modified gold nanoparticles prepared in Example 1, and other aminopyrimidine modified gold nanoparticles prepared by the preparation method provided by the present invention can also be used. Perform a cytotoxicity test.
  • Example 7 The above cytotoxicity test uses the 2-mercapto-4,6-diaminopyrimidine modified gold nanoparticles prepared in Example 1, and other aminopyrimidine modified gold nanoparticles prepared by the preparation method provided by the present invention can also be used. Perform a cytotoxicity test.
  • Example 7 Perform a cytotoxicity test.
  • Escherichia coli (Escherichia col " CGMCC 1.2389 ) was cultured in broth medium for 8-10 hours, and the OD value (600 nm) of the diluted solution was 0.1 with fresh broth medium. 12 parts 1.78 Ml broth medium, divided into 4 groups, 3 parts each (ie three parallel), 1-4 groups were added 4, 6, 8 and 10 g/ml of 2-mercapto-4,6-diaminopyrimidine The modified gold nanoparticles aqueous solution was 200 ⁇ M each, and the three groups in the same group were added at the same concentration. Then, 20 ⁇ M of the diluted E. coli bacteria solution was added to all 12 portions, and cultured at 37 ° C and a shaking rate of 200 rpm.
  • the turbidity of the bacterial liquid was measured after 24 hours, and the lowest nanoparticle concentration in the 4 groups was not turbid (OD value ⁇ 0.05 at 600 nm), and the minimum nanoparticle concentration was 6 g/ml, that is, 2-mercapto-4,6-diaminopyrimidine modified
  • the minimum inhibitory concentration (MIC) of gold nanoparticles to E. coli was 6 g/ml.
  • the above antibacterial activity was detected by using the 2-mercapto-4,6-diaminopyrimidine-modified gold nanoparticle prepared in Example 1, and the 2-mercapto-4,6-diaminopyrimidine preparation prepared in Example 2 was used in the same manner.
  • Gold nanoparticles, 2-mercapto-4-aminopyrimidine modified gold nanoparticles prepared in Example 3, 2,4-diamino-6-mercaptopyrimidine modified gold nanoparticles prepared in Example 4, and Preparation of Example 5 The 2-n-based 4,6-diaminopyrimidine/2-mercapto-4-aminopyrimidine-modified gold nanoparticles were also tested for anti-E. coli activity, and the results showed that the above aminopyrimidine-modified gold nanoparticle aqueous solution also had Bacteriostatic effect.
  • Staphylococcus epidermidis (CGMCC 1.2429) was cultured in broth for 16 hours, and the OD value (600 nm) of the diluted solution was 0.1 with fresh broth medium. Take 12 parts of 1.78 ml of broth culture medium and divide into 4 groups of 3 parts (three parallels), and groups 1-4 add 4, 8 , 16 and 32 g/ml of 2-mercapto-4,6 respectively. - 200 ⁇ M of diaminopyrimidine-modified gold nanoparticle solution, the same concentration of the three drugs in the same group, and then add 20 ⁇ M of diluted S.
  • the culture was carried out at a rate of 200 rpm, and the turbidity of the bacterial liquid was measured after 24 hours.
  • the lowest nanoparticle concentration in the four groups was not turbid (OD value ⁇ 0.05 at 600 nm), and the minimum nanoparticle concentration was 32 g/ml, that is, 2-mercapto-4,6-
  • the minimum inhibitory concentration (MIC) of diaminopyrimidine modified gold nanoparticles against S. epidermidis was 32 g/ml.
  • Detection of antibacterial activity of aqueous solution of aminopyrimidine Escherichia col CGMCC 1.2389 was cultured in broth medium for 8-10 hours, and the OD value (600 nm) of the diluted solution was 0.1 with fresh broth medium. Take 9 parts of 1.78 ml of broth culture medium, divide into 3 groups, 3 parts each (ie three parallel), and add 1-3 groups of 10, 100 and 1000 g/ml 2-mercapto-4,6-two The aqueous solution of aminopyrimidine was 200 ⁇ M each, and the three drugs in the same group were added at the same concentration. Then, 20 ⁇ M of the diluted E.
  • coli bacteria solution was added to all 9 parts, and cultured at 37 ° C, shaking rate of 200 rpm, 24 hours. After that, the turbidity of the bacterial liquid was measured. All three groups of bacteria were turbid (OD values at 1.6 nm were 1.62 ⁇ 0.14, 1.76 ⁇ 0.11 and 1.90 ⁇ 0.11, respectively), so the minimum inhibitory concentration (MIC) of 2-mercapto-4,6-diaminopyrimidine against Escherichia coli More than 1000 g/ml, no antibacterial activity is considered.
  • MIC minimum inhibitory concentration
  • the above antibacterial activity was tested by using the pyrimidine ligand used in the preparation of the 2-mercapto-4,6-diaminopyrimidine modified gold nanoparticle aqueous solution of Example 1, and the same example was used to prepare the corresponding aminopyrimidine modified gold nanoparticle.
  • the aminopyrimidine ligand used in the granules was also tested for antibacterial activity. The results showed that the minimum inhibitory concentration of the above aminopyrimidine ligands was more than 1000 g/ml, and no antibacterial activity was considered.
  • Escherichia coli (Escherichia col " CGMCC 1.2389 ) was cultured in broth medium for 8-10 hours, and the OD value (600 nm) of the diluted liquid solution was 0.1 with fresh broth medium. 12 parts of 1.78 ml of broth was cultured. Base, divided into 4 groups, each group of 3 (ie three parallel), 1 ⁇ 4 groups were added 0.4, 0.8, 1 and 2 g / ml of gentamicin aqueous solution 200 ⁇ 1 each, three in the same group The concentration of the drug was the same, and 20 ⁇ l of the diluted E.
  • the minimum drug concentration of the turbid liquid (OD value ⁇ 0.05 at 600 nm) is 1 g/ml, that is, the minimum inhibitory concentration (MIC) of gentamicin to Escherichia coli is 1 g/ml.
  • the strain is Escherichia coli, and the antibacterial activity of P. aeruginosa, multidrug-resistant Escherichia coli, multidrug-resistant Pseudomonas aeruginosa and Gram-positive bacteria can be detected by the same method.
  • mice infected with E. coli Treatment of mice infected with E. coli:
  • Escherichia coli was cultured overnight, diluted 100-fold with a medium, and diluted 10 times with a medium containing 0.5% of highly active dry yeast.
  • ICR mice Male and female, divided into 5 groups, 10 in each group, 0.5 ml of E. coli culture medium containing high activity yeast, and then intravenously give four groups of mice respectively.
  • Injection doses of 0.001, 0.005, 0.05, 0.5 mg/kg of 2-mercapto-4,6-diaminopyrimidine modified gold nanoparticles were applied once, and the fifth group was the untreated control group. Survival rate. The results showed that all the mice in the control group died, and the gold nanoparticle treatment group showed a therapeutic effect with a cure rate of 20 to 60%.
  • the above bacterial infection treatment method adopts the preparation of 2-mercapto-4,6-diaminopyrimidine-modified gold nanoparticle aqueous solution in Example 1, and similarly, the aminopyrimidine-modified gold nanoparticle prepared by using other examples has a therapeutic effect.
  • the cure rate is 10 to 60%.
  • the above bacterial infection treatment method uses an aqueous solution of gold nanoparticles, and a 5% glucose solution using gold nanoparticles also has a cure rate of 10% or more.
  • the above-mentioned bacterial infection treatment method is administered intravenously, and other methods of intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection or oral administration have a cure rate of more than 10%.
  • the above-mentioned bacterial infection treatment method adopts a single administration method, and other administration methods are administered twice a day, and once a day, three consecutive administrations also have a cure rate of more than 10%.
  • the above-mentioned bacterial infection treatment method is Escherichia coli, and the same method can be used for Pseudomonas aeruginosa, multidrug-resistant Escherichia coli, multidrug-resistant Pseudomonas aeruginosa and Gram-positive bacteria.
  • Treatment of infection in the body It can be concluded that: the invention modifies aminopyrimidine on gold nanoparticles, can exhibit antibacterial activity of aminopyrimidine which does not have antibacterial activity, and can be applied to prepare anti-gram-negative bacteria and anti-gram-positive bacteria Drug or/and anti-multidrug resistant clinical isolates; and no fine-package toxicity.

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Abstract

L'invention concerne des nanoparticules d'or modifiées par un aminopyrimide, leur procédé de préparation et leur utilisation. L'aminopyrimidine est de préférence choisie parmi la 2-thiol-4,6-diaminopyrimidine, la 2-thiol-4-aminopyrimidine et/ou la 2,4-diamino-6-mercaptopyrimidine. La taille de particule des nanoparticules d'or est de 1,5 à 10,0 nm et le rapport molaire entre l'aminopyrimidine et Au est de 0,1:1 à 0,9:1. Les nanoparticules d'or ont un effet antibiotique à large spectre.
PCT/CN2011/000341 2010-03-05 2011-03-02 Nanoparticules d'or modifiées par un aminopyrimide, leur procédé de préparation et leur utilisation WO2011106993A1 (fr)

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