CN105412940B - A kind of composite nano antibacterial material is used for the treatment of drug resistance of vancomycin pathogenic bacteria - Google Patents
A kind of composite nano antibacterial material is used for the treatment of drug resistance of vancomycin pathogenic bacteria Download PDFInfo
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- CN105412940B CN105412940B CN201510874259.3A CN201510874259A CN105412940B CN 105412940 B CN105412940 B CN 105412940B CN 201510874259 A CN201510874259 A CN 201510874259A CN 105412940 B CN105412940 B CN 105412940B
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- vancomycin
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- antibacterial material
- silver piece
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
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- 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
Abstract
The invention discloses a kind of composite nano antibacterial material and preparation method thereof, the problems such as to solve cumbersome synthesis existing for conventional Targeted Photosensitizer, poor biocompatibility.The nano antibacterial material is made of the vancomycin Porphyrin Molecule of the triangle Nano silver piece silica dioxide granule of nucleocapsid and its surface.Wherein, triangle Nano silver piece core forms surface-enhanced Raman (SERS) signal as host material, and silica shells constitute poly pattern as rigid platfor coupling vancomycin Porphyrin Molecule.The nano antibacterial materials synthesis is of low cost, preparation method is simple and has good biocompatibility and tissue penetration performance.It is applied in the Raman image and photodynamic therapy of drug resistance of vancomycin pathogenic bacteria in addition, the present invention further discloses above-mentioned nano antibacterial material.
Description
Technical field:
It is mould the present invention relates to a kind of preparation of composite nano antibacterial material based on triangle Nano silver piece and its through the ages
Application in the treatment of plain drug resistance pathogenic bacteria, belongs to the technical applications of triangle Nano silver piece and photodynamics bacteriostatic treatments.
Background technology:
Vancomycin (Vancomycin, abbreviation Van) is a kind of glycopeptide antibiotics, for preventing and treating gram sun
Property pathogenic infection.It since vancomycin sterilizing activity is strong, and is not easy that bacterium is made to generate drug resistance, in other antibiotic without ruling by law
It is just allowed to use when treating bacterium infection, therefore is referred to as " last line of defense of antibiotic ".However, as vancomycin is in clinic
On excessive use, the whole world occur successively vancomycin-resistant enterococcus (Vancomycin resistant Enterococci,
VRE) the report of this " superbacteria ".Due to lacking effective precautionary measures and oversight mechanism, the rapid wave of VRE antibody-resistant bacterium
And all over the world, quantity increases year by year.Currently, the incidence of China VRE infection is in ascendant trend year by year, in Individual hospitals even
Up to 20%, become the main pathogenic fungi of nosocomial infection and the repository of drug resistant gene.The appearance and propagation of these antibody-resistant bacterium
So that " last line of defense " of this treatment bacterium infection of vancomycin faces collapse, the clinical treatment of bacterium infection is proposed newly
Challenge.Therefore, new, significantly more efficient bacteriostasis method and strategy are researched and developed, to preventing and controlling infection and the biography of VRE drug-fast bacterias
It broadcasts with important clinical meaning.
With the appearance and propagation of VRE pathogenic bacteria, the conventional route of clinical treatment VRE infection is research and development new antibiotic
Drug is used for substituting original vancomycin.However, new drug development work faces, input is big, the period is long, risk is high, and difficulty is big etc.
Many difficulties.Therefore, researcher proposes a relatively simple new drug development strategy, i.e., by vancomycin polymer
Multivalence interacts, it is made to form stronger affinity between VRE bacteriums, achievees the purpose that drug-resistant bacteria is effectively inhibited to grow.
In recent years, the certain achievement obtained about the design of vancomycin polymer and the research of bacteriostatic activity.But it is mould through the ages
Plain polymer still has some problems as antibacterials.For example, and not all vancomycin polymer pharmaceutical efficacy all
It can be improved with the increase of affinity, this just gives the medicament research and development band of vancomycin derivatives great uncertain factor.
Even if in addition, vancomycin polymer enhances the inhibitory effect of VRE bacterial strains, but continuing to use and still may result in
The appearance of the new antibody-resistant bacterium for the vancomycin polymer.Therefore, suppression that is new, not will produce bacterial drug resistance is found
Bacterium strategy is still sciences problems urgently to be resolved hurrily in drug-fast bacteria infection treatment.
In order to solve traditional antibacterials problems faced, photodynamic therapy (Photodynamic therapy, PDT)
It is introduced into the research of bacteriostatic treatments, becomes treatment and controls the simple effective method of drug-fast bacteria infection.Photodynamic therapy
It is to generate active oxygen species (Reactive oxide species, ROS) using the light source activation photosensitizer of certain wavelength to damage
The cytoplasma membrane or DNA of bacterium, to achieve the purpose that kill drug-resistant bacteria.Compared with conventional antibiotic, photodynamic therapy
With two big advantages.First, two principal elements (light and photosensitizer) not no inherent sterilizing ability, sterilization in photodynamics
Activity only just will produce by carrying out illumination to the processed region of photosensitizer, be avoided in this way to other microfloras
The destruction in region.Second, and the most important, bacterium will not be to any resistances of killing effect generation caused by photodynamics
Mechanism.However, photodynamic therapy still has some problems as new departure of VRE pathogenic bacteria bacteriostatic treatments, for example, how
Ensure that accurately targeting is positioned at pathogenic bacteria to photosensitizerHow light exposure treatment region is accurately positionedIn order to solve these problems, it studies
Personnel's design has synthesized the vancomycin dimer of porphyrin connection, the specificity parent enhanced VRE bacterium surfaces using dimer
And ability, photosensitive molecular (porphyrin) targeting is fixed to bacterium surface, positioning and light are carried out by the fluorescence signal of porphyrin itself
Photodynamic therapy.But the vancomycin dimer of porphyrin connection is as Targeted Photosensitizer, however it remains following defect needs
It is further to improve:(1) dimer synthetic reaction take, low yield, and purification process is complicated, of high cost;(2) life of dimer
Object poor compatibility easily is combined to form precipitation with haemocyanin, is not used to the treatment of infection of internal deep tissues;(3) porphyrin point
The fluorescence property of son is bad, and near-infrared light source launching efficiency is low, is not suitable for the internal visualization treatment through Leatherwear and tissue.
Therefore, a kind of of low cost, preparation of research and development is simply, targeting is good and has the light of good biocompatibility and tissue penetration performance
Photodynamics bacteriostatic treatments of the quick composite material for drug resistance pathogenic bacteria are still a sciences problems urgently to be resolved hurrily, to preventing
Infection and propagation with control drug-fast bacteria have important clinical meaning.
Invention content:
First of the present invention is designed to provide a kind of composite nano antibacterial material, and the cost of material is low for this, prepares letter
Single, stable structure and there is good biocompatibility and tissue penetration performance.
Second object of the present invention is to provide the preparation method of above-mentioned composite nano antibacterial material, this method technique
Simply, of low cost and success rate is high.
Third object of the present invention is that provide above-mentioned composite nano antibacterial material moves in the light of VRE drug resistance pathogenic bacteria
Application in mechanics visualization treatment.
First purpose of the present invention is achieved by the following technical solution:A kind of composite nano antibacterial material,
Vancomycin-porphyrin the monomer of triangle Nano silver piece particle and modification including Silica-coated in particle surface.
The triangle Nano silver piece of Silica-coated forms the nano particle of nucleocapsid, the triangle silver nanoparticle inside particle
Strong surface-enhanced Raman (SERS) signal that piece can be generated as matrix, passes through SERS signal effective position lesion portion
Position, specifies light exposure treatment region.It is wrapped on the one hand water solubility and biofacies that outer silicon ball effectively improves entire nano material
Capacitive;On the other hand it is coupled to form poly pattern with vancomycin-porphyrin monomer as rigid platfor, by polymer to VRE
Photosensitive molecular is effectively fixed in bacterium surface, realizes the light power of targeting positioning by the superpower targeting binding ability of pathogenic bacteria
Learn bacteriostatic treatments.
Second object of the present invention is achieved by the following technical solution:Above-mentioned composite nano antibacterial material
Preparation method comprises the steps of:
(1) vancomycin-porphyrin monomer is synthesized;
(2) triangle Nano silver piece (AgNPL) is prepared;
(3) Raman molecular is modified to the surfaces AgNPL;
(4) layer of silicon dioxide is wrapped up on the surfaces AgNPL containing Raman molecular, forms the nano particle of nucleocapsid
(AgNPL@SiO2);
(5) by vancomycin-porphyrin monomer modification to AgNPL@SiO2Surface (AgNPL@SiO2-Por-Van)。
Preferential, in the step (1), the synthesis of the vancomycin-porphyrin monomer uses amino carboxyl condensation method system
It is standby.
Preferably, in the step (2), the triangle Nano silver piece length of side is 20-25nm;
Preferably, in the step (3), the Raman molecular is keyed to triangle Nano silver piece table by covalent chemical
Face;
Preferably, in the step (4), the surfaces AgNPL package layer of silicon dioxide completed by stober methods, and water with
The ratio of isoamyl alcohol is 1:5-1:10;
Preferably, in the step (5), vancomycin-porphyrin monomer is contracted using glutaric acid as attachment by amino carboxyl
It closes reaction modification and arrives AgNPL@SiO2Surface.
Third object of the present invention is achieved by the following technical solution:Above-mentioned composite nano antibacterial material exists
The Raman image of vancomycin-resistant bacteria and the application in photodynamics bacteriostatic treatments.
Advantageous effect:Composite nano antibacterial material provided by the invention and preparation method thereof, compared to existing routine
The photosensitive antibacterial medicines of targeting, has the following advantages:
1, compared with monomer vancomycin, vancomycin poly pattern shows the increase to VRE bacterium surface affinity,
Guarantee is provided for the efficient targeting positioning of photosensitive molecular;
2, silica nanosphere is easily modified, and can not only be used for the connecting platform of vancomycin poly pattern, and can increase
The water solubility and biocompatibility of material avoid being combined generation precipitation with haemocyanin, there is the infection for being easy to internal deep tissues
Treatment;
3, compared with other noble metal nanometer materials, the triangle Nano silver piece inside silicon ball has sharp apex angle, can
Cause strong local plasmon field, generates powerful Electromagnetic enhancement effect, the greatly SERS signal of amplification molecule.With it is traditional
Fluorescent technique positioning is compared, and SERS signal positioning is not easily susceptible to the interference of sample autofluorescence and no photobleaching phenomenon goes out
It is existing, and spectral bandwidth is narrow, is suitble to multicomponent label.
Description of the drawings
Fig. 1 composite Nanos antibacterial material (AgNPL@SiO2- Por-Van) prepare schematic diagram;
The characterization of Fig. 2 composite Nano antibacterial materials, including projection Electronic Speculum, photodynamics scattering result and ultraviolet-visible point
Light spectrogram;
The ex-vivo photodynamic bacteriostatic treatments result of Fig. 3 composite Nanos antibacterial material and its component;
The internal photodynamics bacteriostatic treatments result of Fig. 4 composite Nano antibacterial materials;
The Raman image result to vancomycin resistance pathogenic bacteria of Fig. 5 composite Nano antibacterial materials;
Specific implementation mode
Embodiment 1:
A kind of preparation process of composite Nano antibacterial material is as follows:
(1) synthesis of vancomycin-porphyrin monomer:
52.8mg vancomycins and 20.8mg porphyrins are dissolved in the dimethyl sulfoxide of 2mL dryings, 10min is stirred on ice bath,
Then sequentially add 38.0mg and be dissolved in 1mLN, O- benzotriazole-tetramethylurea hexafluorophosphate of dinethylformamide and
The N of 0.06mL, N- diisopropylethylamine.Ice bath is removed, is stirred to react overnight.At the end of reaction, 20mL acetone is added dropwise, can get
Darkviolet solid precipitates, and can be obtained vancomycin-porphyrin through high performance liquid chromatography separation after then washed once with 5mL acetone
Monomer.
(2) preparation of triangle Nano silver piece (AgNPL):
The sodium citrate solution of the silver nitrate solution of 200 μ L 50mM, 2mL 75mM are added sequentially to 96mL ultra-pure waters
In, the sodium borohydride solution of 3.5mL 30% hydrogen peroxide and 1mL 100mM is quickly rapidly added after stirring successively, is stirred at room temperature
Visible solution rapidly goes to purple from colourless after 5min, and continuing to stir 30min makes it fully react can to obtain triangle Nano silver piece,
It is about 20-25nm that Electronic Speculum, which observes its length of side,.
(3) package of the modification of Raman molecular and silica:
The 4- mercaptobenzoic acids of 6 μ L 5mmol/L are added into 2mL triangles Nano silver piece solution made of step (2), stir
After mixing reaction 1hr, it is slowly added dropwise in 10mL isoamyl alcohols, then the tetrem base silicon of 300 μ L 0.6% is added dropwise successively
After reacting 1hr, then aminopropyltriethoxysilane/second of 60 μ L 0.6% is added dropwise in hydrochlorate/ethanol solution and 90 μ L ammonium hydroxide
Alcoholic solution.After the reaction was continued 1hr, centrifugation terminates reaction, and three horn silver AgXs containing Raman molecular are received to obtain the final product after wash precipitation with ethyl alcohol
Rice piece-nano SiO 2 particle (AgNPL@SiO2)。
(4) functional modification of vancomycin-porphyrin
By AgNPL@SiO made of step (3)2Be dissolved in 1mL anhydrous dimethyl sulfoxides, 10min stirred on ice bath, then according to
Secondary glutaric acid/anhydrous dimethyl sulfoxide solution that 0.5mL 0.2% is added, 2mg are dissolved in 100 μ LN, the benzo of dinethylformamide
The N of triazole-tetramethylurea hexafluorophosphate and 3 μ L, N- diisopropylethylamine.Ice bath is removed, is stirred to react overnight.Centrifugation is eventually
It only reacts, dimethyl sulfoxide washing is used in combination three times, is then redissolved in 1mL anhydrous dimethyl sulfoxides, 10min is stirred on ice bath, so
After to sequentially add vancomycin-porphyrin monomer, 2mg made from the step of 1mg is dissolved in 0.5mL anhydrous dimethyl sulfoxide solution (1) molten
In 100 μ LN, the N of the benzotriazole-tetramethylurea hexafluorophosphate and 3 μ L of dinethylformamide, N- diisopropyl second
Amine.Ice bath is removed, is stirred to react overnight.Centrifugation terminates reaction, dimethyl sulfoxide washing is used in combination three times, you can by vancomycin-porphin
The modification of quinoline obtains composite Nano antibacterial material (AgNPL@SiO to triangle Nano silver piece-silica nanoparticle surface2-
Por-Van)。
Composite Nano antibacterial material and its transmission electron microscope of component prepared by this example, photodynamics scatter diagram and ultraviolet
Visible light light-splitting spectrum is as shown in Figure 2.
Embodiment 2:Composite Nano antibacterial material is used for vancomycin resistance field coccus Enterococcus Faecalis
The ex-vivo photodynamic bacteriostatic treatments of (ATCC 51299).
In from picking monoclonal bacterial strain on LB tablets to 5mL LB culture solutions, 37 DEG C are incubated overnight.4000rpm is collected by centrifugation
After being used in combination sterile PBS buffer to wash 3 times, OD is adjusted after bacterial precipitation is resuspended with PBS buffer solution for bacterium600It is 0.5.Then it takes
After 500 μ L bacterium solutions are mixed with the composite Nano antibacterial material of various concentration and its each component, placed in 37 DEG C of incubators
After 15min, illumination or non-photo-irradiation treatment are carried out.All illumination experiments are all made of xenon lamp (400nm-800nm) progress and illumination
Time is 0-2min.Then after the 100 μ L of bacterium solution that take that treated dilute certain multiple with sterile PBS buffer, LB trainings are incorporated in
It supports in plate, 37 DEG C are incubated overnight, and finally record bacterial clone number.
Composite Nano antibacterial material and its component are to vancomycin resistance field coccus Enterococcus in this example
The photodynamics bacteriostatic treatments result of Faecalis (ATCC 51299) is shown in shown in Fig. 3 left figures.
Embodiment 3:Composite Nano antibacterial material is used for vancomycin resistance field coccus Enterococcus faecalis
The ex-vivo photodynamic bacteriostatic treatments of (ATCC 51299).
In from picking monoclonal bacterial strain on LB tablets to 5mL LB culture solutions, 37 DEG C are incubated overnight.4000rpm is collected by centrifugation
After being used in combination sterile PBS buffer to wash 3 times, OD is adjusted after bacterial precipitation is resuspended with PBS buffer solution for bacterium600It is 0.5.Then it takes
After 500 μ L bacterium solutions are mixed with the composite Nano antibacterial material of various concentration and its each component, placed in 37 DEG C of incubators
After 15min, illumination or non-photo-irradiation treatment are carried out.All illumination experiments are all made of xenon lamp (400nm-800nm) progress and illumination
Time is 0-2min.Then by treated, bacterium solution sterile PBS buffer dilutes 106Multiple draws bacterium solution after 100 μ L dilutions
It is incorporated in LB culture plates, 37 DEG C are incubated overnight, and finally record bacterial clone number.
Composite Nano antibacterial material and its component are to vancomycin resistance field coccus Enterococcus in this example
The photodynamics bacteriostatic treatments result of faecalis (ATCC 51299) is shown in shown in Fig. 3 right figures.
Embodiment 4:Composite Nano antibacterial material is used for vancomycin resistance field coccus Enterococcus faecalis
The internal photodynamics bacteriostatic treatments of (ATCC 51299).
In from picking monoclonal bacterial strain on LB tablets to 5mL LB culture solutions, 37 DEG C are incubated overnight.4000rpm is collected by centrifugation
After being used in combination sterile PBS buffer to wash 3 times, OD is adjusted after bacterial precipitation is resuspended with PBS buffer solution for bacterium600It is 0.5.Take 10mL
Bacterium solution centrifugal concentrating is at for use after 100 μ L.
Healthy kunming mice 9 is taken, weight is about 18-22g, after all sloughing back hair, with cutting at baring skin
Knife cuts the wound of diameter about 0.5cm sizes, and the above-mentioned bacterium solutions of 10 μ L is taken to instill wound.Take 3 mouse as a contrast, in addition 6
The composite Nano antibacterial material of 10 μ L100nM is added dropwise, then it is therein 3 progress photo-irradiation treatment 10min, in addition 3 without
Photo-irradiation treatment.Timing takes pictures to wound location daily, records 5 days altogether.
Composite Nano antibacterial material is to vancomycin resistance field coccus Enterococcus faecalis (ATCC in this example
51299) internal photodynamics bacteriostatic treatments result is as shown in Figure 4.
The above is only a preferred embodiment of the present invention, it should be pointed out that:For the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (7)
1. a kind of composite nano antibacterial material, including the triangle Nano silver piece particle of Silica-coated and modification are in particle
The vancomycin on surface-porphyrin monomer;The composite nano antibacterial material is prepared via a method which to obtain:(1)It closes
At vancomycin-porphyrin monomer;(2)Triangle Nano silver piece AgNPL is prepared, is adopted in the preparation process of the triangle Nano silver piece
The hydrogen peroxide of the 200ppm of antifebrin containing stabilizer is used to be as oxidant, and the ratio of hydrogen peroxide and reducing agent sodium borohydride
3.4:1-3.6:1, the gained triangle Nano silver piece length of side is 20-25nm;(3)By Raman molecular modification to the surfaces AgNPL;(4)
Layer of silicon dioxide is wrapped up on the surfaces AgNPL containing Raman molecular, and the triangle Nano silver piece/silica for forming nucleocapsid is received
Rice grain AgNPL@SiO2, triangle Nano silver piece/nano SiO 2 particle of the nucleocapsid prepared using Stober methods,
And the ratio of water and isoamyl alcohol is 1:5-1:10;By vancomycin-porphyrin monomer modification to AgNPL@SiO2Surface A gNPL@
SiO2- Por-Van, the vancomycin-porphyrin monomer and the mass ratio of triangle Nano silver piece/nano SiO 2 particle are
0.5:1-1:1。
2. a kind of preparation method of composite nano antibacterial material described in claim 1, which is characterized in that it includes following steps
Suddenly:
(1)Synthesize vancomycin-porphyrin monomer;
(2)Triangle Nano silver piece AgNPL is prepared, acetophenone containing stabilizer is used in the preparation process of the triangle Nano silver piece
The hydrogen peroxide of amine 200ppm is as oxidant, and the ratio of hydrogen peroxide and reducing agent sodium borohydride is 3.4:1-3.6:1, gained three
The horn silver AgX nanometer sheet length of side is 20-25nm;
(3)By Raman molecular modification to triangle Nano silver piece surface;
(4)Layer of silicon dioxide is wrapped up on the triangle Nano silver piece surface containing Raman molecular, forms three horn silver AgXs of nucleocapsid
Nanometer sheet/nano SiO 2 particle AgNPL@SiO2, triangle Nano silver piece/nano SiO 2 particle of the nucleocapsid
It is prepared using Stober methods, and the ratio of water and isoamyl alcohol is 1:5-1:10;
(5)Vancomycin-porphyrin monomer modification is formed into compound receive to triangle Nano silver piece/silica nanoparticle surface
Rice antibacterial material AgNPL@SiO2- Por-Van, the vancomycin-porphyrin monomer and triangle Nano silver piece/silica nanometer
The mass ratio of particle is 0.5:1-1:1.
3. a kind of preparation method of composite nano antibacterial material according to claim 2, which is characterized in that step(1)
In, the synthesis of the vancomycin-porphyrin monomer carries out condensation reaction generation by the carboxyl of vancomycin and the amino of porphyrin.
4. a kind of preparation method of composite nano antibacterial material according to claim 2, which is characterized in that step(3)
In, the Raman molecular is the 4- mercaptobenzoic acids or 4- mercaptoanilines of small molecule, and triangle silver is keyed to by covalent chemical
Nanometer sheet surface.
5. a kind of preparation method of composite nano antibacterial material according to claim 2, which is characterized in that step(5)
In, pass through the carboxyl with glutaric acid between the vancomycin-porphyrin monomer and triangle Nano silver piece/nano SiO 2 particle
Condensation reaction is carried out with its respective amino to be attached.
6. a kind of composite nano antibacterial material according to claim 1 the Raman for preparing vancomycin resistance pathogenic bacteria at
As the application in drug.
7. a kind of composite nano antibacterial material according to claim 1 is in the light power for preparing vancomycin resistance pathogenic bacteria
Application in medicine.
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CN106474493B (en) * | 2016-09-22 | 2019-09-03 | 苏州大学 | Diagnosis and treatment integration nano-probe and preparation method thereof for bacterial infections in vivo |
CN109395115B (en) * | 2018-11-30 | 2020-12-08 | 中国科学院长春应用化学研究所 | Photodynamic super-hydrophobic anti-infection coating and preparation method thereof |
CN109701015B (en) * | 2019-01-24 | 2020-12-08 | 华中科技大学 | Triangular prism-shaped nano silver composite probe with stable structure and preparation method and application thereof |
CN111450266A (en) * | 2020-03-30 | 2020-07-28 | 中国科学院大学宁波华美医院 | Magnetic resonance imaging contrast agent targeting gram-positive bacteria, preparation and application |
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