CN112933226B - Preparation and application of targeted antibacterial nanomaterial AuNS-PEG-AMP - Google Patents
Preparation and application of targeted antibacterial nanomaterial AuNS-PEG-AMP Download PDFInfo
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Abstract
The invention belongs to the technical field of nano materials, and particularly discloses preparation and application of a targeted antibacterial nano material AuNS-PEG-AMP. Introducing a gelatinase cutting site at the C end of the antibacterial peptide, introducing a staphylococcus aureus targeted recognition peptide at the N end, and coupling the Cy 7-labeled targeted enzyme-cutting antibacterial peptide to the gold nanostar through a gold-sulfur bond. In vitro experimental results show that gelatinase secreted by staphylococcus aureus can cleave polypeptide and release antibacterial peptide. The released antibacterial peptide has killing capacity to staphylococcus aureus obviously superior to that of escherichia coli due to the existence of the target recognition sequence. In addition, the photothermal effect of the gold nano-star is utilized to generate heat ablation on microorganisms so as to achieve the purpose of cell damage and death. The antibacterial nano material has more stable property, more excellent antibacterial performance, small interference on normal physiological activities of organism cells, low toxicity and high safety.
Description
Technical Field
The invention belongs to the technical field of nano materials, and particularly relates to a preparation method and application of a targeted antibacterial nano material AuNS-PEG-AMP.
Background
With the development of science and technology and the improvement of living standard, the environmental sanitation requirements of people on living, working and living are increasingly improved, so that the rapid development of antibacterial technology and antibacterial materials is promoted, and the nano antibacterial material is an important one of the antibacterial materials. The nano antibacterial material is a novel material with antibacterial property, has the advantages of large specific surface area, high reactivity and the like, and can keep the growth and reproduction of microorganisms including bacteria, fungi, yeasts, algae, viruses and the like at a low level, thereby greatly improving the antibacterial property of the material.
In recent years, a large number of drug-resistant bacteria and multi-drug-resistant bacteria have been continuously developed. The spontaneous drug-resistant mutation of bacteria, the screening of antibiotics and the environmental adaptability of pathogenic bacteria are the basis for the generation of clinical drug-resistant bacteria and multi-drug-resistant bacteria. The antibacterial peptide (AMP) has incomparable advantages of the traditional antibiotics, has unique antibacterial mechanism and rapid bactericidal action, is not easy to cause the drug resistance of bacteria, and is an antibacterial drug with great potential.
Photothermal therapy (PTT), also known as photothermal bacterial lysis, has been used for the past few years for the treatment of localized pathogenic bacterial infections, especially bacterial infections with multiple drug resistant bacteria. Photothermal induced increases in body temperature are beneficial for recovery from certain diseases. When the body temperature rises, the growth rate of pathogens is slowed down and enzymes in the virus may be inactivated because the temperature of the internal environment is no longer suitable for them. Therefore, photothermal effect is widely used for antibacterial and anticancer drug therapy.
Disclosure of Invention
The invention aims to provide a preparation method and application of a targeted antibacterial nano material AuNS-PEG-AMP.
Staphylococcus aureus is one of the pathogens of gelatinase overexpression and is prevalent in infections associated with chronic wounds and implanted medical devices. This feature can distinguish gelatinase-positive pathogenic staphylococcus aureus from gelatinase-negative non-pathogenic staphylococcus epidermidis. Based on the above, the invention introduces gelatinase cutting site (PLGVGG) at the C-terminal of GKRWWKWWRR antibacterial peptide, and introduces staphylococcus aureus target recognition peptide (GLFVD) at the N-terminal. Typically, the nanoprobes are non-fluorescent because of the presence of Fluorescence Resonance Energy Transfer (FRET) between the Cy7 dye and the AuNS core. However, in the pathological enzyme microenvironment of staphylococcus aureus infection, PLGVRG between AuNS and Cy7 will be cleaved by overexpressed gelatinase (cleavage site between G and V), restoring the fluorescence of Cy7, enabling in situ turn-on near-infrared fluorescence imaging of staphylococcus aureus infection. In addition, GLFVD can significantly enhance the targeted accumulation of nanomaterials at the site of Staphylococcus aureus infection and avoid thermal damage to surrounding healthy tissues, thereby enhancing the more specific and localized photothermal treatment of nanomaterials on Staphylococcus aureus infected wounds.
The specific technical scheme of the invention is as follows:
the targeted antibacterial nanomaterial AuNS-PEG-AMP comprises AuNS of a gold nano star, targeted enzyme digestion antibacterial peptide AMP and Cy7 fluorescent dye molecules.
Wherein, the gold nano star AuNS is prepared by reducing chloroauric acid, the maximum absorption wavelength is 690nm, the particle size is 40nm, and the potential is-30 mV.
The sequence of the targeting enzyme digestion antibacterial peptide AMP is GLFVDKGKRWWKWWRRGPLGVRGC, and the antibacterial peptide AMP is synthesized by a conventional solid phase Fmoc method, namely, monomeric amino acid protected by Fmoc on solid phase resin is deprotected to expose amino, and peptide bonds are formed with carboxyl of the amino acid in solution through condensation reaction, so that the amino acid is connected to the resin until a required peptide chain is synthesized. See example 1 for a specific operation.
The prepared AuNS-PEG-AMP has a particle size of 180nm and a potential of +30mV.
Cy 7-labeled antimicrobial peptide through Au-S bond
(GLFVDK (Cy 7) GKRWWKWWRRGPLGVRGC) is coupled to the gold nanostars, PEG is added to increase stability, the particle size of the nano material AuNS-PEG-AMP is stable, the light stability is realized, and the photothermal effect is obvious under 808nm laser irradiation.
The preparation method of the target antibacterial nano material AuNS-PEG-AMP comprises the following steps: and adding SH-PEG into the aureobasidin, putting the aureobasidin into a shaker for reacting at room temperature for 30min, then adding AMP-Cy7, putting the aureobasidin into the shaker for reacting at room temperature for 24h, centrifuging the reactant at 13000rpm for 15min after the reaction is finished, and re-suspending the precipitate with 1mL of ultrapure water to obtain AuNS-PEG-AMP.
The Cy 7-labeled targeting enzyme-cleaved antibacterial peptide (AMP-Cy 7) is purified by HPLC.
The targeted antibacterial nanomaterial AuNS-PEG-AMP can recover fluorescence in a microenvironment where staphylococcus aureus exists, and can target staphylococcus aureus to generate selective sterilization. See example 1 for a specific operation.
Compared with the prior art, the invention has the following beneficial effects:
1. the antibacterial peptide is coupled with the gold nanostar, so that the stability of a fluorescent marker Cy7 on the antibacterial peptide is improved, and the cytotoxicity of the antibacterial peptide is reduced;
2. the antibacterial peptide can be slowly released in a microenvironment where staphylococcus aureus exists due to the existence of the enzyme digestion sequence, and meanwhile, the staphylococcus aureus can be better identified through the targeting sequence, so that the killing capacity to the staphylococcus aureus is improved.
3. The invention introduces gold nanostars for PTT antibiosis, generates photothermal effect under the irradiation of 808nm laser to cause bacterial cell damage and death, and generates synergistic effect with antibacterial peptide, so that AuNS-PEG-AMP has more excellent antibacterial performance.
Description of the drawings:
FIG. 1 is an HPLC chart of GLFVDK- (Cy 7) GKRWWKWWRRGPLGVRGC.
FIG. 2 is a graph of the hydrated particle size of AuNS, auNS-PEG-AMP.
FIG. 3 is a potential diagram of AuNS, auNS-PEG-AMP.
FIG. 4 shows UV absorption spectra of AuNS, AMP-Cy7, and AuNS-PEG-AMP.
FIG. 5 is a graph showing the particle size stability of AuNS-PEG-AMP.
FIG. 6 is a graph showing the evaluation of light stability of AMP-Cy7 (7A) and AuNS-PEG-AMP (7B).
FIG. 7 is a graph of photothermal temperature increase for different concentrations of AuNS-PEG-AMP.
FIG. 8 is a graph of the recovery of fluorescence after co-incubation of AuNS-PEG-AMP with Staphylococcus aureus.
FIG. 9 is a graph of the inhibitory growth of AuNS-PEG-AMP against Staphylococcus aureus under both light and non-light conditions.
FIG. 10 is a graph of the inhibitory growth of AuNS-PEG-AMP against E.coli under both light and non-light conditions.
FIG. 11 is a graph of AuNS-PEG-AMP inhibition of Staphylococcus aureus biofilms.
FIG. 12 is a graph of AuNS-PEG-AMP abrogation of Staphylococcus aureus biofilms.
FIG. 13 is a graph of the inhibition of E.coli biofilms by AuNS-PEG-AMP.
FIG. 14 is a graph of the elimination of E.coli biofilms by AuNS-PEG-AMP.
FIG. 15 is a graphic of bacterial plating after 1 hour of co-incubation of AuNS-PEG-AMP with Staphylococcus aureus.
FIG. 16 is a photograph of bacterial plating after 1 hour incubation of AuNS-PEG-AMP with E.coli.
FIG. 17 is a graph of bacterial plating after 1 hour incubation of AuNS-PEG-AMP mixed with AMP-Cy7.
FIG. 18 is a drawing of bacterial plating after 2 hours of co-incubation of AuNS-PEG-AMP with mixed species of AMP-Cy7.
FIG. 19 is a plot of Live/Dead staining before and after AuNS-PEG-AMP response to Staphylococcus aureus.
FIG. 20 is a graph of cytotoxicity of AuNS-PEG-AMP against HUVEC.
FIG. 21 is a graph of an experiment of E.coli infection in animal wounds with AuNS-PEG-AMP.
FIG. 22 is a graph of animal wound S.aureus infection experiments with AuNS-PEG-AMP.
FIG. 23 is a graph of a wound tissue plating experiment in AuNS-PEG-AMP animals.
FIG. 24 is a graph of AMP-Cy7 plate-plating experiments against Staphylococcus aureus.
FIG. 25 is a graph showing the AMP-Cy7 plate-coating experiment against E.coli.
FIG. 26 is a drawing of an AMP-Cy7 mixed strain antibacterial plating experiment.
Detailed Description
The present invention will be described in detail with reference to examples, but it is not limited to these examples.
Example 1
Synthesis of AuNS-PEG-AMP nanomaterial
1) Synthesis of gold nano star
The synthesis method of the gold nano-star adopts a one-step synthesis method, glassware required before material synthesis is soaked in aqua regia (hydrochloric acid with volume ratio: nitric acid =3: 1) overnight until no bubbles escape, then the aqua regia is recovered, and the soaked glassware is washed with ultrapure water for three times and dried. Preparing a 1M NaOH solution for later use, weighing 3g of HEPES in a 250mL pear-shaped bottle, adding 79.2mL of deionized water, oscillating and ultrasonically dissolving, adding 10.8mL of NaOH solution (1M) to adjust the pH value to about 7.4, and finally obtaining a 90mL HEPES solution (the concentration is 0.14M, and the pH value is = 7.4). mu.L of chloroauric acid aqueous solution (40 mM) is added into the pear-shaped bottle, a 5mL pipette gun is used for rapidly blowing to ensure that the HEPES solution and the chloroauric acid aqueous solution are uniformly mixed until the solution is colorless and transparent, and the mixture is kept stand for 1 hour in a dark place. And after the reaction is finished, the solution turns dark blue, the solution is centrifuged at 13000rpm for 15min to remove supernatant, the precipitate is resuspended by deionized water, the centrifugation is repeated three times, and finally the obtained precipitate is dispersed in 10mL of deionized water, wherein the concentration of AuNS is 0.2nM.
2) Preparation of targeted enzyme digestion antibacterial peptide
The GLFVDK (Mtt) GKRWWKWWRRGPLGVRGC targeted enzyme digestion antibacterial peptide is prepared by taking ChemMatrix resin and amino acid as raw materials and adopting an Fmoc solid-phase synthesis method. The polypeptide sequence was introduced with a lysine protected with Mtt, the Mtt protecting group was cleaved after synthesis of the polypeptide, and 5-fold molar amount of polypeptide of Cy7 and equimolar ratio of EDC, DIEA and HOBT (i.e. molar ratio AMP: cy7: EDC: DIEA: HOBT = 1).
The dye not bound was washed every other day, and 20% piperidine (vol., piperidine: DMF =1: 4) was added to cleave the Fmoc protecting group at the N-terminus of the G amino acid, followed by cleavage with a polypeptide cleavage solution (vol., TFA: EDT: TIS: H 2 O =94:2.5:1:2.5 Reaction at room temperature for 3h to separate the polypeptide from the resin, precipitating with glacial ethyl ether, centrifuging at 4000rpm to remove the supernatant, blowing the precipitated nitrogen gas to dryness, dissolving, purifying by high performance liquid chromatography, and collecting the components which peak at both 220nm and 749nm (figure 1).
3) Preparation of AuNS-PEG-AMP
To 10mL of the gold nanostar (0.2 nM) was added 500. Mu.L of SH-PEG (1 mg/mL), placed in a shaker at room temperature for 30min, then 500. Mu.L of AMP-Cy7 (28. Mu.M) was added, placed in a shaker at room temperature for 24h. After the reaction, the reaction mixture was centrifuged at 13000rpm for 15min, the supernatant was collected, and the precipitate was resuspended in 1mL of ultrapure water, at which time the concentration of AuNS-PEG-AMP was 2nM as the concentration of AuNS.
2. Characterization of AuNS-PEG-AMP nanomaterials
1) Hydrated particle size, zeta potential and ultraviolet absorption change of AuNS-PEG-AMP nano material
20 μ L of AuNS, auNS-PEG and AuNS-PEG-AMP nanophase materials were diluted to 2mL with deionized water. Wherein 1mL is used for measuring the hydrated particle size and 1mL is used for measuring the Zeta potential. Each set of samples was run in triplicate and monitored for changes throughout the process. The results are shown in FIGS. 2 and 3.
And judging whether each step is successful or not according to the change conditions of the hydrated particle size and the Zeta potential of the AuNS, auNS-PEG and AuNS-PEG-AMP nano materials. From fig. 2, it can be seen that the hydrated particle size gradually increased from the initial AuNS nanoparticles to the final AuNS-PEG-AMP, demonstrating the successful coupling of PEG and AMP-Cy7. Fig. 3 is a graph of Zeta potential change, which also demonstrates that AuNS nanoparticles have PEG and AMP-Cy7 attached.
200 μ L of AMP-Cy7, auNS, and AuNS-PEG-AMP were put into a 96-well plate, and ultraviolet absorption was measured at 400-950nm by a microplate reader, the effect is shown in FIG. 4.
As can be seen from FIG. 4, AMP-Cy7 has a distinct absorption peak at 749nm, auNS has an absorption peak around 690nm, and the ultraviolet absorption peak of AuNS-PEG-AMP is red-shifted between AMP-Cy7 and AuNS, which indicates that the ultraviolet absorption changes after AMP-Cy7 is coupled with AuNS.
2) Particle size stability examination experiment of AuNS-PEG-AMP
To investigate the stability of AuNS-PEG-AMP in DI water, 10. Mu.L of AuNS-PEG-AMP was diluted to 1mL with DI water, followed by determination of hydrated particle size using a dynamic light scattering instrument for 7 consecutive days and the dimensional stability of AuNS-PEG-AMP was monitored. The results are shown in FIG. 5.
It is seen from fig. 5 that the hydrated particle size of AuNS-PEG-AMP does not change significantly within 7 days, which indicates that AuNS-PEG-AMP has better stability, mainly because the stability of the nanoprobe in aqueous solution is greatly improved by modifying thiol-polyethylene glycol on the AuNS-PEG-AMP.
3. Evaluation of photostability of AuNS-PEG-AMP
Since the heptamethine bond in the Cy7 near-infrared fluorescent dye is unstable and there is a photobleaching phenomenon upon irradiation with intense light, it is necessary to study the change in light stability when AuNS is coupled with AMP-Cy7. mu.L of AuNS-PEG-AMP (AMP-Cy 7 concentration 6. Mu.M) was pipetted by a pipette, the total wavelength was measured by a microplate reader after 10min of laser irradiation at 808nm, and AMP-Cy7 (6. Mu.M) was used as a control group.
As shown in figure 6, after 808nm laser irradiation, the ultraviolet absorption of AMP-Cy7 alone at 749nm is obviously reduced, while the absorption peak intensity of AuNS-PEG-AMP before and after irradiation is almost unchanged, which shows that the light stability of the near-infrared fluorescent dye Cy7 is improved after AMP-Cy7 is coupled with AuNS.
4. Photothermal effect performance test of AuNS-PEG-AMP
To evaluate the photothermal efficiency of the materials, 200. Mu.L of AuNS-PEG-AMP (0 nM, 0.125nM, 0.25nM, 0.375nM, 0.5 nM) at various concentrations were placed in 2mL centrifuge tubes and irradiated with near infrared laser (808nm, 1.8W/cm) 2 ) And 6 minutes, monitoring the temperature by a thermal imager, and performing data processing by using Origin to obtain a photothermal heating curve.
As shown in FIG. 7, auNS-PEG-AMP has good photo-thermal properties, and the photo-thermal effect is enhanced with the increase of the concentration of AuNS-PEG-AMP.
5. AuNS-PEG-AMP in-vitro bacteria activation near-infrared fluorescence experiment
1mL of staphylococcus aureus in the logarithmic growth phase is taken in a 2mL centrifugal tube, and the staphylococcus aureus is centrifuged at 4000rpm for 5min at low temperature. After centrifugation, the supernatant was collected and 100. Mu.L of AuNS-PEG-AMP (concentration 2 nM) was put into six wells of a 96-well plate, and equal volumes of deionized water and S.aureus supernatant were added thereto, respectively, and the sample was incubated for 30min in a constant temperature shaker at 37 ℃. After the incubation, fluorescence at a specific wavelength (the maximum excitation wavelength was set to 705nm, and the scanning wavelength was 745nm to 850 nm) was detected by a microplate reader three times in parallel, and data was recorded.
As shown in figure 8, after AuNS-PEG-AMP and staphylococcus aureus are incubated together, the originally quenched fluorescence is recovered, and the aim of controlling the fluorescence switch by using gelatinase is fulfilled.
6. In vitro antibacterial experiments of AuNS-PEG-AMP
1) Effect of AuNS-PEG-AMP on bacterial growth Curve
To explore the effect of AuNS-PEG-AMP on bacterial growth, overnight cultured S.aureus, E.coli bacteria were diluted to 10 5 CFU/mL, 150. Mu.L of the bacterial solution and 50. Mu.L of AuNS-PEG-AMP with a concentration of 2nM were added to a 96-well plate, and samples were irradiated with a laser at 808nM for 6min, with three replicates per group being prepared without laser irradiation and water as control groups. And measuring the absorbance at 600nm by using a microplate reader for 12 hours continuously, and then, drawing and observing the growth conditions of the bacteria in the experimental group and the control group according to the measured data. The effect is shown in figures 9 and 10.
As can be seen from the figure, auNS-PEG-AMP has obvious inhibition effect on the growth of staphylococcus aureus and poor inhibition effect on escherichia coli, which is caused by the fact that the inhibition effect on the escherichia coli is reduced due to the targeting sequence on the antibacterial peptide, but the sterilization effect is better after illumination because the escherichia coli is thermolabile.
2) AuNS-PEG-AMP inhibition/disruption of bacterial biofilms
Biofilm inhibition experiments: flat bottom 96 well plates were selected and 200. Mu.L of TSB medium was added to selected 12 wells followed by 2. Mu.L of S in log phase growth.Aureus/E.coli bacteria divided into three groups (PBS control group, auNS-PEG-AMP + illumination group), each group had 3 wells, 20. Mu.L of PBS was added to the first group, 20. Mu.L of AuNS-PEG-AMP was added to the second and third groups, and 808nm laser (1.5W/cm) was used to the third group 2 ) Irradiating for 6min. And (3) putting the 96-well plate into a biochemical incubator at 37 ℃ for incubation for 48h, taking out after the incubation is finished, sucking out the upper culture solution by using an injector, washing the wells for 2-3 times by using PBS (phosphate buffer solution), air-drying for 10min, and adding 100 mu L of crystal violet solution with the concentration of 1% into each well for dyeing for 20min. Gently absorbing the upper layer crystal violet solution, washing with sterile PBS for 3 times, air-drying for 10min, adding 200 μ L of 80% ethanol into each well, placing the 96-well plate on a constant temperature shaking table, oscillating for 2h, detecting the absorbance of each well at 590nm by using an enzyme-labeling instrument, taking an average value and recording data.
Eliminating the biological film experiment: selecting a flat-bottom 96-well plate, adding 200 mu L of TSB culture solution into 12 selected wells, adding 2 mu L of S.aureus bacterial solution/E.coli bacterial solution in a logarithmic growth phase, and placing the well plate in a biochemical incubator at 37 ℃ for incubation for 48h. After incubation was complete, the 9 wells were divided into three groups, the first group was added with 20. Mu.L PBS, the second and third groups were added with 20. Mu.L AuNS-PEG-AMP, wherein the third group used a 808nm laser (1.5W/cm) 2 ) Irradiating for 6min. After the illumination is finished, incubating for 30min again, sucking out the upper culture solution, washing each hole for 2-3 times by using sterilized PBS, air-drying for 10min, and adding 100 mu L of crystal violet solution with the concentration of 1% into each hole for dyeing for 20min. The crystal violet solution was gently aspirated, washed 2-3 times with sterile PBS, and air dried for 10min. Adding 200 mu L of 80% ethanol into each well, placing the wells in a constant temperature shaking table to be vibrated and dissolved for 2h, detecting the absorbance of each well at 590nm by a microplate reader, and taking the average value of three wells as data record.
As shown in the attached figures 11, 12, 13 and 14, auNS-PEG-AMP has the effects of inhibiting and eliminating the biological membranes of two bacteria, and the bacteriostasis effect is more obvious after laser irradiation.
3) AuNS-PEG-AMP bacterial plating experiment
To investigate the effect of AuNS-PEG-AMP on the survival rate of S.aureus bacteria, auNS-PEG-AMP solutions were prepared at concentrations of 0.25, 0.5, 0.75, and 1nM, respectively, and 10 samples were taken 8 CFU/mL S.aureus bacterial liquid 100 muL and 100 muLAnd co-incubating AuNS-PEG-AMP with different concentrations for 2h, co-incubating a negative control group by using 100 mu L of PBS solution, diluting by 8 ten thousand times after incubation, taking 100 mu L of coated plates, putting the coated plates into a biochemical incubator for culturing for 15h, counting the number of colonies growing on the TSA plate, and parallelly measuring each sample for three times.
To investigate the effect of AuNS-PEG-AMP on E.coli survival, auNS-PEG-AMP solutions were prepared at concentrations of 0.25, 0.5, 0.75, and 1nM, respectively, and 10 samples were taken 8 And incubating 100 mu L of CFU/mL E.coli liquid and 100 mu L of AuNS-PEG-AMP with different concentrations for 2h, incubating the negative control group by 100 mu L of PBS solution, diluting the incubation group by 8 ten thousand times, taking 100 mu L of coated plates, putting the coated plates into a biochemical incubator for culturing for 15h, counting the number of colonies growing on the LB agar plate, and measuring each sample in parallel for three times.
To investigate the targeting performance of AuNS-PEG-AMP to s.aureus and e.coli, auNS-PEG-AMP solutions were prepared at concentrations of 1 and 2nM, respectively, and the concentrations of the polypeptides coupled to the gold nanostars were measured at 3 and 6 μ M, respectively, using the BCA protein kit, thus preparing AMP-Cy7 polypeptide solutions at concentrations of 3 and 6 μ M as a control group. Get 10 8 1mL and 10mL of CFU/mL E.coli liquid 8 1mL of S.aureus bacterial liquid of CFU/mL is uniformly mixed, 100 mu L of the S.aureus bacterial liquid and 100 mu L of AuNS-PEG-AMP with different concentrations are taken from the mixed bacterial liquid to be incubated for 2h together, a negative control group is incubated for 1h and 2h by 100 mu L of PBS solution, after the incubation is finished, the 100 mu L of bacterial liquid is taken out and coated after 2 ten thousand times and 8 ten thousand times of the bacterial liquid are respectively diluted, the bacterial liquid is put into a biochemical incubator to be cultured for 15h, the number of colonies growing on an LB agar plate is counted, and each sample is parallelly measured for three times.
As shown in FIG. 15, auNS-PEG-AMP has significant killing effect on Staphylococcus aureus, and the results in FIG. 16 show that AuNS-PEG-AMP has insignificant killing effect on Escherichia coli due to the targeting sequence. The results in fig. 17 show that the selective killing of s.aureus by AuNS-PEG-AMP was not significant at 1 hour co-incubation, probably due to the fact that the gelatinase secreted by staphylococcus aureus did not reach a certain concentration in the bacterial solution. And as the co-incubation time was extended to 2 hours (fig. 18), the effect of AuNS-PEG-AMP on selective killing of s.
4) Live/dead staining of AuNS-PEG-AMP-treated S.aureus bacteria
To explore the staining before and after the AuNS-PEG-AMP was applied to S.aureus bacteria, this was done using live/dead kit. 100 μ L of AuNS-PEG-AMP (1 nM) was added to 100 μ L of 10. Mu.L each 9 In S.aureus of CFU/mL, 30min after incubation with a near infrared laser (808nm, 1.5W/cm) 2 ) The bacteria were irradiated for 10min. The control group used in this experiment was not laser irradiated and not supplemented with AuNS-PEG-AMP. Centrifuging the three groups of samples at 5000rpm for 10min, discarding the supernatant, staining the precipitate with 30 μ L live/dead reagent, blowing uniformly, vortexing, standing in the dark for 20min, and finally dropping 20 μ L of sample on the slide for observation by an inverted fluorescence microscope. The results are shown in FIG. 19.
By adjusting the ratio of the two dyes SYTO 9 and Propidium Iodide, bacteria with intact cell membrane structure can be dyed green, but when the cell membrane of the bacteria is damaged, the bacteria can be dyed red. The comparative experiment results show that the S.aureus bacteria in the PBS group are bright green, which indicates that the bacteria still keep better bacterial viability, the bacterial cell membranes still keep complete structures, but the bacteria are bright red after the AuNS-PEG-AMP is added, which indicates that the S.aureus bacteria are killed after the AuNS-PEG-AMP is added, the cell membranes are destroyed, and the sterilization effect is more obvious after the AuNS-PEG-AMP is illuminated, and visible illumination can trigger the photothermal effect to enhance the sterilization effect of the material.
7. Biocompatibility experiments for AuNS-PEG-AMP
HUVEC cells were digested, counted, and counted at 1 × 10 per well 4 Cells were plated, marginal wells filled with PBS, incubated overnight, and samples placed under uv light overnight. A sample of 300. Mu.L was added to 900. Mu.L of DMEM (-) (-) medium and mixed well to a sample concentration of 0.5nM, and 500. Mu.L each of AuNS-PEG-AMP was prepared at 0.1nM, 0.2nM, 0.3nM and 0.4 nM. Adding into selected 25 wells, incubating for 24h, adding 20 μ L MTT indicator, incubating for 4h, sucking out the culture medium after the incubation is finished, adding 150 μ L DMSO, and shaking to dissolve to measure the absorbance at 490 nm.
As shown in figure 20, the growth state of cells co-incubated by AuNS-PEG-AMP with different concentrations is good, the absorbance of the cells is measured after the MTT indicator is added, and the MTT detection result shows that the biocompatibility of the AuNS-PEG-AMP is good and the cytotoxicity is low.
8. Animal wound experiments with AuNS-PEG-AMP
And establishing a mouse double-wound infection model. Selecting 9 depilation mice, perforating a circular wound with diameter of about 1cm on the upper and lower parts of the back of the depilation mice by using a perforator, and perforating the wound on the upper part by using 10 8 Coli bacteria three times CFU/mL, 10 for lower wounds 8 Three times of CFU/mL Staphylococcus aureus infection. Dividing 9 mice into 3 groups, wherein each group comprises 3 parallel samples, one group comprises PBS group, and 50 mu L of PBS solution is dripped into the back wounds of three mice every day to serve as a control group for five days; one group was AuNS-PEG-AMP group, and 50. Mu.L of AuNS-PEG-AMP solution (0.5 nM) was added dropwise once a day to the back wounds of three mice as experimental group for five days; one group was AuNS-PEG-AMP + light group, 50. Mu.L of AuNS-PEG-AMP solution (0.5 nM) was added dropwise to the dorsal wound of three mice once a day, and a 808nM laser (1.8W/cm) 2 ) Irradiating for 6min while monitoring temperature with thermal imaging instrument to maintain the temperature at 42-43 deg.C to prevent damage of skin tissue of mouse due to excessive temperature, and the group is light irradiation experiment group for five days. The wounds of 9 mice were photographed and the wound area was measured daily for 11 days. After 11 days, 9 mice were sacrificed, wound tissues of the mice were taken and put into PBS solution to be mashed and incubated for half an hour, plates were coated by diluting two ten thousand times, and the number of colonies on the culture medium was observed every other day.
As shown in figure 21, the difference between the wound infection of Escherichia coli in PBS group and AuNS-PEG-AMP group is small in 11 days, which indicates that AuNS-PEG-AMP has poor curative effect on wound infection of Escherichia coli and illumination group has good effect, because Escherichia coli has poor heat resistance and heat generated by laser irradiation has good bactericidal effect, the wound recovery of AuNS-PEG-AMP + illumination group has better effect than the other two groups.
As shown in fig. 22, the wound area infected with staphylococcus aureus in PBS group recovered worse after 11 days, the wound recovery in AuNS-PEG-AMP group was significantly better, and the wound recovery in AuNS-PEG-AMP + light group was the best, indicating that AuNS-PEG-AMP had a targeted bactericidal effect on staphylococcus aureus and had a better antibacterial effect due to photothermal effect under laser irradiation.
As shown in figure 23, the results of the mouse wound tissue plating show that AuNS-PEG-AMP group has lower killing effect on Escherichia coli and better killing effect on Staphylococcus aureus, and AuNS-PEG-AMP + illumination group has very good killing effect on both bacteria due to photothermal effect.
Comparative example 1
The invention carries out comparison experiments of antibacterial performance on AMP-Cy7 polypeptide which is not coupled with the gold nanostars.
1. AMP-Cy7 plate-coating experiment for resisting staphylococcus aureus
Preparing AMP-Cy7 solutions with concentrations of 3, 6, 9, and 12 μ M with sterile water, and collecting 10 8 And incubating 100 mu L of CFU/mL S.aureus bacterial liquid and 100 mu L of AMP-Cy7 with different concentrations for 1h, incubating the negative control group by using 100 mu L of PBS solution, diluting for 2 ten thousand times after incubation, taking 100 mu L of coated plates, putting the coated plates into a biochemical incubator for culturing for 15h, counting the number of colonies growing on the TSA plates, and performing parallel measurement for three times on each sample.
As shown in FIG. 24, AMP-Cy7 has a concentration gradient bacteriostatic tendency towards Staphylococcus aureus, MIC 90 Between 6 and 9. Mu.M, MIC 50 At around 3. Mu.M. MIC of AuNS-PEG-AMP as compared to AuNS-PEG-AMP 50 The concentration of the polypeptide coupled with the gold nanostar is about 2.3 mu M under the concentration of about 0.75nM detected by a BCA protein kit, which shows that the antibacterial performance of the polypeptide is improved after the gold nanostar is coupled. In addition, as the enzyme digestion sequence on the polypeptide sequence is cut by gelatinase generated by staphylococcus aureus, the nano material also has the capability of sustained release, and the capability of resisting staphylococcus aureus is further improved.
2. AMP-Cy7 anti-E.coli plating experiment
Preparing AMP-Cy7 solutions with concentrations of 3, 6, 9, and 12 μ M with sterile water, and collecting 10 8 And incubating 100 mu L of CFU/mL E.coli liquid and 100 mu L of AMP-Cy7 with different concentrations for 1h, incubating the negative control group by using 100 mu L of PBS solution, diluting the incubation solution by 2 ten thousand times, taking 100 mu L of coated plates, putting the coated plates into a biochemical incubator for culturing for 15h, counting the number of colonies growing on an LB agar plate, and measuring each sample in parallel for three times.
As shown in FIG. 25, AMP-Cy7 showed a concentration gradient antibacterial tendency against E.coli, but had a poor inhibitory effect against E.coli at a low concentration (about 3. Mu.M), and MIC 90 At around 9 μ M. After the AMP-Cy7 is coupled with the gold nanostars, the concentration of the AMP-Cy7 coupled with the gold nanostars is 2.3 mu M through the detection of a BCA protein kit, and the AMP-Cy7 has weak damage to escherichia coli no matter whether the AMP-Cy7 is coupled with the gold nanostars or not. In addition, escherichia coli does not release gelatinase, so AuNS-PEG-AMP cannot release AMP-Cy7 continuously, and experiments show that AuNS-PEG-AMP and AMP-Cy7 have weak killing capacity on Escherichia coli and have small difference on Escherichia coli.
3. AMP-Cy7 mixed strain antibacterial plating experiment
In order to study the targeting performance of AMP-Cy7 on S.aureus and E.coli bacteria, the mixed strain antibacterial experiment is carried out when the concentration of AMP-Cy7 is lower by combining the possibility of selective killing on staphylococcus aureus when the concentration of AMP-Cy7 is lower and the killing capability on both bacteria when the concentration is higher. Thus, solutions of AMP-Cy7 polypeptide were prepared in sterile water at concentrations of 1, 2, 3, and 4. Mu.M. Get 10 8 CFU/mL E.coli liquid 1mL and 10mL 8 1mL of CFU/mL S.aureus bacterial liquid is uniformly mixed, 100 mu L of the mixed bacterial liquid and 100 mu L of AMP-Cy7 with different concentrations are incubated for 1h, after the incubation is finished, the mixed bacterial liquid is diluted by 2 ten thousand times, 100 mu L of coated plates are taken and put into a biochemical incubator for culturing for 15h, the number of colonies growing on an LB agar plate is counted, and each sample is tested in parallel for three times.
As shown in fig. 26, AMP-Cy7 showed better killing of staphylococcus aureus at low concentrations, so AuNS-PEG-AMP could target staphylococcus aureus, and AMP-Cy7 played a dominant role in nanomaterials. Compared with AuNS-PEG-AMP, the target ability of AMP-Cy7 coupled with gold nanostar is enhanced, and AuNS-PEG-AMP has better killing ability to staphylococcus aureus as shown in figure 18.
Claims (3)
1.A targeted antibacterial nano material AuNS-PEG-AMP is characterized in that the nano material consists of gold nano star AuNS, targeted enzyme digestion antibacterial peptide AMP and Cy7 fluorescent dye molecules;
the aureobasidin AuNS is prepared by reducing chloroauric acid, the maximum absorption wavelength is 690nm, the particle size is 40nm, and the potential is-30 mV;
the sequence number of the target enzyme digestion antibacterial peptide AMP is GLFVDKGKRWWKWWRRGPLGVRGC, and the target enzyme digestion antibacterial peptide AMP is prepared by an Fmoc solid phase synthesis method;
the particle size of the AuNS-PEG-AMP nano material is 180nm, and the potential is +30 mV;
the preparation method of the target antibacterial nanomaterial AuNS-PEG-AMP comprises the following steps: and adding SH-PEG into the aureobasidin, putting the aureobasidin into a shaker for reacting at room temperature for 30min, then adding AMP-Cy7, putting the aureobasidin into the shaker for reacting at room temperature for 24h, centrifuging the reactant at 13000rpm for 15min after the reaction is finished, and re-suspending the precipitate with 1mL of ultrapure water to obtain AuNS-PEG-AMP.
2. The targeted antibacterial nanomaterial AuNS-PEG-AMP according to claim 1, wherein the AMP-Cy7 is prepared by a method comprising: according to AMP: cy7: EDC: DIEA: HOBT =1:3:1:1: AMP, cy7, EDC, DIEA and HOBT were weighed out at a molar ratio of 1, dissolved in DMF and reacted overnight with exclusion of light.
3. The targeted antibacterial nanomaterial AuNS-PEG-AMP of claim 1, wherein the Cy 7-labeled targeted enzymatic antibacterial peptide AMP-Cy7 is purified by HPLC.
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