CN112618714B - Anti-FLT1 polypeptide mediated synthetic gold cluster and preparation method and application thereof - Google Patents
Anti-FLT1 polypeptide mediated synthetic gold cluster and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a gold cluster synthesized by Anti-FLT1 polypeptide mediation, a preparation method and application thereof, belonging to the field of biomedical materials. The gold cluster is a gold cluster containing Anti-FLT1 polypeptide; the amino acid sequence of the Anti-FLT1 polypeptide is CGNQWFI. The Anti-FLT1 polypeptide-mediated gold cluster is synthesized by a simple one-pot method, and has an excellent Anti-angiogenesis effect and an excellent photodynamic treatment effect; compared with the gold cluster synthesized by singly using the Anti-FLT1 polypeptide or not using the Anti-FLT1 polypeptide as a medium, the gold cluster synthesized by the Anti-FLT1 polypeptide as a medium has a synergistic effect. The gold cluster synthesized by the Anti-FLT1 polypeptide mediated synthesis overcomes the defects of poor Anti-angiogenesis and photodynamic treatment effects of the gold cluster in the prior art, and has good application prospect.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a gold cluster synthesized by Anti-FLT1 polypeptide mediation, a preparation method and application thereof.
Background
Gold clusters (AuNCs) are molecular aggregates formed by combining a core consisting of several to dozens of gold atoms and organic single molecules such as thiol compounds or proteins and the like serving as ligand protection groups, and have a ligand shell-metal core structure. The fluorescent quantum dot has good biocompatibility, excellent fluorescence quantum yield and light stability, and fluorescence adjustability. These characteristics make gold clusters promising for applications. The gold cluster can be used for cell marking, imaging, drug delivery, photodynamic anti-tumor therapy (PDT), heavy metal ion detection, protein and microorganism detection and the like. Meanwhile, research shows that the nanogold also has anti-angiogenesis activity. The gold clusters can inhibit the proliferation of endothelial cells by acting on heparin-binding proteins such as Vascular Endothelial Growth Factor (VEGF) and fibroblast growth factor (bFGF), thereby inhibiting angiogenesis.
In recent years, ligand polypeptide protected gold nanoclusters have received increasing attention. Because the AuNCs synthesized by the polypeptide have the advantages, and more importantly, the diversity and the versatility of the polypeptide sequence make it possible to design the nanoclusters with specific recognition functions.
The Xing topic group at Guangzhou medical university recently reported that the RGD polypeptide sequence achieved specific targeting of gold clusters to cancer cells. The juanjuan and the Qujiabo, etc. utilize the designed reasonable and effective polypeptide sequence to synthesize the nano gold cluster which can target and mark the nucleolus, thereby providing better help for researching the nucleolus and the related process thereof. Further, Wang Xiao Juan, Qujiabo, etc. design a gold nanocluster of targeting marker nucleolus synthesized by KCK polypeptide mediation, reduce background interference of cell imaging of the existing nucleolus marker material, and solve the problems of high manufacturing cost, high toxicity and complex manufacturing process of the existing nucleolus marker material.
However, it has not been found that the use of polypeptide-mediated synthetic gold clusters can enhance anti-angiogenic activity and enhance photodynamic therapeutic effect.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to find a gold cluster synthesized by an Anti-FLT1 polypeptide mediated method and having more definite angiogenesis inhibiting activity, and a preparation method and application thereof. Thereby improving the angiogenesis inhibiting and photodynamic therapy effects of the gold clusters.
The invention provides a gold cluster, which is a gold cluster containing Anti-FLT1 polypeptide; the amino acid sequence of the Anti-FLT1 polypeptide is CGNQWFI.
Further, the Anti-FLT1 polypeptide has a structure shown in formula I:
further, the gold cluster takes gold atoms as a metal core, and Anti-FLT1 polypeptide as a ligand shell;
preferably, the particle size of the gold cluster is 0.5-5 nm;
more preferably, the gold cluster has a particle size of about 2 nm.
Further, the gold cluster is prepared by taking tetrachloroauric acid and Anti-FLT1 polypeptide as raw materials;
preferably, the molar mass ratio of the tetrachloroauric acid to the Anti-FLT1 polypeptide is 1: (0.5 to 4);
more preferably, the molar mass ratio of the tetrachloroauric acid to the Anti-FLT1 polypeptide is 1: 4.
further, the preparation method of the gold cluster comprises the following steps:
(1) preparing Anti-FLT1 polypeptide into suspension by using ultrapure water;
(2) adding a tetrachloroaururic acid solution into the suspension, mixing and reacting to obtain a reaction solution;
(3) and (5) ultrafiltering the reaction solution to obtain the final product.
Further, the air conditioner is provided with a fan,
in the step (1), the concentration of the Anti-FLT1 polypeptide in the suspension is 2-2.5 mg/mL;
and/or in the step (2), the tetrachloroauric acid solution is a tetrachloroauric acid aqueous solution;
and/or in the step (2), the reaction is carried out for 5-10 minutes at 20-25 ℃, and then the temperature is raised to 70-80 ℃ for reaction for 8-12 hours;
and/or, in the step (3), the ultrafiltration uses an ultrafiltration tube with 10 KD;
preferably, the first and second electrodes are formed of a metal,
in the step (2), the concentration of the tetrachloroauric acid solution is 100 mmol/L.
The invention also provides a method for preparing the gold cluster, which comprises the following steps:
(1) preparing Anti-FLT1 polypeptide into suspension by using ultrapure water;
(2) adding a tetrachloroaururic acid solution into the suspension, mixing and reacting to obtain a reaction solution;
(3) and (5) ultrafiltering the reaction solution to obtain the final product.
Further, the air conditioner is provided with a fan,
in the step (1), the concentration of the Anti-FLT1 polypeptide in the suspension is 2-2.5 mg/mL;
and/or in the step (2), the tetrachloroauric acid solution is a tetrachloroauric acid aqueous solution;
and/or in the step (2), the reaction is carried out for 5-10 minutes at 20-25 ℃, and then the temperature is raised to 70-80 ℃ for reaction for 8-12 hours;
and/or, in the step (3), the ultrafiltration uses an ultrafiltration tube with 10 KD;
preferably, the first and second electrodes are formed of a metal,
in the step (2), the concentration of the tetrachloroauric acid solution is 100 mmol/L.
The invention also provides the application of the gold cluster in preparing a photosensitizer;
preferably, the photosensitizer is a photosensitizer used in photodynamic therapy.
The invention also provides the application of the gold cluster in preparing a medicament for inhibiting angiogenesis;
preferably, the medicament is useful for treating tumors.
Compared with the prior art, the Anti-FLT1 polypeptide-mediated synthesized AF @ AuNCs related by the invention has the advantages of simple preparation method, strong operability, low cost, good biocompatibility, low toxicity, good stability and good fluorescence, and can be used for inhibiting the formation of vascular lumens. And enhances the PDT effect. Provides a new idea and method for PDT research of AuNCs.
The Anti-FLT1 polypeptide-mediated gold cluster is synthesized by a simple one-pot method, and the gold cluster has an excellent Anti-angiogenesis effect and an excellent photodynamic treatment effect; compared with the gold cluster synthesized by singly using the Anti-FLT1 polypeptide or not using the Anti-FLT1 polypeptide as a medium, the gold cluster synthesized by the Anti-FLT1 polypeptide as a medium has a synergistic effect. The gold cluster synthesized by the Anti-FLT1 polypeptide mediated synthesis overcomes the defects of poor Anti-angiogenesis and photodynamic treatment effects of the gold cluster in the prior art, and has good application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a transmission electron microscope image of gold clusters synthesized by Anti-FLT1 polypeptide mediation.
FIG. 2 is a graph showing the distribution of hydrated particle sizes of gold clusters synthesized by Anti-FLT1 polypeptide mediated synthesis.
FIG. 3 is a fluorescence spectrum of gold clusters synthesized by Anti-FLT1 polypeptide mediation.
FIG. 4 shows the result of Anti-FLT1 polypeptide-mediated synthesis of gold clusters inhibiting the lumen formation of blood vessels.
FIG. 5 shows the results of Anti-FLT1 polypeptide-mediated synthesis of gold clusters inhibiting vascular endothelial cell migration and invasion: a is a microscopic image of Anti-FLT1 polypeptide mediated synthesized gold cluster inhibiting vascular endothelial cell migration; b is a statistical analysis result of inhibiting the migration of vascular endothelial cells by gold clusters synthesized by Anti-FLT1 polypeptide mediation; c is a microscopic image of inhibiting vascular endothelial cell invasion by gold clusters synthesized by Anti-FLT1 polypeptide mediated; d is the result of statistical analysis that Anti-FLT1 polypeptide-mediated synthesized gold clusters inhibit vascular endothelial cell invasion.
FIG. 6 shows the result of inhibiting chick chorioallantoic membrane angiogenesis by gold clusters synthesized by Anti-FLT1 polypeptide of the present invention: a is a microscope image; and B is a statistical analysis result.
FIG. 7 shows the photodynamic action of gold clusters in Anti-FLT1 polypeptide-mediated synthesis.
FIG. 8 shows the result of staining with gold clusters AO/PI by Anti-FLT1 polypeptide-mediated synthesis.
Detailed Description
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.
In the present invention, the amino acid sequence of the Anti-FLT1 polypeptide is Cys-Gly-Asn-Gln-Trp-Phe-Ile, abbreviated as CGNQWFI (SEQ ID NO. 1); the structural formula of the Anti-FLT1 polypeptide is shown as the formula I:
example 1 preparation of gold clusters for Anti-FLT1 polypeptide mediated synthesis according to the invention
The specific process steps are as follows:
(1) soaking the glass bottle in aqua regia, cleaning, and drying for later use.
(2) 15mL of ultrapure water was taken and put into a treated glass bottle, 34.68mg of Anti-FLT1 polypeptide was weighed and dissolved in the ultrapure water, and the solution was vigorously stirred to be uniformly distributed. Slowly add 100. mu.L, 100mmol/L HAuCl4The solution is put into a heat collection type constant temperature magnetic stirrer with the temperature of 25 ℃ after being evenly mixed for heating and stirring for 5 minutes, then the temperature is raised to 70 ℃, the heating and stirring are continuously carried out for 8 hours, and observation can be carried outThe solution color changed from clear to cloudy white to a glass bottle. ICP-MS determination of Au in solution+The final concentration of (A) was 14.71 mg/L.
(3) After the reaction is finished, the sample is moved to a 10KD ultrafiltration tube and centrifuged at 10000rpm for 20 minutes to obtain the final product Anti-FLT1 polypeptide-mediated synthetic gold cluster (AF @ AuNCs).
Example 2 preparation of gold clusters for Anti-FLT1 polypeptide mediated synthesis according to the invention
Using the preparation method described in example 1, Anti-FLT1 and HAuCl4In a molar mass ratio of 0.5: 1 to 4: 1 (Anti-FLT1 and HAuCl in example 14The molar mass ratio of (A) to (B) is 4: 1) the Anti-FLT1 polypeptide-mediated synthetic gold cluster (AF @ AuNCs) can be prepared within the pH value range of 1-13 and the synthetic time of 8-12 hours.
The advantageous effects of the present invention are demonstrated by specific test examples below.
Experimental example 1 characterization of physicochemical Properties of gold clusters synthesized by Anti-FLT1 polypeptide mediation
1. Test method
AF @ AuNCs prepared in example 1 was diluted 1000 times with ultrapure water, and after 30 minutes of ultrasonic oscillation at room temperature, the solution was put into a cuvette, and the particle size distribution of AF @ AuNCs was measured on a dynamic light scattering instrument (SZ-100Z, Horiba, Japan).
AF @ AuNCs prepared in example 1 was diluted 1000 times with ultrapure water at room temperature, 10. mu.L of the diluted solution was dropped on a clean copper mesh, and after drying by ventilation at room temperature, it was observed by using TEM (JEM-2100F).
1.5mL of AF @ AuNCs prepared in example 1 was added to a rectangular square transparent quartz cuvette, and the emission wavelength of the sample was measured first, and then the appropriate excitation wavelength was measured according to the emission wavelength. A 3-dimensional fluorescence spectrum is obtained.
2. Test results
As can be seen from the image of the particle size distribution in FIG. 1 and the image of the particle size distribution in FIG. 2, the particle size range of AF @ AuNCs prepared by the method is about 2 nm. FIG. 3 shows the three-dimensional UV spectrophotometer detection result of the gold cluster synthesized by Anti-FLT1 polypeptide mediation, as shown in FIG. 3: in 3D fluorescence spectra, the results show that the synthesized AF @ AuNCs has a red emission band centered at about 620nm and a large Stokes shift. An obvious absorption peak is near 330nm, and strong fluorescence emission is realized in the region of 575-635nm, and the emission peak is 605 nm. The excitation and emission wavelengths of the material are determined.
Test example 2 cellular test for inhibiting angiogenesis of Anti-FLT1 polypeptide-mediated synthetic gold clusters
First, tube cavity formation test
1. Test method
To verify the inhibitory effect of AF @ AuNCs prepared in example 1 on angiogenesis, a luminal formation test was performed, the specific test method being as follows:
(1) melting glue: the Matrigel was thawed by placing it in a refrigerator at 4 ℃ overnight. Putting the 96-well plate and the yellow gunpoint in a refrigerator at 4 ℃ for precooling in advance.
(2) Spreading glue: in a 96-well plate, 50. mu.L of the matrix gel was slowly added to each well to avoid generation of air bubbles as much as possible, and the plate was allowed to stand on ice for 10 minutes. Each set of 3 secondary orifices. Then incubated at 37 ℃ for 45 minutes to form a gel.
(3) Plate preparation: taking out the trypsinized HUVECs from the T25 culture flask, and preparing HUVECs cell suspension by using a serum-free culture medium. According to 3.0X 104One/well was seeded on the coagulated matrigel.
(4) Adding medicine: the same dose of AF (Anti-FLT1 polypeptide), AF @ AuNCs, AuNCs and negative control PBS (drug concentrations: 25. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL) were added, respectively. Culturing in a cell culture box for 6 h.
(5) Drawing: the lumen formation images were taken with an inverted fluorescence microscope and 3 fields were collected per well.
(6) And (3) analysis: the indicators of Angiogenesis such as the length of the lumen, the number of nodes, and the mesh were automatically analyzed by using ImageJ software Angiogenesis plug-ins.
AuNCs are traditional commonly-used gold clusters and Glutathione (GSH) -protected gold clusters, and the specific preparation method is as follows:
(1) cleaning a container: soaking the glass bottle in aqua regia, cleaning, and drying for later use.
(2) Preparation of GSH suspension: 3.07mg of L-glutathione was weighed into a glass bottle containing 15mL of ultrapure water, and dissolved by rapid stirring.
(3) Adding HAuCl4: taking HAuCl4The solution (100mM, 0.1mL) was added dropwise to the L-glutathione solution.
(4) Preparing a mixed solution: after being mixed evenly, the mixture is placed on a heat collection type constant temperature magnetic stirrer to be stirred vigorously for 5min (25 ℃).
(5) Synthesizing: and then heating to 70 ℃ for reaction for 12 hours to obtain reaction liquid, transferring the reaction liquid into a 10KD ultrafiltration tube, and centrifuging at 10000rpm for 20 minutes to obtain the AuNCs.
(6) Synthesis, identification and storage: and (3) placing the AuNCs into an ultraviolet illuminator with the wavelength of 365 nm to detect whether the product generates fluorescence or not, cooling the AuNCs which are successfully prepared to room temperature, and then placing the AuNCs into a refrigerator with the temperature of 4 ℃ for long-term storage.
2. Test results
The ability of Anti-FLT1 polypeptide-mediated synthetic gold clusters to inhibit vascular lumen is shown in FIG. 4. FIG. 4 is a representative picture of the effect of different anti-angiogenic drugs AF, AF @ AuNCs, AuNCs at concentrations of 25. mu.g/mL, 100. mu.g/mL and 200. mu.g/mL on the luminal formation of HUVECs. The images show that all three groups of drugs have the ability to inhibit lumen formation, which is affected by changes in drug concentration. At concentrations of 100. mu.g/m and 200. mu.g/mL, the anti-angiogenic effect of AF @ AuNCs was stronger than that of the other groups, and it was seen in the pictures that HUVECs cells were dispersed throughout the visual field with almost no apparent luminal structure formation. Among them, 100. mu.g/mL of AF @ AuNCs had the best effect on tube formation. The Anti-angiogenesis gold cluster synthesized by the Anti-FLT1 polypeptide mediated synthesis is obviously superior to that of the Anti-FLT1 polypeptide and the traditional gold cluster which are singly used under the same dosage, and the synergistic effect is achieved.
Second, scratch test and invasion test
1. Test method
And (3) scratch test:
the scratch test is adopted to research the influence of AF @ AuNCs prepared in example 1 on the migration of HUVECs, and the specific test method is as follows:
(1) plate preparation: HUVECs were incubated in 6-well plates for 24 hours to achieve greater than 90% confluence.
(2) Scratching: the cells were stably drawn with a yellow tip along the long axis in the horizontal and vertical directions of the 6-well plate.
(3) Washing: washed three times with PBS to remove the lesions and scraped cells.
(4) Adding medicine: serum-free culture medium is added to culture the human umbilical vein endothelial cells, and 100 mug/mL of AF, AF @ AuNCs and AuNCs are respectively added into each experimental hole; no drug was added after scratching as a control.
(5) And (3) observation: pictures at the scratch were recorded by taking pictures under a microscope at time points of 0 hour and 24 hours.
(6) Calculating the healing rate: analysis was performed with ImageJ software. Percent healing ═ (initial area-area at a point in time)/initial area.
Invasion test:
an invasion test is carried out by adopting a transwell chamber to detect the influence of AF @ AuNCs prepared in example 1 on the longitudinal migration capacity of HUVECs, and the specific test method is as follows:
(1) starved cells: cells were starved for 12 hours prior to plating.
(2) Plate preparation: the concentration of the digested HUVECs cell suspension was adjusted to 5.0X 105Perml, 200. mu.L of serum-free cell suspension was added to the upper chamber of the transwell.
(3) Adding medicine: in the upper chamber of the transwell, 100. mu.g/mL of AF, AF @ AuNCs and AuNCs were added, respectively, and in the lower chamber 750. mu.L of DMEM containing 10% FBS (3 replicate wells per group) was added, and the well plate was placed in an incubator for 12 hours. No drug was added as a control group.
(4) Fixing: after the culture medium in the upper and lower chambers was aspirated, the cells were washed 3 times with PBS and fixed with 10% paraformaldehyde.
(5) Dyeing: staining was performed with the formulated 1:1000 DAPI dye for 5 minutes.
(6) And (4) observation: images were taken under an inverted fluorescence microscope, and 5 fields were taken per well plate.
(7) And (3) analysis: ImageJ software analyzed the fluorescence area under each field.
The method for preparing AuNCs was the same as in test example 2, "one, lumen formation test".
2. Test results
FIG. 5 shows the results of the ability of Anti-FLT1 polypeptide of the present invention to mediate the synthesis of gold clusters inhibiting the migration of vascular endothelial cells. As shown in FIG. 5A, the gap at the scratch was still wider and did not differ much from the initial gap area in the presence of 100 μ g/mL AF and AF @ AuNCs at the 24 hour observation time point. Indicating that the scratch healed very little. While the gaps of other groups become narrower, the area is reduced, and the cell crawling coverage is more. Indicating that the scratch healed more. This figure demonstrates that AF and AF @ AuNCs can significantly inhibit HUVECs migration. The statistical analysis of FIG. 5B also shows this trend: HUVEC had a healing area that was only one-third of that of the control and AuNCs groups (P <0.01) for the AF and AF @ AuNCs treatment groups using 100. mu.g/mL. The effect of AF @ AuNCs on inhibiting the migration of HUVECs is obviously better than that of AF alone. The Anti-FLT1 polypeptide-mediated synthetic gold cluster obtained after AF and AuNCs are compounded can improve the capability of inhibiting HUVECs migration, and plays a synergistic effect.
The blue fluorescence area in the invasive results reflects the cells that passed through the transwell membrane. The nuclei of these cells were stained blue by DAPI. The larger the area of the blue region, the greater the number of penetrating cells. FIG. 5C shows that the number of migrating cells (blue fluorescent spots) was less for the AF and AF @ AuNCs treated groups than for the remaining groups. In FIG. 5D, the fluorescence area of 100. mu.g/mL AF and AF @ AuNCs was about half that of the control and AuNCs groups (P < 0.01). The data indicate that AF and AF @ AuNCs significantly inhibit the invasion of HUVECs.
The test results show that the gold cluster synthesized by the Anti-FLT1 polypeptide mediated synthesis can effectively inhibit HUVECs cell migration and invasion and play a synergistic effect.
Test example 3 animal test for inhibiting angiogenesis of gold clusters synthesized by Anti-FLT1 polypeptide of the present invention
1. Test method
The inhibition effect of AF @ AuNCs prepared in example 1 on angiogenesis is studied by adopting chick embryo chorioallantoic membrane (CAM) experiment, and the specific test method is as follows:
(1) chicken embryo incubation: after sterilization of fertilized eggs, the fertilized eggs were incubated at 37 ℃ in an incubator with a relative humidity of 60% for 7 days. Then divided into 4 groups of 4 replicates each.
(2) Marking a gas chamber: and illuminating the blunt end of the egg by using an egg illuminator to find the range of the air chamber. The air cell range was marked with a black pen.
(3) Manufacturing an artificial air chamber: making a window (0.3-0.4 cm) on the air bag with a drill2) The ear washing bulb blows off the fallen eggshell fragments. The air cell membrane was torn open, exposing the underlying CAM, where vascularity was visible on the CAM.
(4) Adding medicine: a gelatin sponge 5 mm in diameter was placed over the CAM and 0.1mL of 100. mu.g/mL AF, AF @ AuNCs, AuNCs and 100. mu.L PBS (control) were added.
(5) And (3) sealing an artificial air chamber: and sealing the drilling window by using a sealing film to form an artificial air chamber.
(6) The eggs were incubated for 72 hours.
(7) And (3) observation: the neovascular area around the gelatin sponge was observed with a stereomicroscope and photographed.
(8) And (3) data analysis: ImageJ software analyzed the ratio of the area of the vessels to the total area for each field of view.
The method for preparing AuNCs was the same as in test example 2, "one, lumen formation test".
2. Test results
The result of inhibiting the angiogenesis of chick chorioallantoic membrane by the gold clusters synthesized by the Anti-FLT1 polypeptide mediated is shown in FIG. 6. The anti-angiogenic effect of AF @ AuNCs was verified by classical CAM experiments. As shown in fig. 6A, the number of branched blood vessels was significantly reduced in the AF, AF @ AuNCs, and AuNCs-treated groups compared to the control group. Importantly, AF @ AuNCs inhibited angiogenesis more significantly in fertilized eggs than pure AF and AuNCs. Meanwhile, quantitative analysis of angiogenesis regions (fig. 6B) was consistent with images captured by a stereomicroscope, indicating that AF @ AuNCs has stronger anti-angiogenic activity in vivo than AF and AuNCs, and exerts a synergistic effect.
Experimental example 4 photodynamic effects of gold clusters synthesized by Anti-FLT1 polypeptide of the present invention
1. Test method
CCK8 is adopted to detect the photodynamic effect of AF @ AuNCs prepared in example 1 with different concentrations under the illumination condition compared with AuNCs, and the specific test method is as follows:
(1) plate preparation: the concentration of the digested CAL-27 cell suspension was adjusted to 1.0X 104A hole. Plating onto 96-well plates. Incubate for 24 hours.
(2) Starved cells: cells were starved for 12 hours prior to plating.
(3) Adding medicine: add 25. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL and 500. mu.g/mL AF, AF @ AuNCs or AuNCs, respectively, to the well plate. The well plates were placed in an incubator for 12 hours. No drug composition control was added.
(4) Illumination: and illuminating a 96-well plate needing illumination by using an LED lamp with the excitation wavelength of 360 nanometers. 0.1w,10 minutes. Incubate for 24 hours.
(5) CCK-8 color development: the cells were washed once with PBS and then reacted with the viable cells for 1 hour with CCK-8 solution. At this time, the color of the control group changed to orange of medium brightness.
(6) And (3) detection: and taking out the 96-well plate, and reading the absorbance value of each well at 450 nm displayed on the microplate reader.
(7) And (3) calculating toxicity: higher cell viability indicates less cytotoxicity. Survival (%) × 100% (medicated-blank)/(control-blank)). Cellular activity of CAL-27 was evaluated.
The AO/PI staining is adopted to detect the condition of living and dead cells, and the specific test method is as follows:
(1) plate preparation: adjusting the concentration of the digested CAL-27 cell suspension to 5.0X 104A hole. Plating onto 12-well plates. Incubate for 24 hours.
(2) Starved cells: cells were starved for 12 hours prior to plating.
(3) Adding medicine: add 100. mu.g/mL AF @ AuNCs or AuNCs, respectively, to the well plate. The well plates were placed in an incubator for 12 hours. No drug composition control was added.
(4) Illumination: and illuminating a 96-well plate needing illumination by using an LED lamp with the excitation wavelength of 360 nanometers. 0.1w,10 minutes. Incubate for 24 hours.
(5) AO/PI staining: the medium was discarded, the cells were washed 1 time with PBS, and the cells were then incubated with AO/PI stain (670. mu. mol/L AO, 750. mu. mol/L PI) for 20 minutes in the absence of light.
(6) Cells were washed 3 times with PBS and observed under an inverted fluorescence microscope.
The method for preparing AuNCs was the same as in test example 2, "one, lumen formation test".
2. Test results
FIG. 7 results: under different concentrations and non-lighting conditions, the three materials (AF, AF @ AuNCs and AuNCs) have no reduction of cell activity, which indicates that the three materials have no cytotoxicity. Under the illumination condition, compared with the AF group and the AuNCs group, the AF @ AuNCs group has lower cell activity, which indicates that the AF @ AuNCs kills more CAL-27 cells under the illumination condition. Especially at 25. mu.g/mL and 100. mu.g/mL, the cell activity was the lowest, indicating that the photodynamic effect was the best. The test result shows that compared with the single use of Anti-FLT1 polypeptide or traditional gold clusters (AuNCs), the gold clusters synthesized by Anti-FLT1 polypeptide mediation have the advantages that the photodynamic effect is obviously improved, and the synergistic effect is achieved.
FIG. 8 results: dye AO penetrates the membrane and stains the DNA and RNA of the cell as green fluorescence. Whereas PI stains only dead/apoptotic, staining red. FIG. 8 shows that the AF @ AuNCs group has more red fluorescence than the AuNCs group under the condition of 100 mu g/mL and illumination, which indicates that the group has more apoptotic and dead cells and stronger photodynamic effect.
The above test results demonstrate that: compared with the non-mediated synthesis of gold clusters (AuNCs) under the illumination condition, the Anti-FLT1 polypeptide-mediated synthesis of gold clusters enhances the photodynamic effect and plays a role in synergy.
According to the test results of the test examples, the Anti-angiogenesis activity of the gold cluster synthesized by utilizing the Anti-FLT1 polypeptide in a mediated manner is remarkably improved, the photodynamic therapy (PDT) effect is remarkably improved, and the gold cluster synthesized by utilizing the Anti-FLT1 polypeptide in a mediated manner has a synergistic effect compared with the gold cluster synthesized by utilizing the Anti-FLT1 polypeptide alone or not utilizing the Anti-FLT1 polypeptide in a mediated manner.
In conclusion, the Anti-FLT1 polypeptide-mediated gold cluster is synthesized by a simple one-pot method, and has an excellent Anti-angiogenesis effect and an excellent photodynamic treatment effect; compared with the gold cluster synthesized by singly using the Anti-FLT1 polypeptide or not using the Anti-FLT1 polypeptide as a medium, the gold cluster synthesized by the Anti-FLT1 polypeptide as a medium has a synergistic effect. The gold cluster synthesized by the Anti-FLT1 polypeptide mediated synthesis overcomes the defects of poor Anti-angiogenesis and photodynamic treatment effects of the gold cluster in the prior art, and has good application prospect.
SEQUENCE LISTING
<110> Sichuan university
<120> Anti-FLT1 polypeptide mediated synthetic gold cluster and preparation method and application thereof
<130> GYKH1118-2020P0112147CC
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 7
<212> PRT
<213> Artificial sequence
<400> 1
Cys Gly Asn Gln Trp Phe Ile
1 5
Claims (12)
1. A gold cluster, characterized by: the gold cluster is prepared by taking gold atoms as a metal core, Anti-FLT1 polypeptide as a ligand shell and a gold cluster with the particle size of 0.5-5 nm, and tetrachloroauric acid and Anti-FLT1 polypeptide as raw materials, wherein the molar mass ratio of the tetrachloroauric acid to the Anti-FLT1 polypeptide is 1 (0.5-4), and the preparation method comprises the following steps:
(1) preparing Anti-FLT1 polypeptide into suspension by using ultrapure water;
(2) adding a tetrachloroaururic acid solution into the suspension, mixing and reacting to obtain a reaction solution;
(3) ultrafiltering the reaction solution to obtain the reaction solution;
the amino acid sequence of the Anti-FLT1 polypeptide is CGNQWFI, and the structure is shown as the formula I:
formula I.
2. The gold cluster according to claim 1, wherein: the grain diameter of the gold cluster is 2 nm.
3. The gold cluster according to claim 1, wherein: the molar mass ratio of the tetrachloroauric acid to the Anti-FLT1 polypeptide is 1: 4.
4. The gold cluster according to claim 1, wherein in the step (1) of the preparation method, the concentration of the Anti-FLT1 polypeptide in the suspension is 2-2.5 mg/mL;
and/or in the step (2), the tetrachloroauric acid solution is a tetrachloroauric acid aqueous solution;
and/or in the step (2), the reaction is carried out for 5-10 minutes at 20-25 ℃, and then the temperature is raised to 70-80 ℃ for reaction for 8-12 hours;
and/or, in the step (3), the ultrafiltration uses an ultrafiltration tube with 10 KD.
5. The gold cluster of claim 4, wherein: in the step (2), the concentration of the tetrachloroauric acid solution is 100 mmol/L.
6. A method for producing the gold cluster according to any one of claims 1 to 5, characterized by: it comprises the following steps:
(1) preparing Anti-FLT1 polypeptide into suspension by using ultrapure water;
(2) adding a tetrachloroaururic acid solution into the suspension, mixing and reacting to obtain a reaction solution;
(3) and (5) ultrafiltering the reaction solution to obtain the final product.
7. The method of claim 6, wherein: in the step (1), the concentration of the Anti-FLT1 polypeptide in the suspension is 2-2.5 mg/mL;
and/or in the step (2), the tetrachloroauric acid solution is a tetrachloroauric acid aqueous solution;
and/or in the step (2), the reaction is carried out for 5-10 minutes at 20-25 ℃, and then the temperature is raised to 70-80 ℃ for reaction for 8-12 hours;
and/or, in the step (3), the ultrafiltration uses an ultrafiltration tube with 10 KD.
8. The method of claim 7, wherein: in the step (2), the concentration of the tetrachloroauric acid solution is 100 mmol/L.
9. Use of the gold cluster according to any one of claims 1 to 5 for the preparation of a photosensitizer.
10. Use according to claim 9, characterized in that: the photosensitizer is a photosensitizer used in photodynamic therapy.
11. Use of the gold clusters of any one of claims 1 to 5 in the manufacture of a medicament for inhibiting angiogenesis.
12. Use according to claim 11, characterized in that: the medicine can be used for treating tumors.
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