Disclosure of Invention
The invention aims at providing an aggregation-induced emission nanoparticle modified montmorillonite composite, and a preparation method and application thereof, aiming at the defects of the prior art.
The invention relates to an aggregation-induced emission nanoparticle modified montmorillonite composite, which takes pharmaceutical grade calcium-based montmorillonite as a carrier and loads aggregation-induced emission nanoparticles, wherein the aggregation-induced emission nanoparticles have the structural formula:
Further, the mass ratio of the aggregation-induced emission nanoparticles to the calcium-based montmorillonite is (0.5-2.0): 100.
Further, pharmaceutical grade calcium-based montmorillonite is used as a carrier.
The preparation method of the aggregation-induced emission nanoparticle modified montmorillonite composite comprises the following steps:
S1, dispersing montmorillonite in deionized water, and carrying out ultrasonic configuration to obtain uniformly dispersed montmorillonite suspension;
s2, dissolving aggregation-induced emission nanoparticles by using DMSO (dimethyl sulfoxide), and preparing an aggregation-induced emission nanoparticle solution;
And S3, uniformly mixing the montmorillonite suspension with the aggregation-induced emission nanoparticle solution at a certain temperature and at a certain vibration speed, centrifuging the obtained material, and freeze-drying the precipitate to obtain the aggregation-induced emission nanoparticle modified montmorillonite composite MMT-TTPy.
Further, the concentration of the aggregation-induced emission nanoparticles is 0.01-10mg/mL.
Further, in the step S3, the mixture is uniformly mixed for 2 to 4 hours at a constant speed of between 30 and 40 ℃ and between 800 and 1200 rpm.
Further, in step S3, the mixture is centrifuged at 1300rpm to 1500rpm for 8 to 12 minutes.
Further, in step S3, the precipitate is freeze-dried for 20-28 hours.
The aggregation-induced emission nanoparticle modified montmorillonite composite can be applied to a hemostatic agent.
The preparation method disclosed by the invention has the advantages that the hemostatic effect of the montmorillonite composite hemostatic material can be improved by using a small amount of aggregation-induced emission nano particles loaded by the calcium-based montmorillonite, the prepared MMT-TTPy composite hemostatic material has no obvious cytotoxicity, no hemolysis phenomenon, good biocompatibility, high safety performance and good antibacterial effect, and the preparation method disclosed by the invention is simple in steps, easy to operate and beneficial to large-scale production.
The calcium-based montmorillonite with a lamellar structure is used for loading the aggregation-induced emission nano particles, the aggregation-induced emission nano particles with positive charges are loaded on the surface of the montmorillonite, the surface of the platy montmorillonite is negatively charged, the platy montmorillonite has abundant surface hydroxyl groups, the high-efficiency load of the functional nano particles is facilitated, the encapsulation rate of the aggregation-induced emission nano particles can reach 100%, the aggregation-induced emission nano particles have high biocompatibility, photodynamic therapy sterilization can be realized under the irradiation of white light (100 mW/cm2 and 30 min), the cell safety and the biocompatibility of mineral materials are improved on the premise that the hemostatic performance of the montmorillonite is ensured, and the antibacterial effect of the montmorillonite is improved, so that the calcium-based montmorillonite can be applied to the fields of hemostasis and wound healing, and hemostatic products with social and economic significance are developed.
Detailed Description
The following are specific embodiments of the present invention and the technical solutions of the present invention will be further described with reference to the accompanying drawings, but the present invention is not limited to these embodiments.
The pharmaceutical grade montmorillonite in the examples in this specification is pharmaceutical grade montmorillonite product of Shanghai Ala Biochemical technology Co., ltd, and as shown in FIG. 1, is identical to standard card PDF#13-0135, and has molecular formula Ca0.2(Al,Mg)2Si4O10(OH)2·4H2 O of calcium-based montmorillonite.
The aggregation-induced emission nanoparticles of the comparative example and the examples were prepared according to the structural formula designed by the inventor of the present application from the company of Carbonisatoic biotechnology, inc. of Guangzhou, and the structures are shown in FIGS. 2a, 2b and 2 c.
Preparation of montmorillonite suspension
Example 1:
1g of montmorillonite is dispersed in 10mL of deionized water, and ultrasonic treatment is carried out for 30min, thus obtaining montmorillonite suspension with the concentration of 100 mg/mL.
Examples 2 to 5
The preparation process of the aggregation-induced emission nanoparticle (TTPy structure) modified montmorillonite composite is shown in fig. 9.
Preparation of aggregation-induced emission nanoparticle suspensions
And dissolving 10mg of aggregation-induced emission nano particles (TTPy) with 1mL of DMSO to obtain 10mg/mL of aggregation-induced emission nano particle solution.
Example 2:
The preparation method of the aggregation-induced emission nanoparticle modified montmorillonite composite comprises the following steps of adding 160 mu L of deionized water into 800 mu L of montmorillonite suspension with the concentration of 100mg/mL for dispersion, and adding 40 mu L of aggregation-induced emission nanoparticle solution with the concentration of 10 mg/mL. And placing 1mL of the obtained mixed solution into a constant temperature mixer, uniformly mixing for 3 hours at a constant speed of 1000rpm at 37 ℃, centrifuging the obtained material for 10 minutes at 14000rpm, and freeze-drying the precipitate for 24 hours to obtain the aggregation-induced emission nanoparticle modified montmorillonite composite, wherein the label is 0.5% TTPy.
Example 3:
The preparation method of the aggregation-induced emission nanoparticle modified montmorillonite composite comprises the following steps of adding 560 mu L of deionized water into 400 mu L of montmorillonite suspension with the concentration of 100mg/mL for dispersion, and adding 40 mu L of aggregation-induced emission nanoparticle solution with the concentration of 10 mg/mL. And placing 1mL of the obtained mixed solution into a constant temperature mixer, uniformly mixing for 3 hours at a constant speed of 1000rpm at 37 ℃, centrifuging the obtained material for 10 minutes at 14000rpm, and freeze-drying the precipitate for 24 hours to obtain the aggregation-induced emission nanoparticle modified montmorillonite composite, wherein the label is 1.0% TTPy.
Example 4:
The preparation method of the aggregation-induced emission nanoparticle modified montmorillonite composite comprises the following steps of adding 693 mu L of deionized water into 267 mu L of montmorillonite suspension with the concentration of 100mg/mL for dispersion, and adding 40 mu L of aggregation-induced emission nanoparticle solution with the concentration of 10 mg/mL. And placing 1mL of the obtained mixed solution into a constant temperature mixer, uniformly mixing for 3 hours at a constant speed of 1000rpm at 37 ℃, centrifuging the obtained material for 10 minutes at 14000rpm, and freeze-drying the precipitate for 24 hours to obtain the aggregation-induced emission nanoparticle modified montmorillonite composite, wherein the label is 1.5% TTPy.
Example 5:
the preparation method of the aggregation-induced emission nanoparticle modified montmorillonite composite comprises the following steps of adding 760 mu L of deionized water into 200 mu L of montmorillonite suspension with the concentration of 100mg/mL for dispersion, and adding 40 mu L of aggregation-induced emission nanoparticle solution with the concentration of 10 mg/mL. And placing 1mL of the obtained mixed solution into a constant temperature mixer, uniformly mixing for 3 hours at a constant speed of 1000rpm at 37 ℃, centrifuging the obtained material for 10 minutes at 14000rpm, and freeze-drying the precipitate for 24 hours to obtain the aggregation-induced emission nanoparticle modified montmorillonite composite, wherein the label is 2.0% TTPy.
The prepared suspension and the centrifuged optical pictures of the embodiments 1-5 are shown in fig. 3, and the preparation method of the embodiments 1-5 is simple and easy to implement, the encapsulation efficiency of the aggregation-induced emission nanoparticles can reach 100%, and the utilization rate of the aggregation-induced emission nanoparticles is greatly improved. In addition, as is apparent from the structure of the aggregation-induced emission nanoparticle of fig. 2c, the aggregation-induced emission nanoparticle, as a cationic dye, can be bonded to the surface of montmorillonite through electrostatic interaction, and bonding occurs between the N+ group of the aggregation-induced emission nanoparticle and the si—o group of the surface of montmorillonite.
Preparation of aggregation-induced emission nanoparticle (structure of FIG. 2 a) modified montmorillonite composite
Comparative example 1:
Preparation of aggregation-induced emission nanoparticle (AIE 1) modified montmorillonite composite 50 μl of 0.5mg/mL montmorillonite suspension was dispersed by adding 900 μl of deionized water, and then 50 μl of 0.5mg/mL aggregation-induced emission nanoparticle solution was added. And placing 1mL of the obtained mixed solution into a constant temperature mixer, uniformly mixing for 30min at a constant speed of 1000rpm at 37 ℃, centrifuging the obtained material for 10min at 14000rpm, and freeze-drying the precipitate for 24h to obtain the aggregation-induced emission nanoparticle modified montmorillonite composite, wherein the aggregation-induced emission nanoparticle modified montmorillonite composite is marked as MMT-AIE1.
Preparation of aggregation-induced emission nanoparticle (structure of FIG. 2 b) modified montmorillonite composite
Comparative example 2:
Preparation of aggregation-induced emission nanoparticle (AIE 2) modified montmorillonite composite, adding 700 μl deionized water into 250 μl1.0mg/mL montmorillonite suspension for dispersion, and adding 50 μl1.0mg/mL aggregation-induced emission nanoparticle solution. And placing 1mL of the obtained mixed solution into a constant temperature mixer, uniformly mixing for 30min at a constant speed of 1000rpm at 37 ℃, centrifuging the obtained material for 10min at 14000rpm, and freeze-drying the precipitate for 24h to obtain the aggregation-induced emission nanoparticle modified montmorillonite composite, wherein the aggregation-induced emission nanoparticle modified montmorillonite composite is marked as MMT-AIE2.
In vitro antibacterial experiments:
The antibacterial activity of the samples was evaluated using staphylococcus aureus (ATCC 25923) as a gram positive model. The individual staphylococcus aureus strains were dispersed in 5mL of Luria-Bertani liquid medium and shaken at 37 ℃ for 24 hours to give an initial bacterial concentration of 2 x 109 CFU/mL. Subsequently, the bacterial suspension was serially diluted 106 times with PBS. Then, 100. Mu.L of the diluted bacterial suspension was mixed with 100. Mu.L of the material solution to obtain 500. Mu.g/mL (based on the concentration of montmorillonite) of the material solution. The mixed bacterial sample was incubated in the dark for 5min and then irradiated under 100mw/cm2 white light for 30min. The concentrations of comparative example 1 and comparative example 2 were 2.5. Mu.g/mL and 10. Mu.g/mL (based on the AIE1 and AIE2 concentrations, respectively), the mixed bacterial samples were incubated in the dark for 5min and then irradiated with 100mw/cm2 of white light for 15min. The bacterial suspension was then serially diluted 10-fold with PBS and 100uL of the diluted bacteria were plated onto corresponding solid agar plates and incubated at 37℃for 14-16h. And evaluating the antibacterial activity of the material on bacteria by taking the bacterial survival rate as an index.
Bacterial viability = average number of colonies in sample/average number of colonies in control group x 100%
The bacteriostatic effects of examples 1-5 and comparative examples 1,2 on Staphylococcus aureus are shown in FIGS. 4a, 4b, 4c and 4d, respectively. As can be seen from the results of the bacterial plaque of FIG. 4a, the antibacterial effect of AIE1 and MMT-AIE1 was not significant, and as can be seen from the results of the bacterial plaque of FIG. 4b, AIE2 had a significant antibacterial effect, but the effect of MMT-AIE2 was still not significant. From the results of the bacterial coating in fig. 4c and d, it can be seen that MMT-TTPy has a remarkable antibacterial effect, and the result of the bacterial survival rate can be intuitively seen that MMT has a certain antibacterial effect under illumination, and the bacterial survival rate is 79.46%. After compounding TTPy, the antibacterial effect of MMT-TTPy is significantly improved, wherein the corresponding bacterial survival rates of the materials corresponding to example 4 and example 5, 1.5% TTPy and 2.0% TTPy are respectively 10.64% and 3.73%. This result demonstrates that finding aggregation-induced emission nanoparticles (TTPy structures) suitable for complexing with montmorillonite is critical to improving the antibacterial properties of montmorillonite. Furthermore, excellent antibacterial performance can be achieved at low TTPy compound ratio (1.5%) and low concentration dose.
In vitro reactive oxygen species production experiment:
The samples were tested for ROS formation under 100mW/cm2 white light using the commonly used Reactive Oxygen Species (ROS) indicator 2, 7-dichlorofluorescein diacetate (DCFH-DA). In this experiment, 0.5mL of ethanol-dissolved DCFH-DA (1X 10-3mol·L-1) was added to 2mL of 1X 10-2mol·L-1 NaOH and stirred at room temperature for 30 minutes. The hydrolysate was then neutralized with 10ml of 1×pbs and stored in the dark. At this time, DCFH-DA was hydrolyzed to 2, 7-Dichlorofluorescein (DCFH). ROS indicator (4X 10-5 mol.L-1) in PBS was then further diluted to 5X 10-6mol·L-1 in MMT-TTPy composite sample solution (500 μg/mL) and the fluorescence spectrum of the solution at excitation wavelength 488nm and emission wavelength 490-700 nm was measured immediately after white light irradiation.
The linear plot of relative fluorescence intensity over time for examples 1-5 is shown in FIG. 5, and it can be seen from FIG. 5 that MMT can produce very small amounts of active oxygen under visible light, whereas the active oxygen generating capacity of the MMT-TTPy complex increases with increasing TTPy content. This suggests that the antibacterial mechanism of TTPy-MMT primarily generates reactive oxygen species by irradiation, thereby killing bacteria.
Cytotoxicity test
The test uses Cell Counting Kit-8 (CCK-8) method and uses human immortalized fiber cells (BJ cells) as the research object to analyze the cytotoxicity of the sample. Complete medium for culturing BJ cells was prepared with RPMI-1640 minimal medium and 1% diabody and 10% fetal bovine serum. Taking normally frozen BJ cells, shaking continuously in a 37 ℃ water bath environment until thawing, sucking 1mL of BJ cell frozen stock solution, adding into a 15mL centrifuge tube poured with 10mL of complete culture medium, shaking and mixing uniformly, centrifuging at 1000rpm for 5min, removing supernatant, re-suspending cells with the complete culture medium, and transferring the cell suspension into a culture dish. After the cells were cultured until the adherent growth state was good, digestion was performed with pancreatin to resuspend the cells out of the adherent state. The density of the cell suspension is regulated, cells are inoculated into a 96-well plate at the cell density of 1X 104 cells per well, the cells are incubated under the cell incubator environment of 37 ℃ and 5% CO2 until the adherence production state is good, the original culture solution is discarded, 100 mu L of fresh complete culture medium is added into a control well, and 100 mu L of culture solution containing samples with different concentrations is added into a test well. After further culturing for 24 hours, cytotoxicity was measured by adding complete medium containing 10% CCK-8 reagent to each well, culturing was continued for 1 hour, absorbance (OD) value at 450nm of the culture solution was measured by an enzyme-labeled instrument, and cell viability was calculated by measuring absorbance using wells without added sample material as a blank control group. Cell viability was calculated as follows:
cell viability = (OD experimental well-OD blank well)/(OD control well-OD blank well) ×100%
Toxicity evaluation was performed on examples 1 to 5 by setting concentration gradients of 2.5, 5, 10, 20, 40. Mu.g/mL, and the test results of the experiment are shown in FIG. 6. In the cytotoxicity class, cell viability was assigned to achieve a primary biosafety level when greater than 75%. The cell viability of the five samples of examples 1-5 was higher than 75% over the experimental concentration range, indicating good biocompatibility of both MMT and MMT-TTPy complexes. In addition, compared with MMT, the cell survival rate of the MMT-TTPy compound is obviously improved, which proves that TTPy is combined on the MMT surface, and the biocompatibility of the MMT can be improved.
Hemolysis experiment:
Preparation of 2% erythrocyte suspension 1mL of fresh anticoagulated rabbit blood was centrifuged at 2500rpm for 5min, the supernatant was removed, washed 3 times with Phosphate Buffer (PBS), 500. Mu.L of the washed puree was taken into a 50mL centrifuge tube, and PBS was added to 50mL.
Hemolysis experiments example 1-5 and PBS prepared material solutions with concentrations of 2.5, 5, 10, 20, 40. Mu.g/mL, respectively. 500. Mu.L of each concentration solution was mixed with 500. Mu.L of the prepared 2% erythrocyte suspension. A positive control group of 500. Mu.L of deionized water was mixed with 500. Mu.L of 2% erythrocyte suspension, a negative control group of 500. Mu.L of PBS solution was mixed with 500. Mu.L of 2% erythrocyte suspension, 3 tubes of each group were parallel, and the samples were incubated in a 37℃water bath for 1 hour, centrifuged at 2500rpm, and the supernatant was collected and the OD value thereof was measured by an enzyme-labeled instrument (540 nm). The lower the hemolysis, the higher the biocompatibility. The rate of hemolysis was less than 5%, and it was considered that hemolysis did not occur.
Hemolysis ratio (%) = = (OD experimental group-OD negative group)/(OD positive group-OD negative group) ×100%
The hemolysis results of examples 1-5 are shown in FIG. 7, and it can be seen from FIG. 7 that the MMT-TTPy complex has a lower hemolysis rate than MMT, which indicates that TTPy is combined on the MMT surface, so that the biocompatibility of MMT can be improved.
In vitro bleeding time assay:
8mg of MMT and MMT-TTPy composite hemostatic material are weighed into 2mL centrifuge tubes, placed in a 37 ℃ water bath environment for preheating for 3 minutes, 200 mu L of New Zealand white rabbit anticoagulated whole blood is dripped into sample powder at the bottom of the tubes, and then 2.5 mu L of 0.2mol/L CaCl2 solution is dripped into a mixed system rapidly to calcifie blood for triggering coagulation. The mixed system is rapidly placed into a 37 ℃ water bath environment for culture, the centrifuge tube is shaken every 10 seconds, the flowing condition of blood in the tube is observed until the blood is coagulated, and the hemostatic time is recorded. 3 replicates were run for each material.
TABLE 1 hemorrhage time of MMT and MMT-TTPy composite hemostatic Material
As can be seen from fig. 8 and table 1, the MMT has a better hemostatic effect, and after TTPy is compounded, the hemostatic speed can be effectively improved. This is probably due to the fact that the positively charged TTPy has weak interaction with Ca2+ adsorbed on the surface of montmorillonite after complexing with MMT, which is beneficial to the stabilization of Ca2+, and Ca2+ can activate coagulation factors and promote coagulation cascade reaction, thereby effectively improving the hemostatic efficacy of montmorillonite.
The above is not relevant and is applicable to the prior art.
While certain specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the foregoing examples are provided for the purpose of illustration only and are not intended to limit the scope of the invention, and that various modifications or additions and substitutions to the described specific embodiments may be made by those skilled in the art without departing from the scope of the invention or exceeding the scope of the invention as defined in the accompanying claims. It should be understood by those skilled in the art that any modification, equivalent substitution, improvement, etc. made to the above embodiments according to the technical substance of the present invention should be included in the scope of protection of the present invention.