CN111714636B - Photodynamic and photothermal synergistic sterilized flaky manganese tetraoxide nano material and preparation method thereof - Google Patents
Photodynamic and photothermal synergistic sterilized flaky manganese tetraoxide nano material and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicine, and particularly discloses a photodynamic and photothermal synergistic bactericidal flaky manganese tetraoxide nano material and a preparation method thereof. The method comprises the following steps: adding hydrazine hydrate into KMnO 4 Preparing a precursor solution in the aqueous solution, heating the precursor solution in an autoclave for reaction, cooling the precursor solution to room temperature after the reaction is finished, collecting milky precipitate, centrifugally washing the milky precipitate, and drying the milky precipitate to obtain a brown product; adding brown product, indocyanine green and water into a lightproof reaction bottle for reaction, centrifuging after the reaction is finished, removing supernatant, washing and centrifuging the precipitate to obtain a flaky trimanganese tetroxide nano material ICG@Mn 3 O 4 . ICG@Mn prepared by the method 3 O 4 Has stable photo-thermal performance and higher photo-dynamic activity, and the photo-thermal conversion efficiency is up to 67.5 percent and is 0.33W cm ‑2 The high temperature of 50 ℃ and a large amount of Reactive Oxygen Species (ROS) for killing bacteria can be generated under the irradiation of 808nm laser, the hemolysis rate and the coagulation rate are low, the cell survival rate is high, and the photodynamic/photothermal combined sterilization which can be activated by a low-power single laser is realized.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a sheet-shaped trimanganese tetroxide nano material with photodynamic and photothermal synergistic sterilization and a preparation method thereof.
Background
The highest heat-resistant temperature of most bacteria in organisms is 43 ℃, and when the temperature is higher than 43 ℃ and the organism tissues are not damaged, harmful bacteria can be killed, so that the photothermal treatment of the organisms is realized. Research has found that Mn 3 O 4 Has strong photo-thermal effect, and can replace antibiotics to treat the multi-drug resistant bacterial infection which is difficult to cure at present. Photothermal therapy (PTT) is a process of killing bacteria or cancer cells by converting light energy into heat energy using a material having high photo-thermal conversion rate under irradiation of an external light source. However, if only a single photothermal antimicrobial is relied upon, the required power density is greater, which is far greater than the U.S. safe use of laser marksQuasi (maximum power density allowed to continuously irradiate skin under 808nm laser according to ANSI ZI136.1-2014 is 0.33W/cm) 2 ). In addition, bacterial infection may recur due to uneven heat distribution of the target tissue, which may not completely destroy the bacteria. Thus, current PTT studies mostly employ combination with other therapies to improve efficacy, most commonly synergistic photodynamic therapy (PDT). However, due to the mismatch in the absorption of photosensitizers and photothermal agents, PDT/PTT combination therapies based on photothermal coupling agents typically require long-term irradiation of laser light of a relatively high power density or sequential irradiation of two lasers of different wavelengths, which not only complicates the treatment process but also causes damage to the irradiated tissue. Thus, there is a need for a safer strategy for achieving PDT/PTT co-therapy using low power, single wavelength near infrared lasers.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a sheet-shaped trimanganese tetroxide nanomaterial with synergistic sterilization by photodynamic and photothermal and a preparation method thereof, which are used for solving the problems of complex treatment process, overlarge laser power density, overlong irradiation time required for treatment, damage to irradiated tissues and the like in the PDT/PTT combined therapy in the prior art.
To achieve the above and other related objects, a first aspect of the present invention provides a method for preparing a sheet-shaped trimanganese tetroxide nanomaterial by photodynamic and photothermal co-sterilization, comprising the steps of:
(1) Hydrazine hydrate (N) 2 H 4 ·H 2 O) adding KMnO 4 Preparing a precursor solution in the aqueous solution, slowly heating the precursor solution to 160-200 ℃ in an autoclave, reacting, cooling to room temperature after the reaction is finished, collecting milky precipitate from the bottom of the autoclave, centrifuging, washing and drying to obtain a brown product;
(2) Adding the brown product obtained in the step (1), indocyanine green (ICG) and water into a lightproof reaction bottle for reaction, centrifuging after the reaction is finished, discarding the supernatant, washing and centrifuging the precipitate to obtain the flaky trimanganese tetroxide nano material ICG@Mn 3 O 4 。
Further, the method comprises the steps of,in the step (1), KMnO per millimole 4 The amount of hydrazine hydrate required is 1 to 4mL, preferably 2.5mL.
Further, in the step (1), hydrazine hydrate (N) 2 H 4 ·H 2 O) is 85% N 2 H 4 ·H 2 O hydrazine hydrate liquid.
Further, in the step (1), the reaction time is 8 to 16 hours, preferably 12 hours.
Further, in the step (1), the drying temperature is 80-100 ℃, preferably 80 ℃; the drying time is 6 to 12 hours, preferably 12 hours.
Further, in the step (1), hydrazine hydrate is slowly added to KMnO while stirring 4 In aqueous solution, so as to make the violent reaction between strong reducing agent and strong oxidizing agent more complete.
Further, in the step (1), the autoclave is a polytetrafluoroethylene-lined autoclave.
Further, in the step (2), the weight ratio of the brown product prepared in the step (1), indocyanine green (ICG) and water is 35:3:5000-20000, preferably 35:3:10000.
Further, in the step (2), the brown product obtained in the step (1), indocyanine green (ICG) and water are added into a lightproof reaction bottle to be uniformly mixed, then the mixture is subjected to ultrasonic treatment at normal temperature for 1 to 4 hours, then the mixture is stirred and reacted for 1 to 3 days at normal temperature, after the reaction is finished, the supernatant is removed by centrifugation, and the precipitate is washed and centrifuged to obtain the flaky trimanganese tetroxide nanomaterial ICG@Mn 3 O 4 。
Further, in the step (2), the room temperature ultrasonic treatment time is 2 hours, and the room temperature stirring reaction time is 2 days.
Further, in the step (2), the stirring speed of the stirring reaction at normal temperature is 600 to 1000rpm, preferably 800rpm.
Further, the milky white precipitate obtained in the step (1) is centrifugally washed with ultrapure water and ethanol; the precipitate obtained in the step (2) is subjected to washing centrifugation with an aqueous NaCl solution.
Further, the concentration of the aqueous NaCl solution used in the step (2) was 0.9% (wt).
The second aspect of the invention provides a flaky trimanganese tetroxide nanomaterial ICG@Mn prepared by the method for preparing the flaky trimanganese tetroxide nanomaterial with photodynamic and photothermal synergistic sterilization according to the first aspect 3 O 4 。
Further, the nanomaterial ICG@Mn 3 O 4 Is hexagonal in structure.
As described above, the sheet-shaped manganous-manganic oxide nano material with photodynamic and photothermal synergistic sterilization and the preparation method thereof have the following beneficial effects:
the invention synthesizes a photosensitizer@photothermal agent-indocyanine green@mangano oxide (ICG@Mn) with photodynamic and photothermal synergistic sterilization function 3 O 4 ) Realizes the low-power single-laser activatable PDT/PTT combined sterilization, which is 0.33W cm -2 The high temperature of 50 ℃ and a large amount of reactive oxygen species (including singlet oxygen, superoxide anion free radicals and hydroxyl free radicals) which can kill bacteria are generated under the irradiation of 808nm laser, and the photo-thermal conversion efficiency is as high as 67.5 percent. The size distribution of the hexagonal trimanganese tetroxide nano-sheet is obtained by the statistical analysis of a Scanning Electron Microscope (SEM) image and a transmission electron microscope image (TEM), the thickness of the hexagonal trimanganese tetroxide nano-sheet is 29.8+/-0.6 nm, and the side length is 124.8+/-1.2 nm. High resolution TEM images show that there is a lot of lattice distortion on the surface of hexagonal trimanganese tetroxide nanoplates, which provides a large number of active sites for active oxygen production. Furthermore, cytotoxicity experiments demonstrated icg@mn 3 O 4 The nano structure has low toxicity and can be used for in vitro sterilization and wound repair.
Drawings
Fig. 1 shows an XRD spectrum of hexagonal trimanganese tetroxide nanoplatelets in example 1 of the present invention.
Fig. 2 shows a scanning electron microscope (a), a transmission electron microscope (b) and a statistical chart of size distribution of the hexagonal trimanganese tetroxide nanoplatelets in example 1 of the present invention: side length (c), thickness (d).
FIG. 3 shows experimental evidence (electron spin resonance spectrum) of the generation of three different reactive oxygen species ROS under laser irradiation in example 2 of the present invention.
FIG. 4 shows the present inventionICG@Mn at different concentrations (0, 0.5, 1, 1.5, 2 mg/mL) in example 3 3 O 4 At the same power density (0.33W/cm 2 ) Photo-thermal conversion under laser irradiation.
FIG. 5 shows the power density (0.33, 1.0, 1.5, 2.0 W.cm) at different power densities in example 3 of the present invention -2 ) Under 808nm laser irradiation, 1.5mg/mL ICG@Mn 3 O 4 Is a photo-thermal conversion contrast graph of (2).
FIG. 6 shows the ICG@Mn in example 4 of the present invention 3 O 4 Cytotoxicity evaluation test results of (2) are shown. (a) ICG@Mn at various concentrations (10, 50, 200, 600, 2000, 6000. Mu.g/mL) 3 O 4 An ultraviolet-visible absorption spectrum after mixing with blood; (b) ICG@Mn at different concentrations 3 O 4 Is a graph of the hemolysis rate results; (c) Blank control (0.9% NaCl) and ICG@Mn at different concentrations 3 O 4 Is a photograph of a hemolysis experimental sample; (d) ICG@Mn at various concentrations (0, 25, 50, 600, 2000. Mu.g/mL) 3 O 4 MTS experimental result graph of (2); (e) 1.5mg/mL ICG@Mn 3 O 4 Results of coagulation experiments.
FIG. 7 shows a graph of the results of an in vitro antibacterial test against MRSA (G+: gram positive bacteria) in example 5 of the present invention.
FIG. 8 shows the measurement of ICG@Mn by a broad-spectrum antibacterial test in example 5 of the present invention 3 O 4 The results of the antibacterial experiments on enterococcus faecalis (G+: gram positive bacteria), escherichia coli (G-: gram negative bacteria) and Pseudomonas aeruginosa (G-).
FIG. 9 is a graph showing the change in fluorescence intensity of MRSA cell membrane permeability detected by PI staining assay in example 6 of the present invention.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
The invention provides a sheet-shaped manganous-manganic oxide nano material with photodynamic and photothermal synergistic sterilization and a preparation method thereof. Due to Mn 3 O 4 Has good biocompatibility, photo-thermal stability, higher photo-thermal conversion efficiency and photodynamic activity, and shows excellent performance in PDT/PTT combined sterilization. Indocyanine green (Indocyanine green, ICG) is a potent photosensitizer that can generate reactive oxygen species (cytotoxic reactive oxygen species, ROS) that are toxic to cells. Meanwhile, ICG has a maximum light absorption wavelength at 780nm and has been approved by the FDA for clinical use, and is considered as a nontoxic photodynamic therapeutic agent. In order to realize simultaneous cooperation of PDT/PPT under single low-power near infrared laser irradiation, the invention synthesizes ICG@Mn with good biocompatibility 3 O 4 . Because of ICG@Mn 3 O 4 Can simultaneously generate satisfactory local high temperature and a large amount of active oxygen (namely PTT and PDT synergism) under the irradiation of laser with the wavelength of 808nm, even if the power density is 0.33W cm -2 (meeting ANSI Z136.1-2014, american safety use laser Standard), ICG@Mn 3 O 4 Has good in vitro antibacterial activity on various pathogenic bacteria such as staphylococcus aureus, enterococcus faecalis, escherichia coli and pseudomonas aeruginosa. The problem of toxicity caused by long-term irradiation of laser with high power density and continuous irradiation of laser with different wavelengths is solved.
The specific implementation process is as follows:
example 1
1. Preparation of ICG@Mn 3 O 4 Sheet-like nanomaterial
Preparation of ICG@Mn by hydrothermal method 3 O 4 The specific method of the nano material is as follows:
into a conical flask containing 40mL of ultrapure water was charged 0.316g of KMnO 4 Uniformly stirring to obtain KMnO 4 Aqueous solution, then 5mL of hydrazine hydrate liquid (N) was added to solution 1 2 H 4 ·H 2 O, 85%), and stirring while adding to obtain a precursor solution. The precursor solution obtained was transferred to a polytetrafluoroethylene-lined autoclave of 100mL volume, and slowly heated to 180℃after sealing, and whereThe temperature was maintained for 12h, and then naturally cooled to room temperature. Subsequently, the milky precipitate was collected from the bottom of the autoclave, washed with ultrapure water and ethanol by centrifugation (11000 rpm,6 min) a plurality of times, and dried overnight in an oven at 80℃to give a brown sample. 0.035g of brown sample was taken, 0.003g ICG,10mL H 2 O was added to an opaque brown bottle. Vortex for 2min, ultrasonic for 2h at normal temperature and magnetic stirrer for 2d at 800rpm at normal temperature. Transfer to a 10mL EP tube. Centrifuging at 8000rpm for 8min, and discarding supernatant; adding 0.9% (wt) NaCl, shaking for dispersing to make the osmotic pressure of the solution and bacteria consistent, centrifuging, and removing supernatant; the process is repeated for 4 to 5 times to obtain the trimanganese tetroxide nanosheet ICG@Mn 3 O 4 Finally, the mixture is preserved at 4 ℃ in dark.
2. Adopting XRD, SEM, TEM to detect the manganese tetraoxide nano-sheet prepared by the method, wherein an XRD spectrum of the manganese tetraoxide nano-sheet is shown in figure 1, and a Scanning Electron Microscope (SEM) (figure 2 a) and a Transmission Electron Microscope (TEM) (figure 2 b) of the manganese tetraoxide nano-sheet are shown in figure 2; and obtaining a dimension distribution statistical diagram of the hexagonal trimanganese tetroxide nano sheet according to the statistical analysis of the scanning electron microscope diagram (figure 2 a) and the transmission electron microscope diagram (figure 2 b): side length (fig. 2 c), thickness (fig. 2 d). As can be seen from FIG. 2, the side length of the trimanganese tetroxide nano-sheet is 124.8+ -1.2 nm, and the thickness is 29.8+ -0.6 nm.
Example 2
ICG@Mn 3 O 4 Photodynamic performance evaluation of (2)
1. Experimental method
Test (1) at a power density of 0.33 W.cm -1 Under the laser irradiation condition of 808nm, an electron paramagnetic resonance instrument is used for testing the system: radical scavenger TEMP and ICG@Mn 3 O 4 Singlet oxygen molecules in the mixed aqueous solution 1 O 2 )；
Test (2), test system with electron paramagnetic resonance instrument in the absence of laser irradiation: singlet oxygen molecules in mixed aqueous solution of free radical trapping agent TEMP and ICG@Mn3O4 1 O 2 )；
Test (3) at a power density of 0.33 W.cm -1 Under the laser irradiation condition of 808nm, an electron paramagnetic resonance instrument is used for testing the system: radical scavenger DMPO and ICG@Mn 3 O 4 Superoxide radical (O) in the mixed aqueous solution 2 - ·)；
Test (4), test system with electron paramagnetic resonance instrument in the absence of laser irradiation: radical scavenger DMPO and ICG@Mn 3 O 4 Superoxide radical (O) in the mixed aqueous solution 2 - ·)；
Test (5) at a power density of 0.33 W.cm -1 Under the laser irradiation condition of 808nm, an electron paramagnetic resonance instrument is used for testing the system: radical scavenger DMPO and ICG@Mn 3 O 4 Hydroxyl radical (·oh) in the mixed aqueous solution;
test (6), test system with electron paramagnetic resonance instrument in the absence of laser irradiation: radical scavenger DMPO and ICG@Mn 3 O 4 Hydroxyl radical (·oh) in the mixed aqueous solution;
2. analysis of experimental results
DMPO as a hydroxyl radical (. OH) and superoxide radical specific capture agent (O) 2 - T.C. using TEMP as singlet oxygen molecule 1 O 2 ) Specific capture agents, characteristic absorption of the corresponding adducts was tested using an electron paramagnetic resonance instrument. The test results are shown in FIG. 3, and tests (1), (3) and (5) show that the electron paramagnetic resonance instrument detects ICG@Mn under the illumination condition 3 O 4 Can generate multiple ROS, including 1 O 2 、O 2 - andOH; tests (2), (4) and (6) show that little active oxygen is produced in the system in the absence of light.
In this example DMPO refers to 5, 5-dimethyl-1-pyrroline-N-oxide and TEMP refers to 4-amino-2, 6-tetramethylpiperidine.
Example 3
ICG@Mn 3 O 4 Is used for evaluating the photo-thermal performance of (C)
1. Experimental method
The ICG@Mn prepared in example 1 was taken 3 O 4 Prepared into 0, 0.5, 1, 1.5 and 2mg/mL ICG@Mn 3 O 4 300 mu L of each concentration is placed in a 96-well plate and the power density is 0.33W cm -2 808nm laser radiationIlluminating for 15min.
(1) Several 300. Mu.L of 1.5mg/mL ICG@Mn were taken 3 O 4 Placed in 96-well plates, and laser beams of 808nm are respectively used for measuring the thickness of the plates at 0.33, 0.97, 1.94 and 2.91W cm -2 Irradiation was performed for 15min at four different power densities.
(2) ICG@Mn by ON/OFF cyclic irradiation experiments 3 O 4 The photo-thermal stability of (2) is evaluated by the following steps:
300 mu L of 1.5mg/mL ICG@Mn 3 O 4 Placed in a 96-well plate, and irradiated with a 808nm laser at 0.33 W.cm -2 The laser was turned off for 10min at power density. After the temperature cooled to room temperature, the laser was turned on, re-irradiated with the same power density for 10min, and turned off. The same operation was repeated 4 times. The temperature was measured with an electron thermometer (Lutron TM 917) and the temperature was recorded every 20 s. And calculating the photo-thermal conversion efficiency according to the improved formula of roper.
The modified formula for roper is as follows:
2. analysis of experimental results
(1) As can be seen from FIG. 4, by measuring ICG@Mn at different concentrations (0, 0.5, 1, 1.5, 2 mg/mL) 3 O 4 At the same power density (0.33W/cm 2 ) The temperature change was found to be dependent on ICG@Mn 3 O 4 The concentration increases, the faster the temperature changes, and stabilizes after 10 minutes.
(2) As can be seen from FIG. 5, by measuring 1.5mg/mL ICG@Mn 3 O 4 At different power densities (0.33, 1.0, 1.5, 2.0W cm) -2 ) The temperature change under the condition of ICG@Mn was found 3 O 4 The photo-thermal effect of (2) is proportional to the power density.
(3) For measuring ICG@Mn 3 O 4 In this example, 1.5mg/mL ICG@Mn was used 3 O 4 At a power density of 0.33W/cm 2 An ON/OFF cyclic irradiation experiment was performed as follows. After five times of cyclic irradiation, when the same timeIn the room, the difference between the maximum temperatures that can be reached is less than 5 ℃, and the average loss temperature per time is less than 1 ℃, indicating ICG@Mn 3 O 4 The photo-thermal stability of the (C) is better. 1.5mg/mL ICG@Mn was calculated according to the modified formula of roper 3 O 4 The light-heat conversion efficiency of (2) was 67.5%. At 0.33W/cm 2 Icg@mn at ultra-low power density of (a) 3 O 4 Has higher photo-thermal conversion efficiency than most current PTT studies.
Note that: specific calculation modes of the photo-thermal conversion efficiency can be referred to literature
Roper DK,Ahn W,Hoepfner M.Microscale Heat Transfer Transduced by Surface Plasmon Resonant Gold Nanoparticles.J.Phys.Chem.C 2007,111,3636–3641。
Example 4
ICG@Mn 3 O 4 Cytotoxicity evaluation of (2)
1. Hemolysis experiment
(1) Experimental method
Whole blood of healthy person was taken, 10 times of 0.9% NaCl was added and gently mixed, and after centrifugation at 6000rpm for 5min, the supernatant was removed, and washing was repeated 3 times with 0.9% NaCl until the supernatant became red-free. The lower pellet, red blood cells, was removed and added to 4 volumes of 0.9% nacl to make RBC suspension. ICG@Mn prepared in example 1 was quenched with 0.9% NaCl 3 O 4 Diluted to 10, 50, 200, 600, 2000, 6000 mug/mL.
Sample preparation: several sterile EP tubes were taken and 50uL of RBC suspension was added to each EP tube. The test group EP was supplemented with 700uL of diluted ICG@Mn at various concentrations 3 O 4 . 700uL of H was added to the EP tube of the positive control group 2 O, 700uL of 0.9% NaCl was added to the EP tube of the negative control. All samples were gently mixed.
All prepared samples were incubated at 37℃for 2 hours, and then centrifuged at 6000rpm for 5 minutes, and the supernatant was taken and measured for ultraviolet absorbance at 450 to 700 nm. The haemolysis rate was calculated according to the following formula:
(2) Analysis of experimental results
As can be seen from FIG. 6a, with ICG@Mn 3 O 4 The concentration of (2) increases and the ultraviolet absorption value of 500-600nm gradually increases. As can be seen from FIGS. 6b and 6c, the hemolysis ratio was compared with ICG@Mn 3 O 4 The concentration of (2) shows a positive correlation, and ICG@Mn is 10-2000 mug/mL 3 O 4 The hemolysis rate of (2) is lower than 5%; and, ICG@Mn of 10-2000 μg/mL 3 O 4 No obvious hemolysis was observed, indicating that ICG@Mn was 10-2000. Mu.g/mL 3 O 4 Does not cause serious hemolysis and is safer.
2. Coagulation experiment
(1) Experimental method
200uL of healthy human plasma is taken, 50uL of 1.5mg/mL ICG@Mn is added 3 O 4 After that, the mixture is gently mixed and kept stand for 20min; measurement of ICG@Mn with fully automatic blood coagulometer 3 O 4 Activated Partial Thromboplastin Time (APTT), prothrombin Time (PT) and Thrombin Time (TT) of the blood samples before and after.
(2) Analysis of experimental results
The normal ranges of the thrombin time (APTT), the Prothrombin Time (PT) and the Thrombin Time (TT) of human blood are respectively 15-34 s, 10.5-13.7 s and 14-21 s. As can be seen from FIG. 6e, when ICG@Mn is added 3 O 4 Post-blood sample with no ICG@Mn 3 O 4 The Prothrombin Time (PT), thrombin Time (TT) of the previous blood sample was almost unchanged; adding ICG@Mn 3 O 4 The thromboplastin time (APTT) of the post-blood sample was slightly greater than that of the non-added ICG@Mn 3 O 4 Previous blood samples, but with differences less than 3s, were within normal ranges. The above experimental results demonstrate ICG@Mn 3 O 4 Has less influence on the coagulation function of normal blood.
3. MTS assay
(1) Experimental method
Cell viability was determined by the MTS assay. After seeding HUVEC cells in 96-well plates, the cells were plated in 5% CO 2 Culturing for 24 hours; respectively adding 0 and 25 into different holesICG@Mn of 50, 600, and 2000 μg/mL 3 O 4 Three sets of parallel experiments were set for each concentration; after 24h incubation, MTS/PMS solution was added to each well and then placed at 37℃with 5% CO 2 Incubating for 1 hour; the absorbance of the solution at 490nm was measured for each well using a VERS Amax microplate reader.
(2) Analysis of experimental results
As can be seen from FIG. 6d, the ICG@Mn of 0, 25, 50, 600, 2000. Mu.g/mL was measured 3 O 4 Survival of HUVEC cells after incubation was found when ICG@Mn 3 O 4 At a concentration of 2000. Mu.g/mL, the cell viability was 65%, indicating ICG@Mn 3 O 4 The effect on viability of HUVEC cells was less.
Example 5
In vitro antibacterial experiments
1. In vitro antibacterial experiments against methicillin-resistant Staphylococcus aureus MRSA (G+: gram-positive bacteria)
(1) Experimental method
Diluting bacterial liquid: culturing MRSA inoculated bacterial liquid to OD 600 =0.5, 100-fold dilution.
Preparing a sample: placing 100uL of diluted bacterial liquid into 6 sterile EP pipes, and sequentially adding 100uL of 3mg/mL ICG@Mn into 1-6 # 3 O 4 、2.0mg/mL ICG@Mn 3 O 4 、3mg/mL Mn 3 O 4 、3mg/mL ICG@Mn 3 O 4 、PBS、PBS。
Laser irradiation: the prepared samples are transferred to a 96-well plate after being uniformly mixed. Samples No. 1-3 and No. 5 were subjected to a near infrared laser of 808nm (power density of 0.33W/cm) 2 ) Irradiating for 15min, and standing for 15min without irradiating sample No. 4 and sample No. 6.
Coating a flat plate: after the above treatment, sample No. 1-6 was applied onto sterile petri dishes by pipetting 10uL each with a sterile pipette and incubated in a incubator at 37℃for 12h.
(2) Analysis of experimental results
By measuring ICG@Mn 3 O 4 Antibacterial activity against MRSA (G+: gram positive bacteria) in vitroICG@Mn now 3 O 4 The antibacterial activity of (a) gradually increases with the increase of the concentration thereof.
As can be seen from FIG. 7, "1.5mg/mL ICG@Mn 3 O 4 The colony count of the experimental group of +808nm laser' was significantly less than that of "1.5mg/mL Mn 3 O 4 +808nm laser group and "1.5mg/mL ICG@Mn 3 O 4 "group", this is due to ICG@Mn under near infrared radiation of 808nm 3 O 4 Can convert light and heat into heat energy and generate ROS, thereby killing bacteria. Thus, ICG and Mn 3 O 4 The synergistic effect of the two is larger than that of Mn alone 3 O 4 And does not cause damage to the unirradiated tissue. The colony count of the "PBS+808nm laser" group was not reduced, because PBS did not have photothermal and photoactivating effects, and even if irradiated, bacteria could not be killed.
2. In vitro broad-spectrum antibacterial experiment
(1) Experimental method
Diluting bacterial liquid: bacterial solutions respectively inoculated with enterococcus faecalis (G+ to gram positive bacteria), escherichia coli (G-to gram negative bacteria) and Pseudomonas aeruginosa (G-to gram negative bacteria) are cultured to OD 600 =0.5, 100-fold dilution.
Preparing a sample: taking 9 sterile EP pipes, placing 100uL of diluted enterococcus faecalis bacterial liquid in a No. 1-3 sterile EP pipe, sucking 100uL of diluted escherichia coli bacterial liquid in a No. 4-6 sterile EP pipe, and placing 100uL of diluted pseudomonas aeruginosa bacterial liquid in a No. 7-9 sterile EP pipe. 1. Sterile EP tubes No. 2, 4, 5, 7, 8 were each filled with 3mg/mL ICG@Mn 3 O 4 100uL, sterile EP tubes No. 3, 6, 9 were added to PBS 100uL, respectively.
Laser irradiation: the prepared samples are transferred to a 96-well plate after being uniformly mixed. 2. Sample 5 and sample 8 are not irradiated and are kept stand for 15min; the other samples were subjected to a near infrared laser (power density of 0.33 W.cm) -2 ) Irradiating for 15min.
Coating a flat plate: after the above treatment, samples 1 to 9 were inoculated onto sterile dishes by pipetting 10uL each with a sterile pipette and placed in a 37℃solid incubator for 12 hours.
(2) Analysis of experimental results
FIG. 8 shows ICG@Mn 3 O 4 Antibacterial activity against enterococcus faecalis (G+), escherichia coli (G-), and Pseudomonas aeruginosa (G-). As shown in fig. 8, the colony numbers were ranked as follows: "1.5mg/mL ICG@Mn 3 O 4 +808nm laser "group<“1.5mg/mL ICG@Mn 3 O 4 Group<"PBS" group.
“1.5mg/mL ICG@Mn 3 O 4 The antibacterial activity of the +808nm laser group is obviously greater than that of the ICG@Mn of 1.5mg/mL 3 O 4 The reason for the "group and" PBS "group is: under 808nm laser irradiation, ICG@Mn 3 O 4 Local high temperatures are generated and ROS are generated, thereby killing bacteria; ICG@Mn in the absence of laser irradiation 3 O 4 No photo-thermal and photo-activation effects occur, which indirectly indicate that it does not cause toxicity to the non-irradiated skin or tissue. The above results indicate that ICG@Mn under 808nm near infrared radiation 3 O 4 Has different degrees of antibacterial activity on various pathogenic bacteria, but has different minimum antibacterial concentration on different bacteria, which can be realized by adjusting ICG@Mn 3 O 4 The concentration of the product reaches the aim of complete sterilization.
Example 6
ICG@Mn3O4 sterilization mechanism research
1. Experimental method
Diluting bacterial liquid: culturing bacterial liquid inoculated with MRSA (G+: gram positive bacteria) to OD 600 =0.5, 100-fold dilution.
Preparing a sample: the 9 sterile EP tubes were taken, and 150uL of the diluted bacterial solution was placed in the sterile EP tubes No. 1-9. Sterile Ep tubes No. 1-3 were each filled with 3mg/mL ICG@Mn 3 O 4 100uL, no. 4-6 sterile EP tube was separately added 3mg/mL Mn 3 O 4 100uL, sterile EP 7-9 tubes were each added to PBS 100uL.
Laser irradiation: the prepared samples are transferred to a 96-well plate after being uniformly mixed. 1. Sample Nos. 4 and 7 were subjected to near infrared laser (power density: 0.33 W.cm) -2 ) Irradiating for 15min; 2. 808nm near infrared laser for sample No. 5 and No. 8(Power Density of 0.33 W.cm) -2 ) Irradiating for 5min; 3. samples 6 and 9 were left for 15min without irradiation.
PI staining: sample nos. 1 to 9 were treated as described above, stained with PI (3 μm) and fluorescence of the sample was measured with a fluorescence spectrophotometer.
2. Analysis of experimental results
It is mentioned in the literature that both local overheating and ROS can damage the bacterial cell membrane system leading to its death. (Sun D, liu Y, yu Q, qin X, yang L, zhou Y, et al, inhibition of tumor growth and vasculature and fluorescence imaging using functionalized ruthenium-thiol protected selenium nanomatrics, biomaterials 2014,35,1572-1583;Mao C,Xiang Y,Liu X,Zheng Y,Yeung KWK,Cui Z,et al.Local Photothermal/Photodynamic Synergistic Therapy by Disrupting Bacterial Membrane To Accelerate Reactive Oxygen Species Permeation and Protein Leakage. ACS appl. Mater. Interfaces 2019,11,17902-17914).
To verify ICG@Mn 3 O 4 In this example, the permeability of MRSA cell membrane was measured by PI staining, and ICG@Mn was measured 3 O 4 Destructive to MRSA cell membranes. PI can enter the cell through the broken cell membrane, bind to nucleic acid in the cell, and fluoresce.
The experimental results are shown in FIG. 9, and after PI dyeing, ICG@Mn is obtained in the absence of laser irradiation 3 O 4 Group, mn 3 O 4 The fluorescence of the group and the PBS group is not greatly different; however, with increasing laser irradiation time, ICG@Mn 3 O 4 Group, mn 3 O 4 The fluorescence intensity of the group was significantly higher than that of the PBS group, and ICG@Mn 3 O 4 The fluorescence of the group was the strongest, indicating ICG@Mn 3 O 4 Under the irradiation of laser, the MRSA cell membrane can be destroyed, so that PI can be combined with nucleic acid in cells or excreted, and strong fluorescence can be emitted, and the cell membrane destruction is stronger than that of Mn alone 3 O 4 。
In conclusion, the invention successfully synthesizes ICG@Mn with both photoactivation effect and photothermal effect 3 O 4 Nanomaterial for the preparation of a nanoparticleOvercomes the defect that the traditional PDT/PTT combined treatment needs long-term laser irradiation with larger power density and the sequential irradiation of two lasers with different wavelengths. ICG@Mn 3 O 4 The photodynamic performance evaluation, the photothermal performance evaluation and the cytotoxicity evaluation of the fluorescent dye show satisfactory conclusion, namely Gao Guangre conversion efficiency, stable photothermal performance, capability of simultaneously generating three ROS under light irradiation, lower hemolysis rate, lower coagulation rate and higher cell survival rate. The PDT/PTT combination has a greater antimicrobial activity than the PDT or PTT alone. Such an ICG@Mn-based catalyst 3 O 4 The low-power single-laser activatable PDT/PTT combined strategy of the nano material has wide prospect in clinical treatment.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (15)
1. The preparation method of the sheet-shaped manganous-manganic oxide nano material with photodynamic and photothermal synergistic sterilization is characterized by comprising the following steps of:
(1) Adding hydrazine hydrate into KMnO 4 Preparing a precursor solution in the aqueous solution, slowly heating the precursor solution to 160-200 ℃ in an autoclave, reacting, cooling to room temperature after the reaction is finished, collecting milky precipitate from the bottom of the autoclave, centrifuging, washing and drying to obtain a brown product;
(2) Adding the brown product obtained in the step (1), indocyanine green and water into an opaque reaction bottle for reaction, centrifuging after the reaction is finished, discarding the supernatant, washing and centrifuging the precipitate to obtain the flaky trimanganese tetroxide nanomaterial ICG@Mn 3 O 4 。
2. The preparation method according to claim 1, characterized in thatThe method comprises the following steps: in the step (1), KMnO per millimole 4 The dosage of the required hydrazine hydrate liquid solution is 1-4 mL.
3. The method of manufacturing according to claim 1, characterized in that: in the step (1), the hydrazine hydrate contains 85 percent of N 2 H 4 ·H 2 O hydrazine hydrate liquid.
4. The method of manufacturing according to claim 1, characterized in that: in the step (1), the reaction time is 8-16 h.
5. The method of manufacturing according to claim 1, characterized in that: in the step (1), the drying temperature is 80-100 ℃ and the drying time is 6-12 h.
6. The method of manufacturing according to claim 1, characterized in that: in the step (1), hydrazine hydrate is slowly added into KMnO while stirring 4 In an aqueous solution.
7. The method of manufacturing according to claim 1, characterized in that: in the step (1), the autoclave is a polytetrafluoroethylene lining autoclave.
8. The method of manufacturing according to claim 1, characterized in that: in the step (2), the weight ratio of the brown product prepared in the step (1), indocyanine green (ICG) and water is 35:3:5000-20000.
9. The method of manufacturing according to claim 1, characterized in that: in the step (2), the brown product obtained in the step (1), indocyanine green (ICG) and water are added into a lightproof reaction bottle to be uniformly mixed, then the mixture is subjected to ultrasonic treatment at normal temperature for 1 to 4 hours, then the mixture is stirred and reacts for 1 to 3 days at normal temperature, after the reaction is finished, the supernatant is centrifugally removed, and the precipitate is washed and centrifugally treated to obtain the flaky trimanganese tetroxide nano material ICG@Mn 3 O 4 。
10. The method of manufacturing according to claim 9, wherein: in the step (2), the normal-temperature ultrasonic treatment time is 2 hours, and the normal-temperature stirring reaction time is 2 days.
11. The method of manufacturing according to claim 9, wherein: in the step (2), the stirring speed of stirring reaction at normal temperature is 600-1000 rpm.
12. The method of manufacturing according to claim 1, characterized in that: the milky white precipitate obtained in the step (1) is centrifugally washed by ultrapure water and ethanol; the precipitate obtained in the step (2) is subjected to washing centrifugation with an aqueous NaCl solution.
13. The method of manufacturing according to claim 12, wherein: the concentration of the aqueous NaCl solution used in said step (2) was 0.9% (wt).
14. The sheet-shaped trimanganese tetroxide nanomaterial icg@mn prepared by a method for preparing a photodynamic and photothermal synergistic bactericidal sheet-shaped trimanganese tetroxide nanomaterial according to any one of claims 1-13 3 O 4 。
15. The platy trimanganese tetroxide nanomaterial icg@mn according to claim 14 3 O 4 The method is characterized in that: the nanomaterial ICG@Mn 3 O 4 Is hexagonal in structure.
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