CN114949207B - Low-toxicity zinc-doped carbon dot and application thereof - Google Patents

Low-toxicity zinc-doped carbon dot and application thereof Download PDF

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CN114949207B
CN114949207B CN202210482896.6A CN202210482896A CN114949207B CN 114949207 B CN114949207 B CN 114949207B CN 202210482896 A CN202210482896 A CN 202210482896A CN 114949207 B CN114949207 B CN 114949207B
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edta
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CN114949207A (en
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徐小平
钟卓伶
刘书瑶
张传维
雍智全
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Guangzhou Anhao Pharmaceutical Technology Co ltd
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Abstract

The invention belongs to the technical field of photoinduction sterilization, and particularly relates to a low-toxicity zinc-doped carbon dot and application thereof. The low-toxicity zinc-doped carbon dot consists of the following raw materials in parts by weight: 268-328 parts of zinc doped carbon dots, 335-409.5 parts of EDTA-2 Na; the EDTA covers the surface of the low-toxicity zinc-doped carbon dots. The low-toxicity zinc-doped carbon dot has photoinduction sterilization equivalent to the existing zinc-doped carbon dot, and compared with the existing zinc-doped carbon dot, the biotoxicity of the low-toxicity zinc-doped carbon dot is obviously reduced, so that the low-toxicity zinc-doped carbon dot has good application prospect in clinical application.

Description

Low-toxicity zinc-doped carbon dot and application thereof
Technical Field
The invention belongs to the technical field of photoinduction sterilization, and particularly relates to a low-toxicity zinc-doped carbon dot and application thereof.
Background
Due to the widespread use of antibiotics, many bacteria even develop resistance. Drug-resistant bacteria seriously affect the life health of people in the world. Researchers are looking for a new non-drug resistant antimicrobial regimen or agent. Many physical antimicrobial regimens are of interest to scientists because of their lower probability of developing resistance and lower toxicity to the body, such as: radiation, heat, etc. Among them, photodynamic therapy (photodynamic therapy, PDT) is one of means for treating cancer, especially skin cancer, is a minimally invasive treatment method, and has the advantages of low drug resistance, less damage to peripheral tissues, low toxic and side effects, etc., compared with other conventional therapies such as surgery, radiotherapy, etc. Based on PDT, researchers have proposed an antibacterial photodynamic therapy, which is based on the principle: the Photosensitizer (PS) is activated by visible light of a suitable wavelength to generate Reactive Oxygen Species (ROS) that are cytotoxic to pathogens, thereby achieving a bacterial killing effect.
PDT comprises three elements of photosensitizer, excitation light and molecular oxygen in tissue, the reaction mechanism of which is a nonthermal photochemical reaction between the photosensitizer and a specific wavelength laser. Firstly, the photosensitizer is systematically or locally placed in a target area, after the photosensitizer is absorbed and combined with biological tissues such as cells and the like, the photosensitizer can be excited by irradiation of specific wavelength, so that the photosensitizer can transit from a singlet state with extremely short service life to an excited state through a ground state and interact with surrounding oxygen molecules to generate reactive oxygen (reactive oxygen species, ROS) molecules (such as singlet oxygen, superoxide anions and the like), and then the photosensitizer is subjected to oxidation reaction with the target cell structure to destroy a biological membrane structure or other functional units, so that cells are damaged or dead.
Carbon Dots (CDs) were first discovered in 2004 when single-walled carbon nanotubes were prepared from Xu et al. The fluorescent dye has the advantages of high biosafety, stable chemical property, excellent optical performance, photobleaching resistance, low toxicity, simple and various synthesis modes and the like, is concerned by students, has bright application prospect in various fields such as antibiosis, biological markers, biocatalysis and the like, and opens up a new way for developing novel antibacterial materials and photosensitizers. Ju et al prepared carbon dots using chloroform, diethylamine and ammonium carbonate, found that carbon dots were able to generate strong electrostatic interactions with bacteria and thereby damage the bacterial cell walls or membranes of s.aureus and e.coli. The nitrogen and zinc doped luminescent carbon dots prepared by Das et al can kill escherichia coli under light excitation. The carbon dot composite material prepared by Meziani et al was found to kill E.coli after 30 minutes of visible light irradiation. Although research on the application of carbon dots to photodynamic sterilization therapy is at an early stage, no specific mechanism has been fully clarified. But has the advantages of excellent photosensitizer due to the unique fluorescence performance, stable chemical property, low toxicity, easy combination with bacteria and biological film thereof, good photoinduction electron transfer capability, light absorption capability and the like, and has great significance in developing new antimicrobial therapies.
Many photocatalytically active nanomaterials have been used as photocatalysts for studying electron transfer processes and charge separation. The challenge in this area is to develop more low cost, high efficiency photocatalysts to replace the known limited photocatalysts based on noble metal nanoparticles, such as ruthenium nanoparticles. Fortunately, carbon Dots (CDs) have unique electron transfer and broadband light absorption capabilities, have a large specific surface area, and have the advantages of being rich, inexpensive, nontoxic, and the like. These excellent properties favor intermolecular electron transfer, which plays a key role in many photooxidation reactions. In order to improve the electron transfer and photooxidation reaction efficiency of the photocatalyst, the strategy of co-doping carbon points and heteroatoms attracts attention of students, and has wide application in the fields of biomolecular marking, chemical sensing, photoelectricity, trace element detection and the like. However, the metal atoms are doped into the CDs matrix, and the toxicity is increased to some extent, so that the ideal metal dopant should not cause toxicity to CDs.
The literature, "inhibition of Staphylococcus aureus growth and biofilm formation by Zinc-doped carbon Point in combination with blue light irradiation", discloses that the inhibition of Staphylococcus aureus growth by Zinc-doped carbon Point in combination with blue light irradiation is found in university of Jilin university journal (medical edition), 2020, 46 (3) ". Theoretically, zinc is an important element that aids many electron transfer processes in the environment, it is also a trace element of diverse biological systems, toxicity is negligible, and zinc deficiency can cause many health hazards. Therefore, the zinc-doped carbon dots disclosed in the document are a photosensitizer material which has potential to be applied to the field of sterilization. However, the inventor found in experiments that although the toxicity of zinc element is considered to be negligible, the zinc-doped carbon point still has non-negligible toxicity in practical application, which poses a great obstacle to the clinical transformation application of the material.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a low-toxicity zinc doped carbon dot and application thereof, and aims at: provided is a zinc-doped carbon dot having low toxicity and excellent photoinduced sterilization effect.
A low-toxicity zinc doped carbon dot is prepared from the following raw materials in parts by weight:
268-328 parts of zinc doped carbon dots,
EDTA-2Na 335-409.5 parts;
in the low-toxicity zinc-doped carbon dots, complex ions of EDTA-2Na cover the surfaces of the zinc-doped carbon dots.
Preferably, the zinc-doped carbon point is prepared from o-phenylenediamine, L-tryptophan and zinc ion-containing salt, wherein the molar ratio of the o-phenylenediamine, L-tryptophan and zinc ion is 0.9-1.1:0.24-0.26:0.9-1.1.
Preferably, the zinc ion-containing salt is selected from at least one of zinc chloride, zinc acetate or zinc tartrate.
Preferably, the particle size of the zinc-doped carbon dots is 20nm-40nm.
The invention also provides application of the low-toxicity zinc doped carbon dot as a photosensitizer.
The invention also provides application of the low-toxicity zinc-doped carbon dot in preparation of photoinduced bactericides.
The invention also provides a photoinduced bactericide which is prepared by taking the low-toxicity zinc doped carbon point as a photosensitizer and adding pharmaceutically acceptable auxiliary materials or auxiliary components.
Preferably, the method comprises the following steps:
step 1, mixing zinc doped carbon dots with EDTA-2Na to prepare a mixed solution;
and 2, regulating the pH of the mixed solution to 5.8-6.2 for reaction, thus obtaining the product.
Preferably, in the step 2, the reaction condition is that the reaction is carried out for 1.5 to 2 hours at 58 to 62 ℃;
and/or in the step 2, after the reaction is finished, sterilizing the mixed solution after the reaction by using a filter membrane with the diameter of 0.22 mu m, and freeze-drying to obtain the low-toxicity zinc doped carbon dot.
Preferably, the zinc-doped carbon dots are prepared by the following steps:
step A, mixing and dissolving o-phenylenediamine, L-tryptophan and zinc ion-containing salt in water in a reaction kettle to obtain a reaction solution, and adding 36-38% of concentrated hydrochloric acid by mass percent; the volume ratio of the reaction solution to the concentrated hydrochloric acid is 9-11:0.19-0.21;
and step B, reacting for 2.9-3.1h at 198-202 ℃ to obtain the catalyst.
In the present invention, the "zinc-doped carbon dot" may be expressed in terms of the unit "mmol" in which case one zinc-doped carbon dot as a whole is regarded as one molecular state.
The invention discovers that zinc-doped carbon dots have non-negligible toxicity after being prepared into the photoinduced bactericide for the first time. Based on the method, EDTA is further covered on the surface of the zinc-doped carbon dot, so that a novel low-toxicity zinc-doped carbon dot is prepared, and the toxicity of the zinc-doped carbon dot is greatly reduced. The low-toxicity zinc-doped carbon dot provided by the invention can efficiently generate active oxygen, is used as a photosensitizer for photoinduction sterilization, has a good sterilization effect, and has greatly reduced toxicity compared with the raw material zinc-doped carbon dot. Therefore, the method has good application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a TEM image (a), particle size distribution (b), elemental scans (c, d) and XPS characterization (e) of the EDTA-Zn-RCDs sample, and XPS characterization (f) of the Zn-RCDs sample of Experimental example 1;
FIG. 2 shows TEM images (a) and (b) of the RCDs of comparative example 1, TEM images (c) and (d) of the Zn-RCDs of comparative example 2 and TEM images (e) and (f) of the EDTA-Zn-RCDs of experimental example 1;
FIG. 3 is a graph showing the ultraviolet images (a) of the RCDs of comparative example 1, the ultraviolet images (b) of the Zn-RCDs of comparative example 2, the ultraviolet images (c) of the EDTA-Zn-RCDs of experimental example 1 and the ultraviolet combinations of the above 3 samples;
FIG. 4 is a graph showing the infrared image (a) of the RCDs sample in comparative example 1, the infrared image (b) of the Zn-RCDs sample in comparative example 2, the infrared image (c) of the EDTA-Zn-RCDs sample in experimental example 1, and the infrared combinations of the above 3 samples;
FIG. 5 is a graph showing a combination of a fluorescent image (a) of the RCDs sample in comparative example 1, a fluorescent image (b) of the Zn-RCDs sample in comparative example 2, an infrared image (c) of the EDTA-Zn-RCDs sample in experimental example 1 and the above 3 samples;
FIG. 6 is the result of the cytotoxicity test in Experimental example 2;
FIG. 7 is a graph showing the results of the bactericidal effect experiment in experimental example 2;
FIG. 8 is a graph showing the results of the experiment of the bactericidal effect of EDTA-Zn-RCDs of example 3 with different amounts of zinc incorporation;
FIG. 9 shows the results of the drug resistance test in Experimental example 4;
FIG. 10 is a flow chart of the animal modeling treatment in Experimental example 5;
fig. 11 is a photograph of wound (n=6) of infected mice in experimental example 5 at various time points;
FIG. 12 is an image of bacterial colonies in wound skin tissue of mice in different treatment groups of Experimental example 5, wherein (a) control group, (b) blue light group, (c) EDTA-Zn-RCDs group, (d) EDTA-Zn-RCDs+blue light group, (e) healthy group (n=6);
FIG. 13 is a graph showing statistical data of bacterial viability in Experimental example 5;
FIG. 14 is a graph of HE staining of infected skin tissue from different treatment groups of Experimental example 5, wherein (a) control group, (b) blue light group, (c) EDTA-Zn-RCDs group, (d) EDTA-Zn-RCDs+blue light group, (E) healthy group (n=6);
fig. 15 is a graph of HE staining of organs (heart, liver, spleen, lung and kidney) of infected mice in the different treatment groups of experimental example 5, wherein (a) control group, (b) blue light group, (c) EDTA-Zn-RCDs group, (d) EDTA-Zn-rcds+blue light group, (E) healthy group, scale = 100nm, (n = 6);
FIG. 16 shows the results of stability experiments on EDTA-Zn and EDTA-Zn-RCDs lyophilized powder in Experimental example 6.
Detailed Description
In the following examples and experimental examples, the reagents and materials used were as follows:
ethanol (analytical grade, chengdu GaoXin Kogyo chemical Co., ltd.), o-phenylenediamine (99%, shanghai Ala Biotechnology Co., ltd.), L-tryptophan (99%, shanghai Michelin Biotechnology Co., ltd.), hydrochloric acid (analytical grade, west-Cheng-Sci Co., ltd.), disodium ethylenediamine tetraacetate (EDTA-2 Na, analytical grade, fuchen chemical Co., ltd.), zinc chloride (analytical grade, tianjin Dongtian fine chemical Co., ltd.), sodium hydroxide (analytical grade, tianjin Yongda chemical Co., ltd.), sodium chloride (analytical grade, fuchen chemical Co., ltd.), agar powder (Chengdu Kolong chemical Co., ltd.), yeast extract powder (Chengdu Kolong chemical Co., ltd.), tryptone (Beijing Obock Biotechnology Co., ltd.), anhydrous diethyl ether (analytical grade, west-Chen Sci Co., ltd.). Pancreatin, DMEM and FBS are all imported reagents. Glutaraldehyde fixative (2.5%) was purchased from Shanghai Seiyaka Biotechnology Co., ltd., active oxygen detection kit (DCFDA, solarbio), propidium Iodide (PI) was purchased from Shanghai Biyunshan Biotechnology Co., ltd., singlet oxygen detection kit (SOSG, meilunoo), porphyrin (PpIX, sigma).
Mice (BALB/c, 7 weeks, female, SPF grade, beijing Fukang Biotechnology Co., ltd.) were supplied with L929 cells and Staphylococcus aureus and Escherichia coli from the national emphasis laboratory at university of Sichuan.
Reagents and materials not specifically described are commercially available.
Example 1
The embodiment provides low-toxicity zinc-doped carbon point EDTA-Zn-RCDs, which are prepared from the following raw materials in parts by weight: zinc doped carbon dots 1.0mmol,298mg, EDTA-2Na 1.0mmol,372.24mg.
The preparation method comprises the following steps:
108mg of o-phenylenediamine (1.0 mmol), 54mg of L-tryptophan (0.25 mmol) and 136mg of zinc chloride (1.0 mmol) are respectively weighed into a clean reaction kettle filled with 10ml of deionized water, 200 mu L of concentrated hydrochloric acid is added to be uniformly mixed, reaction is carried out for 3 hours at 200 ℃,372.24mg of EDTA-2Na is added to the obtained liquid mixture, the mixture is uniformly mixed, pH is regulated to be 6.0 to be reacted for 1.5 hours in a water bath at 60 ℃, the obtained mixture is sterilized by a filter membrane with the concentration of 0.22 mu m, and then low-toxicity zinc-doped carbon point EDTA-Zn-RCDs powder is obtained through freeze drying.
Example 2
The preparation method of this example is the same as that of example 1 except that the ratio of the raw materials is adjusted, and the amounts used in this example are: 328mg of zinc-doped carbon point and 335mg of EDTA-2 Na.
Example 3
The preparation method of this example is the same as that of example 1 except that the ratio of the raw materials is adjusted, and the amounts used in this example are: 268mg of zinc doped carbon point and 409.5mg of EDTA-2 Na.
Example 4
The preparation method of this example is the same as that of example 1, except that the zinc doping amount (zinc chloride amount) and EDTA-2Na amount are adjusted, and the amounts in this example are: zinc chloride (0, 0.1, 0.5 and 2.0 mmol) was 0, 13.6, 78 and 272mg, respectively, and the corresponding EDTA-2Na (0, 0.1, 0.5 and 2.0 mmol) was 0, 37.2, 186.12 and 744.48mg, respectively. EDTA-Zn-RCDs powders with different amounts of zinc incorporation were obtained (control samples for Experimental example 2 were obtained when the amounts of zinc chloride and EDTA-2Na were 0).
Comparative example 1
This comparative example provides a Red Carbon Dots (RCDs) of a comparative sample.
The preparation method comprises the following steps:
108mg of o-phenylenediamine and 54mg of L-tryptophan are respectively weighed into a clean reaction kettle filled with 10ml of deionized water, 200 mu L of concentrated hydrochloric acid is added to be uniformly mixed, the mixture is reacted for 3 hours at 200 ℃, the obtained mixture is sterilized by a filter membrane with the thickness of 0.22 mu m, and then freeze-dried to obtain carbon dot powder.
Comparative example 2
This comparative example provides a comparative sample of zinc-doped carbon dots (Zn-RCDs).
The preparation method comprises the following steps:
108mg of o-phenylenediamine, 54mg of L-tryptophan and 136mg of zinc chloride are respectively weighed into a clean reaction kettle filled with 10ml of deionized water, 200 mu L of concentrated hydrochloric acid is added to be uniformly mixed, the mixture is reacted for 3 hours at 200 ℃, the obtained mixture is sterilized by a filter membrane with the thickness of 0.22 mu m, and then the mixture is freeze-dried to obtain zinc-doped carbon dot powder.
The beneficial effects of the present invention are further illustrated by the following experiments conducted on samples prepared in examples 1, 4 (EDTA-Zn-RCDs), comparative example 1 (RCDs) and comparative example 2 (Zn-RCDs).
Experimental examples characterization of 1RCDs, zn-RCDs and EDTA-Zn-RCDs
The samples prepared in example 1 (EDTA-Zn-RCDs), comparative example 1 (RCDs) and comparative example 2 (Zn-RCDs) were used in this experimental example.
1. Experimental method
Taking 5mg of freeze-dried powder in 5ml of PBS solution to prepare 1mg/ml of RCDs, zn-RCDs and EDTA-Zn-RCDs solution, dripping the solution on a copper mesh, observing the morphology and the particle size of the carbon dots under a Transmission Electron Microscope (TEM) after the solution is naturally dried, observing EDTA-Zn-RCDs with the largest particle size under a dark field, and scanning Zn elements.
And uniformly mixing 20mg of lyophilized RCDs, zn-RCDs powder, EDTA-Zn-RCDs and potassium bromide powder according to a certain proper proportion, pressing into a transparent sheet, and measuring infrared absorption spectrograms of the RCDs, zn-RCDs and EDTA-Zn-RCDs by using an infrared spectrometer.
5mg of the lyophilized powder was taken in 5ml of PBS solution to prepare 1mg/ml of RCDs, zn-RCDs and EDTA-Zn-RCDs solutions, which were placed in a quartz cuvette and the ultraviolet absorption spectrum of CDs was measured on an ultraviolet-visible spectrophotometer.
Taking 5mg of freeze-dried powder in 5ml of PBS solution to prepare 1mg/ml of RCDs, zn-RCDs and EDTA-Zn-RCDs solution, placing the solution in a four-way fluorescence cuvette, and measuring fluorescence spectra of the solution under different excitation wavelengths on a fluorescence spectrophotometer; taking 20mg of lyophilized RCDs, zn-RCDs and EDTA-Zn-RCDs powder, sending to an analysis and test center, and performing XPS experiment under dark and vacuum conditions.
2. Experimental results
TEM images of EDTA-Zn-RCDs samples and their particle size distribution are shown in FIGS. 1a, b. TEM images of RCDs and Zn-RCDs samples and their particle size distributions are shown in FIGS. 2a, b and 2c, d. The TEM element scanning results are shown in figures 1c and d, and Zn elements are uniformly distributed in EDTA-Zn-RCDs particles, so that successful doping of zinc ions is proved.
As shown in FIGS. 1e, f, XPS characterization showed that EDTA-Zn-RCDs and Zn-RCDs contain Zn element and that the split peak of Zn2P (FIG. 1 f) shows two strong absorptions at 1020 and 1043eV, indicating Zn 6+ And Zn 3+ And it can be seen that the EDTA-Zn-RCDs and Zn-RCDs have a higher Zn element ratio than N and C elements, again demonstrating successful doping with zinc ions.
As can be seen from FIG. 3 (b), the Zn-RCDs had 11 peaks at 218nm,233nm,248nm,262nm,270nm, 284 nm,380nm,400nm,460nm,552nm and 600nm, which proved to some extent that the successful doping of Zn by RCDs resulted in a large variation in the peak shape of Zn-RCDs. The Zn-RCDs have more absorption peaks in the visible light region than RCDs, and even absorb at 610nm, so that the optical performance of carbon dots doped with metal zinc is improved to a certain extent, and the visible light with abundant resources can be better utilized. As seen in FIG. 3 (C), EDTA-Zn-RCDs have 7 absorption peaks at 219nm,232nm,248nm,261nm,272nm,280nm and 415nm, wherein the 280nm absorption peak is due to absorption by pi-pi transition of C=C bond, and wherein the 415nm absorption may be n-pi * The EDTA-Zn-RCDs are obtained by complexing Zn-RCDs with EDTA due to absorption caused by transition, and the peak shape is changed to a certain extent compared with Zn-RCDs, thus proving the success of the complexing reaction.
As shown in FIG. 4 (a), RCDs are 3386.27cm -1 Characteristic absorption band of the stretching vibration of the center absorption peak belonging to O-H and N-H, 1060.09cm -1 The peak of (C) may be a C-N stretching vibration, at 1457.08cm -1 The absorption peak at this point corresponds to the flexural vibration of C-O. As shown in FIG. 4 (b), zn-RCDs were measured at 3390.56cm -1 Characteristic absorption band of the stretching vibration of the center absorption peak belonging to O-H and N-H, 1085.84cm -1 The peak of (C-N) may represent the stretching vibration of C-N, and the successful doping of Zn by RCDs is proved to a certain extent, so that the peak shape of Zn-RCDs is greatly changed. As shown in FIG. 4 (c), EDTA-Zn-RCDs were measured at 3369.78cm -1 Characteristic absorption band of the stretching vibration of the center absorption peak belonging to O-H and N-H, 1106.23cm -1 The peak of (C) may be a C-N stretching vibration, at 1581.62cm -1 The absorption peak at the position corresponds to the in-plane bending vibration of N-H, EDTA-Zn-RCDs, and the change of peak shape compared with Zn-RCDs proves the success of the complexation reaction to a certain extent. The analysis shows that the three carbon points mainly contain elements such as carbon, oxygen, nitrogen and the like.
As shown in FIG. 5 (a), RCDs are dual-center excitation emission, the excitation wavelength is increased from 260nm to 305nm, the fluorescence intensity is increased along with the increase of the excitation wavelength in the wavelength range of 260-295nm, the fluorescence intensity is reduced along with the increase of the excitation wavelength in the wavelength range of 295-305nm, the first center maximum excitation wavelength of RCDs is 295nm, and the maximum emission wavelength is two, and the fluorescence performance is good at 342nm and 700nm respectively; then the excitation wavelength is increased from 580nm to 620nm, the fluorescence intensity is increased along with the increment of the excitation wavelength in the wavelength range of 580-600nm, and the fluorescence intensity is reduced along with the increment of the excitation wavelength in the wavelength range of 600-620nm, which means that the second center maximum excitation wavelength of the RCDs is 600nm, the maximum emission wavelength is 612nm, and the fluorescence performance is good. As shown in FIG. 5 (b), zn-RCDs are dual-center excitation emission, the excitation wavelength is increased from 240nm to 310nm, the fluorescence intensity is increased along with the increment of the excitation wavelength in the wavelength range of 240-300nm, the fluorescence intensity is reduced along with the increment of the excitation wavelength in the wavelength range of 300-310nm, the first center maximum excitation wavelength of Zn-RCDs is 300nm, and the maximum emission wavelengths are two, namely 344nm and 702nm, so that the fluorescence performance is good; then the excitation wavelength is increased from 540nm to 620nm, the fluorescence intensity is also increased along with the increment of the excitation wavelength in the wavelength range of 540-610nm, and the fluorescence intensity is reduced along with the increment of the excitation wavelength in the wavelength range of 610-620nm, which means that the maximum excitation wavelength of the second center of the Zn-RCDs is 610nm, the maximum emission wavelength is 615nm, and the fluorescence performance is good. The emission peak of Zn-RCDs is slightly red shifted compared with RCDs, and the intensity of emitted light at 615nm is significantly enhanced. The improvement of the optical properties of the carbon dots doped with metallic zinc is demonstrated to some extent. As shown in FIG. 5 (c), EDTA-Zn-RCDs are single-center excitation emission, the excitation wavelength is increased from 240nm to 300nm, the fluorescence intensity is increased along with the increment of the excitation wavelength in the wavelength range of 240-260nm, the fluorescence intensity is reduced along with the increment of the excitation wavelength in the wavelength range of 260-300nm, the maximum excitation wavelength of EDTA-Zn-RCDs is 260nm, and the maximum emission wavelengths are two, namely 345nm and 703nm, and the fluorescence performance is good. The success of EDTA complexation was demonstrated by the disappearance of the light emitted by EDTA-Zn-RCDs at about 610nm compared with Zn-RCDs.
Experimental example 2 cell biocompatibility experiment and bactericidal Effect experiment
The samples prepared in example 1 (EDTA-Zn-RCDs), comparative example 1 (RCDs) and comparative example 2 (Zn-RCDs) were used in this experimental example.
1. Experimental method
Cell culture and biocompatibility experiments:
the cytotoxicity test of RCDs, zn-RCDs and EDTA-Zn-RCDs adopts CCK8 method. L929 cells were resuscitated in H-DMEM medium containing 1% diabody and 10% foetal calf serum and cultured in a cell incubator. L929 cells in logarithmic growth phase were inoculated into 96-well plates at a density of 5000 cells per well at 37℃in 5% CO 2 Culturing in a cell culture incubator. After 24 hours, cell exchange was performed to obtain culture solutions containing RCDs, zn-RCDs and EDTA-Zn-RCDs at different concentrations (5, 10, 25, 50, 100, 200, 400 and 600. Mu.g.ml -1 ) 6 duplicate wells were set for each concentration, while the added cell broth group was set as a blank control group and incubation was continued for 24h.Finally, each well was washed 3 times with PBS, 100. Mu.l of a culture medium containing CCK-8. Mu.l was added, incubated for 2 hours, and the optical density value of each well was measured at a wavelength of 450nm by an enzyme-labeling instrument, and the relative cell viability (%) = (OD value of experimental group/OD value of blank group) ×100% was calculated therefrom.
Bacterial culture and bactericidal effect experiments:
experiments were divided into 2 groups: firstly, taking an S.aureus colony or an E.coli colony on a flat plate by using a sterilization ring, inoculating the S.aureus colony or the E.aureus colony into LB culture solution, culturing for 18-24 hours, taking 100 μl of bacterial solution, placing the bacterial solution at a wavelength of 600nm to measure the optical density value, and obtaining the bacterial concentration corresponding to the OD600 = 0.5 of 1 x 10 8 cfu.ml -1 Diluting according to the ratio of 1:100 for later use. Inoculating 100 μl of Staphylococcus aureus and Escherichia coli in logarithmic growth phase into 96-well plate, adding RCDs, zn-RCDs and EDTA-Zn-RCDs (5, 10, 25, 50, 100, 200, 400 and 600 μg.ml) -1 ) 6 compound holes are arranged at each concentration, shaking is carried out uniformly, incubation is carried out for 15min at 37 ℃, then blue light is irradiated for 30min, the distance between a blue light lamp and a sample is 20cm, light is prevented from being cultured for 24h at 37 ℃, meanwhile, a bacterial liquid group which is not added with medicines and is not subjected to blue light irradiation is set as a blank control group, the optical density value (OD 600) is measured at the wavelength of 600nm, the bacterial liquid concentration of each group is represented by the optical density value, and the relative survival rate (%) = (OD value of an experimental group/OD value of the blank control group) ×100% of bacteria is calculated.
2. Experimental results
As shown in FIGS. 6a and 7a, the concentration of RCDs reached 200. Mu.g.ml -1 When cytotoxicity reached a threshold, the average survival rate of bacteria was 57.85% and the average sterilization rate was 42.15%.
The carbon dots were doped with metallic zinc to increase the photocatalytic efficiency of the surface of the carbon dots, and as a result, the Zn-RCDs concentration reached 25. Mu.g.ml as shown in FIGS. 6b and 7b -1 When the cytotoxicity reached the threshold, the average survival rate of the corresponding bacteria was 59.3%, the average sterilization rate was 40.7%, and the concentration of Zn-RCDs reached 200. Mu.g.ml -1 The average survival rate of the corresponding bacteria is 11.8%, and the average sterilization rate is as high as 88.2%.
EDTA and Zn-RCDs midstreamThe isolated zinc ions were reacted to reduce their toxicity and then the properties of the products were examined, as shown in FIGS. 6c and 7c, at EDTA-Zn-RCDs concentrations of 0-600. Mu.g.ml -1 Within the range, the cell viability is still above 75%, and the average viability of bacteria shows a decreasing trend with increasing concentration of EDTA-Zn-RCDs, and reaches 600 mug.ml at the concentration -1 When the bacterial strain is used, the average survival rate reaches 11.9%, and the average sterilization rate reaches 88.1%.
It can be seen that EDTA-Zn-RCDs have significantly reduced cytotoxicity while maintaining bactericidal effects.
Experimental example 3 zinc doping amount screening experiment
The bactericidal effect was examined by the method of experimental example 2 using the sample prepared in example 4. The sterilization efficiency of EDTA-Zn-RCDs can be improved by doping zinc into carbon dots, and in order to examine the influence of different zinc doping amounts in the EDTA-Zn-RCDs on the sterilization efficiency, EDTA-Zn-RCDs with different Zn doping amounts (0, 0.1, 0.5, 1.0 and 2.0 mmol) are combined with blue light power to observe the sterilization efficiency of the EDTA-Zn-RCDs on staphylococcus aureus. As shown in FIG. 8, the zinc doping amount of EDTA-Zn-RCDs is in the range of 0-2.0mmol, the average survival rate of bacteria shows a tendency of decreasing before leveling with increasing zinc doping amount, and when the zinc doping amount reaches 1.0mmol, the average survival rate is as low as 10.5%, and when the zinc doping amount reaches 2.0mmol, the average sterilization rate is as high as 89.5%, and meanwhile, when the zinc doping amount reaches 2.0mmol, the average survival rate reaches 11%, and the average sterilization rate reaches 89%. While maintaining its highest sterilization rate, we finally selected EDTA-Zn-RCDs with a zinc doping level of 1.0mmol.
Experimental example 4 drug resistance experiment
This experimental example uses the sample prepared in example 1 (EDTA-Zn-RCDs).
1. Experimental method
Experiments were divided into 2 groups: firstly, taking an S.aureus colony on a flat plate by using a sterilization ring, inoculating the colony into LB culture solution, culturing for 18-24 hours, taking 100 mu l of bacterial solution, placing the bacterial solution at a wavelength of 600nm to measure optical density value, and obtaining the corresponding bacterial concentration of 1 multiplied by 10 with OD600 = 0.5 8 cfu.ml -1 Diluting according to the ratio of 1:100 for later use. Taking the mixture in the logarithmic phaseThe staphylococcus aureus was inoculated into 96-well plates at 100 μl per well, and the carbon spot + blue light group was added to a final concentration of 600 μg.ml -1 EDTA-Zn-RCDs liquid medicine, 6 compound holes are arranged, shaking is carried out uniformly, incubation is carried out for 15min at 37 ℃, then blue light is irradiated for 30min, the distance between a blue light lamp and a sample is 20cm, light is prevented from being cultured for 24h at 37 ℃, meanwhile, a bacterial liquid group which is not added with medicines and is not subjected to blue light irradiation is arranged as a blank control group, an optical density value (OD 600) is measured at the wavelength of 600nm, the concentration of each bacterial liquid is represented by the optical density value, and the relative survival rate (%) = (OD value of an experimental group/OD value of the blank control group) multiplied by 100% of the bacterial liquid is calculated. Viable bacterial cells were re-cultured in fresh LB medium for 24 hours, and then subjected to the 7 rounds of treatment described above, each round of relative bacterial viability was calculated.
2. Experimental results
To examine whether the EDTA-Zn-RCDs+blue light antimicrobial regimen breaks through the drug-resistant bacteria infection, this experimental example simulates the bacterial drug-resistant process, and 7 rounds of experiments were repeated, after each round of which surviving Staphylococcus aureus was immediately separated and subjected to the next round of treatment after the viability assay, and as a result, the average bacterial viability for these 7 rounds was 10.32%,10.07%,10.17%,9.86%,9.85%,9.46% and 10.35%, respectively, as shown in FIG. 9. Therefore, it can be inferred that the EDTA-Zn-RCDs+blue light antibacterial scheme does not cause bacteria to generate drug resistance, has the advantages of low drug resistance, little damage to peripheral tissues, low toxic and side effects and the like, and has great clinical transformation potential for treating drug-resistant bacterial infection.
Experimental example 5 in vivo antibacterial experiment
This experimental example uses the sample prepared in example 1 (EDTA-Zn-RCDs).
1. Experimental method
The total of 30 mice are divided into 5 groups, namely, a healthy group is respectively a non-modeling group, and the modeling group is divided into four groups: PBS group, blue light irradiation group, EDTA-Zn-RCDs group and EDTA-Zn-RCDs+blue light irradiation group, 6 mice per group. First, a skin wound model of a bacterial infected mouse was established, and first, the mouse was anesthetized with diethyl ether. Then, a circular mark with a diameter of 1cm is made on the back dehairing area, the skin is cut off along the edge, and then the prepared skin is injected at the periphery of the woundA good concentration is 1.0X10 8 CFU/ml of Staphylococcus aureus liquid 100. Mu.l. The following day, the wound surface of the back of the mouse has liquid secretion, and is red and swollen, which indicates that the modeling is successful. Subsequently, infected mice were randomly divided into 4 groups of 6 mice each. The 4 groups of mice received different treatment regimens: PBS, EDTA-Zn-RCDs (600. Mu.g/ml), blue light irradiation and EDTA-Zn-RCDs (600. Mu.g/ml) +blue light irradiation. Mu.l of EDTA-Zn-RCDs (600. Mu.g/ml) and PBS were each added dropwise to the affected wound, wherein the light group was irradiated with blue light for 30min at a distance of 20cm from the sample. During the treatment, the mice were under anesthesia. Wound pictures were taken at different time points (0 d, 2d, 4d, 6d, 8d and 10 d). After 10d, first, bacteria at the wound site are sampled, and the skin with lesions on the back is taken, and homogenized by adding 10ml of sterile physiological saline into each 50mg of skin. Sequentially diluting the homogenate to 10 as stock solution -1 、10 -2 、10 -3 、10 -4 Mu.l of each dilution was taken in LB plates, and 3 plates were set for each dilution. And collecting skin tissue and viscera (heart, liver, spleen, lung and kidney) from the wound site of the mice for histological analysis (hematoxylin and eosin staining: H&E)。
2. Experimental results
As shown in fig. 10, which shows a flow chart of animal modeling treatment, modeling was performed on day 0, treatment was performed on day 2, wound pictures were taken at various time points (0 d, 2d, 4d, 6d, 8d, and 10 d), mice were sacrificed on day 10, and reverse culture of bacteria at the wound and tissue embedding sections and staining were performed.
To more intuitively monitor the effect of different treatment regimens on mouse wounds, wound pictures were taken at 0d, 2d, 4d, 6d, 8d and 10d, respectively. FIG. 11 shows four animals, EDTA-Zn-RCDs, blue light and EDTA-Zn-RCDs+blue light, with the wounds of the 4 animals becoming smaller over time, and the EDTA-Zn-RCDs and blue light animals showed no significant wound size and healing compared to the control, whereas the EDTA-Zn-RCDs+blue light had significantly smaller wounds and healed all over the 10 th day, and it was evident from the figure that new skin tissue was formed.
The experimental example is also that the skin of the wound of the mouse is subjected to reverse bacterial culture on the 10 th day so as to observe the bacterial number of the wound, as shown in fig. 12, the experimental example is a control group, namely an EDTA-Zn-RCDs group, a blue light group, an EDTA-Zn-RCDs+blue light group and a healthy group, the bacterial colony number of the EDTA-Zn-RCDs group and the blue light group is not obviously reduced compared with the control group, which indicates that the EDTA-Zn-RCDs and the blue light only have no inhibition effect on bacteria, and the bacterial colony number of the EDTA-Zn-RCDs+blue light group is obviously reduced compared with the control group, and is equivalent to the bacterial colony number of the healthy group, which indicates that the novel antibacterial strategy of EDTA-Zn-RCDs+blue light has good antibacterial effect on bacterial infection of wounds in the mouse. As shown in fig. 13, which is a graph of the corresponding bacterial survival rate statistics, the bacterial survival rate of EDTA-Zn-rcds+blue group is significantly different from that of the control group, and the average bacterial survival rate is 8.83% and the average sterilization efficiency is as high as 91.17% similar to that of the healthy group.
Meanwhile, mice were sacrificed on day 10 and their heart, liver, spleen, lung and kidney skin was removed for embedding, sectioning and HE staining, fig. 14 is a histological analysis of the skin at five animal infections of EDTA-Zn-RCDs, blue light, EDTA-Zn-rcds+blue light and healthy groups, the number of animal inflammatory cells (blue cells) of EDTA-Zn-RCDs and blue light alone was not significantly reduced compared to the control, many denser inflammatory cells were still observed, the number of inflammatory cells of EDTA-Zn-rcds+blue light was significantly reduced compared to the control, but some scattered and sparse inflammatory cells were still observed, similar to the number of inflammatory cells of healthy groups, and it could also be demonstrated that the treatment regimen of EDTA-Zn-rcds+blue light effectively alleviated inflammation at the 10 th day. The viscera (heart, liver, spleen, lung and kidney) of each mouse on day 10 are also collected, and histological analysis is carried out on the viscera sections of five groups of animals, namely a control group, an EDTA-Zn-RCDs group, a blue light group, an EDTA-Zn-RCDs+blue light group and a healthy group, so that the toxicity effect of the EDTA-Zn-RCDs in the body of the mouse is examined, as shown in figure 15, no obvious viscera injury or inflammation injury is found in each group, no obvious difference exists between the groups, and the fact that the toxicity of the EDTA-Zn-RCDs in the body of the mouse is small is indicated, and the biosafety is good.
The experimental example shows that the EDTA-Zn-RCDs+blue light irradiation sterilization scheme provided by the invention can generate good sterilization effect in animals, and has low toxicity in animals and good biological safety.
Experimental example 6 stability experiment
This experimental example uses the sample prepared in example 1 (EDTA-Zn-RCDs).
1. Experimental method
Stability experiments of EDTA-Zn and EDTA-Zn-RCDs lyophilized powder:
the same batch of EDTA-Zn and EDTA-Zn-RCDs was subjected to L929 cytotoxicity test every 4 days within 28 days to examine the stability of EDTA-Zn and EDTA-Zn-RCDs lyophilized powder, and the concentrations of EDTA-Zn and EDTA-Zn-RCDs were 600. Mu.g/ml.
2. Experimental results
Stability test of EDTA-Zn and EDTA-Zn-RCDs lyophilized powder
The experimental results are shown in fig. 16. Within 28 days, 600 μg.ml-1EDTA-Zn and EDTA-Zn RCD cytotoxicity was above the threshold. Thus, it can be inferred that toxic zinc ions are not present in biological systems within 28 days, and EDTA-Zn-RCDs are very stable in powder state.
It can be seen from the above examples and experimental examples that the present invention prepares a new low toxicity zinc-doped carbon dot by coating EDTA on the surface of the zinc-doped carbon dot through complexation. The zinc-doped carbon dot has photoinduced sterilization equivalent to the existing zinc-doped carbon dot, and the biotoxicity of the zinc-doped carbon dot is obviously reduced compared with the existing zinc-doped carbon dot. In addition, the low-toxicity zinc-doped carbon dot of the present invention does not cause bacteria to develop resistance, and it can maintain low toxicity and excellent bactericidal effect for a long period of time. Therefore, the invention has good application prospect in clinical application.

Claims (9)

1. The low-toxicity zinc doped carbon dot is characterized by being prepared from the following raw materials in parts by weight:
268-328 parts of zinc doped carbon dots,
EDTA-2Na 335-409.5 parts;
in the low-toxicity zinc-doped carbon dots, complex ions of EDTA-2Na cover the surfaces of the zinc-doped carbon dots;
the particle size of the zinc-doped carbon dots is 20nm-40 nm;
the preparation method of the low-toxicity zinc-doped carbon dot comprises the following steps:
step 1, mixing zinc doped carbon dots with EDTA-2Na to prepare a mixed solution;
and 2, regulating the pH of the mixed solution to 5.8-6.2 for reaction, wherein the reaction condition is that the reaction is carried out at 58-62 ℃ for 1.5-2h, and the preparation method is finished.
2. The low toxicity zinc doped carbon dot of claim 1, wherein: the zinc-doped carbon point is prepared from o-phenylenediamine, L-tryptophan and zinc ion-containing salt, wherein the molar ratio of the o-phenylenediamine to the L-tryptophan to the zinc ion is 0.9-1.1:0.24-0.26:0.9-1.1.
3. The low toxicity zinc doped carbon dot of claim 2, wherein: the zinc ion-containing salt is selected from at least one of zinc chloride, zinc acetate or zinc tartrate.
4. Use of a low toxicity zinc doped carbon dot according to any of claims 1-3 as a photosensitizer for non diagnostic and therapeutic purposes.
5. Use of a low toxicity zinc doped carbon dot according to any of claims 1-3 for the preparation of a light induced fungicide.
6. A light-induced bactericide, characterized in that: it is prepared by taking the low-toxicity zinc-doped carbon dots as the photosensitizer and adding pharmaceutically acceptable auxiliary materials or auxiliary components.
7. A method for preparing a low toxicity zinc doped carbon dot according to any one of claims 1 to 3, comprising the steps of:
step 1, mixing zinc doped carbon dots with EDTA-2Na to prepare a mixed solution;
and 2, regulating the pH of the mixed solution to 5.8-6.2 for reaction, wherein the reaction condition is that the reaction is carried out at 58-62 ℃ for 1.5-2h, and the preparation method is finished.
8. The method of preparing as claimed in claim 7, wherein: and 2, after the reaction is finished, sterilizing the mixed solution after the reaction by using a filter membrane with the diameter of 0.22 mu m, and freeze-drying to obtain the low-toxicity zinc-doped carbon dot.
9. The method of preparing as claimed in claim 7, wherein: the zinc-doped carbon dots are prepared by the following steps:
step A, mixing and dissolving o-phenylenediamine, L-tryptophan and zinc ion-containing salt in water in a reaction kettle to obtain a reaction solution, and adding 36-38% of concentrated hydrochloric acid by mass percent; the volume ratio of the reaction solution to the concentrated hydrochloric acid is 9-11:0.19-0.21;
and step B, reacting for 2.9-3.1h at 198-202 ℃ to obtain the catalyst.
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