A multifunctional antibacterial nanomaterial TCFNPs and its preparation method and application
Through FECL3 and TCFNPs prepared by FECL3 and tetracycline in ethanol, the problem of unstable and insufficient antibacterial effects in photophamotic therapy in photosidth therapy is solved, and its stability and antibacterial effect in photogenic therapy are realized.
Patent Information
- Application Number
- CN202310530828.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-12
- Publication Date
- 2025-01-28
- Estimated Expiration
- 2043-05-12
AI Technical Summary
Tetracycline is unstable in aqueous solution and its antibacterial effect is not ideal in the face of multidrug-resistant superbacterials, limiting its application in photothermal therapy.
By reacting tetracycline with FeCL3 in ethanol, the nano particles TCFNPs that complexy tetracycline complexy tetracycline is formed, and a pot of synthesis is performed under the condition of 0.5-1.5.
The prepared multi -functional antibacterial nanomaterial TCFNPS has good stability and chemical, chemical, and photoresia coordinated antibacterial functions, which significantly improves the bacteriostatic and sterilization capabilities of tetracycline in photocalism.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of metal nanomaterials, and specifically relates to a multifunctional antibacterial nanomaterial TCFNPs and a preparation method and application thereof. Background Art
[0002] Drug delivery system (DDS) has the characteristics of tapping the antibacterial potential of antibiotics, bypassing the original drug resistance of bacteria, and protecting drug molecules. It is an effective means to deal with the threat of super bacteria.
[0003] Tetracycline (TC) is a commonly used broad-spectrum antibiotic. It is unstable in aqueous solution. As the pH of the solution changes, tetracycline undergoes changes such as epimerization, dehydration or esterification. This shortcoming seriously restricts the use of tetracycline. To overcome this shortcoming, the hydrochloride of tetracycline, i.e., tetracycline hydrochloride, is often used. However, tetracycline hydrochloride is still unstable when exposed to light and needs to be stored away from light. At the same time, with the large-scale emergence of multidrug-resistant super bacteria in recent years, the antibacterial effect of tetracycline is very unsatisfactory, and its clinical application has been greatly limited.
[0004] Photothermal therapy (PTT) is a novel and rapidly developing treatment method. Its principle is to use the characteristics of nanoparticles absorbing light energy and converting it into heat energy. It uses near-infrared light (NIR) of a specific wavelength to irradiate the medication site, increase the local temperature, and thus inhibit bacterial proliferation and cause bacterial death. However, as a key carrier of photothermal therapy, the preparation method of nano-antibacterial material TCFNPs has not been fully explored, which greatly limits the application of tetracycline in photothermal therapy. Summary of the invention
[0005] In order to prepare the multifunctional antibacterial nanomaterial TCFNPs, the present invention uses tetracycline (TC), ethanol, polyvinyl pyrrolidone (PVP) and FeCl3 as raw materials to synthesize TCFNPs by a one-pot method. The multifunctional antibacterial nanomaterial TCFNPs prepared by the present invention not only has good stability, but also has the function of chemical and photothermal synergistic antibacterial, realizes the application of tetracycline in the field of photothermal therapy, and has broad application prospects in antibacterial and sterilization.
[0006] The multifunctional antibacterial nanomaterial TCFNPs of the present invention is composed of Fe 3+ Nanoparticles formed by complexing tetracycline.
[0007] The preparation method of the multifunctional antibacterial nanomaterial TCFNPs of the present invention is as follows: dissolving appropriate amounts of tetracycline and polyvinyl pyrrolidone in anhydrous ethanol, adjusting the pH value to a target value, and then adding an appropriate amount of anhydrous ethanol solution of FeCl3, reacting at a temperature of 10-30°C for 1-60 minutes, centrifuging the mixture, and retaining the precipitate to obtain the multifunctional antibacterial nanomaterial TCFNPs.
[0008] Among them, Fe 3+ The molar ratio of tetracycline to 4:1-8:1.
[0009] Polyvinyl pyrrolidone (PVP) was used as a dispersant, and the amount added was 2-5 mg·mL -1 .
[0010] The pH value of the reaction system is 0.5-1.5.
[0011] The present invention utilizes Fe 3+ Strong complexing ability makes Fe 3+ It can be complexed with phenolic compounds to form a metal-phenol network with photothermal effect.
[0012] The multifunctional antibacterial nanomaterial TCFNPs prepared according to the above method has a hydrated particle size of 10-50nm and good stability. It has shown better stability than tetracycline under light and long-term storage conditions. At the same time, the nanomaterial has the ability to carry out photothermal therapy and has shown an antibacterial effect better than tetracycline in in vitro experiments.
[0013] Compared with the prior art, the present invention has the following beneficial effects:
[0014] 1. The preparation process involved in the present invention is simple, the reaction conditions are mild, the production efficiency is high, and it complies with the concept of green environmental protection.
[0015] 2. The multifunctional antibacterial nanomaterial TCFNPs obtained by the present invention has good stability, and its stability under light or long-term storage conditions is better than that of tetracycline.
[0016] 3. The multifunctional antibacterial nanomaterial TCFNPs obtained by the present invention has the function of implementing photothermal therapy and improves the antibacterial and antimicrobial capabilities of tetracycline.
[0017] 4. The raw materials used in the present invention are easy to obtain, low in cost, and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is the appearance of the mixture when the reaction of Example 1 is completed.
[0019] Figure 2 This is the appearance of the TCFNPs precipitate obtained after centrifugation in Example 1.
[0020] Figure 3 This is the morphology of TCFNPs obtained by freeze-drying in Example 1.
[0021] Figure 4 This is the hydrated particle size distribution diagram of TCFNPs in Example 1.
[0022] Figure 5 This is a comparison chart of the changes in TC aqueous solution after storage under different conditions for 14 days.
[0023] Figure 6 This is a comparison chart of the changes in the TCFNPs aqueous dispersion of Example 1 after storage for 14 days under different conditions.
[0024] Figure 7 The inhibition zone diagram of TC against Staphylococcus aureus and Escherichia coli after storage for 14 days under different conditions.
[0025] Figure 8 The antibacterial zone diagrams of TCFNPs of Example 1 against Staphylococcus aureus and Escherichia coli after storage for 14 days under different conditions.
[0026] Fig. 9 The figure is a statistical bar graph of the diameter of the inhibition zone of TC against Staphylococcus aureus and Escherichia coli after storage for 14 days under different conditions.
[0027] Fig.10 Statistical bar graphs of the diameters of the inhibition zones of the TCFNPs of Example 1 against Staphylococcus aureus and Escherichia coli after storage for 14 days under different conditions.
[0028] Fig.11 Graph showing the temperature change of TCFNPs aqueous dispersions of Example 1 at different concentrations under near-infrared light irradiation.
[0029] Fig.12 25 μg mL -1 Temperature change diagram of the TCFNPs aqueous dispersion of Example 1 when repeatedly irradiated with near-infrared light.
[0030] Fig.13 The figure is a bar graph showing the survival rate of Staphylococcus aureus under different conditions.
[0031] Fig.14 The figure is a statistical bar graph of the survival rate of Escherichia coli under different conditions.
[0032] Fig.15 Live / Dead staining images of Staphylococcus aureus under different conditions.
[0033] Fig.16 Live / Dead staining images of Escherichia coli under different conditions.
[0034] Fig.17 Figure 2 shows the biofilm formation inhibition of Staphylococcus aureus under different conditions.
[0035] Fig.18 Figure 2 shows the biofilm formation inhibition of Escherichia coli under different conditions.
[0036] Fig.19 Diagram showing the destruction of biofilm formed by Staphylococcus aureus under different conditions.
[0037] Fig. 20 Diagram showing the destruction of biofilm formed by Escherichia coli under different conditions. DETAILED DESCRIPTION
[0038] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments.
[0039] Example 1
[0040] 1. Preparation and purification of multifunctional antibacterial nanomaterials TCFNPs
[0041] Dissolve 40 mg of polyvinylpyrrolidone (PVP) in 6.625 ml of anhydrous ethanol and add 0.375 ml of 0.4 mol ml -1 Ferric chloride (dissolved in anhydrous ethanol) and mix well, slowly add 3ml 0.01mmol ml -1 Tetracycline hydrochloride (TC) ethanol solution, adjust pH to 1.0, the total volume of the solution is 10mL. React at room temperature for 30 minutes, the resulting mixture is composed of Figure 1 The mixture was centrifuged to obtain a brown-black precipitate (see attached Figure 2 The precipitate was fully washed with anhydrous ethanol, and then dispersed in water and freeze-dried to obtain multifunctional antibacterial nanomaterials TCFNPs, whose morphology is composed of attached Figure 3 shown.
[0042] 2. Determination of hydrated particle size of multifunctional antibacterial nanomaterial TCFNPs
[0043] The hydrated particle size of TCFNPs was determined by dynamic light scattering (DLS). The specific steps were as follows: the brown-black precipitate in step 1 was fully washed with anhydrous ethanol and then added with deionized water. The precipitate was fully shaken to disperse evenly to form a 2 ml TCFNPs aqueous dispersion. The solution was measured at 25°C and a scattering angle of 173°. Each sample was measured three times, and the average particle size was calculated by volume distribution. The hydrated particle size distribution of TCFNPs was determined by the attached Figure 4 shown.
[0044] According to the attached Figure 4 The hydrated particle size of TCFNPs is distributed in a narrow area around 9.95nm, with an average of 9.95nm±3.54nm. The polydispersity index (PdI) is 0.201, which is less than 0.3, indicating that TCFNPs have good dispersion and are relatively stable in water without obvious agglomeration, which meets the research requirements.
[0045] 3. Characterization of the stability of multifunctional antibacterial nanomaterials TCFNPs
[0046] The stability of TCFNPs was characterized by the inhibition zone method. The specific steps were as follows: -1 Tetracycline (TC) aqueous solution and TCFNPs aqueous dispersion were prepared. One portion of TC aqueous solution and one portion of TCFNPs aqueous dispersion were stored at 4°C in the dark, and the other two samples were kept indoors without being protected from light. After 14 days, the changes of TC and TCFNPs were respectively Figure 5 , 6 The samples were diluted to 10, 20, and 30 μg mL -1 For determination of inhibition zone. Place 4 Oxford cups inoculated with 100 μL 10 7 CFU / mL of Staphylococcus aureus and Escherichia coli, add 100 μL of sterile PBS, 10, 20, and 30 μg mL -1 TC or TCFNPs, after 12 hours of culture, the inhibition zone images were obtained by Figure 7 , 8 As shown in the figure, the diameter of the inhibition zone is measured by Fig. 9 , 10 shown.
[0047] By the attached Figure 5 , 6 As shown in the figure, after 14 days of storage, the aqueous solution of TC changed significantly, indicating that TC may have undergone degradation, isomerization and other reactions; while the aqueous dispersion of TCFNPs did not change significantly, preliminarily proving that TCFNPs are more stable than TC under low temperature and light-proof conditions. Figure 7 , 8 As shown in the figure, except for the control group with PBS, all other experimental groups had inhibition zones. On the same culture plate, the diameter of the inhibition zone increased with the increase of sample concentration. Fig. 9 , 10 As shown, when dealing with Staphylococcus aureus and Escherichia coli, the diameter of the inhibition zone formed by adding TC that was not stored at low temperature and away from light was about 25% smaller than that of TC stored at low temperature and away from light, while there was no significant change in the diameter of the inhibition zone between the two groups of TCFNPs. It can be considered that the effective ingredients of TC were significantly reduced after storage for 14 days, while the effective ingredients of TCFNPs were not significantly reduced, further proving that TCFNPs have higher stability than TC.
[0048] 4. Characterization of the photothermal properties of multifunctional antibacterial nanomaterials TCFNPs
[0049] 1) Verification of the photothermal heating capability of TCFNPs
[0050] TCFNPs were formulated into 1, 5, 10, 15, 20, and 25 μg mL -1 The aqueous dispersion was prepared by using a near-infrared light source (NIR, 3.0 W·cm -2 &808nm) continuous irradiation, record the temperature difference (ΔT) of the liquid, and the experimental results are shown in the attached Fig.11 shown.
[0051] By the attached Fig.11 It can be seen that under continuous NIR irradiation, the TCFNPs aqueous dispersions with different concentrations have obvious temperature increases, and ΔT increases with the increase of concentration. Even at low concentrations, TCFNPs still have good photothermal heating ability.
[0052] 2) Verification of the photothermal stability of TCFNPs
[0053] In the study of the photothermal stability of TCFNPs, a near-infrared light source (NIR, 3.0 W cm -2 &808nm) for a concentration of 25μg·mL -1 The TCFNPs aqueous dispersion was irradiated for three cycles, each cycle was irradiated for 480 seconds, and then irradiated again after the liquid cooled to room temperature. The temperature change curve obtained is shown in the attached Fig.12 shown.
[0054] By the attached Fig.12 As shown, in three cycles, the concentration was 25 μg mL -1 The TCFNPs aqueous dispersion can always reach 55°C, and the temperature change curves of the three cycles are basically the same, which proves that the photothermal performance of TCFNPs is still very stable even after repeated irradiation.
[0055] 5. Characterization of antibacterial properties of multifunctional antibacterial nanomaterials TCFNPs
[0056] 1) Determination of the minimum inhibitory concentration (MIC) of TCFNPs
[0057] The MIC of TCFNPs was determined by the culture medium turbidity method. The specific steps were as follows: 180 μL of 10 6 CFU / mL Staphylococcus aureus or Escherichia coli and 20 μL 50-350 μg mL -1 TCFNPs or TC were co-incubated for 16 h, and the TCFNPs+Light group was illuminated with a near-infrared light source (NIR, 3.0 W·cm -2After incubation, the absorbance of each microwell at 600nm (OD600) was measured using a microplate reader. The microwells with only bacterial solution added were used as control wells, and the microwells with only TC or TCFNPs added were used as background wells. Each experiment was repeated three times. The measured OD600 (au) was determined by the attached Fig.13 , 14 shown.
[0058] The minimum concentration at which OD600 (au) was less than 1% was selected as MIC. Fig.13 As shown, in the case of Staphylococcus aureus, the MIC TC MIC TCFNPs and MIC TCFNPs+Light 25 μg·mL -1 , 25 μg·mL -1 and 15 μg mL -1 . Fig.14 As shown, in the case of Escherichia coli, the MIC TC MIC TCFNPs and MIC TCFNPs+Light 25 μg·mL -1 , 25 μg·mL -1 and 20 μg mL -1 In summary, it can be shown that TCFNPs have similar chemical antibacterial properties to TC for both Staphylococcus aureus and Escherichia coli. After combined with photothermal therapy, the antibacterial performance of TCFNPs+Light is significantly better than that of TC and TCFNPs, indicating that TCFNPs can carry out chemical antibacterial and photothermal antibacterial and has good synergistic antibacterial properties.
[0059] 2) Live / Dead staining experiment of bacteria after TCFNPs treatment
[0060] Live / Dead staining reagent was used to detect the survival of bacteria after TCFNPs treatment. The specific steps were as follows: 100 μL of 30 μg mL -1 TC or TCFNPs were added to 100 μL of 10 8 CFU / mL of Staphylococcus aureus or Escherichia coli, TCFNPs+Light group used NIR (3.0W·cm -2 &808nm) for 10 minutes, and PBS was used as the control group. All samples were incubated for 30 minutes, and then centrifuged at 4°C and 5000rpm for 10 minutes. Live / Dead staining reagent was added to the precipitate, and after standing for 15 minutes in the dark, 20μL was dropped on a slide and observed under an inverted fluorescence microscope. The staining images obtained were Fig.15 , 16 shown.
[0061] The Live / Dead staining reagent consists of two fluorescent dyes, SYTO 9 and propidium iodide (PI). SYTO 9 can enter all bacteria, while PI only stains bacteria with damaged membrane structures. When the two dyes appear at the same time, the fluorescence intensity of SYTO 9 will weaken. That is, bacteria with intact membrane structures will show green fluorescence, and bacteria with damaged membrane structures will show red fluorescence. Fig.15 , 16 The results showed that when dealing with Staphylococcus aureus and Escherichia coli, the control group showed evenly dispersed bright green fluorescence, indicating that the bacterial structure was intact and active; the TC and TCFNPs experimental groups showed basically the same obvious red fluorescence, indicating that the membrane structure of some bacteria was destroyed and lost their activity, which shows that TCFNPs without photothermal therapy have similar bactericidal activity as TC.
[0062] The TCFNPs+Light experimental group showed a large area of red fluorescence, indicating that the membrane structure of a large number of bacteria was destroyed, further demonstrating that photothermal therapy using TCFNPs can achieve synergistic antibacterial properties that are superior to TC.
[0063] 6. Experiment on the inhibition and destruction of biofilm by multifunctional antibacterial nanomaterials TCFNPs
[0064] 1) Experiment on inhibition of biofilm formation by TCFNPs
[0065] 180 μL 10 5 CFU / mL Staphylococcus aureus or Escherichia coli and 20 μL 200
[0066] μg·mL -1 TCFNPs were added to a 96-well plate and incubated for 72 h. The TCFNPs+Light group was exposed to NIR (3.0 W·cm -2 &808nm) for 5 minutes. After incubation, the microwells were gently washed twice with PBS, stained with 1% crystal violet for 10 minutes, washed again with PBS and air-dried, 80% ethanol was added to dissolve the crystal violet, and the absorbance of each well at 590nm (OD590) was measured using an ELISA reader. PBS was used as the control group, and each experiment was repeated 3 times. The measured OD590 was determined by the attached Fig.17 , 18 shown.
[0067] By observing the Fig.17 , 18By studying the formation of biofilms in the experiment, it can be found that TCFNPs and TCFNPs+Light effectively inhibited the biofilm formation of Staphylococcus aureus by 22% and 65%, and the biofilm formation of Escherichia coli by 63% and 84%, respectively, indicating that TCFNPs has a good ability to inhibit biofilm formation.
[0068] 2) Experiments on the destruction of established biofilms by TCFNPs
[0069] 200 μL 10 5 CFU / mL Staphylococcus aureus or Escherichia coli was added to a 96-well plate and incubated for 72 h. When a complete biofilm was formed, the medium was discarded and 200 μL of 20 μg mL -1 TCFNPs were incubated for 1 hour. The TCFNPs+Light group was treated with NIR (3.0 W·cm -2 &808nm) for 5 minutes. After incubation, the microwells were gently washed twice with PBS, stained with 1% crystal violet for 10 minutes, washed again with PBS and air-dried, 80% ethanol was added to dissolve the crystal violet, and the absorbance of each well at 590nm (OD590) was measured using an ELISA reader. PBS was used as the control group, and each experiment was repeated 3 times. The measured OD590 was determined by the attached Fig.19 , 20 shown.
[0070] By observing the Fig.19 , 20 From the residual situation of biofilm in the experiment, it can be found that TCFNPs and TCFNPs+Light destroyed 24% and 64% of the biofilm formed by Staphylococcus aureus and 39% and 90% of the biofilm formed by Escherichia coli, respectively, indicating that TCFNPs has a good ability to destroy the formed biofilm.
[0071] Example 2
[0072] Same as step 1 and step 2 of Example 1, except that Fe 3+ The molar ratio of Fe and tetracycline was set to 4:1-8:1, and the measured hydrated particle size of multifunctional antibacterial nanomaterial TCFNPs is shown in Table 1. 3+ The molar ratio of tetracycline to Fe is in the range of 4:1-8:1. 3+ With the increase of the molar ratio of Fe and tetracycline, the hydrated particle size of TCFNPs first decreased and then increased. 3+ When the molar ratio of tetracycline to TCFNPs is 5:1, the hydrated particle size of TCFNPs is the smallest.
[0073] Table 1. Hydration particle size of TCFNPs at different PVP concentrations
[0074]
[0075] Example 3
[0076] The same as step 1 and step 2 of Example 1, only the amount of polyvinyl pyrrolidone (PVP) added was changed to 20, 30, 40, 50 mg, that is, the concentration of PVP in the reaction solution was 2-5 mg ml -1 The measured hydrated particle size of multifunctional antibacterial nanomaterial TCFNPs is shown in Table 2. The results show that when the PVP concentration is 2-5 mg ml -1 Within the range of 4 mg ml -1 When , the hydrated particle size of TCFNPs is the smallest.
[0077] Table 2. Hydration particle size of TCFNPs at different PVP concentrations
[0078]
[0079] Example 4
[0080] The same as step 1 and step 2 of Example 1, only the pH value was set to 0.5, 1.0, and 1.5. The measured hydrated particle size of the multifunctional antibacterial nanomaterial TCFNPs is shown in Table 3. The results show that within the pH range of 0.5-1.5, as the pH increases, the hydrated particle size of TCFNPs first decreases and then increases, and when the pH is 1.0, the hydrated particle size of TCFNPs is the smallest.
[0081] Table 3. Hydration particle size of TCFNPs under different pH conditions
[0082]
Claims
1. A method for preparing multifunctional antibacterial nanomaterial TCFNPs, characterized in that: The preparation method comprises the following steps: dissolving tetracycline and polyvinyl pyrrolidone in anhydrous ethanol, adjusting the pH value, and then adding an anhydrous ethanol solution of FeCl3. After the reaction is completed, centrifuging the mixture and retaining the precipitate to obtain the multifunctional antibacterial nanomaterial TCFNPs.
2. The method for preparing the multifunctional antibacterial nanomaterial TCFNPs according to claim 1, characterized in that: Fe 3+ The molar ratio of tetracycline to 4:1-8:
1.
3. The method for preparing the multifunctional antibacterial nanomaterial TCFNPs according to claim 1, characterized in that: Polyvinyl pyrrolidone (PVP) is used as a dispersant, and its addition amount is 2-5 mg·mL -1 .
4. The method for preparing the multifunctional antibacterial nanomaterial TCFNPs according to claim 1, characterized in that: The pH value of the reaction system is adjusted to 0.5-1.5, the reaction temperature is 10-30°C, and the reaction time is 1-60min.
5. The multifunctional antibacterial nanomaterial TCFNPs prepared by the method according to claim 1, characterized in that: The multifunctional antibacterial nanomaterial TCFNPs is composed of Fe 3+ Nanoparticles formed by complexing tetracycline.
6. The multifunctional antibacterial nanomaterial TCFNPs according to claim 5, characterized in that: The hydrated particle size of TCFNPs is 10-50 nm.
7. The use of the multifunctional antibacterial nanomaterial TCFNPs prepared by the method according to claim 1, characterized in that: The TCFNPs were used as chemical and photothermal antibacterial materials.
8. The use of the multifunctional antibacterial nanomaterial TCFNPs according to claim 7, characterized in that: TCFNPs were used as antibacterial materials for Staphylococcus aureus and Escherichia coli.