Near-infrared responsive antibacterial nanomaterial, its preparation method and application

Abstract

This invention proposes a near-infrared responsive antibacterial nanomaterial, its preparation method, and its application. The near-infrared responsive antibacterial nanomaterial uses CuO2 nanoparticles and ICG as the core and PCM phase change material as the shell. It has good antibacterial properties, no drug resistance, and stability. Moreover, it achieves combined antibacterial activity in three modes: CDT / PDT / PTT, exhibiting broad-spectrum antibacterial properties and good biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of antibacterial materials technology, and in particular to a near-infrared responsive antibacterial nanomaterial, its preparation method, and its application. Background Technology

[0002] Bacteria can cause a variety of skin and soft tissue infections, making them one of the greatest enemies of human health today. Current treatments mainly involve using high-dose antibiotics to combat bacterial infections, but the rapid emergence and evolution of bacterial resistance makes clinical infection treatment extremely difficult.

[0003] In recent years, antibacterial strategies based on the generation of reactive oxygen species (ROS) through chemodynamic therapy (CDT) and photodynamic therapy (PDT) have attracted widespread attention. However, the lack of H2O2 and O2 in the skin infection microenvironment severely reduces their antibacterial efficiency. Metal peroxides can react with H2O to generate H2O2 and can decompose to generate O2 under heating conditions. However, the instability, easy decomposition, and strong toxicity of metal peroxides greatly limit their application in the biomedical field.

[0004] The literature (Self-enhanced ROS generation by responsive co-delivery of H2O2 and O2 based on a versatile composite biomaterial for hypoxia-irrelevant multimodal antibiofilm therapy, Jiayu Xiao et al., Chemical Engineering Journal, Volume 465, June 2023, 142958) loaded CuO2 and ICG onto the surface of polydopamine (PDA) particles to obtain PDA / CP / ICG. The drug loading rate of CuO2 in PDA / CP / ICG was only 41.5%, which was low. When the concentration of PDA / CP / ICG was above 100 μg / mL, the irradiation time using a near-infrared light source in antibacterial applications was 10 minutes. The long irradiation time may cause overheating of nanoparticles, resulting in damage to surrounding tissues. Furthermore, it may cause patient discomfort and affect treatment compliance in later clinical applications. Summary of the Invention

[0005] This invention proposes a near-infrared responsive antibacterial nanomaterial, its preparation method, and its application. The prepared ICG / CuO2@PCM exhibits good antibacterial properties, no drug resistance, and stability. Furthermore, it achieves combined antibacterial activity using three modes: CDT / PDT / PTT, demonstrating broad-spectrum antibacterial activity and good biocompatibility.

[0006] The technical solution of the present invention is implemented as follows: a near-infrared responsive antibacterial nanomaterial, wherein the near-infrared responsive antibacterial nanomaterial (ICG / CuO2@PCM) has CuO2 nanoparticles and indocyanine green (ICG) as the core and PCM as the shell.

[0007] Furthermore, the particle size of CuO2 nanoparticles is 5-20 nm, and the particle size of near-infrared responsive antibacterial nanomaterials is 100-350 nm.

[0008] Furthermore, the melting point of phase change material PCM is 40-45℃.

[0009] Furthermore, the phase change material PCM is prepared by esterification of hexadecyl alcohol and oleic acid.

[0010] Furthermore, the loading of CuO2 nanoparticles is 60%-90%.

[0011] A method for preparing near-infrared responsive antibacterial nanomaterials includes the following steps:

[0012] 1) Copper chloride dihydrate and hydrogen peroxide were mixed and reacted under alkaline conditions, and then ultrafiltered to obtain CuO2 nanoparticles;

[0013] 2) Cetyl alcohol and oleic acid were dispersed in ethanol, ultrasonically treated, and then vacuum dried to obtain phase change material PCM;

[0014] 3) CuO2 nanoparticles, ICG and surface modifier were mixed as solution 1, and phase change material PCM was dissolved in ethanol as solution 2. Solution 1 and solution 2 were mixed, ultrasonically treated, and then immediately placed in an ice bath. After the ice bath, the mixture was dialyzed to obtain near-infrared responsive antibacterial nanomaterials.

[0015] Further, in step 1), the mixing reaction time is 30-60 min, the temperature is 20-30℃, and centrifugation is performed using a 50kD ultrafiltration tube at a speed of 5000-7000 rpm.

[0016] Further, in step 2), the mass ratio of oleic acid to cetyl alcohol is 1:2-5, the ultrasonic treatment is performed for 5-20 minutes, and the vacuum drying temperature is 40-60℃.

[0017] Further, in step 3), in solution 1, the concentration of CuO2 nanoparticles is 1-5 mg / mL, the concentration of ICG is 0.1-5 mg / mL, and the surface modifiers are lecithin and DSPE-mPEG, with the concentration of lecithin being 1-10 mg / mL and the concentration of DSPE-mPEG being 0.5-10 mg / mL; in solution 2, the concentration of phase change material PCM is 5-20 mg / mL; and the volume ratio of solution 1 to solution 2 is 10:0.5-1.

[0018] Furthermore, in step 3), the ultrasonic temperature is 42-50℃, the ultrasonic time is 5-20 min, the ice-water bath time is 5-20 min, the molecular weight cutoff of the dialysis bag is 8-20 kD, and the dialysis time is 24-48 h.

[0019] Application of a near-infrared responsive antibacterial nanomaterial in the preparation of antibacterial drugs.

[0020] The beneficial effects of this invention are:

[0021] The ICG / CuO2@PCM antibacterial nanomaterial prepared by this invention exhibits enhanced photostability of ICG under the protection of PCM. Through the synergistic effect of CuO2 and ICG, the antibacterial rate of ICG / CuO2@PCM against Staphylococcus aureus and MRSA reaches 99.9%, indirectly indicating that MRSA is ineffective against ICG / CuO2@PCM, which is significantly higher than the 77.4% of ICG@PCM. Moreover, the drug loading rate of CuO2 in the ICG / CuO2@PCM nanoparticles prepared by this invention can reach more than 87%, and when the concentration of ICG / CuO2@PCM nanoparticles is above 100 μg / mL, the irradiation time using a near-infrared light source is only 5 minutes.

[0022] The ICG / CuO2@PCM nano-antibacterial material prepared by this invention achieves synergistic effects of chemodynamic therapy, photodynamic therapy, and photothermal therapy through self-supplying hydrogen peroxide and oxygen. It overcomes the limitations of the lack of H2O2 and O2 in the bacterial infection environment, improves the bactericidal effect, and has the low concentration required for antibacterial activity. It also has good biocompatibility and broad-spectrum bactericidal effect.

[0023] The ICG / CuO2@PCM nano-antibacterial material of this invention is prepared by encapsulating CuO2 and ICG in organic phase change material PCM. The preparation method is simple and efficient, the reaction conditions are mild, the cost is low, and it is suitable for large-scale industrial application. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a comparison chart of the stability of ICG / CuO2@PCM and ICG;

[0026] Figure 2 This is a transmission electron microscope (TEM) image of CuO2;

[0027] Figure 3 This is a scanning electron microscope image of ICG / CuO2@PCM;

[0028] Figure 4 This is the UV spectrum of ICG / CuO2@PCM;

[0029] Figure 5 This is the XPS spectrum of ICG / CuO2@PCM;

[0030] Figure 6 This is a graph showing the photothermal properties of ICG / CuO2@PCM;

[0031] Figure 7 This is a chemodynamic therapy performance diagram of ICG / CuO2@PCM;

[0032] Figure 8 It refers to the amount of ·OH generated from CuO2 under heating and non-heating conditions;

[0033] Figure 9 This is a performance graph of photodynamic therapy using ICG / CuO2@PCM;

[0034] Figure 10 This is a graph showing the antibacterial properties of ICG / CuO2@PCM against various bacteria;

[0035] Figure 11 This is a graph showing the antibacterial properties of ICG / CuO2@PCM against various bacteria using different materials;

[0036] Figure 12 These are the results of the biocompatibility of ICG / CuO2@PCM with various cell types;

[0037] Figure 13 This is a scanning electron microscope image of ICG / CuO2@PCM with a CuO2 concentration of 2 mg / mL. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1

[0040] A method for preparing near-infrared responsive antibacterial nanomaterials includes the following steps:

[0041] 1) Synthesis of CuO2 nanoparticles: PVP (2.5g) was dissolved in CuCl2·2H2O (25mL, 0.01M) aqueous solution, and then NaOH (25mL, 0.02M) and 30% H2O2 (500μL) were added sequentially. After stirring at 25℃ for 30 minutes, CuO2 nanoparticles were collected by ultrafiltration and washed with water 3 times.

[0042] 2) Synthesis of phase change material PCM: 40 mg of oleic acid and 100 mg of 1-hexadecyl alcohol were added to 10 mL of anhydrous ethanol, and after ultrasonic treatment for 5 minutes, the mixture was vacuum dried at 40 °C for 6 hours.

[0043] 3) Synthesis of ICG / CuO2@PCM: 9 mg of phase change material PCM was dissolved in 0.5 mL of ethanol to obtain solution 2. CuO2 nanoparticles (1 mg / mL) were added to 10 mL of a 4% (v / v) ethanol aqueous solution as solvent. Then, 5 mg of DSPE-mPEG-2000, 20 mg of lecithin, and 3 mg of ICG were added to obtain solution 1. Solution 1 was added to solution 2, and the mixture was ultrasonically heated at 48 °C for 5 min, immediately placed in an ice bath for 10 min, and dialyzed in DI water for 24 h. The drug loading rate of CuO2 in the ICG / CuO2@PCM nanoparticles prepared in this example reached 87.27%.

[0044] Example 2

[0045] A method for preparing near-infrared responsive antibacterial nanomaterials includes the following steps:

[0046] 1) Synthesis of CuO2 nanoparticles: PVP (2.5g) was dissolved in CuCl2·2H2O (25mL, 0.01M) aqueous solution, and then NaOH (25mL, 0.02M) and 30% H2O2 (500μL) were added sequentially. After stirring at 25℃ for 30 minutes, CuO2 nanoparticles were collected by ultrafiltration and washed with water 3 times.

[0047] 2) Synthesis of phase change material PCM: 40 mg of oleic acid and 160 mg of 1-hexadecyl alcohol were added to 10 mL of anhydrous ethanol. After ultrasonic treatment for 5 minutes, the mixture was vacuum dried at 40 °C for 5 hours.

[0048] 3) Synthesis of ICG / CuO2@PCM: 18 mg PCM was dissolved in 1 mL of ethanol to obtain solution 2; CuO2 nanoparticles were added to 10 mL of 4% (v / v) ethanol aqueous solution as solvent, with a CuO2 nanoparticle concentration of 1 mg / mL, and then 10 mg DSPE-mPEG-2000, 20 mg lecithin and 10 mg ICG were added to obtain solution 1. Solution 1 was added to solution 2, and the mixture was heated under ultrasound at 48 °C for 5 min, immediately placed in an ice bath for 10 min, and dialyzed in DI water for 24 h.

[0049] Comparison of the stability of ICG / CuO2@PCM prepared in Example 1 with that of ICG alone (free ICG) Figure 1 As shown, by Figure 1 It is known that when ICG is encapsulated in a phase change material PCM, the ICG is unaffected by external water and oxygen, effectively reducing photodegradation and improving its photostability.

[0050] 1. Electron micrographs of CuO2 nanoparticles and ICG / CuO2@PCM nanoparticles

[0051] The CuO2 nanoparticles prepared in Example 1 and their transmission electron microscopy images are shown below. Figure 2 As shown, by Figure 2 It can be seen that the prepared CuO2 nanoparticles have a uniform particle size of approximately 10 nm.

[0052] The scanning electron microscope image of ICG / CuO2@PCM prepared in Example 1 is shown below. Figure 3 As shown in (Bar = 200nm), by Figure 3 As can be seen, ICG / CuO2@PCM is uniformly distributed, has a regular shape, and a particle size of approximately 120 nm (left figure). However, under laser irradiation, ICG / CuO2@PCM begins to melt, causing the shape to gradually become irregular and the particle size to decrease (right figure).

[0053] 2. UV and XPS spectra of ICG / CuO2@PCM nanoparticles

[0054] CuO2 nanoparticles and ICG / CuO2@PCM nanoparticles were dispersed in distilled water and ultrasonically dispersed. The spectra from 300 to 900 nm were measured using a UV spectrophotometer. Figure 4As shown, the UV-Vis absorption spectrum indicates that the loading of ICG was successful. The characteristic absorption peak of ICG redshifted from 780 nm to 800 nm, which may be due to the enrichment of ICG, but it is more conducive to the excitation of nanoparticles at 808 nm.

[0055] XPS spectra of ICG / CuO2@PCM nanoparticles are shown below. Figure 5 As shown, the characteristics of C, N, Cu, O and S elements are displayed. The Cu 2p peak indicates that CuO2 loading was successful, while the N1s peak indicates the presence of a PVP coating on the CuO2 surface. The appearance of the S2p peak confirms that ICG loading was successful.

[0056] 3. Photothermal performance testing of ICG / CuO2@PCM nanoparticles

[0057] ICG / CuO2@PCM was dispersed in deionized water to form dispersions of different concentrations. 1 mL of each dispersion was placed in an EP tube and tested with an 808 nm laser (1 W / cm²). 2 Irradiation was performed for 5 minutes (NIR), and the temperature was measured and recorded every half minute using an electronic thermometer. The results are as follows: Figure 6 As shown, the dispersion with a drug concentration of 0, serving as a control, showed no significant photothermal effect; the temperature did not rise significantly after 5 minutes of light exposure. In contrast, the temperature of the 200 μg / mL dispersion increased from 23.5°C to 55.6°C after 5 minutes of light exposure. This temperature is sufficient to reach the melting point of PCM, thereby promoting the release of its contents and also achieving a photothermal bactericidal effect.

[0058] 4. Chemodynamic Performance Testing of ICG / CuO2@PCM

[0059] ICG / CuO2@PCM can decompose under weakly acidic conditions to produce hydrogen peroxide and Cu. 2+ Then hydrogen peroxide and Cu 2+ The hydroxyl radical ·OH was generated through a Fenton-like reaction. ·OH reacts with 3,3',5,5'-tetramethylbenzidine (TMB) to form TMB oxide. The TMB oxide product exhibits a characteristic peak at 652 nm. The absorbance at 652 nm was measured using a microplate reader to evaluate its chemodynamic therapeutic performance. In a pH 5.5 buffer solution, 200 μg / mL of ICG / CuO2@PCM was mixed with 200 μg / mL of TMB, using water as a control, and the absorbance at 652 nm was measured. Results are as follows... Figure 7As shown (+INR, referring to 808 nm near-infrared irradiation for 5 min), the control group (ultrapure water) showed no color change, and the UV-Vis absorption peak of TMB at 652 nm did not change significantly at pH 7.4. However, under near-infrared irradiation at pH 5.5, the peak value at 652 nm increased significantly compared to the control group (ultrapure water), indicating that ICG / CuO2@PCM can release CuO2 in response to 808 nm near-infrared irradiation. Subsequently, CuO2 undergoes a Fenton-like reaction to generate ·OH in an acidic microenvironment (pH 5.5).

[0060] The amount of ·OH generated by the CuO2 nanoparticles prepared in Example 1 under heated and unheated conditions is as follows: Figure 8 As shown, by Figure 8 It can be seen that under heating conditions, the amount of ·OH produced by CuO2 increases significantly, indicating that the photothermal effect of ICG in this system can improve the CDT efficiency of CuO2, thereby increasing the yield of ·OH.

[0061] 5. Photodynamic therapy performance testing of ICG / CuO2@PCM nanoparticles

[0062] ICG / CuO2@PCM can generate singlet oxygen under near-infrared irradiation at 808 nm. 1 O2, once 1 When O2 combines with DPBF, DPBF undergoes irreversible oxidation, resulting in a rapid decrease in absorbance in the UV-Vis region. Different materials (200 μg / mL) were mixed with DPBF (40 μg / mL), and then subjected to an 808 nm near-infrared laser (1 W / cm²). 2 The absorbance at 417 nm was measured under INR (Inductively Coupled Reflectance) for 5 minutes. The results are as follows: Figure 9 As shown, the absorbance of the control group (ultrapure water), the CuO2@PCM group, and the ICG / CuO2@PCM group showed almost no change. However, the absorbance of both the ICG@PCM+NIR group and the ICG / CuO2@PCM+NIR group at 410 nm decreased significantly with increasing irradiation time, indicating that... 1 O2 is generated by ICG after near-infrared irradiation. It is noteworthy that the O2 generated by the ICG / CuO2@PCM+NIR group... 1 The CuO2 content was significantly higher than that of the ICG@PCM+NIR group. This enhancement is due to the fact that CuO2 can provide more O2 to the PDT under heating conditions, thereby improving the efficiency of the PDT.

[0063] 6. Antibacterial properties of ICG / CuO2@PCM nanoparticles at different concentrations

[0064] Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and methicillin-resistant Staphylococcus aureus (MRSA) were selected as model strains. Different concentrations of the drug were mixed with fresh bacterial suspensions (1×10⁻⁶). 6 The mixture was then divided into two groups: with and without laser irradiation (1 W / cm²). 2 (5 min). Take 100 μL of the above-treated suspension from each group and spread it evenly on an agar plate. After incubating at 37℃ for 16 h, count the number of colonies on the agar plate. The results are as follows: Figure 10 As shown, at the same concentration, the number of bacterial colonies in the near-infrared treatment group was significantly lower than that in the non-near-infrared treatment group, indicating that ICG / CuO2@PCM only exhibits antibacterial properties under near-infrared response. Furthermore, the number of bacterial colonies decreased significantly with increasing ICG / CuO2@PCM concentration. At a concentration of 100 μg / mL, ICG / CuO2@PCM achieved a 99.9% inhibition rate against Staphylococcus aureus and MRSA, indirectly indicating that MRSA is ineffective against ICG / CuO2@PCM. Meanwhile, for Escherichia coli, the ICG / CuO2@PCM concentration needed to be increased to 125 μg / mL to achieve a similar inhibition rate, which may be related to the complex cell wall composition of Escherichia coli.

[0065] The antibacterial properties of ICG / CuO2@PCM prepared in Example 1 against various bacteria are as follows: Figure 11 As shown, CuO2@PCM exhibits low antibacterial activity. In contrast, ICG / CuO2@PCM achieves a bactericidal effect of 99.9%, significantly higher than ICG@PCM's 77.4%. This further demonstrates that CuO2 can supply O2 to improve the PDT efficiency of ICG, thereby playing a synergistic bactericidal role.

[0066] 7. Biocompatibility of ICG / CuO2@PCM nanoparticles with various cell types

[0067] The cytotoxicity of ICG / CuO2@PCM against mouse embryonic fibroblasts (NIH-3T3), human umbilical vein endothelial cells (HUVEC), and cervical cancer cells (HeLa) was evaluated using the CCK-8 assay. Cells were seeded into 96-well cells (1 × 10⁶ cells per well). 5 Cells were incubated at 37°C for 24 h in a 5% CO2 incubator, followed by treatment with different concentrations of ICG / CuO2@PCM for 24 and 72 h. After treatment, the cells were washed three times with sterile PBS, each time with fresh culture medium containing 100 μL of CCK8 reagent. After incubation at 37°C for 4 h, the absorbance at 495 nm was measured in each well. Results are shown below. Figure 12As shown, when the concentration of ICG / CuO2@PCM reached 150 μg / mL, the survival rate of the three cells remained above 80%, indicating that ICG / CuO2@PCM has good biocompatibility.

[0068] In summary, the ICG / CuO2@PCM nanoparticles prepared by this invention have excellent antibacterial properties, good safety, high biocompatibility, low required concentration, and broad-spectrum bactericidal effect, and can be used to prepare antibacterial drugs.

[0069] Because CuO2 acts as the "core" in ICG / CuO2@PCM, in step 3), when the CuO2 concentration is below 1 mg / mL, such as 0.5 mg / mL, the mixture of CuO2, ICG, and PCM will not form a solid shape. When the CuO2 concentration is above 1 mg / mL, such as 2 mg / mL, the ICG / CuO2@PCM will have irregular shapes and varying sizes (e.g., ...). Figure 13 (As shown).

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A near-infrared responsive antibacterial nanomaterial, characterized in that: Near-infrared responsive antibacterial nanomaterials use CuO2 nanoparticles and ICG as the core and PCM phase change material as the shell. The preparation method of the near-infrared responsive antibacterial nanomaterial includes the following steps: 1) Copper chloride dihydrate and hydrogen peroxide were mixed and reacted under alkaline conditions, and then ultrafiltered to obtain CuO2 nanoparticles; 2) Cetyl alcohol and oleic acid were dispersed in ethanol, ultrasonically treated, and then vacuum dried to obtain phase change material PCM; 3) Mix CuO2 nanoparticles, ICG and surface modifier as solution 1, dissolve phase change material PCM in ethanol as solution 2, mix solution 1 and solution 2, sonicate, and immediately place in an ice bath. After the ice bath, dialyze to obtain near-infrared responsive antibacterial nanomaterials. In step 3), in solution 1, the concentration of CuO2 nanoparticles is 1-5 mg / mL, the concentration of ICG is 0.1-5 mg / mL, and the surface modifiers are lecithin and DSPE-mPEG, with the concentration of lecithin being 1-10 mg / mL and the concentration of DSPE-mPEG being 0.5-10 mg / mL; in solution 2, the concentration of phase change material PCM is 5-20 mg / mL; the volume ratio of solution 1 to solution 2 is 10:0.5-1. In step 3), the ultrasonic temperature is 42-50℃, the ultrasonic time is 5-20min, and the ice water bath time is 5-20min.

2. The near-infrared responsive antibacterial nanomaterial according to claim 1, characterized in that: The particle size of CuO2 nanoparticles is 5-20 nm, while the particle size of near-infrared responsive antibacterial nanomaterials is 100-350 nm; the loading of CuO2 nanoparticles is 60%-90%.

3. The near-infrared responsive antibacterial nanomaterial according to claim 1, characterized in that: The melting point of phase change material PCM is 40-45℃.

4. A near-infrared responsive antibacterial nanomaterial according to claim 1 or 3, characterized in that: Phase change material PCM is prepared by esterification of hexadecyl alcohol and oleic acid.

5. The near-infrared responsive antibacterial nanomaterial according to claim 4, characterized in that, In step 1), the mixing reaction time is 30-60 min and the temperature is 20-30℃.

6. The near-infrared responsive antibacterial nanomaterial according to claim 5, characterized in that, In step 2), the mass ratio of oleic acid to cetyl alcohol is 1:2-5, and the ultrasonic treatment lasts for 5-20 minutes.

7. The use of a near-infrared responsive antibacterial nanomaterial according to any one of claims 1-6 in the preparation of antibacterial drugs.

Images