Nanoparticles loaded with curcumin and antibacterial peptide as well as preparation method and application of nanoparticles

Abstract

The invention belongs to the technical field of biological medicine and nanotechnology, and particularly relates to nanoparticles loaded with curcumin and antibacterial peptide as well as a preparation method and application of the nanoparticles. The nanoparticles loaded with the curcumin and the antibacterial peptide are prepared by the following steps: taking PEI-PLGA and the curcumin as raw materials, and preparing PEI-PLGA-Cur nanoparticles; pEI-PLGA-Cur nanoparticles and the antibacterial peptide are used as raw materials, and the nanoparticles loaded with the curcumin and the antibacterial peptide are prepared. The nanoparticles loaded with the curcumin and the antibacterial peptide provided by the invention have a photodynamic therapy effect, the drug loading capacity of the curcumin in the nanoparticles is relatively high, the loading rate of the antibacterial peptide is relatively large, auxiliary materials are safe, the preparation process is simple, the particle size of the nanoparticles is relatively small and uniform, the blood compatibility is good, the nanoparticles can be used for treating infection of drug-resistant bacteria, and the application prospect is wide. The potential of becoming a novel nano antibacterial drug and the clinical application prospect are realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and nanotechnology, and particularly relates to nanoparticles loaded with curcumin and antimicrobial peptides, and a preparation method and application thereof. Background Art

[0002] Burns are tissue damage caused by exposure to high-temperature gases, high-temperature liquids (such as boiling water, hot oil, molten steel), or high-temperature solids (such as hot metal). These injuries primarily involve skin or mucous membranes, and in severe cases, subcutaneous tissue may be damaged. In the early stages of burns, infection caused by pathogens is a major cause of disease and mortality. Due to a weakened skin barrier, wounds are immediately colonized by numerous pathogens. Furthermore, acute inflammation and immunosuppression make it difficult for patients to combat infection. Furthermore, burn eschars provide an ideal environment for bacterial growth and reproduction, maintaining an ideal growth temperature and providing a rich source of nutrients, including denatured proteins and lipids. Burn patients are highly susceptible to infection due to prolonged hospitalization, long-term anti-scar treatment, and decreased immunity.

[0003] In recent years, the inappropriate use of antibiotics has led to a decline in their effectiveness against bacteria and a gradual increase in bacterial resistance. The therapeutic efficacy of single traditional antibiotics has failed to meet clinical needs, making infections caused by drug-resistant bacteria difficult to treat. Multidrug resistance (MDR) refers to the phenomenon in which microorganisms become insensitive or resistant to multiple structurally unrelated antimicrobial drugs with different molecular targets. This is typically due to intracellular mutations and the accumulation of multiple resistance genes. Bacterial culture, isolation, and identification of burn wounds revealed that multidrug-resistant Staphylococcus aureus and Escherichia coli were the most common strains isolated from these samples. The recurring and complex nature of multidrug-resistant bacteria presents significant challenges in the clinical treatment of infections caused by these bacteria. Therefore, the search for novel antimicrobial agents to combat resistant infections is crucial, and the development of novel antimicrobial agents has become a top priority. Antimicrobial peptides offer a promising option.

[0004] Antimicrobial peptides (AMPs) are a class of small peptides with antimicrobial activity. Their unique mechanism of disrupting bacterial cell membranes makes them potential candidates for novel antimicrobial therapeutics. However, natural AMPs often face limitations in their translational applications as clinical antimicrobial agents due to their weak antimicrobial activity, instability, and high cytotoxicity. Studies have shown that the combination of nanoparticles with AMPs can effectively increase the duration of action and antimicrobial activity of AMPs while also reducing their cytotoxic effects. Therefore, the use of AMP-modified nanoparticles to combat infections with drug-resistant bacteria has become a research hotspot.

[0005] Nanoparticles typically range in size from 1 to 1000 nm. Some nanoparticles have the ability to encapsulate drugs of varying solubility or attach therapeutic agents, enhancing their antimicrobial activity by improving their solubility and stability. The rapid development of nanotechnology and photodynamic therapy has made the translational application of novel antimicrobial peptides possible. Photodynamic therapy (PDT) is a novel non-thermal sterilization technique based on the reaction of photosensitizers with ground-state oxygen molecules in the environment upon irradiation with a specific wavelength of light, producing free radicals that kill microorganisms. PDT offers advantages such as low toxicity, minimal invasiveness, high selectivity, and controllability. It is widely used not only in the treatment of precancerous lesions and tumors but also in antibacterial therapies. Compared with conventional antimicrobial agents, antimicrobial agents with PDT properties require only controlled light dose to achieve control of their antibacterial effect. The concentration of the agent used and the duration of the drug's antimicrobial effect are also lower than those of conventional antimicrobial agents. Therefore, nanoparticles with PDT properties have enormous clinical value and potential for antimicrobial applications.

[0006] Curcumin (Cur) is a polyphenol extracted from the rhizome of turmeric, with a wide range of pharmacological activities and medicinal value, including anti-inflammatory, anti-tumor, and hypoglycemic effects. Existing studies have shown that curcumin can shorten the inflammatory phase by reducing the levels of pro-inflammatory factors. During wound granulation and tissue remodeling, curcumin can promote the proliferation of fibroblasts and collagen, thereby shortening the epithelialization phase of the wound and accelerating wound healing. At the same time, curcumin, as a photosensitizer, can induce the production of free radicals under irradiation of a specific wavelength, thereby killing pathogens around the wound and reducing the infection of the wound by pathogens. However, in actual application, the poor water solubility, low dissolution rate, rapid metabolism, and low bioavailability of curcumin have limited its clinical application. Therefore, it is urgent to develop a pharmaceutical preparation that combines curcumin with nanomaterials to improve the water solubility, dissolution rate, and drug loading of curcumin. This preparation not only allows curcumin to maintain its characteristics as a photosensitizer, but also has the characteristics of anti-inflammatory and low cytotoxicity.

[0007] Polyethyleneimine (PEI) is a cationic polymer rich in amino groups, often used as a gene transfection vector, surface modification vector, and drug delivery vehicle to enhance drug targeting and improve the efficacy of gene therapy. Due to its high cationic charge, PEI exhibits a certain degree of cytotoxicity to cells. Numerous studies have demonstrated that PEI cytotoxicity can be significantly reduced by modifying it. Poly(lactic-co-glycolic acid) (PLGA) is a high-molecular-weight organic compound formed by the polymerization of lactic acid and glycolic acid, exhibiting excellent biocompatibility and biodegradability. PLGA is a US Food and Drug Administration-approved medical material and one of the most widely used drug delivery vehicles, capable of encapsulating substances such as peptides, viral DNA, and anticancer drugs. PLGA-modified PEI not only exhibits reduced cytotoxicity but also enables drug co-delivery.

[0008] Existing anti-infection drugs for burns, such as silver sulfadiazine, have low solubility and are photosensitivity. During clinical use, multiple doses are required to maintain an effective therapeutic concentration at the wound site, and frequent debridement and dressing changes are inconvenient for patients. Free antimicrobial peptides are generally unstable and have some hemolytic properties, which limits their clinical application. Curcumin, which exhibits anti-inflammatory and photodynamic therapeutic properties, has poor water solubility and a slow dissolution rate, making it difficult to achieve sufficient levels for application. Therefore, there is currently no method for preparing photodynamic therapy nanoparticles that combine curcumin and antimicrobial peptides with high solubility, good stability, excellent biocompatibility, and anti-inflammatory and antimicrobial properties.

[0009] Therefore, there is an urgent need to develop photodynamic therapy nanoparticles with high solubility, good stability, good biocompatibility, and anti-inflammatory and antibacterial capabilities to solve the above problems. Summary of the Invention

[0010] To solve the problem that there is currently no existing technology for preparing photodynamic therapy nanoparticles with high solubility, good stability, good biocompatibility, and anti-inflammatory and antibacterial capabilities by loading curcumin and antimicrobial peptides, the present invention adopts the following technical solutions to achieve the above objectives:

[0011] The invention provides nanoparticles loaded with curcumin and antimicrobial peptides. PEI-PLGA and curcumin are used as raw materials to prepare PEI-PLGA-Cur nanoparticles; and PEI-PLGA-Cur nanoparticles and antimicrobial peptides are used as raw materials to prepare the nanoparticles loaded with curcumin and antimicrobial peptides.

[0012] The present invention also provides a method for preparing the nanoparticles loaded with curcumin and antimicrobial peptides, comprising the following steps:

[0013] Preparation of PEI-PLGA-Cur nanoparticles: PEI-PLGA and dimethylformamide were mixed to completely dissolve the PEI-PLGA, and then curcumin, triethanolamine, and PBS were added to obtain a mixed solution; the mixed solution was heated and mixed, and dialyzed to obtain a PEI-PLGA-Cur solution;

[0014] Preparation of PEI-PLGA-Cur@32C nanoparticles: PEGDA is added to a PEI-PLGA-Cur solution to make a final concentration of PEGDA in the solution of 0.08-0.12 mg / mL, the solution is heated and mixed, and dialyzed to obtain a PEI-PLGA-Cur solution loaded with PEGDA; an antimicrobial peptide is added to the PEI-PLGA-Cur solution loaded with PEGDA to a final concentration of 48-52 μg / mL, the solution is heated and mixed, and dialyzed to obtain PEI-PLGA-Cur@32C nanoparticles, i.e., the nanoparticles loaded with curcumin and antimicrobial peptide.

[0015] Preferably, the volume ratio of the PEI-PLGA to dimethylformamide is 1:48-52.

[0016] Preferably, after PEI-PLGA is completely dissolved, curcumin and triethanolamine are added first, and then PBS is added dropwise at 22-28° C. while shaking.

[0017] Preferably, the heating and mixing conditions are: shaking reaction at 36-38° C. for 22-26 hours.

[0018] Preferably, the dialysis conditions are: dialysis with deionized water in a 3500Da dialysis bag for 22 to 26 hours.

[0019] Preferably, the final concentration of the antimicrobial peptide added to the PEI-PLGA-Cur solution of PEGDA is 50 μg / mL.

[0020] Preferably, the antimicrobial peptide is AMP-32C.

[0021] The present invention also provides the use of the nanoparticles loaded with curcumin and antimicrobial peptides in the preparation of antimicrobial drugs, wherein the antimicrobial drugs are antimicrobial sprays.

[0022] Preferably, the antibacterial spray comprises the nanoparticles loaded with curcumin and antimicrobial peptides according to claim 1 and PBS.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention provides nanoparticles loaded with curcumin and an antimicrobial peptide. These nanoparticles exhibit high solubility, excellent stability, good biocompatibility, and anti-inflammatory and antimicrobial properties for photodynamic therapy. These nanoparticles utilize safe excipients, are simple to manufacture, have a small and uniform particle size, and exhibit good blood compatibility. They can be used to treat infections caused by drug-resistant bacteria, and have the potential to become a new type of nano-antimicrobial drug with promising clinical applications.

[0025] 2. The present invention utilizes hydrophilic PEI and hydrophobic PLGA to self-assemble to form PEI-PLGA nanoparticles with a hydrophilic shell and a hydrophobic core. This allows PLGA, as the hydrophobic core, to encapsulate hydrophobic drugs, while PEI, as the hydrophilic shell, can bind to negatively charged substances. By utilizing PEI-PLGA nanoparticles to encapsulate the more hydrophobic curcumin, curcumin is not only more evenly dispersed but also reduces the cytotoxicity of PEI. Through a Schiff base reaction, the aldehyde group of polyethylene glycol diacrylate (PEGDA) reacts with the amino group of PEI-PLGA to obtain PEGylated nanoparticles (PEGDA-PEI-PLGA-Cur).

[0026] AMP-32 (LKKLAGKIYRVLAKL) is an antimicrobial peptide with broad-spectrum antimicrobial activity discovered by our research group from the UniProt database using our self-developed antifungal webserver (http: / / www.chemoinfolab.com / antifungal), a model for antimicrobial peptide drugs. Targeting the membrane-bound mechanisms of antimicrobial peptides, we combined antimicrobial peptides with nanotechnology. AMP-32 was selected as the main component, and a cysteine ​​residue was added to the amino acid sequence of AMP-32 to synthesize AMP-32C, with the amino acid sequence LKKLAGKIYRVLAKLC. The thiol group of the cysteine ​​was designed to act as a "linker" to facilitate attachment to PEGylated nanoparticles (PEGDA-PEI-PLGA-Cur), resulting in nanoparticles loaded with curcumin and the antimicrobial peptide (PEI-PLGA-Cur@32C nanoparticles).

[0027] 3. This invention constructs nanoparticles loaded with curcumin and antimicrobial peptides (PEI-PLGA-Cur@32C nanoparticles), simultaneously addressing issues such as drug stability, water solubility, and antimicrobial activity. Combined with photodynamic inactivation technology, this method achieves synergistic sterilization. The antimicrobial efficacy and wound healing ability of the nanoparticles were validated using a mouse burn infection model. This invention provides a research strategy for achieving a highly efficient, controllable, and low-toxic nanoantimicrobial system, offering a new breakthrough and solution to addressing the serious challenges of drug-resistant bacterial infections.

[0028] 4. The preparation method of nanoparticles loaded with curcumin and antimicrobial peptides provided by the present invention has a simple production process and has the potential for mass production. No additional lyoprotectant is required during the low-temperature freeze-drying process, and the product is easy to store and transport.

[0029] 5. The nanoparticles loaded with curcumin and antimicrobial peptides provided by this invention can be used to prepare novel nano-antimicrobial drugs with photodynamic therapy capabilities, which can be used clinically to treat microbial infections. Curcumin, acting as a photosensitizer, also has a certain anti-inflammatory effect, and the antimicrobial activity of the nanoparticles is further enhanced after exposure to light. Transdermal administration provides a new approach for treating burn infections and offers a new antimicrobial drug option for the clinical treatment of patients with burn infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Characterization of the PEI-PLGA-Cur@32C nanoparticles in the present invention, wherein A is the particle size distribution; B is the Zeta potential distribution diagram; C is the solution morphology observation diagram; a: curcumin solution; b: nanoparticle solution; D is the transmission electron microscopy image of the PEI-PLGA-Cur@32C nanoparticles;

[0031] Figure 2 This is the test of blood compatibility and in vitro antibacterial activity of PEI-PLGA-Cur@32C nanoparticles in the present invention, wherein A is the hemolysis of red blood cells by different substances; B is the in vitro hemolysis rate of nanoparticles; C is the effect of nanoparticles on growth curve (no photodynamic therapy group); D is the bactericidal kinetics of nanoparticles; E is the effect of nanoparticles on bacterial survival rate (no photodynamic therapy group); F is the effect of nanoparticles on bacterial survival rate (photodynamic therapy group);

[0032] Figure 3 The effect of PEI-PLGA-Cur@32C nanoparticles on the generation of reactive oxygen species in the present invention, wherein A is the group without photodynamic therapy; B is the group with photodynamic therapy; C is the result of flow cytometry detection of the accumulation of reactive oxygen species in bacterial cells caused by nanoparticles;

[0033] Figure 4Figure 3 shows the therapeutic effect of the PEI-PLGA-Cur@32C nanoparticles of the present invention on a mouse scald infection model. ... wound images of mice on days 0, 3, and 7; Figure 3 shows the wound area; Figure 3 shows the wound healing rate; Figure 4 shows the bacterial load on the skin of mice; Figure 4 shows the bacterial load on the skin of mice; Figure 4 shows the bacterial load on the skin of mice; Figure 4 shows the pathological section of the mouse skin tissue. DETAILED DESCRIPTION

[0034] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0035] The materials, reagents, instruments, experimental cells, bacteria and animals used in the following examples are as follows:

[0036] (1) Main reagents

[0037] PEI-PLGA (polyethyleneimine-poly(lactic-co-glycolic acid)) was purchased from Guangzhou Carbon Technology Co., Ltd.; DMF (dimethylformamide) was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.; curcumin (Cur) was purchased from Beijing Solebow Technology Co., Ltd.; TEA (triethanolamine) was purchased from Thermo Fisher Scientific; Triton X-100 was purchased from Beijing Solebow Technology Co., Ltd.; silver sulfadiazine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; PI stain was purchased from Beijing Solebow Technology Co., Ltd.; SYTO9 stain was purchased from Beijing Solebow Technology Co., Ltd.; and hematoxylin and eosin (H&E) were purchased from Beijing Solebow Technology Co., Ltd.

[0038] (2) Main instruments

[0039] Particle size analyzer: Brookhaven Instrument Company, USA; HT7800 transmission electron microscope: Hitachi; SynergyH1 multifunctional microplate reader: Berton Instrument Co., Ltd., USA; Scientz-10N vacuum freeze dryer: Ningbo Xinzhi Biotechnology Co., Ltd.; Spin SR10 laser confocal microscope: Olympus Corporation.

[0040] (3) Experimental cells, bacteria, and animals

[0041] Fresh blood cells from healthy individuals were obtained from the Laboratory Department of Guihang Guiyang Hospital. Escherichia coli ATCC 35218, methicillin-resistant Staphylococcus aureus MRSA.01, Candida auris CBS12766, Candida tropicalis ATCC 20962, and azole-resistant Cryptococcus neoformans Cne.01 were obtained from the Key Laboratory of Modern Pathogenic Biology, Guizhou Medical University. Balb / c mice were purchased from Spefox (Beijing) Biotechnology Co., Ltd.

[0042] (4) Culture medium

[0043] MHB culture medium was prepared with 5 g beef extract powder, 1.5 g starch, and 17.5 g casein hydrolyzate, with a pH of 7.2 ± 0.2.

[0044] LB solid medium was prepared with 10.0 g of peptone, 5.0 g of yeast powder, 5.0 g of sodium chloride, and 16.0 g of agar powder, with a pH of 7.0 ± 0.1.

[0045] Example 1

[0046] A method for preparing nanoparticles loaded with curcumin and antimicrobial peptides comprises the following steps:

[0047] (1) Preparation of PEI-PLGA-Cur nanoparticles

[0048] Accurately weigh 20 mg of PEI-PLGA into a 10 mL centrifuge tube, add 1 mL of DMF, and sonicate until completely dissolved. Separately, add 5 mg of curcumin and 10 μL of TEA to the PEI-PLGA solution. Add 0.5 mL of PBS dropwise while shaking at 25°C. The solution is then shaken at 37°C for 24 hours. After shaking, dialyze the reaction solution against deionized water in a 3500 Da dialysis bag for 24 hours. Collect the solution in the bag to obtain PEI-PLGA-Cur nanoparticles. The PBS concentration is 0.01 M and the pH is 7.4.

[0049] (2) Preparation of PEI-PLGA-Cur@32C nanoparticles

[0050] Prepare a 10 mg / mL polyethylene glycol diacrylate (PEGDA, 1000 Da) solution and add it to the PEI-PLGA-Cur solution to a final PEGDA concentration of 0.1 mg / mL. Incubate the solution at 37°C with shaking for 24 hours. After shaking, dialyze the reaction mixture against deionized water in a 14,000 Da dialysis bag for 24 hours. Collect the solution in the bag to obtain the PEGylated PEI-PLGA-Cur solution. The 10 mg / mL polyethylene glycol diacrylate solution was prepared by accurately weighing 10 mg of polyethylene glycol diacrylate and adding 1 mL of PBS.

[0051] AMP-32C was added to the PEGylated PEI-PLGA-Cur solution at a final concentration of 50 μg / mL, and the reaction was shaken at 37°C for 24 hours. After shaking, the solution was dialyzed against deionized water using a 14,000 Da dialysis bag for 24 hours. The solution in the bag was collected to obtain PEI-PLGA-Cur@32C nanoparticles, i.e., the nanoparticles loaded with curcumin and antimicrobial peptides.

[0052] AMP-32C, also known as 32C peptide, is synthesized by adding cysteine ​​residues to the amino acid sequence of AMP-32 (LKKLAGKIYRVLAKLC).

[0053] AMP-32 (LKKLAGKIYRVLAKL) is an antimicrobial peptide with broad-spectrum antimicrobial activity discovered by our research team using the antifungal webserver (http: / / www.chemoinfolab.com / antifungal), a model for antimicrobial peptide drugs. Targeting the membrane-bound mechanisms of antimicrobial peptides against pathogens, we combined antimicrobial peptides with nanotechnology, synthesizing AMP-32C (LKK LAGKIYRVLAKLC) by adding a cysteine ​​residue to the amino acid sequence of AMP-32.

[0054] In Example 1 of the present invention, nanoparticles loaded with curcumin and antimicrobial peptides were prepared. To illustrate their performance, the present invention used the nanoparticles loaded with curcumin and antimicrobial peptides (PEI-PLGA-Cur@32C nanoparticles) prepared in Example 1 as an example and performed characterization analysis. The specific research is as follows:

[0055] (1) Particle size of PEI-PLGA-Cur@32C nanoparticles

[0056] The particle size distribution of nanoparticles was detected by a particle size analyzer to determine the size and distribution of the particles. The particle size of PEI-PLGA-Cur@32C nanoparticles was detected by a particle size analyzer. The results are as follows: Figure 1 shown.

[0057] Depend on Figure 1 It can be seen that the particle size of PEI-PLGA-Cur@32C nanoparticles is 145.47±7.31nm, the particle size distribution is uniform, the polydispersity coefficient is 0.296±0.033, and the particle size distribution presents a single peak.

[0058] (2) Zeta potential of PEI-PLGA-Cur@32C nanoparticles

[0059] The zeta potential of PEI-PLGA-Cur@32C nanoparticles was measured using a potentiostat. The measurement conditions were: a temperature of 25°C, water as the medium, a 90° angle, and red light as the excitation source, with the intensity adjusted according to the sample.

[0060] The Zeta potential of PEI-PLGA-Cur@32C nanoparticles was detected by particle size analyzer, and the results are shown in Table 1 and Figure 1 As shown in B.

[0061] Table 1 Zeta potential of PEI-PLGAPEI-PLGA-Cur@32C nanoparticles

[0062]

[0063] From Table 1 and Figure 1 As shown in Figure 2, the zeta potential of the PEI-PLGA-Cur@32C nanoparticles is -45.80 ± 2.23 mV. A larger absolute value of the zeta potential indicates greater nanoparticle stability. These results suggest that the PEI-PLGA-Cur@32C nanoparticles prepared in this invention have good stability and are less prone to aggregation.

[0064] (3) Morphological observation of PEI-PLGA-Cur@32C nanoparticles

[0065] 3 mL of a 5 mg / mL curcumin (Cur) solution and 3 mL of a PEI-PLGA-Cur@32C nanoparticle solution were precisely measured and placed in glass bottles to observe the differences in their appearance. The morphology of the PEI-PLGA-Cur@32C nanoparticles was observed using transmission electron microscopy. The curcumin (Cur) solution was prepared by precisely weighing 5 mg of curcumin and adding 1 mL of PBS. The PEI-PLGA-Cur@32C nanoparticle solution was prepared by precisely weighing 5 mg of PEI-PLGA-Cur@32C nanoparticles and adding 1 mL of PBS.

[0066] Transmission electron microscopy: Use a pipette to draw 20 μL of the sample onto a carbon-coated copper grid and leave it for 2 minutes. Use filter paper to absorb excess liquid. Place 2% phosphotungstic acid on a carbon-supported copper grid and leave it for 2 minutes for negative staining. Collect images after drying at room temperature.

[0067] The appearance of curcumin (Cur) solution and PEI-PLGA-Cur@32C nanoparticle solution is shown in Figure 2. Figure 1 As shown in C. Figure 1C: A large amount of particles in the curcumin solution settled at the bottom of the glass bottle. The particles are almost insoluble in water. The PEI-PLGA-Cur@32C nanoparticle solution appears as a clear brown-red solution, and no particle sedimentation is observed with the naked eye.

[0068] Transmission electron microscopy results are as follows Figure 1 As shown in Figure 4, the image shows that the PEI-PLGA-Cur@32C nanoparticles are spherical in shape, with uniform size and good dispersion, and the sizes are all within the nanometer size range.

[0069] 1. The present invention provides nanoparticles loaded with curcumin and antimicrobial peptides (PEI-PLGA-Cur@32C nanoparticles). The above experimental results show that the nanoparticles loaded with curcumin and antimicrobial peptides (PEI-PLGA-Cur@32C nanoparticles) provided by the present invention have a particle size of 145.47±7.31nm, a Zeta potential of -45.80±2.23mV, good particle stability and uniform distribution. Taking the nanoparticles loaded with curcumin and antimicrobial peptides (PEI-PLGA-Cur@32C nanoparticles) as an example, the in vitro hemolytic activity, in vitro antibacterial activity, and in vivo antibacterial activity of the nanoparticles were tested: In vitro hemolytic activity test of PEI-PLGA-Cur@32C nanoparticles

[0070] 1.1 Preparation of red blood cell suspension

[0071] Wash 1 mL of fresh healthy human blood cells with 0.01 M phosphate buffered solution (PBS). Centrifuge at 1000 g for 10 minutes to remove plasma and white blood cells. Repeat this process three times to obtain washed red blood cells. Pipette 4 volumes of red blood cells and add 96 volumes of PBS. Mix thoroughly and prepare a 4% red blood cell suspension.

[0072] 1.2 Hemolytic assay

[0073] PEI-PLGA-Cur@32C nanoparticles containing AMP-32C at concentrations of 0.0875, 0.175, 0.35, 0.7, 1.4, 2.8, 5.6, and 11.2 μg / mL were incubated with a 4% red blood cell suspension at 37°C for 1 hour. The supernatants were centrifuged at 1000 × g for 10 minutes at 4°C. The absorbance of the supernatants was collected and measured at 540 nm using a multifunctional microplate reader. The absorbance of 0.01 M PBS was used as a negative control (A blank), and the absorbance of a 0.1% Triton X-100-treated red blood cell suspension was used as a positive control for 100% hemolysis (A Triton).

[0074] The hemolysis rate was calculated according to the following formula:

[0075] Hemolysis rate (%) = [(A sample - A blank) / (A Triton - A blank)] × 100%

[0076] The blood compatibility of PEI-PLGA-Cur@32C nanoparticles was tested using fresh red blood cells. The results are shown in Figure 2 A and 2B. Figure 2 As shown in Figures A and 2B, the nanoparticle group showed no significant hemolysis compared to the positive control group. The absorbance of the solution was measured at 540 nm, and the in vitro hemolysis rate was calculated. The results, shown in Table 2, showed that the in vitro hemolysis rate of the PEI-PLGA-Cur@32C nanoparticles was less than 5%, indicating good hemocompatibility of the nanoparticles.

[0077] Table 2 In vitro hemolysis rate of PEI-PLGA-Cur@32C nanoparticles

[0078] AMP-32C (μg / mL) in PEI-PLGA-Cur@32C Hemolysis rate (%) 11.2 3.68±0.93 5.6 -2.61±0.58 2.8 -2.65±0.52 1.4 -3.12±0.13 0.7 -3.14±0.42 0.35 -3.17±0.21 0.175 -3.09±0.04 0.0875 -2.90±0.70

[0079] 2. In vitro antibacterial activity assay of PEI-PLGA-Cur@32C nanoparticles

[0080] 2.1 Minimum inhibitory concentration and minimum bactericidal concentration

[0081] To test the antibacterial effect of PEI-PLGA-Cur@32C nanoparticles and whether photodynamic therapy can enhance their antibacterial ability, the experimental groups were divided into the following steps:

[0082] (1) Curcumin group (Curwithout PDT): 1, 2, 4, 8, 16, 32, 64, 128, 256 μg / mL of curcumin (Cur) solution was added to each group and protected from light. (2) Curcumin-photodynamic therapy group (Cur with PDT): 1, 2, 4, 8, 16, 32, 64, 128, 256 μg / mL of curcumin (Cur) solution was added to each group and irradiated with a 450-460 nm wavelength light source for 1 h (irradiation dose of 144 J / cm 2(3) Sulfadiazine Silver without PDT group: 1, 2, 4, 8, 16, 32, 64, 128, 256 μg / mL of sulfadiazine Silver solution was added to each group and protected from light. (4) Sulfadiazine Silver with PDT group: 1, 2, 4, 8, 16, 32, 64, 128, 256 μg / mL of sulfadiazine Silver solution was added to each group and irradiated with a 450-460 nm wavelength light source for 1 h (the irradiation dose was 144 J / cm 2 (5) Antimicrobial peptide group (AMP-32Cwithout PDT): 1, 2, 4, 8, 16, 32, 64, 128, 256 μg / mL of AMP-32C (AMP-32C) solution was added to each group and protected from light. (6) Antimicrobial peptide-photodynamic therapy group (AMP-32C with PDT): 1, 2, 4, 8, 16, 32, 64, 128, 256 μg / mL of AMP-32C (AMP-32C) solution was added to each group and irradiated with a 450-460 nm wavelength light source for 1 h (the irradiation dose was 144 J / cm 2 (7) Physical mixing group (Cur+AMP-32C without PDT): 1, 2, 4, 8, 16, 32, 64, 128, 256 μg / mL of AMP-32C (AMP-32C) solution was mixed with 1, 2, 4, 8, 16, 32, 64, 128, 256 μg / mL of curcumin (Cur) solution, and protected from light. (8) Physical mixing-photodynamic therapy group (Cur+AMP-32Cwith PDT): 1, 2, 4, 8, 16, 32, 64, 128, 256 μg / mL of AMP-32C (AMP-32C) solution was mixed with 1, 2, 4, 8, 16, 32, 64, 128, 256 μg / mL of curcumin (Cur) solution, and irradiated with a 450-460 nm wavelength light source for 1 h (the irradiation dose was 144 J / cm 2(9) Nanoparticle group (PEI-PLGA-Cur@32C without PDT): PEI-PLGA-Cur@32C solution containing AMP-32C at concentrations of 0.35, 0.7, 1.4, 2.8, 5.6, and 11.2 μg / mL was added to each group and protected from light. (10) Nanoparticle-photodynamic therapy group (PEI-PLGA-Cur@32C with PDT): PEI-PLGA-Cur@32C solution containing AMP-32C at concentrations of 0.35, 0.7, 1.4, 2.8, 5.6, and 11.2 μg / mL was added to each group and irradiated with a 450-460 nm wavelength light source for 1 h (irradiation dose of 144 J / cm 2 (11) PBS group (PBS without PDT): 0.01M PBS was added to each group and protected from light. (12) PBS-photodynamic therapy group (PBS with PDT): 0.01M PBS was added to each group and irradiated with a 450-460nm wavelength light source for 1 h (the irradiation dose was 144J / cm 2 ).

[0083] After the experimental groups were treated as above, they were treated with 1.0×10 6 A suspension of Escherichia coli ATCC 35218 (CFU / mL) was mixed and incubated in a 37°C incubator protected from light. Silver sulfadiazine served as a positive control, MHB medium supplemented with E. coli ATCC 35218 suspension served as a negative control, and sterile MHB medium served as a blank control. The absorbance at 630 nm was measured using a multifunctional microplate reader to observe bacterial growth in each well. By comparing the measured values, the lowest concentration that showed no significant difference from the value in the positive control wells was defined as the minimum inhibitory concentration (MIC) of PEI-PLGA-Cur@32C nanoparticles against the bacteria. 100 μL of sample from wells with no significant difference from the positive control wells was plated onto LB agar plates and incubated at 37°C for 24 hours to determine the minimum bactericidal concentration. The results are shown in Table 3.

[0084] Table 3 MIC and MBC of PEI-PLGA-Cur@32C against ATCC 35218

[0085]

[0086] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PEI-PLGA-Cur@32C nanoparticles against Escherichia coli ATCC 35218 were determined using the microdilution and agar plating methods. The results are shown in Table 3. Without light treatment, a 256 μg / mL curcumin solution had no inhibitory or bactericidal effect against the bacterium. However, after light treatment, curcumin exhibited both inhibitory and bactericidal effects, with both an MIC and MBC of 32 μg / mL, consistent with the results for the positive control, silver sulfadiazine. Regardless of whether the samples were light-treated, the MIC and MBC were both 32 μg / mL for both the antimicrobial peptide alone (AMP-32C) and the physical mixture of antimicrobial peptide and curcumin (Cur + AMP-32C). After photodynamic therapy, the MIC and MBC of the nanoparticle group (PEI-PLGA-Cur@32C) were significantly reduced. This result suggests that the nanoparticles have good antibacterial activity and that light can enhance the antibacterial efficacy of the nanoparticles.

[0087] 2.2 Growth curve

[0088] The patients were divided into curcumin-photodynamic therapy group, silver sulfadiazine-photodynamic therapy group, antimicrobial peptide-photodynamic therapy group, physical mixture-photodynamic therapy group, nanoparticle-photodynamic therapy group, and PBS-photodynamic therapy group according to the above grouping treatment. 32 μg / mL curcumin, 32 μg / mL silver sulfadiazine, 32 μg / mL physical mixture (containing a final concentration of 32 μg / mL Cur and a final concentration of 32 μg / mL AMP-32C), 0.7 μg / mL PEI-PLGA-Cur@32C, 1.4 μg / mL PEI-PLGA-Cur@32C preparations were mixed with 1.0×10 6 The bacterial suspension of CFU / mL Escherichia coli ATCC 35218 was mixed and incubated, and the absorbance at 630nm was measured at 0h, 1h, 2h, 4h, 6h, 8h, 10h, and 12h using a multifunctional microplate reader to observe the changes in the bacterial growth curve. The positive control group was added with silver sulfadiazine, the blank control group was MHB medium without antimicrobial peptides, and the negative control group was added with bacterial suspension of Escherichia coli ATCC 35218 and MHB medium to draw a growth curve. The results are shown in Figure 2 C.

[0089] 2.3 Bactericidal kinetics

[0090] With reference to the above grouping treatment, the cells were divided into curcumin group, curcumin-photodynamic therapy group, silver sulfadiazine group, silver sulfadiazine-photodynamic therapy group, antimicrobial peptide group, antimicrobial peptide-photodynamic therapy group, physical mixing group, physical mixing-photodynamic therapy group, nanoparticle group, nanoparticle-photodynamic therapy group, PBS group, and PBS-photodynamic therapy group.6 A suspension of Escherichia coli ATCC 35218 (CFU / mL) was incubated with PEI-PLGA-Cur@32C nanoparticles at concentrations of 1.4, 2.8, 5.6, and 11.2 μg / mL for 12 hours. At various time points (0, 0.5, 1, 2, 4, 6, 8, 10, and 12 hours), the mixture was diluted to the appropriate multiple. The diluted suspension was inoculated onto LB solid medium and incubated in an inverted manner at 37°C. The number of single bacterial colonies was counted, and a time-kill curve was plotted.

[0091] The bactericidal kinetic curve of PEI-PLGA-Cur@32C nanoparticles is shown in Figure 2. Figure 2 As shown in Figure D, the colony count of Escherichia coli ATCC35218 decreased to varying degrees after treatment with PEI-PLGA-Cur@32C nanoparticles, with a dose-dependent relationship between colony count and nanomaterial dosage. In the absence of photodynamic therapy (PDT), no colonies were detected on plates containing LB solid medium after 10 hours of exposure to 11.2 μg / mL PEI-PLGA-Cur@32C nanoparticles, suggesting that PEI-PLGA-Cur@32C nanoparticles can act as a bactericidal agent and exert a bactericidal effect within 10 hours. After photodynamic therapy, the bacterial colony count decreased significantly, with no colonies detected on plates containing LB solid medium within 1 hour. These results suggest that PEI-PLGA-Cur@32C nanoparticles can exert a rapid bactericidal effect within 1 hour after photodynamic therapy.

[0092] 2.4 Live to dead bacteria ratio

[0093] The cells were divided into the nanoparticle group, nanoparticle-photodynamic therapy group, PBS group, and PBS-photodynamic therapy group. Fluorescence staining was used to detect the live-dead ratio of Escherichia coli ATCC35218 after the treatment of PEI-PLGA-Cur@32C nanoparticles. 1.4 μg / mL and 11.2 μg / mL PEI-PLGA-Cur@32C were respectively combined with 1.0×10 8 CFU / mL of Escherichia coli ATCC 35218 suspension and 1.0×10 8A MRSA bacterial suspension containing 100 CFU / mL of PBS was incubated at 37°C for 1 hour. A control group containing the bacterial suspension in sterile PBS served as the control. Samples were stained with 10 μM PI and 5 μM SYTO9 dyes, and images were captured using a laser confocal microscope. The staining process involved adding 10 μM PI and 5 μM SYTO9 dyes to each sample group and incubating at 37°C in the dark for 15 minutes. After incubation, 1 mL of 0.01 M PBS was added, mixed, and centrifuged at 2000 g for 5 minutes. The supernatant was discarded to remove unbound fluorescent dye. The samples were then applied to glass slides and mounted with anti-fluorescence decay mounting medium.

[0094] The present invention uses SYTO9 and PI fluorescent dyes to detect the bacterial membrane integrity of Escherichia coli ATCC 35218, and uses membrane integrity as a detection indicator to determine the antibacterial properties of nanoparticles and the survival of bacteria. Figure 2 E and 2F.

[0095] like Figure 2 As shown in Figures E and 2F, no bacterial cells in the PBS group (PBS without PDT) or the PBS-PDT group (PBS with PDT) were stained with PI and exhibited red fluorescence, indicating intact bacterial cell membranes, good bacterial viability, and high survival rates. After treatment with 1.40 μg / mL nanoparticles, no red fluorescence was observed in the non-PDT group, while most bacterial cells in the PDT group were stained with PI, though some cells only exhibited green fluorescence. As the nanoparticle concentration increased to 11.2 μg / mL, a small number of bacterial cells in the non-PDT group were stained with PI, while all bacterial cells in the PDT group were stained with PI.

[0096] The above results suggest that the number of dead cells of Escherichia coli ATCC 35218 increased significantly after treatment with PEI-PLGA-Cur@32C nanoparticles, the mortality rate was positively correlated with the dosage of nanoparticles, and light irradiation could further increase the cell mortality rate.

[0097] 2.5 Active oxygen content detection

[0098] Based on the aforementioned grouping, the samples were divided into a nanoparticle group, a nanoparticle-photodynamic therapy group, a PBS group, and a PBS-photodynamic therapy group. Fluorescence staining was used to detect the reactive oxygen species (ROS) content in E. coli ATCC35218 samples treated with PEI-PLGA-Cur@32C nanoparticles. PEI-PLGA-Cur@32C at concentrations of 0.7 μg / mL, 1.4 μg / mL, 2.8 μg / mL, 5.6 μg / mL, and 11.2 μg / mL was incubated with a 1.0 × 108 CFU / mL suspension of E. coli ATCC 35218 at 37°C for 1 hour. Sterile PBS supplemented with the suspension served as the control group. The samples were stained with a final concentration of 10 μM DCFH-DA dye. Fluorescence intensity was measured using a fluorescence microplate reader, and the number of fluorescent cells was counted by flow cytometry.

[0099] The staining process involved adding DCFH-DA dye to a final concentration of 10 μM to each sample group and incubating at 37°C in the dark for 15 minutes. After incubation, 1 mL of 0.01 M PBS was added, mixed, and centrifuged at 2000 g for 5 minutes. The supernatant was discarded to remove unbound fluorescent dye and analyzed.

[0100] The present invention uses DCFH-DA fluorescent dye to detect the content of active oxygen accumulation in Escherichia coli ATCC 35218 bacteria, and uses the active oxygen content as a detection indicator to determine the ability of nanoparticles to induce the production of active oxygen in the bacterial environment. The results show that Figure 3 .

[0101] 3. In vivo antibacterial activity detection of PEI-PLGA-Cur@32C nanoparticles

[0102] 3.1 Establishment of a mouse burn infection model

[0103] Forty-two SPF-grade Balb / c mice were selected and fixed in the prone position. The hair on the back of the mice was removed with a depilatory cream, covering an area of ​​approximately 4 cm × 4 cm. The mice were then rinsed with sterile PBS. A 1.5 cm diameter constant temperature electric soldering iron was set to 100°C and placed without pressure on the left and right sides of the lumbar vertebrae on the back of the mice to create a burn wound. This was repeated three times on each wound. 40 μL of 1.0 × 10 8 A bacterial suspension of 100 CFU / mL of Escherichia coli ATCC 35218 was applied to the wound surface and bandaged with sterile gauze after the bacterial suspension was absorbed. Mice were sacrificed 24, 48, and 72 hours after bacterial inoculation. 0.5 cm × 1.0 cm of subscab tissue was excised under sterile conditions and the colony count was calculated using the dilution smear method. Quantification of subscab bacteria > 1.0 × 10 7 CFU / g was used as the success standard for establishing a mouse burn infection model.

[0104] Pus appeared at the wound site of the mouse 24 hours after infection, and a foul odor appeared when the scab was removed. The tissue from the mouse wound site was homogenized and the bacterial count under the scab was detected by agar spot plate method. 7 CFU / g, indicating that the mouse burn infection model was successfully established.

[0105] 3.2 Experimental Grouping and Treatment

[0106] Forty-two mice with successfully established burn infection models were randomly divided into seven groups as follows: (1) PBS group; (2) 32 μg / mL silver sulfadiazine group; (3) 32 μg / mL curcumin group; (4) 32 μg / mL antimicrobial peptide group; (5) physical mixing group (final concentrations of curcumin and antimicrobial peptide were both 32 μg / mL); (6) nanoparticle group (final concentration of antimicrobial peptide was 32 μg / mL); (7) nanoparticle-photodynamic therapy group (final concentration of antimicrobial peptide was 32 μg / mL, and the light dose was 144 J / cm 2 Subsequently, 40 μL of the drug of each treatment group was evenly applied to the burn wound under a sterile environment. After the drug was absorbed, the wound was bandaged with sterile gauze and an Elizabethan collar was placed on the head to prevent the mouse from licking the gauze. Each group received drug administration or light exposure once a day and observed for 7 consecutive days.

[0107] 3.3 Observation of wound healing sites in mice

[0108] Observe and record the wound healing status of mice in each treatment group on days 0, 3, and 7. Figure 4 A. Observe and record the wound healing area and healing rate. The wound is measured by the horizontal and vertical lengths. The wound area (cm 2 ) = horizontal length (cm) × vertical length (cm); wound healing rate (%) = (area before administration - area after administration) / area before administration × 100%, see the results Figure 4 B and 4C.

[0109] like Figure 4 As shown: In the mouse scald infection model, in order to further evaluate the ability of nanoparticles to promote wound healing in vivo, the wound area of ​​mice on day 0, day 3, and day 7 during the wound healing process was quantitatively counted (see Figure 4B and 4C). On days 1-2, the wound area of ​​the PBS group (PBS without PDT) continued to increase, indicating that the severity of the burn infection in the mice gradually worsened. The wound area reached its maximum on day 2, and the wound areas of the other treatment groups decreased to varying degrees. On day 7, without treatment, the wound area of ​​the PBS group only slightly shrank, and the wound healing rate was (20.02±0.08)%. The wound healing rate of the 32μg / mL curcumin group (Curwithout PDT) treated with curcumin was (35.64±0.07)%, and the healing rate was only increased by 15.62%. In addition, the wound healing rate of the antimicrobial peptide group (AMP-32C without PDT) at the same concentration was (67.63±0.02)%, which was better than the wound healing rate of the 32μg / mL sulfadiazine silver group (Sulfadiazine Silver without PDT) (57.51±0.02)%, indicating that AMP-32C has a better wound healing effect on this burn infection model than sulfadiazine silver. The wound healing rate of the physical mixture group (Cur+AMP-32C without PDT) was (46.58±0.05)%, which was inferior to the wound healing effect of the antimicrobial peptide alone. In contrast, the nanoparticle group (PEI-PLGA-Cur@32C without PDT) and the nanoparticle-photodynamic therapy group (PEI-PLGA-Cur@32C with PDT) had higher wound healing rates. The wound healing rate without PDT was (91.14±0.02)%, while the wound healed almost completely after PDT, with a healing rate of (94.31±2.71)%. These results suggest that PEI-PLGA-Cur@32C nanoparticles promote wound healing during the healing process of infected burns in mice, and that PDT has a certain effect in accelerating wound healing.

[0110] 3.4 Quantitative detection of bacteria

[0111] On the seventh day after the mice were scalded and infected, mice in each group were anesthetized and sacrificed. Under sterile conditions, a 0.5 cm × 0.5 cm skin tissue block, reaching the fascia, was obtained. The tissue was weighed in a sterile centrifuge tube and homogenized at 4°C. The homogenate was diluted to an appropriate concentration with normal saline and plated on a plate containing LB solid medium containing 256 μg / mL penicillin for colony count. The bacterial count per gram of tissue was calculated using the following formula:

[0112] Bacterial content (CUF / g) = dilution factor × number of colonies / tissue weight.

[0113] Depend on Figure 4As shown in D and 4E, the bacterial load in the skin tissue of mice was detected on the 7th day, and the bacterial culture identification showed that the main bacteria were Escherichia coli. The bacterial count in the PBS group was (17.991±6.334)×10 6 The bacterial count in the 32 μg / mL curcumin group was (8.072±1.579)×10 6 The bacterial count in the 32 μg / mL silver sulfadiazine group was (1.686±0.572)×10 6 CFU / g, the bacterial quantification of the antimicrobial peptide group was (1.102±0.593)×10 6 CFU / g, the bacterial quantity of the physical mixing group was (5.231±1.436)×10 6 CFU / g, the bacterial quantification of the nanoparticle group was (0.041±0.018)×10 6 CFU / g, the bacterial quantification in the nanoparticle-photodynamic therapy group was (0.002±0.001)×10 6 CFU / g.

[0114] Statistical analysis of the above results showed that there was no statistically significant difference in bacterial quantification between the 32 μg / mL antimicrobial peptide group and the 32 μg / mL sulfamethoxazole nanosilver group (P>0.05), indicating that the antibacterial effect of antimicrobial peptide AMP-32C on skin tissue was similar to that of sulfamethoxazole silver at the same concentration. The bacterial quantification on the wound surface of mice in the nanoparticle group was significantly lower than that in the PBS group, and the difference was statistically significant (P<0.0001). In addition, the samples in the nanoparticle group after photodynamic therapy (nanoparticle-photodynamic therapy group) carried less bacteria than the samples in the nanoparticle group without photodynamic therapy (nanoparticle group), indicating that photodynamic therapy can accelerate the clearance of bacteria in the wound.

[0115] 3.5 Histomorphological observation

[0116] Refer to the method in 3.4 and take tissue blocks from each group of mice in a sterile environment and fix them in 4% paraformaldehyde overnight. Hematoxylin and eosin (H&E) were used to evaluate the growth of granulation tissue and skin maturity. Fibroblasts, capillaries, extracellular matrix, etc. constitute the new granulation tissue. The thickness of granulation tissue and the number of hair follicles are the basis for evaluating the growth of new tissue and skin maturity. Masson trichrome staining was used to observe the degree of new epithelium and collagen fiber formation to evaluate collagen deposition. Results are shown in Figure 4 F.

[0117] Figure 4Figure F shows H&E and Masson stain images of corresponding skin samples from the wounded areas of the different treatment groups on day 7. The PBS group suffered severe skin damage due to burns and bacterial infection, with epidermal loss, dermal necrosis, scab formation accompanied by collagen degradation, and a decrease or disappearance of skin appendages (such as hair follicles and sweat glands) (indicated by the yellow arrows). Similar findings were observed in the 32 μg / mL curcumin group. Compared with the PBS group, wound defects were covered with new epithelium to varying degrees in all other treatment groups. Compared with the PBS group, collagen fibers proliferated in the silver sulfadiazine and antimicrobial peptide groups, with partial collagen deposition and the gradual emergence of skin appendages. All wounds in the nanoparticle group showed healing, with complete formation of the epidermis and dermis, a significant increase in the number of skin appendages, and significant collagen fiber deposition compared to the other treatment groups. Furthermore, the number of skin accessory organs in the nanoparticle-photodynamic therapy group was significantly increased compared to the nanoparticle group without photodynamic therapy. The epidermis recovered with the formation of the stratum corneum, revealing a complete skin structure, a phenomenon that suggests near-complete skin healing. These results are consistent with the wound healing rate, further confirming the effectiveness of photodynamic therapy.

[0118] The present invention provides the use of PEI-PLGA-Cur@32C nanoparticles in the preparation of an antibacterial drug. The antibacterial drug has good biocompatibility, anti-inflammatory and antibacterial activity, and photodynamic therapy effects. The antibacterial drug can be prepared into an antibacterial spray or antibacterial particles.

[0119] Let's take antibacterial spray as an example to illustrate its effect. The specific research is as follows:

[0120] An antibacterial spray with PEI-PLGA-Cur@32C nanoparticles as a main component is composed of the following raw materials: 3 g of nanoparticles loaded with curcumin and antimicrobial peptides and 60 mL of PBS.

[0121] The preparation method of the antibacterial spray comprises the following steps:

[0122] 3 g of PEI-PLGA-Cur@32C nanoparticles were added to 60 mL of PBS to prepare a PEI-PLGA-Cur@32C aqueous solution with a concentration of 50 μg / mL, which is the above-mentioned antibacterial spray.

[0123] We conducted in vitro antibacterial activity testing on the PEI-PLGA-Cur@32C aqueous solution (antibacterial spray) prepared above. The specific research is as follows: The antibacterial properties of the PEI-PLGA-Cur@32C aqueous solution were tested using methicillin-resistant Staphylococcus aureus, Escherichia coli ATCC 35218, Candida tropicalis ATCC 20962, Candida auris, and azole-resistant Cryptococcus neoformans. The bacteria or fungi in this experiment were prepared with PBS to a bacterial concentration of 1.0×10 6 CFU / mL bacterial suspension. A PEI-PLGA-Cur@32C aqueous solution was mixed with a bacterial suspension and incubated at 37°C for 16 hours. A PEI-PLGA-Cur@32C aqueous solution was mixed with a fungal suspension and incubated at 28°C for 16 hours. After incubation, 100 μL of the mixture was spread onto LB plates. The bacterial plates were incubated at 37°C for 24 hours before colony counts were performed, while the fungal plates were incubated at 28°C for 24 hours before colony counts were performed. The results are shown in Table 4.

[0124] The antibacterial rate was calculated according to the following formula:

[0125] Table 4 MIC and MBC of PEI-PLGA-Cur@32C against ATCC 35218

[0126]

[0127] From the above results, it can be seen that the antibacterial spray provided by the present invention with PEI-PLGA-Cur@32C nanoparticles as the main component has a good antibacterial effect.

[0128] The present invention provides nanoparticles loaded with curcumin and an antimicrobial peptide. These nanoparticles, prepared by loading curcumin and an antimicrobial peptide, exhibit high solubility, good stability, excellent biocompatibility, and anti-inflammatory and antimicrobial properties for photodynamic therapy. These nanoparticles utilize safe excipients, a simple production process, a small and uniform particle size, and excellent blood compatibility. They can be used to treat infections caused by drug-resistant bacteria, and have the potential to become a new type of nano-antimicrobial drug with promising clinical applications.

[0129] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0130] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0131] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A nanoparticle loaded with curcumin and antimicrobial peptide, characterized in that: PEI-PLGA and curcumin are used as raw materials to prepare PEI-PLGA-Cur nanoparticles; PEI-PLGA-Cur nanoparticles and antimicrobial peptides are used as raw materials to prepare the nanoparticles loaded with curcumin and antimicrobial peptides.

2. The method for preparing nanoparticles loaded with curcumin and antimicrobial peptide according to claim 1, wherein: The steps include: Preparation of PEI-PLGA-Cur nanoparticles: PEI-PLGA and dimethylformamide were mixed to completely dissolve the PEI-PLGA, and then curcumin, triethanolamine, and PBS were added to obtain a mixed solution; the mixed solution was heated and mixed, and dialyzed to obtain a PEI-PLGA-Cur solution; Preparation of PEI-PLGA-Cur@32C nanoparticles: PEGDA is added to a PEI-PLGA-Cur solution to make a final concentration of PEGDA in the solution of 0.08-0.12 mg / mL, the solution is heated and mixed, and dialyzed to obtain a PEI-PLGA-Cur solution loaded with PEGDA; an antimicrobial peptide is added to the PEI-PLGA-Cur solution loaded with PEGDA to a final concentration of 48-52 μg / mL, the solution is heated and mixed, and dialyzed to obtain PEI-PLGA-Cur@32C nanoparticles, i.e., the nanoparticles loaded with curcumin and antimicrobial peptide.

3. The method for preparing nanoparticles loaded with curcumin and antimicrobial peptide according to claim 2, wherein: The volume ratio of the PEI-PLGA to dimethylformamide is 1:48-52.

4. The method for preparing nanoparticles loaded with curcumin and antimicrobial peptide according to claim 2, wherein: After PEI-PLGA is completely dissolved, curcumin and triethanolamine are added first, and then PBS is added dropwise at 22-28°C while shaking.

5. The method for preparing nanoparticles loaded with curcumin and antimicrobial peptide according to claim 2, wherein: The conditions for heating and mixing are: shaking reaction at 36-38°C for 22-26 hours.

6. The preparation method and application of the nanoparticles loaded with curcumin and antimicrobial peptide according to claim 2, characterized in that: The dialysis conditions were as follows: dialysis with deionized water in a 3500 Da dialysis bag for 22 to 26 hours.

7. The method for preparing nanoparticles loaded with curcumin and antimicrobial peptides according to claim 2, wherein: The final concentration of antimicrobial peptide added to the PEI-PLGA-Cur solution of PEGDA is 50 μg / mL.

8. The method for preparing nanoparticles loaded with curcumin and antimicrobial peptide according to claim 7, wherein: The antimicrobial peptide is AMP-32C.

9. Use of the nanoparticles loaded with curcumin and antimicrobial peptides in the preparation of antibacterial drugs according to claim 1, characterized in that: The antibacterial drug is an antibacterial spray.

10. The use according to claim 9, characterized in that The antibacterial spray comprises the nanoparticles loaded with curcumin and antibacterial peptides according to claim 1 and PBS.

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