Multifunctional nano-drug as well as preparation method and antibacterial application thereof

Abstract

The invention relates to the field of chemical synthesis and biomedical materials, and discloses a multifunctional nano-drug and a preparation method and antibacterial application thereof, and the multifunctional nano-drug is mainly composed of a copper sulfide nano-carrier, a bacterial targeting photosensitizer and glucose oxidase (GOx). Wherein the copper sulfide nano-carrier (CuS NPs) has a hollow mesoporous structure and can effectively load drug molecules. The bacterium targeting photosensitizer, namely, D-cysteine functional modified pentafluorophenyl mycogreen (FBC-Cys), can be used for targeting bacteria by utilizing the characteristic that free D-type amino acid is integrated to the end of peptidoglycan on the surface of a cell wall in the cell wall synthesis process of the bacteria. Glucose oxidase (GOx) can efficiently consume glucose in the bacterial microenvironment, and the hunger treatment effect is achieved. The hollow mesoporous copper sulfide nanoparticles are prepared by adopting a one-pot hydrothermal method, synthesis raw materials are easy to obtain, and the method is simple and rapid. And the FBC-Cys and the GOx are loaded to the cavities and the surfaces of the copper sulfide nanoparticles through a physical adsorption effect. The multifunctional nano-drug can target bacterial cell walls, change the bacterial microenvironment, integrate photodynamic therapy and hunger therapy, and achieve a multifunctional antibacterial effect. The antibacterial hydrogel provided by the invention has excellent antibacterial effect and biocompatibility, and can play an excellent role in treatment of future bacterial infection.

Description

Technical Field

[0001] The invention belongs to the field of chemical synthesis and biomedical materials, and specifically relates to a multifunctional nano drug and a preparation method and antibacterial application thereof. Background Art

[0002] Bacterial infection is one of the most important factors affecting wound healing, and can induce a variety of infectious diseases ranging from surface infection to diseases affecting internal organs and even systemic diseases. Antibiotics are now the main way to prevent and treat bacterial infections in clinical practice. Common antibiotics can effectively inhibit the growth of pathogenic bacteria and reduce many postoperative symptoms caused by bacterial infections. However, the overuse of antibiotics has caused bacteria to develop drug resistance, and some strains have even gradually evolved into "super bacteria", which seriously threatens human and animal health. Therefore, it is crucial to develop treatments that have excellent antibacterial effects but do not produce bacterial resistance.

[0003] In recent years, photodynamic therapy (PDT) has attracted attention and developed rapidly. Antimicrobial photodynamic therapy (aPDT) uses photosensitizers to absorb light of a specific wavelength and be excited. The excited photosensitizers can transfer their energy to the surrounding oxygen molecules, thereby generating highly toxic reactive oxygen species (ROS) for antibacterial treatment. It has the characteristics of broad-spectrum antibacterial properties, low drug resistance, and good selectivity, and has broad application prospects. However, the complex microenvironment of the bacterial infection site and the shortcomings of the photosensitizers at the core of photodynamic therapy, such as poor water solubility, poor stability, easy aggregation quenching, and short action distance, greatly limit the effect of phototherapy.

[0004] Starvation therapy is an emerging anti-tumor method that has attracted increasing attention due to its promising clinical applications. Bacterial proliferation is highly dependent on glucose glycolysis, and starvation therapy inhibits bacterial proliferation by consuming glucose to remove the nutrient supply of bacteria. Glucose oxidase (GOx)-mediated starvation therapy has received increasing attention. GOx consumes nutrients in the bacterial microenvironment by catalyzing the reaction between glucose and oxygen, thereby inhibiting bacterial proliferation. At the same time, the reaction also produces hydrogen peroxide (H2O2) and D-gluconic acid, both of which have certain antibacterial effects. H2O2 can cause oxidative damage to bacterial DNA, proteins, and membrane lipids, and the pH-lowering effect caused by the accumulation of D-gluconic acid may also inhibit bacterial growth. However, due to the limited oxygen content in the bacterial microenvironment, a single GOx-mediated starvation therapy is difficult to achieve a significant antibacterial effect.

[0005] In recent years, the innovative development and application fields of nanomaterials have been continuously expanded, and many nanomaterials suitable for biomedical diagnosis and treatment have been developed. Nanomaterials are an important branch of materials science, with the characteristics of small size, high specific surface area, high surface activity, simple and controllable synthesis, etc. In the inflammatory area, these nanomaterials usually show enhanced permeability and retention effects. In aPDT, nanomaterials can act as nanocarriers while ensuring the effective delivery of these photosensitizers. There have been many attempts to combine nanomaterials with aPDT, which has increased the stability and effectiveness of aPDT and improved the efficiency of ROS generation. Researchers hope that the use of nanomaterial carriers in the aPDT process can improve the required solubility and dispersibility, while the multi-drug loading capacity can further synergize the treatment and improve the efficacy of nanoplatform antibacterial therapy. The development of a multifunctional nanoplatform for antibacterial treatment has certain practical application value. . Summary of the invention

[0006] In view of this, the present invention provides a multifunctional nanomedicine for achieving synergistic and efficient antibacterial effects of photodynamic therapy and starvation therapy. Nanoscale copper sulfide nanocarriers (CuS NPs) are prepared as an inorganic nanocarrier platform, which can enhance the permeability and retention effect in the inflammatory area, and load the bacteria-targeted photosensitizer D-cysteine ​​functionalized modified pentafluorophenyl bacteriochlorin (FBC-Cys) for effective delivery for targeted photodynamic antibacterial therapy. The modified groups of the photosensitizer molecules can target bacteria and produce reactive oxygen species (ROS) under 750 nm light to achieve the effect of precise killing. Glucose oxidase (GOx) can change the bacterial microenvironment by consuming glucose, achieve the effect of starvation therapy, and achieve better antibacterial effects in synergy with photodynamic therapy. The multifunctional nanomedicine has good water solubility, good biocompatibility, significant antibacterial effect and is not easy to cause bacteria to develop drug resistance. It can be used to solve the problem that antibiotics in the current antibacterial field can cause bacteria to develop drug resistance, and traditional photosensitizers have poor water solubility, easy aggregation, and poor retention, which reduces the effect of photodynamic therapy.

[0007] In order to achieve the above technical objectives, this invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a multifunctional nano drug, the preparation process and method of which are as follows:

[0009] S1: First, a certain amount of CuCl2·2H2O and polyvinyl pyrrolidone were weighed and dissolved in water, and sodium L-ascorbate and sodium hydroxide were added under stirring at room temperature, and the temperature was raised to 60°C, and a certain amount of Na2S was added, and the mixture was stirred at 60°C for 3 hours, and then washed and dried by high-speed centrifugation to obtain CuS NPs;

[0010] S2: Then, a certain amount of CuS NPs was weighed and dissolved in water, a certain amount of polyethyleneimine was added, and the FBC-Cys aqueous solution was slowly added to the CuS NPs solution under mechanical stirring conditions, stirred for a period of time, and washed and dried by high-speed centrifugation to obtain CuS@FBC-Cys powder;

[0011] S3: Finally, a certain amount of CuS@FBC-Cys was weighed and dissolved in water. Under mechanical stirring conditions, a certain amount of GOx aqueous solution was slowly added to the CuS@FBC-Cys solution. After stirring for a period of time, the solution was washed and dried by high-speed centrifugation to obtain the multifunctional nanodrug CuS@FBC-Cys@GOx powder.

[0012] The second aspect of the present invention provides the application of multifunctional nanomedicine in antibacterial aspect.

[0013] This invention provides a multifunctional nanomedicine. The nanoplatform has excellent drug loading capacity, water dispersibility and biocompatibility. The loaded FBC-Cys has excellent ability to generate singlet oxygen, the coated GOx consumes glucose for starvation therapy, and synergistic photodynamic therapy shows outstanding antibacterial effect, which is suitable for antibacterial therapy.

[0014] The application includes the following tests:

[0015] S1: CuS NPs, CuS@FBC-Cys and CuS@FBC-Cys@GOx were prepared into aqueous solutions, and the UV absorption spectra of the samples were tested by UV-visible spectrophotometer, and the particle size distribution of the multifunctional nanodrug particle samples was tested by dynamic light scattering.

[0016] S2: The singlet oxygen test agent 1,3-diphenylisobenzofuran (DPBF) solution was mixed with the multifunctional nanodrug particles, irradiated with 750 nm light, and the ultraviolet absorption of the test solution at 425 nm was measured. The decrease rate of its absorbance was used to characterize the singlet oxygen generation ability of the multifunctional nanodrug.

[0017] S3: Mix the hydrogen peroxide detector Ti(SO4)2 solution, glucose solution and multifunctional nanomedicine, test the absorbance of the solution at 405 nm at regular intervals, and obtain the content of hydrogen peroxide based on the relationship between the absorbance in the concentration-absorbance standard curve. The increase in hydrogen peroxide content is used to characterize the ability of the multifunctional nanomedicine to degrade glucose.

[0018] S4: Using methicillin-resistant Staphylococcus aureus and Escherichia coli as experimental strains, the plate count method was used to test the antibacterial effect of the multifunctional nanomedicine.

[0019] S5: L929 cells were used as experimental cells, and the CCK-8 kit was used to detect the biocompatibility of the multifunctional nanomedicine.

[0020] Beneficial effects of the present invention:

[0021] 1. Experiments have shown that the multifunctional nanomedicine provided by the present invention has excellent water solubility, presents a green solution, and has no obvious precipitation.

[0022] 2. Experiments have shown that the multifunctional nanomedicine provided by the present invention can effectively inhibit the growth of bacteria.

[0023] 3. The CuS NPs, FBC-Cys and GOx used in the present invention are relatively easy to synthesize and obtain, and the preparation method is simple and easy to operate, suitable for large-scale implementation.

[0024] 4. The CuS NPs in the present invention are obtained by chemical preparation, have good drug loading capacity and water dispersibility, and enhance the permeability and retention effect in the inflammatory area, thereby enhancing the drug delivery effect; FBC-Cys is loaded into the hollow mesoporous CuS NPs cavity, and the outer layer is coated with GOx, and the prepared multifunctional nano drug powder particles are stable and have good water dispersibility; the loaded photosensitizer can target bacteria for precise treatment and improve the treatment efficiency. This nano-platform uses photosensitizer-mediated photodynamic therapy and GOx-mediated starvation therapy for combined antibacterial treatment, significantly enhancing the antibacterial effect under co-culture conditions, and showing broad application prospects. The present invention not only improves the effect and efficiency of treatment, but also opens up a new direction for synergistic photodynamic antibacterial therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 UV absorption spectra of CuS NPs, FBC-Cys and CuS@FBC-Cys@GOx;

[0026] Figure 2 This is the particle size distribution diagram of the multifunctional nanomedicine;

[0027] Figure 3 UV absorption degradation curves of DPBF after treatment with H2O, CuS NPs, CuS@FBC-Cys and CuS@FBC-Cys@GOx;

[0028] Figure 4 This is the hydrogen peroxide production curve after multifunctional nanomedicine treatment;

[0029] Figure 5 This is the result of the antibacterial test of the multifunctional nanomedicine (methicillin-resistant Staphylococcus aureus and Escherichia coli);

[0030] Figure 6This is the result of the biocompatibility experiment of multifunctional nanomedicine; DETAILED DESCRIPTION

[0031] The following drawings introduce the preferred embodiments in the specification to demonstrate the feasibility of the present invention, comprehensively demonstrate the present invention to the technicians, and make the technical content clearer and easier to understand. In order to better understand the essence of the present invention, the concept of the present invention is explained through specific operation processes, but it is not a limitation of the present invention. The technicians can optimize and improve according to the basic concept of the present invention, which is always within the scope of the present invention.

[0032] Example 1

[0033] This embodiment 1 provides a method for preparing a multifunctional nano drug, and the preparation process is as follows:

[0034] Firstly, nanoscale copper sulfide nanocarriers (CuS NPs) were prepared as an inorganic nanocarrier platform, and then the bacteria-targeted photosensitizer D-cysteine ​​functionalized modified pentafluorophenylbacteriochlorin (FBC-Cys) was loaded into the cavity of CuS NPs. Finally, glucose oxidase (GOx) was coated on the outside to prepare a multifunctional nanodrug.

[0035] The preparation method of the multifunctional nano drug is as follows:

[0036] S1: Synthesis of CuS NPs

[0037] CuCl2·H2O (85.2 mg, 0.5 mmol) and polyvinyl pyrrolidone (500 mg) were dissolved in water (200 ml), stirred at room temperature for 5 min, and then 2.5 mL of sodium hydroxide aqueous solution (0.2 M) was added to the solution, stirred at room temperature for 5 min, and then 2.5 mL of L-sodium ascorbate aqueous solution (0.1 M) was added to the solution, heated to 60 °C in a water bath, and then 2.0 mL of Na2S solution (1.0 M) was added to the solution, and stirred at 60 °C for 3 hours. After the reaction was completed, it was cooled to room temperature, and the precipitate was collected by high-speed centrifugation (11000 rpm, 5 min), washed three times with deionized water, and the precipitate was collected by high-speed centrifugation (11000 rpm, 5 min), and vacuum dried to obtain CuS NPs;

[0038] S2: Synthesis of CuS@FBC-Cys

[0039] CuS NPs (10 mg) were dispersed in water (15 mL), polyethyleneimine (200 mg) was added, and FBC-Cys aqueous solution (2 mg / mL, 5 mL) was slowly added to the above solution under mechanical stirring conditions, and stirred at room temperature for 24 h. After the reaction was completed, the precipitate was collected by high-speed centrifugation (11000 rpm, 5 min), washed three times with deionized water, and the precipitate was collected by high-speed centrifugation (11000 rpm, 5 min), and vacuum dried to obtain CuS@FBC-Cys powder;

[0040] S3: Synthesis of CuS@FBC-Cys@GOx

[0041] CuS@FBC-Cys (10 mg) was dissolved and dispersed in water (15 mL). Under mechanical stirring, GOx aqueous solution (2 mg / mL, 5 mL) was slowly added to the above solution and stirred at room temperature for 24 h. After the reaction, the precipitate was collected by high-speed centrifugation (11000 rpm, 5 min), washed three times with deionized water, and the precipitate was collected by high-speed centrifugation (11000 rpm, 5 min). The multifunctional nanomedicine CuS@FBC-Cys@GOx powder was obtained by vacuum drying.

[0042] Example 2

[0043] This example 2 provides a characterization of the properties of the multifunctional nanomedicine in Example 1

[0044] S1: UV absorption test of CuS NPs, FBC-Cys and CuS@FBC-Cys@GOx

[0045] 1. Experimental materials: CuS NPs, FBC-Cys and CuS@FBC-Cys@GOx

[0046] 2. Experimental method: CuS NPs, FBC-Cys and CuS@FBC-Cys@GOx were prepared into deionized aqueous solutions respectively. The baseline was first scanned with deionized water, and then the UV absorption spectra of CuS NPs, FBC-Cys and CuS@FBC-Cys@GOx aqueous solutions were scanned in the wavelength range of 500 nm-850 nm.

[0047] 3. Experimental results: UV absorption spectrum Figure 1 As shown, the absorption peak of FBC-Cys photosensitizer appeared at 750 nm on CuS@FBC-Cys@GOx, indicating that FBC-Cys was successfully loaded.

[0048] S2: Particle size detection of multifunctional nanomedicines

[0049] 1. Experimental materials: Multifunctional nanodrug CuS@FBC-Cys@GOx aqueous solution

[0050] 2. Experimental method: Use dynamic light scattering to determine the particle size distribution of the multifunctional nanodrug solution.

[0051] 3. Experimental results: The particle size of multifunctional nanomedicine is Figure 2 As shown, the hydrodynamic diameter of the multifunctional nano drug is 157 nm, and the polydispersity index is 0.245. The particle size of the multifunctional nano drug is less than 200 nm and is evenly distributed.

[0052] Example 3

[0053] This Example 3 provides a test for the singlet oxygen generation ability of the multifunctional nanomedicine in Example 1.

[0054] 1. Experimental materials: CuS NPs, CuS@FBC-Cys and CuS@FBC-Cys@GOx aqueous solutions

[0055] 2. Experimental method: The CuS NPs, CuS@FBC-Cys and multifunctional nanodrug CuS@FBC-Cys@GOx in Example 1 were mixed with the singlet oxygen detection reagent 1,3-diphenylisobenzofuran (DPBF) to form a solution. Each group was irradiated with a light source with a wavelength of 750 nm. The ultraviolet absorption of the solution at 425 nm was detected for 120 seconds each time. The experiment was repeated 3 times.

[0056] 3. Experimental results: The singlet oxygen generation capacity of the UV absorption is shown in Table 1 and Figure 3 shown.

[0057] Table 1: Singlet oxygen production capacity results 0 s 120 s 240 s 360 s 480 s 600s <![CDATA[H2O]]> 1 0.92 0.88 0.84 0.81 0.77 CuS NPs 1 0.91 0.87 0.84 0.81 0.78 CuS@FBC-Cys 1 0.80 0.69 0.62 0.54 0.49 CuS@FBC-Cys@GOx 1 0.82 0.71 0.64 0.58 0.52

[0058] From Table 1 and Figure 3 It can be seen that the multifunctional nanomedicine has a good ability to produce singlet oxygen, and the coated GOx molecules do not significantly cause a decrease in singlet oxygen yield.

[0059] Example 4

[0060] This Example 4 provides a method for detecting the GOx enzyme activity of the multifunctional nanomedicine in Example 1.

[0061] 1. Experimental materials: CuS@FBC-Cys@GOx, 20mM glucose aqueous solution

[0062] 2. Experimental method: The CuS@FBC-Cys@GOx (3 mg) in Example 1 was mixed with a 20 mM glucose aqueous solution (20 mL). 1 mL of the above solution was taken every 15 seconds to prepare a detection solution with Ti(SO4)2 detection solution (1 mL). The ultraviolet absorption at 405 nm was detected, and the hydrogen peroxide production content was calculated according to the absorbance-concentration standard curve. The experiment was repeated 3 times.

[0063] 3. Experimental results: The results of GOx enzyme activity of multifunctional nanomedicine are as follows Figure 4 As shown, H2O2 gradually increases with time, indicating that the multifunctional nanomedicine is successfully loaded with GOx and can play the role of GOx in consuming glucose to produce hydrogen peroxide.

[0064] Example 5

[0065] This Example 5 provides an antibacterial evaluation of the multifunctional nanomedicine in Example 1.

[0066] Antibacterial testing of multifunctional nanomedicine under light conditions

[0067] 1. Experimental materials: Multifunctional nanomedicine CuS@FBC-Cys@GOx prepared in Example 1

[0068] 2. Experimental method: In this experiment, methicillin-resistant Staphylococcus aureus and Escherichia coli were used as test bacteria to test the antibacterial effect of CuS@FBC-Cys@GOx. 100 μL of 2 × 10 6 CFU / mL bacterial solution and 100 µL of 50 µM CuS@FBC-Cys@GOx solution diluted with culture medium. Each well was irradiated with a 750 nm wavelength laser for 15 min and cultured at 37 °C for 24 h. Then, different groups were diluted by the same multiple with sterile PBS, and 20 µL was smeared on a pre-prepared solid culture medium plate. After culturing for 18 hours, the live bacterial colony counting method was used to determine the bacterial concentration and calculate the antibacterial rate.

[0069] The antibacterial rate calculation formula is: X=(AB) / A×100%

[0070] Where: X-inhibition rate%; A-average colony count of control samples; B-average colony count of test samples.

[0071] 3. Experimental results: The antibacterial test results are shown in Table 2 and Figure 5 shown.

[0072] Table 2: Antibacterial test results

[0073] From Table 2 and Figure 5It can be seen from the results that the multifunctional nanomedicine prepared in Example 1 of the present invention can effectively kill methicillin-resistant Staphylococcus aureus and Escherichia coli, and has high antibacterial activity. This also shows that under the synergistic effect of FBC-Cys and GOx loaded by CuSNPs, a better effect against common bacteria and drug-resistant bacteria is produced.

[0074] Example 6

[0075] This Example 6 provides an evaluation of the biocompatibility of the multifunctional nanomedicine in Example 1.

[0076] 1. Experimental materials: Multifunctional nanomedicine prepared in Example 1

[0077] 2. Experimental method: In this experiment, L929 cells were used as test cells to test the biocompatibility effect of multifunctional nanomedicines. L929 cells were dispersed in a 96-well plate, and multifunctional nanomedicine solutions diluted with culture medium at concentrations of 0, 12.5, 25, 50, 100, and 200 µg / mL were added, and cultured in an incubator for 24 hours. Then the old culture medium was removed and 100 µL of new culture medium containing CCK-8 (5 µg / mL) was added. After 1 hour, the OD value of each well at 450 nm was measured by an enzyme reader to determine the cell survival rate.

[0078] 3. Experimental results: The antibacterial test results are shown in Table 3 and Figure 6 shown.

[0079] Table 3: Biocompatibility test results

[0080] From Table 3 and Figure 6 It can be seen from the results that the multifunctional nanomedicine prepared in Example 1 of the present invention does not produce cytotoxicity under the condition of no light, indicating that the multifunctional nanomedicine has good biocompatibility and stability. With its good biocompatibility and excellent antibacterial potential, the multifunctional nanomedicine demonstrates important application value in the field of antibacterial therapy, providing a new option for antibacterial photodynamic therapy in the future.

[0081] The above description is only a preferred embodiment of the present invention. It should be pointed out that other embodiments obtained by ordinary technicians in this field without making creative work should also be regarded as within the protection scope of the present invention.

Claims

1. A multifunctional nano drug, characterized in that: It is composed of copper sulfide nanocarriers (CuS NPs), bacteria-targeted photosensitizer D-cysteine ​​functionalized modified pentafluorophenyl bacteriochlorin (FBC-Cys) and glucose oxidase (GOx), and its preparation process is as follows:

2. The method for preparing the multifunctional nano drug according to claim 1, characterized in that: The following steps are involved: S1: First, a certain amount of CuCl2·2H2O and polyvinyl pyrrolidone were weighed and dissolved in water, and sodium L-ascorbate and sodium hydroxide were added under stirring at room temperature, and the temperature was raised to 60°C, and a certain amount of Na2S was added, and the mixture was stirred at 60°C for 3 hours, and then washed and dried by high-speed centrifugation to obtain CuS NPs; S2: Then, a certain amount of CuS NPs was weighed and dissolved in water, a certain amount of polyethyleneimine was added, and the FBC-Cys aqueous solution was slowly added to the CuS NPs solution under mechanical stirring conditions, stirred for a period of time, and washed and dried by high-speed centrifugation to obtain CuS@FBC-Cys powder; S3: Finally, a certain amount of CuS@FBC-Cys was weighed and dissolved in water. Under mechanical stirring conditions, a certain amount of GOx aqueous solution was slowly added to the CuS@FBC-Cys solution. After stirring for a period of time, the solution was washed and dried by high-speed centrifugation to obtain the multifunctional nanodrug CuS@FBC-Cys@GOx powder.

3. The multifunctional nano drug according to claim 1, characterized in that: The CuS NPs are hollow mesoporous copper sulfide nanoparticles.

4. The multifunctional nano drug according to claim 1, characterized in that: It has good drug loading capacity and water dispersibility and the size of the nanoparticles is less than 200 nm.

5. The FBC-Cys according to claim 1, characterized in that It is able to target bacteria and generate reactive oxygen species (ROS) under 750 nm light irradiation.

6. The GOx according to claim 1, characterized in that Glucose in the bacterial microenvironment can be consumed for starvation therapy, and the produced hydrogen peroxide can synergistically enhance the antibacterial effect.

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