Application of antibacterial tannin-modified silver nanoparticles as antitumor drug carriers in the preparation of antibacterial and antitumor nanomedicines

Abstract

This invention provides an application of antibacterial tannin-modified silver nanoparticles as antitumor drug carriers in the preparation of antibacterial and antitumor nanomedicines. The method involves adding an antitumor drug to an aqueous dispersion of tannin-modified silver nanoparticles, thoroughly mixing them under dark conditions to load the antitumor drug onto the surface of the antibacterial silver nanoparticles, and then centrifuging to obtain nanomedicines with both antibacterial and antitumor properties. The tannin-modified silver nanoparticles not only possess good biocompatibility and excellent antibacterial properties, but also efficiently load antitumor drugs through electrostatic interactions. The resulting nanomedicines exhibit pH- and glutathione-sensitive antitumor drug release behavior, simultaneously exerting antitumor and antibacterial effects, and are expected to address the problem of reduced efficacy of chemotherapy drugs due to patients' susceptibility to bacterial infections during cancer treatment.

Description

Technical Field

[0001] This invention relates to the application of antibacterial tannin-modified silver nanoparticles as antitumor drug carriers in the preparation of antibacterial and antitumor nanomedicines, belonging to the fields of composite materials technology and biomedical technology. Background Technology

[0002] Bacteria are closely related to the development and progression of cancer. The role of bacterial infection in cancer cells and how bacteria interfere with cancer treatment have received widespread attention in recent years. Many cancers, including stomach, colorectal, and pancreatic cancers, have been found to be caused by bacterial infections. Bacteria can exacerbate tumor development through inflammatory responses, the secretion of bacterial enzymes, toxins, and oncogenic peptides. Polym. Chem. , 2021, 12 (3992–4005). Studies have shown that due to the formation of a bacterial-friendly environment in cancer tissues and the highly compromised immune state of cancer patients, bacterial infections can occur in cancer patients after surgery, radiotherapy, or chemotherapy. Microbial infections can significantly reduce the effectiveness of cancer treatment and lead to serious complications. Currently, the overuse and long-term abuse of antibiotics have led to the rapid emergence of multidrug-resistant pathogens. Therefore, developing drug delivery systems with both antibacterial and antitumor properties is crucial for the treatment of cancers associated with bacterial infections.

[0003] Nanomaterial-based drug delivery systems have many advantages in the field of cancer treatment, and among them, nanomedicine carriers with antibacterial properties are receiving increasing attention for solving the problem of bacterial infection during cancer treatment. Biomater. Sci. , 2020, 8 , 6814-6824). Among the antibacterial nanocarriers developed in recent years, silver nanoparticles (AgNPs) have attracted great attention in the treatment of bacterial infections due to their broad-spectrum antibacterial activity, good biocompatibility, and resistance to drug resistance. Biomater. Sci. , 2020, 8 (4852–4860). AgNPs release Ag + As a special element, AgNPs can exert their powerful bactericidal activity by inhibiting bacterial wall synthesis, disrupting cell membranes, and interfering with nucleic acid function. However, traditional AgNPs have relatively inert surfaces, making it difficult to directly load anticancer drugs. They can only exert their antibacterial effect by being loaded together with anticancer drugs into delivery materials such as micelles and gels. ACS Appl. Nano Mater. 2019, 2 , 7393−7408; ACS Appl. Mater. Interfaces 2017, 9(16880−16889). Developing silver nanoparticles with drug-loading properties for anticancer drug delivery remains challenging.

[0004] Plants provide us with a variety of secondary metabolites, including natural compounds and polymers with diverse biological activities and functions. Tannins are a class of natural polyphenolic compounds widely distributed in various plants. Their molecular structure contains abundant catechol or pyrogallol groups, exhibiting excellent ability to reduce noble metal ions and significant antibacterial, antioxidant, and antiviral activities. Rev. Med. Chem. ,2008, 8 , 1179–1187). Summary of the Invention

[0005] The purpose of this invention is to provide an application of antibacterial tannin-modified silver nanoparticles as an antitumor drug carrier in the preparation of antibacterial and antitumor nanomedicines. Specifically, the prepared antibacterial tannin-modified silver nanoparticles are used as an antitumor drug carrier for tumor-targeted antitumor and antibacterial sustained-release drugs.

[0006] I. Preparation and Properties of Tannin-Modified Silver Nanoparticles

[0007] 1. Preparation of Tannin-Modified Silver Nanoparticles

[0008] Silver nitrate and tannin were dissolved in water to form solutions. The pH of the tannin aqueous solution was adjusted to 7.5-8.5 with sodium carbonate. The pH-adjusted tannin aqueous solution was quickly added to the boiling silver nitrate aqueous solution, and the mixture was heated and stirred to obtain a tannin-modified silver nanoparticle dispersion. The precipitate of the nanoparticle dispersion was collected by centrifugation and washed with deionized water to obtain antibacterial tannin-modified silver nanoparticles.

[0009] The tannin is any one of tannic acid, chebulic acid, chebulic acid, and gallic acid, and the molar ratio of silver nitrate to tannin is 0.5:1 to 5:1.

[0010] In order to fully reduce Ag with tannins + To form nanoparticles, the mixed solution is heated and stirred at 95~105℃ for 1~2 h.

[0011] The centrifugation is performed at 12000 rpm for 10-20 minutes.

[0012] Morphological characterization of tannin-modified silver nanoparticles was performed using a FEI-Tecnai G2 transmission electron microscope (TEM). Figure 1Transmission electron microscopy (TEM) images of silver nanoparticles modified with tannic acid, chebulic acid, and gallic acid reduction are shown. As can be seen, the three tannin-modified silver nanoparticles all exhibit typical spherical structures with diameters of approximately 25, 10.4, and 37.5 nm, respectively. The absorption wavelengths were investigated using a Perkin-Elmer Lambda 35 ultraviolet spectrophotometer. Figure 2 The UV absorption spectra of silver nanoparticles modified by reduction with tannic acid, chebulic acid, and gallic acid are shown. It can be seen that the maximum absorption wavelengths of the three tannin-modified silver nanoparticles are 420, 407, and 422 nm, respectively.

[0013] 2. Antibacterial properties of tannin-modified silver nanoparticles

[0014] Taking tannic acid-modified silver nanoparticles (TA-AgNPs) as an example, the inhibitory effect of TA-AgNPs on Gram-negative bacteria (Escherichia coli) was investigated using the minimum inhibitory concentration experiment.

[0015] Experimental methods: *E. coli* was cultured in beef extract peptone liquid medium for 24 hours (37℃), and the absorbance (optical density, OD) was measured at a wavelength of 600 nm. 600 Dilute the bacterial culture to 1×10⁻⁶. 5 CFU / mL. Add 100 μL of bacterial dispersion (10 CFU / mL) to a 96-well plate. 5 CFU / mL), then add a series of 2-fold diluted TA-AgNPs samples (100 μL). Incubate the 96-well plate at 37°C for 24 hours, and measure the OD using a microplate reader (SpectraMax®, Molecular Devices, USA). 600 Evaluate the survival rate of bacteria.

[0016] Figure 3 This graph shows the antibacterial effects of different concentrations of TA-AgNPs on Escherichia coli. The results indicate that the survival rate of E. coli decreased in a concentration-dependent manner in the presence of TA-AgNPs. When the concentration of TA-AgNPs was 8 μg / mL, the survival rate of E. coli was higher than 60%, while at a concentration of 16 μg / mL, the survival rate dropped sharply to below 20%. Figure 3 The results showed that the minimum inhibitory concentration of TA-AgNPs against Escherichia coli was approximately 16 μg / mL.

[0017] 3. Biocompatibility of Tannin-Modified Silver Nanoparticles

[0018] Taking TA-AgNPs as an example, the biocompatibility of tannin-modified silver nanoparticles was investigated using the Live-Dead colorimetric method. The cytotoxicity of TA-AgNPs at concentrations of 5 and 50 μg / mL on normal Vero cells (African green monkey kidney cells) was examined. Vero cells were seeded in 24-well plates (1 × 10⁶ cells per well). 6 Cells were cultured in wells containing 5 μg / mL TA-AgNPs for 24 hours. The old medium was replaced with medium containing 5 μg / mL TA-AgNPs, and cultured at 37°C for 24 hours. The medium was removed, and fluorescent dye for live / dead cells was added to each well, and the cells were cultured at 37°C for 30 minutes. Images of the stained cells were captured using a laser confocal microscope (Olympus Fluoview 1000, Japan).

[0019] Figure 4 This image shows the live / dead results of Vero cells cultured with different concentrations of TA-AgNPs. Laser confocal microscopy images show that when treated with 5 or 50 μg / mL TA-AgNPs and phosphate-buffered saline (PBS, control), the morphology of Vero cells remained almost unchanged, and almost no red fluorescence (dead cells) was observed in either the TA-AgNPs group or the control group. These results indicate that TA-AgNPs have low cytotoxicity to normal cells and are a biocompatible drug carrier.

[0020] II. Preparation and Properties of Nanomedicines with Antibacterial and Antitumor Properties

[0021] 1. Preparation of nanomedicines with antibacterial and antitumor properties

[0022] Antitumor drugs were added to an aqueous dispersion of tannin-modified silver nanoparticles and thoroughly mixed at room temperature in the dark to load the antitumor drugs onto the surface of the antibacterial silver nanoparticles. After centrifugation, nanomedicines with both antibacterial and antitumor properties were obtained.

[0023] The antitumor drug is any one of doxorubicin hydrochloride, epirubicin hydrochloride, or irinotecan hydrochloride. The mass ratio of tannin-modified silver nanoparticles to the antitumor drug is 10:1 to 1:1.

[0024] The mixing method under the dark conditions is stirring or oscillation, and the mixing time is 12~72 h.

[0025] The centrifugation was performed at 12,000 rpm for 10-20 minutes.

[0026] Taking epirubicin hydrochloride as an example, the surface potential changes of TA-AgNPs before and after epirubicin loading were characterized using a Zeta potential instrument, such as... Figure 5As shown, the average surface potential of TA-AgNPs is -38.9 mV, while the surface potential of the nanomedicine after loading epirubicin is -30.5 mV. This significant difference in surface potential indicates that epirubicin has been successfully loaded onto the surface of TA-AgNPs through electrostatic interactions.

[0027] 2. Antibacterial and antitumor properties of nanomedicines: pH and glutathione-sensitive (GSH) drug release behavior.

[0028] Taking TA-AgNPs nanomedicine loaded with epirubicin hydrochloride (EPI) (TA-AgNPs / EPI) as an example, the in vitro release behavior of EPI in the nanomedicine was investigated in three different PBS buffers: pH 7.4 PBS buffer (simulating normal physiological environment), pH 5.0 PBS buffer (simulating the slightly acidic environment of tumor cells), and pH 7.4 PBS buffer containing GSH (10 mM, simulating the high GSH content environment of tumor cells). 5.0 mL of TA-AgNPs / EPI dispersed in different buffers was placed in a dialysis bag (molecular weight cutoff 6000-8000 Da), and then the dialysis bag was immersed in different buffers (30 mL), and shaken at 37°C for 24 hours. Each release condition included three parallel samples. At predetermined time intervals, 5.0 mL of buffer was removed, and then 5.0 mL of fresh buffer was added. The EPI content in each sample was analyzed using a fluorescence spectrophotometer (Perkin-Elmer LS-55).

[0029] Figure 6 The figure shows the release behavior of EPI in TA-AgNPs / EPI nanomedicine in three different buffer solutions. In a buffer solution at pH 7.4, less than 12% of EPI was released within 24 hours. In a buffer solution at pH 5.0 or containing GSH, approximately 20% and 37% of EPI were released within 24 hours, respectively. This pH-sensitive release behavior is mainly attributed to the protonation of the phenolic hydroxyl groups of tannic acid on the surface of TA-AgNPs under acidic conditions, which weakens the electrostatic interaction between EPI and the nanoparticles, triggering EPI release. Furthermore, the thiol groups in GSH undergo ligand-exchange reactions with the tannic acid on the surface of silver nanoparticles, also leading to the dissociation of TA on the silver nanoparticle surface and triggering EPI release. These results indicate that TA-AgNPs / EPI nanomedicine exhibits good stability in normal physiological environments, while the slightly acidic tumor environment with high GSH content can effectively trigger EPI release through protonation of the phenolic hydroxyl groups of TA and ligand-exchange reactions with GSH.

[0030] 3. In vitro cytotoxic activity of TA-AgNPs / EPI nanomedicines

[0031] A cell model of coexistence between *E. coli* and HeP G2 liver cancer cells was established to investigate the cytotoxic activity of EPI and TA-AgNPs / EPI nanomedicines in the presence of bacteria. *E. coli* colonies were inoculated into 5 mL of LB medium and cultured at 37°C for 24 h (100 rpm) in a shaker. After centrifugation for 5 min (5000 rpm), the medium was discarded, and the bacteria were resuspended in PBS. The cytotoxicity was measured by OD... 600 Adjust the bacterial concentration to approximately 2 × 10⁻⁶. 4 CFU / mL. HepG2 cells were seeded into 96-well plates (200 μL, cell density approximately 5 × 10⁶ CFU / mL). 3 (cells / well), after culturing for 12 h, 2 μL of bacterial culture and 20 µg / mL of EPI and nanomedicine were added, and after co-culturing for 24 h, the cell and bacterial viability was determined by the MTS method.

[0032] Figure 7 The graph shows the cell viability of Hep G2 cells co-cultured with EPI and TA-AgNPs / EPI under sterile and aseptic conditions. It is evident that at an EPI concentration of 20 µg / mL, the presence of *E. coli* significantly inhibited the killing effect of EPI on Hep G2 cells, causing almost complete inactivation of EPI. However, for the nanomedicine group (TA-AgNPs / EPI), at the same EPI concentration, the presence of *E. coli* no longer affected the therapeutic effect. Both under sterile and aseptic conditions, the TA-AgNPs / EPI nanomedicine exhibited excellent cytotoxic activity. These results indicate that the TA-AgNPs / EPI nanomedicine can achieve a combined antitumor and antibacterial effect, effectively avoiding the impact of microbial infection on the efficacy of EPI.

[0033] In summary, this invention combines the advantages of tannins and silver nanoparticles to construct tannin-modified silver nanoparticles, which enhances their antibacterial activity. Crucially, the abundant phenolic hydroxyl groups in tannins readily interact with drugs through complexation, hydrogen bonding, and electrostatic interactions, thereby achieving highly efficient loading of anticancer drugs. Using tannin-modified silver nanoparticles as carriers for antitumor drugs can effectively broaden the application of silver nanoparticle materials in the field of biomedical materials. Furthermore, by efficiently loading antitumor drugs, nanomedicines with both antibacterial and antitumor properties can be prepared, potentially addressing the problem of reduced chemotherapy efficacy due to bacterial infections during cancer treatment, and providing a new approach for the simultaneous treatment of bacterial infections and tumors. Simultaneously, the method for preparing nanomedicines with both antibacterial and antitumor properties in this invention is simple, rapid, and efficient. Attached Figure Description

[0034] Figure 1 Transmission electron microscopy image of silver nanoparticles modified by reduction of tannic acid, chebulic acid, and gallic acid;

[0035] Figure 2 The UV-Vis absorption spectra of silver nanoparticles modified by reduction with tannic acid, chebulic acid, and gallic acid;

[0036] Figure 3 The graph shows the antibacterial effect of different concentrations of TA-AgNPs on Escherichia coli.

[0037] Figure 4 Live / dead results of Vero cells cultured with different concentrations of TA-AgNPs;

[0038] Figure 5 Surface potential diagrams of TA-AgNPs before and after loading with epirubicin hydrochloride;

[0039] Figure 6 The graph shows the release behavior of EPI in three different buffer solutions for TA-AgNPs / EPI nanomedicine.

[0040] Figure 7 The graph shows the cell viability of Hep G2 cells co-cultured with EPI and TA-AgNPs / EPI nanomedicines in the presence of Escherichia coli and under aseptic conditions. Detailed Implementation

[0041] To enable those skilled in the art to better understand the technical solution of the present invention, the preparation of tannin-modified silver nanoparticles and antibacterial and antitumor nanomedicines of the present invention will be further explained below with reference to implementation examples.

[0042] Example 1

[0043] (1) Preparation of tannic acid-modified silver nanoparticles (TA-AgNPs): 17 mg of tannic acid was dissolved in 1 mL of deionized water to prepare a 0.01 M aqueous solution. The pH of the tannic acid aqueous solution was then adjusted to 8.0 with 0.5 M sodium carbonate. The pH-adjusted tannic acid aqueous solution was quickly added to boiling silver nitrate aqueous solution (1 mM, 50 mL), and the mixture was stirred at 100 °C for 1.5 h to obtain a dispersion of tannic acid-modified silver nanoparticles. The nanoparticle dispersion was centrifuged at 12000 rpm for 20 minutes, the precipitate was collected, and the precipitate was washed with deionized water to obtain antibacterial tannic acid-modified silver nanoparticles.

[0044] (2) Preparation of TA-AgNPs-loaded epirubicin hydrochloride nanomedicine: 5 mg TA-AgNPs were ultrasonically dispersed in 50 mL deionized water, and 1 mL of epirubicin hydrochloride aqueous solution (1.0 mg / mL) was added to the solution. The mixture was stirred in the dark for 24 hours. The product was collected by centrifugation (12,000 rpm, 20 min) to obtain the target substance—TA-AgNPs-loaded epirubicin hydrochloride nanomedicine. The release behavior of this drug of epirubicin hydrochloride in three different buffer solutions is shown in [the figure]. Figure 6 Its in vitro cytotoxic activity is shown in Figure 7 .

[0045] Example 2

[0046] (1) Preparation of tannic acid-modified silver nanoparticles (TA-AgNPs): 17 mg of tannic acid was dissolved in 1 mL of deionized water to prepare a 0.01 M aqueous solution. The pH of the tannic acid aqueous solution was then adjusted to 8.0 with 0.5 M sodium carbonate. The pH-adjusted tannic acid aqueous solution was quickly added to boiling silver nitrate aqueous solution (1 mM, 50 mL), and the mixture was stirred at 100 °C for 1.5 h to obtain a dispersion of tannic acid-modified silver nanoparticles. The nanoparticle dispersion was centrifuged at 12000 rpm for 20 minutes, the precipitate was collected, and the precipitate was washed with deionized water to obtain antibacterial tannic acid-modified silver nanoparticles.

[0047] (2) Preparation of TA-AgNPs-loaded irinotecan hydrochloride nanomedicine: 5 mg of TA-AgNPs were ultrasonically dispersed in 50 mL of deionized water, and 1 mL of irinotecan hydrochloride aqueous solution (1.0 mg / mL) was added to the solution. The mixture was stirred in the dark for 24 hours. The product was collected by centrifugation (12,000 rpm, 20 minutes) to obtain the target substance—TA-AgNPs-loaded irinotecan hydrochloride nanomedicine. The drug release behavior and in vitro cytotoxic activity of this drug were similar to those in Example 1.

[0048] Example 3

[0049] (1) Preparation of thiamethoxam-modified silver nanoparticles: 19 mg of thiamethoxam was dissolved in 1 mL of deionized water to prepare a 0.02 M aqueous solution. The pH of the tannin aqueous solution was then adjusted to 8.0 with 0.5 M sodium carbonate. The pH-adjusted thiamethoxam aqueous solution was quickly added to boiling silver nitrate aqueous solution (1 mM, 50 mL), and the mixture was stirred at 100 °C for 1.5 h to obtain a dispersion of thiamethoxam-modified silver nanoparticles. The nanoparticle dispersion was centrifuged at 12000 rpm for 20 minutes, the precipitate was collected, and the precipitate was washed with deionized water to obtain antibacterial thiamethoxam-modified silver nanoparticles.

[0050] (2) Preparation of nanomedicine containing epirubicin hydrochloride loaded on silver nanoparticles modified with chellic acid: 5 mg of silver nanoparticles modified with chellic acid were ultrasonically dispersed in 50 mL of deionized water, and 2 mL of epirubicin hydrochloride aqueous solution (1.0 mg / mL) was added to the solution. The mixture was shaken in the dark for 24 hours. The product was collected by centrifugation (12,000 rpm, 15 min) to obtain the target substance—silver nanoparticles modified with chellic acid loaded with epirubicin hydrochloride. The drug release behavior and in vitro cytotoxic activity of this drug were similar to those in Example 1.

[0051] Example 4

[0052] (1) Preparation of gallic acid-modified silver nanoparticles: 8.5 mg of gallic acid was dissolved in 1 mL of deionized water to prepare a 0.05 M aqueous solution. Then, the pH of the tannin aqueous solution was adjusted to 8.0 with 0.5 M sodium carbonate. The pH-adjusted gallic acid aqueous solution was quickly added to boiling silver nitrate aqueous solution (1 mM, 50 mL), and the mixture was stirred at 100 °C for 1.5 h to obtain a dispersion of gallic acid-modified silver nanoparticles. The nanoparticle dispersion was centrifuged at 12000 rpm for 10 minutes, the precipitate was collected, and washed with deionized water to obtain antibacterial gallic acid-modified silver nanoparticles.

[0053] (2) Preparation of gallic acid-modified silver nanoparticles loaded with doxorubicin hydrochloride nanomedicine: 5 mg of gallic acid-modified silver nanoparticles were ultrasonically dispersed in 50 mL of deionized water, and 1 mL of doxorubicin hydrochloride aqueous solution (1.0 mg / mL) was added to the solution. The mixture was stirred in the dark for 24 hours. The product was collected by centrifugation (12,000 rpm, 10 minutes) to obtain the target substance—gallic acid-modified silver nanoparticles loaded with doxorubicin hydrochloride nanomedicine. The drug release behavior and in vitro cytotoxic activity of this drug were similar to those in Example 1.

Claims

1. The application of antibacterial tannin-modified silver nanoparticles as an antitumor drug carrier in the preparation of antibacterial and antitumor nanomedicines, characterized in that: The antibacterial tannin-modified silver nanoparticles were prepared by the following method: (1) Dissolve silver nitrate and tannin in water to form a solution, and adjust the pH of the tannin aqueous solution to 7.5~8.5 with sodium carbonate. The tannin is tannic acid. (2) The tannin aqueous solution after pH adjustment is quickly added to the boiling silver nitrate aqueous solution. The molar ratio of silver nitrate to tannin is 0.5:1~5:

1. Stir at 95~105℃ for 1~2 h to obtain a dispersion of tannin-modified silver nanoparticles. (3) Centrifuge the dispersion at 12000 rpm for 10-20 minutes, collect the precipitate, wash with deionized water to obtain antibacterial tannin-modified silver nanoparticles; An antitumor drug was added to an aqueous dispersion of tannin-modified silver nanoparticles and thoroughly mixed in the dark to load the antitumor drug onto the surface of the antibacterial silver nanoparticles. After centrifugation, a nanomedicine with both antibacterial and antitumor properties was obtained; the antitumor drug was epirubicin hydrochloride.

2. The application of the antibacterial tannin-modified silver nanoparticles as described in claim 1 as an antitumor drug carrier in the preparation of antibacterial and antitumor nanomedicines, characterized in that: The mass ratio of tannin-modified silver nanoparticles to antitumor drugs is 10:1 to 1:

1.

3. The application of the antibacterial tannin-modified silver nanoparticles as described in claim 1 as an antitumor drug carrier in the preparation of antibacterial and antitumor nanomedicines, characterized in that: The mixing method under the dark conditions is stirring or shaking, and the mixing time is 12~72 h; the centrifugation is performed at 12000 rpm for 10~20 minutes.

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