Method for green synthesis of silver nanoparticles from rhubarb extract and application of silver nanoparticles in preparation of anti-tumor nano-drugs

Abstract

The invention discloses a method for green synthesis of silver nanoparticles from a rheum officinale extract and application of the silver nanoparticles in preparation of anti-tumor nano-drugs. According to the method, an extract obtained after rheum tanguticum is extracted through ethyl alcohol and ethyl acetate serves as a reducing agent and a stabilizing agent and reacts with a boiling silver nitrate solution under the alkaline condition, and the silver nanoparticles uniform in size and good in dispersity are efficiently prepared. The silver nanoparticles have good biocompatibility and remarkable antibacterial activity. On the basis, anti-tumor drugs such as doxorubicin hydrochloride or epirubicin hydrochloride can be further loaded through electrostatic interaction, and an anti-tumor nano-drug is constructed. The prepared nano-drug can realize intelligent responsive release of the drug under the conditions of simulating acidity and high glutathione of a tumor microenvironment, and simultaneously plays an antibacterial and anti-tumor synergistic treatment role, and has a wide application prospect in treatment of tumors accompanied by bacterial infection.

Description

Technical Field

[0001] This invention relates to a method for the green synthesis of silver nanoparticles from rhubarb extract and the application of silver nanoparticles in the preparation of antitumor nanomedicines, belonging to the fields of composite materials technology and biomedical technology. Background Technology

[0002] As an indispensable component of the body's microecology, bacteria play a crucial role in regulating cancer susceptibility and tumor progression. Helicobacter pylori (Helicobacter pylori) H. pylori It has been confirmed that it is a major driving factor for gastric cancer; and Salmonella typhi (Salmonella typhi) has been identified as a major driving factor for gastric cancer. S. typhi Bacterial microorganisms (Bacteria) are potent carcinogens of gallbladder cancer (GBC) (Nat. Rev. Cancer, 2023, 23:600-618). Numerous studies have shown that bacteria can promote tumor induction and development through various mechanisms, including direct DNA damage from bacterial toxins, bacterial metabolites, direct physical interactions with host cells, inflammatory responses induced by chronic infection and invasive biofilm formation, and suppression of antitumor immune responses (CancerDiscov., 2021, 11: 2378-2395). Furthermore, in cancer treatment, bacterial infections can reduce the efficacy of anticancer drugs, weaken drug internalization, and cause toxic side effects leading to various fatal complications. Currently, antibiotics are the main strategy for treating bacterial infections associated with tumors, but the overuse of antibiotics has increased the spread of drug-resistant bacteria, leading to multidrug resistance and extreme drug resistance. Antibacterial nanomaterials have received widespread attention as a potential alternative. Therefore, developing novel nanomedicine delivery systems with both antibacterial and antitumor functions based on antibacterial nanomaterials is a key research direction for overcoming the challenges of treating cancers related to bacterial infections and has significant practical implications.

[0003] Among numerous antibacterial materials, silver nanoparticles (AgNPs) are a class of nanomaterials with advantages such as broad-spectrum antibacterial activity and resistance to drug resistance. AgNPs can exert strong bactericidal activity through mechanisms such as membrane damage, electron transport chain disruption, catalytic killing, cell division arrest, prolonged ion killing, and cell capture mediated by nanoparticle aggregation (Angew. Chem. Int. Ed., 2023, 62: e202217345). However, AgNPs prepared using traditional chemical reducing agents such as sodium borohydride have a strong inert surface, making it difficult to directly achieve effective loading of anticancer drugs, and the prepared AgNPs have relatively limited bioactivity. Developing efficient preparation methods for multifunctional silver nanoparticles and antitumor drug delivery systems based on silver nanoparticles remains challenging.

[0004] Rhubarb is the medicinal rhubarb plant of the Lycium family. Rheum officinale Rhubarb palmatum R. palmatu Or Tangut rhubarb R. tanguticum The dried roots and rhizomes of rhubarb possess purgative, heat-clearing, fire-purging, blood-cooling, detoxifying, blood-stasis-removing, menstruation-regulating, dampness-reducing, and jaundice-relieving effects. Rhubarb is rich in phenolic compounds, mainly including anthraquinones, anthrones, tannins, and stilbenesides. These phenolic compounds exhibit excellent ability to reduce noble metal particles and significant antibacterial, antitumor, anti-inflammatory, and antioxidant activities, providing an important material basis for the green and efficient preparation of multifunctional silver nanoparticles. Furthermore, the phenolic compounds in rhubarb can bind to various substances through π-π stacking, electrostatic interactions, hydrogen bonding, and coordination, providing numerous interaction sites for loading antitumor drugs onto the surface of silver nanoparticles.

[0005] However, current research mainly focuses on utilizing the direct drug activity of rhubarb extract. How to combine the multiple bioactivities of rhubarb with the characteristics of silver nanoparticles through ingenious preparation process design, and further construct a nanomedicine delivery system that can simultaneously respond to the tumor microenvironment, achieve controlled drug release, and have both antibacterial and antitumor functions, remains a huge challenge, and no relevant reports have been found. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the green synthesis of silver nanoparticles from rhubarb extract; Another object of the present invention is to provide the application of the above-mentioned green synthetic silver nanoparticles from rhubarb extract in the preparation of antitumor nanomedicines. I. Preparation method and performance evaluation of silver nanoparticles synthesized from rhubarb extract in a green manner. 1. A method for the green synthesis of silver nanoparticles from rhubarb extract Silver nitrate and rhubarb extract were dissolved in water to form solutions. The pH of the rhubarb extract aqueous solution was adjusted to 7.5-8.5 with sodium carbonate. The pH-adjusted rhubarb extract aqueous solution was quickly added to boiling silver nitrate aqueous solution, and the mixture was heated and stirred to obtain a silver nanoparticle dispersion. The precipitate of the silver nanoparticle dispersion was collected by centrifugation and washed with deionized water to obtain green synthesized silver nanoparticles (RHT-AgNPs) from rhubarb extract.

[0007] The rhubarb extract is a mixture obtained by crushing Tangut rhubarb slices, extracting with 30% ethanol and ethyl acetate, concentrating, and drying. The mass ratio of silver nitrate to rhubarb extract is 0.5:1 to 5:1.

[0008] In order to fully reduce Ag by rhubarb extract + To form nanoparticles, the mixed solution was heated and stirred at 95~105℃ for 1~2 h; the centrifugation was carried out at 12000 rpm for 10~20 minutes.

[0009] The morphology of RHT-AgNPs was characterized using a FEI-Tecnai G2 transmission electron microscope (TEM). Figure 1 The image shows a TEM image of RHT-AgNPs. As can be seen from the image, RHT-AgNPs have a typical spherical structure with a diameter of approximately 15 nm and excellent dispersibility. The absorption wavelength was investigated using a Perkin-Elmer Lambda35 ultraviolet spectrophotometer. Figure 2 The study found that the maximum absorption wavelength of RHT-AgNPs was 426 nm.

[0010] 2. Antibacterial properties of RHT-AgNPs The bactericidal effect of RHT-AgNPs against Gram-negative bacteria (Escherichia coli) was investigated using a plating experiment.

[0011] Experimental method: Escherichia coli (concentration of 10) 5 -10 6 RHT-AgNPs (CFU / mL) were cultured using the shake-flask method and calibrated at 600 nm wavelength using a microplate reader (SpectraMax®, Molecular Devices, USA). 20 mg and 5 mg of RHT-AgNPs were added to 10 mL of E. coli solution in Erlenmeyer flasks, and the flasks were sealed to prevent air exposure. All flasks were incubated at 37°C and 100 rpm for 0–24 h (2, 4, 6, 12, and 24 h). Afterward, 100 μL of bacterial culture was taken at each of the above specific time points and diluted 10-fold and 100-fold in 24-well plates, respectively. The diluted 100 μL of bacterial culture was spread onto agar plates. All agar plates were incubated at 37°C until colonies appeared. Finally, the number of bacterial colonies on the agar plates was counted using ImageJ software and compared with the control group to determine the clearance rate.

[0012] Figure 3 This graph shows the clearance rates of *E. coli* by different concentrations of RHT-AgNPs. The results indicate that the clearance rate of *E. coli* exhibits a time-dependent increase in the presence of RHT-AgNPs. When the concentration of RHT-AgNPs was 0.5 mg / mL and the culture time was 2 h, the clearance rate of *E. coli* was 2.05%, while when the culture time was increased to 24 h, the clearance rate increased sharply to 68.99%. When the concentration of RHT-AgNPs was 2 mg / mL and the culture time was 2 h, the clearance rate of *E. coli* was 4.14%, while when the culture time was increased to 24 h, the clearance rate increased sharply to 89.47%.

[0013] 3. Biocompatibility of RHT-AgNPs The biocompatibility of RHT-AgNPs was investigated using the Live-Dead assay, examining the cytotoxicity of RHT-AgNPs at concentrations of 5 and 50 μg / mL against normal MRC-5 cells (human embryonic lung fibroblasts). MRC-5 cells were seeded in 24-well plates (1 × 10⁶ cells per well). 6 Cells were cultured in wells containing 5 μg / mL RHT-AgNPs for 24 hours. The old medium was replaced with medium containing 5 μg / mL RHT-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).

[0014] Figure 4 This image shows the live / dead results of MRC-5 cells cultured with different concentrations of RHT-AgNPs. Laser confocal microscopy images show that when treated with 5 or 50 μg / mL RHT-AgNPs and phosphate-buffered saline (PBS, control), the morphology of MRC-5 cells remained almost unchanged, and almost no red fluorescence (dead cells) was observed in either the RHT-AgNPs group or the control group. These results indicate that RHT-AgNPs have low cytotoxicity to normal cells and are a biocompatible drug carrier.

[0015] II. Preparation and Performance Evaluation of Antitumor Nanomedicines 1. Preparation of antitumor nanomedicines Antitumor drugs were added to an aqueous solution of silver nanoparticles synthesized from rhubarb extract. The mixture was thoroughly mixed at room temperature and in the dark to load the antitumor drugs onto the surface of the silver nanoparticles. The antitumor nanomedicine was obtained by centrifugation.

[0016] The antitumor drug is either doxorubicin hydrochloride or epirubicin hydrochloride. The mass ratio of silver nanoparticles to the antitumor drug is 10:1 to 1:1.

[0017] The mixing method under the dark conditions is stirring or oscillation, and the mixing time is 12~72 h.

[0018] The centrifugation was performed at 12,000 rpm for 10-20 minutes.

[0019] Taking epirubicin hydrochloride as an example, the surface potential changes of RHT-AgNPs before and after epirubicin loading were characterized using a Zeta potential instrument. Figure 5As shown, the average surface potential of RHT-AgNPs is -44.24 mV, while the surface potential of the nanomedicine after loading epirubicin is -38.58 mV. This significant difference in surface potential indicates that epirubicin has been successfully loaded onto the surface of RHT-AgNPs through electrostatic interactions.

[0020] 2. pH- and glutathione (GSH)-sensitive drug release behavior of antitumor nanomedicines Taking RHT-AgNPs nanomedicine loaded with epirubicin hydrochloride (EPI) (RHT-AgNPs / EPI) as an example, the in vitro release behavior of EPI in the nanomedicine was investigated in three different PBS buffers: PBS buffer at pH 7.4 (simulating normal physiological environment), PBS buffer at pH 5.0 (simulating the slightly acidic environment of tumor cells), and PBS buffer at pH 7.4 containing GSH (10 mM, simulating the high GSH content environment of tumor cells). 5.0 mL of RHT-AgNPs / EPI dispersed in different buffers was placed in a dialysis bag (molecular weight cutoff of 6000-8000 Da), and then the dialysis bag was immersed in different buffers (30 mL), and shaken at 37°C for 30 hours. Each release condition included three parallel samples. At predetermined time intervals (1, 2, 3, 4, 5, 6, 7, 11, 22, and 30 h), 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).

[0021] Figure 6 The figure shows the release behavior of EPI from RHT-AgNPs / EPI nanomedicine in three different buffer solutions. In a buffer solution at pH 7.4, 11.23% of EPI was released within 30 hours. In a buffer solution at pH 5.0 or containing GSH, 41.54% and 36.98% of EPI were released within 30 hours, respectively. This pH-sensitive release behavior is mainly attributed to the protonation of the abundant phenolic hydroxyl groups on the surface of RHT-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 surface of silver nanoparticles, also leading to the dissociation and release of EPI from the silver nanoparticle surface. These results indicate that RHT-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 phenolic hydroxyl groups and ligand-exchange reactions with GSH.

[0022] 3. In vitro cytotoxic activity of RHT-AgNPs / EPI nanomedicines in bacterial coexistence A cell model of coexistence between *E. coli* and breast cancer cells MCF-7 was established to investigate the cytotoxic activities of EPI and RHT-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. 5 CFU / mL. MCF-7 cells were seeded into 96-well plates (200 μL, cell density approximately 5 × 10⁶ cells / mL). 3 After culturing for 12 h, 2 μL of bacterial culture and 20 µg / mL of EPI and nanomedicine were added, and the cells and bacteria were co-cultured for 24 h. The viability of cells and bacteria was determined by the MTS method.

[0023] Figure 7 The graph shows the cell viability of MCF-7 cells co-cultured with EPI and RHT-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 MCF-7 cells, causing most of the EPI to become inactive. However, for the nanomedicine group (RHT-AgNPs / EPI), at the same EPI concentration, the presence of *E. coli* did not affect the therapeutic effect of the drug. Both under sterile and aseptic conditions, the RHT-AgNPs / EPI nanomedicine exhibited excellent cytotoxic activity. These results indicate that the RHT-AgNPs / EPI nanomedicine can achieve a synergistic effect of antitumor and antibacterial activity, effectively avoiding the impact of microbial infection on the efficacy of EPI.

[0024] In summary, this invention utilizes rhubarb extract as a reducing agent and stabilizer to synthesize novel silver nanoparticles with uniform size, excellent dispersibility, and superior antibacterial activity in a green manner, providing an efficient preparation method for multifunctional silver nanoparticles. This method is simple, efficient, and environmentally friendly. The prepared silver nanoparticles exhibit uniform size, excellent dispersibility and stability, and combine the excellent bioactivity of rhubarb, demonstrating significant antibacterial activity. Most importantly, the phenolic compounds abundant in rhubarb are efficiently modified on the surface of the silver nanoparticles, and anticancer drugs are efficiently loaded through electrostatic interactions, showing great potential for the preparation of antitumor nanomedicines. The prepared antitumor nanomedicines exhibit pH- and glutathione-responsive drug release behavior and simultaneously possess antibacterial and antitumor effects, potentially addressing the key issue of decreased chemotherapy efficacy due to bacterial infection in tumor treatment, and providing a new approach for the synergistic treatment of bacterial infection and tumors. Attached Figure Description

[0025] Figure 1 This is a transmission electron microscope image of the green synthetic silver nanoparticles (RHT-AgNPs) of rhubarb extract prepared in Example 1 of the present invention.

[0026] Figure 2 The image shows the UV-Vis absorption spectrum of the RHT-AgNPs prepared in Example 1 of this invention.

[0027] Figure 3 The graph shows the results of the clearance rate of Escherichia coli by different concentrations of RHT-AgNPs over time.

[0028] Figure 4 Fluorescence microscopy images of co-cultured normal human embryonic lung fibroblasts (MRC-5) with different concentrations of RHT-AgNPs using the Live / Dead staining method.

[0029] Figure 5 The graph shows the change in zeta potential of RHT-AgNPs before and after loading with epirubicin hydrochloride (EPI).

[0030] Figure 6 The cumulative drug release curves of the nanomedicine loaded with epirubicin hydrochloride (RHT-AgNPs / EPI) in three different release media are shown.

[0031] Figure 7 This is a comparison of the in vitro cytotoxicity of free EPI and RHT-AgNPs / EPI nanomedicines on breast cancer MCF-7 cells under conditions of coexistence with and without E. coli. Detailed Implementation

[0032] The present invention will be further described below with reference to the embodiments, but the scope of the present invention is not limited to the following embodiments.

[0033] Example 1: Green synthesis of silver nanoparticles (RHT-AgNPs) from rhubarb extract 25.5 mg of rhubarb extract powder was dissolved in 1.5 mL of deionized water, and the pH of the rhubarb extract aqueous solution was adjusted to 8.0 with 0.5 M sodium carbonate solution. The pH-adjusted rhubarb extract aqueous solution was quickly added to boiling silver nitrate aqueous solution (silver nitrate mass: 12.75 mg, dissolved in 75 mL of deionized water), and the mixture was stirred at 100 °C for 1.5 h to obtain a silver nanoparticle dispersion. The obtained silver nanoparticle dispersion was centrifuged at 12000 rpm for 20 minutes, the precipitate was collected, and washed three times with deionized water to obtain RHT-AgNPs.

[0034] Example 2: Preparation of RHT-AgNPs-loaded epirubicin hydrochloride nanomedicine (RHT-AgNPs / EPI) 28 mg of RHT-AgNPs prepared in Example 1 were ultrasonically dispersed in 15 mL of deionized water, and 3 mL of an aqueous solution of epirubicin hydrochloride with a concentration of 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 (12000 rpm, 20 min) to obtain the RHT-AgNPs nanomedicine loaded with epirubicin hydrochloride (RHT-AgNPs / EPI).

[0035] Example 3: Synthesis of RHT-AgNPs with different mass concentration ratios To illustrate the range of the mass concentration ratio of silver nitrate to rhubarb extract, while keeping the mass concentration of rhubarb extract constant, the amount of silver nitrate added was varied during synthesis. The specific steps are as follows: A fixed mass of 25.5 mg of rhubarb extract was dissolved in 1.5 mL of water. 6.375 mg, 12.75 mg, 76.5 mg, and 127.5 mg of silver nitrate were weighed and dissolved in 75 mL of water to prepare silver nitrate solutions of different concentrations. RHT-AgNPs were then synthesized according to the same method as in Example 1 (pH=8.0, reaction at 100℃ for 1.5 h).

[0036] Characterization results showed that silver nanoparticles could be generated when the mass concentration ratio of silver nitrate to rhubarb extract was in the range of 0.25:1 to 5:1. Within the range of 0.5:1 to 5:1, the prepared silver nanoparticles exhibited uniform size and good dispersibility, demonstrating the effectiveness of this ratio range.

Claims

1. A method for the green synthesis of silver nanoparticles from rhubarb extract, characterized in that, Silver nanoparticles were prepared by mixing and reacting an aqueous solution of rhubarb extract with an aqueous solution of silver nitrate, using rhubarb extract as a reducing agent and stabilizer.

2. The method according to claim 1, characterized in that, The rhubarb extract is a mixture obtained by crushing Tangut rhubarb slices, extracting with 30% ethanol, extracting with ethyl acetate, concentrating, and drying.

3. The method according to claim 1, characterized in that, Before the mixing reaction, the pH of the rhubarb extract aqueous solution was adjusted to 7.5-8.5 using an alkaline reagent.

4. The method according to claim 1, characterized in that, The mixing reaction involves adding the rhubarb extract aqueous solution to the boiling silver nitrate aqueous solution and stirring at 95-105°C for 1-2 hours.

5. The method according to claim 1, characterized in that, The mass ratio of silver nitrate to rhubarb extract is 0.5:1 to 5:

1.

6. The use of silver nanoparticles prepared by the method according to any one of claims 1-5 in the preparation of antitumor nanomedicines.

7. The application according to claim 6, characterized in that, The preparation method of the antitumor nanomedicine includes: An antitumor drug was added to an aqueous solution of the silver nanoparticles and mixed at room temperature and in the dark to load the antitumor drug onto the surface of the silver nanoparticles; centrifugation was performed to obtain nanomedicine loaded with the antitumor drug.

8. The application according to claim 7, characterized in that, The antitumor drug is selected from doxorubicin hydrochloride or epirubicin hydrochloride.

9. The application according to claim 7, characterized in that, The mass ratio of the silver nanoparticles to the anti-tumor drug is 10:1 to 1:

1.

10. The application according to claim 7, characterized in that, The mixing method under the dark conditions is stirring or shaking, and the mixing time is 12 to 72 hours.

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