A GPS / EGCG / CM system nano drug-loaded particle for liver cancer treatment and a preparation method thereof

Abstract

The application relates to the technical field of nano drug loading, and provides a nano drug loading particle based on a GPS / EGCG / CM system and a preparation method thereof. The nano drug loading particle is co-assembled by a drug GPS / EGCG / CM and a nano carrier carboxymethylated chitosan (CMCTS), and the assembled nano drug loading particle has a spherical structure. The natural drug EGCG and GPS are used to adjust the chemotherapeutic drug CM, so that the CM can effectively resist liver cancer and further reduce the toxicity of the CM to human cells. In addition, the CMCTS nano carrier with high biological safety is used to load the GPS / EGCG / CM, so that the biological safety of the liver cancer treatment drug is further improved, and the GPS / EGCG / CM@CMCTS is beneficial to the application and development of the liver cancer treatment drug.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine delivery, specifically to a GPS / EGCG / CM system of nanomedicine-carrying particles for liver cancer treatment and its preparation method. Background Technology

[0002] Currently, commonly used chemotherapy drugs (CMs) for liver cancer treatment include 5-fluorouracil (5-FU), cisplatin (DDP), oxaliplatin (L-OHP), and capecitabine (CAPE). However, all of these CMs have strong side effects, leading to significant differences in individual patient tolerance. Furthermore, liver cancer cells grow within the liver and erode normal liver cells, damaging healthy cells and impairing their metabolic, detoxification, and bile production functions, thus affecting liver function. Drug metabolism requires normal liver function; if liver function is impaired, drug metabolism and clearance may be affected, impacting efficacy. Therefore, compared to other cancers, liver cancer treatment requires a more comprehensive consideration of the patient's physical condition and careful selection of CMs to ensure efficacy and safety. Thus, there is an urgent clinical need to find natural components with anti-cancer activity against liver cancer cells but high safety for normal human cells and tissues to harmonize CMs, which has profound significance for the clinical treatment of liver cancer patients.

[0003] In China, ginseng is considered a natural medicinal herb with nourishing properties and is widely used in traditional Chinese medicine. In modern medicine, ginseng is also widely used in the clinical treatment of cancer, cardiovascular diseases, and nervous system disorders. The chemical components of ginseng mainly include saponins, polysaccharides, polypeptides, and trace elements. Among them, ginsenosides are the main active ingredients of ginseng, possessing various pharmacological effects such as anti-tumor, antioxidant, anti-inflammatory, and immunomodulatory effects. However, ginsenosides generally suffer from insolubility in water and poor lipid solubility. In addition, ginseng polysaccharides (GPS) have similar therapeutic effects, but unlike ginsenosides, GPS is rich in hydroxyl groups and has better water solubility. Currently, some researchers are conducting research and surveys on the therapeutic technology of ginseng polysaccharides synergistic with chemotherapy, finding that GPS can induce endogenous tumor necrosis factor, enhance the activity of NK cells and CTL cells, and activate the body's immune function to reduce the side effects caused by tumor chemotherapy. However, compared with the control group, the incidence of adverse reactions in GPS synergistic with CM remains high.

[0004] Epigallocatechin gallate (EGCG) is also a naturally occurring compound found in tea leaves. Belonging to the catechin flavonoid family, it possesses various biological activities and pharmacological effects, exhibiting promising anti-cancer activity against a range of cancers, including lung cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, thyroid cancer, oral cancer, and pancreatic cancer. Furthermore, EGCG demonstrates good biocompatibility and excellent water solubility, similar to GPS. Therefore, utilizing GPS in conjunction with EGCG to develop novel CMs may yield unexpected advancements. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned technical problems by providing a drug-loaded nanoparticle based on a GPS / EGCG / CM system and its preparation method. This drug-loaded nanoparticle is co-assembled from a drug—GPS / EGCG / CM—and a nanocarrier—carboxymethylated chitosan (CMCTS), resulting in a spherical structure. Furthermore, the CM in GPS / EGCG / CM is 5-FU.

[0006] This invention also provides a method for preparing drug-loaded nanoparticles based on a GPS / EGCG / CM system, specifically including the following steps:

[0007] (1) Drug pretreatment: CM, EGCG and GPS were dissolved in deionized water and then transferred to 125mL round bottom flasks. The mixture was pre-stirred in a 60℃ constant temperature water bath magnetic stirrer at 150rpm. After stirring, the drug solution was poured out and centrifuged at 10000rpm for 10min. The mixture was washed twice with deionized water and the precipitate was freeze-dried to obtain the compound drug GPS / EGCG / CM.

[0008] (2) Carboxymethylated chitosan: Chitosan was dissolved in isopropanol and stirred to swell for 1 hour. Then, 40% NaOH solution was added and stirred to alkalize for 2 hours. Then, 4.5 g of chloroacetic acid was added to the isopropanol solution. After the chloroacetic acid dissolved, it was slowly added dropwise to the alkalized chitosan solution. The reaction was carried out in a water bath at 60°C for 4 hours. After the reaction was completed, the mixture was washed with ethanol solution and filtered to obtain a white precipitate. 60 mL of deionized water was added to dissolve the precipitate. Finally, the mixture was dried in a vacuum drying oven at 60°C to obtain CMCTS.

[0009] (3) Carboxymethylated modified chitosan drug loading: CMCTS was dissolved in deionized water and sonicated for 10 min; GPS / EGCG / CM was dissolved in 5 mL of isopropanol solution, and the isopropanol solution of GPS / EGCG / CM was added dropwise to the above CMCTS solution. After sonication for 30 min, it was transferred to a dialysis bag and dialyzed overnight at room temperature to obtain GPS / EGCG / CM@CMCTS solution. The solution was washed with ethanol and deionized water in sequence, centrifuged, and dried in a vacuum drying oven at 60℃ to obtain GPS / EGCG / CM@CMCTS.

[0010] Furthermore, the mass ratio of CM, EGCG and GPS in step (1) is 0.025:(1-3):(1-3).

[0011] Furthermore, the mass-to-volume ratio of chitosan, NaOH, and chloroacetic acid in step (2) is 1g:10mL:4.5g.

[0012] Furthermore, the mass ratio of CMCTS, GPS / EGCG / CM in step (3) is 1:3.

[0013] The advantages of this invention are:

[0014] This invention utilizes the natural drug EGCG and GPS to harmonize the chemotherapy drug CM, thereby maintaining its effective anti-liver cancer efficacy while further reducing the toxicity of CM to human cells. Furthermore, this invention employs the highly biosafety-compliant CMCTS nanocarrier to load GPS / EGCG / CM, further enhancing the biosafety of the liver cancer treatment drug and facilitating its application and development in liver cancer treatment. Attached Figure Description

[0015] Figure 1 This is a morphology characterization diagram of the nanoparticles used in this invention; Figure 1 (ab) are scanning electron microscope images of the drug-loaded nanoparticles at different magnifications.

[0016] Figure 2 These are data on the effects of the nano-drug-loaded particles in Examples 1-3 of this invention on the activity of liver cancer cells; Figure 2 (af) is a graph showing the cell viability of liver cancer cells at different concentrations of drug-loaded nanoparticles (0, 25, 50, 50, 100, 200 mg / mL) and different incubation times (24, 48, 72 h).

[0017] Figure 3 This data represents the effects of nanoparticles (200 mg / mL, 72 h) on the activity of liver cancer cells in Example 1 and Comparative Examples 1-2 of this invention.

[0018] Figure 4 This data represents the effects of the drug-loaded nanoparticles of Example 1 and Comparative Examples 1-5 on the activity of normal human hepatocytes under different incubation times (24, 48, 72 h). Detailed Implementation

[0019] The technical solutions described in this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only some feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0020] Example 1: Preparation of drug-loaded nanoparticles, wherein CM is 5-FU, and the mass ratio of 5-FU, EGCG and GPS is 0.025:2:2.

[0021] (1) Drug pretreatment: Weigh 2.5 mg of 5-FU solid powder and add it to 10 mL of deionized water. Use a 1 mL pipette to repeatedly blow and stir to fully dissolve the drug. Weigh 0.2 g of EGCG and add it to 20 mL of deionized water. Use a vortex shaker to fully dissolve the drug (referred to as solution A). Weigh 0.2 g of GPS solid powder in the same way and dissolve it in 20 mL of deionized water (referred to as solution B). Transfer the above solution A to a 125 mL round bottom flask and pre-stir at 150 rpm for 2 min in a 60 °C constant temperature water bath with magnetic stirring. Then add 20 mL of solution B. Then, while stirring magnetically for 2 min, quickly add the 5-FU solution prepared above. Continue stirring under the same conditions for 3 min. After stirring, carefully pour out the drug solution. After the reaction is finished, centrifuge the reaction solution at 10000 rpm for 10 min. Wash twice with deionized water by centrifugation. Freeze-dry the precipitate to obtain the compound drug GPS / EGCG / 5-FU.

[0022] (2) Carboxymethylated modified chitosan: Dissolve 1g of chitosan in 10mL of isopropanol, stir and swell for 1h, add 10mL of 40% NaOH solution, stir and alkalize for 2h, then add 4.5g of chloroacetic acid to 10mL of isopropanol solution, and slowly add it to the alkalized chitosan solution after the chloroacetic acid dissolves. React in a water bath at 60℃ for 4h. After the reaction, wash with ethanol solution and filter to obtain a white precipitate, add 60mL of deionized water to dissolve, and finally dry in a vacuum drying oven at 60℃ to obtain CMCTS;

[0023] (3) Carboxymethylated modified chitosan drug loading: Weigh 30 mg CMCTS and add it to 5 mL of deionized water, and sonicate for 10 min; dissolve 90 mg GPS / EGCG / 5-FU in 5 mL of isopropanol solution, add GPS / EGCG / 5-FU isopropanol solution dropwise to the above CMCTS solution, and then transfer it to a dialysis bag after sonication for 30 min. Dialyze overnight at room temperature to obtain GPS / EGCG / CM@CMCTS solution. Wash with ethanol and deionized water in sequence, centrifuge, and dry in a vacuum drying oven at 60℃ to obtain GPS / EGCG / 5-FU@CMCTS.

[0024] Comparative Example 1

[0025] Based on Example 1, Comparative Example 1 was prepared. The difference between Comparative Example 1 and Example 1 is that no chemotherapy drug 5-FU was added. All other steps were the same, and GPS / EGCG@CMCTS was finally obtained.

[0026] Comparative Example 2

[0027] Based on Example 1, Comparative Example 2 was prepared; the difference between Comparative Example 2 and Example 1 is that CMCTS was not added, but the other steps were the same, and GPS / EGCG / 5-FU was finally obtained.

[0028] Comparative Example 3

[0029] Based on Example 1, Comparative Example 3 was prepared. The difference between Comparative Example 3 and Example 1 is that the natural component GPS / EGCG was not added. All other steps were the same, and 5-FU@CMCTS was finally obtained.

[0030] Comparative Example 4

[0031] Based on Example 1, Comparative Example 4 was prepared. The difference between Comparative Example 4 and Example 1 is that GPS was not added, but all other steps were the same, and EGCG / 5-FU@CMCTS was finally obtained.

[0032] Comparative Example 5

[0033] Based on Example 1, Comparative Example 5 was prepared. The difference between Comparative Example 5 and Example 1 is that EGCG was not added. All other steps were the same, and GPS / 5-FU@CMCTS was finally obtained.

[0034] Example 2: Preparation of drug-loaded nanoparticles, wherein 5-FU was selected as CM, and the mass ratio of 5-FU, EGCG and GPS was 0.025:3:1. All other steps were the same, and GPS / EGCG / 5-FU@CMCTS was finally obtained.

[0035] Example 3: Preparation of drug-loaded nanoparticles, wherein 5-FU was selected as CM, and the mass ratio of 5-FU, EGCG and GPS was 0.025:1:3. All other steps were the same, and GPS / EGCG / 5-FU@CMCTS was finally obtained.

[0036] Example 4: Morphological characterization of the GPS / EGCG / 5-FU@CMCTS prepared in Example 1

[0037] The GPS / EGCG / 5-FU@CMCTS drug-loaded nanoparticles prepared in Example 1 were analyzed using scanning electron microscopy, and the results are as follows: Figure 1 As shown in (ab), the synthesized drug-carrying nanoparticles have a spherical structure with a size of approximately 500 nm.

[0038] Example 5: Determination of drug loading and encapsulation efficiency

[0039] Add 10 mg of GPS / EGCG / 5-FU@CMCTS and 10 mL of 3% hydrochloric acid sequentially to a 25 mL round-bottom flask. Then, with magnetic stirring, maintain the reaction temperature at 60 °C and react for 4 hours. Transfer the mixture to a 25 mL volumetric flask and dilute to volume for later use. Then, determine the drug content using a UV-Vis spectrophotometer. The formulas for drug loading and encapsulation efficiency are as follows:

[0040] The results showed that when m(CMCTS):m(GPS / EGCG / 5-FU) = 1:3, the drug loading was 17.53% and the encapsulation efficiency was 14.27%.

[0041] Example 6: Evaluation of in vitro toxicity of drug-loaded nanoparticles to human hepatocellular carcinoma cells HepG2 and Hep3b using the CCK8 assay

[0042] (1) Cell activity detection was performed on the drug-loaded nanoparticles prepared in Examples 1-3 of the present invention. The specific steps are as follows: HepG2 and Hep3b were respectively loaded with 5×10 4 Cells were seeded at a density of [number] cells / well in different 96-well plates and then cultured overnight in a cell culture incubator. After incubation, the culture medium was removed and the cells were washed with PBS solution. HepG2 and Hep3b cells were then placed in an environment containing different concentrations (0, 25, 50, 100, and 200 μg / mL) of drug-loaded nanoparticles and cultured at 37°C in a 5% CO2 incubator for 24, 48, and 72 h. Afterward, 100 μL of fresh culture medium (containing 10% CCK-8 solution) was added to the washed cells, and after incubation for 3 h, the optical density (OD) of each well was measured at 450 nm using a microplate reader, and cell viability was calculated. The toxicity of each nanogroup to cells at different concentrations was determined, and the optimal embodiment was selected based on this for subsequent experimental testing.

[0043] Experimental results are as follows Figure 2 As shown, with increasing drug concentration and prolonged co-incubation time, the GPS / EGCG / 5-FU@CMCTS nanoparticles prepared in Examples 1-3 exhibited significant inhibitory effects on both HepG2 and Hep3b cells, with the inhibition rate increasing accordingly. Specifically, the optimal inhibition rate against HepG2 and Hep3b tumor cells was achieved at a concentration of 200 mg / mL and an incubation time of 72 h, reaching 52.3% and 77.8%, respectively. These results demonstrate that the GPS / EGCG / 5-FU@CMCTS nanoparticles possess excellent ability to kill liver cancer cells in vitro.

[0044] (2) In this experiment, human liver cancer cells HepG2 and Hep3b were selected as experimental subjects. The cell activity of the nano-drug-loaded particles prepared in Example 1 and Comparative Examples 1-2 of this invention was detected by the CCK8 method. The specific steps were the same as above.

[0045] Experimental results are as follows Figure 3 As shown, Comparative Example 1, without the chemotherapy drug 5-FU, exhibited the highest cell bioactivity. Under the nanoparticle treatment conditions of Comparative Example 1, the inhibition rates of HepG2 and Hep3b cells did not exceed 25%. This also indirectly demonstrates that EGCG / GPS@CMCTS has a certain inhibitory effect on the growth of HepG2 and Hep3b cells. In Comparative Example 2, the composite drug was not embedded in the nanocarrier, but Comparative Example 2 showed a similar inhibition rate of liver cancer cells as Example 1. This can be attributed to the good biocompatibility of the CMCTS nanocarrier, thus ensuring that CMCTS does not affect the bioactivity of HepG2 and Hep3b cells.

[0046] Example 7: Evaluation of the biosafety of drug-loaded nanoparticles to normal human LO2 cells based on the CCK8 assay

[0047] Human normal cells LO2 were used as experimental subjects in this experiment. The cell activity of the drug-loaded nanoparticles prepared in Example 1 and Comparative Examples 1-3 of this invention was detected by the CCK8 method. The concentration of the drug-loaded nanoparticles prepared in Example 1 and Comparative Examples 1-3 of this invention was 200 mg / mL, and the incubation time was 24, 48 and 72 h, respectively. The remaining steps were the same as above.

[0048] Experimental results are as follows Figure 4As shown, compared to Example 1 and Comparative Example 2, Comparative Example 1 exhibits superior cellular biocompatibility. This can be attributed to the fact that Comparative Example 1 did not contain the chemotherapy drug 5-FU, while the original natural components EGCG and GPS are highly biocompatible, thus giving Comparative Example 1 over 90% cellular bioactivity. Comparative Example 2, on the other hand, showed similar cell-killing data to Example 1, only slightly higher in the initial 24 hours of incubation. This can be attributed to the fact that Comparative Example 1 was not encapsulated by the nanocarrier CMCTS, allowing it to kill cells more rapidly than Example 1.

[0049] Furthermore, with increasing incubation time, although the drug-loaded nanoparticles prepared in Example 1 showed some cytotoxic activity against LO2 cells, it was far less effective than their ability to kill liver cancer cells. This may be because the natural drug components EGCG and GPS have anti-cancer activity against cancer cells, while exhibiting high biocompatibility with normal cells and enhancing cellular immune function. Moreover, compared to Example 1, Comparative Example 3, which did not incorporate the natural drug GPS / EGCG, showed poorer cellular bioactivity than Example 1, which also indirectly verifies that GPS / EGCG can modulate the cytotoxic effect of 5-FU on LO2 cells. Figure 4 In the data from Comparative Examples 4-5, we can further analyze the moderating effect of GPSE / GCG on 5-FU. Compared to Example 1 (GPS / EGCG / 5-FU@CMCTS), Comparative Examples 4 (EGCG / 5-FU@CMCTS) and 5 (GPS / 5-FU@CMCTS), which contain only a single natural ingredient, showed poorer cell bioactivity, basically similar to the data of Comparative Example 3 (5-FU@CMCTS). This indicates that the combination of a single natural ingredient GPS or EGCG with 5-FU does not produce a regulatory effect on the cytotoxicity of 5-FU to LO2 cells; a synergistic effect is only achieved when GPS / EGCG is combined to reduce the cytotoxicity of 5-FU to LO2 cells. Specifically, after 72 hours of incubation, the viability of LO2 cells in Example 1 (GPS / EGCG / 5-FU@CMCTS) was approximately 75%, indicating a relatively good cellular physiological environment. Compared to Comparative Example 3 (5-FU@CMCTS) without the added natural drug, Comparative Example 4 (EGCG / 5-FU@CMCTS) with a single natural component, and Comparative Example 5 (GPS / 5-FU@CMCTS), Example 1 improved cell bioactivity by approximately 15%.

[0050] In summary, this invention successfully prepared a novel chemotherapy drug based on a natural drug compound, which retains the high biocompatibility of natural drugs, helps reduce the toxicity of the chemotherapy drug 5-FU to human LO2 cells, and at the same time can play a good inhibitory role against liver cancer cells.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A drug-loaded nanoparticle based on a GPS / EGCG / CM system, characterized in that, The drug-loaded nanoparticles are assembled from a drug—GPS / EGCG / CM and a nanocarrier—carboxymethylated chitosan (CMCTS). The drug-loaded nanoparticles are GPS / EGCG / CM@CMCTS, and the assembled drug-loaded nanoparticles have a spherical structure. The CM in GPS / EGCG / CM is 5-FU. The method for preparing the drug-loaded nanoparticles includes the following steps: (1) Drug pretreatment: CM, EGCG and GPS were dissolved in deionized water and then transferred to 125 mL round bottom flasks. The mixture was pre-stirred in a 60℃ constant temperature water bath magnetic stirrer at 150 rpm. After stirring, the drug solution was poured out and centrifuged at 10000 rpm for 10 min. The mixture was washed twice with deionized water and the precipitate was freeze-dried to obtain the compound drug GPS / EGCG / CM. (2) Carboxymethylated modified chitosan: Chitosan was dissolved in isopropanol and stirred to swell for 1 h. 40% NaOH solution was added and stirred to alkalize for 2 h. Then 4.5 g chloroacetic acid was added to the isopropanol solution. After the chloroacetic acid was dissolved, it was slowly added dropwise to the above-mentioned alkalized chitosan solution. The reaction was carried out in a water bath at 60℃ for 4 h. After the reaction was completed, the mixture was washed with ethanol solution and filtered to obtain a white precipitate. 60 mL of deionized water was added to dissolve the precipitate. Finally, the mixture was dried in a vacuum drying oven at 60℃ to obtain CMCTS. (3) Carboxymethylated modified chitosan drug loading: CMCTS was dissolved in deionized water and sonicated for 10 min; GPS / EGCG / CM was dissolved in 5 mL isopropanol solution, and GPS / EGCG / CM isopropanol solution was added dropwise to the above CMCTS solution. After sonication for 30 min, it was transferred to a dialysis bag and dialyzed overnight at room temperature to obtain GPS / EGCG / CM@CMCTS solution. The solution was washed with ethanol and deionized water in sequence, centrifuged, and dried in a vacuum drying oven at 60℃ to obtain GPS / EGCG / CM@CMCTS.

2. The drug-loaded nanoparticles based on the GPS / EGCG / CM system according to claim 1, characterized in that, The mass ratio of CM, EGCG and GPS in step (1) is 0.025:(1-3):(1-3).

3. The drug-loaded nanoparticles based on the GPS / EGCG / CM system according to claim 1, characterized in that, In step (2), the mass-to-volume ratio of chitosan, NaOH, and chloroacetic acid is 1 g: 10 mL: 4.5 g.

4. The drug-loaded nanoparticles based on the GPS / EGCG / CM system according to claim 1, characterized in that, The mass ratio of CMCTS, GPS / EGCG / CM in step (3) is 1:

3.

5. The application of the drug-loaded nanoparticles based on the GPS / EGCG / CM system as described in claim 1 in the preparation of a drug for the treatment of liver cancer, wherein the drug can improve the safety of liver cancer treatment.

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