Ginsenoside Rg3-coated PLGA-HA nano-composite as well as preparation method and application thereof

Abstract

The invention provides a ginsenoside Rg3-coated PLGA-HA nano-composite as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: dissolving hyaluronic acid in water, and adding DMSO to obtain a hyaluronic acid solution; adding the EDC. HCl solution and the NHS solution into the hyaluronic acid solution; dissolving cetylamine in DMSO (dimethylsulfoxide), adding into the mixed solution, reacting for 20-28 hours, dialyzing, freezing and drying to obtain hyaluronic acid-cetylamine powder, and dissolving the hyaluronic acid-cetylamine powder in water to obtain hyaluronic acid-cetylamine aqueous solutions with different concentrations; the method comprises the following steps: dissolving Rg3 and PLGA in trichloromethane, adding into hyaluronic acid-cetylamine aqueous solutions with different concentrations, reacting in a dark place, and removing an organic solvent to obtain ginsenoside Rg3-coated PLGA-HA. The Rg3 coated PLGA-HA provided by the invention improves the treatment effect of Rg3, and possibly improves the prognosis of gallbladder cancer patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to ginsenoside Rg3@PLGA-HA nanocomposites and their preparation methods and applications. Background Art

[0002] Gallbladder cancer (GBC) is the most common malignant tumor of the biliary tract, accounting for 80% - 95% of global biliary tract tumors. It is characterized by high lethality, late diagnosis, and chemotherapy resistance, ranking sixth among gastrointestinal tumors. Surgery is the only treatment method for GBC patients. Most patients are diagnosed with advanced GBC, at which stage resection treatment is impossible. In addition, most patients have frequent postoperative relapses and poor prognoses after chemotherapy or radiotherapy. The prognoses of stage 0 and stage 1 GBC patients are optimistic, with a 5-year survival rate of 50% - 80%, but the prognoses of stage II-IV are poor, with a 5-year survival rate of 2% - 28%. 5-fluorouracil has been shown to have only a 20% response rate, and the limited response rate of gemcitabine is 36%. Therefore, there is an urgent need to develop new and effective treatment regimens for GBC patients.

[0003] Traditional Chinese medicine has the characteristics of low toxicity and effectiveness and has a history of thousands of years in tumor prevention and treatment. Ginsenoside Rg3 is a new autophagy inhibitor. Fei Zhang et al. studied the effects of Rg3 on cell growth and survival in human GBC cell lines to evaluate its anti-tumor activity. The data showed that Rg3 was able to inhibit the growth of GBC cells. The effect of Rg3 on GBC growth was confirmed in vivo through a mouse xenograft model. However, Rg3 has hemolytic effects and extremely poor water solubility, severely limiting its application. At present, there is an urgent need for a new method of drug administration to avoid the hemolytic effect of ginsenosides and improve its effectiveness. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of ginsenoside Rg3@PLGA-HA nanocomposites.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a preparation method of ginsenoside Rg3@PLGA-HA nanocomposites, and the preparation method comprises the following steps:

[0007] 1) Dissolve hyaluronic acid in water, add DMSO to obtain a hyaluronic acid solution;

[0008] 2) Add the EDC·HCl solution dissolved in DMSO and the NHS solution dissolved in water to the hyaluronic acid solution to obtain a mixed solution;

[0009] 3) Dissolve hexadecylamine in DMSO and add it dropwise to the mixed solution. React at 20 - 25 °C for 20 - 28 h to obtain a reaction solution. Dialyze the reaction solution with distilled water. After dialysis, take the retentate and freeze-dry it to obtain hyaluronic acid-hexadecylamine powder;

[0010] 4) Dissolve the hyaluronic acid-hexadecylamine powder in water to obtain hyaluronic acid-hexadecylamine aqueous solutions with concentrations of 0.5 - 3 mg / mL and 0.1 - 1 mg / mL respectively;

[0011] 5) Dissolve ginsenoside Rg3 and PLGA in chloroform to obtain a mixed solution A. Add it to the hyaluronic acid-hexadecylamine aqueous solution with a concentration of 0.5 - 3 mg / mL, and ultrasonically mix to obtain a mixed solution B. Then add the mixed solution B to the hyaluronic acid-hexadecylamine aqueous solution with a concentration of 0.1 - 1 mg / mL. After reacting in the dark, remove the organic solvent to obtain ginsenoside Rg3@PLGA-HA.

[0012] Preferably, in step 1), the mass-volume ratio of the hyaluronic acid to water and DMSO is 10 - 20 mg:1 mL:1 mL.

[0013] Preferably, in step 2), the mass-volume ratio of EDC·HCl to DMSO is 10 - 30 mg:1 mL, and the mass-volume ratio of NHS to water is 11 - 13 mg:1 mL; the volume ratio of the hyaluronic acid solution to the EDC·HCl solution and the NHS solution is (30 - 50):(1 - 2):(1 - 2).

[0014] Preferably, in step 3), the mass-volume ratio of HDA to DMSO is 5 - 7 mg:1 mL, the mass ratio of the hyaluronic acid to hexadecylamine is (10 - 20):(0.5 - 1.5), the molecular weight of the dialysis membrane used for dialysis is 3 - 4 KDa, the dialysis duration is 2 days, and the liquid is changed every 6 h.

[0015] Preferably, in step 5), the mass-volume ratio of ginsenoside Rg3 to PLGA and chloroform is 1 - 3 mg:90 - 100 mg:1 mL, the volume ratio of the mixed solution A to the hyaluronic acid-hexadecylamine aqueous solution with a concentration of 0.5 - 3 mg / mL is 1:(5 - 7), the volume ratio of the mixed solution B to the hyaluronic acid-hexadecylamine aqueous solution with a concentration of 0.1 - 1 mg / mL is 1:(2 - 3), the ultrasonic mixing condition is to use an ultrasonic probe at 50%, 3S on / 3S off for ultrasonic mixing for 1 - 10 min, and the PLGA is polymerized from 75% polylactic acid and 25% polyglycolic acid, with a molecular weight of 20,000 - 40,000.

[0016] Preferably, in step 5), the time for the reaction in the dark is 8 - 10 h.

[0017] The present invention also provides a ginsenoside Rg3@PLGA-HA nanocomposite.

[0018] The present invention also provides the use of a ginsenoside Rg3@PLGA-HA nanocomposite in the preparation of a drug for treating gallbladder cancer.

[0019] The preparation method of the ginsenoside Rg3@PLGA-HA nanocomposite provided by the present invention encapsulates ginsenoside Rg3 in PLGA nanomaterials, realizing the carrier of Rg3. It not only improves the water solubility of Rg3, but also avoids the contact between Rg3 and red blood cells, weakening the hemolytic effect of Rg3. The ginsenoside Rg3@PLGA-HA nanocomposite provided by the present invention is an innovative drug delivery system. It not only increases the accuracy of treatment by targeted drug delivery to the target, improves the therapeutic effect of ginsenoside Rg3, reduces side effects, but also improves the prognostic effect of Rg3 on gallbladder cancer patients. Moreover, the present invention also verifies through cell experiments that Rg3 inhibits autophagy through the MOB1A--IL6 / STAT3 pathway, thereby inhibiting the growth and proliferation of GBC cells. Description of the Drawings

[0020] Figure 1 1HNMR spectrum of HA@PLGA in Example 1;

[0021] Figure 2 FT-IR images of HA-HDA, PLGA-HA, and Rg3@PLGA-HA in Example 1;

[0022] Figure 3 Hydrated particle size detection results of Rg3@PLGA-HA nanoparticles in Example 1;

[0023] Figure 4 TEM Mapping images of Rg3@PLGA-HA nanoparticles in Example 1;

[0024] Figure 5 Zeta potential detection results of Rg3@PLGA-HA nanoparticles measured in Example 1;

[0025] Figure 6 HPLC original data for detecting the content of Rg3 in Rg3@PLGA-HA nanoparticles in Example 1;

[0026] Figure 7 Drug release efficiency characterization of Rg3 at pH = 6.5 and 37 °C in Example 1;

[0027] Figure 8Cell morphology pictures of the GBC-SD+Rg3 group in Example 2 under different culture conditions;

[0028] Figure 9 Result analysis diagrams of the GBC-SD+Rg3 group in Example 2 affecting cell proliferation under different culture conditions;

[0029] Figure 10 Cell morphology pictures of the GBC-SD+PLGA-HA group in Example 2 under different culture conditions;

[0030] Figure 11 Result analysis diagrams of the GBC-SD+PLGA-HA group in Example 2 affecting cell proliferation under different culture conditions;

[0031] Figure 12 Cell morphology pictures of the GBC-SD+Rg3@PLGA-HA group in Example 2 under different culture conditions;

[0032] Figure 13 Result analysis diagrams of the GBC-SD+Rg3@PLGA-HA group in Example 2 affecting cell proliferation under different culture conditions;

[0033] Figure 14 Cell morphology pictures of the HIBEpiC+Rg3 group in Example 2 under different culture conditions;

[0034] Figure 15 Result analysis diagrams of the HIBEpiC+Rg3 group in Example 2 affecting cell proliferation under different culture conditions;

[0035] Figure 16 Cell morphology pictures of the HIBEpiC+PLGA-HA group in Example 2 under different culture conditions;

[0036] Figure 17 Result analysis diagrams of the HIBEpiC+PLGA-HA group in Example 2 affecting cell proliferation under different culture conditions;

[0037] Figure 18 Result analysis diagrams of the HIBEpiC+Rg3@PLGA-HA group in Example 2 affecting cell proliferation under different culture conditions;

[0038] Figure 19 Result analysis diagrams of the HIBEpiC+Rg3@PLGA-HA group in Example 2 affecting cell proliferation under different culture conditions;

[0039] Figure 20 Hemolysis experiment result diagrams of Rg3, PLGA-HA and Rg3@PLGA-HA in Example 3;

[0040] Figure 21 Intuitive diagram of the effects of Rg3, PLGA-HA, and Rg3@PLGA-HA on the colony formation of GBC-SD cells in Example 4;

[0041] Figure 22 Graph of the counting results of the effects of Rg3, PLGA-HA, and Rg3@PLGA-HA on the colony formation of GBC-SD cells in Example 4;

[0042] Figure 23 Graph of the results of the effects of Rg3, PLGA-HA, and Rg3@PLGA-HA on the migration ability of GBC-SD cells in Example 5;

[0043] Figure 24 Graph of the quantitative results of the effects of Rg3, PLGA-HA, and Rg3@PLGA-HA on the migration rate of GBC-SD cells in Example 5;

[0044] Figure 25 Graph of the results of the effects of Rg3, PLGA-HA, and Rg3@PLGA-HA on the invasion ability of GBC-SD cells in Example 6;

[0045] Figure 26 Graph of the quantitative results of the effects of Rg3, PLGA-HA, and Rg3@PLGA-HA on the invasion ability of GBC-SD cells in Example 6;

[0046] Figure 27 Graph of the results of the effects of Rg3, PLGA-HA, and Rg3@PLGA-HA on the anaphase of GBC-SD cell division in Example 7;

[0047] Figure 28 Graph of the quantitative results of the effects of Rg3, PLGA-HA, and Rg3@PLGA-HA on the anaphase of GBC-SD cell division in Example 7;

[0048] Figure 29 Graph of the results of the effects of Rg3, PLGA-HA, and Rg3@PLGA-HA on the apoptosis of GBC-SD cells in Example 8;

[0049] Figure 30 Graph of the quantitative results of the effects of Rg3, PLGA-HA, and Rg3@PLGA-HA on the apoptosis rate of GBC-SD cells in Example 8;

[0050] Figure 31 Graph of the results of detecting the effects of Rg3, PLGA-HA, and Rg3@PLGA-HA on the apoptosis rate of GBC-SD cells by the TUNEL method in Example 9;

[0051] Figure 32Results graph showing the effects of Rg3, PLGA-HA, and Rg3@PLGA-HA on the apoptosis rate of HIBEpic cells detected by the TUNEL method in Example 9;

[0052] Figure 33 Quantification results graph showing the effects of Rg3, PLGA-HA, and Rg3@PLGA-HA on the apoptosis rate of GBC-SD cells and HIBEpic cells detected by the TUNEL method in Example 9;

[0053] Figure 34 Results graph showing the detection of autophagy by GFP-LC3 fusion protein after treating GBC-SD cells with Rg3, PLGA-HA, and Rg3@PLGA-HA respectively in Example 10;

[0054] Figure 35 Results graph showing the detection of LC3II / LC3I, MOB1A, p-STAT3, TG, NF-κB, and IL-6 by Western Blot after treating GBC-SD cells with Rg3, PLGA-HA, and Rg3@PLGA-HA respectively in Example 11;

[0055] Figure 36 Quantification results graph of LC3II / LC3I, MOB1A, p-STAT3, TG, NF-κB, and IL-6 after treating GBC-SD cells with Rg3, PLGA-HA, and Rg3@PLGA-HA respectively in Example 11. Detailed implementation mode

[0056] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0057] Example 1

[0058] Preparation and characterization of Rg3@PLGA-HA related nanomaterials

[0059] 1. Experimental materials and instruments

[0060] 1.1 Experimental materials

[0061] Table 1 Main experimental materials

[0062]

[0063] 2. Preparation of nanomaterials:

[0064] 2.1 Synthesis of HA-HDA

[0065] First, dissolve 300 mg of HA in 20 ml of ultrapure water. Subsequently, add 20 ml of DMSO and stir for 10 min to obtain an HA solution. Dissolve EDC·HCl (30.6 mg) in 1.50 ml of DMSO, dissolve NHS (18.4 mg) in 1.5 ml of ultrapure water, and then add both to the HA solution. Stir at room temperature for 1 h to obtain a reaction solution. Then, dissolve HDA (19.3 mg) in 3 ml of DMSO, sonicate (50%, 3S on / 3S off, 3 min), and then add it dropwise to the above reaction solution. After reacting at room temperature for 24 h, dialyze with distilled water (dialysis membrane molecular weight is 3.5 KDa) for 2 days, changing the dialysis fluid every 6 h. After dialysis is completed, take the retentate and perform freeze-drying for 48 h. Finally, collect the sample and store it in a -20°C refrigerator for standby. Use 1H NMR to characterize its structure, and the results are as Figure 1 shown. The structure of the HA-HDA material was verified to be correct by 1H NMR spectra.

[0066] 2.2 Synthesis of Rg3@PLGA-HA nanoparticles

[0067] First, dissolve 2.0 mg of Rg3 and 96.0 mg of PLGA (75:25, 20,000 - 40,000) in 1.0 ml of chloroform and sonicate for 3 min to assist dissolution. Then, add the above solution to an aqueous solution of HA-HDA (2 mg / mL, 6 mL) under ultrasonic conditions (50%, 3S on / 3S off). Subsequently, sonicate with an ultrasonic probe (50%, 3S on / 3S off) for 3 min to obtain a mixed solution A. Then, under ultrasonic conditions (50%, 3S on / 3S off), continue to add mixed solution A dropwise to an aqueous solution of HA-HDA (0.5 mg / mL, 15 mL), and stir in the dark at room temperature for 9 h to obtain a mixed solution B. Then, remove the organic solvent inside. Wash the sample 3 times with distilled water, centrifuge (12,000 rpm, 5 min) to collect the sample, redissolve it with a 5% lactose aqueous solution, and perform freeze-drying, and store it in the dark at 4°C for standby. Repeat the above steps without adding Rg3 to obtain PLGA-HA.

[0068] Use Fourier transform infrared spectroscopy (FT-IR) to characterize the structures of HA-HDA, PLGA-HA, and Rg3@PLGA-HA. The results are as Figure 2 shown. It was verified by Fourier transform infrared spectroscopy (FT-IR) that the material structures of HA-HDA, PLGA-HA, and Rg3@PLGA-HA are correct.

[0069] Figure 3To further evaluate the size of Rg3@PLGA-HA nanoparticles using DLS, the results showed that the main distribution was around 350 nm; Figure 4 To determine the morphology of Rg3@PLGA-HA nanoparticles by TEM, the results showed nanospheres with a diameter of about 300 nm. The reason for this difference is that the hydrated particle size of nanoparticles detected by DLS in liquid is slightly larger than the actual size. As Figure 5 The zeta potential of the measured Rg3@PLGA-HA nanoparticles was -30.88 mV. The nanoparticles were negatively charged and had a stable structure.

[0070] 2.3 Preparation of the standard curve

[0071] Preparation of the Rg3 standard curve: Using DMSO as the solvent, solutions with concentrations of 100, 80, 60, 40, 20, 10, and 0 μg / mL were prepared respectively, and their absorbance values were detected by ultraviolet method. The peak areas of the Rg3 standard solution with gradient concentrations were detected by HPLC, and the standard concentration curve (y = 29.835x - 11.365, R 2 = 0.9983) was plotted.

[0072] 2.4 Determination of the drug encapsulation efficiency and loading capacity of NPs

[0073] Take 1 mg of the freeze-dried sample, dilute it with DMSO to a concentration within the range of the standard curve, then detect its absorbance value by ultraviolet, and determine the content of Rg3 in the nanoparticles according to the pre-established standard curve of Rg3 in DMSO. The encapsulation efficiency and loading capacity of Rg3 were calculated according to the following formulas:

[0074] Loading capacity (%) = [Amount of drug in NPs] / [Mass of carrier + Mass of drug] × 100%

[0075] Encapsulation efficiency (%) = [Mass of drug in NPs] / [Mass of added drug] × 100%

[0076] The results are as Figure 6 shown. After detection and calculation, the loading capacity of Rg3 in Rg3@PLGA-HA was 0.45%, and the encapsulation efficiency was 33%.

[0077] 2.5 Determination of drug release behavior

[0078] Dissolve 1 mg of Rg3@PLGA-HA in 1 mL of phosphate buffer saline (PBS) and transfer it into a dialysis bag (MW cut-off value 3.5 kDa). Then place the dialysis bag into a centrifuge tube containing 30 mL of PBS under different conditions: pH = 7.4, pH = 7.4 + HAase (hyaluronidase) (1000 units / mL), and pH = 5.5 + HAase (1000 units / mL), and conduct dialysis in a water bath thermostatic oscillator (rotation speed 100 rpm, 37 °C). At each designated point, take out 3 mL of the dialysis solution and supplement it with an equal volume of fresh medium. The results are as Figure 7 shown that under the condition of pH = 6.5, the drug is rapidly released within 12 hours, and the cumulative release rate exceeds 60%. The cumulative release reaches 80% at 72 h.

[0079] Example 2

[0080] Evaluate the effect of Rg3@PLGA-HA nanoparticles on gallbladder cancer cells and the regulatory pathway at the cellular level.

[0081] 2.1 Experimental materials

[0082] Table 2 Main reagents involved in the experiment

[0083] Name Manufacturer GBC-SD human gallbladder cancer cells Wuhan Pricella Biotechnology HIBEpiC human intrahepatic bile duct epithelial cells Ningbo Mingzhou Bio CCK8 detection kit Beyotime RPMI-1640 medium Shanghai Yuanpei Fetal Bovine Serum Hyclone Cell culture dishes, cell culture flasks Corning 0.25% Trypsin CTCC PBS phosphate buffer solution CTCC Other chemical reagents Domestic analytical pure

[0084] 2.2 Experimental grouping

[0085] ① GBC-SD + Rg3 (0, 2.5, 5, 10, 20, 50 μM)

[0086] ② HIBEpiC + Rg3 (0, 2.5, 5, 10, 20, 50 μM)

[0087] ③ GBC-SD + PLGA-HA (0, 2.5, 5, 10, 20, 50 μM)

[0088] ④ HIBEpiC + PLGA-HA (0, 2.5, 5, 10, 20, 50 μM)

[0089] ⑤ GBC-SD + Rg3@PLGA-HA (0, 2.5, 5, 10, 20, 50 μM)

[0090] ⑥ HIBEpiC + Rg3@PLGA-HA (0, 2.5, 5, 10, 20, 50 μM)

[0091] 2.3 Experimental methods

[0092] 1) Take GBC-SD and HIBEpiC cells in the logarithmic growth phase, and adjust the cell density to 1×10 4Cells / well were inoculated into a 96-well culture plate according to the experimental groups, 100 μL per well, and cultured at 37 °C in a 5% CO 2 incubator for 24 h.

[0093] 2) The culture supernatant in the culture dish was aspirated, and the cells were washed with PBS; 3) Rg3 (0, 2.5, 5, 10, 20, 50 μM), PLGA-HA (0, 2.5, 5, 10, 20, 50 μM), and Rg3@PLGA-HA (0, 2.5, 5, 10, 20, 50 μM) were added respectively according to the experimental groups, and cultured at 37 °C in a 5% CO 2 incubator for 24 h, 48 h, 72 h, and 96 h; 4) At the end of the culture, the cell morphology was observed by microscope photography, and the results were as shown in Figure 8 、 10 、12, 14, 16, 18; 5) 10 μL of 5 mg / ml CCK8 was added to each well, and incubated in the dark at 37 °C in a 5% CO 2 incubator for 1 h; 6) The OD value at a wavelength of 450 nm was measured with an enzyme-labeled instrument, and the OD value measured was used for the analysis of the effect on cell proliferation. The results were as shown in Figure 9 、 11 、13, 15, 17, 19.

[0094] 2.4 Experimental results

[0095] It can be seen from Figures 8 - 19 that compared with the 0 μM group, Rg3, PLGA-HA, and Rg3@PLGA-HA had no significant effect on the proliferation ability of HIBEpiC cells. PLGA-HA had no significant effect on the proliferation ability of GBC-SD cells. After 48 h of treatment with 50 μM Rg3, 72 h of treatment with 20 μM Rg3, and 96 h of treatment with 10 μM Rg3, the proliferation ability of GBC-SD cells was significantly inhibited. After 24 h of treatment with 50 μM Rg3@PLGA-HA, 48 h of treatment with 20 μM Rg3@PLGA-HA, 72 h of treatment with 10 μM Rg3@PLGA-HA, and 96 h of treatment with 5 μM Rg3@PLGA-HA, the proliferation ability of GBC-SD cells was significantly inhibited. (*p < 0.05, **p < 0.01, compared with the 0 μM group).

[0096] Example 3

[0097] Hemolysis experiment

[0098] 3.1 Experimental materials: Blood collection needle, manufacturer: Hyclone, product number: SV30087.02; Blood collection tube, manufacturer: domestic analytical pure.

[0099] 3.2 Experimental groups

[0100] ① CK ② Rg3 ③ PLGA-HA ④ Rg3@PLGA-HA

[0101] 3.3 Experimental methods

[0102] 1) Take 500 μl of whole blood, add 5 ml of PBS, centrifuge at 1200 rpm for 5 min, and discard the supernatant; 2) Add PBS to make up to 10 ml and mix well, centrifuge at 1200 rpm for 5 min, discard the supernatant, and repeat this step until the supernatant is clear; 3) Resuspend the red blood cells with 10 ml of PBS. 4) According to the experimental groups, take 200 μl of the red blood cell suspension each and add 50 μM of Rg3, PLGA-HA, and Rg3@PLGA-HA respectively. 5) Observe the cell morphology under the microscope after 30 min.

[0103] The results are as Figure 20 shown. The figure shows that compared with the CK group, hemolysis occurred after the cells were treated with Rg3, while no hemolysis occurred after the cells were treated with PLGA-HA and Rg3@PLGA-HA.

[0104] Example 4

[0105] Colony formation assay

[0106] 4.1 Experimental materials: 4% paraformaldehyde, Beyotime, P0099; crystal violet, Beyotime, C0121.

[0107] 4.2 Experimental groups: ① CK ② Rg3 ③ PLGA-HA ④ Rg3@PLGA-HA

[0108] 4.3 Experimental methods: 1) Take the GBC-SD monolayer cultured cells in the logarithmic growth phase, digest them with 0.25% trypsin and pipette them into single cells, and suspend the cells in the complete culture medium with 10% fetal bovine serum for standby; 2) Dilute the cell suspension in gradient multiples and inoculate it into a 6-well plate at an appropriate cell density; 3) Gently rotate to disperse the cells evenly, and incubate at 37 °C, 5% CO 2 and saturated humidity for 24 h; 4) According to the experimental groups, add 50 μM of Rg3, PLGA-HA, and Rg3@PLGA-HA respectively, and incubate in an incubator at 37 °C, 5% CO 2 for 48 h. 5) Aspirate the culture supernatant in the 6-well plate, change to normal culture medium, and incubate at 37 °C, 5% CO 2Cultured in an incubator; 6) Change the culture medium once every 2 days. 7) Observe regularly. When visible clones appear in the culture dish, terminate the culture; 8) Discard the supernatant, and carefully wash twice with PBS; 9) Add 4% paraformaldehyde and fix at room temperature for 15 minutes; 10) Remove the fixative, add an appropriate amount of crystal violet staining solution and stain for 10 minutes, then slowly wash off the staining solution with running water and air dry; 11) Invert the petri dish and take pictures, directly count the clones with the naked eye, and the results are as Figure 21 shown, or count the number of clones with more than 10 cells under a microscope (low power), and the results are as Figure 22 shown. Figures 21 - 22 showed that compared with the CK group, the number of colony formations in the Rg3 and Rg3@PLGA-HA groups was significantly reduced, and the reduction in the Rg3@PLGA-HA group was more significant. (*P<0.05, **P<0.01, compared with the CK group).

[0109] Example 5

[0110] Scratch assay

[0111] 5.1 Experimental grouping: ①CK ②Rg3 ③PLGA-HA ④Rg3@PLGA-HA

[0112] 5.4 Experimental method: 1) First, use a marker pen to draw horizontal lines evenly on the back of a 3.5 cm dish with a ruler, about one line every 0.5 - 1 cm; 2) Take GBC-SD cells in the logarithmic growth phase with good growth status, digest and count about 5×10 5 cells with 0.25% trypsin, inoculate them in a 3.5 cm dish, and culture overnight at 37°C, 5% CO 2 under saturated humidity conditions; 3) When the cell density reaches about 90% and covers the bottom of the 3.5 cm dish, use a 200 μl pipette tip against the ruler to make scratches as perpendicular as possible to the horizontal lines on the back. The pipette tip should be perpendicular and not tilted; 4) Wash the cells 3 times with PBS to remove the scratched cells, observe and take pictures under a microscope; 5) According to the experimental grouping, add 50 μM of Rg3, PLGA-HA, and Rg3@PLGA-HA respectively, and culture under the conditions of 37°C, 5% CO 2 ; 6) After 48 h, take pictures under a microscope to observe the cell migration, and the results are as Figure 23 , 24 shown.

[0113] 5.5 Experimental results: The results of the scratch assay showed that compared with the CK group, the migration ability of GBC-SD cells was significantly inhibited after the cells were treated with Rg3 and Rg3@PLGA-HA for 48 h, and the inhibitory effect of Rg3@PLGA-HA was more significant. (*P<0.05, **P<0.01, compared with the CK group).

[0114] Example 6

[0115] Transwell invasion assay

[0116] 6.1 Experimental grouping: ① CK ② Rg3 ③ PLGA-HA ④ Rg3@PLGA-HA

[0117] 6.2 Experimental method: 1) Take the treated GBC-SD cells, add 3 ml of PBS to wash the cells, digest and collect them with 0.25% trypsin respectively, centrifuge at 1000 rpm for 5 min, discard the supernatant, wash twice with PBS to remove the residual serum; 2) Resuspend the cells with serum-free RPMI-1640 medium, count the cells with a cell counting chamber, and dilute the cell concentration to 3×10 5 cells / ml with serum-free RPMI-1640 medium for standby; 3) Pre-add 800 μl of RPMI-1640 medium containing 10% FBS (with double antibodies) to a 24-well plate, and place a transwell chamber in it. After 1 h, add 200 μl of the cell suspension of each group to the upper chamber of the transwell, and incubate in a 37 °C, 5% CO 2 incubator for 24 h; 4) According to the experimental grouping, add 50 μM of Rg3, PLGA-HA, and Rg3@PLGA-HA respectively, and incubate at 37 °C and 5% CO 2 for 24 h and 48 h respectively; 5) Take out the transwell, carefully wash the chamber once with PBS, and fix the cells with 70% ice ethanol solution for 1 h; 6) Stain with 0.5% crystal violet staining solution, place at room temperature for 20 min, wash with PBS, wipe off the non-migrated cells on one side of the upper chamber with a clean cotton ball, and observe and photograph under a microscope; as Figure 25 、 Figure 26 shown.

[0118] 6.3 Experimental results: The results of the Transwell migration assay showed that compared with the CK group, the migration ability of GBC-SD cells was significantly inhibited after the cells were treated with Rg3@PLGA-HA for 24 h, and the migration ability of GBC-SD cells was significantly inhibited after the cells were treated with Rg3 for 48 h. (*P<0.05, **P<0.01, compared with the CK group).

[0119] Example 7

[0120] Flow cytometry detection of the cell division cycle of gallbladder cancer cells

[0121] Main equipment and reagents involved in the experiment: Flow cytometer, manufacturer: BD, model: FACSVerse. AnnexinV-FITC / PI kit: manufacturer: BestBio, product number: 401006.

[0122] 7.1 Experimental grouping: ① CK ② Rg3 ③ PLGA-HA ④ Rg3@PLGA-HA

[0123] 7.2 Experimental method: 1) Take GBC-SD cells in the logarithmic growth phase, adjust the cell density to 5×10 5 cells / well, inoculate into a 6-well culture plate, add 2 mL of complete medium to each well, and culture in an incubator at 37°C and 5% CO 2 for 24 h. 2) According to the experimental grouping, add 50 μM of Rg3, PLGA-HA, and Rg3@PLGA-HA respectively, and culture in an incubator at 37°C and 5% CO 2 for 48 h. 3) Digest and collect the cells with trypsin without EDTA, and wash them three times with PBS. 4) Add pre-cooled 70% ethanol and fix overnight at 4°C. 5) Centrifuge to collect the cells and wash them with PBS. 6) Resuspend the cells with 500 μL of PBS, add PI at a ratio of 1:200, and incubate in the dark at 37°C for 30 min. 7) Wash the cells once with pre-cooled PBS. 8) Resuspend the cells with 300 μL of PBS and detect them with a flow cytometer.

[0124] 7.3 Experimental results: Figure 27 、 Figure 28 The results of the flow cytometry cycle showed that compared with the CK group, the G0 / G1% and G2 / M% in the Rg3@PLGA-HA group were significantly decreased, and the S% was significantly increased; the G0 / G1% in the Rg3 group was significantly decreased, and the S% was significantly increased. (*P<0.05, **P<0.01, compared with the CK group).

[0125] Example 8

[0126] Detection of cell apoptosis rate by AnnexinV and PI double staining method

[0127] 8.1 Experimental grouping: ① CK ② Rg3 ③ PLGA-HA ④ Rg3@PLGA-HA

[0128] 8.2 Experimental method: 1) Take GBC-SD cells in the logarithmic growth phase, adjust the cell density to 5×10 5 cells / well, inoculate into a 6-well culture plate, add 2 mL of complete medium to each well, and culture in an incubator at 37°C and 5% CO 2 for 48 h. 2) According to the experimental grouping, add 50 μM of Rg3, PLGA-HA, and Rg3@PLGA-HA respectively, and culture in an incubator at 37°C and 5% CO 2Cultivate in an incubator for 48 h. 3) Digest and collect the cells with trypsin without EDTA, and wash twice with pre-cooled PBS. 4) Resuspend the cells with 100 μL PBS, add PI (5 μL) and AV (5 μL), and incubate at 37 °C in the dark for 30 min. 5) Add PBS and wash twice. 6) Add 300 μL PBS to resuspend the cells, and detect with a flow cytometer.

[0129] 8.3 Experimental results: Figure 29 、 30 The results of flow cytometry apoptosis showed that compared with the CK group, the apoptosis rates of the Rg3 group and the Rg3@PLGA-HA group were significantly increased. (*P < 0.05, **P < 0.01, compared with the CK group).

[0130] Example 9

[0131] Detection of cell apoptosis by TUNEL method

[0132] 9.1 Experimental reagents: One-step TUNEL Cell Apoptosis Detection Kit, manufacturer: Beyotime, product number: C1088.

[0133] 9.2 Experimental groups: ① CK ② Rg3 ③ PLGA-HA ④ Rg3@PLGA-HA

[0134] 9.3 Experimental method: 1) Take GBC-SD and HIBEpiC cells in the logarithmic growth phase, adjust the cell density to 5×10 3 cells / well, inoculate into a 96-well culture plate, 100 μL per well, and cultivate in an incubator at 37 °C with 5% CO 2 for 24 h. 2) According to the experimental groups, add 50 μM of Rg3, PLGA-HA and Rg3@PLGA-HA respectively, and cultivate in an incubator at 37 °C with 5% CO 2 for 48 h. 3) Wash the cells once with PBS, and fix the cells with 4% paraformaldehyde for 30 min. 4) Wash the cells once with PBS, add PBS containing 0.3% Triton X-100, and incubate at room temperature for 5 min. 5) Wash the cells twice with PBS. 6) Prepare an appropriate amount of TUNEL detection according to the ratio of 5 μL TDT enzyme + 45 μL fluorescence labeling solution. 7) Add 50 μL of TUNEL detection solution to each well, and incubate at 37 °C in the dark for 60 min. 8) Wash the cells three times with PBS. 9) Take pictures and observe under a fluorescence microscope.

[0135] 9.4 Experimental results: Figure 31 、 32, The TUNEL results of 33 showed that compared with the CK group, the apoptosis rates of each group of HIBEpic cells had no significant change, and the apoptosis rates of the Rg3 group and the Rg3@PLGA-HA group of GBC-SD cells were significantly increased. (*P<0.05, **P<0.01, compared with the CK group)

[0136] Example 10

[0137] Detection of autophagy by GFP-LC3 fusion protein

[0138] Experimental reagents: LC3, manufacturer: abcam; Hoechst 33342, manufacturer: Beyotime.

[0139] Experimental grouping: ①CK ②Rg3 ③PLGA-HA ④Rg3@PLGA-HA

[0140] 10.1 Experimental method: Take GBC-SD cells in the logarithmic growth phase, adjust the cell density to 5×10 3 cells / well, inoculate in a 96-well culture plate, 100 μL per well, and culture in an incubator at 37°C with 5% CO 2 for 24 h. According to the experimental grouping, add 50 μM of Rg3, PLGA-HA, and Rg3@PLGA-HA respectively, and culture in an incubator at 37°C with 5% CO 2 for 48 h. Wash the cells with PBS and fix the cells with 4% paraformaldehyde for 15 min; wash the cells with PBS, permeabilize with PBS containing 0.1% Triton X-100 at room temperature for 5 min; wash the cells with PBS, block with PBS containing 5% FBS at 37°C for 1 h; wash the cells with PBS, dilute the LC3 antibody with PBS containing 2% FBS at a ratio of 1:200, and incubate overnight at 4°C; wash the cells with PBS, dilute the fluorescent secondary antibody with PBS containing 2% FBS at a ratio of 1:200, and incubate at 37°C in the dark for 1 h; wash the cells with PBS, dilute Hoechst33342 with PBS at a ratio of 1:1000, and incubate at 37°C in the dark for 10 min; wash the cells with PBS. Observe by fluorescence microscopy photography.

[0141] 10.2 Experimental results: Figure 34 In, the laser confocal results showed that compared with the CK group, the apoptosis rates of the Rg3 group and the Rg3@PLGA-HA group were significantly increased. (*P<0.05, **P<0.01, compared with the CK group)

[0142] Example 11

[0143] Western Blot experiment

[0144] Reagents required in Table 2

[0145]

[0146]

[0147] 11.1 Experimental grouping: ① CK ② Rg3 ③ PLGA-HA ④ Rg3@PLGA-HA

[0148] 11.2 Experimental procedures:

[0149] 11.2.1. Sample preparation

[0150] 1) Take GBC-SD cells in the logarithmic growth phase, adjust the cell concentration to 5×10 5 cells / dish, inoculate into 6 cm culture dishes, 3 ml per well, culture in an incubator at 37 °C and 5% CO 2 for 24 h. 2) According to the experimental grouping, add 50 μM of Rg3, PLGA-HA and Rg3@PLGA-HA respectively, and culture in an incubator at 37 °C and 5% CO 2 for 48 h. 3) Discard the culture medium in each dish, add 1 ml of trypsin to each well and digest for 20 s, then add 1 ml of complete medium to terminate the digestion. Pipette the cells at the bottom of the plate and collect them into a centrifuge tube. Centrifuge at 1200 rpm for 3 min, discard the supernatant, add 100 μl of RIPA lysis buffer to the cell pellet respectively, and add 1 μl of PMSF, place on ice and lyse for 30 min; centrifuge at 12000 rpm at 4 °C for 10 min; transfer the supernatant to a new EP tube, perform protein quantification and store in a -20 °C refrigerator after quantification.

[0151] 11.2.2 Protein quantification

[0152] a. Preparation of the standard curve: Take an enzyme-linked immunosorbent assay (ELISA) plate and add reagents according to Table 3 below:

[0153] Table 3 Reagent addition amounts

[0154] Well number 1 2 3 4 5 6 7 8 Protein standard solution (μl) 0 1 2 3 4 5 6 7 Deionized water (μl) 20 19 18 17 16 15 14 13

[0155] b. Prepare an appropriate amount of BCA working solution by mixing BCA reagent A and reagent B at a volume ratio of 50:1, and mix well; add 200 μl of BCA working solution to each well;

[0156] c. Place the ELISA plate on an oscillator and shake for 30 s, incubate at 37 °C for 30 min, and then measure the absorbance at 562 nm. Use the absorbance value as the ordinate and the protein concentration (mg / ml) as the abscissa to plot the standard curve y = 1.7044x - 2.029, R 2 = 0.9983.

[0157] d. Based on the absorbance value of the measured sample, the corresponding protein concentration (mg / ml) can be calculated on the standard curve, and multiplying it by the sample dilution factor of 8 gives the actual sample concentration (unit: mg / ml). Determine the sample loading amount according to the sample concentration.

[0158] 11.2.3 Preparation of PAGE Gel

[0159] Operate according to the formula:

[0160] Table 4 Preparation of Separating Gel and Stacking Gel

[0161] Reagent 5% Stacking gel Separating gel Separating gel ddH2O 1.4ml 1.6ml 1.1ml 30% Acrylamide 0.33ml 2.0ml 2.5ml 1.5M — 1.3ml 1.3ml 1.0M 0.25ml — — 10% SDS 0.02ml 0.05ml 0.05ml 10% Ammonium persulfate 0.02ml 0.05ml 0.05ml TEMED 0.002ml 0.002ml 0.002ml Total volume 2ml 5ml 5ml

[0162] 11.2.4 Sample Loading and Electrophoresis

[0163] a. Sample Loading

[0164] The mass of protein loaded per well is 30 μg. Place the prepared PAGE gel in the electrophoresis tank, add an appropriate amount of electrophoresis buffer, remove the comb, and gently blow the loading wells with a pipette to avoid residual gel in the wells affecting the results. Load the samples. Slowly add the prepared samples to the corresponding wells with a pipette gun, taking care not to overflow the wells.

[0165] b. Electrophoresis

[0166] Generally, for the stacking gel, run at 80 V for 20 min, and for the separating gel, run at 120 V for 1 h. The voltage and time can be adjusted according to specific experimental requirements. When the dye reaches the bottom of the gel, cut off the power supply, stop electrophoresis, and proceed to the next step of membrane transfer.

[0167] 11.2.5 Membrane Transfer

[0168] Carefully transfer the gel into the transfer buffer. Cut a PVDF membrane of the same size, soak it in methanol for 1 min, then wash it with water for 2 min. Cut 6 pieces of filter paper of the same size as the PVDF membrane and equilibrate them in the transfer buffer for 15 min. Place the gasket, filter paper, gel, membrane, filter paper, and gasket on the transfer device from the negative electrode to the positive electrode, removing air bubbles. Transfer at a voltage of 200 mA for 1 min per 1 KD.

[0169] 11.2.6 Membrane Blocking and Antibody Incubation

[0170] a. Blocking: Block with 5% skim milk powder at room temperature for 1 h.

[0171] b. Primary Antibody: Dilute the antibody in the blocking solution to the required concentration and incubate with the membrane overnight at 4°C.

[0172] c. Secondary antibody: The membrane incubated with the primary antibody was washed 5 times with TBST for 10 minutes each time. Subsequently, the secondary antibody corresponding to the primary antibody was diluted 1:5000 according to the dosage and incubated with the membrane at 37°C for 1 hour. Then it was washed 5 times with TBST for 10 minutes each time. 11.2.7. Photograph was taken with an integrated chemiluminescence instrument

[0173] The chemiluminescent solution (Solution A and Solution B) was prepared by mixing at a ratio of 1:1 (pay attention to light protection). An appropriate amount of the chemiluminescent solution was aspirated with a pipette to cover the PVDF membrane, and the image was exposed and collected on an ECL chemiluminescence instrument.

[0174] 11.2.8 Experimental results

[0175] Figure 35 、 36 The results of Western Blot showed that compared with the CK group, the expression levels of LC3II / LC3I and MOB1A in the Rg3 group and the Rg3@PLGA-HA group were significantly increased, while the expression levels of p-STAT3, TG, NF-κB, and IL-6 were significantly decreased. (*P<0.05, **P<0.01, compared with the CK group)

[0176] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a ginsenoside Rg3@PLGA-HA nanocomposite, characterized in that: The preparation method comprises the following steps: 1) dissolving hyaluronic acid in water and adding DMSO to obtain a hyaluronic acid solution; 2) adding an EDC˙HCl solution dissolved in DMSO and an NHS solution dissolved in water to the hyaluronic acid solution to obtain a mixed solution; 3) dissolving hexadecylamine in DMSO, adding the mixture dropwise to the mixed solution, reacting at 20-25° C. for 20-28 hours to obtain a reaction solution, dialyzing the reaction solution with distilled water, and freeze-drying the retentate after the dialysis to obtain hyaluronic acid-hexadecylamine powder; 4) dissolving the hyaluronic acid-hexadecylamine powder in water to obtain hyaluronic acid-hexadecylamine aqueous solutions with concentrations of 0.5-3 mg / mL and 0.1-1 mg / mL, respectively; 5) Ginsenoside Rg3 and PLGA are dissolved in chloroform to obtain a mixed solution A, which is added to the hyaluronic acid-hexadecylamine aqueous solution with a concentration of 0.5-3 mg / mL, and ultrasonically mixed to obtain a mixed solution B, and the mixed solution B is further added to the hyaluronic acid-hexadecylamine aqueous solution with a concentration of 0.1-1 mg / mL, and after the reaction is protected from light, the organic solvent is removed to obtain ginsenoside Rg3@PLGA-HA.

2. The method for preparing the ginsenoside Rg3@PLGA-HA nanocomposite according to claim 1, characterized in that: Step 1) The mass volume ratio of the hyaluronic acid to water and DMSO is 10-20 mg: 1 mL: 1 mL.

3. The method for preparing the ginsenoside Rg3@PLGA-HA nanocomposite according to claim 1, characterized in that: Step 2) The mass volume ratio of the EDC˙HCl to the DMSO is 10-30 mg:1 mL, and the mass volume ratio of the NHS to water is 10-20 mg:1 mL; the volume ratio of the hyaluronic acid solution to the EDC˙HCl solution and the NHS solution is (30-50):(1-2):(1-2).

4. The method for preparing the ginsenoside Rg3@PLGA-HA nanocomposite according to claim 1, characterized in that: Step 3) The mass volume ratio of the HDA to the DMSO is 5-7 mg:1 mL, the mass ratio of the hyaluronic acid to hexadecylamine is (10-20): (0.5-1.5), the molecular weight of the dialysis membrane used for dialysis is 3-4 KDa, the dialysis time is 2 days, and the liquid is changed every 6 hours.

5. The method for preparing the ginsenoside Rg3@PLGA-HA nanocomposite according to claim 1, characterized in that: Step 5) The mass volume ratio of the ginsenoside Rg3 to the PLGA and the chloroform is 1-3 mg:90-100 mg:1 mL, the volume ratio of the mixed solution A to the 0.5-3 mg / mL hyaluronic acid-hexadecylamine aqueous solution is 1:(5-7), the volume ratio of the mixed solution B to the 0.1-1 mg / mL hyaluronic acid-hexadecylamine aqueous solution is 1:(2-3), the ultrasonic mixing conditions are 50% with an ultrasonic probe, 3S on / 3S off for 1-10 min, the PLGA is polymerized by 75% polylactic acid and 25% polyglycolic acid, and has a molecular weight of 20,000 to 40,000.

6. The method for preparing the ginsenoside Rg3@PLGA-HA nanocomposite according to claim 1, characterized in that: Step 5) The light-proof reaction time is 8-10 hours.

7. A ginsenoside Rg3@PLGA-HA nanocomposite obtained by the preparation method as claimed in claim 1.

8. Use of the ginsenoside Rg3@PLGA-HA nanocomposite as claimed in claim 7 in the preparation of a drug for treating gallbladder cancer.

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