Echinacoside and folic acid-loaded functionalized polylactic acid-glycolic acid copolymer nanoparticles as well as preparation method and application thereof

Abstract

The invention provides echinacoside and folic acid-loaded functionalized polylactic acid-glycolic acid copolymer nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of antitumor drugs. The E-coated PLGA-FA nanoparticles are prepared by a double emulsification method, echinacoside is wrapped by a polylactic acid-glycolic acid copolymer, and folic acid is modified to the surface of the polylactic acid-glycolic acid copolymer. As folic acid has high binding affinity with folic acid receptors on the surfaces of hepatoma carcinoma cells, the obtained E-coated PLGA-FA shows specific targeting to HepG2 hepatoma carcinoma cells, and the bioavailability of echinacoside is effectively improved. An in-vitro cell experiment shows that the E-coated PLGA-FA can be effectively internalized by HepG2 cells, so that cell apoptosis is obviously increased. In-vivo experiments show that the E-coated PLGA-FA has remarkable anti-tumor activity compared with a non-targeted nano-drug E-coated PLGA.

Description

Technical Field

[0001] The invention relates to the technical field of anti-tumor drugs, and in particular to a polylactic acid-glycolic acid copolymer nanoparticle functionalized with echinacoside and folic acid, and a preparation method and application thereof. Background Art

[0002] Primary liver cancer, which mainly includes hepatocellular carcinoma (HCC) cases and intrahepatic cholangiocarcinoma, is the third leading cause of cancer death. Common causes of HCC include chronic infection with HBV or HCV, aflatoxin exposure, excessive weight, alcohol abuse, type 2 diabetes, and smoking. Current treatment options for HCC include liver resection and transplantation and systemic therapy based on disease stage progression. In the past decade, HCC management has made great progress, and 6 systemic therapies have been approved for the treatment of advanced HCC. Treatments such as atezolizumab, regorafenib, cabozantinib, bevacizumab, sorafenib, and lenvatinib have shown good results in terms of overall improvement in progression-free survival outcomes in HCC patients. However, HCC treatment is complex and multifaceted, as the expected outcome often depends on the time of diagnosis and any pre-existing comorbidities, so the challenges in HCC treatment cannot be ignored.

[0003] Considering that HCC is often accompanied by other diseases, understanding the underlying mechanisms of liver tumor microenvironment (TME) through its signaling pathways and selecting tumor cell targets safely and effectively remain the main goals of research in this field. In recent years, nanotechnology has obviously developed into a rapidly developing therapeutic field due to its unprecedented advantages over traditional therapies. Nanoparticles (NPs, with a size range of 10 to 100 nm) can deliver active therapeutic moieties (e.g., drugs, proteins, peptides, and nucleic acids) from the blood to target cells and subcellular compartments, depending on their excellent properties in controlling physiological surface properties and structures, thereby achieving optimal therapeutic targeting without any off-target toxicity. So far, only a few nanodrugs have been approved by the FDA. Therefore, expanding the types of nanodrugs and exerting good antitumor activity is of great significance for the treatment of HCC. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a polylactic acid-co-glycolic acid nanoparticle functionalized with echinacoside and folic acid and a preparation method and application thereof. The nanoparticle prepared by the present invention has significant anti-tumor activity.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing echinacoside- and folic acid-functionalized polylactic acid-glycolic acid copolymer nanoparticles, comprising the following steps:

[0007] The aqueous phase solution of echinacea glycoside, polylactic acid-glycolic acid and an organic solvent are mixed and subjected to a first ultrasonic treatment to obtain a primary emulsion;

[0008] The primary emulsion is mixed with a sodium cholate solution and subjected to a second ultrasonic treatment to obtain a secondary emulsion;

[0009] removing the organic solvent in the secondary emulsion, and obtaining the polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside after dialysis;

[0010] The polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside are mixed with a DSPE-mPEG5000-folic acid solution and functionalized with folic acid to obtain polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside and folic acid.

[0011] Preferably, the mass ratio of echinacoside to polylactic acid-glycolic acid is 1:1-3;

[0012] The mass concentration of the aqueous solution of echinacoside is 10-20 mg / mL.

[0013] Preferably, the mass ratio of echinacoside to sodium cholate is 1:30-50;

[0014] The mass concentration of the sodium cholate solution is 5-8%.

[0015] Preferably, the power of the first ultrasonic treatment and the second ultrasonic treatment are independently 37.5-50W, and the time is independently 2-3 minutes.

[0016] Preferably, the molecular weight cut-off of the dialysis is 12000Da, and the dialysis time is 22 to 24 hours.

[0017] Preferably, the mass concentration of the DSPE-mPEG5000-folic acid solution is 1-2 mg / mL;

[0018] The folic acid functionalization is carried out under stirring conditions, and the folic acid functionalization time is 10 to 12 hours.

[0019] The present invention provides polylactic acid-glycolic acid copolymer nanoparticles functionalized with echinacoside and folic acid prepared by the above preparation method, comprising polylactic acid-glycolic acid copolymer nanoparticles loaded with echinacoside and folic acid modified on the surface of the polylactic acid-glycolic acid copolymer nanoparticles loaded with echinacoside;

[0020] The polylactic acid-glycolic acid copolymer nanoparticles carrying echinacoside comprise echinacoside and the polylactic acid-glycolic acid copolymer encapsulating the echinacoside.

[0021] Preferably, the particle size of the polylactic acid-glycolic acid copolymer nanoparticles functionalized with echinacoside and folic acid is 90 to 100 nm.

[0022] Preferably, the drug loading amount of the poly(lactic acid-co-glycolic acid) nanoparticles functionalized with echinacoside and folic acid is 12.05-12.3%.

[0023] The present invention provides the use of the above-mentioned polylactic acid-glycolic acid copolymer nanoparticles functionalized with echinacoside and folic acid in the preparation of anti-liver cancer drugs.

[0024] The invention provides a preparation method of polylactic acid-glycolic acid copolymer nanoparticles (abbreviated as E@PLGA-FA) functionalized with echinacoside and folic acid, comprising the following steps: mixing an aqueous phase solution of echinacoside, polylactic acid-glycolic acid and an organic solvent, performing a first ultrasonic treatment to obtain a primary emulsion; mixing the primary emulsion with a sodium cholate solution, performing a second ultrasonic treatment to obtain a secondary emulsion; removing the organic solvent in the secondary emulsion, dialyzing to obtain polylactic acid-glycolic acid copolymer nanoparticles loaded with echinacoside; mixing the polylactic acid-glycolic acid copolymer nanoparticles loaded with echinacoside with a DSPE-mPEG5000-folic acid solution, performing folic acid functionalization, and obtaining polylactic acid-glycolic acid copolymer nanoparticles loaded with echinacoside and folic acid. Echinacoside is a natural styrene glycoside, which is found and separated from the garden plant Echinacea purpurea and the Chinese medicine Cistanche deserticola and Echinacea purpurea. Echinacoside can effectively inhibit HCC cell invasion and migration in vitro and effectively prevent HCC metastasis in vivo. Its mechanism is related to upregulating miR-30c-5p, which in turn downregulates FOXD1, thereby reducing the transcriptional activity of KLF12. The present invention adopts a double emulsification method to prepare E@PLGA-FA nanoparticles, uses polylactic acid-glycolic acid copolymer to encapsulate echinacoside, and modifies folic acid on the surface of polylactic acid-glycolic acid copolymer. Since folic acid has a high binding affinity with the folic acid receptor on the surface of liver cancer cells, the obtained E@PLGA-FA shows specific targeting to HepG2 liver cancer cells, effectively improving the bioavailability of echinacoside. The present invention evaluates the anti-tumor effect of E@PLGA-FA in vitro and in vivo. In vitro cell experiments show that E@PLGA-FA can be effectively internalized by HepG2 cells, resulting in a significant increase in cell apoptosis. In vivo experiments show that compared with the non-targeted nanomedicine E@PLGA, E@PLGA-FA has significant anti-tumor activity.

[0025] At the same time, the preparation method provided by the present invention is simple to operate, low in cost, and easy to realize industrial mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The characterization results of E@PLGA and E@PLGA-FA nanoparticles;

[0027] Figure 2 The inhibition of HepG2 cell proliferation by E@PLGA-FA;

[0028] Figure 3 The absorption of E@PLGA and E@PLGA-FA nanoparticles in HepG2 cells;

[0029] Figure 4 The effect of E@PLGA-FA nanoparticles on the behavior of HepG2 cells;

[0030] Figure 5 The antitumor effect of E@PLGA-FA in liver cancer model mice carrying HepG2 cells. DETAILED DESCRIPTION

[0031] The present invention provides a method for preparing echinacoside- and folic acid-functionalized polylactic acid-glycolic acid copolymer nanoparticles, comprising the following steps:

[0032] The aqueous phase solution of echinacea glycoside, polylactic acid-glycolic acid and an organic solvent are mixed and subjected to a first ultrasonic treatment to obtain a primary emulsion;

[0033] The primary emulsion is mixed with a sodium cholate solution and subjected to a second ultrasonic treatment to obtain a secondary emulsion;

[0034] removing the organic solvent in the secondary emulsion, and obtaining the polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside after dialysis;

[0035] The polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside are mixed with a DSPE-mPEG5000-folic acid solution and functionalized with folic acid to obtain polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside and folic acid.

[0036] Unless otherwise specified, the raw materials used in the present invention are all commercially available.

[0037] The present invention mixes an aqueous solution of echinacoside, polylactic acid-glycolic acid and an organic solvent, and performs a first ultrasonic treatment to obtain a primary emulsion. In the present invention, the aqueous solution of echinacoside is preferably a PBS solution of echinacoside. In the present invention, the mass concentration of the aqueous solution of echinacoside is preferably 10 to 20 mg / mL, more preferably 20 mg / mL.

[0038] In the present invention, the polylactic acid-glycolic acid is preferably provided in the form of a solution, the solvent of the polylactic acid-glycolic acid solution is preferably dichloromethane, and the mass concentration is preferably 10 mg / mL. In the present invention, the mass ratio of echinacoside to polylactic acid-glycolic acid is preferably 1:1 to 3, more preferably 1:1.

[0039] In the present invention, the organic solvent is preferably dichloromethane.

[0040] In the present invention, the power of the first ultrasonic treatment is preferably 37.5-50W, more preferably 37.5-40W; the time is preferably 2-3min, more preferably 2min. In the first ultrasonic treatment, the present invention preferably repeats the process of turning on for 3 seconds and turning off for 3 seconds. The present invention obtains a W / O primary emulsion through the first ultrasonic treatment.

[0041] After obtaining the primary emulsion, the present invention mixes the primary emulsion with a sodium cholate solution, performs a second ultrasonic treatment, and obtains a secondary emulsion. In the present invention, the sodium cholate solution is preferably an aqueous solution of sodium cholate, and the mass concentration of the sodium cholate solution is preferably 5-8%, more preferably 5-6%. In the present invention, the mass ratio of echinacoside to sodium cholate is preferably 1:30-50, more preferably 1:30-40. In the present invention, the role of the sodium cholate is to avoid the stratification of the aqueous phase and the oil phase during the secondary ultrasonic treatment.

[0042] In the present invention, the power of the second ultrasonic treatment is preferably 37.5 to 50 W, more preferably 37.5 to 40 W; the time is preferably 2 to 3 min, more preferably 2 min. In the second ultrasonic treatment, the present invention preferably repeats the process of turning on for 3 seconds and turning off for 3 seconds. The present invention obtains a W / O / W secondary emulsion through the second ultrasonic treatment.

[0043] After obtaining the secondary emulsion, the present invention removes the organic solvent in the secondary emulsion, and obtains polylactic acid-glycolic acid copolymer nanoparticles loaded with echinacoside after dialysis. In the present invention, the method of removing the organic solvent is preferably rotary evaporation. In the present invention, the dialysis is preferably carried out at 4° C. and in the dark. In the present invention, the solvent used for the dialysis is preferably a PBS solution, the molecular weight cutoff of the dialysis is preferably 12000Da, and the time is preferably 24h. In the present invention, the PBS solution is replaced every 8 hours during the dialysis process to remove unencapsulated free drugs.

[0044] After obtaining the polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside, the present invention mixes the polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside with a DSPE-mPEG5000-folic acid solution for folic acid functionalization to obtain polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside and folic acid functionalized. In the present invention, the solvent of the DSPE-mPEG5000-folic acid solution is preferably a PBS solution, and the concentration of the DSPE-mPEG5000-folic acid solution is preferably 1 to 2 mg / mL, more preferably 1 mg / mL.

[0045] In the present invention, the folic acid functionalization is preferably carried out under stirring conditions, the time of the folic acid functionalization is preferably 10 to 12 hours, more preferably 12 hours; the temperature of the folic acid functionalization is preferably room temperature.

[0046] The present invention provides polylactic acid-glycolic acid copolymer nanoparticles functionalized with echinacoside and folic acid prepared by the above preparation method, comprising polylactic acid-glycolic acid copolymer nanoparticles loaded with echinacoside and folic acid modified on the surface of the polylactic acid-glycolic acid copolymer nanoparticles loaded with echinacoside;

[0047] The polylactic acid-glycolic acid copolymer nanoparticles carrying echinacoside comprise echinacoside and the polylactic acid-glycolic acid copolymer encapsulating the echinacoside.

[0048] In the present invention, the particle size of the polylactic acid-co-glycolic acid nanoparticles functionalized with echinacoside and folic acid is preferably 90 to 100 nm.

[0049] In the present invention, the drug loading amount of the poly(lactic acid-co-glycolic acid) nanoparticles functionalized with echinacoside and folic acid is preferably 12.05-12.3%.

[0050] The present invention provides the use of the above-mentioned polylactic acid-co-glycolic acid nanoparticles functionalized with echinacoside and folic acid in the preparation of anti-liver cancer drugs. In the present invention, the liver cancer is preferably hepatocellular carcinoma (HCC).

[0051] The following is a detailed description of the polylactic acid-glycolic acid copolymer nanoparticles functionalized with echinacoside and folic acid provided by the present invention, as well as the preparation method and application thereof, in conjunction with the embodiments. However, they should not be construed as limiting the scope of protection of the present invention.

[0052] In the following examples, echinacoside was purchased from Aladdin Biochemical Technology;

[0053] Poly(lactic-co-glycolic acid) was purchased from Sigma-Aldrich.

[0054] Example 1

[0055] Preparation of poly(lactic-co-glycolic acid) nanoparticles functionalized with echinacoside and folic acid (E@PLGA-FA) was performed by the following steps:

[0056] Add 200 μL of PBS solution of echinacoside (20 mg / mL) to 400 μL of dichloromethane (DCM) solution dissolved with polylactic acid-glycolic acid (PLGA, 10 mg / mL). After adding another 200 μL of dichloromethane, use a SCIENTZIID ultrasonic cell crusher to ultrasonically treat the mixture at 37.5 W for 2 minutes, turn on for 3 seconds, and turn off for 3 seconds. Next, add 2.4 mL of sodium cholate solution (5%, w / w), and continue to ultrasonically treat the mixture for 2 minutes (37.5 W, turn on for 3 seconds, turn off for 3 seconds). After that, use a rotary evaporator to remove the organic solvent. Then, use dialysis (M WCO =12,000Da) for 24 hours, and PBS was replaced every 8 hours to remove unencapsulated free drugs. The obtained nanoparticles were dispersed in a PBS solution containing DSPE-mPEG5000-folic acid (1 mg / mL) and stirred for 12 hours to obtain E@PLGA-FA.

[0057] Comparative Example 1

[0058] The difference from Example 1 is that the PBS solution of DSPE-mPEG5000-folic acid is replaced with a PBS solution of DSPE-mPEG5000 (1 mg / mL), thereby obtaining nanoparticles without folic acid functionalization, which are denoted as E@PLGA.

[0059] Structural characterization

[0060] (1) Characterization of Nanoparticles

[0061] The nanoparticles of Example 1 and Comparative Example 1 were dispersed in PBS solution (0.2 mg / mL), and the particle size was characterized using a ZetaSizerPro instrument (Malvern, UK) and a dynamic light scattering (DLS) instrument. The nanoparticles were dispersed in distilled water, and then their zeta potential was measured using DLS. The morphology of the nanoparticles was characterized using a transmission electron microscope (TEM).

[0062] (2) Determination of encapsulation efficiency and drug loading

[0063] To evaluate the encapsulation efficiency of echinacoside by nanoparticles, 200 μL of nanoparticle solution was mixed with 800 μL of DMSO and dialyzed, which was recorded as sample 1. Another 200 μL of nanoparticle solution that was not dialyzed was fully mixed with 800 μL of PBS and centrifuged at 12000 rpm for 30 minutes to obtain the supernatant, which was recorded as sample 2. The content of echinacoside in each solution was determined by HPLC, and a standard curve was prepared using an echinacoside standard. The encapsulation efficiency of echinacoside was calculated as follows: EE (%) = (the content of echinacoside in sample 2 / the content of echinacoside in sample 1) × 100%.

[0064] To calculate the drug loading, 5 mL of the purified nanoparticle solution obtained by dialysis was taken, freeze-dried, and 2 mg of the resulting powder was weighed. Then, 1 ml of DMSO was added to break the nanoparticles, and the echinacoside content was quantified by HPLC, and a standard curve was prepared using the echinacoside standard. The drug loading calculation formula for echinacoside is: DL (%) = (m / total mass of nanoparticles) × 100%, where m represents the content of echinacoside.

[0065] (3) Drug release

[0066] The nanoparticles were dispersed in a PBS solution of pH 5.5 or pH 7.4 (1 mg / mL), gently stirred (250 rpm) at 37°C, and centrifuged at 12,000 rpm for 30 min at a predetermined time point to precipitate the nanoparticles. The echinacoside content in the supernatant was then analyzed by HPLC (Waters, USA) to plot the drug release curve.

[0067] (4) Colloidal stability of nanoparticles

[0068] To evaluate the colloidal stability of the nanoparticles, the nanoparticles were dispersed in PBS solution containing 10% FBS and gently stirred at 37°C (250 rpm). At predetermined time points, samples were taken and the particle size of the nanoparticles was characterized using a dynamic light scattering instrument to evaluate their colloidal stability.

[0069] Figure 1 are the characterization results of E@PLGA and E@PLGA-FA nanoparticles. Figure 1 (a) is the TEM image of E@PLGA and E@PLGA-FA, (b) is the particle size distribution of E@PLGA and E@PLGA-FA, (c) is the Zeta potential of E@PLGA and E@PLGA-FA, (d) is the colloidal stability test results of E@PLGA and E@PLGA-FA nanoparticles, and (e) is the drug release curve of E@PLGA and E@PLGA-FA nanoparticles incubated under different pH conditions.

[0070] Transmission electron microscopy images showed that both E@PLGA and E@PLGA-FA formed monodisperse spherical structures with uniform morphology (see Figure 1 Dynamic light scattering data showed that the particle sizes of E@PLGA and E@PLGA-FA were approximately 91 nm and 100 nm, respectively (see Figure 1 b), the particle size observed by transmission electron microscopy is similar to that obtained by dynamic light scattering. After folic acid modification on the surface of the nanoparticles, the potential of E@PLGA decreased from -22.50 mV to -31.89 mV (see Figure 1 c). The encapsulation efficiency of E@PLGA-FA for echinacoside was 28.78%, and the drug loading was 12.05%, which were slightly lower than the encapsulation efficiency (40.34%) and drug loading (16.12%) of E@PLGA nanoparticles. In vitro colloidal stability experiments showed that E@PLGA and E@PLGA-FA maintained good colloidal stability in PBS containing 10% FBS and pure PBS, and the particle size and PDI did not change significantly (see Figure 1 d in the figure). The in vitro cumulative drug release test showed that at pH = 7.4, the drug release rate of echinacoside in E@PLGA-FA was about 22%; at pH = 5.5, the release rate of echinacoside was accelerated, and the drug release rate within 12 hours was about 67% (see Figure 1 e) in.

[0071] Performance Testing

[0072] (1) Effect on cell proliferation

[0073] In the in vitro experiment, the present invention first studied the effect of E@PLGA-FA on the proliferation of HepG2 cells. The specific experimental method is as follows:

[0074] ① Cell culture

[0075] Human HepG2 hepatoma cells (Chinese Academy of Sciences, Shanghai, China) were routinely cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2. Cells were harvested using trypsin and routinely passaged at a ratio of 1:3 when the cell confluence reached approximately 90%. The medium was changed every 2–3 days.

[0076] ②Cellular uptake

[0077] Fluorescence microscopy was used to detect the uptake capacity of HepG2 cells for E@PLGA and E@PLGA-FA. 4HepG2 cells and A549 cells were exposed to 50 g / mL E@PLGA or E@PLGA-FA for 6 hours. E@PLGA or E@PLGA-FA was prepared using a DCM solution with DIR probe added to obtain DIR-labeled nanoparticles. After 6 hours of incubation, the cell nuclei were stained with DAPI (Shanghai Biotech Co., Ltd.) and then observed with a fluorescence microscope (Olympus, Japan). DIR-labeled nanoparticles appeared red and the cell nuclei were stained blue.

[0078] ③Cell viability, cell cycle and apoptosis assay

[0079] The cell proliferation was detected by CCK-8 method. HepG2 cells were cultured at 1×10 4 The cells were inoculated in a 96-well plate at a concentration of 10 cells / well and cultured as usual. The cells were then treated with 0, 0.1, 1, 10, 50 and 100 μg / mL E@PLGA-FA for 72 h, and the cell viability at 0 h, 24 h, 48 h and 72 h after treatment was detected using a CCK-8 detection kit (Shanghai Biotech Biotech) according to the instructions. The absorbance of each well was measured at 450 nm. HepG2 cells were cultured at 3×10 5 The cells were seeded in 12-well plates at a density of 100 μg / well, and then treated with normal control nanoparticles, E@PLGA, and E@PLGA-FA at a dose of 50 μg / mL and maintained at 37°C for 48 h. For cell cycle assay, cells were finally collected by centrifugation at 1000 × g for 5 min. Then they were fixed with 700 μL of pre-cooled anhydrous ethanol, incubated with 1 mg / mL RNase A (100 μL) for 30 min in the dark, and stained with 50 μg / mL propidium iodide (PI, 400 μL) for 10 min. For apoptosis assay, cells treated for 48 h were centrifuged at 1000 × g for 5 min, incubated with 5 μL Annexin V-FITC for 15 min at 4°C, and incubated with 5 μL PI for 5 min. Cell cycle and apoptosis were determined on a flow cytometer (Becton-Dickinson FACSCalibur, San Joes, CA, USA).

[0080] E@PLGA-FA inhibits the proliferation of HepG2 cells Figure 2 shown. Figure 2 ***P<0.001 compared with E@PLGA-FA (0 μg / mL).

[0081] The absorption of E@PLGA and E@PLGA-FA nanoparticles in HepG2 cells is shown in Figure 2. Figure 3 shown. Figure 3The blue color in the figure represents the cell nucleus stained with DAPI, and the red color represents E@PLGA and E@PLGA-FA stained with DIR probe.

[0082] The results showed that after E@PLGA-FA treatment, cell viability decreased in a dose-dependent manner (see Figure 2 ). In addition, at concentrations above 10 μg / mL, E@PLGA-FA significantly inhibited cell proliferation at 24, 48, and 72 h after treatment (see Figure 2 ). In addition, the cellular uptake of E@PLGA and E@PLGA-FA in HepG2 cells showed that both types of nanoparticles were easily captured by HepG2 cells and they were mainly localized in the cytoplasm (see Figure 3 ).

[0083] (2) Effects on cell behavior

[0084] Next, HepG2 cells were treated with 50 μg / mL of nanoparticles and cell behavior was measured. The specific experimental method is as follows:

[0085] ① Colony formation test

[0086] Colony formation test: 1×10 3 HepG2 cells were seeded in 6 cm culture dishes and treated with different nanoparticles (at a dose of 50 μg / mL) for 6 days. At the end of the experiment, the colonies were fixed with paraformaldehyde for 15 minutes and stained with 0.5% crystal violet for 30 minutes. The colonies were then counted.

[0087] ②Cell migration

[0088] Cell migration was assessed using a chamber with an 8 μm pore size filter (Corning, New York, NY, USA). 50% confluent HepG2 cells were cultured in serum-free medium in the upper chamber and treated with different nanoparticles at a dose of 50 μg / mL. The medium containing 10% FBS was transferred to the lower chamber. The cells were incubated at 37°C for 24 hours, and then all non-migrating cells were completely removed. Then, the migrated cells were fixed with 4% paraformaldehyde and then stained with 0.5% crystal violet, developed under a microscope and counted.

[0089] ③Wound healing test

[0090] HepG2 cells (1×10 6) were seeded into 6-well plates and the cells were grown to 100% confluence. The plates were scratched using a P200 pipette tip. Different nanoparticles were then added at a dose of 50 μg / mL and the cells were cultured at 37°C without FBS. Images were collected at 0 h and 48 h under an inverted microscope (Olympus, Japan). Cell migration was analyzed using imageJ software (NIH).

[0091] Effects of E@PLGA-FA nanoparticles on the behavior of HepG2 cells Figure 4 shown. Figure 4 In the figure, (a) is the cell viability of HepG2 cells evaluated by clone formation, (b) is the result of apoptosis measurement, (c) is the result of cell cycle measurement, (d) is the migration result of HepG2 cells exposed to NC-NPs, E@PLGA and E@PLGA-FA, and (e) is the mobility result of HepG2 cells exposed to NC-NPs, E@PLGA and E@PLGA-FA. Figure 4 *p<0.05, **p<0.01, ***p<0.001 compared with control group. #p<0.05, ##p<0.01, ###p<0.001 compared with NC-NPs. ^^p<0.01, ^^^p<0.001 compared with E@PLGA.

[0092] Colony formation assays showed that E@PLGA and E@PLGA-FA treatments significantly reduced the colony formation growth of HepG2 cells compared with NC-NPs (control nanoparticles, i.e., poly(lactic-co-glycolic acid) nanoparticles without echinacea glycoside and folic acid) (see Figure 4 a), and the colony formation growth of HepG2 cells in the E@PLGA-FA group was reduced compared with that in the E@PLGA group. Meanwhile, E@PLGA-FA treatment induced apoptosis of HepG2 cells compared with the E@PLGA group and NC-NPs, but there was no difference between the E@PLGA group and the NC-NPs group (see Figure 4 b). In addition, cell cycle assessment showed that E@PLGA-FA and E@PLGA arrested the cell cycle of HepG2 cells at the G0 / G1 phase, and the proliferation index (PI) of the E@PLGA-FA group: (S+G2M) / (G0 / 1+S+G2M) was lower than that of the E@PLGA group (see Figure 4 c). In addition, the present invention compared the migration of HepG2 cells after exposure to NC-NPs, E@PLGA-FA and E@PLGA. Compared with the normal control, NC-NPs and E@PLGA had no effect on the migration of HepG2 cells, while E@PLGA-FA significantly reduced the migration of HepG2 cells (see Figure 4d in the figure). In addition, the migration rate of HepG2 cells after exposure to NC-NPs, E@PLGA-FA, and E@PLGA was measured using a well-established wound healing assay. After 48 hours of scraping, the wound closure rates of NC-NPs, E@PLGA, and E@PLGA-FA were 86.42%, 66.91%, and 50.44%, respectively, indicating that E@PLGA-FA significantly inhibited the migration ability of HepG2 cells (see Figure 4 e) in.

[0093] (3) Animal experiments

[0094] To verify the anticancer effect of E@PLGA-FA, a subcutaneous liver cancer tumor model was established by injecting HepG2 cells into the left posterior abdomen. After 12 days, the nanoparticles were administered to the mice by subcutaneous injection. The specific method is as follows:

[0095] Twenty-four male Balb / c mice (8 weeks old) were obtained from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. (certificate number: 20240428Abba0105000597, license number: SCXK (Zhejiang) 2019-0004) and were housed under specific pathogen-free conditions with free access to food and water. This study was approved by the Animal Ethics Committee of Shanghai Shushu Biotechnology Co., Ltd. Neuroblastoma HepG2 cells (1×10 6 ). Twelve days after subcutaneous injection, the nanoparticles (normal control nanoparticles, E@PLGA-FA and E@PLGA, respectively. 20 mg / kg, twice a week) were injected into mice for 3 weeks, and the mouse specimens (six mice per group) were killed. During this period, the growth curve of the subcutaneous tumor was recorded. Then, the collected tumor tissues were fixed with 10% formalin and embedded in paraffin. Histological examination was performed by staining the tissue with hematoxylin and eosin. Tumor cell death was assessed using TUNEL labeling according to established procedures.

[0096] The antitumor effect of E@PLGA-FA in liver cancer model mice carrying HepG2 cells Figure 5 shown. Figure 5 In the figure, (a) is an in vivo tumor image, (b) is an in vitro tumor image, (c) is the tumor weight at the end of the animal experiment, (d) is the tumor growth curve, and (e) is a representative in vivo fluorescence image at 0, 2, and 4 hours after subcutaneous injection of E@PLGA and E@PLGA-FA. (f) is a HE-stained section image obtained from the tumor tissue, (g) is an image of the tumor tissue evaluated by TUNEL staining, and (h) is the result of cell apoptosis in the tumor tissue evaluated by TUNEL staining. Figure 5#p<0.05, ##p<0.01, ###p<0.001 compared with NC-NPs. ^p<0.05, ^^^p<0.001 compared with E@PLGA.

[0097] The results showed that both E@PLGA and E@PLGA-FA inhibited tumor growth, and E@PLGA-FA was more effective than E@PLGA (see Figure 5 a~d in). In addition, Figure 5 Figure e shows the bioluminescence of HepG2 cells and the fluorescence (red) signals of E@PLGA and E@PLGA-FA at the indicated times, clearly indicating that E@PLGA-FA can target HepG2 cells 2 and 4 h after subcutaneous injection. Figure 5 HE staining in the figure f showed that there was no obvious pathological change in the tumor tissue of the control group and NC-NPs group, while the tumor tissue of the E@PLGA-FA group showed obvious structural destruction and necrosis. In addition, the TUNEL test results also showed that both E@PLGA and E@PLGA-FA could cause tumor cell death, and the effect of E@PLGA-FA was better than that of E@PLGA.

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing poly(lactic acid-co-glycolic acid) nanoparticles functionalized with echinacoside and folic acid, characterized in that: The following steps are involved: The aqueous phase solution of echinacea glycoside, polylactic acid-glycolic acid and an organic solvent are mixed and subjected to a first ultrasonic treatment to obtain a primary emulsion; The primary emulsion is mixed with a sodium cholate solution and subjected to a second ultrasonic treatment to obtain a secondary emulsion; removing the organic solvent in the secondary emulsion, and obtaining the polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside after dialysis; The polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside are mixed with a DSPE-mPEG5000-folic acid solution and functionalized with folic acid to obtain polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside and folic acid.

2. The preparation method according to claim 1, characterized in that: The mass ratio of echinacoside to polylactic acid-glycolic acid is 1:1-3; The mass concentration of the aqueous solution of echinacoside is 10-20 mg / mL.

3. The preparation method according to claim 1, characterized in that: The mass ratio of echinacoside to sodium cholate is 1:30-50; The mass concentration of the sodium cholate solution is 5-8%.

4. The preparation method according to claim 1 or 2, characterized in that: The power of the first ultrasonic treatment and the second ultrasonic treatment are independently 37.5-50W, and the time is independently 2-3min.

5. The preparation method according to claim 1, characterized in that: The molecular weight cut-off of the dialysis is 12000Da, and the dialysis time is 22 to 24 hours.

6. The preparation method according to claim 1, characterized in that: The mass concentration of the DSPE-mPEG5000-folic acid solution is 1-2 mg / mL; The folic acid functionalization is carried out under stirring conditions, and the folic acid functionalization time is 10 to 12 hours.

7. The poly(lactic acid-co-glycolic acid) nanoparticles functionalized with echinacoside and folic acid prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The invention comprises polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside and folic acid modified on the surface of the polylactic acid-co-glycolic acid nanoparticles loaded with echinacoside; The polylactic acid-glycolic acid copolymer nanoparticles carrying echinacoside comprise echinacoside and the polylactic acid-glycolic acid copolymer encapsulating the echinacoside.

8. The poly(lactic acid-co-glycolic acid) nanoparticles functionalized with echinacoside and folic acid according to claim 7, characterized in that: The particle size of the echinacoside- and folic acid-functionalized polylactic acid-glycolic acid copolymer nanoparticles is 90-100 nm.

9. The poly(lactic acid-co-glycolic acid) nanoparticles functionalized with echinacoside and folic acid according to claim 7, characterized in that: The drug loading amount of the echinacoside- and folic acid-functionalized polylactic acid-glycolic acid copolymer nanoparticles is 12.05-12.3%.

10. Use of the poly(lactic acid-co-glycolic acid) nanoparticles functionalized with echinacoside and folic acid as claimed in any one of claims 7 to 9 in the preparation of anti-liver cancer drugs.

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