CN113893229B

CN113893229B - Bionic nanoparticle with gambogic acid wrapped by erythrocyte membrane, preparation method and application thereof

Info

Publication number: CN113893229B
Application number: CN202110905801.2A
Authority: CN
Inventors: 白兰; 师健友; 童荣生
Original assignee: Sichuan Peoples Hospital of Sichuan Academy of Medical Sciences
Current assignee: Sichuan Peoples Hospital of Sichuan Academy of Medical Sciences
Priority date: 2021-08-06
Filing date: 2021-08-06
Publication date: 2023-09-05
Anticipated expiration: 2041-08-06
Also published as: CN113893229A

Abstract

The invention discloses bionic nano particles of gambogic acid coated by erythrocyte membranes, a preparation method and application thereof, and relates to the technical field of nano particle preparation. The environment-friendly red blood cell comprises an inner core and an outer shell, wherein the outer shell is coated on the periphery of the inner core, the inner core comprises gamboge acid and an encapsulation component, and the outer shell comprises an erythrocyte membrane; the entrapping component is selected from amorphous block polymers. The drug delivery system combining RBC membrane (bionic erythrocyte membrane) and polymer is adopted, so that the purposes of increasing the aqueous solubility of gambogic acid and prolonging the half life of gambogic acid are achieved, and the safety of GA is improved.

Description

Bionic nanoparticle with gambogic acid wrapped by erythrocyte membrane, preparation method and application thereof

Technical Field

The invention relates to the technical field of nanoparticle preparation, in particular to bionic nanoparticles of gambogic acid wrapped by erythrocyte membranes, a preparation method and application thereof.

Background

At present, the number of new liver cancer diseases is increased year by year, and the data of the international cancer research institution indicate that the number of new liver cancer cases in China reaches 41 ten thousand in 2020. The major subtype of liver cancer is hepatocellular carcinoma (hepatocellular carcinoma, HCC) with a relatively high incidence, which is the fourth leading cause of cancer death in humans. For the treatment of liver cancer, surgical treatment and local area strategy are still main means for maximizing survival rate of early and medium stage liver cancer patients. Whereas systemic treatment is the only option for patients with advanced HCC. Clinically, the selection of anti-liver cancer drugs is less, the first-line drugs are only sorafenib and lenvatinib, and the second-line drugs are Regorafenib, cabozantinib and ramucirumab. The existing anti-advanced liver cancer drugs have obvious toxic and side effects, and the survival time of patients is prolonged to be short, for example, patients taking sorafenib have various adverse reactions such as diarrhea, skin diseases, anorexia, alopecia and the like, and the average survival time can only be prolonged to be less than 3 months. Therefore, development of a new drug against advanced liver cancer is urgent.

Gamboge is a gum-like resin of gamboge (Garcinia hamburgy hook. F.) of the family gambogaceae. In 1973, gamboge was prepared as an injection for anti-tumor clinical study. Gambogic Acid (GA) is one of the main active substances in gamboge, and GA has been found to have activity against transplanted tumors such as liver cancer. However, the solubility in GA water is only about 10. Mu.g/mL; the elimination half-life in Beagle canine plasma was about 60min. The two major drawbacks of poor water solubility and short half-life of GA have greatly limited its clinical application.

The erythrocyte membrane has the advantages of good stability, long circulation in vivo, escape immunity and the like for the wrapped medicine by the erythrocyte membrane by virtue of the natural advantages, and plays an important role in the delivery of the medicine.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide bionic nano particles of gambogic acid wrapped by erythrocyte membranes, a preparation method and application thereof, which are used for solving the problems of short half-life and poor water solubility of Gambogic Acid (GA) and enriching the selectivity of antitumor drugs.

The invention adopts a drug delivery system combining RBC membrane (bionic erythrocyte membrane) and polymer, thereby achieving the purposes of increasing the aqueous solubility of gambogic acid and prolonging the half-life period of gambogic acid, and improving the safety of GA.

The invention is realized in the following way:

the invention provides bionic nano particles with gambogic acid wrapped by erythrocyte membranes, which comprise an inner core and an outer shell, wherein the outer shell is wrapped on the periphery of the inner core, the inner core comprises gambogic acid and a wrapping component, and the outer shell comprises erythrocyte membranes; the entrapping component is selected from amorphous block polymers.

The inventor achieves the purposes of enhancing the water solubility of GA, prolonging the circulation time of GA and improving the safety of GA by preparing a bionic Red Blood Cell (RBC) membrane coated GA nano drug delivery system (RBC@GPP-NPs).

Gamboge is a gelatinous resin of gamboge (Garcinia hamburgy hook. F.) of the family gambogaceae, and is mainly produced in tropical regions such as vietnam. Gamboge is used as pigment, and is also a traditional Chinese medicinal material. Various effects of gamboges are recorded in the medical works of China: 1) Detoxify and kill parasites. Tang Dynasty Li, written in the "herbal medicine for sea: "treat dental caries and fall immediately after the point". Qing dynasty Wang Zi connects "Deyi Bencao" in a book: san Huang Bao wax pill and Li Dongwan are good at detoxication. 2) Treating stubborn dermatitis. Write in the schema for Chan Yi of Qing Dynasty Zhao Xuemin: wuhuang san (containing gamboge) is used for treating chicken feet. 3) Traumatic injury. The "surgical complete tissue collection" of Qing dynasty Wang Hongxu describes: the medicines such as gamboge and bezoar are prepared into pills, and can be taken orally or applied externally for treating traumatic injury and symptoms of severe swelling and toxicity. 4) Diminishing inflammation. Gamboge tincture and gamboge kuh-seng wine are used for local inflammation, such as folliculitis, etc. In abroad, tabasheer is used for treating the rise of blood pressure in edema and cerebral hemorrhage. With the development of traditional Chinese medicine chemistry, single chemical components in gamboge, such as Gambogic Acid (GA), are extracted, and the chemical structure is shown as follows. Since then, anti-tumor studies on active ingredients in gamboge have been started, and GA and neogambogic acid are still more studied at present.

GA is one of main active substances in gamboge, has been found to have activity against transplanted tumors such as liver cancer, and has been found through pharmaceutical test research, compared with common antitumor drugs, GA has good antitumor effect and good safety. In small dosage, GA has strong killing power to tumor cells, but has no obvious influence on the hematopoietic system and immune function of animals. The inventor selects GA as a drug carrier to realize the killing of tumor cells by utilizing the anti-tumor activity of GA.

The bionic cell membrane has good biocompatibility, long circulation effect and the like, and the nanoparticle wrapped by the bionic cell membrane has the advantages of 1) inherent biocompatibility and biodegradability; 2) Avoiding some of the inherent toxicity of nano-preparation; 3) The stability is improved, the in vitro storage time is prolonged, and aggregation is prevented; 4) Because of the large number of cell membranes, high load capacity is easily achieved. Among these biological membranes, red Blood Cell (RBC) membranes have the advantages of abundant sources, simple extraction, escape of immune system and life span up to 120 days, and can realize long-term circulation of nanoparticles in vivo.

The inventors have found that the long circulation and controlled release effects can be achieved by combining the advantages of RBC membranes with the advantages of nanomaterials.

In a preferred embodiment of the present invention, the amorphous block polymer is polyethylene glycol-polylactic acid.

Polymeric polylactic acid (PLA) is approved for clinical use by the U.S. food and drug administration (Food and Drug Administration, FDA) and has been successfully used in medicine as a suture material. In vivo, PLA can be degraded by simple hydrolysis of the esterified backbone, and the degradation product lactic acid can be metabolized and excreted without residue in vivo. PLA is typically linked to polyethylene glycol (Polyethylene glycol, PEG) when used as a micelle carrier material. PEG is also an FDA approved material for internal consumption, has good water solubility, is nontoxic, and can be rapidly removed from the body. PEG-PLA is an amorphous block polymer, has higher hydrophilicity than PLA and higher drug loading rate. In mouse experiments, it was found that the PEG-PLA coated vaccine nanoparticles induced a significantly enhanced immune response compared to PLA coated vaccine nanoparticles.

The polyethylene glycol-polylactic acid is used as a drug carrier material in the nanoparticles, can wrap the drug, can prolong the blood circulation time of the drug in vivo and improve the bioavailability of the drug. The structural formula of the polyethylene glycol-polylactic acid is as follows:

In one embodiment, the polyethylene glycol in the polyethylene glycol-polylactic acid has a molecular weight of 2000-5000 and the polylactic acid has a molecular weight of 2000-3000.

In one embodiment, the molecular weight of polyethylene glycol in polyethylene glycol-polylactic acid is 3400, and the molecular weight of polylactic acid is 2000, and in other embodiments, the molecular weight of the polymer may be adaptively adjusted as needed, and is not limited to the molecular weight listed above.

In a preferred embodiment of the present invention, the mass ratio of gambogic acid to entrapped components in the core is 1:2-20. Preferably 1:10-15; for example, it may be 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15.

The inventors found that following PEG ₃₄₀₀ -PLA ₂₀₀₀ Increasing the ratio to GA, decreasing the particle size, increasing the EE, decreasing the EE, and increasing the polydispersity index (polydispersity index, PDI), decreasing the EE. At 10:1, particle size is minimum, EE is maximum, PDI is small, PEG ₃₄₀₀ -PLA ₂₀₀₀ The optimal dosage ratio of the catalyst to GA is 10:1.

EE refers to the encapsulation efficiency of the core, and refers to the mass of gambogic acid relative to the mass percent of gambogic acid in the core prior to centrifugation.

In a preferred embodiment of the present invention, the volume ratio of the inner core to the outer shell is 1-1.2:1. Optionally, the mass ratio of the inner core to the outer shell is 1:1, 1.1:1 or 1.2:1.

The encapsulation efficiency of the inner core is 86.37+/-0.84%, and the encapsulation efficiency of the bionic nano-particles is 79.11+/-1.42%; preferably, the drug loading of the core is 3.76±0.07%.

In a preferred embodiment of the present invention, the average particle size of the core is 80-96nm;

preferably, the average particle size of the biomimetic nanoparticles is 98.48.+ -. 0.72nm.

The invention provides a preparation method of bionic nano particles of gambogic acid wrapped by erythrocyte membranes, which comprises the following steps: the inner core is prepared by adopting a film dispersion method, and then the inner core is mixed with erythrocyte membranes and is extruded together.

In a preferred embodiment of the present invention, the method for preparing the inner core by using the film dispersing method refers to: mixing gambogic acid, amorphous block polymer and organic solvent, removing the organic solvent to obtain medicinal film, adding physiological saline into the medicinal film, and performing ultrasonic treatment.

The inventors found that the average particle diameter of the inner core was related to the power of the ultrasonic treatment, the ultrasonic time, the amount of physiological saline, the amount of the organic solvent, and the amount of polyethylene glycol-polylactic acid.

Preferably, the ratio of the organic solvent to the amorphous block polymer is from 0.15mL to 0.25mL to 1mg. The ratio of organic solvent to amorphous block polymer added was 0.15mL:1mg and 0.25mL: at 1mg, the particle size and EE did not change much. The polydispersity index remains substantially unchanged.

Preferably, the ratio of the physiological saline to the addition amount of the amorphous block polymer is 0.12mL-0.20mL:1mg; preferably 0.15-0.17 mL/1 mg. When the ratio of the amount of physiological saline to the addition amount of the amorphous block polymer is 0.15-0.17mL:1mg, the particle size is minimum and EE is large. The organic solvents include, but are not limited to, methylene chloride, chloroform, acetone or methanol, as long as the dissolution of the drug and polymer is satisfied.

Preferably, the ultrasound is performed under ice bath conditions; preferably, the ultrasonic power is 10-15%; the ultrasonic time is 5-20min, preferably 10min. When the ultrasonic power of the probe reaches 10%, the particle size of the prepared kernel is minimum, and EE is large.

Preferably, the ultrasonic treatment further comprises a centrifugation and filtration step; the free GA was removed by centrifugation and filtration steps to give the core (GPP-NPs).

The ultrasonic treatment time has little influence on the particle size and PDI of GPP-NPs, and EE change is small when the particle size and PDI are more than or equal to 10min.

The rotational speed of the centrifugation is 3000-10000rpm/min, preferably 7000rpm/min.

The encapsulation efficiency of the obtained GPP-NPs is higher when the rotating speed is 7000rpm/min.

In a preferred embodiment of the present invention, the steps of diluting and crushing the erythrocyte membrane before mixing the erythrocyte membrane with the inner core, and then extruding;

Preferably, the dilution is 4-5 times diluted with isotonic physiological saline. The inventors found that RBC membranes failed to pass through 0.4 μm polycarbonate membrane (PC) membrane when they were diluted with 1, 2 and 3 times physiological saline, respectively, but that RBC membranes were very easy to pass through 0.4 μm PC membrane and were able to pass through 0.2 μm PC membrane when they were increased 4 times. Because the lower the amount of physiological saline is, the lower the dilution factor of the final drug is, the dilution factor of physiological saline should be controlled as much as possible.

The crushing is carried out by adopting an ultrasonic crushing method, wherein the crushing time of the ultrasonic crushing method is 2-3min. The inventors found that the average particle size of the erythrocyte membrane is less concentrated in particle size distribution when the crushing time is 5 and 7 min. The average particle size of RBC membrane vesicles (RVs) obtained by crushing for 3min is about 158.3nm, the particle size is more than 50nm, and the size distribution is good.

Preferably, extrusion refers to extrusion of the disrupted red blood cell membrane with a polycarbonate membrane to obtain RBC membrane vesicles.

In one embodiment, RBCs are extruded sequentially with extruders equipped with 0.4. Mu.M and 0.2. Mu.M PC films, with repeated extrusion at least 10 times per particle size.

In a preferred embodiment of the invention, after the inner core is mixed with the erythrocyte membrane, ultrasound is also included, and the bionic nano particles are obtained by extruding a polycarbonate membrane;

Preferably, the ultrasonic treatment time is 10-12min, the treatment frequency is 35-40Hz, and the treatment power is 100-120W;

the pore size of the polycarbonate membrane was 0.2. Mu.m.

The invention also provides application of the bionic nano particles of the gambogic acid wrapped by the erythrocyte membrane or the bionic nano particles prepared by the preparation method in preparing tumor therapeutic drugs;

preferably, the tumor is liver cancer, lung cancer, stomach cancer, breast cancer or colon cancer.

The invention provides an anti-tumor drug, which comprises bionic nano particles of gambogic acid wrapped by erythrocyte membranes;

preferably, the antineoplastic agent is an anti-liver cancer agent.

The invention has the following beneficial effects:

aiming at the defects of poor GA water solubility, short half-life and the like, the invention adopts a drug delivery system combining erythrocyte membranes and polymers to achieve the purposes of increasing GA water solubility and prolonging GA half-life. Experiments prove that the bionic nano particles can inhibit the activity of liver cancer cells in vitro, and have good stability, longer in-vitro release time, stronger anti-liver cancer activity and higher safety. The invention provides better medicine selection for the anti-tumor medicine.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and that other related drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram of the design concept of the present invention;

FIG. 2 shows the purified gambogic acid;

FIG. 3 is a 1H NMR spectrum of GA;

FIG. 4 is a 13C NMR spectrum of GA;

FIG. 5 is a response surface diagram;

FIG. 6 is a graph showing particle size distribution of red blood cell membrane vesicles at different disruption times;

FIG. 7 is a particle size distribution of GPP-NPs (a) and RBC@GPP-NPs (b);

FIG. 8 is a TEM image of GPP-NPs and RBC@GPP-NPs;

FIG. 9 is the stability of GPP-NPs and RBC@GPP-NPs;

FIG. 10 is an in vitro release profile of GPP-NPs and RBC@GPP-NPs;

FIG. 11 is a graph showing the proliferation results of HepG2 cells;

FIG. 12 is a statistical plot of HepG2 cell viability after 24h (a) and 48h (b) of treatment with different concentrations of GA, GPP-NPs and RBC@GPP-NPs;

FIG. 13 is a graph showing comparison of hemolysis ratio of GA, GPP-NPs, RBC@GPP-NPs;

FIG. 14 is a graph showing the results of hemolysis tests of GA, GPP-NPs and RBC@GPP-NPs;

FIG. 15 is a graph showing the effect of GA, GPP-NPs and RBC@GPP-NPs on LO2 cell viability.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

Referring to fig. 1, in this embodiment, the preparation of the biomimetic nanoparticles is performed according to the design concept of fig. 1. In this example, GA was extracted from gamboge by itself, and in other embodiments, GA may be selected from commercially available GA.

1. GA was extracted from gamboge and purified.

The equipment and reagents used are shown in the following table:

(1) Extracting: grinding resina Garciniae into powder, and sieving with No. 2 sieve. Weighing 200g of gamboge powder and 300g of diatomite, and uniformly stirring in a beaker. The pre-dried column was taken, the mixture was packed into the column and washed with ethyl acetate until the effluent was nearly colorless. The liquid was removed by rotary evaporation at 40 ℃ to give a percolate.

(2) Purification

Pure water is prepared in advance and heated to 40 ℃ for standby. The percolate was dissolved with pyridine in a 1:1 (mL: g) ratio and stirred well to give GA as a pyridinium salt. 100mL of pure water is added, when part of substances begin to precipitate, 200mL of petroleum ether is added, the mixture is cooled, yellow substances precipitate and are distributed in the middle layer, after filtration, the solid is washed by a small amount of methanol, and the crude product GA pyridine salt is obtained by vacuum drying at room temperature.

Adding 40 ℃ methanol into the crude GA pyridinium until the crude GA pyridinium is just completely dissolved, cooling until a large amount of crystals are separated out, filtering, and repeating the above operation for a plurality of times to obtain GA pyridinium crystals.

GA pyridine salt is dissolved in a 2N hydrochloric acid-diethyl ether mixture (4:5, v/v), stirred for 30min, and the mixture is left to stand, the upper layer solution is taken and washed 2 times with saturated sodium chloride solution, water-soluble impurities are removed, anhydrous sodium sulfate is added to remove water in the diethyl ether layer, filtration is performed, and the filtrate is evaporated under reduced pressure at 35 ℃ to obtain a solid substance (see FIG. 2).

10mg of GA and 0.6mL of deuterated chloroform are weighed, dissolved and then are filled into a nuclear magnetic tube, and nuclear magnetic resonance detection is carried out by using a nuclear magnetic resonance spectrometer ¹ H and ¹³ C-NMR)。

extracts of the plant ¹ The H NMR chart is shown in FIG. 3. ¹ H NMR data: (400 MHz, CDCl) ₃ )δ12.75(s,1H),7.54(d,J＝6.9Hz,1H, 10-H),6.57(d,J＝10.1Hz,1H,4-H),6.11(td,J＝7.4,1.6Hz,1H,27-H),5.36(d,J＝10.2Hz,1H,3-H), 5.04(qt,J＝6.9,3.7Hz,2H,32-H,37-H),3.48(s,1H,11-H),3.29(dd,J＝14.6,8.0Hz,1H,31-H),3.13 (dd,J＝14.7,5.2Hz,1H,31-H),3.01-2.93(m,2H,26-H),2.51(d,J＝9.3Hz,1H,21-H),2.31(dd,J＝ 13.4,4.8Hz,1H),2.01(q,J＝8.1Hz,2H),1.78–1.52(m,20H),1.46–1.18(m,8H)。 ¹ The H NMR data are consistent with those of GA in the literature.

Extracts of the plant ¹³ C NMR chart is shown in FIG. 4. ¹³ The C NMR data were: (76 MHz, chloroform-d) delta 203.35 (12-C), 178.85 (8-C), 171.37 (30-C), 161.46 (18-C), 157.53 (6-C), 157.34 (16-C), 138.21 (27-C), 135.29 (10-C), 133.35 (9-C), 131.76 (38-C), 131.47 (33-C), 127.61 (28-C), 124.44 (3-C), 123.86, (37-C) 123.83 (32-C), 115.87 (4-C), 107.57 (17-C), 102.71 (5-C), 100.42 (7-C), 90.93 (14-C), 83.86 (23-C), 83.80 (13-C), 81.27 (2-C), 49.00 (22-C), 46.80 (11-C), 41.97 (20-C), 29.87 (25-C), 29.71 (26-C), 29.27 (24-C), 28.86 (19-C), 39-25-C), 35.27 (35-C), 35.31-C, 35.27 (21-C), 35.31-C (21-35-40-C), and (21.31-40-C. ¹³ C NMR data are consistent with GA in literature by ¹ H NMR ¹³ C NMR data analysis results confirm that the material is GA.

(3) Precisely weighing GA 10mg,3 parts, preparing a solution with a certain concentration, precisely weighing 10.72mg of GA reference substance, dissolving in methanol, and fixing the volume to 50mL to obtain 214.4 mug.mL ^-1 The control is stored in solution. The detection wavelength of GA by high performance liquid chromatography (High performance liquid chromatography, HPLC) was determined by full wavelength scanning. And (3) taking a certain amount of GA reference substance solution, diluting methanol to a proper concentration, performing full-wavelength scanning in a wavelength range of 190-900 nm, and finally selecting 360nm as the detection wavelength of HPLC.

Instrument: waters e2695 high performance liquid chromatograph (Waters 2998 ultraviolet detector)

Chromatographic column: diamond C18 (250 mm. Times.4.6 mm,5 μm)

Detection wavelength: 360nm of

Column temperature: 25 DEG C

Mobile phase: methanol-0.1% acetic acid aqueous solution (93:7)

Flow rate: 1.0 mL/min ^-1

Sample injection amount: 20 mu L

Precisely sucking 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0mL of GA reference stock solution, respectively fixing volume to 10mL with methanol to obtain serial reference solutions, measuring according to the method under 2.4.1, and linearly regressing GA mass concentration (C) with peak area integral value (A) to obtain regression equation A=25048C-28165 (R) ² =0.9994). The results show that 21.44-85.76 mu g.mL ^-1 The linearity of GA is good in the range.

The GA content was calculated to give an average GA content of 99.01% in 3 groups of samples (see Table below).

The extraction process is adopted to extract 10.23g of GA from 200g of gamboge, and the yield is 5.12%. The purity was 99.01% by HPLC.

2. Preparing the bionic nanoparticle of gambogic acid coated by erythrocyte membrane.

The kit materials used are shown in the following table:

reagent(s)

Material for container

Kunming mice (18-22 g), available from Du Da laboratory animals Co.

2.1 preparation of Kernel (GPP-NPs)

GPP-NPs were prepared using thin film dispersion. Precisely weighing a certain amount of GA and PEG ₃₄₀₀ -PLA ₂₀₀₀ Adding dichloromethane, stirring at room temperature for dissolution, and removing dichloromethane by rotary evaporation to obtain a mixed medicinal film. Adding a certain amount of physiological saline into the medicinal membrane for ultrasonic dissolution, performing ultrasonic treatment with a probe under ice bath, centrifuging at low temperature, filtering with a 0.22 μm microporous filter membrane, and removing free GA to obtain GPP-NPs. Under the condition of no GA, the blank nanoparticle 1 (PP-NPs) is prepared by adopting the same method.

2.2 The determination of the GPP-NPs encapsulation efficiency was performed as follows:

taking 0.2mL of GPP-NPs solution before centrifugation, fixing the volume of methanol to 10mL, measuring the GA content by HPLC, and calculating the GA mass as W _{Total (S)} . Collecting filtered GPP-NPs 0.2mL, processing by the same method, measuring GA content, and calculating GA mass as W _Package 。

Each group was run 3 times in parallel. EE is calculated as follows.

W _Package Quality of GA in 0.2mL GPP-NPs; w (W) _{Total (S)} : mass of GA in GPP-NPs before centrifugation of 0.2 mL.

2.3 Determination of GPP-NPs particle size

The particle size and polydispersity index (polydispersity index, PDI) of GPP-NPs were measured using a particle size meter. GPP-NPs were diluted 5-fold with physiological saline to avoid the generation of bubbles, 1mL of diluted liquid was taken in a disposable sample cell, the instrument temperature was set to 25℃and the measurement was performed using the default parameters of the instrument, 3 samples were prepared in parallel for each sample, and the average value was calculated.

2.4 GPP-NPs prescription order factor investigation

2.4.1 PEG ₃₄₀₀ -PLA ₂₀₀₀ Inspection of the amount of usage

PEG ₃₄₀₀ -PLA ₂₀₀₀ The ratio of the mixture to GA is 2, 5, 10, 15 and 20 (mg: mg), GPP-NPs are prepared according to the method of 2.1, and particle size, PDI and EE are used as evaluation indexes to examine different PEG ₃₄₀₀ -PLA ₂₀₀₀ Effect of usage on GPP-NPs.

PEG ₃₄₀₀ -PLA ₂₀₀₀ The results of the amount investigation are shown in Table 1. The results show that following PEG ₃₄₀₀ -PLA ₂₀₀₀ Increasing the ratio to GA, decreasing the particle size first and then increasing, increasing EE first and then decreasing and then increasing, and decreasing PDI first and then increasing. At 10:1, particle size is smallest, EE is largest, and PDI is smaller, therefore PEG ₃₄₀₀ -PLA ₂₀₀₀ The optimal dosage ratio of the catalyst to GA is 10:1.

TABLE 1 PEG ₃₄₀₀ -PLA ₂₀₀₀ Effect of the amount of use on nanoparticle size, PDI and EE

2.4.2 investigation of the dichloromethane dosage.

Dichloromethane (mL) with PEG ₃₄₀₀ -PLA ₂₀₀₀ The ratio of the usage amounts is 0.1,0.15,0.2 and 0.25 (mL: mg), GPP-NPs are prepared according to the method under item 2.1, and the effects of the usage amount of methylene dichloride on GPP-NPs are examined by taking particle size, PDI and EE as evaluation indexes.

The results of the dichloromethane dosage investigation are shown in Table 2. The results showed that dichloromethane and PEG ₃₄₀₀ -PLA ₂₀₀₀ The ratio of 0.15 to 0.25 (mL: mg) showed little change in particle size and EE, so methylene chloride and PEG were selected ₃₄₀₀ -PLA ₂₀₀₀ Is the most significant of (3)The preferred dosage ratio is 0.15:1 (mL: mg).

Table 2 effect of dichloromethane usage on GPP-NPs particle size, PDI and EE.

2.4.3 investigation of the amount of physiological saline.

Physiological saline dosage and PEG ₃₄₀₀ -PLA ₂₀₀₀ According to the ratio 0.05,0.1,0.15,0.2,0.25 (mL: mg), GPP-NPs were prepared according to the method of 2.1, and the influence of the amount of physiological saline on GPP-NPs was examined by taking particle size, PDI and EE as evaluation indexes.

The results of the physiological saline dose investigation are shown in Table 3. The results show that the physiological saline dosage and PEG ₃₄₀₀ -PLA ₂₀₀₀ When the ratio is 0.15 (mL: mg), the particle size is the smallest and EE is large, so physiological saline and PEG are selected ₃₄₀₀ -PLA ₂₀₀₀ The optimal dosage ratio of (C) is 0.15:1 (mL: mg).

TABLE 3 Effect of physiological saline usage on GPP-NPs particle size, PDI and EE

2.4.4 probe ultrasound power investigation.

The ultrasonic power of the probe is 5, 10, 15, 20 and 25 percent respectively, GPP-NPs are prepared according to the method under the item 2.1, and the particle size, PDI and EE are used as evaluation indexes to examine the influence of the ultrasonic power of the probe on the GPP-NPs.

The ultrasonic power inspection results of the probe are shown in table 4. The results show that when the ultrasonic power of the probe reaches 10%, the particle size is minimum, and EE is larger. Thus, the optimal probe ultrasound power is 10%.

TABLE 4 influence of probe ultrasonic Power on GPP-NPs particle size, PDI and EE

2.4.5 probe ultrasound time investigation.

The ultrasonic time of the probe is 5, 10, 15, 20 and 25min respectively, GPP-NPs are prepared according to the method under the 2.1 item, particle size, PDI and EE are used as evaluation indexes, and the influence of the ultrasonic time of the probe on the GPP-NPs is examined.

The ultrasonic time inspection results of the probe are shown in table 5. The results show that the ultrasonic time of the probe has little influence on the particle size and PDI of GPP-NPs, and EE change is small when the ultrasonic time of the probe is more than or equal to 10 minutes. Therefore, the probe ultrasound time was chosen to be 10min.

TABLE 5 influence of probe ultrasound time on GPP-NPs particle size, PDI and EE

2.4.6 centrifugal revolutions.

12 parts of 1mL RBC@GPP-NPs were taken at 1.5 The mL EP tube was centrifuged for 5min at 3000, 5000, 7000 and 10000rpm/min respectively, and filtered through a 0.22. Mu.M microporous membrane, and the GA content was measured by HPLC to calculate the EE condition.

The results of the centrifugal revolution investigation are shown in Table 6. As can be seen from the experimental results, the above 4 groups can reach higher EE, and the encapsulation efficiency of GPP-NPs obtained in the 3000rpm/min group is maximum, but the loss thereof in the filtration process is large, so that a group with the rotation speed of 7000rpm/min is selected. The group of EEs with the rotation speed of 5000rpm/min is slightly smaller, probably because part of substances with poor morphology block the filter holes, so that part of nano particles slightly smaller than the filter holes cannot penetrate the filter holes and are throttled on the filter. Whereas a group with a rotational speed of 10000rpm/min may sink a small amount of GPP-NPs due to the large centrifugal force. During the filtration process, it is notable that, because the filter membrane area of the 0.22 μm filter head is small, the filter head needs to be replaced multiple times during the operation process, if the same filter head is used multiple times, part of substances may block the filter holes, so that part of nano particles are trapped in the filter head, thereby reducing EE.

TABLE 6 influence of centrifugal rotational speed on EE of GPP-NPs

2.5 prescription process optimization.

2.5.1 star point design.

According to the single factor investigation result, PEG which significantly influences GPP-NPs particle size and EE ₃₄₀₀ -PLA ₂₀₀₀ Ratio of GA to dosage (A), physiological saline to PEG ₃₄₀₀ -PLA ₂₀₀₀ The dosage ratio (B) and the ultrasonic power (C) of the probe are independent variables, and each factor 3 level is respectively-1, 0 and 1 for coding. Particle size and EE are respectively Y ₁ And Y ₂ The composite score is expressed as OD value as a final evaluation index. The star point design factor level codes are shown with reference to table 7.

TABLE 7 Star Point design factor level encoding

The Hassan method is adopted to divide the Y pairs first ₁ 、Y ₂ Standardized, Y ₁ The factor of smaller and better value is calculated as d _min Value [ d ] ₁ ＝ (Y _max －Y _i )/(Y _max －Y _min )]，Y ₂ The factor of the higher the value, the better, i.e. calculate d _max Value [ d ] ₂ ＝(Y _i －Y _min )/(Y _max －Y _min )]Calculate OD value [ od= (d) ₁ *d ₂ ) ^1/2 ]. The particle size, EE and OD values are correspondingly changed under different process conditions by utilizing the Box-Behnken center combination design optimization according to the OD value, and the response surface diagram is shown in a table 8 and a graph in a figure 5.

TABLE 8 Box-Behnken test design scheme and response values

According to the star Design test result, model fitting is carried out on the result by adopting Design-experered 8.0.6, so that a fitting equation is OD=0.95-0.034+0.18+B-0.090+0.025+A+B+0.058+A-0.11+B+C-0.40+A ² -0.23*B ² -0.35*C ² ， R ² ＝0.9701，P<0.05, with good correlation. As can be seen from the analysis of variance data in table 9, B, A in the fitted equation ² 、B ² 、C ² The P values of the terms are all less than 0.05, indicating PEG ₃₄₀₀ -PLA ₂₀₀₀ Ratio to GA dosage, physiological saline and PEG ₃₄₀₀ -PLA ₂₀₀₀ The dosage ratio and the ultrasonic power of the probe have larger influence on the particle size and EE.

TABLE 9 analysis of variance

The results in the star point Design test are processed and optimized by adopting Design-Expert V8.0.6 software, and the simulated optimal process is as follows: PEG (polyethylene glycol) ₃₄₀₀ -PLA ₂₀₀₀ Ratio of GA to physiological saline to PEG ₃₄₀₀ -PLA ₂₀₀₀ The dosage ratio is 9.75:1 (mg: mg) and 0.17:1 (mL: mg); the ultrasonic power of the probe is 10.12%. The particle size of the optimum recipe calculated by fitting the equation was 85.81nm and EE was 87.35%. Rounding, correcting the optimal prescription process, and PEG ₃₄₀₀ -PLA ₂₀₀₀ Ratio to GA dosage, physiological saline and PEG ₃₄₀₀ -PLA ₂₀₀₀ The dosage ratio is respectively 10:1 (mg: mg) and 1.7:10 (mL: mg); the ultrasonic power of the probe is 10%.

2.5.2 verification test.

According to the method of item 2.1, 3 batches of GPP-NPs are prepared by adopting the optimal prescription obtained by simulation, the particle size and EE of the GPP-NPs are measured, and the result is compared with the model prediction result.

Three batches of GPP-NPs prepared using the simulated optimal recipe had EE (86.57.+ -. 0.73)%, and an average particle size of (85.42.+ -. 0.29) nm. Compared with the model prediction results, the deviation is less than 5%, which shows that the model established by the fitting equation has good predictability and the process is stable and feasible (refer to table 10).

Table 10 star design-verification of effect plane optimization (n=3, )

2.6 And (3) examining the preparation process of RBC@GPP-NPs.

(1) Extraction and membrane treatment of erythrocyte membranes

Erythrocyte membranes were extracted by hypotonic method (see Chai, Z, 2019), mice were intraperitoneally injected with about 0.1mL of 10% chloral hydrate, and after the mice were anesthetized, hearts were taken and blood was placed in anticoagulation tubes, 2mL per tube. The anticoagulation tube is subjected to low-temperature centrifugation (4 ℃,1000 Xg, 5 min), RBC layers are taken, the RBC layers are washed 3 times by pre-cooled isotonic normal saline, the supernatant is discarded, hypotonic normal saline (0.25 Xnormal saline, pH=7.4) is added, the mixture is fully suspended, the mixture is placed in a refrigerator at 4 ℃ for 2h, after centrifugation (4 ℃,15000rpm,7 min), pre-cooled hypotonic normal saline is continuously added into the lower sediment, the supernatant is washed to be nearly colorless, and then the isotonic normal saline is added to be washed to be nearly colorless, a certain amount of isotonic normal saline is dispersed and placed at 4 ℃ for standby.

RBC membranes were diluted several times with isotonic physiological saline, sonicated with a probe for several minutes (probe ultrasound power 15%), RBC were sequentially extruded with an extruder fitted with 0.4 μm and 0.2 μm polycarbonate membranes (polycarbonate membrane, PC membranes), and repeated extrusion at least 10 times at each particle size to give RBC membrane vesicles (RBC membrane vesicles, RVs) for use.

(2) Dilution fold investigation.

RVs were resuspended in different volumes of saline and extruded separately using extruders equipped with PC membranes with pore sizes of 0.4 μm and 0.2 μm to allow the minimum saline dose to pass through the extruder as the optimal dose.

RBC membranes cannot pass through 0.4 μm PC membrane when diluted with 1, 2 and 3 fold saline, respectively, but when increased by 4 fold, RBC membranes pass through 0.4 μm PC membrane very easily and can pass through 0.2 μm PC membrane. The lower the amount of physiological saline, the lower the dilution factor of the final drug, and the dilution factor of physiological saline should be controlled as much as possible. Therefore, the dilution factor of RBC was chosen to be 4.

(3) Investigation of the crushing time.

4mL of RVs suspension is taken in a 5mL Eppendorf (EP) tube, probe ultrasound is carried out under ice bath, the power is 15%, the crushing time is 3,5 and 7min respectively, the particle size of RVs is measured after extrusion, and the particle size of RVs is concentrated to be used as a judging standard to determine the optimal crushing time.

RVs particle size diagram obtained by crushing RBC membrane solution diluted 4 times with physiological saline for 3,5,7min respectively is shown in figure 6. As can be seen from FIG. 6, when the crushing times were 5 and 7min, the average particle sizes thereof were 136.9nm and 134.0nm, respectively, but the particle size distribution thereof was less concentrated. The RVs obtained by crushing for 3min have an average particle diameter of about 158.3nm, the particle diameter is more than 50nm, and the size distribution is good, so that the crushing time is 3min.

2.7 Preparation of RBC@GPP-NPs

GPP-NPs prepared as per 2.1 were combined with RVs as per 1: mixing at a ratio of 1, performing ultrasonic treatment for 10min (100 w, 40 Hz), and extruding with an extruder with a 0.2 μm PC film for at least 10 times to obtain RBC@GPP-NPs. And preparing the PP-NPs and RVs by adopting the same method to obtain blank nanoparticles.

The inventor adopts a film dispersion method to prepare GPP-NPs, takes the particle size and EE of GPP-NPs as evaluation indexes, inspects and optimizes the preparation conditions of GPP-NPs, and determines the optimal process as follows: precisely weighing a certain amount of GA and PEG ₃₄₀₀ -PLA ₂₀₀₀ The medicine carrying ratio is 1: 10, adding a certain amount of dichloromethane for dissolution, and removing the solvent by rotary evaporation to form a medicinal film. Physiological saline is added into the medicinal film, and the dosage of the physiological saline and PEG is equal to that of the medicinal film ₃₄₀₀ -PLA ₂₀₀₀ The ratio of (2) is 1.7:10 (mL: mg), ultrasonic dissolution, probe ultrasonic under ice bath for 10min (power 10%), 7000 rpm/min low temperature centrifugation 5min, filtering with 0.22 μm microporous membrane, and removing free GA to obtain GPP-NPs.

Based on GPP-NPs, the preparation process of RBC@GPP-NPs is examined, and the use level of physiological saline and the crushing time of RBC membranes are determined to be 4 times and 3 minutes respectively. The preparation process comprises the following steps: extracting RBC membrane by osmotic pressure hypotonic method, diluting RBC membrane with isotonic physiological saline 4 times, ultrasonic crushing with probe for 3min (ultrasonic power of probe 15%), sequentially extruding RBC with extruder equipped with 0.4 μm and 0.2 μm PC membrane, repeatedly extruding at least 10 times under each particle size, and obtaining RVs. GPP-NPs and RVs were set to 1: 1:1, sonicated for 10min (100 w, 40 HZ), and extruded at least 10 times with an extruder equipped with a 0.2. Mu.M PC film to give RBC@GPP-NPs.

There are some more notable points in the preparation of RBC@GPP-NPs. GA may affect the stability of GA at 60 deg.C, and therefore, the temperature of spin steaming, sonication, especially crushing sonication, needs to be controlled. During the crushing ultrasound, very high temperatures are generated and therefore need to be carried out in an ice bath.

The inventors have also established methods for determining the GA content in GPP-NPs and RBC@GPP-NPs. The chromatographic conditions are finally determined as follows: diamond C18 (250 mm. Times.4.6 mm,5 μm) column; the mobile phase is methanol-0.1% acetic acid aqueous solution (93:7); the detection wavelength is 360nm; column temperature of 25 ℃;1.0 mL/min ^-1 The amount of the sample introduced was 20. Mu.L. GA concentration is 0.816-65.280 mug.mL ^-1 The linear relationship in the range is good.

Example 2

This example is characterized by GPP-NPs and RBC@GPP-NPs.

(1) Measurement of particle size and Zeta potential:

1mL GPP-NPs and RBC@GPP-NPs solutions were taken separately, and diluted to a certain concentration with physiological saline. 1mL of diluted solution is taken and added into a sample cell, the generation of bubbles is avoided in the process, and the particle size and PDI of the nanoparticles are detected. For the measurement of the Zeta potential of the nano particles, diluted GPP-NPs or RBC@GPP-NPs are added into a folded capillary sample cell, the liquid needs to be higher than the lowest point of electrodes at two sides, and the Zeta potentials of the GPP-NPs and the RBC@GPP-NPs are measured in an instrument for 3 times.

The particle size distribution is shown in FIG. 7. As can be seen from the experimental results, the particle size distribution of GPP-NPs and RBC@GPP-NPs is concentrated, and the average particle sizes are (85.70 + -0.47) nm and (98.48+ -0.72) nm, respectively. The increased particle size of RBC@GPP-NPs compared to GPP-NPs suggests that RBC membranes are encapsulated on the surface of GPP-NPs. In addition, the Zeta potential of RBC membrane is (-11.27+ -0.67) mV, the potentials of GPP-NPs and RBC@GPP-NPs are respectively (3.40+ -0.29) mV and (-6.96+ -0.60) mV, and RBC and GPP-NPs have certain electrostatic action.

Table 11 particle size and Zeta potential data sheet for RBC and RBC@GPP-NPs

(2) And (5) morphological observation.

0.2mL GPP-NPs and RBC@GPP-NPs were taken separately, and saline was diluted to about 100. Mu.g/mL with GA. Placing the common carbon film of the copper mesh on a piece of filter paper, dripping 1 drop of supernatant on the copper mesh to enable the supernatant to enter into small holes of the copper mesh, dyeing with 2% phosphotungstic acid, standing for 10min, naturally drying, and observing the shape by a transmission electron microscope.

The morphology of GPP-NPs and RBC@GPP-NPs nanoparticles is shown in FIG. 8. The nanoparticle has a regular sphere shape, uniform size and good dispersion, and the size of the nanoparticle is slightly smaller than the particle size, which is probably the result of drying the nanoparticle on a copper mesh.

Example 3

The present example makes measurements of the encapsulation efficiency and drug loading of GPP-NPs and RBC@GPP-NPs.

Taking 1mL of GPP-NPs solution before centrifugation, fixing methanol to 10mL, measuring GA content by HPLC, and calculating the GA mass as W _{Total (S)} . Respectively taking 1mL GPP-NPs and RBC@GPP-NPs, processing by the same method, measuring the GA content, and calculating the mass of GA as W _Package . 1mL GPP-NP was taken separately _S Weighing after lyophilization (W) ₁ ) Dissolving in methanol, fixing volume to 10mL, measuring GA content, and calculating GA mass (W ₂ ). Each group was run 3 times in parallel. The calculation formulas of EE and drug loading are respectively as follows:

W _package GA mass measured in 1mL GPP-NPs or RBC@GPP-NPs; w (W) _{Total (S)} : GA mass measured in GPP-NPs before centrifugation at 1 mL; w (W) ₁ : total mass of 1mL GPP-NPs or RBC@GPP-NPs; w (W) ₂ : the total mass of GA was measured in 1mL GPP-NPs or RBC@GPP-NPs.

The EE and drug loading results for GPP-NPs and RBC@GPP-NPs are shown in Table 12. Experimental results indicate that the EE of GPP-NPs and RBC@GPP-NPs are (86.37.+ -. 0.84)% and (79.11.+ -. 1.42)%, respectively, and the EE of RBC@GPP-NPs is decreased, probably because part of GPP-NPs or RBC@GPP-NPs are broken during extrusion, GA is released and separated out, is trapped by PC film, and the GA content is decreased. The GPP-NPs drug loading was small, 3.76.+ -. 0.07)%. This is because 1mL of physiological saline is contained in about 1mL of the nanoparticle, and sodium chloride remains in the lyophilized sample after lyophilization, which increases the sample mass and makes the drug loading smaller.

Table 12 encapsulation efficiency and drug loading results

Example 4

This example performed GPP-NPs and RBC@GPP-NPs nanoparticle stability assays.

The GPP-NPs and RBC@GPP-NPs prepared in example 1 were stored at 4℃and the particle sizes and PDI were measured at 0, 1, 2, 3, 4, 6, 8, and 16 days, respectively.

From the particle size and PDI data (table 13 and shown in fig. 9) of GPP-NPs and rbc@gpp-NPs stored at 4 ℃ for 16 days, it can be seen that during this period, the particle size variation of GPP-NPs and rbc@gpp-NPs is small, indicating good stability of GPP-NPs and rbc@gpp-NPs within 16 days in a 4 ℃ environment.

Table 13 stability of GPP-NPs and rbc@gpp-NPs.

Example 5

In vitro release assays for GPP-NPs and RBC@GPP-NPs were performed in this example.

Membrane dialysis was used. Taking 6 dialysis bags (with the molecular weight cut-off of 3.5 KD) treated in advance, dividing into 2 groups, respectively adding a certain amount of GPP-NPs and RBC@GPP-NPs, wherein the mass of GA contained in each dialysis bag is 2mg, immersing the dialysis bags in PBS (1% Tween 80) with the pH of 7.4 to release external liquid (200 mL) and placing the external liquid in a constant-temperature shaking table (37 ℃ at 100 rpm/min), respectively sucking 1mL of fresh release external liquid from the bottom of the release external liquid at 1, 2, 4, 8, 12, 24, 48 and 72h, detecting by adopting a second chapter GA content measuring method, and calculating the cumulative release degree of GA.

From the in vitro release results of GPP-NPs and RBC@GPP-NPs (FIG. 10), the in vitro cumulative release was 20.70%, 38.46% and 53.17% for GPP-NPs at 1, 12, 72h, respectively, while the cumulative release was 5.36%, 15.22, 30.53% for GPP@RBC-NP at 1, 12, 72h, respectively. Experimental results show that the encapsulation of the bionic RBC membrane may have the effect of prolonging GA release time for GPP-NPs.

Example 6

In this example, the in vitro anti-liver cancer activity and safety evaluation were performed on the red cell membrane-coated gambogic acid biomimetic nanoparticle prepared in example 1. Evaluation was performed by an in vitro hemolysis test and a biocompatibility test.

Reagent:

and (3) cells:

and (3) subpackaging the reagent:

to avoid repeated freeze thawing of fetal bovine serum (fetal bovine serum, FBS) and the diabody (Penicillin/Streptomycin Solution), the FBS and the diabody were separately dispensed. A500 mL FBS was dispensed into 50mL centrifuge tubes, 50mL each. Because the empty volume of the 15mL centrifuge tube is smaller, the volume of the liquid is increased to a certain extent after freezing, and the volume of the added liquid is reduced in order to avoid the damage of the centrifuge tube due to the increase of the volume after freezing; since 5mL of the diabody was required per 500mL of the medium when the culture broth was prepared. Thus, 10mL of diabody was dispensed per tube for ease of use. And (5) placing the centrifuge tube into a temperature of minus 20 ℃ for storage for later use.

Preparation of cell culture solution

One tube of FBS and the double antibody were thawed in a refrigerator at 4 ℃ and the next day FBS, double antibody and medium were mixed at 10:1: 100. is mixed evenly and split-packed in 50mL centrifuge tubes, and is preserved in a refrigerator at 4 ℃ for standby.

Preparation of frozen stock solution

The frozen stock solution is prepared from FBS and DMSO according to the proportion of 1: 9. Melting a tube of 50mL FBS at 4 ℃ or normal temperature, taking a clean 50mL centrifuge tube, adding 45mL FBS,5mL DMSO, uniformly mixing to obtain frozen stock solution, and preserving at-20 ℃ for later use.

Configuration of MTT

A 50mL centrifuge tube was closed with aluminum foil paper, and 50mL PBS was placed in the centrifuge tube for MTT dissolution and formulation. Dispersing MTT in a bottle containing 250mg MTT by taking 2mL of PBS in a centrifuge tube, transferring the dispersed MTT into a centrifuge tube, continuously dispersing MTT in the bottle by absorbing PBS in the centrifuge tube, and repeating the above operation until no obvious yellow substance exists in the bottle. After complete dissolution by ultrasound (MMT dissolved slowly in PBS), 0.22. Mu.M was filtered and stored at-20deg.C for further use. The whole operation process needs to be protected from light.

1. Culture of adherent cells

Cell resuscitation

In order to avoid the damage of the crystal ice generated in the thawing process of the frozen stock solution to cells, the thawing time of the frozen stock solution needs to be shortened as much as possible. The specific operation is as follows: the water bath was opened to raise the temperature and kept at 37℃and a centrifuge tube containing 9mL of culture medium was prepared in the operating table, the cells were removed from the liquid nitrogen tank, the horse was quickly melted in the water bath, and the cells were removed to the standby centrifuge tube on a clean bench, mixed well, centrifuged at 1000rpm/min for 3min to collect the cells, and resuspended in 1mL of culture medium. A clean culture dish with a diameter of 10cm was taken, 9mL of the culture solution was added with a 10mL pipette, and 1mL of the cell suspension was added thereto, and the mixture was stirred uniformly and placed in an incubator (37 ℃ C., 5% CO 2) for cultivation. Wherein HepG2 and LO2 cells were cultured in DMEM and RPMI 1640 medium, respectively.

Cell exchange liquid

The resuscitated cells were cultured overnight and the fluid was changed. The petri dish was taken out and placed on an ultra clean bench, the liquid was sucked off with a pipette, washed with PBS, fresh culture solution was added, and the cells were returned to the incubator for further culture. In addition, if the number of cells is less than 90% in the process of culturing the cells, the cells need to be replaced when the color of the culture solution in the culture dish is yellow or more floating cells exist.

Cell passage

Cells were propagated to a confluency of about 90% at the bottom of the dish and passaged. Pouring out the liquid in the culture dish, washing off residual culture solution and the like by using PBS, adding 1mL of pancreatin to digest for 2-4 min, and adding 2mL of culture solution to stop the digestion process when most cells lose the adherent form. Tilting the culture dish, sucking the liquid in the dish and repeatedly blowing off to make the cells at the bottom of the dish fall off and blow off into single cells, so that the single cells can be more easily dispersed during resuspension. The liquid in the dish was transferred to a 15mL centrifuge tube and the cells were collected by centrifugation. Slowly pouring out the liquid in a tilting way, and adding 2-3 mL of fresh complete culture solution to uniformly disperse the cells. Taking 2-3 culture dishes of 10cm, respectively adding 9mL of culture solution and 1mL of cell suspension, blowing and mixing uniformly by a pipette, marking, and placing the culture dishes into a incubator for culture.

Cell cryopreservation

Cells with good growth state are needed to be adopted in the experiment. And (3) placing the program cooling box containing isopropanol at room temperature in advance, and writing cell names, culture medium names, freezing dates and the like on the freezing tube in an ultra-clean bench. When the cells grow at the bottom of the dish, the cells are collected, the liquid is poured out, and a proper amount of frozen stock solution is added and mixed uniformly. Every 1mL of the cell suspension is transferred to a new freezing tube, and the freezing tube is placed in a freezing box which is placed at room temperature in advance, and the cell suspension is preserved at-80 ℃. If the cells are not used for a long time, the cells placed in the freezing box for a period of time are placed in a liquid nitrogen tank for preservation.

Cell count

Collecting cells, adding a proper amount of culture solution to uniformly disperse the cells, adding 10 mu L of the culture solution to one end of a glass plate of a counting plate, counting under a microscope, and dividing the total cell number in 4 16 square grids on the counting plate by 4 to obtain the concentration of the cell suspension.

HepG2 cell proliferation assay

In the ultra clean bench, the cells were collected and counted, and the cell concentration was adjusted to 3X 10 ⁴ 、4×10 ⁴ 、5×10 ⁴ 、6×10 ⁴ And each mL. First, it was examined whether E-Plate 16 could be used normally on the RTCA Station. 50. Mu.l of the culture medium was added to each well of E-Plate 16, and the mixture was placed on the RTCA Station to perform detection, and the cell index was required to be 0.063 or less. E-Plate 16 was removed and 100. Mu.l of 5X 10 was added to the wells ⁴ HepG2 cell suspension at a concentration of each mL was allowed to stand E-Plate 16 for 30min, and then placed on RTCA Station to monitor cell proliferation.

The assay measures the growth of different numbers of HepG2 cells and determines the cell concentration for the cytotoxicity assay. FIG. 11 shows proliferation of several cell concentrations, and it can be seen from the graph that the proliferation rate of cells in the 3K and 4K groups was slow, and the cell index was less than 1 after 20 hours. The proliferation rate of a group of cells with the cell concentration of 6K is too high, the cells grow for 10 hours, the cell index is over 1, and the proliferation is hardly carried out again about 70 hours. For a group with a cell concentration of 5K, the cell proliferation 13h cell index reached 1 and also had a tendency to proliferate at 72 h. The instrument measures cytotoxicity test, generally adding medicine when cell index reaches more than 1, and finally selecting 5K as plating concentration of HepG2 cells.

3. In vitro antiproliferative activity

3.1 cell seeding

When the cells grow to the bottom of the culture dish and the fusion rate is about 90%, collecting the cells, counting, taking part of cell suspension, diluting with fresh culture solution, mixing uniformlyHomogenizing to give final concentration of 5×10 ⁴ Each of the cells/mL was inoculated into a 96-well plate at 100. Mu.L/well, and a round of the periphery was not inoculated with cells, and 200. Mu.L of PBS was added to protect the cells. Labeling on 96-well plate, and culturing in incubator at 37 deg.C.

3.2 administration of drugs

And adding the medicine for culturing after the cells grow to about 70% of fusion rate at the bottom of the hole. The concentrations of GPP-NPs, RBC@GPP-NPs and free GA were all diluted to 5.60, 2.80, 1.40, 0.70, 0.35 and 0.175. Mu.g/mL (concentrations calculated as GA content) with culture medium, and the blank group contained no drug. mu.L of drug was added to each of the cell plated 96 well plates, at least 3 wells in parallel. And (5) marking the 96-well plate, and then returning the 96-well plate to the incubator for continuous culture.

3.3 CCK8 detection

The effect of the drug on cell growth was examined at 24 and 48 hours of dosing, respectively. After adding 20. Mu.L of CCK8 and co-incubating for 1-2 hours when reaching the action time, the optical density OD value (optical density: OD) at the wavelength of 450nm is measured, and the cell growth survival rate is calculated by the following formula:

as can be seen from Table 14 and the experimental results in FIG. 12, the activities of GA, GPP-NPs and RBC@GPP-NPs were positively correlated with the concentrations for HepG2 cells. 24 hours of administration, RBC@GPP-NPs inhibited less than GA and GPP-NPs; after 48h of drug action, the inhibition of low concentration of RBC@GPP-NPs was also lower than that of GA and GPP-NPs, probably due to incomplete release of GA in RBC@GPP-NPs and lower concentration of free GA in the system. In addition, at low concentrations, the inhibition of GPP-NPs was also lower than GA, probably because GPP-NPs and RBC@GPP-NPs had some sustained release effect, consistent with the in vitro release results.

Table 14 effect of different concentrations of nanoparticles on HepG2 cell proliferation.

4 hemolytic test

Preparation of erythrocyte suspensions

Mice were intraperitoneally injected with about 0.1mL of 10% chloral hydrate, and after the mice were anesthetized, the hearts were bled and the blood was placed in anticoagulation tubes with 2mL of blood per tube. Fresh whole blood of a mouse is taken for 4mL, low-temperature centrifugation (4 ℃,1000 Xg, 5 min) is carried out, a white membrane layer and upper liquid are sucked and removed, RBC is washed for 3 times by pre-cooled isotonic normal saline, supernatant is removed, 2mL of compacted RBC is taken for constant volume at 100 mL, 2% of red blood cell suspension is obtained, and the red blood cell suspension is preserved at 4 ℃ for standby.

Measurement of hemolysis Rate

The experiment uses a direct measurement of free hemoglobin. A series of gradient solubility RBC@GPP-NPs, GPP-NPs and GA solutions were prepared, respectively, with GA contents of 0.35, 0.7, 1.4, 2.8, 5.6 μg/mL. 500. Mu.L of the drug was added to 500. Mu.L of 2% RBC suspension, mixed well, incubated at 37℃for 3 hours, and double distilled water and saline groups were used as positive and negative controls, respectively, with 3 samples in parallel. All samples were removed and centrifuged at 12000rpm for 15min. 100 μl of supernatant was aspirated and added to the 96-well plate, 3 wells per group, and the OD at 570nm was measured by an microplate reader. The Haemolysis Ratio (HR) is calculated as follows:

The experiment judges the hemolysis rate of the medicine by a direct hemolysis rate measurement method. A hemolysis ratio exceeding 5% is regarded as hemolysis. As can be seen from FIGS. 13 and 14, with increasing GA, the hemolysis rate increased, and the RBC@GPP-NPs group had a slightly lower hemolysis rate than the free GA and GPP-NPs at GA concentrations of 1.4 μg/mL or less. At GA concentrations of 2.8. Mu.g/mL and 5.6. Mu.g/mL, there was a significant difference in the hemolysis rate of the three groups. At 2.8 μg/mL, none of the three groups reached hemolysis; when the drug concentration was increased to 5.6. Mu.g/mL, hemolysis occurred in both the free GA and GPP-NPs groups, with hemolysis rates of 11.98% and 7.49% respectively for the GA group and GPP-NPs group, which were significantly higher than for the RBC@GPP-NPs group (4.03%). The hemolysis rate of the GPP-NPs and RBC@GPP-NPs groups was significantly reduced compared to the free GA group, demonstrating their better biocompatibility. Experimental results show that the use of the carrier material obviously improves the biocompatibility of the medicine in blood, so that the safety of the medicine in vivo is improved.

6. Biocompatibility test

Cell seeding

L02 cells were plated 5X 10 per well ³ Individual cells.

Administration of drugs

The concentrations of GPP-NPs, RBC@GPP-NPs and free GA were all diluted to 22.40, 11.20, 5.60, 2.80, 1.40 and 0.70 μg/mL (concentrations calculated as GA content) with culture medium and the blank group contained no drug. And adding the medicine to continue culturing after the cells grow to the concentration of about 70%. mu.L of the prepared drug was added to each of the cell-plated 96-well plates, and 3 wells were arranged in parallel. The 96-well plate is marked and put back into the incubator for culture.

MTT assay

Cells were incubated with drug for 24 and 48h, respectively, and 20 μl MTT was added to each well of the 96-well plate and incubation was continued for 4h. Then, the liquid in the plate was poured off, 150. Mu.L of dimethyl sulfoxide was dissolved by shaking for 10min, OD value was measured at a wavelength of 490nm, and the growth and survival rate of each group of cells was calculated by the following formula:

GA. GPP-NPs and RBC@GPP-NPs vs LO ₂ The results of the cell compatibility test are as follows (FIG. 15 and Table 15). From the experimental results, it can be seen that RBC@GPP-NPs vs. LO ₂ The biocompatibility of (C) is significantly higher than that of the GA group. At 24h of drug action, the administration group with GA content of 5.6 mug/mL in RBC@GPP-NPs and below is almost nontoxic to LO2 cells; when the concentration was increased to 11.2. Mu.g/mL, the inhibition did not reach 50%. With prolonged drug action time, at 48h, low concentration of RBC@GPP-NPs vs LO ₂ The inhibition of cells remains small. From three sets of data with GA content of 5.6. Mu.g/mL, it can be seen that RBC@GPP-NPs vs. LO ₂ The inhibition of cells is obviously reduced; when the administration concentration was increased to 11.2. Mu.g/mL, three pairs of LOs were added ₂ The cells have obvious inhibition effect.

Table 15 effect of different concentrations of nanoparticles on LO2 cell proliferation.

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According to in vitro experimental study, the invention discovers that GPP-NPs and RBC@GPP-NPs still retain the activity on liver cancer HepG2 cells. While RBC@GPP-NPs are slightly less active than GA and GPP-NPs, this is probably due to the long circulation and slow release effects of RBC@GPP-NPs, the GA concentration in the cell viability was not as high as that of GA group, consistent with the in vitro release results in chapter four. In an in vitro hemolysis experiment, the hemolysis rate of RBC@GPP-NPs on red blood cells is reduced, which indicates that the safety is improved. In addition, in the biocompatibility experiment, compared with GA and GPP-NPs, the inhibition effect of RBC@GPP-NPs on normal liver LO2 cells is obviously reduced, and the results show that the RBC@GPP-NPs have good biosafety. In vitro experiments all show that RBC@GPP-NPs have superior properties to GA and GPP-NPs, and can possibly overcome the problems of water solubility and half-life of GA, so that the RBC@GPP-NPs have the opportunity of being applied to disease treatment. RBC@GPP-NPs also require further investigation and verification by in vivo experiments, which is also one of the directions of investigation of the subject after the subject group.

In conclusion, the nano-particle RBC@GPP-NPs prepared by the method has good stability, longer in-vitro release time, stronger anti-liver cancer activity and higher safety.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The bionic nanoparticle is characterized by comprising an inner core and an outer shell, wherein the outer shell is coated on the periphery of the inner core, the inner core comprises gambogic acid and a coating component, and the outer shell comprises an erythrocyte membrane; the entrapping component is selected from amorphous block polymers; the amorphous block polymer is polyethylene glycol-polylactic acid; the mass ratio of the gambogic acid to the entrapped components in the inner core is 1:5-15, the average particle size of the inner core is 80-96nm, the molecular weight of the polyethylene glycol in the polyethylene glycol-polylactic acid is 2000-5000, and the molecular weight of the polylactic acid is 2000-3000.

2. The biomimetic nanoparticle of red blood cell membrane-encapsulated gambogic acid of claim 1, wherein the volume ratio of the inner core to the outer shell is 1-1.2:1.

3. The biomimetic nanoparticle of red blood cell membrane-encapsulated gambogic acid according to claim 2, wherein the encapsulation efficiency of the inner core is 86.37±0.84%, and the encapsulation efficiency of the biomimetic nanoparticle is 79.11±1.42%.

4. The biomimetic nanoparticle of red blood cell membrane-encapsulated gambogic acid according to claim 2, wherein the drug loading of the inner core is 3.76±0.07%.

5. The biomimetic nanoparticle of red blood cell membrane-encapsulated gambogic acid according to claim 1, wherein the average particle size of the biomimetic nanoparticle is 98.48±0.72nm.

6. A method for preparing the biomimetic nanoparticle of the red blood cell membrane-coated gambogic acid according to any one of claims 1 to 5, comprising: preparing a kernel by adopting a film dispersion method, diluting and crushing erythrocyte membranes, and then extruding; mixing the inner core with erythrocyte membrane, and extruding, wherein the dilution is 4-5 times of the dilution by using isotonic physiological saline; the crushing is carried out by adopting an ultrasonic crushing method, and the crushing time of the ultrasonic crushing method is 2-3min.

7. The method according to claim 6, wherein the film dispersion method for preparing the core means: mixing gambogic acid, amorphous block polymer and organic solvent, removing the organic solvent to obtain medicinal film, adding physiological saline into the medicinal film, and performing ultrasonic treatment.

8. The method according to claim 7, wherein the ratio of the organic solvent to the addition amount of the amorphous block polymer is 0.15 to mL mL to 0.25 mL/1 mg.

9. The method according to claim 8, wherein the organic solvent is dichloromethane, chloroform, acetone or methanol.

10. The method according to claim 7, wherein the ratio of the amount of physiological saline to the amorphous block polymer added is 0.12 to mL ml/1 mg.

11. The method according to claim 10, wherein the ratio of the amount of physiological saline to the amorphous block polymer added is 0.15 to 0.17 mL/1 mg.

12. The method of claim 7, wherein the ultrasound is performed under ice bath conditions; the ultrasonic power is 10-15%; the ultrasonic time is 5-20min.

13. The method of claim 12, wherein the sonication time is 10 minutes.

14. The method according to claim 12, wherein the ultrasonic treatment is followed by centrifugation and filtration steps; the rotational speed of the centrifugation is 3000-10000rpm/min.

15. The method of claim 14, wherein the centrifugation is at 7000 rpm/min.

16. The method according to claim 6, wherein the extrusion is extrusion of the crushed red cell membrane with a polycarbonate membrane to obtain RBC membrane vesicles.

17. The method according to claim 6, wherein the mixing of the inner core with the erythrocyte membrane further comprises ultrasonic treatment, and then extruding with polycarbonate membrane to obtain bionic nano particles;

the ultrasonic treatment time is 10-12min, the treatment frequency is 35-40Hz, and the treatment power is 100-120W;

the pore size of the polycarbonate membrane was 0.2. Mu.m.

18. Use of the biomimetic nanoparticle of red blood cell membrane-coated gambogic acid according to any one of claims 1-5 or the biomimetic nanoparticle prepared by the preparation method according to any one of claims 6-17 in the preparation of a medicament for treating tumor.

19. The use according to claim 18, wherein the tumour is liver cancer, lung cancer, stomach cancer, breast cancer or colon cancer.

20. An antitumor drug comprising the biomimetic nanoparticle of the red blood cell membrane-coated gambogic acid of any one of claims 1-5.

21. The antitumor drug of claim 20, wherein the antitumor drug is an anti-liver cancer drug.