CN115252560B

CN115252560B - Self-assembled nanoparticle based on natural product and preparation method and application thereof

Info

Publication number: CN115252560B
Application number: CN202210898520.3A
Authority: CN
Inventors: 曲玮; 张可欣; 朱嘉新; 王如意; 冯锋; 王璨
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2024-02-13
Anticipated expiration: 2042-07-28
Also published as: CN115252560A

Abstract

The invention discloses a self-assembled nanoparticle based on natural products, which is prepared from a first component and a second component, wherein the first component is triterpene, anthraquinone, flavonoid, alkaloid, polyphenol, coumarin or lignan compounds except gambogic acid, and the second component is gambogic acid, and the gambogic acid induces the self-assembly of the first component to form the nanoparticle. The invention also discloses a self-assembled nanoparticle with mitochondrial targeting combined metabolism blocking effect based on natural products, which is prepared by self-assembling berberine, lonidamine and gambogic acid, or further coating vitamin E polyethylene glycol succinate outside the nanoparticle to improve stability. The invention also discloses application of the nano-particles in preparing anticancer drugs.

Description

Self-assembled nanoparticle based on natural product and preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical preparations, relates to self-assembled nanoparticles based on natural products and a preparation method and application thereof, and in particular relates to self-assembled nanoparticles with mitochondrial targeting combined metabolism blocking effect based on natural products and a preparation method and application thereof.

Background

At present, the treatment means of cancers used clinically mainly comprise surgery, chemotherapy, radiotherapy, targeted therapy and immunotherapy. However, these treatments often do not have tumor targeting, which can lead to serious systemic side effects for the patient, and can cause great pain to the patient. Thus, people are beginning to focus on natural anticancer drugs. Natural products are generally biocompatible, less toxic and highly selective as antitumor agents, and many antitumor natural product molecules have proven to be clinically effective. However, the natural products have poor water solubility, low bioavailability and poor patentability, so that further clinical application of the natural products is limited.

Compared with normal tissues, tumors have unique pathological characteristics. The well-known Warburg effect (Warburg effect) indicates: unlike normal differentiated cells, most tumor cells rely primarily on aerobic glycolysis for energy rather than oxidative phosphorylation of mitochondria. Therefore, the compound can be used as a potential anticancer drug action target to regulate and control tumor energy metabolism, change tumor growth microenvironment, inhibit or even cut off energy required by tumor cell biological activity, and further realize inhibition of tumor proliferation and migration and killing of malignant cancer cells. However, metabolic inhibitors have poor efficacy due to poor water solubility and low bioavailability.

In recent years, with the progress and development of nanomaterial chemistry, nano-drug delivery systems are often used for improving drug formation, prolonging circulation time of drugs in vivo, imparting active or passive targeting effects to drugs, and the like. However, the traditional nano preparation still has the problems of low drug loading, poor stability and the like, so that the function and the final efficiency of the nano preparation in clinical application are still not satisfactory.

Disclosure of Invention

The inventor discovers through research that the cytotoxic compound gambogic acid can induce natural products to self-assemble to form nano particles, thereby improving the application of the natural products in the field of tumor treatment.

The self-assembled nanoparticle is prepared from a first component and a second component, wherein the first component is triterpene, anthraquinone, flavonoid, alkaloid, polyphenol, coumarin or lignan compounds except gambogic acid, the second component is gambogic acid, and the first component is induced to self-assemble by the gambogic acid.

Preferably, the mass ratio of the first component to the gambogic acid is 2:1-1:2.

Preferably, the triterpene compound is artemisinin, dihydroartemisinin, oleanolic acid, glycyrrhizic acid; the anthraquinone compound is rhein and hypericin; the flavonoid compound is quercetin; the alkaloid compound is berberine; the polyphenol compound is curcumin; the coumarin compound is coumarin; the lignin compound is magnolol and honokiol; the self-assembled nanoparticles are artemisinin-gambogic acid nanoparticles, dihydroartemisinin-gambogic acid nanoparticles, oleanolic acid-gambogic acid nanoparticles, glycyrrhizic acid-gambogic acid nanoparticles, rhein-gambogic acid nanoparticles, hypericin-gambogic acid nanoparticles, quercetin-gambogic acid nanoparticles, berberine-gambogic acid nanoparticles, curcumin-gambogic acid nanoparticles, coumarin-gambogic acid nanoparticles, magnolol-gambogic acid nanoparticles, and honokiol-gambogic acid nanoparticles.

Another object of the present invention is to provide a method for preparing the self-assembled nanoparticle, comprising: respectively dissolving the first component and gambogic acid in a benign organic solvent, and then mixing the two solutions according to the mass ratio of the first component to the gambogic acid of 2:1-1:2 to obtain a mixed solution, or dissolving the first component and the gambogic acid in the benign organic solvent according to the mass ratio of 2:1-1:2 to form a mixed solution; and (3) dropwise adding the mixed solution into pure water with the constant temperature of 50-60 ℃, stirring during dropwise adding, standing after dropwise adding is finished, and removing the organic solvent through dialysis to obtain the self-assembled nanoparticle.

Preferably, the first component and the gambogic acid are respectively dissolved in benign organic solvents to prepare a first component solution with the concentration of 10mg/mL and a gambogic acid solution with the concentration of 10mg/mL, and then the first component solution and the gambogic acid solution are uniformly mixed according to the mass ratio of the first component to the gambogic acid of 2:1-1:2 to obtain a mixed solution.

Preferably, the benign organic solvent is one or more of dimethyl sulfoxide, absolute ethyl alcohol, methanol or tetrahydrofuran, and preferably is dimethyl sulfoxide. The volume ratio of benign organic solvent to pure water is 1:9 to 1:50, preferably 1:9.

The standing time is generally 0.5 to 3 hours.

The molecular weight cut-off of the dialysis bag used for dialysis is 1000-10000 Da, preferably 3000Da.

The self-assembled nanoparticle is uniformly distributed in a sphere-like shape, has uniform particle size and narrow particle size distribution range, and has good stability. Experiments show that the self-assembled nanoparticle has good in vitro cytotoxicity to breast cancer cells, is superior to or equivalent to gambogic acid, has obviously smaller toxicity to normal cells than breast cancer cells, has smaller toxicity to normal cells than gambogic acid, has lower cytotoxicity to normal cells, and has good biocompatibility and selectivity.

The invention also aims to provide the application of the self-assembled nanoparticle in anti-tumor drugs.

On the basis of improving poor targeting property of chemotherapeutic drugs, reducing toxic and side effects and improving the drug effect of metabolic inhibitors, in order to enrich nano-preparations at tumor sites and target mitochondria, the effective killing of tumors is realized by realizing multiple metabolic inhibition of glycolysis, oxidative phosphorylation and glutamine metabolism and synergistic treatment with the chemotherapeutic drugs. The invention also aims to provide a natural product-based self-assembled nanoparticle with a mitochondrial targeting combined metabolism blocking effect, which is prepared by self-assembling berberine, lonidamine and gambogic acid.

The mass ratio of the berberine to the lonidamine to the gambogic acid is (1-10): 1-10, preferably (1-7): 1:4, more preferably (1-5): 1:4, and most preferably 3:1:4.

Another aspect of the present invention is to provide a method for preparing the natural product-based self-assembled nanoparticle having a mitochondrial targeting co-metabolism blocking effect, comprising: dissolving berberine, lonidamine and gambogic acid in benign organic solvent to form mixed solution, or dissolving berberine, lonidamine and gambogic acid in benign organic solvent to form solution, and mixing the solution to form mixed solution; dripping the mixed solution into pure water with the constant temperature of 50-60 ℃, stirring during dripping, standing after dripping, dialyzing to remove the organic solvent, and preparing the nano particles.

The mass volume ratio of the berberine to the benign organic solvent is 1:0.1-1:0.65 mg/mL, preferably 1:0.1-1:0.25 mg/mL.

The benign organic solvent is one or more of dimethyl sulfoxide, absolute ethyl alcohol, methanol or tetrahydrofuran, and is preferably a mixed solvent of dimethyl sulfoxide and methanol in a volume ratio of 1:4-1:12. The volume ratio of the benign organic mixed solvent to the pure water is 1:10-1:50, preferably 1:10-1:25.

The standing time is generally 0.5 to 3 hours.

In order to further improve the stability of the nanoparticle, the self-assembled nanoparticle with the mitochondrial targeting combined metabolism blocking effect based on the natural product is further preferably prepared from berberine, lonidamine, gambogic acid and a hydrophilic material vitamin E polyethylene glycol succinate (VE-TPGS) serving as raw materials, wherein the nanoparticle is prepared from berberine, lonidamine and gambogic acid through self-assembly, and the nanoparticle is coated with the vitamin E polyethylene glycol succinate to obtain the nanoparticle with a core-shell structure. The self-assembled nanoparticles with mitochondrial targeting combined metabolism blocking effect based on natural products are uniformly distributed in a sphere-like shape, have uniform particle size, narrow particle size distribution range and particle size distribution of 170-280 nm, preferably about 200 nm. The stability is better, the particle size is not obviously changed when the product is placed for 48 hours at room temperature.

The mass ratio of the total amount of berberine, lonidamine and gambogic acid to the vitamin E polyethylene glycol succinate is 5:1-15:1, preferably 5:1.

The molecular weight of polyethylene glycol contained in the vitamin E polyethylene glycol succinate is 1000-10000, preferably 2000.

Another aspect of the present invention is to provide a method for preparing the natural product-based self-assembled nanoparticle having a mitochondrial targeting co-metabolism blocking effect, comprising: dissolving berberine, lonidamine, gambogic acid and vitamin E polyethylene glycol succinate in benign organic solvent to form mixed solution, or dissolving berberine, lonidamine, gambogic acid and vitamin E polyethylene glycol succinate in benign organic solvent to form solution respectively, and mixing the solutions to form mixed solution; dripping the mixed solution into pure water with the constant temperature of 50-60 ℃, stirring during dripping, standing after dripping, dialyzing to remove the organic solvent, and preparing the nano particles.

The mass volume ratio of the vitamin E polyethylene glycol succinate to the benign organic solvent is 1:1-3:1 mg/mL, preferably 2:1-3:1 mg/mL.

The standing time is generally 0.5 to 3 hours.

The self-assembled nanoparticle with the mitochondrial targeting combined metabolism blocking effect based on the natural product can improve the bioavailability of metabolic inhibitors and cytotoxic drugs, and comprises the purposes of improving in vivo circulation time, tumor mitochondrial targeting, cell uptake and intracellular site-specific release; can be used for blocking a plurality of energy metabolism pathways of tumor cells, including glycolysis, oxidative phosphorylation and glutamine metabolism, interfering mitochondria simultaneously, and synergistically improving the anti-tumor capability of cytotoxic substances. The inventor performs in-vitro anticancer activity screening, which shows that the self-assembled nanoparticle with the mitochondrial targeting combined metabolism blocking effect based on the natural product can obviously inhibit proliferation of breast cancer cells, has obvious anticancer activity on the breast cancer, and can more comprehensively block substance synthesis and energy supply of tumor cells. Therefore, another object of the present invention is to provide the use of the self-assembled nanoparticle having a mitochondrial targeting combined metabolic blocking effect based on natural products in anti-tumor drugs.

Preferably, the application is the application of preparing a mitochondria targeting antitumor drug for blocking energy supply of tumor cells and/or inhibiting growth of tumor cells.

The tumor is lung cancer, liver cancer and breast cancer.

The berberine is respiratory chain complex I and glutamine metabolism inhibitor, lonidamine is hexokinase 2 inhibitor, the combination of berberine and lonidamine simultaneously plays roles in inhibiting glycolysis, mitochondrial respiratory chain complex and glutamine metabolism, the berberine provides mitochondrial targeting function, and the gambogic acid provides cytotoxicity. According to the invention, the metabolic inhibitor and the cytotoxic compound are self-assembled to form nanoparticles by adopting a nano precipitation method, the synergistic effect of the metabolic inhibitor and the cytotoxic compound is exerted, the advantages of the nano preparation are combined while the targeting and treatment effects of tumors are enhanced, and the targeting, the drug property and the bioavailability of the drug are improved; and further, the hydrophilic vitamin E polyethylene glycol succinate is coated outside the nano-particles, so that the stability can be improved.

The self-assembled nanoparticle based on the joint metabolism blocking effect with mitochondrial targeting has the advantages of a nano drug delivery system (such as passive targeting capability, in vivo circulation time extension, pharmacokinetic behavior improvement and the like), and is specifically expressed as the following 4 advantages:

1) In the aspect of drug effect, compared with physical mixing of gambogic acid or berberine, lonidamine and gambogic acid, in vitro cytotoxicity experiments show that the nanoparticles can obviously inhibit proliferation of breast cancer cells, have obvious anticancer activity, but have lower cytotoxicity to normal cells;

2) Compared with free berberine or a physical mixed group, in the aspect of targeting, the nanoparticle not only has EPR (enhanced permeability and retention) effect of dosage form, but also can accurately target the mitochondria of tumor subcellular organelles, thereby laying a foundation for realizing metabolic inhibition besides improving tumor specific targeting;

3) In the aspect of energy metabolism, the nano preparation with the combined metabolism blocking effect can realize multiple metabolism inhibition of glycolysis, oxidative phosphorylation and glutamine metabolism, and can be used for synergistic treatment with chemotherapeutics, and various key proteins (such as hexokinase) in a metabolic pathway are used as targets to comprehensively cut off energy supply of tumors and kill tumor cells;

4) In terms of pharmacy, the advantages of the nano dosage form are utilized, and the bioavailability of the chemotherapeutic drugs and the metabolic inhibitors is improved, including the improvement of in vivo circulation time, tumor targeting, cell uptake and the like.

Drawings

Fig. 1: dynamic light scattering histogram of nanoparticles (BLG NPs) prepared in example 2.

Fig. 2: buddha effect plot of self-assembled nanoparticles (BLG@TPGS NPs) prepared in example 2.

Fig. 3: dynamic light scattering histogram of self-assembled nanoparticles (blg@tpgs NPs) prepared in example 2.

Fig. 4: transmission Electron Microscopy (TEM) images of self-assembled nanoparticles (blg@tpgs NPs) prepared in example 2.

Fig. 5: stability examination results of self-assembled nanoparticles (blg@tpgs NPs) prepared in example 2.

Fig. 6: the self-assembled nanoparticles (blg@tpgs NPs) prepared in example 2 mimic the drug release profile of physiological environments.

Fig. 7: drug release profile of self-assembled nanoparticles (blg@tpgs NPs) prepared in example 2 in an acidic environment mimicking tumors.

Fig. 8: the self-assembled nanoparticle (BLG@TPGS NPs), berberine-lonidamine-gambogic acid physical mix group and cytotoxicity (12 h incubation) of gambogic acid on breast cancer cells MDA-MB-231 prepared in example 2.

Fig. 9: the self-assembled nanoparticle (BLG@TPGS NPs), berberine-lonidamine-gambogic acid physical mix group and cytotoxicity (24 h incubation) of gambogic acid on breast cancer cells MDA-MB-231 prepared in example 2.

Fig. 10: the self-assembled nanoparticle (blg@tpgs NPs), berberine-lonidamine-gambogic acid physical mix group and gambogic acid cytotoxicity (24 h incubation) on normal hepatocytes L02 prepared in example 2.

Fig. 11: ATP content measurement results.

Fig. 12: hexokinase activity assay results.

Fig. 13: glutamine content determination results.

Fig. 14: mitochondrial respiratory chain complex type i activity assay results.

Fig. 15: results of examination of mitochondrial targeting ability of self-assembled nanoparticles (blg@tpgs NPs) prepared in example 2.

Fig. 16: and (5) examining the mitochondrial targeting ability of the berberine-lonidamine-gambogic acid physical mixed group.

Fig. 17: mitochondrial membrane potential fluorescence microscopy images.

Detailed Description

The technical scheme of the present invention is further described by the following specific examples, but the following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1

Dissolving the first component and gambogic acid in dimethyl sulfoxide (DMSO) respectively to prepare a mother solution of 10 mg/mL; according to Table 1, the two solutions are fully mixed according to the mass ratio of the first component to gambogic acid to obtain a mixed solution, the mixed solution is slowly dripped into pure water with the temperature of 60 ℃ according to the volume ratio of DMSO to the pure water of 1:9, the mixed solution is stirred while being added, the mixture is placed for 2 hours after the dripping is finished, a dialysis bag (molecular retention of 3000 Da) is filled in the mixture, the mixture is placed in the pure water for dialysis for 12 hours, water is changed every 3 hours, and the organic solvent is removed, so that the self-assembled nanoparticle is obtained, and the self-assembled nanoparticle has opalescence and Tyndall effect.

TABLE 1 preparation conditions of self-assembled nanoparticles

The prepared 12 self-assembled nanoparticles are uniformly distributed in a sphere-like shape. Referring to example 2, "stability of blg@tpgs NPs", stability of 12 self-assembled nanoparticles under physiological conditions was examined for 3 hours, 48 hours, stability was examined by a malvern particle sizer, and as can be seen from table 2, the self-assembled nanoparticles had uniform particle size, 3 hours after preparation, a narrow particle size distribution range, a particle size distribution of 100 to 210nm, and a polydisperse coefficient PDI range of 0.05 to 0.0.28; the particle size distribution of self-contained nano particles is 154-300 nm within 48 hours after preparation, and the polydisperse coefficient PDI range is 0.05-0.22. The self-assembled nanoparticle has good stability within 48 hours.

Cytotoxicity of self-assembled nanoparticles on breast cancer cells 4T1 and human normal hepatocytes L02

Freeze-drying 12 self-assembled nanoparticles; precisely weighing self-assembled nanoparticles, and preparing a solution with the concentration of the nanoparticles of 1mg/mL by using a serum-free DMEM culture medium in an ultrasonic manner. Before the experiment, the self-assembled nanoparticle is diluted into a solution with the concentration of 2.5 mug/mL, 5 mug/mL, 10 mug/mL and 20 mug/mL by fresh DMEM incomplete culture medium under the aseptic condition.

Gambogic acid group: gambogic acid is precisely weighed, gambogic acid solution with the concentration of 10mg/mL is prepared by using dimethyl sulfoxide ultrasonic, and before an experiment, the gambogic acid solution is diluted by fresh DMEM incomplete culture medium under the aseptic condition to obtain gambogic acid gradient solutions with the concentration of 0.5 mug/mL, 1 mug/mL, 2 mug/mL, 4 mug/mL and 7.5 mug/mL.

Taking logarithmic phase breast cancer cell 4T1 and human normal liver cell L02, and collecting cells by pancreatin digestion, wherein the two cells are 5×10 ³ Density of each well was inoculated into 96-well plates, 100 μl of DMEM medium containing 10% fetal bovine serum was added to each well, and 5 parallel wells were set per group. Incubation was performed in an incubator, after 80% of the cells had adhered, the supernatant was carefully aspirated with a pipette and replaced with 100. Mu.L of incomplete medium (blank) in LDMEM or 100. Mu.L of mediumThe DMEM incomplete culture medium (test group) of the samples with different concentrations is continuously incubated for 24 hours, a 96-well plate is taken out, 10 mu L of MTT solution with the concentration of 5mg/mL is added under the light-proof condition, the incubation is continuously carried out for 2 hours under the light-proof condition, the supernatant is carefully discarded, and 100 mu LDMSO is added.

The absorbance value of each well was measured at 492nm using an enzyme-labeled instrument, the results were recorded, and the viability of the cells was calculated according to the following formula.

Cell viability (%) = test group absorbance value/blank group absorbance value ×100%

The cytotoxicity results after 12 self-assembled nanoparticles are incubated with 4T1 and L02 cells for 24 hours are shown in Table 2, and the prepared nanoparticles are proved to have good in vitro cytotoxicity on breast cancer cells 4T 1. In addition, nanoparticles are significantly less toxic to human normal hepatocytes than breast cancer cells, and nanoparticles are more toxic to normal cells than gambogic acid (IC ₅₀ = 6.074 μg/mL) is smaller. The nano particles have low cytotoxicity to normal cells and good biocompatibility and selectivity.

TABLE 2 stability of self-contained nanoparticles and in vitro cytotoxicity results

Example 2

Preparation of BLG NPs:

weighing 1.5mg of Berberine (B), 0.5mg of Lonidamine (L) and 2mg of Gambogic Acid (GA), and dissolving in a mixed solvent of 250 mu L of methanol (MeOH) and 25 mu L of dimethyl sulfoxide (DMSO) to obtain a mixed solution; the mixed solution is added into 3.725mL of pure water preheated to 55 ℃ dropwise, stirred while adding, kept stand for 2h after the dripping is finished, put into a dialysis bag (molecular cut-off of 3000 Da), put into pure water for dialysis for 12h, change water every 3h, remove organic solvent, and obtain nano particles, which are marked as BLG NPs and have opalescence and Tyndall effects. The dynamic light scattering histogram of BLG NPs is shown in FIG. 1, and the average particle size is about 170 nm.

Because gambogic acid and lonidamine are insoluble in water, berberine is insoluble in water, and the prepared nano-particle BLG NPs have strong hydrophobic acting force, so that the BLG NPs cannot be stabilized in water for a long time, and obvious yellow flocculent precipitate is separated out after 36 hours. To improve the stability of BLG NPs, the inventors tried to encapsulate hydrophilic vitamin E polyethylene glycol succinate outside the nanoparticle BLG NPs to prepare blg@tpgs NPs.

Preparation of BLG@TPGS NPs:

weighing 1.5mg of berberine (B), 0.5mg of lonidamine (L), 2mg of Gambogic Acid (GA) and 0.8mg of vitamin E polyethylene glycol succinate (the molecular weight of the contained polyethylene glycol is 2000), and dissolving in a mixed solvent of 290 mu L of methanol (MeOH) and 25 mu L of dimethyl sulfoxide (DMSO) to obtain a mixed solution; and (3) dropwise adding the mixed solution into 3.685mL of pure water preheated to 55 ℃, stirring while adding, standing for 2 hours after the dropwise adding is finished, filling into a dialysis bag (molecular retention of 3000 Da), dialyzing in the pure water for 12 hours, changing water every 3 hours, and removing an organic solvent to obtain self-assembled nanoparticles with mitochondrial targeting combined metabolism blocking effect based on natural products, namely BLG@TPGS NPs, wherein the self-assembled nanoparticles have opalescence.

Preliminary determinations of blg@tpgs NPs were made by the tyndall effect, as shown in fig. 2, blg@tpgs NPs had a significant tyndall effect.

The dynamic light scattering histogram of BLG@TPGS NPs is shown in FIG. 3, and the average particle size is about 200nm, and the particle size is relatively small; the particle size of the blg@tpgs NPs is slightly increased compared to BLG NPs due to the encapsulation of the hydrophilic shell. The PDI value of BLG@TPGS NPs is about 0.15, which indicates that the particle size of the nanoparticles is high at one time.

The morphology of the BLG@TPGS NPs is observed by adopting a transmission electron microscope, and the method specifically comprises the following steps: and (3) dripping 10 mu LBLG@TPGS NPs liquid into a copper net, settling for 10min, dripping one drop of phosphotungstic acid into the liquid to dye the sample, and observing the morphological characteristics of the sample by adopting a transmission electron microscope. As shown in fig. 4, the appearance of blg@tpgs NPs is spherical, and has an obvious core-shell structure and uniform morphology.

Example 3

Stability of BLG@TPGS NPs and drug release experiments

(1) The stability of blg@tpgs NPs in physiological state was studied using phosphate buffer (PBS buffer, ph=7.4) to simulate physiological environment.

The blg@tpgs NPs liquid prepared in example 2 was taken and placed in 10 volumes of PBS buffer, and the change in particle size of the nanoparticles incubated in this environment was monitored by dynamic light scattering using a malvern particle sizer at different time points for 48 h.

The stability test results are shown in FIG. 5, and in the PBS buffer solution, the particle size of the BLG@TPGS NPs does not change obviously within 48 hours, which indicates that the BLG@TPGS NPs have good stability.

(2) The in vitro drug release condition of BLG@TPGS NPs is studied by adopting a dialysis method.

Taking the BLG@TPGS NPs prepared in example 2, adding Tween 80 to ensure that the concentration of the BLG@TPGS NPs is 1mg/mL and the volume fraction of the Tween 80 is 0.1%; transferring to dialysis bags (mwco=3000 Da), immersing the dialysis bags in 100mL of PBS buffer solutions with different pH (pH 6.5 and pH 7.4), incubating at 37 ℃ in a shaker at a rotation speed of 200rpm; the media in the 20 μl dialysis bag were removed at 0h, 0.25h, 0.5h, 1h, 3.5h, 5.5h, 8h, 12h, 24h, respectively, and replenished with an equal amount of fresh media. The 20. Mu.L of the medium was taken out and added with 5-fold volume chromatography methanol to break the emulsion, and the release amounts of berberine, lonidamine and gambogic acid in the samples were measured by HPLC.

HPLC conditions: mobile phase: 0.1% glacial acetic acid (mobile phase a) -methanol (mobile phase B); elution conditions: 20% mobile phase B; flow rate: 1mL/min; detection wavelength: 298nm; sample injection volume: 10 mu L.

Drug release results showed that blg@tpgs NPs released at a slower rate under physiological conditions (PBS buffer, ph=7.4, fig. 6) and under weak acid conditions (PBS buffer, ph=6.5, fig. 7). This prolongs the nanoparticle circulation time to some extent, avoiding premature release of the drug. In addition, the characteristic of BLG@TPGS NPs is beneficial to the therapeutic effect of the BLG@TPGS NPs on tumor sites because the tumor microenvironment presents weak acidity.

Example 4

Evaluation of in vitro inhibition effect of BLG@TPGS NPs on growth of breast cancer cells

Control group: gambogic acid, berberine-lonidamine-gambogic acid physical mixture group.

After freeze-drying the BLG@TPGS NPs prepared in example 2, precisely weighing the BLG@TPGS NPs, and preparing a solution with the nanoparticle concentration of 1mg/mL by ultrasonic treatment with a serum-free DMEM medium. Before the experiment, the solution with BLG@TPGS concentration of 1. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL, 8. Mu.g/mL and 15. Mu.g/mL was diluted with fresh DMEM incomplete medium under aseptic conditions.

Berberine-lonidamine-gambogic acid physical Mix group (Mix): precisely weighing berberine, lonidamine and gambogic acid, and respectively preparing into berberine solution with concentration of 10mg/mL, lonidamine solution with concentration of 10mg/mL and gambogic acid solution with concentration of 10mg/mL by using dimethyl sulfoxide ultrasound. Before the experiment, the berberine gradient solution with the concentration of 1.125 mug/mL, 2.25 mug/mL, 4.5 mug/mL, 9 mug/mL, 16.875 mug/mL, 0.375 mug/mL, 0.75 mug/mL, 1.5 mug/mL, 3 mug/mL, 5.625 mug/mL and the lonidamine gradient solution with the concentration of 1.5 mug/mL, 3 mug/mL, 6 mug/mL, 12 mug/mL and 22.5 mug/mL are respectively diluted by fresh DMEM incomplete culture medium under the sterile condition; mixing berberine gradient solution, lonidamine gradient solution and gambogic acid gradient solution according to a volume ratio of 1:1:1, for example: the berberine gradient solution with the concentration of 1.125 mug/mL and the lonidamine gradient solution with the concentration of 0.375 mug/mL are mixed according to the volume ratio to obtain the physical mixed solution of berberine-lonidamine-gambogic acid with the total concentration of 1 mug/mL, and the physical mixed solution of berberine-lonidamine-gambogic acid with the total concentration of 1 mug/mL, 2 mug/mL, 4 mug/mL, 8 mug/mL and 15 mug/mL is obtained by analogy.

Taking the logarithmStage breast cancer cells MDA-MB-231 and human normal hepatocytes L02, and collecting cells by pancreatin digestion, and then culturing at 5×10 ³ Density of each well was inoculated into 96-well plates, 100 μl of DMEM medium containing 10% fetal bovine serum was added to each well, and 5 parallel wells were set per group. After 80% of the cells are attached to the wall, carefully sucking the supernatant by a pipette, replacing the supernatant with 100 mu LDMEM incomplete medium (blank group) or 100 mu L DMEM incomplete medium (test group) containing samples with different concentrations, continuously incubating for 12h and 24h respectively, taking out the 96-well plate, adding 10 mu L MTT solution with the concentration of 5mg/mL under the dark condition, continuously incubating for 2h under the dark condition, carefully discarding the supernatant, and adding 100 mu LDMSO.

Cytotoxicity results after 12h incubation of drug with MDA-MB-231 cells are shown in FIG. 8, IC ₅₀ About 15.88. Mu.g/mL.

The cytotoxicity results after the drug and MDA-MB-231 cells are incubated for 24 hours are shown in FIG. 9 and Table 3, and the Gambogic Acid (GA), the berberine-lonidamine-gambogic acid physical mixture (Mix) and the berberine-lonidamine-gambogic acid self-assembly nanoparticle (BLG@TPGS NPs) have cytotoxicity on breast cancer cells MDA-MB-231 in vitro. In particular, after self-assembled nanoparticles (BLG@TPGS NPs) are formed, cytotoxicity to cancer cells is enhanced, and better anti-tumor activity is achieved.

TABLE 3 toxicity results of the drugs on Normal hepatocytes L02 and breast cancer cells MDA-MB-231 (24 h incubation)

FIG. 10 shows that the berberine-lonidamine-gambogic acid self-assembled nanoparticle (BLG@TPGS NPs) still maintains more than 70% of cell survival rate to normal liver cancer cells of human beings at the same administration concentration as breast cancer cells. The BLG@TPGS NPs are low in cytotoxicity to normal cells and good in biocompatibility.

Example 5

Inhibition of in vitro metabolism

After freeze-drying the BLG@TPGS NPs prepared in example 2, precisely weighing the BLG@TPGS NPs, and preparing a solution with the nanoparticle concentration of 1mg/mL by ultrasonic treatment with a serum-free DMEM medium. Before the experiment, the solution was diluted to a concentration of BLG@TPGS of 5. Mu.g/mL in fresh DMEM incomplete medium under aseptic conditions.

Berberine-lonidamine-gambogic acid physical mix group (mix): precisely weighing berberine, lonidamine and gambogic acid, and respectively preparing into berberine solution with concentration of 10mg/mL, lonidamine solution with concentration of 10mg/mL and gambogic acid solution with concentration of 10mg/mL by using dimethyl sulfoxide ultrasound. Before the experiment, the berberine gradient solution with the concentration of 5.625 mug/mL and the lonidamine gradient solution with the concentration of 1.875 mug/mL and the gambogic acid gradient solution with the concentration of 7.5 mug/mL are respectively diluted by fresh DMEM incomplete culture medium under the aseptic condition; and mixing the berberine gradient solution, the lonidamine gradient solution and the gambogic acid gradient solution according to the volume ratio of 1:1:1 to obtain physical mixed solutions of berberine-lonidamine-gambogic acid with the total concentration of 5 mug/mL respectively.

a) Intracellular Adenosine Triphosphate (ATP) content determination

Taking logarithmic phase human breast cancer cells MDA-MB-231, collecting cells by pancreatin digestion, and collecting cells at 1×10 ⁶ The density of each well was inoculated into a 6-well plate and cultured for 12 hours. The medium was removed, 1mL of buffer (ph=7.4) was added as a Control, physical mixture of berberine-lonidamine-gambogic acid (5 μg/mL), blg@tpgs NPs (5 μg/mL), and incubated for 12h under serum-free conditions, and intracellular ATP content was determined using ATP Assay Kit (beyotide).

As shown in fig. 11, blg@tpgs NPs were able to significantly reduce intracellular ATP levels, cutting off tumor cell energy sources.

b) Hexokinase Activity assay

The metabolic inhibitor lonidamine can be combined with hexokinase to inhibit the activity of hexokinase, thereby inhibiting the conversion of glucose into glucose 6 phosphate and blocking the glycolytic pathway.

Taking logarithmic phase human breast cancer cells MDA-MB-231, collecting cells by pancreatin digestion, and collecting cells at 1×10 ⁶ The density of each well was inoculated into a 6-well plate and cultured for 12 hours. The medium was removed, 1mL of buffer (ph=7.4, control as Control), physical berberine-lonidamine-gambogic acid mix (5 μg/mL), blg@tpgs NPs (5 μg/mL) were added to each well, incubated for 12h under serum-free conditions, and enzyme activity was determined using hexokinase activity assay kit (Hexokinase Colorimetric Assay Kit, abcam).

As shown in fig. 12, blg@tpgs NPs were able to significantly inhibit hexokinase activity.

c) Intracellular glutamine assay

Glutamine is the main source of alpha-ketoglutarate, an intermediate of the TCA cycle, and is an important substance for nucleic acid and other amino acid synthesis and energy production. In particular, in cancer cells, glutamine as a substrate promotes glutamine metabolism to supply energy.

Taking breast cancer cells MDA-MB-231 in logarithmic phase, and collecting cells by pancreatin digestion at 1×10 ⁶ The density of each well was inoculated into a 6-well plate and cultured for 12 hours. The medium was removed, 1mL of buffer (ph=7.4, control as Control), physical berberine-lonidamine-gambogic acid mix (5 μg/mL), blg@tpgs NPs (5 μg/mL) were added to each well, incubated for 12h under serum-free conditions, and intracellular glutamine was measured using glutamine detection kit (Glutamine Assay Kit-WST, dojindo).

As shown in fig. 13, blg@tpgs NPs were able to significantly reduce intracellular glutamine levels.

d) Activity measurement of intracellular mitochondrial respiratory chain complex I

Taking breast cancer cells MDA-MB-231 in logarithmic phase, and collecting cells by pancreatin digestion at 1×10 ⁶ The density of each well was inoculated into a 6-well plate and cultured for 12 hours. The medium was removed, 1mL of buffer (ph=7.4), physical mixing of berberine-lonidamine-gambogic acid (5 μg/mL), blg@tpgs NPs (5 μg/mL) were added to each well, incubated for 12h under serum-free conditions, and the mitochondrial respiratory chain complex i activity detection reagent was usedThe kit (mitochondrial complex I activity detection kit, solarbio) determines intracellular mitochondrial respiratory chain complex I activity.

As shown in fig. 14, blg@tpgs NPs were able to significantly reduce intracellular mitochondrial respiratory chain complex I activity.

Example 6

Mitochondrial targeting ability investigation

The preparation of the physical mixture of BLG@TPGS solution, berberine-lonidamine-gambogic acid was the same as in example 5.

The distribution of the nanoparticle and the physical mixed group in the cell can be judged by observing the green fluorescence of the berberine (BBR channel) by utilizing the self-fluorescence signal of the berberine in the nanoparticle and the physical mixed group.

Taking breast cancer cells MDA-MB-231 in logarithmic phase, and collecting cells by pancreatin digestion at 1×10 ⁶ The density of each well was inoculated into a 6-well plate and cultured for 12 hours. The medium was removed, 1mL of buffer (ph=7.4), physical mixture of berberine-lonidamine-gambogic acid (5 μg/mL), blg@tpgs NPs (5 μg/mL) were added to each well, and after incubation for 12h in serum-free conditions, the cells were washed 3 times with PBS. Mitotacker with 50nM concentration of commercial mitochondrial dye ^TM Red dyes mitochondria (Red fluorescence) for 15min, hoechst333442 dyes nuclei (blue fluorescence) for 5min, and then a fluorescence microscope is used for observing a berberine fluorescence channel (green fluorescence), so as to ascertain the distribution condition of the drug in the cells after entering the tumor cells.

BBR represents the green fluorescent signal of the nanoparticle or physical mixture group, mitosracker Red represents the Red fluorescent signal of the cell mitochondria, hoechst represents the blue fluorescent signal of the cell nucleus, and Merge represents the coincidence of the three fluorescent signals. If obvious yellow appears, namely, the superposition of red and green fluorescent signals, the nanoparticle can target mitochondria; if the yellow fluorescence is weak or the red and green fluorescence signals do not overlap, this indicates that the preparation does not have the ability to target mitochondria.

As shown in fig. 15, the nanoparticle group clearly sees that the nanoparticle group that shows green fluorescence (BBR channel) coincides with the mitochondria that shows Red fluorescence (Mitotracker Red channel), and the nanoparticle group shows yellow after the coincidence (mere). The yellow fluorescence of the nanoparticle group was stronger and the overlap of the red and green fluorescence signals was higher compared to the physical mix group (fig. 16), indicating that blg@tpgs NPs could target mitochondria effectively.

Example 7

Mitochondrial membrane potential investigation

After freeze-drying the BLG@TPGS NPs prepared in example 2, precisely weighing the BLG@TPGS NPs, and preparing a solution with the nanoparticle concentration of 1mg/mL by ultrasonic treatment with a serum-free DMEM medium. Before the experiment, the culture medium was diluted with fresh DMEM incomplete medium to obtain BLG@TPGS gradient solutions with a concentration of 1. Mu.g/mL and 2. Mu.g/mL, respectively, under aseptic conditions.

Berberine-lonidamine-gambogic acid physical mix group (mix): precisely weighing berberine, lonidamine and gambogic acid, and respectively preparing into berberine solution with concentration of 10mg/mL, lonidamine solution with concentration of 10mg/mL and gambogic acid solution with concentration of 10mg/mL by using dimethyl sulfoxide ultrasound. Before the experiment, the berberine gradient solution with the concentration of 1.125 mug/mL and 2.25 mug/mL and the lonidamine gradient solution with the concentration of 0.375 mug/mL and 0.75 mug/mL and the gambogic acid gradient solution with the concentration of 1.5 mug/mL and 3 mug/mL are respectively diluted by fresh DMEM incomplete culture medium under the aseptic condition; the berberine gradient solution with the concentration of 1.125 mug/mL, the lonidamine gradient solution with the concentration of 0.375 mug/mL and the gambogic acid gradient solution with the concentration of 1.5 mug/mL are mixed according to the volume ratio of 1:1:1, the berberine gradient solution with the concentration of 2.25 mug/mL, the lonidamine gradient solution with the concentration of 0.75 mug/mL and the gambogic acid gradient solution with the concentration of 3 mug/mL are mixed according to the volume ratio of 1:1:1, and the physical mixed solution of the berberine-lonidamine-gambogic acid with the total concentration of 1 mug/mL and 2 mug/mL is respectively obtained.

Taking breast cancer cells MDA-MB-231 in logarithmic phase, and collecting cells by pancreatin digestion at 1×10 ⁶ The density of each well was inoculated into a 6-well plate and cultured for 12 hours. The medium was removed and 1mL of buffer (ph=7.4), physical mixture of berberine-lonidamine-gambogic acid (1 μg/mL, 2 μg/mL), blg@tpgs NPs (1 μg/mL, 2 μg/mL), CCCP (carbonyl cyanide-3-chlorophenyl hydrazone, 10 mM) were added to each well, and the cells were incubated for 12h in serum-free conditions and washed 3 times with PBS. Benefit (benefit)Mitochondrial membrane potential changes under different drug treatments were measured using a mitochondrial membrane potential detection kit (JC-1, beyotime). At higher mitochondrial membrane potentials, JC-1aggregates in the matrix of mitochondria (matrix), forming polymers (JC-1 aggregates), which can produce red fluorescence; at low mitochondrial membrane potential, JC-1 cannot aggregate in the matrix of mitochondria, and JC-1 is a monomer (JC-1 monomer) and green fluorescence can be generated. Merge is the overlap of JC-1aggregate state and JC-1monomer fluorescence signal, in which the degree of mitochondrial depolarization is measured by the relative proportion of red-green fluorescence. That is, the transition of JC-1 from the aggregated state (red fluorescence) to the monomer (green fluorescence) can be used as a detection index for mitochondrial membrane potential and early apoptosis.

CCCP is a positive control group, namely the mitochondrial membrane potential of the cells after CCCP treatment is obviously reduced, and the cells are shown to have larger red fluorescence ratio; control group was a Control group given only PBS (ph=7.4), where JC-1 was present in the form of polymer in the cell mitochondria and was brightly red fluorescent and very weak in green fluorescent.

As shown in fig. 17, the proportion of green fluorescence in the blg@tpgs NPs group was greater than that in the other two groups, and dose-dependency was exhibited, as compared to the CCCP positive control group and the PBS blank group. Indicating that the mitochondrial membrane potential of the tumor cells after BLG@TPGS NPs treatment is reduced, and early apoptosis is shown.

Example 8

Weighing 0.5mg of berberine (B), 0.5mg of lonidamine (L) and 2mg of Gambogic Acid (GA) and dissolving in a mixed solvent of 100 mu L of methanol (MeOH) and 25 mu L of dimethyl sulfoxide (DMSO) to obtain a mixed solution; and (3) dropwise adding 2.875mL of the mixed solution into pure water preheated to 55 ℃, stirring while adding, standing for 2 hours after the dropwise adding is finished, filling into a dialysis bag (molecular cut-off of 3000 Da), dialyzing for 12 hours in the pure water, changing water every 3 hours, and removing the organic solvent to obtain the BLG NPs with opalescence and Tyndall effect.

Example 9

1mg of berberine (B), 0.5mg of lonidamine (L) and 2mg of Gambogic Acid (GA) are weighed and dissolved in a mixed solvent of 200 mu L of methanol (MeOH) and 25 mu L of dimethyl sulfoxide (DMSO) to obtain a mixed solution; the mixed solution is added into 3.275mL of pure water preheated to 55 ℃ dropwise, stirred while adding, kept stand for 2h after the dripping is finished, put into a dialysis bag (molecular retention of 3000 Da), put into pure water for dialysis for 12h, change water every 3h, and remove organic solvent, thus obtaining BLG NPs with opalescence and Tyndall effect.

Example 10

Weighing 2.5mg of berberine (B), 0.5mg of lonidamine (L) and 2mg of Gambogic Acid (GA) and dissolving in a mixed solvent of 250 mu L of methanol (MeOH) and 25 mu L of dimethyl sulfoxide (DMSO) to obtain a mixed solution; the mixed solution is added into 3.725mL of pure water preheated to 55 ℃ dropwise, stirred while adding, kept stand for 2h after the dripping is finished, put into a dialysis bag (molecular retention of 3000 Da), put into pure water for dialysis for 12h, change water every 3h, and remove organic solvent, thus obtaining BLG NPs with opalescence and Tyndall effect.

Claims

1. A natural product-based self-assembled nanoparticle characterized by: the self-assembled nanoparticle is prepared from a first component and a second component, wherein the first component is triterpene, anthraquinone, flavonoid, alkaloid, polyphenol, coumarin or lignan compounds except gambogic acid, and the second component is gambogic acid, and the gambogic acid induces the self-assembly of the first component to form the nanoparticle;

the preparation method comprises the following steps: respectively dissolving the first component and gambogic acid in a benign organic solvent, and then mixing the two solutions according to the mass ratio of the first component to the gambogic acid of 2:1-1:2 to obtain a mixed solution, or dissolving the first component and the gambogic acid in the benign organic solvent according to the mass ratio of 2:1-1:2 to form a mixed solution; dripping the mixed solution into pure water with the constant temperature of 50-60 ℃ while stirring, standing after dripping, and removing the organic solvent by dialysis to obtain self-assembled nanoparticles;

wherein the triterpene compound is artemisinin, dihydroartemisinin, oleanolic acid, and glycyrrhizic acid; the anthraquinone compound is rhein and hypericin; the flavonoid compound is quercetin; the alkaloid compound is berberine; the polyphenol compound is curcumin; the coumarin compound is coumarin; the lignanoid compound is magnolol and honokiol;

the benign organic solvent is dimethyl sulfoxide; the volume ratio of benign organic solvent to pure water was 1:9.

2. A self-assembled nanoparticle with mitochondrial targeting for joint metabolic blocking based on natural products, characterized in that: the nano-particle is prepared by taking berberine, lonidamine, gambogic acid and vitamin E polyethylene glycol succinate as raw materials, self-assembling berberine, lonidamine and gambogic acid to prepare nano-particles, and wrapping the vitamin E polyethylene glycol succinate outside the nano-particles;

the preparation method comprises the following steps: dissolving berberine, lonidamine, gambogic acid and vitamin E polyethylene glycol succinate in benign organic solvent to form mixed solution, or dissolving berberine, lonidamine, gambogic acid and vitamin E polyethylene glycol succinate in benign organic solvent to form solution respectively, and mixing the solutions to form mixed solution; dripping the mixed solution into pure water with the constant temperature of 50-60 ℃, stirring during dripping, standing after dripping, dialyzing to remove the organic solvent, and preparing the nano particles;

wherein the mass ratio of the berberine to the lonidamine to the gambogic acid is (1-5) 1:4;

the mass ratio of the total amount of berberine, lonidamine and gambogic acid to the vitamin E polyethylene glycol succinate is 5:1;

the mass volume ratio of the vitamin E polyethylene glycol succinate to the benign organic solvent is 2:1-3:1 mg/mL;

the benign organic solvent is a mixed solvent of dimethyl sulfoxide and methanol in a volume ratio of 1:4-1:12;

the volume ratio of the benign organic mixed solvent to the pure water is 1:10-1:25;

the dialysis bag used for dialysis had a molecular weight cut-off of 3000Da.

3. Use of the nanoparticle according to claim 1 or 2 for the preparation of an antitumor drug.

4. A use according to claim 3, characterized in that: the tumor is lung cancer, liver cancer and breast cancer.

5. Use of the nanoparticle of claim 2 for the preparation of a mitochondria-targeted antitumor drug that blocks the energy supply of tumor cells and/or inhibits the growth of tumor cells.

6. The use according to claim 5, characterized in that: the tumor is lung cancer, liver cancer and breast cancer.