CN120661678A - A natural product-based mitochondrial-targeted self-assembly PROTAC nanomaterial and its preparation method and application - Google Patents

Abstract

The invention discloses a natural product-based mitochondrion-targeted self-assembled PROTAC nanometer material, which is a nanoparticle prepared by a nano precipitation method by taking a mitochondrion-targeted molecule, a photosensitizer and PROTAC as raw materials, wherein the mitochondrion-targeted molecule is berberine, the photosensitizer is hypericin, PROTAC is dBET57, the mass ratio of the dBET57 to the berberine is (1-10): 1-10, and the mass ratio of the dBET57 to the hypericin is (1-10). The nano material can be used for blocking a plurality of energy metabolism pathways of tumor cells, including glycolysis and oxidative phosphorylation, damaging mitochondria, inducing iron death and improving the anti-tumor capability of pharmacodynamic molecules, and can efficiently degrade BRD4 and downstream oncogenic protein c-Myc thereof in the tumor cells. The invention also discloses application of the nano material in preparing antitumor drugs.

Description

Mitochondria-targeted self-assembled PROTAC nano material based on natural product, and preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical preparations, relates to a natural product mitochondrion-targeted self-assembled PROTAC nanometer material, a preparation method and an application thereof, and in particular relates to a self-assembled nanoparticle with a mitochondrial-targeted self-assembled PROTAC nanometer material combined photodynamic therapy/metabolism blocking effect, and a preparation method and an application thereof.

Background

At present, the first-line clinical antitumor treatment means mainly comprise surgery, chemotherapy, radiotherapy, targeted therapy and immunotherapy. However, the traditional radiotherapy and chemotherapy drugs may be unevenly distributed in the body, and the tumor targeting is poor, so that serious systemic side effects are usually caused for patients, and the quality of life is greatly reduced. Thus, people are beginning to focus on botanical anticancer drugs. Natural products are abundant in source, generally have better biocompatibility, lower toxicity and higher selectivity. A great deal of researches show that various photosensitive substances exist in natural products and derivatives thereof, such as tetrapyrroles, polyphenols, anthraquinone, thiophene, perylenequinone compounds and the like, and the chemical structure diversity provides abundant choices for developing photodynamic therapy (Photodynamic therapy, PDT) photosensitizers. However, the natural products have poor water solubility, low bioavailability and poor patentability, so that further clinical application of the natural products is limited.

The technology of proteolytic targeting chimera (Proteolysis-TARGETING CHIMERAS, PROTAC) is an emerging targeting protein degradation strategy, which induces ubiquitination and subsequent proteasome degradation of specific proteins by small molecule compounds, and can realize continuous protein degradation even under the condition of low drug concentration, and remarkably improve the curative effect. PROTACs are expected to overcome some of the limitations of conventional small molecule inhibitors. However, its practical application is still limited by low bioavailability caused by poor water solubility and off-target toxicity caused by lack of target organ specificity, and there is a need for improvement in the design of intelligent drug delivery systems.

In recent years, with the progress and development of nanomaterial chemistry, nano-drug delivery systems are often used to improve drug formation, extend the in vivo circulation time of drugs, and impart active or passive targeting effects to drugs. Among other things, organelle targeted delivery strategies represent a great potential in cancer treatment. Mitochondria act as an "energy factory" for cells, maintaining the normal physiological function of organisms by providing ATP, and thus nanomedicine delivery systems for simultaneously targeting and damaging mitochondria-induced cell death are highly desirable in cancer treatment. However, the traditional carrier-assisted nano-drug delivery system still has the problems of low drug loading, poor stability and the like, so that the function and the final efficiency of the traditional carrier-assisted nano-drug delivery system in clinical application are still not satisfactory. The carrier-free self-assembled nanoparticles are mainly self-assembled by prodrugs, pure drugs (such as single drugs or multiple drugs) or amphiphilic drug conjugates, and have higher drug loading, longer blood circulation time and lower systemic toxicity compared with the prior art.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a natural product-based mitochondrion-targeted self-assembled PROTAC nano material, a preparation method and application thereof, wherein the nano material is nanoparticles prepared by self-assembling berberine, hypericin and dET 57 in a water system by a nano precipitation method. The nanoparticle can be enriched at a tumor part, target mitochondria, and kill tumor cells cooperatively by hunger therapy, photodynamic therapy and targeted protein degradation technology, thereby improving the application of natural products and PROTAC in the field of tumor treatment.

The technical scheme of the invention is as follows:

A self-assembled PROTAC nanometer material based on natural product mitochondrion targeting is prepared from mitochondrion targeting molecule, photosensitizer and PROTAC as raw materials by nanometer precipitation method.

Wherein the mitochondrial targeting molecule is berberine, the photosensitizer is hypericin, and PROTAC is dBET57 (CAS number: 1883863-52-2).

The mass ratio of the dET 57 to the berberine is (1-10): (1-10), preferably 0.8:1-1.5:1, and most preferably 1:1. The ratio of the dET 57 to hypericin is (1-10): (1-10), preferably 1:0.8-1:1.5, more preferably 1:1. According to the ratio of the amounts of the substances, the nanoparticles can be prepared, and according to a preferred scheme, the nanoparticles with average particle diameters of about 110-115 nm and high average particle diameters of about 0.13 of polydispersion coefficient (PDI) can be prepared.

The berberine, hypericin and dBET57 are self-assembled to form nano particles mainly through hydrogen bond interaction, pi-pi stacking and various hydrophobic interactions.

The natural product-based mitochondria-targeted self-assembled PROTAC nanometer material has a uniform spherical appearance.

The nano precipitation method is to dissolve berberine, hypericin and dBET57 in benign organic solvent to form mixed solution, or dissolve berberine, hypericin and dBET57 in benign organic solvent to prepare berberine solution, hypericin solution and dBET57 solution respectively, then mix the three solutions to form mixed solution, drop the mixed solution into water at room temperature, stir the mixed solution during drop, stand, ultrafiltrate or dialyze to remove organic solvent after drop, and obtain nano particles.

The invention further aims to provide a preparation method of the natural product-based mitochondrion-targeted self-assembled PROTAC nanometer material, which comprises the steps of dissolving berberine, hypericin and dBET57 in benign organic solvents to form a mixed solution, or respectively dissolving berberine, hypericin and dBET57 in benign organic solvents to prepare berberine solution, hypericin solution and dBET57 solution, mixing the three solutions to form the mixed solution, dripping the mixed solution into water at room temperature, stirring during dripping, standing, and removing the organic solvents through ultrafiltration or dialysis to obtain the nanoparticle.

In the mixed solution, the dosage ratio of dBET57 to benign organic solvent is 1:25-1:35 mol/L, preferably 1:33mol/L.

The benign organic solvent is one or more of dimethyl sulfoxide, absolute ethyl alcohol, methanol or tetrahydrofuran, preferably a mixed solvent with the volume ratio of dimethyl sulfoxide to methanol being 4:1-10:1, and more preferably a mixed solvent with the volume ratio of dimethyl sulfoxide to methanol being 10:1.

The water is used in an amount greater than that of the benign organic solvent. Generally, the volume ratio of benign organic solvent to water is 1:10 to 1:50, preferably 1:40 to 1:50, more preferably 1:45 to 1:46.

The standing time is generally 0.5-3 hours.

The molecular weight cut-off of the ultrafiltration tube used for ultrafiltration is 1000-10000 Da, preferably 3500Da.

The room temperature of the invention is 25+/-5 ℃.

The natural product mitochondrion-based targeted self-assembled PROTAC nano material can improve the bioavailability of pharmacodynamic molecules, including improving the internal circulation time, accurately targeting tumor mitochondria, cell uptake and intracellular site release, can be used for blocking a plurality of energy metabolism pathways of tumor cells, including glycolysis and oxidative phosphorylation, damaging mitochondria at the same time, inducing iron death and improving the anti-tumor capability of pharmacodynamic molecules, and can efficiently degrade BRD4 and downstream oncogenic protein c-Myc thereof in the tumor cells by utilizing PROTAC strategy. The inventor carries out in-vitro and in-vivo anti-triple negative breast cancer activity evaluation, shows that the natural product mitochondrion-targeted self-assembled PROTAC nano material can obviously inhibit proliferation of triple negative breast cancer cells MDA-MB-231, has obvious anticancer activity and excellent tumor targeting on breast cancer, can induce iron death while damaging mitochondria to block tumor energy supply, and cooperatively amplifies the anti-tumor activity. Therefore, another object of the invention is to provide the application of the natural product-based mitochondria-targeted self-assembled PROTAC nanometer material in preparing antitumor drugs.

The invention also aims to provide the application of the natural product-based mitochondria-targeted self-assembled PROTAC nanometer material in preparing anti-tumor drugs for photodynamic therapy and cooperative starvation therapy.

The invention also aims to provide the application of the natural product-based mitochondria-targeted self-assembled PROTAC nanometer material in preparing a mitochondria-targeted antitumor drug for cooperatively blocking energy supply of tumor cells and inhibiting growth of the tumor cells by photodynamic therapy.

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

The breast cancer is triple negative breast cancer.

The natural product-based mitochondria-targeted self-assembled PROTAC nano-material has the advantages of a carrier-free nano-drug delivery system (such as passive targeting capability, prolonged in vivo circulation time, high drug loading, improved pharmacokinetic behavior and the like), and has the following 4 advantages:

1) Compared with free medicines or physical mixing, in-vitro cytotoxicity experiments show that the nanoparticles can obviously inhibit proliferation of tumor cells such as breast cancer and the like under the irradiation of near infrared laser, and have obvious anticancer activity;

2) Compared with a physical mixed group, in the aspect of targeting, the nanoparticle not only has EPR (enhanced permeability and retention) effect of a dosage form, but also can accurately target the mitochondria of tumor subcellular organelles, thereby laying a foundation for starvation therapy;

3) In the aspect of energy metabolism, the nano preparation with the combined metabolism blocking effect can inhibit glycolysis and oxidative phosphorylation, and is used for cooperatively treating PROTAC/PDT, and can target and degrade BRD4 protein to inhibit c-Myc expression while damaging mitochondria, so that the energy supply of tumors is comprehensively cut off, and tumor cells are killed;

4) In the aspect of pharmacy, the advantages of the nanometer dosage form are utilized to improve the bioavailability of the photosensitizer and PROTAC, including improving tumor accumulation, amplifying treatment effect and reducing toxic and side effects on normal tissues.

Drawings

FIG. 1 shows a dynamic light scattering histogram of nanoparticles (BHP NPs) prepared in example 1.

FIG. 2 is a diagram showing the Tyndall effect of self-assembled nanoparticles (BHP NPs) prepared in example 1.

FIG. 3 is a Transmission Electron Microscope (TEM) image of self-assembled nanoparticles (BHP NPs) prepared in example 1.

FIG. 4 comparison of particle size of self-assembled nanoparticles (BHP NPs) of berberine, hypericin and dBET57 in different molar ratios.

FIG. 5 shows the results of stability investigation of self-assembled nanoparticles (BHP NPs) prepared in example 1, wherein PBS represents the particle size change of BHP NPs in PBS and FBS represents the particle size change of BHP NPs in PBS containing 10% FBS.

FIG. 6 shows the results of molecular dynamics simulation of self-assembled nanoparticles (BHP NPs) prepared in example 1.

FIG. 7 cytotoxicity (24 h incubation) of the self-assembled nanoparticle (BHP NPs) and berberine-hypericin-dBET 57 physical mixture group prepared in example 1 on breast cancer cells MDA-MB-231 in the absence of light.

FIG. 8 shows the measurement results of ATP content.

FIG. 9 shows the results of measurement of lactic acid content.

FIG. 10 shows the results of measurement of mitochondrial respiratory chain complex I activity.

FIG. 11 shows the results of examination of mitochondrial targeting ability of self-assembled nanoparticles (BHP NPs) prepared in example 1.

FIG. 12 results of mitochondrial targeting in berberine-hypericin-dBET 57 physical blends.

FIG. 13 shows the degradation effects of the self-assembled nanoparticles (BHP NPs), free drug and berberine-hypericin-dBET 57 physical mixture prepared in example 1 on the target proteins BRD4 and c-Myc in the triple negative breast cancer MDA-MB-231 in the absence of light.

FIG. 14 shows morphology observations of iron-death-induced organelles of self-assembled nanoparticles (BHP NPs) prepared in example 1.

FIG. 15 is a graph showing the results of examining the ability of self-assembled nanoparticles (BHP NPs) prepared in example 1 to induce iron death.

FIG. 16 is the weight effect of the self-assembled nanoparticle (BHP NPs), free drug, and berberine-hypericin-dBET 57 physical mixture prepared in example 1 on MDA-MB-231 tumor-bearing nude mice in the absence of light.

FIG. 17 shows the effect of the physical mixture of self-assembled nanoparticles (BHP NPs), free drug and berberine-hypericin-dBET 57 prepared in example 1 on tumor suppression rate of MDA-MB-231 tumor-bearing nude mice in the absence of light.

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

The mitochondrial targeting self-assembled PROTAC nanometer material (marked as BHP NPs) based on natural products is prepared by the following preparation method, and the specific process is as follows:

The berberine 3.36mg (Berberine, BBR,0.01 mmol), hypericin 5.04mg (HYPERICIN, HPC,0.01 mmol) and dBET 57.00 mg (0.01 mmol) are weighed according to the mol ratio of berberine, hypericin and dBET57 of 1:1, dissolved in 300 mu L of dimethyl sulfoxide (DMSO) and 30 mu L of methanol (MeOH) to obtain a mixed solution, the mixed solution is dropwise added into 15.07mL of pure water at room temperature, the mixed solution is stirred while being dropwise added, after the dropwise addition is finished, the mixed solution is kept stand for 2h, and then is added into a ultrafilter tube (molecular cut-off 3500 Da), the solution is centrifuged for 15min at 2000rpm, the organic solvent is removed, the berberine-hypericin-dBET 57 self-assembled nanoparticle (1 mg/mL) is obtained by re-suspending with 15.4mL of pure water, a dynamic light scattering histogram of BHP NPs is shown in a graph of FIG. 1, the Average particle size (Z-Average particle size) of P NPs is about 111.51nm, and the particle size of PDP NPI is about 0.13 ℃ is higher than that of the Average particle size of NPI. The nanoparticles also have opalescence and tyndall effects (see fig. 2).

And carrying out morphological observation on the BHP NPs by adopting a transmission electron microscope, namely taking 10 mu L of BHP NPs liquid to drop on a copper net, settling for 10min, dropwise adding one drop of phosphotungstic acid to dye a sample, and adopting the transmission electron microscope to observe morphological characteristics of the sample. As shown in FIG. 3, the BHP NPs are spherical in appearance, have obvious core-shell structures and are uniform in appearance.

Comparative example 1

Unlike example 1, the molar ratio of the drugs is different:

Weighing berberine 3.36mg (0.01 mmol), hypericin 5.04mg (0.01 mmol) and dBET57 14.00mg (0.02 mmol) according to the mol ratio of berberine, hypericin and dBET57 of 1:1:2, dissolving in a mixed solvent of 300 mu L dimethyl sulfoxide and 30 mu L methanol to obtain a mixed solution, dropwise adding the mixed solution into 22.37mL pure water at room temperature while stirring, standing for 2h after dropwise adding, adding into a ultrafilter tube (molecular cut-off 3500 Da), centrifuging at 2000rpm for 15min, removing the organic solvent, and re-suspending with 22.4mL pure water to obtain BHP NPs (1 mg/mL). The dynamic light scattering histogram of BHP NPs is shown in FIG. 4, and the average particle size is about 1100nm, the PDI value of BHP NPs is about 0.82, and the method can not form uniform nano particles, and has larger size and poor biocompatibility.

Comparative example 2

Unlike example 1, the molar ratio of the drugs is different:

Weighing berberine 3.36mg (0.01 mmol), hypericin 10.08mg (0.02 mmol) and dBET 57.00 mg (0.01 mmol) according to the mol ratio of berberine, hypericin and dBET57 of 1:2:1, dissolving in a mixed solvent of 300 mu L dimethyl sulfoxide and 30 mu L methanol to obtain a mixed solution, dropwise adding the mixed solution into 20.41mL pure water at room temperature, stirring while dropwise adding, standing for 2h after dropwise adding, adding into a ultrafilter tube (molecular cut-off 3500 Da), centrifuging at 2000rpm for 15min, removing the organic solvent, and re-suspending with 20.44mL pure water to obtain BHP NPs (1 mg/mL). The dynamic light scattering histogram of BHP NPs is shown in FIG. 4, and the average particle size of the BHP NPs is about 400nm, the PDI value of the BHP NPs is about 0.36, and the nanoparticle prepared by the method has larger size and poor biocompatibility.

Example 2

Stability and self-assembly mechanism study of BHP NPs:

(1) The stability of BHP NPs in physiological state was studied using phosphate buffer (PBS, pH 7.4), 10% Fetal Bovine Serum (FBS) DMEM medium to simulate physiological environment, PBS containing 0.1mM H ₂O₂ to simulate oxidative stress tumor microenvironment.

The BHP NPs liquid prepared in example 1 was placed in 10 volumes of PBS or 10% FBSDMEM medium or PBS containing 0.1mM H ₂O₂, and the particle size change of the nanoparticles at different time points (4, 12, 24, 36, 48H) was monitored by dynamic light scattering using a malvern particle sizer.

The stability inspection result is shown in fig. 5, the particle size of the BHP NPs has no obvious change within 48 hours, and the BHP NPs has no obvious change under the simulated physiological condition, so that the BHP NPs have good stability.

(2) Molecular dynamics simulation was used to study the self-assembly mechanism of BHP NPs.

Kinetic simulations were performed using the Gromacs 2021.7 program. The small molecule uses ORCA to calculate the force field, which is GAFF. The system size of the dynamic simulation construction system is 10nm by 10nm. Three small molecules were randomly placed in the system, each 20 in number, then filled with water molecules, and the charges were neutralized using Na ions and Cl ions. Then 50000 steps of energy minimization are performed on the system to eliminate bad contacts in the initial structure, then 100ps NVT (constant particle count, volume and temperature) balancing is performed, and 100ps NPT pre-balancing is performed, finally 100ns production simulation is performed. The temperature during the simulation was 298.15K (25 degrees Celsius) and the pressure was one standard atmosphere.

FIG. 6 is an analysis of interactions in the last frame of kinetic simulation, and it can be seen that three molecules in the BHP NPs prepared in example 1 are gathered together mainly by Hydrogen bond interactions (Hydrogen bond), pi-pi stacking (pi-pi stacking), and various hydrophobic interactions (Hydrophobic).

Example 3

BHP NPs in vitro inhibition of breast cancer cell growth effect evaluation:

a) The medicine groups are blank group, berberine (BBR) non-illumination group, dBET57 non-illumination group, hypericin non-illumination (HPC) group, berberine-hypericin-dBET 57 physical mixed non-illumination (Mix) group, BHP NPs non-illumination group, hypericin illumination group (HPC+laser, i.e. HPC (+) group in FIG. 7), berberine-hypericin-dBET 57 physical mixed illumination group (mix+laser, i.e. Mix (+) group in FIG. 7), BHP NPs illumination group (BHP NPs+laser, i.e. NPs (+) group in FIG. 7).

B) Pharmaceutical formulation

After freeze-drying the BHP NPs prepared in example 1, precisely weighing the BHP NPs, and preparing a solution with the nanoparticle concentration of 1mg/mL by ultrasonic treatment with a serum-free DMEM culture medium. Prior to the experiment, the BHP NPs were diluted with fresh DMEM incomplete medium to solutions with a concentration of 0.5. Mu.g/mL, 1. Mu.g/mL, 2. Mu.g/mL, 5. Mu.g/mL, 15. Mu.g/mL, 25. Mu.g/mL, respectively, under sterile conditions.

Precisely weighing berberine, preparing into berberine solution with concentration of 10mg/mL by dimethyl sulfoxide ultrasonic, and diluting with fresh DMEM incomplete culture medium under aseptic condition to obtain berberine gradient solutions with concentrations of 0.5 μg/mL, 1 μg/mL, 2 μg/mL, 5 μg/mL, 15 μg/mL, and 25 μg/mL.

Hypericin is precisely weighed, and prepared into hypericin solution with concentration of 10mg/mL by dimethyl sulfoxide ultrasonic, and before experiments, the hypericin solution is diluted with fresh DMEM incomplete culture medium under aseptic condition to obtain hypericin gradient solutions with concentration of 0.5 mug/mL, 1 mug/mL, 2 mug/mL, 5 mug/mL, 15 mug/mL and 25 mug/mL respectively.

DBET57 is precisely weighed, dBET57 solution with the concentration of 10mg/mL is prepared by dimethyl sulfoxide ultrasonic, and before experiments, dBET57 gradient solutions with the concentration of 0.5 mug/mL, 1 mug/mL, 2 mug/mL, 5 mug/mL, 15 mug/mL and 25 mug/mL are obtained by dilution with fresh DMEM incomplete culture medium under aseptic conditions.

1.17Mg of berberine, 1.60mg of hypericin and 57.22 mg of dBET are precisely weighed, and 50 mu L of dimethyl sulfoxide is used for preparing a physical mixed solution with the concentration of 10mg/mL by ultrasonic treatment. Before the experiment, the physical mixture was diluted with fresh DMEM incomplete medium under sterile conditions to give physical mixtures with concentrations of 0.5. Mu.g/mL, 1. Mu.g/mL, 2. Mu.g/mL, 5. Mu.g/mL, 15. Mu.g/mL, 25. Mu.g/mL, respectively.

C) Three negative breast cancer cells MDA-MB-231 in log phase were collected by pancreatin digestion, and inoculated into 96-well plates at a density of 5X 10 ³ cells/well, 100. Mu.L of DMEM medium containing 10% fetal bovine serum was added to each well, and 5 parallel wells were set up for each group. Incubation was performed in an incubator, after 80% cell attachment, the supernatant was carefully aspirated with a pipette and the medium was changed.

The blank group and 5 non-illuminated groups are replaced by 100 mu L of DMEM incomplete culture medium (blank group) or DMEM incomplete culture medium (non-illuminated group) containing samples with different concentrations, incubation is continued 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 condition of keeping away from light, incubation is continued for 2 hours, the supernatant is carefully discarded, and 100 mu LDMSO is added.

3 Illumination groups, namely, 100 mu L of DMEM incomplete culture medium containing samples with different concentrations is replaced, after incubation is carried out for 4 hours, the 96-well plates are taken out, each well is irradiated for 3 minutes by 635nm (5 mW.cm ^-2) laser, and then the culture medium is returned to an incubator for continuous incubation for 20 hours. The 96-well plate was removed, 10. Mu.L of MTT solution at a concentration of 5mg/mL was added under light-shielding conditions, incubation was continued for 2 hours under light-shielding conditions, the supernatant was carefully discarded, and 100. Mu.L of DMSO was added.

The absorbance value of each well was measured at 492nm using an enzyme-labeled instrument, and 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%

After the drug is incubated with MDA-MB-231 cells for 24 hours, the cytotoxicity results are shown in figure 7, and the physical mixture (Mix) of Hypericin (HPC), berberine-hypericin-dBET 57 and berberine-hypericin-dBET 57 self-assembled nanoparticles (BHP NPs) have cytotoxicity to breast cancer cells MDA-MB-231 in vitro. Particularly, the effect of the berberine-hypericin-dBET 57 self-assembled nanoparticles (BHP NPs) on killing triple negative breast cancer cells is obviously stronger than that of pure physical mixing or free drugs under the same administration concentration, and the effect of the nanoparticles is further enhanced by laser irradiation. The synergistic treatment strategy based on the nano-delivery system is shown to have a significant treatment effect on triple negative breast cancer.

Example 4

BHP NPs in vitro metabolic inhibition evaluation:

The in vitro metabolism inhibition capacity of the nanoparticles is evaluated by measuring the Adenosine Triphosphate (ATP) content, the lactic acid content of glycolytic metabolites and the activity of mitochondrial respiratory chain complex I in MDA-MB-231 cells after different drug treatments.

The drug formulations of each group were the same as in example 3, with the following specific groupings and drug administration concentrations:

The control group (PBS group), the berberine non-illuminated group (BBR group, berberine concentration of 1.17. Mu.g/mL), the dBET57 non-illuminated group (dBET 57 group, dBET57 concentration of 2.22. Mu.g/mL), the hypericin non-illuminated group (HPC group, hypericin concentration of 1.60. Mu.g/mL), the berberine-hypericin-dBET 57 physically mixed non-illuminated group (Mix group, mass ratio of berberine, hypericin and dBET57 of 1.17:1.60:2.22, total concentration of 5. Mu.g/mL), the BHP NPs non-illuminated group (BHP NPs group, BHP NPs concentration of 5. Mu.g/mL), the hypericin illuminated group (HPC+L group, hypericin concentration of 1.60. Mu.g/mL), the berberine-hypericin-dBET 57 physically mixed illuminated group (mix+L group, mass ratio of berberine, hypericin and dB57 of 1.17:1.60:2.60:2.22, total concentration of BHP NPs and total NPP concentration of 5. Mu.g/mL), the BHP NPS non-illuminated group (BHP NPS).

A) Intracellular Adenosine Triphosphate (ATP) content determination

The logarithmic phase of human breast cancer cells MDA-MB-231 was taken, the cells were collected by pancreatin digestion, inoculated into 6-well plates at a density of 1X 10 ⁶ cells/well, and cultured for 12 hours.

The blank group and 5 non-illuminated groups were replaced with 1mL of PBS (blank group) or DMEM incomplete medium (non-illuminated group) containing samples at different concentrations, incubation was continued for 12h, 6-well plates were removed, and intracellular ATP content was determined using an ATP detection kit (ATP ASSAY KIT, beyotime).

3 Illumination groups, namely, 1mL of DMEM incomplete culture medium containing samples with different concentrations is replaced, after incubation is carried out for 4 hours, a 6-hole plate is taken out, each hole is irradiated for 3 minutes by 635nm (5 mW.cm ^-2) laser, and then the culture medium is put back into an incubator to be incubated for 8 hours. Intracellular ATP content was determined using ATP assay kit (ATP ASSAY KIT, beyotime).

As shown in fig. 8, BHP NPs were able to reduce intracellular ATP levels, cutting off tumor cell energy sources, with the combined photodynamic therapy under light conditions most pronounced to suppress ATP levels.

B) Glycolytic Activity assay

The logarithmic phase of human breast cancer cells MDA-MB-231 was taken, the cells were collected by pancreatin digestion, inoculated into 6-well plates at a density of 1X 10 ⁶ cells/well, and cultured for 12 hours.

The blank group and the 5 non-illuminated groups are replaced by 1mL of DMEM incomplete culture medium (blank group) or DMEM incomplete culture medium (non-illuminated group) containing samples with different concentrations, the incubation is continued for 12 hours, a 6-pore plate is taken out, and the lactic acid measuring kit (LACTATE ASSAY KIT-WST, dojindo) is used for measuring the content change of the lactic acid of the glycolytic metabolites of the cells after the drug treatment.

3 Illumination groups, namely, 1mL of DMEM incomplete culture medium containing samples with different concentrations is replaced, after incubation is carried out for 4 hours, a 6-hole plate is taken, each hole is irradiated for 3 minutes by 635nm (5 mW.cm ^-2) laser, and then the culture medium is put back into an incubator to be incubated for 8 hours. The 6-well plate was removed, and the change in the content of lactate, a cell glycolytic metabolite after drug treatment, was measured using a lactate measuring kit (LACTATE ASSAY KIT-WST, dojindo).

As shown in fig. 9, BHP NPs were able to inhibit glycolytic metabolic pathways, reducing the formation of the metabolite lactate, which was most pronounced with combination photodynamic therapy inhibition under light conditions.

D) Activity measurement of intracellular mitochondrial respiratory chain complex I

The breast cancer cells MDA-MB-231 in logarithmic phase were collected by pancreatin digestion, inoculated into 6-well plates at a density of 1X 10 ⁶ cells/well, and cultured for 12 hours.

The blank group and the 5 non-illuminated groups are replaced by 1mL of DMEM incomplete culture medium (blank group) or DMEM incomplete culture medium (non-illuminated group) containing samples with different concentrations, incubation is continued for 12h, a 6-hole plate is taken out, and the mitochondrial respiratory chain complex I activity in cells is measured by using a mitochondrial respiratory chain complex I activity detection kit (Solarbio).

3 Illumination groups, namely, 1mL of DMEM incomplete culture medium containing samples with different concentrations is replaced, after incubation is carried out for 4 hours, a 6-hole plate is taken, each hole is irradiated for 3 minutes by 635nm (5 mW.cm ^-2) laser, and then the culture medium is put back into an incubator to be incubated for 8 hours. The 6-hole plate is taken out, and the mitochondrial respiratory chain complex I activity in the cell is measured by using a mitochondrial respiratory chain complex I activity detection kit (Solarbio).

As shown in fig. 10, BHP NPs were able to significantly reduce intracellular mitochondrial respiratory chain complex I activity, with the combined photodynamic therapy inhibition effect being most pronounced under light conditions.

Example 5

BHP NPs mitochondrial targeting ability investigation

The preparation of the physical mixture of BHP NPs solution and berberine-hypericin-dBET 57 was the same as in example 4, with a concentration of 5. Mu.g/mL.

The distribution of the nanoparticles and the physical mixed medicine in the cells can be judged by observing the red fluorescence of hypericin (HPC channel) by utilizing the self-fluorescence signal of hypericin in the nanoparticles and the physical mixture.

The breast cancer cells MDA-MB-231 in logarithmic phase were collected by pancreatin digestion, inoculated into 6-well plates at a density of 1X 10 ⁶ cells/well, and cultured for 12 hours. The culture medium was removed, a berberine-hypericin-dBET 57 physical mixed group (Mix group, 5. Mu.g/mL) and a BHP NPs group (5. Mu.g/mL) were set, 1mL of the berberine-hypericin-dBET 57 physical mixed solution and the BHP NPs solution were added to each well, and after incubation for 12 hours under serum-free conditions, the cells were washed 3 times with PBS. Commercial mitochondrial dye MitoBright LT Green (Dojindo) with a concentration of 50nM is used for dying mitochondrial 15min,Hoechst 333442 dye for dying cell nuclei (blue fluorescence) for 5min, hypericin (red fluorescence) is observed by a fluorescence microscope, the distribution condition of the drug in the cells after entering tumor cells is detected, and Merge represents the coincidence of the three fluorescence signals. If obvious yellow, namely the superposition of red and green fluorescence signals, the nanoparticle can target mitochondria, and if the yellow fluorescence is weaker or the red and green fluorescence signals are not overlapped, the preparation does not have the capacity of targeting mitochondria.

As shown in fig. 11, the BHP NPs group clearly seen that nanoparticles that fluoresce red (HPC channel) and mitochondria that fluoresce green (Mitotracker Green channel) overlap together, and that appear yellow after the overlap (Merge). The yellow fluorescence of the BHP NPs group was stronger and the overlap of the red and green fluorescence signals was higher compared to the physical mix group (fig. 12), indicating that BHP NPs were able to target mitochondria more effectively.

Example 6

Investigation of degradation ability of BHP NPs on target protein

The ability of nanoparticles to degrade BRD4 and c-Myc proteins in MDA-MB-231 cells was evaluated by Western Blot experiments, and the drug groupings and formulations were as in example 4.

The breast cancer cells MDA-MB-231 in logarithmic phase were collected by pancreatin digestion, inoculated into 6-well plates at a density of 1X 10 ⁶ cells/well, and cultured for 12 hours. Removing the culture medium, adding 1mL PBS buffer solution (pH=7.4) into each hole of a blank control group, adding 1mL into each hole of a berberine non-illumination group, a hypericin non-illumination group, a dBET57 non-illumination group, a berberine-hypericin-dBET 57 physical mixed non-illumination group, a BHP NPs non-illumination group, a hypericin illumination group, a berberine-hypericin-dBET 57 physical mixed illumination group and a BHP NPs illumination group, and continuously culturing for 12h. After incubation, the various samples were assayed according to standard protocols.

FIG. 13 shows the degradation effects of the target proteins BRD4 and c-Myc in MDA-MB-231 cells after different drug treatments, and the nanoparticles can effectively degrade the target proteins, and the degradation of the target proteins under the illumination condition is more remarkable.

Example 7

BHP NPs induce iron death

A) TEM detects organelle morphology

Preparation of BHP NPs solution the concentration of BHP NPs was 5. Mu.g/mL as in example 4.

MDA-MB-231 cells (1X 10 ⁸) were treated with BHP NPs solution (5. Mu.g/mL) for 12h, the cells were collected, centrifuged at 1500rpm, the supernatant was discarded, the cell pellet was collected in a 10mL centrifuge tube, and pre-chilled 4℃fixative was slowly added along the tube wall until the pellet was completely absent. The fixed cells were sectioned using an ultra-thin microtome and stained with lead citrate and uranium acetate in ethanol. The samples were observed using a biological transmission electron microscope, and mitochondria were observed and photographed. Normal cells were used as controls.

As can be seen from fig. 14, compared with normal cells (Control), BHP NPs treated cells showed abnormal mitochondrial morphology, increased mitochondrial membrane density, darkened mitochondrial inner membrane color, decreased mitochondrial cristae, and widened space between mitochondrial cristae, indicating that the major cell death mode of tumor cells was iron death.

B) Quantitative index of lipid peroxidation associated with iron death and qualitative image capture

Iron death is a regulated cell death characterized by peroxidation of intracellular lipids. The most common trigger for lipid oxidation is GSH consumption with reduced GPX4 expression or inactivation of GPX 4. Therefore, the invention confirms that BHP NPs treatment induces iron death by detecting GSH content, GPX4 content and lipid peroxidation degree.

The drug formulation was as in example 3, with the following specific groupings and drug administration concentrations:

A blank control group (PBS group), a berberine-hypericin-dBET 57 physical mixed non-illumination group (Mix group, mass ratio of berberine, hypericin and dBET57 is 1.17:1.60:2.22, total concentration is5 mug/mL), a BHP NPs non-illumination group (BHP NPs group, BHP NPs concentration is5 mug/mL), a berberine-hypericin-dET 57 physical mixed illumination group (mix+L group or Mix (+) group, mass ratio of berberine, hypericin and dET 57 is 1.17:1.60:2.22, total concentration is5 mug/mL), a BHP NPs illumination group (BHP NPs+L group or BHP NPs (+) group, BHP NPs concentration is5 mug/mL).

The breast cancer cells MDA-MB-231 in logarithmic phase were collected by pancreatin digestion, inoculated into 6-well plates at a density of 1X 10 ⁶ cells/well, and cultured for 12 hours.

The blank and 2 non-illuminated groups were replaced with 1mL PBS (blank) or DMEM incomplete medium (non-illuminated group) containing samples at different concentrations, and incubation was continued for 12h.

2 Illumination groups, namely, 1mL of DMEM incomplete culture medium containing different samples is replaced, after incubation is carried out for 4 hours, a 6-hole plate is taken, each hole is irradiated for 3 minutes by 635nm (5 mW.cm ^-2) laser, and then the culture medium is put back into an incubator for further incubation for 8 hours.

The 6-well plate was removed and the Malondialdehyde (MDA), glutathione (GSH) and lipid peroxide Level (LPO) concentrations in the cells after incubation were detected using MDA detection kit, GSH detection kit and Liperfluo-cell lipid peroxide detection kit, respectively.

As can be seen from fig. 15a, the intracellular antioxidant substance GSH content was reduced after BHP nps+l treatment compared to the Control group (Control) and other treatment groups, and there was a significant difference, indicating that BHP nps+l could directly cause the lipid antioxidant substance content to be reduced.

The results of the detection of the final lipid oxidation product MDA capable of reacting with the content of oxidized lipid in cells in the present invention are shown in FIG. 15b, which shows the increase of oxidized lipid caused by BHP NPs+L.

Liperfluo is oxidized specifically by lipid peroxide and emits strong fluorescence in organic solvent such as ethanol, and can be used for detecting Lipid Peroxide (LPO). The treated cells were observed under CLSM (fig. 15 c) and the LPO signal (green fluorescence) was significantly increased in BHP nps+l groups compared to other treatment groups or the placebo group.

The above results demonstrate that BHP nps+l treated cells undergo iron death with typical morphological and biochemical characteristics. Meanwhile, compared with BHP NPs and mix+L, the BHP NPs+L has synergistic iron death enhancement effect.

Example 8

BHP NPs in vivo antitumor ability investigation:

After freeze-drying the BHP NPs prepared in example 1, precisely weighing the BHP NPs, and preparing a solution with a nanoparticle concentration of 1mg/mL by using sterile physiological saline.

Precisely weighing berberine, preparing into 10mg/mL berberine solution with dimethyl sulfoxide by ultrasonic treatment, and diluting with sterile physiological saline under aseptic condition to obtain 0.234mg/mL berberine solution before experiment.

Precisely weighing hypericin, preparing hypericin solution with concentration of 10mg/mL by using dimethyl sulfoxide ultrasound, and diluting with sterile physiological saline under sterile condition to obtain hypericin solution with concentration of 0.320mg/mL before experiment.

DBET57 is precisely weighed, dBET57 solution with the concentration of 10mg/mL is prepared by using dimethyl sulfoxide ultrasound, and before an experiment, dBET57 solution with the concentration of 0.444mg/mL is obtained by dilution with sterile physiological saline under sterile conditions.

Precisely weighing 1.17mg of berberine, 1.60mg of hypericin and 57.22 mg of dBET, and preparing a physical mixed solution with the concentration of 50mg/mL by using 100 mu L of dimethyl sulfoxide ultrasound. Before the experiment, the mixture was diluted with sterile physiological saline under aseptic conditions to give a physical mixture having a concentration of 1 mg/mL.

BALB/c female nude mice, all animal experiments were in accordance with the animal ethics committee standard of the university of Fujian, traditional Chinese medicine.

Taking MDA-MB-231 cells in the logarithmic growth phase, centrifuging, collecting the cells, and suspending the cells by PBS to obtain a cell suspension. The cell suspension was subcutaneously injected into the right mammary gland of BALB/c female nude mice at a density of 10 ⁶ cells/mouse. Observation was started 7 days after inoculation, and when the tumor volume reached 100mm ³, which indicates that the MDA-MB-231 tumor-bearing nude mice model was successfully constructed, mice were randomly divided into 9 groups (n=4), and each mouse was injected with 0.2mL of drug by tail vein:

Physiological saline (salt) group, berberine group (BBR group, 0.234 mg/mL), dBET57 group (0.444 mg/mL), hypericin non-illuminated group (HPC group, 0.320 mg/mL), berberine-hypericin-dBET 57 physical mixed non-illuminated group (Mix group, 1 mg/mL), BHP NPs non-illuminated group (BHP NPs group, 1 mg/mL), hypericin illuminated group (HPC+L group, 0.320 mg/mL), berberine-hypericin-dBET 57 physical mixed illuminated group (mix+L group, 1 mg/mL), BHP NPs illuminated group (BHP NPs+L group, 1 mg/mL).

Each group of mice was dosed on day 1, day 3, and day 5, respectively, wherein the mice tumor sites were irradiated with a 638nm (0.5 mW cm ^-2) laser for 5min after 24h of each dose. The tumor volume of the mice is measured every three days by using a vernier caliper, the tumor volume is calculated, and a tumor volume change curve is drawn.

Tumor volume = major axis x minor axis/2

The weight of the mice was recorded throughout the dosing cycle, the relative rate of change of the weight of the mice to the weight of the mice on day 1 was weighed and calculated, and a weight-time curve was drawn, if the rate of change of weight was reduced by more than 15%, the test drug was considered to have greater toxicity.

Fig. 16 is a graph showing the change in body weight of mice during the treatment period, and shows that the body weight of BHP NPs was not significantly reduced in both the light-irradiated and non-light-irradiated groups compared with the normal saline group, indicating that BHP NPs have better safety.

FIG. 17 is a graph showing the change in tumor volume of mice during the treatment cycle, wherein the tumor volume of each treatment group was reduced to a different extent after the treatment was completed, and the tumor inhibition effect was most remarkable and the treatment group was gradually reduced by BHP NPs.

In summary, the synergistic therapeutic approach based on nano-delivery systems has the best anti-tumor activity and safety compared to either a purely co-administration strategy or a single administration strategy.

Claims (10)

1. A mitochondrial-targeting self-assembling PROTAC nanomaterial based on a natural product, characterized in that it is a nanoparticle prepared by a nanoprecipitation method using a mitochondrial targeting molecule, a photosensitizer, and a PROTAC as raw materials; Wherein, the mitochondrial targeting molecule is berberine, the photosensitizer is hypericin, and the PROTAC is dBET57; the molar ratio of dBET57 to berberine is (1-10):(1-10), and the molar ratio of dBET57 to hypericin is (1-10):(1-10). 2. The natural product mitochondrial-targeted self-assembly PROTAC nanomaterial according to claim 1, characterized in that the molar ratio of the dBET57 to berberine is 0.8:1 to 1.5:1, preferably 1:1; the molar ratio of the dBET57 to hypericin is 1:0.8 to 1:1.5, preferably 1:1. 3. The natural product mitochondrial-targeted self-assembly PROTAC nanomaterial according to claim 1, characterized in that: the nanoprecipitation method is to dissolve berberine, hypericin and dBET57 in a benign organic solvent to form a mixed solution, or dissolve berberine, hypericin and dBET57 in a benign organic solvent to prepare a berberine solution, a hypericin solution and a dBET57 solution, and then mix the three solutions to form a mixed solution; at room temperature, the mixed solution is added dropwise to water, stirred during the addition, and after the addition is completed, the mixture is allowed to stand, and the organic solvent is removed by ultrafiltration or dialysis to obtain nanoparticles. 4. The natural product mitochondrial-targeted self-assembly PROTAC nanomaterial according to claim 3, characterized in that in the mixed solution, the dosage ratio of dBET57 to the benign organic solvent is 1:25 to 1:35 mol/L, preferably 1:33 mol/L. 5. The natural product mitochondrial-targeted self-assembly PROTAC nanomaterial according to claim 3, characterized in that the benign organic solvent is a mixture of one or more of dimethyl sulfoxide, anhydrous ethanol, methanol or tetrahydrofuran, preferably a mixed solvent of dimethyl sulfoxide and methanol with a volume ratio of 4:1 to 10:1, more preferably a mixed solvent of dimethyl sulfoxide and methanol with a volume ratio of 10:1. 6. The natural product-based mitochondrial-targeted self-assembly PROTAC nanomaterial according to claim 3, characterized in that the ultrafiltration tube used in the ultrafiltration has a molecular weight cutoff of 1000 to 10000 Da, preferably 3500 Da. 7. A method for preparing a natural product mitochondrial-targeted self-assembly PROTAC nanomaterial, comprising: dissolving berberine, hypericin, and dBET57 in a benign organic solvent to form a mixed solution, or dissolving berberine, hypericin, and dBET57 in a benign organic solvent to prepare a berberine solution, a hypericin solution, and a dBET57 solution, respectively, and then mixing the three solutions to form a mixed solution; at room temperature, adding the mixed solution dropwise to water with stirring during the addition, and after the addition is complete, allowing the mixture to stand, and removing the organic solvent by ultrafiltration or dialysis to obtain nanoparticles. 8. Use of the natural product mitochondria-targeted self-assembly PROTAC nanomaterial according to claim 1 in the preparation of anti-tumor drugs. 9. Use of the natural product mitochondria-targeted self-assembly PROTAC nanomaterial according to claim 1 in the preparation of anti-tumor drugs for photodynamic therapy synergistic with starvation therapy. 10. Use of the natural product mitochondria-targeted self-assembly PROTAC nanomaterial according to claim 1 in the preparation of mitochondria-targeted anti-tumor drugs that synergistically block the energy supply of tumor cells and inhibit tumor cell growth in photodynamic therapy.