Self-assembly PROTAC nano material based on mitochondria targeting of natural product as well as preparation method and application of self-assembly PROTAC nano material

Abstract

The invention discloses a self-assembled PROTAC nano material based on natural product mitochondria targeting, which is a nano particle prepared from mitochondria targeting molecules, a photosensitizer and PROTAC as raw materials through a nano precipitation method. The mitochondrial targeting molecule is berberine, the photosensitizer is hypericin, and the PROTAC is dBET57; the ratio of the amount of substance of the dBET57 to the amount of substance of the berberine is (1-10): (1-10), and the ratio of the amount of substance of the dBET57 to the amount of substance of the hypericin is (1-10): (1-10). The nano material can be used for blocking multiple energy metabolism pathways of tumor cells, including glycolysis and oxidative phosphorylation, meanwhile, mitochondria is damaged, ferroptosis is induced, and the anti-tumor capacity of pharmacodynamic molecules is improved; bRD4 in tumor cells and downstream carcinogenic protein c-Myc of the BRD4 can be efficiently degraded. The invention also discloses an application of the nano material in preparation of antitumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations and relates to a preparation method and application of a mitochondrial-targeted self-assembly PROTAC nanomaterial based on a natural product, and a preparation method and application thereof, and specifically relates to a self-assembled nanoparticle of a mitochondrial-targeted self-assembly PROTAC nanomaterial combined with photodynamic therapy / metabolic blocking effect, and a preparation method and application thereof. Background Art

[0002] Currently, the main first-line clinical anti-tumor treatments include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. However, traditional radiotherapy and chemotherapy drugs may be unevenly distributed in the body and have poor tumor targeting, which often leads to severe systemic side effects in patients and greatly reduces their quality of life. Therefore, people have begun to focus on plant-derived anticancer drugs. Natural products are abundant in sources and generally have good biocompatibility, low toxicity, and high selectivity. A large number of studies have shown that there are many photosensitizing substances in natural products and their derivatives, such as tetrapyrroles, polyphenols, anthraquinones, thiophenes, and perylenequinone compounds. Their chemical structural diversity provides a rich selection for the development of photodynamic therapy (PDT) photosensitizers. However, natural products have poor water solubility, low bioavailability, and poor drugability, which limits their further application in clinical practice.

[0003] Proteolysis-targeting chimeras (PROTAC) technology is an emerging targeted protein degradation strategy that uses small molecule compounds to induce ubiquitination and subsequent proteasomal degradation of specific proteins. This allows for sustained protein degradation even at low drug concentrations, significantly improving efficacy. The discovery of PROTACs is expected to overcome some of the limitations of traditional small molecule inhibitors. However, their practical application is still limited by low bioavailability due to poor water solubility and off-target toxicity due to lack of target organ specificity, requiring the design of intelligent drug delivery systems for improvement.

[0004] In recent years, with the progress and development of nanomaterial chemistry, nano drug delivery systems are often used to improve the drugability of drugs, prolong the circulation time of drugs in the body, and give drugs active or passive targeting effects. Among them, organelle-targeted delivery strategies have shown great potential in cancer treatment. Mitochondria, as the "energy factory" of cells, maintain the normal physiological functions of organisms by providing ATP. Therefore, in cancer treatment, nano drug delivery systems that simultaneously target and damage mitochondria to induce cell death are very desirable. However, traditional carrier-assisted nano drug delivery systems still have problems such as low drug loading and poor stability, making their functions and ultimate efficiency in clinical applications still unsatisfactory. Carrier-free self-assembled nanoparticles are mainly self-assembled from 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. Summary of the Invention

[0005] To overcome the shortcomings of the existing technology, the present invention provides a mitochondrial-targeting self-assembling PROTAC nanomaterial based on a natural product, as well as its preparation method and application. This nanomaterial is composed of berberine, hypericin, and dBET57, which are self-assembled into nanoparticles in an aqueous system via a nanoprecipitation method. These nanoparticles can accumulate at tumor sites, target mitochondria, and synergistically kill tumor cells through starvation therapy, photodynamic therapy, and targeted protein degradation, thereby improving the application of natural products and PROTACs in tumor treatment.

[0006] The technical solutions of the present invention are as follows:

[0007] A mitochondrial-targeting self-assembled PROTAC nanomaterial based on natural products. It is a nanoparticle prepared by nanoprecipitation method using mitochondrial targeting molecules, photosensitizers and PROTAC as raw materials.

[0008] Wherein, the mitochondrial targeting molecule is berberine, the photosensitizer is hypericin, and the PROTAC is dBET57 (CAS No.: 1883863-52-2).

[0009] The molar ratio of dBET57 to berberine is (1-10):(1-10), preferably 0.8:1-1.5:1, and most preferably 1:1. The molar ratio of dBET57 to hypericin is (1-10):(1-10), preferably 1:0.8-1:1.5, and more preferably 1:1. Nanoparticles can be prepared according to the above molar ratios. According to the preferred embodiment, nanoparticles with an average particle size of about 110-115 nm and a polydispersity index (PDI) of about 0.13 can be prepared.

[0010] The berberine, hypericin and dBET57 are self-assembled to form nanoparticles mainly through hydrogen bond interaction, π-π stacking and various hydrophobic interactions.

[0011] The natural product mitochondria-targeted self-assembly PROTAC nanomaterial has a uniform spherical appearance.

[0012] The nanoprecipitation method comprises 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; adding the mixed solution dropwise into water at room temperature, stirring during the addition, and after the addition is completed, standing, ultrafiltration or dialysis to remove the organic solvent to obtain nanoparticles.

[0013] Another object of the present invention is to provide a method for preparing the 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, stirring during the addition, and after the addition is completed, standing, ultrafiltration or dialysis to remove the organic solvent to obtain nanoparticles.

[0014] In the mixed solution, the ratio of dBET57 to the benign organic solvent is 1:25 to 1:35 mol / L, preferably 1:33 mol / L.

[0015] The benign organic solvent is a mixture of one or more of dimethyl sulfoxide, anhydrous ethanol, methanol or tetrahydrofuran, preferably a mixed solvent in which the volume ratio of dimethyl sulfoxide to methanol is 4:1 to 10:1, more preferably a mixed solvent in which the volume ratio of dimethyl sulfoxide to methanol is 10:1.

[0016] The amount of water used is greater than that of the benign organic solvent. Generally, the volume ratio of the benign organic solvent to water is 1:10 to 1:50, preferably 1:40 to 1:50, and more preferably 1:45 to 1:46.

[0017] The standing time is generally 0.5 to 3 hours.

[0018] The ultrafiltration tube used in the ultrafiltration has a molecular weight cut-off of 1000 to 10000 Da, preferably 3500 Da.

[0019] The room temperature described in the present invention is 25±5°C.

[0020] The natural product mitochondria-targeted self-assembly PROTAC nanomaterials described in the present invention can improve the bioavailability of drug molecules, including increasing in vivo circulation time, precisely targeting tumor mitochondria, cellular uptake, and intracellular specific release; can be used to block multiple energy metabolism pathways in tumor cells, including glycolysis and oxidative phosphorylation, while damaging mitochondria and inducing ferroptosis to enhance the anti-tumor ability of drug molecules; PROTAC strategies can be used to efficiently degrade BRD4 and its downstream oncogenic protein c-Myc in tumor cells. The inventors conducted in vitro and in vivo anti-triple-negative breast cancer activity evaluations, indicating that the natural product mitochondria-targeted self-assembly PROTAC nanomaterials can significantly inhibit the proliferation of triple-negative breast cancer cells MDA-MB-231, and have significant anti-cancer activity and excellent tumor targeting against breast cancer; can damage mitochondria and block tumor energy supply while inducing ferroptosis, synergistically amplifying anti-tumor activity. Therefore, another object of the present invention is to provide the use of the natural product mitochondria-targeted self-assembly PROTAC nanomaterials in the preparation of anti-tumor drugs.

[0021] Another object of the present invention is to provide the use of the natural product mitochondria-targeted self-assembly PROTAC nanomaterial in the preparation of photodynamic therapy synergistic starvation therapy anti-tumor drugs.

[0022] Another object of the present invention is to provide the use of the natural product mitochondria-targeted self-assembled PROTAC nanomaterial 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.

[0023] The tumor is lung cancer, colon cancer, or breast cancer.

[0024] The breast cancer is triple-negative breast cancer.

[0025] The natural product mitochondrial-targeting self-assembly PROTAC nanomaterials described in the present invention have the advantages of a carrier-free nanodrug delivery system (such as passive targeting ability, prolonged in vivo circulation time, high drug loading and improved pharmacokinetic behavior), which are specifically manifested in the following four advantages:

[0026] 1) In terms of efficacy, compared with free drugs or physical mixtures, in vitro cytotoxicity experiments showed that nanoparticles can significantly inhibit the proliferation of breast cancer and other tumor cells under near-infrared laser irradiation, showing significant anti-cancer activity;

[0027] 2) In terms of targeting, compared with the physical mixture group, in addition to the EPR (enhanced permeability and retention) effect of the dosage form, the nanoparticles can accurately target the tumor subcellular organelle mitochondria, laying the foundation for starvation therapy;

[0028] 3) In terms of energy metabolism, the nanoformulation of the present invention with combined metabolic blocking effects can inhibit glycolysis and oxidative phosphorylation, and synergize with PROTAC / PDT to damage mitochondria while simultaneously targeting the degradation of BRD4 protein to inhibit c-Myc expression, thereby more comprehensively cutting off the energy supply of the tumor and killing tumor cells.

[0029] 4) In terms of drugability, the advantages of nano dosage forms can be utilized to improve the bioavailability of photosensitizers and PROTACs, including increasing tumor accumulation, amplifying therapeutic effects, and reducing toxic side effects on normal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : Dynamic light scattering histogram of the nanoparticles (BHP NPs) prepared in Example 1.

[0031] Figure 2 : Tyndall effect diagram of the self-assembled nanoparticles (BHP NPs) prepared in Example 1.

[0032] Figure 3 : Transmission electron microscopy (TEM) image of the self-assembled nanoparticles (BHP NPs) prepared in Example 1.

[0033] Figure 4 : Comparison of particle size of self-assembled nanoparticles (BHP NPs) with different molar ratios of berberine, hypericin and dBET57.

[0034] Figure 5 : Results of stability study of the 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.

[0035] Figure 6 : Molecular dynamics simulation results of self-assembled nanoparticles (BHP NPs) prepared in Example 1.

[0036] Figure 7 : Cytotoxicity of the self-assembled nanoparticles (BHP NPs) prepared in Example 1 and the berberine-hypericin-dBET57 physical mixture group on breast cancer cells MDA-MB-231 under the conditions of light irradiation (incubation for 24 hours).

[0037] Figure 8 : Determination results of ATP content.

[0038] Figure 9 : Determination results of lactic acid content.

[0039] Figure 10:The results of the determination of mitochondrial respiratory chain complex I activity.

[0040] Figure 11 : Results of investigation on the mitochondrial targeting ability of the self-assembled nanoparticles (BHP NPs) prepared in Example 1.

[0041] Figure 12 : Results of investigation on mitochondrial targeting ability of berberine-hypericin-dBET57 physical mixture group.

[0042] Figure 13 : The degradation effects of the self-assembled nanoparticles (BHP NPs), free drugs and berberine-hypericin-dBET57 physical mixture prepared in Example 1 on the target proteins BRD4 and c-Myc in triple-negative breast cancer MDA-MB-231 under the conditions of light and without light.

[0043] Figure 14 : Observation results of the morphology of organelles induced by ferroptosis by self-assembled nanoparticles (BHP NPs) prepared in Example 1.

[0044] Figure 15 : Results of the investigation on the ability of the self-assembled nanoparticles (BHP NPs) prepared in Example 1 to induce ferroptosis.

[0045] Figure 16 : Effects of the self-assembled nanoparticles (BHP NPs), free drug, and berberine-hypericin-dBET57 physical mixture prepared in Example 1 on the body weight of MDA-MB-231 tumor-bearing nude mice with and without light exposure.

[0046] Figure 17 : Effects of the self-assembled nanoparticles (BHP NPs), free drug, and berberine-hypericin-dBET57 physical mixture prepared in Example 1 on the tumor inhibition rate in MDA-MB-231 tumor-bearing nude mice with and without light irradiation. DETAILED DESCRIPTION

[0047] The technical solutions of the present invention are further described below through specific examples. However, the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0048] Example 1

[0049] A mitochondrial-targeting self-assembled PROTAC nanomaterial based on a natural product (denoted as BHP NPs) was prepared by the following preparation method, the specific process is as follows:

[0050] Berberine (BBR, 0.01 mmol), hypericin (HPC, 5.04 mg) and dBET57 (0.01 mmol) were weighed according to a molar ratio of berberine, hypericin and dBET57 of 1:1:1, and dissolved in a mixed solvent of 300 μL dimethyl sulfoxide (DMSO) and 30 μL methanol (MeOH) to obtain a mixed solution. The mixed solution was added dropwise to 15.07 mL of pure water at room temperature with stirring. After the addition was completed, the mixture was allowed to stand for 2 h, then loaded into an ultrafiltration tube (molecular cutoff 3500 Da), centrifuged at 2000 rpm for 15 min, the organic solvent was removed, and the mixture was resuspended in 15.4 mL of pure water to obtain berberine-hypericin-dBET57 self-assembled nanoparticles (1 mg / mL), denoted as BHP. The dynamic light scattering histogram of NPs and BHP NPs is shown in Figure 1 The average particle size (Z-Average) is about 111.51 nm, and the PDI value of BHP NPs is about 0.13, indicating that the particle size uniformity of the nanoparticles is high. The nanoparticles also have opalescence and Tyndall effect (see Figure 2 ).

[0051] The morphology of BHP NPs was observed using a transmission electron microscope. Specifically, 10 μL of BHP NPs liquid was dropped onto a copper mesh, allowed to settle for 10 minutes, and a drop of phosphotungstic acid was added to stain the sample. The morphology was then observed using a transmission electron microscope. Figure 3 As shown in the figure, BHP NPs are spherical in appearance, have obvious core-shell structure and uniform morphology.

[0052] Comparative Example 1

[0053] The difference from Example 1 is that the molar ratio of the drugs is different:

[0054] According to the molar ratio of berberine, hypericin and dBET57 of 1:1:2, berberine 3.36 mg (0.01 mmol), hypericin 5.04 mg (0.01 mmol) and dBET57 14.00 mg (0.02 mmol) were weighed and dissolved in a mixed solvent of 300 μL dimethyl sulfoxide and 30 μL methanol to obtain a mixed solution; at room temperature, the mixed solution was added dropwise to 22.37 mL of pure water while stirring. After the addition was completed, it was allowed to stand for 2 hours, and then loaded into an ultrafiltration tube (molecular retention capacity 3500 Da), centrifuged at 2000 rpm for 15 minutes, the organic solvent was removed, and BHP NPs (1 mg / mL) were resuspended with 22.4 mL of pure water. The dynamic light scattering histogram of BHP NPs is shown in Figure 4It can be seen that the average particle size is about 1100 nm and the PDI value of BHP NPs is about 0.82. This method cannot form uniform nanoparticles, and the size is large and the biocompatibility is poor.

[0055] Comparative Example 2

[0056] The difference from Example 1 is that the molar ratio of the drugs is different:

[0057] According to the molar ratio of berberine, hypericin and dBET57 of 1:2:1, berberine 3.36 mg (0.01 mmol), hypericin 10.08 mg (0.02 mmol) and dBET57 7.00 mg (0.01 mmol) were weighed and dissolved in a mixed solvent of 300 μL dimethyl sulfoxide and 30 μL methanol to obtain a mixed solution; at room temperature, the mixed solution was added dropwise to 20.41 mL of pure water while stirring. After the addition was completed, it was allowed to stand for 2 hours, and then loaded into an ultrafiltration tube (molecular retention capacity 3500 Da), centrifuged at 2000 rpm for 15 minutes, the organic solvent was removed, and BHP NPs (1 mg / mL) were resuspended with 20.44 mL of pure water. The dynamic light scattering histogram of BHP NPs is shown in Figure 4 It can be seen that the average particle size is about 400 nm and the PDI value of BHP NPs is about 0.36. The nanoparticles prepared by this method are large in size and have poor biocompatibility.

[0058] Example 2

[0059] Study on the stability and self-assembly mechanism of BHP NPs:

[0060] (1) Phosphate buffered saline (PBS, pH 7.4) and 10% fetal bovine serum (FBS) DMEM culture medium were used to simulate the physiological environment, and PBS containing 0.1 mM H2O2 was used to simulate the tumor microenvironment of oxidative stress to study the stability of BHP NPs under physiological conditions.

[0061] The BHP NPs liquid prepared in Example 1 was placed in 10 volumes of PBS or 10% FBSDMEM culture medium or PBS containing 0.1 mM H2O2. The particle size changes of the nanoparticles at different time points (4, 12, 24, 36, and 48 h) were monitored by dynamic light scattering using a Malvern particle size analyzer under room temperature.

[0062] The stability test results are shown in Figure 5 , the particle size of BHP NPs did not change significantly within 48 h, and also did not change significantly under simulated physiological conditions, indicating that BHP NPs have good stability.

[0063] (2) Molecular dynamics simulation was used to study the self-assembly mechanism of BHP NPs.

[0064] Dynamic simulations were performed using the Gromacs 2021.7 program. ORCA was used to calculate the force field for small molecules, and the force field was GAFF. The system size of the dynamic simulation construction system is 10nm*10nm*10nm. Three small molecules were randomly placed in the system, with 20 of each small molecule, and then filled with water molecules, and Na ions and Cl ions were used to neutralize the charge. The system was then subjected to 50,000 steps of energy minimization to eliminate poor contacts in the initial structure, followed by 100ps NVT (constant number of particles, volume, and temperature) equilibrium, 100ps NPT pre-equilibrium, and finally a 100ns production simulation. The temperature during the simulation was 298.15K (25 degrees Celsius) and the pressure was one standard atmosphere.

[0065] Figure 6 This is the interaction analysis of the last frame of the dynamics simulation. It can be seen that the three molecules in the BHP NPs prepared in Example 1 are mainly aggregated by hydrogen bond interactions, π-π stacking, and various hydrophobic interactions.

[0066] Example 3

[0067] Evaluation of the inhibitory effect of BHP NPs on breast cancer cell growth in vitro:

[0068] a) Drug groups: blank control group, berberine (BBR) non-illumination group, dBET57 non-illumination group, hypericin non-illumination (HPC) group, berberine-hypericin-dBET57 physical mixture non-illumination (Mix) group, BHP NPs non-illumination group, hypericin illumination group (HPC+Laser, i.e. Figure 7 HPC(+) group), berberine-hypericin-dBET57 physical mixed illumination group (Mix+Laser, i.e. Figure 7 Mix (+) group), BHP NPs illumination group (BHP NPs+Laser, i.e. Figure 7 NPs(+) group).

[0069] b) Drug preparation

[0070] After freeze-drying the BHP NPs prepared in Example 1, the BHP NPs were accurately weighed and sonicated in serum-free DMEM medium to prepare a solution with a nanoparticle concentration of 1 mg / mL. Prior to the experiment, under sterile conditions, fresh incomplete DMEM medium was used to dilute the BHP NPs to solutions with concentrations of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 15 μg / mL, and 25 μg / mL, respectively.

[0071] Berberine was accurately weighed and ultrasonically prepared into a berberine solution with a concentration of 10 mg / mL using dimethyl sulfoxide. Before the experiment, under sterile conditions, it was diluted with fresh DMEM incomplete culture medium to obtain a berberine gradient solution with concentrations of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 15 μg / mL, and 25 μg / mL.

[0072] Hypericin was accurately weighed and ultrasonically prepared into a 10 mg / mL hypericin solution using dimethyl sulfoxide. Before the experiment, under sterile conditions, it was diluted with fresh DMEM incomplete culture medium to obtain a hypericin gradient solution with concentrations of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 15 μg / mL, and 25 μg / mL, respectively.

[0073] Accurately weigh dBET57 and prepare a dBET57 solution with a concentration of 10 mg / mL using dimethyl sulfoxide by ultrasonication. Before the experiment, under sterile conditions, dilute it with fresh DMEM incomplete culture medium to obtain dBET57 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, respectively.

[0074] Accurately weigh 1.17 mg of berberine, 1.60 mg of hypericin, and 2.22 mg of dBET57 and sonicate them in 50 μL of dimethyl sulfoxide to create a 10 mg / mL physical mixture. Prior to the experiment, aseptically dilute the mixture with fresh DMEM incomplete medium to obtain concentrations of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 15 μg / mL, and 25 μg / mL, respectively.

[0075] c) Triple-negative breast cancer cells MDA-MB-231 in the logarithmic phase were collected by trypsin digestion and the cells were cultured at 5×10 3 Cells were seeded into a 96-well plate at a density of 100 μL / well. 100 μL of DMEM medium containing 10% fetal bovine serum was added to each well. Five parallel wells were set up for each group. Incubate in an incubator. After 80% of the cells have attached to the wall, the supernatant was carefully aspirated and the medium was replaced.

[0076] Blank control group and 5 non-illumination groups: replace with 100 μL DMEM incomplete medium (blank control group) or DMEM incomplete medium containing samples at different concentrations (non-illumination group), continue incubation for 24 hours, remove the 96-well plate, add 10 μL of 5 mg / mL MTT solution under light-proof conditions, continue incubation for 2 hours in the dark, carefully discard the supernatant, and add 100 μL DMSO.

[0077] Three illumination groups: replace with 100 μL DMEM incomplete medium containing samples of different concentrations, incubate for 4 h, remove the 96-well plate, and illuminate each well with 635 nm (5 mW·cm -2 ) laser irradiation for 3 minutes, then return to the incubator and continue incubation for 20 hours. Remove the 96-well plate and add 10 μL of 5 mg / mL MTT solution in the dark. Incubate for another 2 hours in the dark. Carefully discard the supernatant and add 100 μL of DMSO.

[0078] Use a microplate reader to measure the absorbance of each well at a wavelength of 492 nm, record the results, and calculate the cell survival rate according to the following formula.

[0079] Cell survival rate (%) = absorbance value of test group / absorbance value of blank group * 100%

[0080] After the drug was incubated with MDA-MB-231 cells for 24 h, the cytotoxicity results were as follows: Figure 7 As shown, hypericin (HPC), a berberine-hypericin-dBET57 physical mixture (Mix), and berberine-hypericin-dBET57 self-assembled nanoparticles (BHP NPs) all exhibited in vitro cytotoxicity against breast cancer MDA-MB-231 cells. In particular, at the same dose concentration, berberine-hypericin-dBET57 self-assembled nanoparticles (BHP NPs) showed significantly greater cytotoxicity against triple-negative breast cancer cells than either the physical mixture alone or the free drug, and laser irradiation further enhanced the cytotoxicity of the nanoparticles. This suggests that a synergistic treatment strategy based on a nanodelivery system has significant therapeutic efficacy against triple-negative breast cancer.

[0081] Example 4

[0082] In vitro metabolic inhibition evaluation of BHP NPs:

[0083] The in vitro metabolic inhibition ability of nanoparticles was evaluated by measuring the intracellular adenosine triphosphate (ATP) content, glycolytic metabolite lactate content, and mitochondrial respiratory chain complex I activity of MDA-MB-231 cells after treatment with different drugs.

[0084] The drug preparations for each group were the same as those in Example 3, and the specific groupings and dosing concentrations were as follows:

[0085] The blank control group (PBS group); berberine non-illumination group (BBR group, berberine concentration was 1.17 μg / mL); dBET57 non-illumination group (dBET57 group, dBET57 concentration was 2.22 μg / mL); hypericin non-illumination group (HPC group, hypericin concentration was 1.60 μg / mL); berberine-hypericin-dBET57 physical mixture non-illumination group (Mix group, berberine, hypericin and dBET57 mass ratio was 1.17:1.60:2.22, total concentration was 5 μg / mL); BHP NPs non-illumination group (BHP NPs group, BHPNPs concentration was 5 μg / mL); hypericin illumination group (HPC+L group, hypericin concentration was 1.60 μg / mL); berberine-hypericin-dBET57 physical mixed illumination group (Mix+L group, the mass ratio of berberine, hypericin and dBET57 was 1.17:1.60:2.22, and the total concentration was 5 μg / mL); BHP NPs illumination group (BHP NPs+L group, BHP NPs concentration was 5 μg / mL).

[0086] a) Determination of intracellular adenosine triphosphate (ATP) content

[0087] Human breast cancer cells MDA-MB-231 in the logarithmic phase were collected by trypsin digestion and the cells were concentrated at 1×10 6 The cells were seeded into 6-well plates at a density of 1000 cells / well and cultured for 12 h.

[0088] Blank control group and five non-illumination groups: replace with 1 mL PBS (blank control group) or DMEM incomplete medium containing different concentrations of samples (non-illumination group), continue incubation for 12 h, remove the 6-well plate, and use the ATP Assay Kit (Beyotime) to determine the intracellular ATP content.

[0089] Three illumination groups: 1 mL of DMEM incomplete medium containing samples of different concentrations was replaced, and after incubation for 4 h, the 6-well plate was removed and each well was illuminated with 635 nm (5 mW·cm -2 The cells were irradiated with laser for 3 min and then returned to the incubator for another 8 h. The intracellular ATP content was determined using an ATP Assay Kit (Beyotime).

[0090] like Figure 8 As shown in the figure, BHP NPs can reduce the intracellular ATP level and cut off the energy source of tumor cells, among which the inhibition of ATP level is most significant when combined with photodynamic therapy under light conditions.

[0091] b) Glycolytic activity assay

[0092] Human breast cancer cells MDA-MB-231 in the logarithmic phase were collected by trypsin digestion and the cells were concentrated at 1×10 6 The cells were seeded into 6-well plates at a density of 1000 cells / well and cultured for 12 h.

[0093] Blank control group and 5 non-illumination groups: replace with 1 mL of DMEM incomplete medium (blank control group) or DMEM incomplete medium containing different concentrations of samples (non-illumination group), continue incubation for 12 hours, remove the 6-well plate, and use the lactate assay kit (Lactate Assay Kit-WST, Dojindo) to determine the changes in the content of cellular glycolysis metabolites after drug treatment.

[0094] 3 illumination groups: replace with 1 mL of DMEM incomplete medium containing samples of different concentrations, incubate for 4 h, take out the 6-well plate, and use 635 nm (5 mW·cm -2 The cells were irradiated with laser for 3 minutes and then returned to the incubator for another 8 hours. The 6-well plate was removed and the lactate assay kit (Lactate Assay Kit-WST, Dojindo) was used to measure the changes in the content of lactate, a glycolytic metabolite in the cells after drug treatment.

[0095] like Figure 9 As shown in the figure, BHP NPs can inhibit the glycolysis metabolic pathway and reduce the production of the metabolite lactate, among which the inhibitory effect is most obvious when combined with photodynamic therapy under light conditions.

[0096] d) Intracellular mitochondrial respiratory chain complex I activity assay

[0097] Logarithmic phase breast cancer cells MDA-MB-231 were collected by trypsin digestion and the cells were collected at 1×10 6 The cells were seeded into 6-well plates at a density of 1000 cells / well and cultured for 12 h.

[0098] Blank control group and five non-illumination groups: replace with 1 mL of DMEM incomplete medium (blank control group) or DMEM incomplete medium containing different concentrations of samples (non-illumination group), continue incubation for 12 h, remove the 6-well plate, and measure the intracellular mitochondrial respiratory chain complex I activity using the mitochondrial respiratory chain complex I activity detection kit (Solarbio).

[0099] 3 illumination groups: replace with 1 mL of DMEM incomplete medium containing samples of different concentrations, incubate for 4 h, take out the 6-well plate, and use 635 nm (5 mW·cm -2 ) laser irradiation for 3 minutes, and then returned to the incubator for another 8 hours. The 6-well plate was removed and the intracellular mitochondrial respiratory chain complex I activity was measured using the Mitochondrial Respiratory Chain Complex I Activity Assay Kit (Solarbio).

[0100] like Figure 10 As shown in the figure, BHP NPs can significantly reduce the activity of intracellular mitochondrial respiratory chain complex I, among which the inhibitory effect is most obvious when combined with photodynamic therapy under light conditions.

[0101] Example 5

[0102] Investigation of mitochondrial targeting ability of BHP NPs

[0103] The preparation of BHP NPs solution and berberine-hypericin-dBET57 physical mixture was the same as in Example 4, with a concentration of 5 μg / mL.

[0104] By utilizing the autofluorescence signal of hypericin in nanoparticles and physical mixtures, the intracellular distribution of nanoparticles and physical mixture drugs can be determined by observing the red fluorescence of hypericin (HPC channel).

[0105] Logarithmic phase breast cancer cells MDA-MB-231 were collected by trypsin digestion and the cells were collected at 1×10 6 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured for 12 hours. The culture medium was removed, and 1 mL of the berberine-hypericin-dBET57 physical mixture (Mix group, 5 μg / mL) and the BHP NPs group (5 μg / mL) were added to each well. After incubation for 12 hours in serum-free conditions, the cells were washed three times with PBS. Mitochondria were stained with the commercial mitochondrial dye MitoBright LT Green (Dojindo) at a concentration of 50 nM for 15 minutes, and cell nuclei were stained with Hoechst 333442 dye (blue fluorescence) for 5 minutes. Hypericin (red fluorescence) was then observed under a fluorescence microscope to detect the intracellular distribution of the drug after entering the tumor cells. Merge represents the overlap of the three fluorescence signals mentioned above. If a clear yellow color appears, that is, the red and green fluorescence signals overlap, it means that the nanoparticles can target mitochondria; if the yellow fluorescence is weak or the red and green fluorescence signals do not overlap, it means that the preparation does not have the ability to target mitochondria.

[0106] like Figure 11 As shown in the figure, in the BHP NPs group, the red fluorescent nanoparticles (HPC channel) and the green fluorescent mitochondria (Mitotracker Green channel) can be clearly seen to overlap with each other, and the two appear yellow after being merged. Figure 12 ), the yellow fluorescence of the BHP NPs group was stronger, and the overlap between the red and green fluorescence signals was higher, indicating that BHP NPs could target mitochondria more effectively.

[0107] Example 6

[0108] Investigation of the degradation ability of BHP NPs on target proteins

[0109] Western Blot experiments were performed to evaluate the ability of the nanoparticles to degrade BRD4 and c-Myc proteins in MDA-MB-231 cells. The drug grouping and formulation were the same as in Example 4.

[0110] Logarithmic phase breast cancer cells MDA-MB-231 were collected by trypsin digestion and the cells were collected at 1×10 6 Cells were seeded at a density of 100 cells / well in a 6-well plate and incubated for 12 hours. The culture medium was removed, and 1 mL of PBS buffer (pH 7.4) was added to each well of the blank control group. 1 mL of PBS buffer was added to each well of the berberine non-illumination group, hypericin non-illumination group, dBET57 non-illumination group, berberine-hypericin-dBET57 physical mixture non-illumination group, BHP NPs non-illumination group, hypericin illumination group, berberine-hypericin-dBET57 physical mixture illumination group, and BHP NPs illumination group, and the culture was continued for 12 hours. After incubation, various samples were measured according to standard operating procedures.

[0111] Figure 13 The degradation effects of target proteins BRD4 and c-Myc in MDA-MB-231 cells after treatment with different drugs show that nanoparticles can effectively degrade target proteins, and the degradation of target proteins is more significant under light conditions.

[0112] Example 7

[0113] BHP NPs induce ferroptosis

[0114] a) TEM examination of organelle morphology

[0115] The preparation of BHP NPs solution was the same as in Example 4, and the concentration of BHP NPs was 5 μg / mL.

[0116] MDA-MB-231 cells (1×10 8 ) were treated with a BHP NP solution (5 μg / mL) for 12 hours. Cells were harvested and centrifuged at 1500 rpm. The supernatant was discarded, and the cell clumps were collected in a 10 mL centrifuge tube. Pre-chilled 4°C fixative was slowly added along the tube wall until the cell clumps were completely submerged. The fixed cells were sectioned using an ultramicrotome and stained with lead citrate and uranyl acetate in ethanol. The samples were observed using a biological transmission electron microscope, and mitochondria were observed and photographed. Normal cells were used as a control.

[0117] Depend on Figure 14It can be seen that compared with normal cells (Control), cells treated with BHP NPs showed abnormal mitochondrial morphology, increased mitochondrial membrane density, darker mitochondrial inner membrane color, reduced mitochondrial cristae, and wider spaces between mitochondrial cristae, indicating that the main cell death mode of tumor cells is ferroptosis.

[0118] b) Quantitative indicators and qualitative image capture of ferroptosis-related lipid peroxidation

[0119] Ferroptosis is a regulated cell death characterized by intracellular lipid peroxidation. The most common trigger for lipid oxidation is GSH depletion, accompanied by decreased GPX4 expression or GPX4 inactivation. Therefore, the present invention confirmed that BHP NPs treatment induced ferroptosis by measuring GSH content, GPX4 content, and the degree of lipid peroxidation.

[0120] The drug preparation was the same as in Example 3, and the specific grouping and administration concentrations were as follows:

[0121] Blank control group (PBS group); berberine-hypericin-dBET57 physical mixing non-illumination group (Mix group, the mass ratio of berberine, hypericin and dBET57 was 1.17:1.60:2.22, and the total concentration was 5 μg / mL); BHP NPs non-illumination group (BHP NPs group, the concentration of BHP NPs was 5 μg / mL); berberine-hypericin-dBET57 physical mixing illumination group (Mix+L group or Mix(+) group, the mass ratio of berberine, hypericin and dBET57 was 1.17:1.60:2.22, and the total concentration was 5 μg / mL); BHP NPs illumination group (BHP NPs+L group or BHP NPs(+) group, the concentration of BHP NPs was 5 μg / mL).

[0122] Logarithmic phase breast cancer cells MDA-MB-231 were collected by trypsin digestion and the cells were collected at 1×10 6 The cells were seeded into 6-well plates at a density of 1000 cells / well and cultured for 12 h.

[0123] Blank control group and two non-illumination groups: replace with 1 mL PBS (blank control group) or DMEM incomplete medium containing different concentrations of samples (non-illumination group), and continue incubation for 12 h.

[0124] 2 illumination groups: replace with 1 mL of DMEM incomplete medium containing different samples, incubate for 4 h, take out the 6-well plate, and use 635 nm (5 mW·cm -2 ) laser irradiation for 3 min, and then returned to the incubator for further incubation for 8 h.

[0125] The 6-well plate was taken out and the concentrations of malondialdehyde (MDA), glutathione (GSH) and lipid peroxide (LPO) in the cells after incubation were detected using an MDA detection kit, a GSH detection kit and a Liperfluo-cell lipid peroxide detection kit, respectively.

[0126] Depend on Figure 15 It can be seen from a that compared with the control group (Control) and other treatment groups, the content of antioxidant GSH in cells was reduced after BHP NPs+L treatment, and there was a significant difference, indicating that BHP NPs+L can directly cause a decrease in the content of anti-lipid oxidation substances.

[0127] The present invention detects the lipid oxidation end product MDA which can reflect the content of oxidized lipids in cells. Figure 15 As shown in b, it shows that BHP NPs+L caused an increase in oxidized lipids.

[0128] Liperfluo will be specifically oxidized by lipid peroxides and emit strong fluorescence in organic solvents such as ethanol, which can be used to detect lipid peroxides (LPO). Figure 15 c), LPO signal (green fluorescence) in the BHP NPs+L group was significantly increased compared with other treatment groups or the blank control group.

[0129] These results confirmed that cells treated with BHP NPs+L underwent ferroptosis, with typical morphological and biochemical characteristics. Furthermore, compared with BHP NPs and Mix+L, BHP NPs+L exhibited a synergistically enhanced ferroptosis effect.

[0130] Example 8

[0131] Investigation of the anti-tumor ability of BHP NPs in vivo:

[0132] After freeze-drying the BHP NPs prepared in Example 1, the BHP NPs were accurately weighed and prepared into a solution with a nanoparticle concentration of 1 mg / mL using sterile physiological saline.

[0133] Berberine was accurately weighed and ultrasonically prepared into a berberine solution with a concentration of 10 mg / mL using dimethyl sulfoxide. Before the experiment, under sterile conditions, it was diluted with sterile saline to obtain a berberine solution with a concentration of 0.234 mg / mL.

[0134] Hypericin was accurately weighed and ultrasonically prepared into a 10 mg / mL hypericin solution using dimethyl sulfoxide. Before the experiment, the solution was diluted with sterile saline under sterile conditions to obtain a 0.320 mg / mL hypericin solution.

[0135] Accurately weigh dBET57 and ultrasonically prepare a dBET57 solution with a concentration of 10 mg / mL using dimethyl sulfoxide. Before the experiment, dilute it with sterile saline under sterile conditions to obtain a dBET57 solution with a concentration of 0.444 mg / mL.

[0136] Accurately weigh 1.17 mg of berberine, 1.60 mg of hypericin, and 2.22 mg of dBET57 and ultrasonically prepare a 50 mg / mL physical mixture solution with 100 μL of dimethyl sulfoxide. Before the experiment, aseptically dilute the solution with sterile saline to a 1 mg / mL concentration.

[0137] BALB / c female nude mice were used, and all animal experiments complied with the standards of the Animal Ethics Committee of Fujian University of Traditional Chinese Medicine.

[0138] Take MDA-MB-231 cells in the logarithmic growth phase, centrifuge, collect the cells, and suspend them with PBS to obtain a cell suspension. 6 The cells were injected subcutaneously into the right mammary gland of BALB / c female nude mice at a density of 100 cells / mouse. Observation began 7 days after inoculation and the tumor volume reached 100 mm. 3 When the MDA-MB-231 tumor-bearing nude mouse model was successfully established, the mice were randomly divided into 9 groups (n=4), and each mouse was injected with 0.2 mL of drug through the tail vein:

[0139] Saline group; berberine group (BBR group, 0.234 mg / mL); dBET57 group (0.444 mg / mL); hypericin non-illumination group (HPC group, 0.320 mg / mL); berberine-hypericin-dBET57 physical mixture non-illumination group (Mix group, 1 mg / mL); BHP NPs non-illumination group (BHP NPs group, 1 mg / mL); hypericin illumination group (HPC+L group, 0.320 mg / mL); berberine-hypericin-dBET57 physical mixture illumination group (Mix+L group, 1 mg / mL); BHP NPs illumination group (BHPNPs+L group, 1 mg / mL).

[0140] Each group of mice was administered with 638 nm (0.5 mW cm -2 The mouse tumors were irradiated with a laser for 5 minutes. Every three days, the tumor volumes were measured using a vernier caliper. Tumor volumes were calculated and plotted.

[0141] Tumor volume = long axis × short axis × short axis / 2

[0142] The weight of the mice was recorded throughout the dosing cycle. The relative change rate of the mice's weight compared to the weight of the mice on day 1 was weighed and calculated, and a weight-time curve was drawn. If the weight change rate decreased by more than 15%, the test drug was considered to be highly toxic.

[0143] Figure 16 Figure 3 is the curve of mouse body weight changes during the treatment period. Compared with the saline group, there was no significant decrease in body weight in the BHP NPs light-irradiated or non-light-irradiated groups, indicating that BHP NPs have good safety.

[0144] Figure 17 Figure 3 is the curve of mouse tumor volume changes during the treatment cycle. It can be seen that after the treatment, the tumor volume of each treatment group decreased to varying degrees, among which the tumor inhibition effect of the BHP NPs light irradiation group was the most significant, and showed a trend of gradual shrinkage.

[0145] In summary, compared with simple combined administration strategy or single administration strategy, the synergistic treatment method based on nanodelivery system has the best anti-tumor activity and safety.

Claims

1. A mitochondrial-targeting self-assembly PROTAC nanomaterial based on natural products, characterized by: It is a nanoparticle prepared by nanoprecipitation method using mitochondrial targeting molecules, photosensitizers and 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 comprises 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; adding the mixed solution dropwise into water at room temperature, stirring during the addition, and after the addition is completed, standing, ultrafiltration or dialysis to remove the organic solvent 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 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 in which the volume ratio of dimethyl sulfoxide to methanol is 4:1 to 10:1, more preferably a mixed solvent in which the volume ratio of dimethyl sulfoxide to methanol is 10:

1.

6. The natural product mitochondrial-targeted self-assembly PROTAC nanomaterial according to claim 3, characterized in that: The ultrafiltration tube used in the ultrafiltration has a molecular weight cut-off of 1000 to 10000 Da, preferably 3500 Da.

7. A method for preparing a natural product mitochondrial-targeting self-assembly PROTAC nanomaterial, comprising: Berberine, hypericin and dBET57 are dissolved in a benign organic solvent to form a mixed solution, or berberine, hypericin and dBET57 are dissolved in a benign organic solvent respectively to prepare a berberine solution, a hypericin solution and a dBET57 solution, and then the three solutions are mixed to form a mixed solution; the mixed solution is added dropwise to water at room temperature with stirring during the addition, and after the addition is completed, the mixture is allowed to stand, and the organic solvent is removed by ultrafiltration or dialyzation 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.

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