Preparation method and application of nanoparticles co-assembled with triterpenoid small molecules wrapped in cancer cell membranes and loaded with Ce6 and copper ions

Abstract

A method for preparing nanoparticles of triterpenoid small molecules co-assembled with Ce6 and copper ions wrapped in cancer cell membranes and its application. The present invention prepares a pure natural co-assembled nanotransmission system with new morphological characteristics and multiple biological activities - terpenoid compound and photosensitizer co-assembled nanoparticles. In the composite nanoassembly, triterpenoid compounds and photosensitizer Ce6 are assembled into nanoparticles, providing effective and safe chemotherapy and photodynamic therapy. Subsequently, Cu is complexed on the surface of the nanoparticles. 2+ , when Cu is introduced 2+ When activated, the reduced PDT effect caused by excessive glutathione in the tumor consuming reactive oxygen species can be counteracted. Furthermore, it can enhance CDT therapy through a Fenton-like catalytic reaction with overexpressed H₂O₂ at the tumor site. Finally, these molecules are encapsulated by the tumor cell membrane. By constructing CM@OABACe6 / CuNPs with homologous targeting, a triple synergistic platform for cancer treatment using PDT, chemotherapy, and CDT has been created.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterials, and specifically relates to a preparation method and application of cancer cell membrane-wrapped terpenoid compound co-assembled nanoparticles loaded with photosensitizer Ce6 and copper ions. Background Art

[0002] It is well known that cancer cells can spread to many different tissues and organs, making treatment very difficult. The clinical efficacy of traditional cancer chemotherapy (chemo) is seriously affected by the easy development of drug resistance and high toxic side effects. Photodynamic therapy (PDT) is a promising cancer treatment modality that can be combined with chemotherapy to improve overall survival. By using visible light and photosensitizers, PDT kills tumor cells in the presence of oxygen. Compared with traditional therapies, PDT has low toxicity, high responsiveness, low invasiveness and good selectivity. However, photosensitizers (PSs) often encounter some limitations in their applications, including poor water solubility, low bioavailability and low selectivity for tumors. Therefore, effective PDT requires an excellent drug delivery system (DDS) to assist.

[0003] To address these deficiencies, nanocarriers made from natural compounds (such as polysaccharides and proteins) have been widely used to construct drug delivery systems (DDS) due to their low toxicity, good biocompatibility, and long blood retention. Among them, nanocarriers derived from self-assembling natural small molecules (NSMs) have recently emerged as a promising approach for cancer treatment. Some NSMs based on triterpenes, diterpenes, and sterols can be assembled into nanocarriers with excellent bioactivity (e.g., anticancer, antioxidant) and biocompatibility. However, most NSMs fail to self-assemble into suitable nanocarrier morphologies to meet the requirements of DDS. To control the physical size and morphology of NSM nanocarriers, researchers have utilized supramolecular co-assembly strategies. Using this co-assembly strategy, it is possible to construct a multifunctional nanodelivery platform with synergistic antitumor effects based on different NSMs. This strategy has greatly expanded the variety and range of NSMs that can be used to create multifunctional nanocarriers. However, due to the complexity of tumor pathogenesis, single chemotherapeutic approaches often fail to achieve the desired therapeutic effect. Therefore, the use of multiple synergistic therapies rather than single therapies has become a fundamental component of recent cancer treatment.

[0004] Chemodynamic therapy utilizes the tumor microenvironment to activate the Fenton-like reaction to produce strong oxidative hydroxyl radicals for tumor-specific treatment. Some metal ion-based nanomaterials show great potential because they can produce ROS through Fenton-like reactions, such as copper ions (Cu 2+). More importantly, under appropriate conditions, the reaction rate is much greater than that of the iron-based Fenton reaction. 2+ It is the third most abundant transition metal ion in the human body and plays an important role in biological systems. As an important trace element, Cu 2+ Due to its active chemical properties and high catalytic ability, it has unique advantages in tumor treatment. It is generally believed that the tumor microenvironment (TME) plays an important role in tumor progression, invasion, metastasis and drug resistance, and is also the source and residence of cancer cells. However, excessive GSH in the TME will consume ROS, resulting in a decrease in the efficacy of PDT. It has been proven that the use of GSH containing metal ions (i.e., Cu 2+ ) nanocarriers for stimuli-responsive anticancer therapy, which consumes excess GSH in cells through redox reactions, thereby improving the efficacy of PDT. 2+ A Fenton-like catalysis occurs for chemical dynamics (CDT). Therefore, multiple synergistic therapies based on Chemo-PDT-CDT nanodelivery systems have become a strategy to improve the clinical efficacy of cancer.

[0005] In biomedicine, one of the major challenges is to design a nano-DDS with active targeting capabilities that can effectively actively transport drugs to specific locations while also having therapeutic effects. As tumor-targeting properties, nanocarriers encapsulated on cell membranes have better biodegradability, lower toxicity, lower immunogenicity, and higher biocompatibility than single NPs. In addition, complex nanocarriers using cell membranes as biomimetic can circulate for longer periods of time without being detected and eliminated by the immune system. They can also be delivered to specific sites with high efficiency and active transport, resulting in higher accumulation at the tumor site. 4T1 (mouse breast cancer cells) are a type of superficial cancer cell that can maximize the therapeutic effect of PDT. Therefore, we chose 4T1 cell membranes to construct nanocarriers with homologous targeting. Summary of the Invention

[0006] The purpose of the present invention is to solve the problems of poor water solubility, low bioavailability, low selectivity for tumors and large toxic side effects of the existing methods, and to provide a method for preparing nanoparticles of triterpenoid small molecules co-assembled and loaded with Ce6 and copper ions wrapped by cancer cell membranes and its application. The triterpenoid small molecules co-assembled and loaded with photosensitizer Ce6, Cu 2+ Nanoparticles are prepared from natural small molecule triterpenoid compounds oleanolic acid and betulinic acid and photosensitizer Ce6, loaded with Cu 2+ The cell membrane is wrapped on the surface. 2+A nano-drug delivery platform targeting tumor cells and tumor cell membranes, a major feature of which is that it provides tumor destination targeting, triple synergistic treatment with chemotherapy, PDT and CDT, and TME stimulation responsiveness.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing cancer cell membrane-wrapped triterpene small molecule co-assembled nanoparticles loaded with photosensitizer Ce6 and copper ions, the method comprising:

[0009] Step 1: Preparation of nanoparticles co-assembled with oleanolic acid, betulinic acid and photosensitizer Ce6:

[0010] (1) Dissolve oleanolic acid, betulinic acid and Ce6 in dimethyl sulfoxide;

[0011] (2) adding a solution of oleanolic acid, betulinic acid, and Ce6 into water;

[0012] (3) Stirring at 400-600 rpm for 10-60 min, and centrifuging at 10,000-15,000 rpm for 10-30 min;

[0013] Step 2: Co-assembly of oleanolic acid and betulinic acid with photosensitizer Ce6 to form nanoparticles complexed with Cu 2+ Preparation:

[0014] (1) Oleanolic acid and betulinic acid were co-assembled with photosensitizer Ce6 and dissolved in water;

[0015] (2) adding CuCl2 aqueous solution to the aqueous solution of nanoparticles co-assembled with oleanolic acid, betulinic acid and photosensitizer Ce6;

[0016] (3) Stirring at 400-600 rpm for 10-60 min, and centrifuging at 10,000-15,000 rpm for 10-30 min;

[0017] Step 3: Oleanolic acid and betulinic acid wrapped in cancer cell membranes are co-assembled with photosensitizer Ce6 to form nanoparticles complexed with Cu 2+ Preparation:

[0018] (1) Lyse 4T1 breast cancer cells using hypotonic lysis buffer;

[0019] (2) Under the action of vortex, the cell membrane is added to the nanoparticle solution (the solution obtained by dissolving the precipitate obtained by the final centrifugation in step 2 in water), and oleanolic acid and betulinic acid are co-assembled with the photosensitizer Ce6 to form nanoparticles complexed with Cu 2+ The nanoparticle concentration is 1 mg / mL to 50 mg / mL;

[0020] (3) subjecting the mixture to probe ultrasound for 1 to 10 minutes; ultrasound provides external force to wrap the cell membrane around the nanoparticles;

[0021] (4) Stir at 400-600 rpm for 10-60 min, and centrifuge at 10,000-15,000 rpm for 10-30 min.

[0022] Furthermore, in step one (1), the concentrations of oleanolic acid, betulinic acid and Ce6 in dimethyl sulfoxide are 5 mg / mL to 50 mg / mL.

[0023] Furthermore, in step 1 (2), the volume ratio of the solution of oleanolic acid, betulinic acid and Ce6 to water is 50 to 100:1.

[0024] Furthermore, in step 2 (1), the concentration of the co-assembled nanoparticles in water is 1 mg / mL to 50 mg / mL.

[0025] Furthermore, in step 2 (2), the concentration of the CuCl2 aqueous solution is 5 mg / mL to 50 mg / mL.

[0026] Furthermore, in step 2 (3), the volume ratio of the CuCl2 aqueous solution to the nanoparticle aqueous solution is 50-100:1.

[0027] Furthermore, in step 3 (1), the 4T1 breast cancer cells are from 10 7 ~10 9 cells.

[0028] Furthermore, in step three (2), the concentration of the nanoparticles is 1 mg / mL to 50 mg / mL.

[0029] Furthermore, in step three (3), the ultrasonic time is 1 min to 10 min.

[0030] The triterpene small molecules wrapped in the cancer cell membrane prepared above are co-assembled into nanoparticles loaded with Ce6 and copper ions for anti-tumor treatment.

[0031] The beneficial effects of the present invention compared to the prior art are:

[0032] (1) Triterpenoids are a class of natural products composed of isoprene as a structural unit. Oleanolic acid and betulinic acid are the main active ingredients in traditional Chinese medicine and natural botanicals, exhibiting multiple therapeutic and tissue-protective properties, including anti-tumor, antioxidant, anti-inflammatory, and cardioprotective effects.

[0033] (2) The preparation method of the present invention is simple and convenient, and the preparation of nanoparticles co-assembled with terpenoid compounds and photosensitizer Ce6 can be achieved at room temperature, saving energy. In addition, no other organic reagents are added during the preparation process, eliminating the need for subsequent impurity removal steps.

[0034] (3) The nanodrug delivery platform prepared by the present invention has the advantages of biocompatibility and biosafety, minimal side effects, enhanced accumulation ability at the tumor site, and significant antitumor activity.

[0035] (4) The present invention broadens the horizon of multi-component NSMs synergistically treating cancer and expands the medical application of bioactive NSMs. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a scanning electron micrograph of the co-assembled nanoparticles of Comparative Example 2;

[0037] Figure 2 is a scanning electron micrograph of the co-assembled nanoparticles of Comparative Example 1;

[0038] Figure 3 is a scanning electron micrograph of the co-assembled nanoparticles of Example 1;

[0039] Figure 4 TEM image of the co-assembled nanoparticles of Comparative Example 1;

[0040] Figure 5 is a transmission electron microscopy image of the co-assembled nanoparticles of Example 1;

[0041] Figure 6 The UV spectra of the co-assembled nanoparticles of Example 1, Comparative Example 1 and Comparative Example 2 are shown;

[0042] Figure 7 This is the XPS experimental result of the co-assembled nanoparticles in Example 1;

[0043] Figure 8 This is a high-resolution XPS experimental result diagram of the co-assembled nanoparticles in Example 1;

[0044] Figure 9 This is a diagram showing the MTT test results of the co-assembled nanoparticles of Example 1;

[0045] Figure 10 This is a graph showing the in vivo distribution experimental results of the co-assembled nanoparticles of Example 1;

[0046] Figure 11 This is a diagram showing the distribution results of the co-assembled nanoparticles in various tissues and organs of Example 1;

[0047] Figure 12 This is a graph showing the experimental results of the anti-tumor activity of the co-assembled nanoparticles of Example 1;

[0048] Figure 13 This is a diagram showing the in vivo safety test results of the co-assembled nanoparticles of Example 1. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments, but the technical solution of the present invention is not limited to the specific implementation methods listed below. Any modification or equivalent replacement of the technical solution of the present invention that does not depart from the core and content of the technical solution of the present invention should be included in the scope of protection of the present invention.

[0050] The present invention implements a new synergistic strategy to improve the safety and efficacy of cancer treatment. Nanocarriers formed by co-assembling two-component triterpenoid compounds (oleanolic acid and betulinic acid) were screened as chemotherapeutic drugs and combined with photodynamic therapy (PDT). Since NSMs were co-assembled with photosensitizer (Ce6), multifunctional DDSs were obtained, which can exert synergistic effects when combined with different treatment methods. Cu was then introduced into the DDSs. 2+ The composite nanocombination not only depletes excess GSH at the tumor site but also unleashes the power of CDT. Finally, by coating the NP surface with the cell membrane of 4T1 (a mouse breast cancer cell), the NPs can be targeted and aggregated at the tumor site due to the homing properties of cancer cells, further enhancing the tumor therapeutic efficacy of the Chemo-CDT-PDT combination. This invention establishes a facile method for a synergistic tumor therapy platform using actively targeted natural nano-DDSs and provides a promising combination strategy that expands the medical applications of NSMs.

[0051] The present invention uses a co-assembly strategy to prepare a pure natural co-assembled nanotransmission system with new morphological characteristics and multiple biological activities - terpenoid compound and photosensitizer co-assembled nanoparticles. In the composite nanoassembly, triterpenoid compounds (OA, and BA) are assembled into nanoparticles together with the photosensitizer chloride 6 (Ce6), providing effective and safe chemotherapy (Chemo) and photodynamic therapy (PDT). Subsequently, Cu is complexed on the surface of OABACe6 NPs. 2+ , when Cu is introduced 2+When the drug is injected into the tumor, the effect of reducing PDT due to the consumption of reactive oxygen species (ROS) by excessive glutathione (GSH) in the tumor can be offset. In addition, it can also confer chemodynamic therapy (CDT) by undergoing a Fenton-like catalytic reaction with H2O2 overexpressed in the tumor site. Finally, these molecules are wrapped by the tumor cell membrane. Studies have shown that nanoparticles encapsulated on the tumor cell membrane achieve good tumor targeting. By constructing CM@OABACe6 / CuNPs with homologous targeting, the present invention creates a triple synergistic platform for cancer treatment using PDT, chemotherapy and CDT, and proposes a new combination strategy for the development of multifunctional synergistic therapies using small molecules.

[0052] Example 1:

[0053] A cancer cell membrane-wrapped triterpenoid small molecule co-assembly loaded with photosensitizer Ce6, Cu 2+ The preparation method of nanoparticles is carried out according to the following steps:

[0054] Step 1: Dissolve 5 mg each of oleanolic acid, betulinic acid, and Ce6 (15 mg total) in 1 mL of dimethyl sulfoxide and sonicate until the solid powder is completely dissolved.

[0055] Step 2: Add 0.01 mL of the oleanolic acid, betulinic acid, and Ce6 solution to 1 mL of water and stir at 400 rpm for 10 minutes. Centrifuge at 10,000 rpm for 20 minutes. The volume ratio of water to dimethyl sulfoxide is 1:100, and the concentration of oleanolic acid, betulinic acid, and Ce6 is 15 mg / mL.

[0056] Step 3: Dissolve 1 mg of oleanolic acid and betulinic acid co-assembled nanoparticles with photosensitizer Ce6 in 1 mL of deionized water;

[0057] Step 4: Dissolve 5 mg of CuCl2 in 1 mL of deionized water and sonicate until the solid powder is completely dissolved; the concentration of CuCl2 in water is 5 mg / mL.

[0058] Step 5: Add 0.01 mL of CuCl2 solution to 1 mL of co-assembled nanoparticle solution, stir at 400 rpm for 10 min, and centrifuge at 10,000 rpm for 20 min.

[0059] Step 6: 1 mg of oleanolic acid and betulinic acid were combined with photosensitizer Ce6 to form Cu 2+ The co-assembled nanoparticles were dissolved in 1 mL of deionized water; the concentration of the nanoparticles in water was 1 mg / mL.

[0060] Step 7: Lyse 4T1 breast cancer cells (usually from 10 7cells):

[0061] Step 8: Add the cell membranes to the nanoparticle solution while vortexing. Then, perform probe ultrasound on the mixture for 1 to 10 minutes. Stir at 400 rpm for 10 minutes. Centrifuge at 10,000 rpm for 20 minutes.

[0062] Comparative Example 1:

[0063] A triterpene small molecule co-assembly loaded with photosensitizer Ce6, Cu 2+ The preparation method of nanoparticles is carried out according to the following steps:

[0064] Step 1: Dissolve 5 mg each of oleanolic acid, betulinic acid, and Ce6 (15 mg total) in 1 mL of dimethyl sulfoxide and sonicate until the solid powder is completely dissolved.

[0065] Step 2: Add 0.01 mL of the oleanolic acid, betulinic acid, and Ce6 solution to 1 mL of water and stir at 400 rpm for 10 minutes. Centrifuge at 10,000 rpm for 20 minutes. The volume ratio of water to dimethyl sulfoxide is 1:100, and the concentration of oleanolic acid, betulinic acid, and Ce6 is 15 mg / mL.

[0066] Step 3: Dissolve 1 mg of oleanolic acid and betulinic acid co-assembled nanoparticles with photosensitizer Ce6 in 1 mL of deionized water;

[0067] Step 4: Dissolve 5 mg of CuCl2 in 1 mL of deionized water and sonicate until the solid powder is completely dissolved; the concentration of CuCl2 in water is 5 mg / mL.

[0068] Step 5: Add 0.01 mL of CuCl2 solution to 1 mL of co-assembled nanoparticle solution, stir at 400 rpm for 10 min, and centrifuge at 10,000 rpm for 20 min.

[0069] Comparative Example 2:

[0070] A method for preparing triterpene small molecule co-assembled photosensitizer Ce6 nanoparticles is carried out according to the following steps:

[0071] Step 1: Dissolve 5 mg each of oleanolic acid, betulinic acid, and Ce6 (15 mg total) in 1 mL of dimethyl sulfoxide and sonicate until the solid powder is completely dissolved.

[0072] Step 2: Add 0.01 mL of the oleanolic acid, betulinic acid, and Ce6 solution to 1 mL of water and stir at 400 rpm for 10 minutes. Centrifuge at 10,000 rpm for 20 minutes. The volume ratio of water to dimethyl sulfoxide is 1:100, and the concentration of oleanolic acid, betulinic acid, and Ce6 is 15 mg / mL.

[0073] Detection test:

[0074] Test 1: Microstructure test:

[0075] The oleanolic acid and betulinic acid obtained in Comparative Example 2, Comparative Example 1 and Example 1 were co-assembled with photosensitizer Ce6 to form nanoparticles, and the oleanolic acid and betulinic acid were complexed with photosensitizer Ce6 to form Cu 2+ Co-assembled nanoparticles and cancer cell membranes encapsulated oleanolic acid and betulinic acid with photosensitizer Ce6 complexed with Cu 2+ After the co-assembled nanoparticles are evenly dispersed in water, a drop of solution is taken and dropped on aluminum foil. After the water is evaporated, the aluminum foil with the sample is fixed with conductive glue and sprayed with gold. The microscopic morphology of the hydrogel is then observed using a scanning electron microscope. Figures 1 to 3 shown.

[0076] Depend on Figures 1 to 3 It can be seen that oleanolic acid and betulinic acid co-assemble nanoparticles with photosensitizer Ce6, and oleanolic acid and betulinic acid complex with photosensitizer Ce6 to form Cu 2+ Co-assembled nanoparticles and cancer cell membranes encapsulated oleanolic acid and betulinic acid with photosensitizer Ce6 complexed with Cu 2+ The co-assembled nanoparticles are uniform spherical nanoparticles with relatively uniform size and dense arrangement.

[0077] Experiment 2: Transmission electron microscopy characterization:

[0078] The oleanolic acid and betulinic acid obtained in Comparative Example 1 and Example 1 were complexed with photosensitizer Ce6 to form Cu 2+ Co-assembled nanoparticles and cancer cell membranes encapsulated oleanolic acid and betulinic acid with photosensitizer Ce6 complexed with Cu 2+ After the co-assembled nanoparticles are evenly dispersed in water, a drop of solution is taken and dropped on aluminum foil. After the water is evaporated, the aluminum foil with the sample is fixed with conductive glue and sprayed with gold. The microscopic morphology of the hydrogel is then observed using a scanning electron microscope. Figure 4 and 5 shown.

[0079] Depend on Figure 4 and 5 It can be seen that oleanolic acid and betulinic acid complexed Cu with photosensitizer Ce6 2+ Co-assembled nanoparticles and cancer cell membranes encapsulated oleanolic acid and betulinic acid with photosensitizer Ce6 complexed with Cu2+ The co-assembled nanoparticles are uniform spherical nanoparticles. It can be seen that the cancer cell membrane prepared by the present invention wraps oleanolic acid and betulinic acid and the photosensitizer Ce6 complexes Cu 2+ The co-assembled nanoparticles showed a double-layer membrane structure, confirming that the 4T1 cell membrane was successfully wrapped onto the surface of NPs.

[0080] Experiment 3: UV characterization:

[0081] Oleanolic acid and betulinic acid obtained in Comparative Example 2, Comparative Example 1 and Example 1 were co-assembled into nanoparticles with photosensitizer Ce6. Oleanolic acid and betulinic acid were complexed with photosensitizer Ce6 to form Cu 2+ Co-assembled nanoparticles and cancer cell membranes encapsulated oleanolic acid and betulinic acid with photosensitizer Ce6 complexed with Cu 2+ After the co-assembled nanoparticles were evenly dispersed in water, full wavelength scanning was performed using an ultraviolet spectrometer. Oleanolic acid, betulinic acid solutions, and Ce6 solutions were also scanned.

[0082] Depend on Figure 6 It can be seen that Ce6 has absorption peaks at 405nm and 660nm in UV / Vis. After being assembled into OA-BA-Ce6 NPs, the absorption peak of Ce6 has red-shifted. This is due to the ππ conjugation effect between OABA and Ce6, which causes red-shift. However, when OABACe6 NPs are complexed with Cu 2+ After that, the absorption peak of Ce6 shifted to the blue. This is because Cu 2+ It is an electron-deficient system that weakens the Ce6 conjugation, resulting in a blue shift.

[0083] Test 4: Particle size test:

[0084] Oleanolic acid and betulinic acid obtained in Comparative Example 2, Comparative Example 1 and Example 1 were co-assembled into nanoparticles with photosensitizer Ce6. Oleanolic acid and betulinic acid were complexed with photosensitizer Ce6 to form Cu 2+ Co-assembled nanoparticles and cancer cell membranes encapsulated oleanolic acid and betulinic acid with photosensitizer Ce6 complexed with Cu 2+ After the co-assembled nanoparticles were uniformly dispersed in water, they were measured using a Malvern particle size analyzer.

[0085] As can be seen from Table 1, oleanolic acid and betulinic acid co-assembled nanoparticles with photosensitizer Ce6, and oleanolic acid and betulinic acid complexed with photosensitizer Ce6 to form Cu 2+ Co-assembled nanoparticles and cancer cell membranes encapsulated oleanolic acid and betulinic acid with photosensitizer Ce6 complexed with Cu 2+The particle sizes of the co-assembled nanoparticles were 146.6±2.1nm, 152.9±1.5nm, and 175.1±1.1nm, respectively. The particle size of CM@OABACe6 / Cu NPs increased to 175.1±1.1nm. This is because the 4T1 cell membrane has a certain thickness, and the particle size will increase significantly after being wrapped with the cell membrane.

[0086] Table 1 Particle size and PDI of NPs

[0087] Nanoparticles Particle size (nm) PDI OABACe6 146.6±2.1 0.143±0.009 OABACe6 / Cu 152.9±1.5 0.159±0.006 CM@OABACe6 / Cu 175.1±1.1 0.122±0.001

[0088] Test 5, XPS test:

[0089] Oleanolic acid and betulinic acid obtained in Comparative Example 2, Comparative Example 1 and Example 1 were co-assembled into nanoparticles with photosensitizer Ce6. Oleanolic acid and betulinic acid were complexed with photosensitizer Ce6 to form Cu 2+ Co-assembled nanoparticles and cancer cell membranes encapsulated oleanolic acid and betulinic acid with photosensitizer Ce6 complexed with Cu 2+ The co-assembled nanoparticles were determined using X-ray photoelectron spectroscopy.

[0090] Figure 7 The results confirmed the chemical composition of Example 1. The spectrum showed four characteristic peaks of C1s, N1s, O1s and Cu 2p at 284.9eV, 399.3eV, 531.9eV and 934.2eV respectively. The high-resolution XPS spectrum of Cu 2p showed two main peaks at 934.6 and 954.2eV, which were Cu 2p3 / 2 and Cu 2p1 / 2 respectively. The results showed that Example 1 was composed of C, N, O and Cu 2+ composition.

[0091] Experiment 6: MTT test:

[0092] The specific test methods are as follows:

[0093] (1) Cell culture: 4T1 cells in the logarithmic growth phase were digested with trypsin to obtain a cell suspension, which was inoculated into a 96-well plate with a volume of 200 μL. The cells were cultured overnight at 37°C and 5% CO2 to allow the cells to adhere to the plate.

[0094] (2) Oleanolic acid and betulinic acid were co-assembled into nanoparticles with photosensitizer Ce6, and oleanolic acid and betulinic acid were complexed with Cu 2+ Co-assembled nanoparticles and cancer cell membranes encapsulated oleanolic acid and betulinic acid with photosensitizer Ce6 complexed with Cu 2+Preparation of co-assembled nanoparticle solution: According to the ratio of 1 mg of co-assembled nanoparticles of Comparative Example 2, Comparative Example 1, and Example 1 to 1 mL of culture medium, 1640 culture medium was added to the nanoparticle samples to prepare a solution with a concentration of 1 mg / mL;

[0095] (3) Mix MTT solid with PBS solution to prepare a 5 mg / mL solution, and then add 1640 medium for dilution to obtain a 0.5 mg / mL MTT solution;

[0096] (4) The 1 mg / mL co-assembled nanoparticle solutions of Comparative Example 2, Comparative Example 1, and Example 1 were diluted to 500 ug / mL, 250 ug / mL, 100 ug / mL, 50 ug / mL, 10 ug / mL, and 5 ug / mL, respectively. The culture medium in the 96-well plate was discarded, and 200 μL of the diluted nanoparticle solutions of different concentrations were added. Six replicate wells were set for each concentration. At the same time, a control group was set up with 200 μL of 1640 culture medium added. The culture was continued at 37°C, 5% CO2 for 24 hours. The laser group was incubated for 4 hours, irradiated with laser for 10 minutes, and incubated for another 20 hours. Then, the solution in the 96-well plate was removed, and 200 μL of the 0.5 mg / mL MTT solution obtained in step (3) was added. The cells were cultured at 37°C for 4 h, and then the liquid in the wells was removed to terminate the culture. 150 μL of DMSO was added to each well and incubated for 10 min. The absorbance of each well at 492 nm was measured by an enzyme-labeled instrument, and the cell survival rate was calculated. The test results were as follows: Figure 9 shown.

[0097] Figure 9 The results show that different concentrations of oleanolic acid and betulinic acid were co-assembled with photosensitizer Ce6 to form nanoparticles. Oleanolic acid and betulinic acid were complexed with photosensitizer Ce6 to form Cu 2+ Co-assembled nanoparticles and cancer cell membranes encapsulated oleanolic acid and betulinic acid with photosensitizer Ce6 complexed with Cu 2+ The toxic and side effects of the co-assembled nanoparticle solution on 4T1 cells show that the survival rate of 4T1 cells decreases rapidly with the increase of the equivalent Ce6 concentration. That is, when the equivalent Ce6 concentration in Comparative Example 2, Comparative Example 1, and Example 1 is 2 μg / mL, the survival rates of 4T1 cells are 41% and 31%, respectively. This shows that the nanoparticles wrapped with cancer cell membranes are more cytotoxic to cancer cells than the nanoparticles that are not wrapped. This is mainly attributed to the tumor homing properties of the cell membrane, which can make the nanoparticles gather more around the cells. Importantly, the survival rate of the nanoparticles after illumination is significantly lower than that of the nanoparticles that have not been illuminated. When the equivalent Ce6 concentration is 2 μg / mL, the survival rate of CM@OABACe6 / CuNP after illumination is 26.06%.

[0098] Experiment 7: In vivo distribution test:

[0099] Oleanolic acid and betulinic acid obtained in Comparative Example 2, Comparative Example 1 and Example 1 were co-assembled into nanoparticles with photosensitizer Ce6. Oleanolic acid and betulinic acid were complexed with photosensitizer Ce6 to form Cu 2+ Co-assembled nanoparticles and cancer cell membranes encapsulated oleanolic acid and betulinic acid with photosensitizer Ce6 complexed with Cu 2+ The co-assembled nanoparticles were evenly dispersed in physiological saline. Mice were randomly divided into two groups (n=3): (1) free Ce6 group (equal amount of Ce6 in each group: 4.5 mg / kg), (2) CM@OABACe6 / Cu NPs group. The tail vein injection volume was 150 μL. Mice were killed by cervical incision at 0, 2, 4, 8, 12, and 24 hours, and tumors and major organs were collected. The fluorescence signals of tumors and organs were obtained using a mouse in vivo optical imaging system. The experimental results are as follows Figure 10 and 11 shown.

[0100] from Figure 10 and 11 As can be seen, after intravenous injection of CM@OABACe6 / Cu NPs, CM@OABACe6 / Cu NPs rapidly accumulated within the tumor. A strong fluorescence signal was detected from the tumor 2 hours after injection, likely due to prolonged blood circulation and an enhanced permeability retention (EPR) effect. Even at the end of observation (24 hours), weak fluorescence emission persisted at the tumor site, confirming the effective accumulation of the nanoassembly within the tumor. In vitro imaging of mouse tumors and major organs 24 hours after injection further demonstrated the good targeting and long retention of CM@OABACe6 / Cu NPs. The liver and kidneys also exhibited significant fluorescence signals, especially at 24 hours after injection, suggesting that CM@OABACe6 / Cu NPs can be cleared from the mouse body via renal and hepatic pathways. Notably, the photosensitizer Ce6 also exhibited selective accumulation in the tumor due to its lower tumor targeting, but the fluorescence intensity was weaker than that of CM@OABACe6 / Cu NPs.

[0101] Experiment 8: In vivo anti-tumor test:

[0102] Oleanolic acid and betulinic acid obtained in Comparative Example 2, Comparative Example 1 and Example 1 were co-assembled into nanoparticles with photosensitizer Ce6. Oleanolic acid and betulinic acid were complexed with photosensitizer Ce6 to form Cu 2+ Co-assembled nanoparticles and cancer cell membranes encapsulated oleanolic acid and betulinic acid with photosensitizer Ce6 complexed with Cu 2+The co-assembled nanoparticles were evenly dispersed in physiological saline. Tumor-bearing mice were randomly divided into eight groups (n=5): (1) blank group: PBS, (2) control group: Ce6+laser (Ce6 equivalent: 4.5 mg / kg), (3) chemotherapy group: OABACe6 NPs (Ce6 equivalent: 4.5 mg / kg), (4) chemotherapy+PDT: OABACe6 NPs+laser (Ce6 equivalent: 4.5 mg / kg), (5) chemotherapy+CDT group: OABACe6 / CuNPs (Ce6 equivalent: 4.5 mg / kg), (6) chemotherapy+PDT+CDT group: OABACe6 / CuNPs+laser (Ce6 equivalent: 4.5 mg / kg), (7) targeted+chemotherapy+CDT group: CM@OABACe6 / Cu NPs (Ce6 equivalent: 4.5 mg / kg), (8) targeted+Chemo+CDT+PDT group: C / OA-BA-Ce6-Cu NPs+laser (Ce6 equivalent: 4.5 mg / kg). The drug was injected into the body through the tail vein, three times every other day. Six hours after administration, mice in the light group were irradiated with 675±10 nm laser for 15 minutes. Within 14 days after the initial treatment, tumor volume and body weight were measured every day to evaluate the antitumor activity and toxicity of the drug carrier. The test results are as follows Figure 12 shown.

[0103] from Figure 12 Compared with the control group, tumors in the non-laser irradiated groups (OABACe6 NPs, OABACe6 / Cu NPs, and CM@OABACe6 / Cu NPs) all showed some growth inhibition, with the CM@OABACe6 / CuNPs group showing a significant inhibitory effect, with a tumor inhibition rate of 47.7%, higher than those in the OA-BA-Ce6 NPs group (36.0%) and the OABACe6 / CuNPs group (34.4%). Due to the Chemo-CDT effect and targeted effect of CM@OABACe6 / CuNPs, mice in the targeted + Chemo+CDT group exhibited a significant tumor inhibition effect. Furthermore, after laser irradiation, tumor growth in mice in the Chemo+PDT and Chemo+CDT+PDT groups was significantly inhibited, with tumor inhibition rates of 51.6% and 63.4%, respectively, demonstrating the significant anti-tumor effect of the combined Chemo-CDT and PDT groups. As expected, the targeted + Chemo + CDT + PDT group achieved the highest tumor inhibition rate, at 85.9%. This confirmed the superior efficacy of the combined therapy. Tumor autopsy photos 14 days after treatment showed that tumors in mice treated with CM@OABACe6 / Cu NPs light were also smaller than those in the other groups.

[0104] Experiment 9: In vivo safety test:

[0105] In order to study the toxicity of nanoparticles, the present invention stained the tissues and organs of tumor-bearing mice at the end of treatment. The main organs such as tumor, heart, liver, spleen, kidney, lung were collected and stained with H&E. At the same time, blood analysis was performed to evaluate the in vivo safety of nanoparticles. The test results are as follows: Figure 13 shown.

[0106] The results showed that compared with the blank control group, the irradiated CM@OABACe6 / CuNPs group showed no significant inflammatory response or damage, further demonstrating the low in vivo toxicity of CM@OABACe6 / CuNPs. Furthermore, no significant differences were observed in hematological parameters, indicating that the combined therapy was biosafe in vivo. In summary, CM@OABACe6 / CuNPs exhibit improved biocompatibility and biodegradability, as well as low in vivo toxicity, and hold great potential as a promising synergistic anti-tumor agent.

Claims

1. A method for preparing nanoparticles of triterpenoid small molecules co-assembled and loaded with Ce6 and copper ions wrapped in cancer cell membranes, characterized by: The method is: Step 1: Preparation of nanoparticles co-assembled with oleanolic acid, betulinic acid and photosensitizer Ce6: (1) Dissolve oleanolic acid, betulinic acid and Ce6 in dimethyl sulfoxide; (2) adding a solution of oleanolic acid, betulinic acid, and Ce6 into water; (3) Stirring at 400-600 rpm for 10-60 min, and centrifuging at 10,000-15,000 rpm for 10-30 min; Step 2: Co-assembly of oleanolic acid and betulinic acid with photosensitizer Ce6 to form nanoparticles complexed with Cu 2+ Preparation: (1) Oleanolic acid and betulinic acid were co-assembled with photosensitizer Ce6 and dissolved in water; (2) adding CuCl2 aqueous solution to the aqueous solution of nanoparticles co-assembled with oleanolic acid, betulinic acid and photosensitizer Ce6; (3) Stirring at 400-600 rpm for 10-60 min, and centrifuging at 10,000-15,000 rpm for 10-30 min; Step 3: Oleanolic acid and betulinic acid wrapped in cancer cell membranes are co-assembled with photosensitizer Ce6 to form nanoparticles complexed with Cu 2+ Preparation: (1) Lyse 4T1 breast cancer cells using hypotonic lysis buffer; (2) Under the action of vortex, the cell membrane was added to the nanoparticle solution, and oleanolic acid and betulinic acid were co-assembled with photosensitizer Ce6 to form nanoparticles complexed with Cu 2+ The nanoparticle concentration is 1 mg / mL to 50 mg / mL; (3) subjecting the mixture to probe ultrasound for 1 to 10 minutes; (4) Stir at 400-600 rpm for 10-60 min, and centrifuge at 10,000-15,000 rpm for 10-30 min.

2. The method for preparing nanoparticles of triterpenoid small molecules co-assembled and loaded with Ce6 and copper ions wrapped by cancer cell membranes according to claim 1, characterized in that: In step 1 (1), the concentrations of oleanolic acid, betulinic acid and Ce6 in dimethyl sulfoxide are 5 mg / mL to 50 mg / mL.

3. The method for preparing nanoparticles of triterpenoid small molecules co-assembled and loaded with Ce6 and copper ions wrapped by cancer cell membranes according to claim 1, characterized in that: In step 1 (2), the volume ratio of the solution of oleanolic acid, betulinic acid and Ce6 to water is 50 to 100:

1.

4. The method for preparing nanoparticles of triterpenoid small molecules co-assembled and loaded with Ce6 and copper ions wrapped by cancer cell membranes according to claim 1, characterized in that: In step 2 (1), the concentration of the co-assembled nanoparticles in water is 1 mg / mL to 50 mg / mL.

5. The method for preparing nanoparticles of triterpenoid small molecules co-assembled and loaded with Ce6 and copper ions wrapped by cancer cell membranes according to claim 1, characterized in that: In step 2 (2), the concentration of the CuCl2 aqueous solution is 5 mg / mL to 50 mg / mL.

6. The method for preparing nanoparticles of triterpenoid small molecules co-assembled and loaded with Ce6 and copper ions wrapped by cancer cell membranes according to claim 1, characterized in that: In step 2 (3), the volume ratio of the CuCl2 aqueous solution to the nanoparticle aqueous solution is 50-100:

1.

7. The method for preparing nanoparticles of triterpenoid small molecules co-assembled and loaded with Ce6 and copper ions wrapped by cancer cell membranes according to claim 1, characterized in that: In step 3 (1), the 4T1 breast cancer cells are from 10 7 ~10 9 cells.

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