Tumor mineralization-inducing phosphorus-containing dendrimer / protein compound loaded with tirapazamine as well as preparation method and application of tumor mineralization-inducing phosphorus-containing dendrimer / protein compound

Abstract

The invention relates to a tirapazamine-loaded phosphorus-containing dendrimer / protein compound for inducing tumor mineralization as well as a preparation method and application thereof, the compound takes diphosphonate-terminated phosphorus-containing dendrimer AK176 as a carrier, and the carrier is loaded with protein and medicine. The nano-drug can target tumor cells through FN and form a biomineralization layer in the peripheral area of tumor tissues, and a tumor hypoxic microenvironment is intensified, so that the pharmaceutical activity of tirapazamine is activated, and cooperative treatment of blocking treatment / chemotherapy of triple-negative breast cancer is realized.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to a phosphorus-containing dendrimer / protein complex loaded with tirapazamine for inducing tumor mineralization, and a preparation method and application thereof. Background Art

[0002] Breast cancer is one of the most common cancers in women, affecting approximately 2 million to 2.5 million women worldwide each year. Among all known breast cancer subtypes, nearly 20% of breast cancers lack the expression of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2, which are called triple-negative breast cancer (TNBC). Because TNBC has similar characteristics to basal-like (about 75%) and lacks the expression of three biomarkers, it exhibits high invasiveness, easy metastasis, and difficult to cure, making TNBC have a high mortality rate (J. Controlled Release, 2020, 326, 628–647). Chemotherapy combined with surgery and / or radiotherapy is the main treatment for early and advanced TNBC, but tumor recurrence and metastasis are still difficult to suppress, resulting in an increase in the recurrence rate and mortality rate of patients year by year (J. Controlled Release 2018, 219, 184–195). Therefore, how to effectively curb tumor proliferation and metastasis is the key to the efficient treatment of TNBC to save patients' lives. Among the many anti-cancer drugs currently available, there is a serious shortage of drugs that inhibit tumor metastasis. Drug carriers based on biomimetic mineralization can overcome the drug resistance of cancer cells and improve the delivery efficiency of drugs, showing good application prospects (Adv. Mater. 2018, 30, 1704876).

[0003] In nature, organisms regulate the formation of inorganic minerals by utilizing organic matrices, a process called biomineralization. Biomineralization can provide protection and support for organisms, but abnormal pathological mineralization in soft tissues can also lead to morbidity and even death. Clinically, secondary calcification foci are often seen in tumor tissues after anti-tumor treatments such as chemotherapy and radiotherapy, and these ectopic mineralizations have been shown to be associated with a benign prognosis to some extent (Anti-Cancer Drugs 2019, 30, 195), which means that deliberately inducing the formation of biomimetic minerals in tumor areas has great prospects in delaying tumor growth. Existing studies have introduced carboxyl-containing folic acid molecules or folic acid-modified polysaccharides into tumor sites by identifying overexpressed folic acid receptors on cancer cell membranes, and used the metal ion adsorption properties of carboxyl groups to build a mineral barrier around tumor tissues and induce tumor biomineralization (Angew. Chem. Int. Ed. 2016, 55, 5225–5229; Angew. Chem. Int. Ed. 2021, 60, 6509–6517). However, due to the limited ion attraction properties of carboxyl groups, it is difficult to build a tight mineral barrier around tumor tissues. According to the solubility product constant order of anionic groups, it can be inferred that bisphosphonic acid groups have stronger cation adsorption properties. Therefore, the introduction of bisphosphonic acid groups into tumor tissues is more likely to promote the formation of a complete biomineral layer. It has been reported that polymers functionalized with bisphosphonic acid groups show strong ion chelation and tumor mineralization induction capabilities, effectively inhibiting the growth of primary tumors and lung metastases (Adv. Mater. 2022, 34, 2110094). In conclusion, this anti-tumor therapeutic approach that forms a biomineralized layer in the peripheral area of ​​tumor tissue is expected to become a blocking therapy to inhibit the proliferation and metastasis of TBNC.

[0004] Inspired by the induction of mineralization by chelating metal ions with bisphosphonic acid groups, phosphorus-containing dendrimers modified with sodium phosphite salts having similar peripheral functional groups are also expected to be used as drug carriers for inducing biomineralization. Importantly, phosphorus-containing dendrimers or crown macromolecules have been developed as nano-delivery platforms for a variety of therapeutic agents, including gene drugs, chemotherapeutic drugs, protein drugs, etc. (Biomacromolecules 2020, 21, 502-2511; ACSNano 2024, 18, 2195-2209). Fibronectin (FN) is known to have an Arg-Gly-Asp (RGD) polypeptide sequence on its central cell-binding domain, which can target the αVβ3 integrin receptor highly expressed on the surface of tumor cells (ACSNano 2022, 16, 984-996). Its combination with phosphorus-containing dendrimers can be used for targeted biomineralization of tumor tissues. In addition, considering that the formation of biomineralization barrier can reduce the blood supply in tumor tissue, thus aggravating the tumor hypoxia microenvironment (Adv.Mater.2023,35,2203291). This aggravation of the microenvironment after hypoxia is conducive to hypoxia activation prodrug tirapazamine (TPZ) and intracellular DNA interaction, showing 300 times higher toxicity than normoxic environment for enhancing anti-tumor therapeutic effect (Nat.Rev.Cancer 2004,4,437). Therefore, the present invention intends to construct the phosphorus-containing dendrimer / fibronectin complex of load tirapazamine for targeting TNBC tumor tissue, inducing the formation of a dense biomineralization layer on the periphery of tumor tissue, blocking the supply of oxygen and nutrients required for tumor growth and activating hypoxia prodrug TPZ, thereby inhibiting tumor proliferation and invasion to achieve TNBC blocking / chemotherapy synergistic treatment.

[0005] A search of domestic and foreign literature and patents has not yet found any relevant reports on the use of bisphosphonate-terminated phosphorus-containing dendrimers as carriers to simultaneously load fibronectin and tirapazamine for the synergistic treatment of blocking therapy / chemotherapy for triple-negative breast cancer. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a phosphorus-containing dendrimer / protein complex loaded with tirapazamine that induces tumor mineralization and its preparation method and application. The present invention uses the phosphorus-containing dendrimer AK176 capped with bisphosphonate as a carrier, and forms a complex with FN by electrostatic force, hydrogen bond, cation-II and hydrophobic interaction, and loads TPZ by electrostatic interaction to obtain a nano drug. The prepared nano drug can target tumor cells overexpressing integrin αvβ3 on the one hand; on the other hand, the special bisphosphonate group at the end of the phosphorus-containing dendrimer AK176 can be used as an induction site for biomineralization, forming a biomineralization layer around the tumor tissue, and the mineral barrier not only achieves efficient tumor blocking by hindering the material exchange between the tumor and the surrounding microenvironment; it also aggravates the hypoxic microenvironment of the tumor to activate the hypoxia-activated prodrug tirapazamine in the nano drug, and realizes the synergistic treatment of blocking therapy / chemotherapy of triple-negative breast cancer.

[0007] The invention provides a drug-loaded phosphorus-containing dendrimer / protein complex. The complex uses bisphosphonate-terminated phosphorus-containing dendrimer AK176 as a carrier, and the carrier loads protein and drugs.

[0008] Preferably, the protein is fibronectin FN; and the drug is tirapazamine TPZ.

[0009] The protein is the targeting agent fibronectin (FN); the drug is the chemotherapy drug tirapazamine (TPZ).

[0010] The structural formula of bisphosphonate-terminated phosphorus-containing dendrimer AK176 is:

[0011] The molecular formula is C 168 H 210 N 15 Na 24 O 90 P 27 , molecular weight is 5267.59 g / mol.

[0012] The present invention provides a method for preparing a drug-loaded phosphorus-containing dendrimer / protein complex, comprising:

[0013] (1) mixing bisphosphonate-terminated phosphorus-containing dendrimer AK176, protein, and solvent, and stirring at room temperature to obtain an AK176 / protein complex (AF);

[0014] (2) Mixing the drug, the AK176 / protein complex prepared in step (1), and the solvent, stirring at room temperature, ultrafiltration, and collecting the supernatant to obtain a drug-loaded phosphorus-containing dendrimer / protein complex (AFT).

[0015] Preferably, the protein in step (1) is fibronectin FN; the solvent is water; and the structural formula of AK176 is:

[0016]

[0017] Preferably, in step (1), the mass ratio of AK176 to protein is 1 to 4:1.

[0018] Preferably, the mixing method in step (1) comprises: dissolving AK176 and protein in solvents respectively, and then dropping the AK176 solution into the protein solution.

[0019] The concentration of AK176 solution is 2-4 mg / mL, and the concentration of protein solution is 1-2 mg / mL.

[0020] Preferably, in step (2), the drug is tirapazamine TPZ; the solvent is water;

[0021] Preferably, in step (2), the mass ratio of AK176 / protein complex to drug is 1 to 4:1.

[0022] Preferably, the mixing method in step (2) comprises dissolving the drug and the AK176 / protein complex in solvents respectively, and then dropping the drug solution into the AK176 / protein complex solution.

[0023] The concentration of the drug solution is 0.5 mg / mL.

[0024] Preferably, the stirring at room temperature in steps (1) and (2) is stirring at room temperature for 12-24 hours;

[0025] Preferably, the ultrafiltration in step (2) is performed using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3000-5000, centrifuging at a centrifugal force of 3000-3500 g for 10-15 min at room temperature, and collecting the upper layer solution of the ultrafiltration centrifuge tube after ultrafiltration centrifugation 3-4 times.

[0026] The present invention provides an application of the drug-loaded phosphorus-containing dendrimer / protein complex in the preparation of nanomedicines for treating triple-negative breast cancer.

[0027] The present invention provides an application of the drug-carrying phosphorus-containing dendrimer / protein complex in preparing a synergistic therapeutic drug for blocking therapy / chemotherapy of tumors.

[0028] The present invention first combines a bisphosphonate-terminated phosphorus-containing dendrimer (AK-176) with fibronectin (FN) to form an AF nanocomposite through electrostatic forces, hydrogen bonds, cation-II, hydrophobic interactions and the like, and further loads tirapazamine (TPZ) with a positive charge on the surface onto the AF nanocomposite through electrostatic interactions to construct a tirapazamine-loaded phosphorus-containing dendrimer / fibronectin complex AK176@FN / TPZ (AFT) for synergistic treatment of blocking therapy / chemotherapy of triple-negative breast cancer.

[0029] The present invention uses Zeta potential and dynamic light scattering analysis (DLS), ultraviolet visible absorption spectrum (UV-vis), transmission electron microscopy (TEM), SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and other means to characterize the physical and chemical properties of the prepared phosphorus-containing dendrimer / protein complex loaded with tirapazamine. The cytotoxicity of AFT was evaluated by CCK-8 analysis; the uptake of materials by human triple-negative breast cancer cells MDA-MB-231 was detected by flow cytometry; the optimal time for incubation of cells with calcium chloride was verified by ICP-OES; the performance of mineralization induced by the material was verified by laser confocal microscopy, scanning electron microscopy (SEM-EDS) equipped with an energy spectrometer, etc.; the apoptosis of cells after co-incubation of different material groups with MDA-MB-231 was detected by flow cytometry; the inhibitory effect of different materials on cancer cell migration and invasion was detected by scratch test and Transwell invasion test. Finally, an in situ triple-negative breast cancer model was established for anti-tumor experiments and evaluation of lung metastasis inhibition.

[0030] Beneficial Effects

[0031] (1) The process of the present invention is simple, the cost is low, the operation and separation are easy, and it has a good development prospect.

[0032] (2) The nanomedicine prepared by the present invention has good stability, water solubility and biocompatibility, providing a new idea for constructing a safe and efficient drug carrier.

[0033] (3) The phosphorus-containing dendrimer / protein complex loaded with tirapazamine prepared by the present invention can, on the one hand, target tumor cells overexpressing integrin αvβ3; on the other hand, the special bisphosphonate group at the end of the phosphorus-containing dendrimer AK176 can serve as an induction site for biomineralization, forming a biomineralization layer around the tumor tissue. This mineral barrier not only achieves efficient tumor blocking by hindering the material exchange between the tumor and the surrounding microenvironment, but also aggravates the hypoxic microenvironment of the tumor, thereby activating tirapazamine to achieve synergistic treatment of blocking therapy / chemotherapy, providing a new strategy for the efficient treatment of triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 (a) and (b) are schematic diagrams of the synthesis and application of the nanomedicine AFT prepared by the present invention;

[0035] Figure 2 The hydrodynamic size (a) and surface potential (b) of AF complexes with different mass ratios;

[0036] Figure 3 UV-visible absorption spectra of TPZ, AF, and AFT;

[0037] Figure 4 The SDS-PAGE electrophoresis diagram of FN, AF, AFT, BSA, ABT, and AK176;

[0038] Figure 5 TEM image (a) of AFT prepared in the present invention and its particle size distribution histogram (b);

[0039] Figure 6 The graph of the hydrodynamic size of the AFT prepared by the present invention in water, PBS and DMEM culture medium over time and the sample photo on the seventh day;

[0040] Figure 7 This is a graph showing cell viability after AFT prepared in the present invention was co-incubated with L929 and MDA-MB-231 cells for 24 hours;

[0041] Figure 8 Representative flow cytometry graphs (a) and mean fluorescence intensity quantitative graphs (b) of MDA-MB-231 cells treated with AFT prepared by the present invention for 5, 15, 30 and 60 minutes before and after trypan blue quenching;

[0042] Fig. 9 Representative flow cytometry graphs (a) and mean fluorescence intensity quantitative graphs (b) of MDA-MB-231 cells treated with AFT and FN-blocked AFT prepared in the present invention;

[0043] Fig.10 The intracellular Ca content of MDA-MB-231 cells after incubation with 10 mM CaCl2 for different time periods;

[0044] Fig.11 Laser confocal images of MDA-MB-231 cells treated with PBS (as control), Ca, AFT or AFTC; CaP was stained with calcein (green), cell membrane was stained with DIR (red), and cell nucleus was stained with DAPI (blue); the scale bar in the figure is 20 μm;

[0045] Fig.12SEM calcium analysis results of normal L929 cells and cancer cell MDA-MB-231 treated with PBS (as control), Ca, AFT or AFTC (a) and EDX analysis of the MDA-MB-231 cell surface treated with PBS (b) and AFTC (c);

[0046] Fig.13 Figure 2 shows the flow cytometry analysis (a) and quantitative results (b) of apoptosis in MDA-MB-231 cells after treatment with PBS (as control), Ca, AFT or AFTC for 24 h.

[0047] Fig.14 Representative microscopic images (a) and relative cell migration rate (b) of MDA-MB-231 cells scratched after treatment with PBS (as control), Ca, AFT or AFTC for 0 h, 12 h, and 24 h; the scale bar in the figure is 80 μm;

[0048] Fig.15 Cell staining (a) and quantitative statistics of cell number (b) in Transwell invasion assay and cell staining (c) and quantitative statistics of cell number (d) in Transwell migration assay of MDA-MB-231 cells after treatment with PBS (as control), Ca, AFT or AFTC for 24 h; scale bar in the figure is 40 μm;

[0049] Fig.16 The changes in tumor volume (a) and weight of mice (b) within 16 days after peritumoral injection of PBS, Ca, AFT and AFTC.

[0050] Fig.17 Micro-CT images of MDA-MB-231 orthotopic tumors (a) and mineral volumes of tumors in each group (b) after peritumoral injection of PBS, Ca, AFT, and AFTC into mice; scale bars in the figures are 1 mm;

[0051] Fig.18 Representative lung tissue photos (a) and H&E staining results (b) of mice after peritumoral injection of PBS, AFT and AFTC; the scale bar in the figure is 2 mm. DETAILED DESCRIPTION

[0052] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0053] Unless otherwise specified, all chemical reagents are commercially available and used directly without further purification. Bisphosphonate-terminated phosphorus-containing dendrimer AK-176 was obtained from the group of Professor JP Majoral of the Coordination Chemistry Laboratory of the French National Center for Scientific Research. Fibronectin (FN) was obtained from Shanghai FiberLink Biotechnology Co., Ltd. Tirapazamine (TPZ) and trypan blue were purchased from Sigma-Aldrich. MDA-MB-231 cells (human triple-negative breast cancer cell line) were obtained from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. DMEM medium, fetal bovine serum, penicillin-streptomycin double antibody and trypsin were purchased from Hangzhou Gino Biomedical Technology Co., Ltd. Cell Counting Kit-8 (CCK-8) and calcium content colorimetric detection kit were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Calcein was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. AnnexinV-FITC / PI apoptosis detection kit was purchased from Jiangsu Keygen Biotechnology Co., Ltd. Regenerated cellulose dialysis membrane with a molecular weight cutoff (MWCO) of 3000 was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Nude mice were purchased from Tianjin Jikang Pharmaceutical Technology Co., Ltd. Ultrafiltration centrifuge tubes with a molecular weight cutoff (MWCO) of 3000 were purchased from Thermo Fisher Scientific Inc. Water with a resistivity higher than 18.2 MΩ.cm used in all experiments was purified by a laboratory water purification system (PURIST UV Ultrapure, Shanghai Lefeng Biotechnology Co., Ltd., Shanghai).

[0054] Example 1

[0055] FN and AK176 were dissolved in aqueous solution, and AK176 was added dropwise to the FN solution at different mass ratios (AK176: FN = 1:1, 2:1, 4:1). The mixture was stirred at room temperature for 24 hours to obtain the nanocomposite AK176@FN (AF). The surface potential, hydration kinetic diameter and polydispersity index (PDI) of AF at different mass ratios were measured. The results are shown in Figure 2. Figure 2 As shown in ab, the hydration kinetic diameter of AK176 is 242.7nm, and the hydration kinetic size of FN alone is 360.6nm. When AK176 is mixed with FN, the hydration kinetic size and potential of the complex change significantly compared with FN, and the particle size of the AF complex increases with the increase of the mass of AK176. Especially when the mass ratio is 2:1, the Zeta potential of the AF nanocomplex is -34.7mV, and it has a smaller hydration kinetic size (229.5nm) and a suitable PDI (0.34). Therefore, the condition of a mass ratio of AK176 to protein of 2:1 was selected to prepare AF and the control material AK176@BSA (ABT) nanocomplex for subsequent experiments.

[0056] Example 2

[0057] AF and TPZ prepared in Example 1 were dissolved in water respectively, and the TPZ solution was added dropwise to the AF complex solution according to different mass ratios (AF:TPZ = 1:1, 2:1, 4:1), and stirred at room temperature for 24 hours. The reaction mixture was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 3000, centrifuged at 3000g for 10 minutes to remove the unloaded TPZ, and the ultrafiltration centrifugation was repeated 3 times, and the upper layer solution of the centrifuge tube was collected to obtain the nano drug AK176@FN / TPZ (AFT). The drug loading rate of the material at different mass ratios was determined by ultraviolet spectrophotometry, and the specific operation was as follows: prepare different concentrations of TPZ to test its absorbance value at a wavelength of 460nm, and draw a TPZ standard curve; collect the TPZ that was not loaded in the lower layer of the centrifuge tube after ultrafiltration centrifugation, and use formula (1, 2) to calculate the encapsulation rate and loading rate of TPZ in the AF nanocomposite at different feed ratios (as shown in Table 1). When the mass ratio of AF to TPZ was 2:1, the AFT nanocomposite had a suitable TPZ loading rate (17.18%) and encapsulation rate (41.48%). Therefore, the mass ratio of AF to TPZ was selected as 2:1 as the optimal feed ratio to prepare AFT and control material AK176@BSA / TPZ (ABT) for subsequent experiments.

[0058] Drug encapsulation efficiency (EE%) = M t / M0×100% (1);

[0059] Drug loading rate (LC%) = M t / Mz×100% (2);

[0060] Where Mt is the mass of the loaded drug, M0 is the mass of the total input drug, and Mz is the total mass of the material after drug loading.

[0061] Table 1

[0062] AF:TPZ (mass ratio) Encapsulation rate (%) Upload rate (%) 1∶1 21.21％ 17.50％ 2∶1 41.48％ 17.18％ 4∶1 42.35％ 9.57％

[0063] Example 3

[0064] In order to further verify the successful loading of TPZ in AFT nanocomposites, the AF and AFT prepared in Examples 1 and 2 were subjected to UV-vis tests. Figure 3 As shown in Figure 2, AFT shows a characteristic absorption peak of TPZ at 460 nm, while AF does not, proving the successful loading of TPZ in the AFT nanocomposite. In addition, as shown in Table 2, the hydration kinetic diameter of AFT after loading TPZ increases to 244.2 nm, and the surface potential increases to -10.8 mV, further indicating the successful loading of TPZ.

[0065] Table 2

[0066] sample Hydration kinetic size (nm) Polydispersity Index (PDI) Surface potential (mV) AF 229.5±28.67 0.34±0.07 -34.7±1.46 TPZ / / 9.74±0.51 AFT 244.2±13.56 0.21±0.1 -10.8±0.81

[0067] Example 4

[0068] In order to further illustrate the retention of FN in the AFT nanocomplex, AF, AFT and ABT prepared in Examples 1 and 2 were subjected to SDS-PAGE characterization. 5 μL of protein marker was added to the last protein lane, and then 10 μL of FN, AF, AFT, BSA, ABT and AK176 solution (protein concentration was 1 mg / mL) was mixed with 2 μL of loading buffer and heated at 100°C for 5 min. Subsequently, 10 μL of each sample was added to the corresponding lane and run at a current of 100-120A for 50 min. The results are shown in FIG. Figure 4 As shown, the AK176 group did not run out a lane with protein streaks, while the AF group and the AFT group both ran out protein streaks lanes similar to those of the pure FN group, and the ABT group ran out protein streaks lanes similar to those of the pure BSA group, which proved the successful complexation of FN and BSA in the nanocomplex.

[0069] Example 5

[0070] The AFT prepared in Example 2 was subjected to TEM characterization to observe its size and morphology. Figure 5 As shown in the figure, the size distribution of AFT is uniform and the morphology is spherical. The average size of AFT is about 134.1nm calculated by the particle size distribution histogram. 0.1mg of AFT was dispersed in 1mL H2O, PBS, and DMEM culture medium respectively. The hydrodynamic size of AFT in various solutions did not change significantly within one week (such as Figure 6 The prepared AFT has good colloidal stability.

[0071] Example 6

[0072] CCK-8 was used to evaluate the effects of AFT and related materials prepared in Example 2 on the vitality of normal cells (L929) and cancer cells (MDA-MB-231). L929 and MDA-MB-231 cells in the logarithmic growth phase were collected and seeded in a 96-well plate at a cell density of 1×104 cells per well. The volume of each well was 100 μL. After culturing for 24 hours at 37°C and 5% CO2, 90 μL of serum-free culture medium was replaced in each well, and 10 μL of different concentrations of AFT (4.125, 8.25, 16.5, 33, 66, 132, 264 μg / mL) was added. PBS was added to the control group. After culturing for 24 hours, the original culture medium was discarded, and after washing with PBS, fresh serum-free culture medium containing 10% (v / v) CCK-8 was added, and the cells were incubated in the incubator for another 3 hours. The absorbance of each well was tested at a wavelength of 450nm using a multifunctional microplate reader. The results are as follows Figure 7 As shown, the cell viability of L929 cells incubated with AFT was higher than 70% within the set concentration range. When the AFT concentration exceeded 16.5 μg / mL, the toxicity of AFT to MDA-MB-231 cells was significantly higher than that to L929 cells, which may be attributed to the targeting of FN in the AFT nanocomplex to MDA-MB-231 cells with high expression of integrin αvβ3, thereby promoting the uptake of AFT by MDA-MB-231 cells. Finally, an AFT concentration of 33 μg / mL was selected for subsequent experiments.

[0073] Example 7

[0074] MDA-MB-231 cells were used as a model to evaluate the cell membrane localization ability of AFT. Cells in the logarithmic growth phase were collected and cultured at 1×10 5 The cells were seeded at a density of 10 cells per well in a 12-well cell culture plate and cultured at 5% CO2 and 37°C for 24 hours. The culture medium was replaced with fresh serum-free DMEM medium containing AFT (FN was labeled with FITC and the concentration of AFT was 33 μg / mL) and cultured for 5, 15, 30 and 60 minutes. Subsequently, 1 mL of trypan blue (1 mg / mL) was added to half of the wells and incubated for 1 minute to quench the fluorescent signal on the cell membrane surface. The trypan blue-untreated group was set as the control. Finally, the trypan blue or culture medium was removed, and the cells were collected after trypsin digestion, centrifugation, and PBS washing three times, and the intracellular fluorescence intensity was detected by flow cytometry. The results are shown in the figure. Figure 8As shown, the cells in the group not treated with trypan blue have the ability to increase the uptake of AFT over time. Since trypan blue can specifically quench the fluorescence on the cell membrane, the average fluorescence intensity of MDA-MB-231 cells co-cultured with AFT for 5, 15, 30 and 60 minutes after trypan blue treatment decreased by 5.4%, 25.5%, 40.2% and 30.1%, respectively, indicating that AFT has the strongest cell membrane retention performance after incubation with cells for 30 minutes. Therefore, in subsequent experiments, 30 minutes will be selected as the optimal incubation time for cell membrane insertion to induce the formation of a mineralized layer on the cell membrane surface.

[0075] Example 8

[0076] MDA-MB-231 cells were used as a cell model to verify the targeting ability of FN. MDA-MB-231 cells in the logarithmic growth phase were collected and cultured at a rate of 1×10 5 The cells were seeded at a density of 10 cells per well in a 12-well cell culture plate and cultured at 5% CO2 and 37°C for 24 hours. Then, the FN-blocked group pretreated the cells with free FN (10 μg / mL) for 2 hours, removed the old culture medium, and replaced it with fresh serum-free DMEM culture medium containing AFT (FN labeled with FITC, the concentration of AFT was 33 μg / mL), and incubated in the incubator for 30 minutes. The control group was treated with PBS. The culture medium was discarded, and the cells were collected after trypsin digestion, centrifugation, and washing three times with PBS. The fluorescence intensity of each group of cell samples was detected by flow cytometry. The results are shown in Fig. 9 As shown in Figure 2, the fluorescence intensity of the AFT group was significantly higher than that of the FN blocking group, indicating that the AFT nanocomplex can activate RGD-integrin α through FN-mediated v The β3 receptor ligand recognition pathway is efficiently engulfed by MDA-MB-231 cells.

[0077] Example 9

[0078] The localization of AFT nanocomplexes on the cell membrane surface facilitates the binding of the bisphosphonate groups in the phosphorus-containing dendrimer AK176 to extracellular cations (Ca 2+ ) to induce mineral deposition on the cell surface. To verify this hypothesis, cells in the logarithmic growth phase were collected and cultured at a rate of 5×10 4 The cells were seeded at a density of 10 cells per well in a 24-well cell culture plate and cultured at 5% CO2 and 37°C for 24 h. The culture medium was replaced with fresh serum-free DMEM medium containing AFT (concentration: 33 μg / mL) and incubated for 30 min. The culture medium was discarded and replaced with DMEM medium containing 10 mM Ca 2+The cells were treated with DMEM medium at 37°C for 5 min, 15 min, 30 min, 1 h, 2 h and 4 h. The medium was discarded, and the cells were washed three times with PBS and digested overnight with 1 mL of aqua regia. The calcium content was measured by ICP-OES. The results are shown in Fig.10 As shown in the figure, the calcium content of MDA-MB-231 cells increased with the extension of the incubation time of exogenous calcium ions, reaching saturation and maximum calcium adsorption after 2 hours. Therefore, the combination of AFT and MDA-MB-231 cells cultured for 30 minutes and assisted by exogenous calcium ions for an additional 2 hours (denoted as AFTC) was used as the optimal cell surface mineralization induction condition.

[0079] Example 10

[0080] AFT retained on the cell membrane surface can recruit positive ions (mainly Ca) through electrostatic interactions. 2+ , derived from the additional addition of 10 mM Ca 2+ DMEM mineralization solution) and negative ions (mainly PO4 3- , derived from DMEM culture medium), thereby forming calcium phosphate precipitation on the cell surface. In order to verify the effect of calcium deposition on the cell surface treated with AFTC, MDA-MB-231 cells in the logarithmic growth phase were collected and cultured at 1×10 5 The cells were seeded in a laser confocal microplate at a density of 10 cells per well and incubated at 5% CO2 and 37°C for 24 h. The Ca group was replaced with a plate containing 10 mM Ca 2+ The AFT group was incubated with DMEM containing 33 μg / mL AFT for 30 min. The AFTC group was first incubated with DMEM containing 33 μg / mL AFT for 30 min, and then replaced with DMEM containing 10 mM Ca 2+ DMEM medium was added and incubated at 37℃ for 2h. PBS was added to the control group. The medium was discarded, the cells were washed three times with PBS, 500μL of DIR dye (50μM) was added and incubated for 15min, the cells were washed three times with PBS, 1mL of 2.5% glutaraldehyde was added and fixed at room temperature for 15min, the cells were washed three times with PBS, 1mL of DAPI (2μg / mL) was added and incubated with the cells for 5min, the cells were washed three times with PBS, 1mL of Calcein (1μg / mL) was added and incubated for 20min, the cells were washed three times with PBS, 200μL of PBS was added and the changes of cell fluorescence intensity were observed under a laser confocal microscope. Fig.11 As shown in Figure 2, cells treated with AFT had a weak green fluorescence, which may be attributed to the fact that AFT chelated a small amount of Ca in the DMEM medium. 2+ In the AFTC group, due to the additional supplementation of Ca 2+The green fluorescence intensity of this group was the highest and significantly higher than that of the Ca group. Some calcein-stained biomineralized layers co-localized with the red fluorescence of the cancer cell membrane, indicating that AFTC can significantly induce the formation of a mineralized layer and embed it on the cell membrane surface.

[0081] In order to further confirm the phenomenon of AFT-induced calcium deposition on the cell surface, MDA-MB-231 cells were observed using a scanning electron microscope equipped with an energy dispersive spectrometer (SEM-EDS). After incubating the cells with the same materials as above, the old culture medium was discarded, washed three times with PBS, and fixed at room temperature for 2 hours with 1 mL of 2.5% glutaraldehyde. The cells were then dehydrated for 30 minutes using different concentrations of ethanol solutions (10%, 30%, 50%, 70%, 90%, 100%). At the end of the procedure, the cells were dried at 4°C and subjected to SEM imaging after gold spraying. Fig.12 As shown in a, the surface of L929 cells and MDA-MB-231 cells in the PBS group, Ca group and AFT group were flat and smooth, and the cells were evenly spread on the silicon wafer without the formation of other substances. There was no obvious change in the morphology of L929 cells before and after AFTC treatment, while after AFTC treatment of MDA-MB-231 cells, it was observed that the cell surface was covered with a thick mineral layer. Similarly, by comparing the EDS results of the MDA-MB-231 cell surface treated with PBS and AFTC, it was found that the calcium content on the surface of MDA-MB-231 cells after AFTC treatment was higher, indicating that AFTC treatment can adsorb exogenous calcium ions and deposit them on the cell membrane surface, which provides an important guarantee for subsequent anti-tumor blocking therapy ( Fig.12 bc).

[0082] Embodiment 11

[0083] In order to study the mechanism of biomineralization in inhibiting tumor cells, MDA-MB-231 cells were used as a cell model to evaluate the effects of different treatments on cell apoptosis levels. MDA-MB-231 cells in the logarithmic growth phase were collected and cultured at a rate of 1×10 5 The cells were seeded in a 12-well cell culture plate at a density of 10 cells per well and cultured at 5% CO2 and 37°C for 24 h. The Ca group was replaced with a cell culture medium containing 10 mM Ca 2+ The AFT group was incubated with DMEM containing 33 μg / mL AFT for 30 min. The AFTC group was first incubated with DMEM containing 33 μg / mL AFT for 30 min, and then replaced with DMEM containing 10 mM Ca 2+DMEM medium was incubated at 37°C for 2h. PBS was added to the control group. The medium was discarded, washed three times with PBS, and fresh medium was replaced and cultured for another 24h. The supernatant was collected and washed three times with PBS. 1mL of trypsin was added to each well to digest the cells. The digestion was terminated, the cells were collected by centrifugation, and 500μL of Binding Buffer was added and gently blown into a single cell suspension. Subsequently, the pre-mixed AnnexinV-FITC and Propidium Iodide were added, incubated at room temperature in the dark for 5-10min, and then transferred to a flow tube to detect cell apoptosis by flow cytometry. Fig.13 As shown, the AFTC-treated group had the highest proportion of apoptotic and necrotic cells (19.22%), which was due to the cell surface calcification-inducing ability of AFTC, which blocked the supply of intercellular nutrients and activated tirapazamine, thereby triggering an enhanced apoptotic effect.

[0084] Example 12

[0085] In order to verify the effect of biological calcification on tumor cell migration and invasion, cell scratch assay, Transwell migration and invasion assay were performed on MDA-MB-231 cells. For the scratch assay, three parallel lines were drawn on the back of a 12-well plate with a sterilized marker pen and ruler. MDA-MB-231 cells in the logarithmic growth phase were collected and plated at 1×10 5 The cells were seeded at a density of 10 cells per well on a 12-well cell culture plate with lines drawn on it and cultured at 5% CO2 and 37°C for 24 h. Part of the culture medium was discarded and a straight scratch was made with a 200 μL pipette tip perpendicular to the well plate and the parallel line on the back of the well plate so that the scratch intersected the marked line. The culture medium was discarded and the cells were washed three times with PBS. Fresh serum-free culture medium was added and the scratch image was taken at 0 h using a Leica DM IL LED inverted phase contrast microscope. The Ca group was replaced with a 10 mM Ca 2+ The AFT group was incubated with DMEM containing 33 μg / mL AFT for 30 min. The AFTC group was first incubated with DMEM containing 33 μg / mL AFT for 30 min, and then replaced with DMEM containing 10 mM Ca 2+ The cells were incubated at 37°C for 2 h with DMEM medium. PBS was added to the control group. The medium was discarded, the cells were washed three times with PBS, and fresh serum-free medium was added. The scratch images were taken at 12 h and 24 h using a Leica DM IL LED inverted phase contrast microscope. Finally, the relative migration area was quantitatively analyzed using ImageJ. The results are shown in Figure 2. Fig.14As shown in the figure, the scratches in the PBS group, Ca group and AFT group became significantly narrower, and the cells tended to migrate toward the scratches, and the cell migration rates reached 48.1%, 43.2% and 33.3% at 24h, respectively, indicating that AFT has limited ability to inhibit the migration of MDA-MB-231 cells. In contrast, since AFTC can induce the deposition of biominerals on the cell surface, the scratch width did not change significantly, and the migration rate at 24h was only 14.2%.

[0086] Transwell migration and invasion experiments were further performed to explore the ability of biological calcification to inhibit the migration and invasion of MDA-MB-231 cells. MDA-MB-231 cells in the logarithmic growth phase were collected and cultured at 1×10 5 The cells were seeded in a 12-well cell culture plate at a density of 10 cells per well and cultured at 5% CO2 and 37°C for 24 h. The Ca group was replaced with a cell culture medium containing 10 mM Ca 2+ The AFT group was incubated with DMEM containing 33 μg / mL AFT for 30 min. The AFTC group was first incubated with DMEM containing 33 μg / mL AFT for 30 min, and then replaced with DMEM containing 10 mM Ca 2+ DMEM medium was incubated at 37℃ for 2h. PBS was added to the control group. The old medium was discarded, washed 3 times with PBS, 1mL of trypsin was added to each well to digest the cells, digestion was terminated, and the cells were collected by centrifugation and labeled. The collected cells were resuspended with 100μL of serum-free DMEM medium and added to the upper chamber, and 500μL of DMEM medium containing 20% ​​serum was added to the lower chamber. After 24h of culture, the medium was discarded, washed 3 times with PBS, 500μL of 2.5% glutaraldehyde was added to each well to fix at room temperature for 30min, washed three times with PBS, 200μL of 1% crystal violet was added to the upper chamber of each well, and 500μL of 1% crystal violet was added to the lower chamber, and stained at room temperature for 10min, washed three times with PBS, and the cells on the chamber were wiped off with a wet cotton ball. The chamber was placed on a slide and the image was taken at 100 times with a Leica DM IL LED inverted phase contrast microscope. The process of Transwell invasion experiment is basically the same as that of migration experiment. The only difference is that before paving cells, BD matrix gel: serum-free DMEM medium = 1:3 was prepared and mixed, and 25 μL was added to the upper chamber of Transwell (24-well plate, chamber inner diameter 6.5 mm, bottom 8.00 μm polycarbonate membrane), and incubated in 37°C incubator for 30 min to solidify the diluted liquid matrix gel. The Transwell results showed that compared with the control group, Ca group and AFT group, the invasion of cells treated with AFTC ( Fig.15 ab) or migration ( Fig.15cd) The number of cells was significantly reduced, which further indicated that AFTC damaged the migration and invasion ability of cells by inducing cell calcification.

[0087] Embodiment 13

[0088] 5×10 6 MDA-MB-231 cells were inoculated into the second mammary pad of female nude mice and the tumor volume reached 100 mm. 3 At around 4 pm, the mice were randomly divided into 4 groups (6 mice in each group). For the mice in the control group, the treatment included peritumoral injection of 100 μL PBS and a second peritumoral injection of 100 μL PBS 30 minutes later. For the mice in the Ca group, the treatment included peritumoral injection of 100 μL PBS and a second peritumoral injection of 100 μL PBS containing 10.0 mM CaCl2 30 minutes later. For the mice in the AFT group, the treatment included peritumoral injection of 100 μL PBS containing 2.5 mg / mLAFT and a second peritumoral injection of 100 μL PBS 30 minutes later. For the mice in the AFTC group, the treatment included peritumoral injection of 100 μL PBS containing 2.5 mg / mLAFT and a second peritumoral injection of 100 μL PBS containing 10.0 mM CaCl2 30 minutes later. The day the treatment started was recorded as day 0, and treatment was performed every 2 days. The body weight and tumor size of the mice were recorded every 2 days. After eight treatments, the mice were euthanized. Fig.16 a It can be seen that compared with the control group and the Ca group, the tumor volume of the AFT group grew slowly and showed significant inhibitory ability against primary tumors. This may be attributed to the fact that AFT can adsorb calcium ions in the physiological environment to induce the formation of a partial biomineralization layer to block tumor growth. It is worth noting that since the introduction of exogenous calcium ions amplifies the biomineralization ability of AFT, it can effectively block the nutrient and oxygen supply process between tumor tissue and the outside world, which is conducive to the activation of the hypoxic prodrug TPZ, so that AFTC has the strongest tumor growth inhibitory ability. In addition, there was no significant change in the body weight of mice treated with different materials, which proves the biosafety of the biomineralization strategy ( Fig.16 b).

[0089] Embodiment 14

[0090] The MDA-MB-231 nude mouse orthotopic tumor constructed in Example 13 was used as a model. After a 16-day treatment, the tumor-bearing mice were killed and dissected, and the tumor tissue was harvested and fixed in a tissue fixative at room temperature for 48 hours. The mouse tumor tissue was observed by a Micro-CT system (SCANCOMedicalAG, Zurich, Switzerland) and the volume of calcified tissue was quantified by image analysis software. The CT scanning parameters were set to a voltage of 45kV, a current of 200μA, and an exposure time of 300ms. Micro-CT images showed that the tumor tissues of the control group and the Ca group remained uniform and had no compaction period, indicating that the CaCl2 injection alone could not produce mineral deposition in the tumor tissue ( Fig.17 a). In contrast, a large number of scattered high-density spots were observed in the tumor tissues of the AFT and AFTC treatment groups, especially in the AFTC group with additional CaCl2, where many mineralized sites appeared at the edge of the tumor tissue and had the highest mineralized volume ( Fig.17 b), which indicates that AFT has the ability to chelate calcium ions in vivo to induce mineralization of tumor tissues. At the same time, the generation of these calcification foci may be an important reason why AFTC can effectively inhibit tumor growth and prolong the survival time of mice.

[0091] Embodiment 15

[0092] Using the MDA-MB-231 nude mouse orthotopic tumor constructed in Example 13 as a model, after 16 days of treatment, representative lung tissues of mice in the PBS, AFT, and AFTC groups were collected on the 22nd, 28th, and 32nd days for photography and H&E tissue section staining, and the anti-tumor metastasis performance of the material was evaluated by observing the lung nodules and section staining results. Fig.18 As shown in the figure, the lung tissue lesions in the PBS group were obvious and accompanied by a large number of lung metastatic nodules, while AFT could partially prevent tumor metastasis by inducing limited biomineralization by chelating calcium ions in the physiological environment in vivo, and a reduced number of lung nodules could be observed. In addition, due to the introduction of exogenous calcium ions, the ability of AFTC to induce tumor biomineralization was enhanced, thereby significantly blocking the lung metastasis process of the tumor, showing a nearly complete lung tissue morphology and the least number of lung nodules.

Claims

1. A drug-loaded phosphorus-containing dendrimer / protein complex, characterized in that: The complex uses bisphosphonate-terminated phosphorus-containing dendrimer AK176 as a carrier, and the carrier is loaded with proteins and drugs.

2. The drug-loaded phosphorus-containing dendrimer / protein complex according to claim 1, characterized in that: The protein is fibronectin FN; the drug is tirapazamine TPZ.

3. A method for preparing a drug-loaded phosphorus-containing dendrimer / protein complex, comprising: (1) mixing a phosphorus-containing dendrimer AK176 capped with bisphosphonate, a protein, and a solvent, and stirring at room temperature to obtain an AK176 / protein complex; (2) Mixing the drug, the AK176 / protein complex of step (1), and the solvent, stirring at room temperature, ultrafiltration, and collecting the supernatant to obtain a drug-loaded phosphorus-containing dendrimer / protein complex.

4. The preparation method according to claim 3, characterized in that: In step (1), the protein is fibronectin FN; the solvent is water; the structural formula of AK176 is: In the step (1), the mass ratio of AK176 to protein is 1 to 4:

1.

5. The preparation method according to claim 3, characterized in that: The mixing method in step (1) includes: dissolving AK176 and protein in solvents respectively, and then dropping the AK176 solution into the protein solution.

6. The preparation method according to claim 3, characterized in that: In the step (2), the drug is tirapazamine TPZ; the solvent is water; In the step (2), the mass ratio of AK176 / protein complex to drug is 1 to 4:

1.

7. The preparation method according to claim 3, characterized in that: The mixing method in step (2) includes: dissolving the drug and the AK176 / protein complex in solvents respectively, and then dropping the drug solution into the AK176 / protein complex solution.

8. The preparation method according to claim 3, characterized in that: The stirring at room temperature in steps (1) and (2) is performed for 12-24 hours; In the step (2), the ultrafiltration is carried out using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3000-5000, centrifuging at a centrifugal force of 3000-3500 g for 10-15 min at room temperature, and the upper layer solution of the ultrafiltration centrifuge tube is collected after ultrafiltration centrifugation 3-4 times.

9. Use of the drug-loaded phosphorus-containing dendrimer / protein complex according to claim 1 in the preparation of nanomedicines for treating triple-negative breast cancer.

10. Use of the drug-loaded phosphorus-containing dendrimer / protein complex according to claim 1 in the preparation of a synergistic therapeutic drug for blocking therapy / chemotherapy of tumors.

Images