ZIF-8 composite material targeting CD326 and preparation method and application thereof

Abstract

The invention provides a ZIF-8 composite material targeting CD326, and a preparation method of the ZIF-8 composite material comprises the following steps: 1) suspending ZIF-8 NPs in a protein coating solution, preserving the suspension overnight by using probe ultrasound, centrifuging and washing, and collecting to obtain ZIF-8 NPs-Protein A; and 2) suspending the collected ZIF-8 NPs at Protein A in a PBS buffer solution again, carrying out ultrasonic treatment by using a probe, adding a CD326 antibody solution into the suspension, incubating, and carrying out centrifugal collection to obtain the ZIF-8 NPs at CD326Ab. The invention belongs to the technical field of biological medicines, and the provided CD326-targeting ZIF-8 composite material can specifically destroy actin cytoskeleton of cancer cells, prevent the actin cytoskeleton from migrating or invading and inhibit cancer cell proliferation, and is expected to play an important role in medicines for preventing and treating breast cancer and / or prostatic cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a CD326-targeted ZIF-8 composite material and a preparation method and application thereof. Background Art

[0002] Epithelial cell adhesion molecule (CD326), also known as EpCAM, is a type I transmembrane glycoprotein and homologous to Ca 2+ Independent cell adhesion molecules are expressed at low levels in the basement membrane of a variety of normal epithelial cells, but are overexpressed in a variety of cancer cells (such as breast cancer and prostate cancer). Their expression levels are closely related to the migration and invasion of cancer cells, which can easily lead to cancer metastasis and poor prognosis of patients.

[0003] Zeolite imidazolate framework-8 nanoparticles (ZIF-8NPs) are a special type of metal-organic framework material formed by the coordination of zinc ions and 2-methylimidazole. They have the characteristics of large specific surface area, porous structure and high porosity, and show good biocompatibility and low toxicity. Zeolite imidazolate framework-8 nanoparticles are often used as drug delivery systems. For example, Chinese patent application CN108524935A discloses a quercetin drug delivery system based on copper sulfide-metal organic framework materials, by selecting photothermal preparation copper sulfide nanoparticles and coordinating them with metal organic framework zeolite imidazolate framework materials (ZIF-8) to form CuS@ZIF-8 carriers as quercetin drug delivery carriers; Chinese patent application CN 117503960A discloses a photoacoustic molecular probe, including a zeolite imidazolate framework material as a metal organic framework and a small molecule dye, and the small molecule dye is loaded in the metal organic framework.

[0004] There are no reports on the direct effects of zeolitic imidazole framework-8 nanoparticles on cancer cells. Therefore, it is of great significance to provide a CD326-targeted ZIF-8 composite material and its preparation method that can prevent cancer cell migration and invasion. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a CD326-targeted ZIF-8 composite material (ZIF-8NPs@CD326Ab), and it is found that the ZIF-8 composite material can be used as the only active ingredient to specifically destroy the actin cytoskeleton of cancer cells and prevent their migration or invasion, inhibit cancer cell proliferation, and is expected to play an important role in the prevention and treatment of breast cancer and / or prostate cancer drugs. Based on the above findings, the present invention is completed.

[0006] The purpose of the present invention will be further illustrated by the following detailed description.

[0007] In one aspect, the present invention provides a ZIF-8 composite material targeting CD326, and the schematic diagram of its preparation route is as follows Figure 1 As shown, the following steps are included:

[0008] 1) suspending ZIF-8NPs in a protein coating solution, using probe ultrasound, storing the suspension overnight, centrifuging and washing, and collecting to obtain ZIF-8NPs@Protein A; the protein coating solution is prepared by dissolving protein A and bovine serum albumin in a PBS buffer at a mass ratio of (0.8-1.2):(2.5-3.5), and the total protein concentration in the protein coating solution is 1.8-2.2 mg / mL;

[0009] 2) The collected ZIF-8NPs@ProteinA was resuspended in PBS buffer, probe sonicated, and CD326 antibody solution was added to the suspension. After incubation, the suspension was collected by centrifugation to obtain a CD326-targeted ZIF-8 composite material, namely, ZIF-8NPs@CD326Ab.

[0010] The above technical scheme is adopted to give ZIF-8NPs the ability to target CD326. First, through the coordination interaction between the amino end of the protein and the zinc center of ZIF-8, a layer of protein A (PA) mixed with bovine serum albumin (BSA) is coated on the surface of ZIF-8NPs, i.e., ZIF-8@ProteinA, and then a subsequent combination with CD326 antibody is performed to obtain a ZIF-8 composite material targeting CD326, i.e., ZIF-8NPs@CD326Ab (also represented as ZIF-8NPs@Ab in the embodiments and drawings of the present invention). Here, each PA molecule contains several binding sites for the Fc region of the antibody, ensuring the optimal orientation of the surface antibody so that the Fab region faces outward, while BSA acts as a spacer to control the density of PA and antibody on the surface of the nanoparticle to prevent steric hindrance of the Fab region of the adjacent antibody.

[0011] Preferably, the dosage ratio of the ZIF-8 NPs to the protein coating solution is 180-220 μg: 0.8-1.2 mL. More preferably, the dosage ratio of the ZIF-8 NPs to the protein coating solution is 200 μg: 1 mL.

[0012] Preferably, the centrifugal speed in step 1) is 7800-8200 r / min, and the time is 8-12 min; the centrifugal speed in step 2) is 6800-7200 r / min, and the time is 8-12 min. More preferably, the centrifugal speed in step 1) is 8000 r / min, and the time is 10 min; the centrifugal speed in step 2) is 7000 r / min, and the time is 10 min.

[0013] In another aspect, the present invention provides a method for preparing a ZIF-8 composite material targeting CD326, comprising the following steps:

[0014] 1) suspending ZIF-8NPs in a protein coating solution, using probe ultrasound, storing the suspension overnight, centrifuging and washing, and collecting to obtain ZIF-8NPs@ProteinA; the protein coating solution is prepared by dissolving protein A and bovine serum albumin in a PBS buffer at a mass ratio of (0.8-1.2):(2.5-3.5), and the total protein concentration in the protein coating solution is 1.8-2.2 mg / mL;

[0015] 2) The collected ZIF-8NPs@ProteinA was resuspended in PBS buffer, probe sonicated, and CD326 antibody solution was added to the suspension. After incubation, the suspension was collected by centrifugation to obtain a CD326-targeted ZIF-8 composite material, namely, ZIF-8NPs@CD326Ab.

[0016] Preferably, the dosage ratio of the ZIF-8 NPs to the protein coating solution is 180-220 μg: 0.8-1.2 mL. More preferably, the dosage ratio of the ZIF-8 NPs to the protein coating solution is 200 μg: 1 mL.

[0017] Preferably, the centrifugal speed in step 1) is 7800-8200 r / min, and the time is 8-12 min; the centrifugal speed in step 2) is 6800-7200 r / min, and the time is 8-12 min. More preferably, the centrifugal speed in step 1) is 8000 r / min, and the time is 10 min; the centrifugal speed in step 2) is 7000 r / min, and the time is 10 min.

[0018] In addition, the present invention also provides use of the CD326-targeting ZIF-8 composite material or the CD326-targeting ZIF-8 composite material prepared by the preparation method in preparing drugs for preventing and treating breast cancer and / or prostate cancer.

[0019] Preferably, the CD326-targeting ZIF-8 composite material is used as the only active ingredient.

[0020] Compared with the prior art, the beneficial effects of the present invention include:

[0021] (1) The present invention provides a ZIF-8 composite material targeting CD326, which is prepared by suspending ZIF-8NPs in a protein coating solution and using probe ultrasound to obtain ZIF-8NPs@ProteinA, which is then resuspended in PBS buffer and ultrasound is used. A CD326 antibody solution is added to the suspension, and the suspension is incubated and centrifuged to obtain the ZIF-8NPs@ProteinA.

[0022] (2) The present invention has experimentally found that the CD326-targeted ZIF-8 composite material can specifically destroy the actin cytoskeleton of cancer cells and prevent their migration or invasion, and the effect of preventing cancer cell migration or invasion is significantly better than that of non-specific BSA-coated ZIF-8 nanoparticles. Its working mechanism involves the synergistic effect of depleting transmembrane CD326 and destroying actin assembly. These zinc ions come from the intracellular uptake and degradation of CD326-targeted ZIF-8 nanoparticles, inhibiting cancer cell proliferation, and are expected to play an important role in the prevention and treatment of breast cancer and / or prostate cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the preparation route of ZIF-8 composites targeting CD326.

[0024] Figure 2 Transmission electron microscopy images and diameter statistics of the prepared ZIF-8NPs and gold nanoparticles.

[0025] Figure 3 The hydrodynamic diameter and zeta potential test results of ZIF-8NPs after each step of surface functionalization in aqueous solution.

[0026] Figure 4 X-ray diffraction analysis results of freshly prepared ZIF-8 NPs and CD326 antibody functionalized ZIF-8 NPs (ZIF-8 NPs@CD326Ab) stored in PBS buffer for 1 week.

[0027] Figure 5 The hydrodynamic diameter and zeta potential detection results of AuNPs in aqueous solution after each step of surface functionalization.

[0028] Figure 6 Proliferation detection results of MDA-MB-231 cells under different treatment conditions.

[0029] Figure 7 Proliferation detection results of PC3M cells under different treatment conditions.

[0030] Figure 8 Colloidal stability results of different nanoparticles in cell culture medium.

[0031] Fig. 9 Immunofluorescence staining results of CD326 on the surface of MDA-MB-231 and PC3M cells.

[0032] Fig.10 XTT cytotoxicity assay evaluated the survival results of MDA-MB-231 and PC3M cells after 12 h incubation with different concentrations of ZIF-8NPs@Ab and other nanoparticle controls; A. 30 μg / mL ZIF-8NPs@BSA; B. 1.5 nM AuNPs@BSA; C. 1.5 nM AuNPs@Ab.

[0033] Fig.11 LDH cytotoxicity assay evaluated the membrane damage results of MDA-MB-231 and PC3M cells after 12 h incubation with different concentrations of ZIF-8NPs@Ab and other nanoparticle controls; A. 30 μg / mL ZIF-8NPs@BSA; B. 1.5 nM AuNPs@BSA; C. 1.5 nM AuNPs@Ab.

[0034] Fig.12 Fluorescence tracking results of MDA-MB-231 cell uptake of FITC-labeled ZIF-8NPs@BSA and ZIF-8NPs@Ab.

[0035] Fig.13 use Fig.12 The total fluorescence intensity of individual cells was used to quantify the uptake of nanoparticles by MDA-MB-231 cells.

[0036] Fig.14 Fluorescence tracking results of PC3M cell uptake of FITC-labeled ZIF-8NPs@BSA and ZIF-8NPs@Ab.

[0037] Fig.15 use Fig.14 The total fluorescence intensity of individual cells was used to quantify the uptake of nanoparticles by PC3M cells.

[0038] FIG162D is the imaging results of a wound healing experiment evaluating the migration ability of MDA-MB-231 cells under different treatment conditions.

[0039] FIG. 172D shows the results of a wound healing assay evaluating the percentage of gap closure of MDA-MB-231 cells under different treatment conditions.

[0040] Figure 182D shows imaging results of a wound healing experiment evaluating the migration ability of PC3M cells under different treatment conditions.

[0041] FIG. 192D shows the results of a wound healing experiment evaluating the percentage of gap closure of PC3M cells under different treatment conditions.

[0042] Figure 203D shows the imaging results of invasion experiment evaluating the invasion ability of MDA-MB-231 cells under different treatment conditions.

[0043] Figure 213D shows the results of invasion assay to evaluate the number of invasive cells of MDA-MB-231 cells under different treatment conditions.

[0044] Figure 223D shows the imaging results of invasion experiment evaluating the invasion ability of PC3M cells under different treatment conditions.

[0045] Figure 233D shows the results of invasion assay to evaluate the number of invasive cells of PC3M cells under different treatment conditions. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0047] In the present invention, the reagents and kits involved are all conventional commercial products, or can be obtained by conventional technical means in the art, such as protein A purchased from Thermo scientific company, item number 77673; CD326 antibody purchased from Cell signaling technology company, item number 2929S; CyQUANTTM NF kit purchased from Thermo Fisher company, item number C35007. % content, if not clearly stated, is understood according to the common practice in the art.

[0048] Example 1 ZIF-8 composite material targeting CD326

[0049] Schematic diagram of the preparation route of ZIF-8 composite material targeting CD326 Figure 1 As shown, the specific steps include:

[0050] 1) 200 μg ZIF-8NPs were suspended in 1 mL protein coating solution, probe sonicated for 10 min, the suspension was stored at 4°C overnight, centrifuged at 8000 r / min for 10 min, and washed three times with PBS buffer to collect ZIF-8NPs@ProteinA; the protein coating solution was prepared by dissolving protein A and bovine serum albumin in a PBS buffer at a mass ratio of 1:3, and the total protein concentration in the protein coating solution was 2 mg / mL;

[0051] 2) The collected ZIF-8NPs@ProteinA was resuspended in 1 mL of PBS buffer, probe sonicated, and 100 μL of 50 μg / mL CD326 antibody solution was added to the suspension. After incubation at room temperature for 30 min, the suspension was centrifuged at 7000 r / min for 10 min to obtain ZIF-8NPs@CD326Ab, i.e., ZIF-8 composite material targeting CD326.

[0052] ZIF-8NPs can be commercially available or prepared as follows: 150 mg of zinc nitrate hexahydrate (dissolved in 7.5 mL of methanol) and 330 mg of 2-methylimidazole (dissolved in 7.5 mL of methanol) were dissolved in HPLC grade methanol. For FITC-labeled ZIF-8NPs, 60 μg of FITC was additionally added to the zinc nitrate solution. The two solutions were mixed under continuous stirring for 10 min, centrifuged at 7000 r / min, washed three times with methanol for 10 min each time, and ZIF-8NPs were collected. The transmission electron microscopy images and diameter statistical analysis are shown in Figure 2. Figure 2 -A and 2-B, it can be seen that the main particle size distribution is around 40 nm. To determine the mass concentration, ZIF-8 NPs were dried at 60 °C for 24 h, weighed, and resuspended in 5 mL of methanol for storage.

[0053] The hydrodynamic diameter and zeta potential of ZIF-8NPs in aqueous solution after each step of surface functionalization are shown in Figure 2. Figure 3 As shown. By gradually increasing the hydrodynamic diameter ( Figure 3 -A) and the zeta potential changes of ZIF-8NPs in aqueous solution ( Figure 3 -B), verifying the two-step surface functionalization: bare ZIF-8NPs (58.8nm, 18.5mV), ZIF-8NPs@PA (85.9nm, -12.1mV), and ZIF-8NPs@CD326Ab (106.6nm, -9.5mV). ZIF-8NPs@BSA (78.8nm, -22.9mV) as nonspecific ZIF-8NPs is a control for ZIF-8NPs@CD326Ab. Data are expressed as mean ± standard deviation, n = 3.

[0054] X-ray diffraction analysis results of freshly prepared ZIF-8NPs and CD326 antibody functionalized ZIF-8NPs (ZIF-8NPs@CD326Ab) stored in PBS buffer for 1 week are shown in Figure 2. Figure 4 As shown, similar diffraction peaks indicate that the CD326-targeted ZIF-8 composites (i.e., CD326 antibody-functionalized ZIF-8 NPs) are chemically stable.

[0055] Comparative Example 1 Gold nanoparticle composite material targeting CD326

[0056] The 10 nm seed solution was prepared by adding an ice-cold sodium borohydride solution (10 mM, 0.45 mL) to hexadecyltrimethylammonium chloride CTAC (200 mM, 5 mL) and tetrachloroauric acid HAuCl4 solution (10 nM, 0.25 mL) for 10 min under vigorous stirring. The mixed solution was then incubated at room temperature for 1 h to obtain 10 nm seed gold nanoparticles. In order to synthesize 20 nm gold nanoparticles, a 20 nm growth solution was prepared by mixing CTAC (200 mM, 5 mL), HAuCl4 (10 nM, 0.25 mL) and 4.765 mL of pure water for 10 min under vigorous stirring. After adding ascorbic acid (40 mM, 0.075 mL) and a 10 nm seed solution (0.025 mL), the 20 nm growth solution was incubated at room temperature for 2 days to obtain 20 nm gold nanoparticles. To synthesize 40 nm gold nanoparticles, a 40 nm growth solution was prepared by mixing CTAC (200 mM, 5 mL), HAuCl4 (10 nM, 0.25 mL) and 4.765 mL of pure water under vigorous stirring for 10 min. After adding ascorbic acid (40 nM, 0.075 mL) and 20 nm seed solution (0.01 mL), the 40 nm growth solution was incubated overnight at room temperature to obtain gold nanoparticles, whose transmission electron microscopy images and diameter statistical analysis are shown in Figure 2. Figure 2 -C and 2-D, it can be seen that the main particle size distribution is around 40 nm; then three centrifugations (each time: 10000r / min, 10min) were performed to remove excess chemicals; the synthesized CTAC-coated gold nanoparticles were stored in deionized water (18.2MΩ-cm).

[0057] Gold nanoparticles targeting CD326 (AuNPs@CD326Ab) were prepared by ligand exchange method: CTAC-coated gold nanoparticles were added to 2 mg / mL protein A / BSA (1 / 3 weight ratio) solution (0.02% citric acid, pH 3) and sonicated for 20 min. Then, the gold nanoparticles were centrifuged at 5000 r / min for 20 min and resuspended in 0.5 mg / mL protein A / BSA solution (0.02% citric acid + sodium hydroxide, pH 12) and incubated overnight at 4 ° C. Next, AuNPs@ProteinA was washed three times with alkaline water at pH 11, each time at a centrifugal speed of 5000 r / min for 10 min. To attach the antibody, AuNPs@ProteinA was resuspended in 1 mL of PBS solution (0.5 nM gold nanoparticles), and 100 μL (50 μg / mL) of CD326 antibody solution was added to the suspension and incubated at room temperature for 30 min. Finally, AuNPs@CD326Ab was collected by centrifugation at 7000 r / min for 10 min.

[0058] The hydrodynamic diameter and zeta potential of AuNPs after each step of surface functionalization in aqueous solution are shown in Figure 2. Figure 5 As shown. By gradually increasing the hydrodynamic diameter ( Figure 5 -A) and the zeta potential change of gold nanoparticles in aqueous solution ( Figure 5 -B), verifying the two-step surface functionalization: bare gold nanoparticles (69.5nm, 38.2mV), AuNPs@PA (81.1nm, -15.9mV), and AuNPs@CD326Ab (97.5nm, -11.6mV). AuNPs@BSA (79.8nm, -28mV) as nonspecific gold nanoparticles is a control for AuNPs@CD326Ab (i.e., gold nanoparticle composites targeting CD326). Data are expressed as mean ± standard deviation, n = 3.

[0059] Example 2 Cell proliferation detection

[0060] Human breast cancer cells MDA-MB-231 and human prostate cancer cells PC3M were cultured in DMEM:F-12 medium containing 10% bovine serum, 1% penicillin and streptomycin at 37°C and 5% CO2; the two cancer cells were inoculated in 96-well plates at a density of 5000 cells / well and cultured with 100 μL of culture medium for 24 h; the culture medium was then replaced with fresh culture medium containing 0 / 10 / 20 / 30 / 40 / 50 μg / mL ZIF-8@CD326Ab, 30 μg / mL ZIF-8@BSA, 1.5 nM AuNPs@CD326Ab or 1.5 nM AuNPs@BSA, respectively. After incubation for 12 h, cell proliferation was detected (CyQUANTTM NF, ThermoFisher, C35007), the culture medium in each well was discarded, and the cells were washed 3 times with DPBS. Then 100 μL of the 1x dye solution in the kit was added to the cells and incubated at 37°C for 30 min. Finally, the fluorescence intensity of each well was measured using a fluorescence microplate reader with an excitation wavelength of 485 nm and an emission detection wavelength of 530 nm. Each condition included 8 wells in duplicate.

[0061] The proliferation detection results of MDA-MB-231 cells and PC3M cells under different treatment conditions are shown in Figure 2 Figure 6 , Figure 7 As shown, the data are mean ± SD, n = 6, and the statistical significance is determined by one-way analysis of variance and Tukey's post hoc test. *** indicates p < 0.001 compared with the corresponding group. From the results, it can be seen that the CD326-targeting ZIF-8 composite material provided by the present invention can significantly inhibit the proliferation of MDA-MB-231 and PC3M cells.

[0062] The colloidal stability results of different nanoparticles in cell culture medium containing 10% fetal bovine serum are shown in Figure 8 As shown, the test time was 5 days, and the data are expressed as mean ± standard deviation, n = 3. Figure 8 It can be seen that the hydrodynamic diameters of ZIF-8NPs@Ab, ZIF-8@BSA, AuNPs@Ab, and AuNPs@BSA changed very little within 5 days, indicating that these NPs have excellent colloidal stability.

[0063] Example 3 Immunostaining of CD326 on the Cell Surface

[0064] MDA-MB-231 and PC3M cells were cultured at 5000 cells / cm 2The cells were seeded at a density of 100 μg / cm2 in a 50 mm glass bottom dish and cultured for 36 h, then fixed with 4% formaldehyde solution for 5 min at room temperature and washed three times with DPBS. The fixed cells were blocked with BlockAid solution (Thermo Fisher) for 30 min at 37 °C and incubated with anti-CD326 primary antibody diluted 1:400 in BlockAid solution at 4 °C overnight. After washing three times with DPBS to remove excess primary antibody, the cells were incubated in a dark room with Alexa Fluor 488-labeled anti-mouse IgG secondary antibody (Cell Signaling Technology, 4408S) diluted 1:1000 in BlockAid at room temperature for 1 h. The cell nuclei were stained with NucBlue ReadyProbes reagent (ThermoFisher) and fluorescent imaging was performed using an Olympus IX71 inverted fluorescence microscope. The results are shown in Figure 2. Fig. 9 As shown, imaging area: 133×133μm 2 The results showed that there was a large amount of CD326 on the surface of MDA-MB-231 and PC3M cancer cells.

[0065] Example 4 Cytotoxicity Detection

[0066] MDA-MB-231 and PC3M cells were cultured in DMEM:F-12 medium containing 10% FBS, 1% penicillin and streptomycin at 37°C and 5% CO2. The two cancer cells were inoculated in 96-well plates at a density of 10,000 cells / well and cultured with 100 μL of culture medium for 24 h; the culture medium was then replaced with fresh culture medium containing 0 / 10 / 20 / 30 / 40 / 50 μg / mL ZIF-8@CD326Ab, 30 μg / mL ZIF-8@BSA, 1.5 nM AuNPs@CD326Ab or 1.5 nM AuNPs@BSA, respectively, and incubated for 12 h before performing an XTT assay (Sigma-Aldrich, 11465015001). The culture medium in each well was discarded, and the cells were washed three times with DPBS. Then, 100 μL of culture medium and 50 μL of freshly prepared XTT detection solution were added to the cells and incubated at 37 °C for 4 h. Finally, the absorbance of each well was measured at 450 nm and 650 nm wavelengths using an EPOCH2 microplate reader to quantify cell viability. The results are shown in Table 1. Fig.10As shown, the surface area and antibody amount of groups B and C were equivalent to those of group A. Therefore, ZIF-8NPs@Ab at a concentration of 30 μg / mL or lower, as well as the corresponding concentrations in groups B and C, were determined as non-cytotoxic doses and applied in subsequent experiments to study their effects on cancer cell motility. Data are expressed as mean ± standard deviation, n = 5; statistical significance was determined by one-way analysis of variance and Tukey's post hoc test, *** indicates p < 0.001 compared with group A. For the LDH experiment, after the washing step, the 96-well plate was incubated with 100 μL of culture medium at 37 °C for 45 min. Then, 50 μL of culture medium in each well was transferred to a new 96-well plate, and the CyQUANT LDH Cytotoxicity Assay Kit (Thermo Fisher, C20300) was used to quantify LDH release, and the absorbance of each well was measured at 490 nm and 680 nm wavelengths using an EPOCH2 microplate reader, respectively. The results are shown in Fig.11 As shown, this result is consistent with Fig.10 Each condition consisted of five replicates.

[0067] Example 5 Microscope imaging detection

[0068] Cellular uptake of ZIF-8NPs was imaged by fluorescence microscopy. MDA-MB-231 and PC3M cells were seeded in 6-well plates containing plasma-cleaned 25 mm coverslips and cultured overnight at 37 °C. Then, both cells were incubated with 30 μg / mL FITC-labeled ZIF-8NPs@CD326Ab or ZIF-8NPs@BSA for 4 h, respectively, which were dispersed in pre-warmed culture medium to allow cellular uptake of NPs. As a control group, MDA-MB-231 and PC3M cells were pretreated with free anti-CD326 antibody (1:400 dilution) before adding ZIF-8NPs@CD326Ab. After fixing the cells with 4% PFA, fluorescence imaging was performed using an Olympus fluorescence microscope (IX73), imaging area: 133×133μm 2 The fluorescence tracking results of MDA-MB-231 and PC3M cell uptake are as follows: Fig.12 , Fig.14 As shown. To demonstrate the targeting specificity, in the third group (from left to right), the cells were pre-blocked with CD326 antibody for 30 min and then co-incubated with ZIF-8NPs@Ab for 12 h before imaging. MDA-MB-231 and PC3M cells showed the highest uptake of ZIF-8NPs@Ab, indicating that ZIF-8NPs@Ab has targeting specificity for MDA-MB-231 and PC3M cells. Fig.12The total fluorescence intensity of individual cells was used to quantify the uptake of nanoparticles by MDA-MB-231 cells. Fig.13 As shown. Fig.14 The total fluorescence intensity of individual cells was used to quantify the uptake of nanoparticles by PC3M cells. Fig.15 Data are presented as mean ± SD, n = 10; statistical significance was determined by one-way ANOVA and Tukey's post hoc test. **** indicates p < 0.0001 compared with the corresponding group.

[0069] Example 6 2D migration experiment

[0070] 300,000 MDA-MB-231 and PC3M cells were seeded in 12-well plates to form monolayer cells. After treatment with 30μg / mL ZIF-8NPs@CD326Ab, 30μg / mL ZIF-8NPs@BSA, 1.5nMAuNPs@CD326Ab or 1.5nMAuNPs@BSA for 12h, the monolayer cells were scraped with a 200μL pipette tip to create linear gaps. Floating cells were removed by three PBS washes before imaging. Gap images were collected using an Olympus microscope (IX71) and a 10x objective. After waiting for 24h in normal cell culture medium to allow cell migration, the healed gaps of MDA-MB-231 and PC3M cells were imaged, and the results are shown as follows. Fig.16 , Fig.18 As shown; further analysis of the intercellular area (wound healing) of MDA-MB-231 and PC3M cells was performed, and the results were as follows: Fig.17 , Fig.19 As shown. In the wound healing experiment, the percentage of gap closure within a given time window was used as an indicator of migration ability. For untreated cells, complete gap closure (100%) could be observed within 24 h, indicating that the cell line has a strong migration ability. Cells treated with AuNPs@BSA, AuNPs@Ab, and ZIF-8NPs@BSA showed moderate gap closure. It is worth noting that cells treated with ZIF-8NPs@CD326Ab showed only a small gap closure, indicating that the migration of MDA-MB-231 and PC3M cells almost stopped after treatment with ZIF-8NPs@CD326Ab, and the percentage of gap closure was significantly lower than that of the ZIF-8NPs@BSA group. Data are expressed as mean ± standard deviation, n = 3. Statistical significance was determined by one-way ANOVA and Tukey's post hoc test; *** indicates p < 0.001 compared with the ZIF-8NPs@BSA group.

[0071] Example 7 3D invasion assay

[0072] MDA-MB-231 and PC3M cells were cultured in 6-well plates to form a monolayer of adherent cells. After 12 h of treatment with 30 μg / mL ZIF-8NPs@CD326Ab, 30 μg / mL ZIF-8NPs@BSA, 1.5 nM AuNPs@CD326Ab or 1.5 nM AuNPs@BSA, the cells were lifted and suspended in serum-free medium. 6.5 mm Transwell chamber inserts (pore size: 8 μm, Corning) were pre-coated with 50 μL 4 mg / mL Matrigel matrix (Corning) at 37 °C for 1 h to form a cell invasion gel. The above cell suspension was seeded into the upper insert of each Transwell, with a cell density of 7.5 × 10 cells per well. 4 Cells. Cell culture medium containing 10% FBS was used as a chemoattractant for the lower layer. After waiting for 24 h, the invading cells at the bottom of the insert were fixed with 4% PFA, stained with crystal violet, and imaged with a 10x objective lens for the invasion of MDA-MB-231 and PC3M cells. The results are shown in Figure 2. Fig. 20 , Fig. 22 The number of invasive cells was quantitatively analyzed and the results were shown as follows: Fig.21 , Fig.23 As shown. The results showed that the invasion ability of MDA-MB-231 and PC3M cells was significantly blocked after treatment with ZIF-8NPs@CD326Ab, and the number of invasive cells was significantly lower than that of the ZIF-8NPs@BSA group. The data are expressed as mean ± standard deviation, n = 3. Statistical significance was determined by one-way ANOVA and Tukey's post hoc test; **** or *** indicates p < 0.0001 or 0.001 compared with the ZIF-8NPs@BSA group, respectively.

[0073] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A ZIF-8 composite material targeting CD326, characterized in that: The preparation method thereof comprises the following steps: 1) suspending ZIF-8NPs in a protein coating solution, using probe ultrasound, storing the suspension overnight, centrifuging and washing, and collecting to obtain ZIF-8NPs@Protein A; the protein coating solution is prepared by dissolving protein A and bovine serum albumin in a PBS buffer at a mass ratio of (0.8-1.2):(2.5-3.5), and the total protein concentration in the protein coating solution is 1.8-2.2 mg / mL; 2) The collected ZIF-8NPs@ProteinA was resuspended in PBS buffer, probe sonicated, and CD326 antibody solution was added to the suspension. After incubation, the suspension was collected by centrifugation to obtain a CD326-targeted ZIF-8 composite material, namely, ZIF-8NPs@CD326Ab.

2. The ZIF-8 composite material targeting CD326 according to claim 1, characterized in that: The dosage ratio of the ZIF-8NPs to the protein coating solution is 180-220 μg: 0.8-1.2 mL.

3. The ZIF-8 composite material targeting CD326 according to claim 1, characterized in that: The centrifugal speed in step 1) is 7800-8200 r / min, and the time is 8-12 min; the centrifugal speed in step 2) is 6800-7200 r / min, and the time is 8-12 min.

4. A method for preparing a CD326-targeting ZIF-8 composite material, characterized in that: The steps include: 1) suspending ZIF-8NPs in a protein coating solution, using probe ultrasound, storing the suspension overnight, centrifuging and washing, and collecting to obtain ZIF-8NPs@Protein A; the protein coating solution is prepared by dissolving protein A and bovine serum albumin in a PBS buffer at a mass ratio of (0.8-1.2):(2.5-3.5), and the total protein concentration in the protein coating solution is 1.8-2.2 mg / mL; 2) The collected ZIF-8NPs@ProteinA was resuspended in PBS buffer, probe sonicated, and CD326 antibody solution was added to the suspension. After incubation, the suspension was collected by centrifugation to obtain a CD326-targeted ZIF-8 composite material, namely, ZIF-8NPs@CD326Ab.

5. The method for preparing the CD326-targeting ZIF-8 composite material according to claim 4, characterized in that: The dosage ratio of the ZIF-8NPs to the protein coating solution is 180-220 μg: 0.8-1.2 mL.

6. The method for preparing the CD326-targeting ZIF-8 composite material according to claim 4, characterized in that: The centrifugal speed in step 1) is 7800-8200 r / min, and the time is 8-12 min; the centrifugal speed in step 2) is 6800-7200 r / min, and the time is 8-12 min.

7. Use of the CD326-targeting ZIF-8 composite material according to any one of claims 1 to 3 or the CD326-targeting ZIF-8 composite material prepared by the preparation method according to any one of claims 4 to 6 in the preparation of drugs for preventing and treating breast cancer and / or prostate cancer.

8. Use of the CD326-targeting ZIF-8 composite material according to claim 7 in the preparation of a drug for preventing and treating breast cancer and / or prostate cancer, characterized in that: The CD326-targeted ZIF-8 composite material serves as the sole active ingredient.

Images