CN115089726B - A tumor targeted diagnosis and treatment probe and its preparation method and application - Google Patents

Abstract

The invention discloses a tumor targeting diagnosis and treatment probe and a preparation method and application thereof, and belongs to the technical field of medicines. The probe (N) ₃ TMPs@NAP) tumor derived microparticles (TMPs) are used as nano-carriers, and the TMPs are loaded with Napabucasin (NAP) and are surface-modified with azide groups (N ₃ ). The invention synthesizes the probe N with the lipid bilayer ₃ TMPs@NAP with a particle size of 220.13 +/-4.52 nm has strong tumor binding capacity and anti-tumor effect. The probe can be used as a tracer for diagnosing tumors by PET/CT, and can be used for displaying the positions, sizes, metastasis and other conditions of the tumors. In the treatment experiments, N ₃ Average tumor volumes and weights at day 14 of the TMPs@NAP treatment group were 270.55.+ -. 107.59mm, respectively ³ And 0.30+ -0.12 g, significantly smaller than other treatment groups, and N ₃ TMPs@NAP can inhibit liver metastasis of colon cancer.

Description

A tumor targeted diagnosis and treatment probe and its preparation method and application

Technical field

The invention belongs to the field of medical technology, and in particular relates to a tumor targeted diagnosis and treatment probe and its preparation method and application.

Background technique

In recent years, cancer diagnosis and treatment options have developed rapidly, but no improvement in survival rates has been observed in colorectal cancer. The insidious nature and high recurrence rate of colorectal cancer have always affected the survival of patients. With the continuous deepening of molecular biology research, the existence of cancer stem cells (CSCs) explains an important reason why colorectal cancer is prone to recurrence. CSCs are called tumor-initiating cells and have strong self-renewal and continuous differentiation capabilities. , are insensitive to conventional chemotherapy drugs, and can serve as "seeds" of malignant tumors to promote tumor recurrence and metastasis. Therefore, how to achieve efficient diagnosis of colorectal cancer and treatment focusing on inhibiting the stemness of colorectal cancer is a huge challenge currently faced.

Napabucasin (NAP), also known as BBI608, is an oral cancer stem cell inhibitor that has been used to treat a variety of cancers, including pancreatic ductal adenocarcinoma, non-small cell lung cancer, etc. Standard chemotherapy drugs, such as gemcitabine or carboplatin, kill tumor cells in large quantities, leading to the enrichment of cancer stem cell subpopulations and ultimately causing tumor recurrence. NAP can inhibit both CSCs and ordinary tumor cells in tumors without significant impact on the survival of normal cells. Although NAP has ideal anti-tumor effects in in vitro studies, its clinical application is limited due to its low bioavailability and large gastrointestinal side effects. Therefore, improving the tumor targeting and bioavailability of NAP will help improve its efficacy and enhance clinical application.

Tumor-derived microparticles (TMPs) are ideal drug delivery carriers. TMPs are extracellular vesicles with a diameter of 100-1000 nanometers, which are isolated from the supernatant of ultraviolet-irradiated tumor cells. As natural nanocarriers, TMPs have the following unique advantages. 1. As carriers, TMPs have tumor homologous targeting capabilities, that is, they can specifically target homologous cancer cells through membrane surface antigens that mediate inherent homotypic adhesion properties. 2. TMPs can also induce anti-tumor immune responses by carrying tumor antigen libraries, co-stimulatory molecules and DNA fragments similar to parental cells, activate antigen-presenting cells, and facilitate the activation of the body's immune system. 3.TMPs have high biological safety and clinical application potential. TMPs extracted from patients are currently in clinical trials and have been shown to slow the progression of malignant pleural effusions in lung cancer patients.

Contents of the invention

One aspect of the present invention provides a tumor-targeted diagnostic and therapeutic probe. The probe (N ₃ -TMPs@NAP) uses TMPs as nanocarriers, and the TMPs load Napabucasin (NAP) and modify the azide group (N ₃ ). N ₃ -TMPs@NAP is pre-injected into tumor-bearing mice. N ₃ -TMPs@NAP uses the enhanced penetration effect and homologous targeting ability to specifically reach tumor cells. NAP is injected into tumor cells through membrane fusion, and The N ₃ group was modified to the surface of the tumor cell membrane, and then the radioactive compound ⁶⁸ Ga-L-NETA-DBCO was injected into the mice. DBCO and N ₃ performed a click chemical reaction in vivo, and the radionuclide ⁶⁸ Ga was labeled on the surface of the tumor cells. PET/CT imaging traces nanoprobes and monitors anti-tumor efficacy.

Another aspect of the present invention is to provide a method for preparing a tumor-targeted diagnostic and therapeutic probe. The method includes the following steps: adding 4% (v/v) dimethyl sulfide to PBS. sulfone and encapsulate NAP, mix TMP and NAP to prepare TMPs@NAP, and then 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[azido (polyvinyl ethanol)-2000] N ₃ -TMPs@NAP probe can be prepared by stirring with TMPs@NAP at 35-38℃.

Preferably, the encapsulation process of the NAP is: add 4% (v/v) dimethyl sulfoxide in PBS, mix TMPs and NAP in the solution, react at 37°C, and then pass through a permeable bag (12,000Da) Dialysis and purification.

Preferably, the reaction time between TMPs and NAP is 12 hours.

Preferably, the mass ratio of TMPs to NAP is 1:1.

Preferably, the mass ratio of the 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[azido (polyvinyl ethanol)-2000] and TMPs@NAP is 1:5.

Another aspect of the present invention is to provide the use of the above-mentioned probe in the preparation of anti-tumor drugs.

Another aspect of the present invention is to provide the application of the above probe preparation method in preparing anti-tumor drugs.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention synthesizes a probe N ₃ -TMPs@NAP with a lipid bilayer, its particle size is 220.13±4.52nm, and it has strong tumor binding ability and in vitro anti-tumor effect. PET/CT imaging can be used to diagnose tumors in vivo. In the treatment experiment, the average tumor volume and weight of N ₃ -TMPs@NAP on day 14 were 270.55±107.59mm ³ and 0.30±0.12g respectively, which were significantly smaller than other groups. N3-TMPs@NAP can also inhibit liver metastasis of colon cancer.

Description of the drawings

Figure 1A is a schematic diagram of the synthesis of N ₃ -TMPs@NAP in Example 1.

Figure 1B is a transmission electron microscope image of TMPs in Example 2.

Figure 1C is a diagram of the average hydrated particle size of TMPs and N ₃ -TMPs@NAP in Example 2.

Figure 1D is the Zeta potential diagram of TMPs and N ₃ -TMPs@NAP in Example 2.

Figure 1E is a graph showing changes in the average hydrated particle size of TMPs and N ₃ -TMPs@NAP over 4 days in Example 2.

Figure 1F is the standard curve of NAP in Example 2.

Figure 1G is the in vitro release curve of NAP from N ₃ -TMPs@NAP in Example 2.

Figure 1H is a fluorescence image of CT26 cells treated with Cy5-TMPs in Example 2, scale bar = 200 μm.

Figure 1I is a graph showing the in vitro cell survival rate of CT26 colon cancer cells after incubation with different concentrations of NAP and N ₃ -TMPs@NAP in Example 4.

Figure 1J shows the in vitro cell survival rate of CT26 colon cancer cells after incubation with different concentrations of TMPs in Example 4.

Figure 1K shows the in vitro anti-tumor effect of each group (Control group, TMPs group, NAP group, N ₃ -TMPs@NAP group) detected by the EdU method in Example 4, scale bar = 200 μm. Data are expressed as mean±SD(n).

Figure 2A shows the PET/CT images of CT26 tumor-bearing mice in Example 5 after pre-injection of N ₃ -TMPs@NAP 24 hours into the tail vein and 1 hour and 2 hours after injection of ⁶⁸ Ga-L-NETA-DBCO.

Figure 2B shows the PET/CT images of CT26 tumor-bearing mice in Example 5 after pre-oral administration of N ₃ -TMPs@NAP for 24 hours and then injection of ⁶⁸ Ga-L-NETA-DBCO 1 hour and 2 hours into the tail vein.

Figure 2C is a radioactive uptake map of various tissues of CT26 tumor-bearing mice 2 hours after injection of ⁶⁸ Ga-L-NETA-DBCO in Example 5.

Figure 2D is a graph of the tumor uptake ratio 2 hours after injection of ⁶⁸ Ga-L-NETA-DBCO in Example 5.

Figure 2E is a graph of the uptake ratio between tumors and muscles 2 hours after injection of ⁶⁸ Ga-L-NETA-DBCO in Example 5.

Figure 2F is the fluorescence image of tumor tissue after tail vein injection or oral administration of Cy5/N ₃ -TMPs@NAP in Example 5. Scale bar = 200 μm. Data are expressed as mean ± SD (n = 3; *P<0.05**P<0.01***P<0.001).

Figure 3A is a diagram of the treatment groups of mice in Example 5.

Figure 3B is a graph of tumor growth curves of each group at given time points in Example 5.

Figure 3C is a graph showing the average tumor weight at the treatment endpoint of each group in Example 5.

Figure 3D is a representative gross image of tumor tissue at the treatment endpoint of each group in Example 5.

Figure 3E is an immunohistochemical staining diagram of Ki67 in tumor tissue in Example 5.

Figure 4A shows the treatment plan and grouping of the colon cancer liver metastasis model in Example 5.

Figures 4B-4E are PET/CT images of liver morphology and radioactivity uptake in each group in Example 5.

Figure 4F is a graph showing the number of liver metastases detected in the N ₃ -TMPs@NAP group in Example 5.

Figures 4G and 4H show the lowest average liver and spleen weight in the N3-TMPs@NAP group in Example 5.

Figures 4I and 4J are H&E staining and corresponding quantitative analysis pictures in Example 5.

Figure 5A is a graph showing changes in body weight of mice in Example 6.

Figures 5B-5D are graphs showing the detection results of liver function indicators (ALT, AST and ALP) and renal function indicators (BUN and CRE) in each group after treatment in Example 6.

Figure 5E is H&E staining images of major organs of mice in each group in Example 6. Scale bar = 100 μm. Data are expressed as mean ± SD, (n=5, ***P<0.001).

Figures 6A-6D show the immune activation status of each group of mice in Example 7, that is, CD3 ⁺ CD4 ⁺ , CD3 ⁺ CD8 ⁺ T cell subsets.

Detailed ways

The 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[azido (polyvinyl ethanol)-2000] used in the following examples was purchased from Xi'an Ruixi Biotechnology Co., Ltd., the product number is R- 0047.

Example 1

This embodiment provides a preparation method for N ₃ -TMPs@NAP. The steps are as follows:

(1) Cell culture

The mouse colon cancer cell line CT26 was obtained from the Hubei Provincial Key Laboratory of Molecular Imaging. Cells were cultured in RPMI-1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) (37°C, 5% CO ₂ ).

(2)Separation of TMPs

CT26 colon cancer cells were irradiated with ultraviolet light (UBV, 300J/m ² ) for 1 hour, and then cultured in RPMI-1640 medium containing 10% FBS (without TMPs) for 24 hours. The supernatant was then used to isolate TMPs. The supernatant was first centrifuged at 3000 g for 30 min to remove cells, and then centrifuged at 14000 g for 60 min to obtain TMPs. TMPs were washed three times and resuspended in culture medium. TMPs were filtered using a 0.45 μm filter, and then the protein concentration of TMPs was determined using a BCA protein analysis kit (Beyotime, Shanghai, China). TMPs can be stored at -80°C.

(3)Synthesis of N ₃ -TMPs@NAP

Dimethyl sulfoxide (DMSO, Solarbio, China) can increase the solubility of NAP and promote the permeability of TMPs lipid membrane, so it is used as a permeability enhancer. NAP was encapsulated using 4% (v/v) dimethyl sulfoxide in PBS, TMPs and NAP were mixed in the solution, and incubated for 12 h to prepare TMPs@NAP, and then 1,2-distearoyl-sn- Glycerol-3-phosphoethanolamine-N-[azido(polyvinyl ethanol)-2000] (DSPE-PEG-N3) and TMPs@NAP were incubated at 37°C for 30 min to form N ₃ -TMPs@NAP, and then the sample was passed through N ₃ -TMPs@NAP was obtained by centrifugation and dialysis in a permeable bag. The synthesis schematic diagram of N ₃ -TMPs@NAP is shown in Figure 1A.

Example 2

This example provides the characterization of N3-TMPs@NAP. The steps are as follows:

N3-TMPs@NAP was detected using transmission electron microscopy (TEM, Hitachi, Japan) and dynamic light scattering (DLS, Malvern Instruments Ltd., Worcestershire, UK). In order to detect the stability of N ₃ -TMPs@NAP, DLS was used to continuously monitor the average hydrated particle size within 4 days in vitro. High-performance liquid chromatography (HPLC) was used to determine the concentration of NAP in N ₃ -TMPs@NAP. Briefly, NAP was dissolved in DMSO and then diluted with PBS to different concentrations (0, 0.05, 0.1, 0.2, 0.3, and 0.5 μM), HPLC was used to measure the UV peaks of NAP at different concentrations, and a standard curve of NAP was drawn. N ₃ -TMPs@NAP was placed in a dialysis bag (MW: 12000Da) and incubated with PBS to quantitatively determine the release curve of NAP and detect the UV peaks at different time points at 0, 24, 48 and 72 h.

TEM showed that TMPs presented a vesicle-like structure called lipid bilayer (see Figure 1B, scale bar = 100 μm). DLS analysis showed that the average hydrated particle size of the isolated TMPs was 180.50±2.38 nm, while the hydrated particle size of N ₃ -TMPs@NAP ranged from 40 to 970 nm, with an average hydrated particle size of 220.13±4.52 nm (see Figure 1C). The zeta potentials of TMPs and N ₃ -TMPs@NAP are –38.40±0.93mV and –36.92±1.01mV, respectively (see Figure 1D). The average hydrated particle size of TMPs and N3-TMPs@NAP did not change significantly within 4 days, thus indicating that the probe has good stability (see Figure 1E).

The standard curve of NAP is shown in Figure 1F. The amount of NAP in TMPs was analyzed using high-performance liquid chromatography. The efficiency of TMPs encapsulating NAP is 31.17±2.50%. We measured the NAP release curve of N ₃ -TMPs@NAP in PBS at 37°C. The results showed that about 35% of NAP was released from N ₃ -TMPs@NAP after 72 h, indicating the stability of N ₃ -TMPs@NAP. The sex is better (see Figure 1G). As shown in the fluorescence images, the uptake of TMPs by CT26 gradually increased over time, indicating that N ₃ -TMPs@NAP has good tumor targeting (see Figure 1H). These data indicate that TMPs are ideal nanocarriers and N ₃ -TMPs@NAP has been successfully constructed.

Example 3

This example provides the synthesis method of ⁶⁸ Ga-L-NETA-DBCO and the detection of its binding ability to N ₃ -TMPs@NAP. The steps are as follows:

⁶⁸ GaCl3 was obtained from the ⁶⁸ Ge/ ⁶⁸ Ga generator using hydrochloric acid (0.05 M) as eluent, and sodium acetate was added to 500-μL ⁶⁸ GaCl3 (187 MBq) to adjust the pH to 3.7. The radionuclide ⁶⁸ Ga and L-NETA-DBCO (5 nmol) were chelated at 100°C for 10 min. After cooling the mixture, ⁶⁸ Ga-L-NETA-DBCO was purified using a C18 column. ⁶⁸ The conjugation of Ga-L-NETA-DBCO and N ₃ -TMPs@NAP was traced using PET/CT imaging through an in vivo click reaction.

Example 4

This example uses fluorescence imaging to detect the in vitro binding ability of TMPs to tumor cells. The steps are as follows:

(1) TMPs and Cy5-NHS were incubated at 37°C for 30 minutes to form Cy5-TMPs. Cy5-TMPs (30 μg/mL) were incubated with CT26 cells at 37°C for 12 hours. The cell nuclei were stained with 4',6-diamino-2- After counterstaining with phenylindole (DAPI), the cells were fixed with paraformaldehyde and observed under a fluorescence microscope (Olympus, Japan).

(2) Cytotoxicity detection (CCK-8) analysis

CT26 cells were seeded in 96-well plates (4000 cells/well) and cultured at 37°C overnight, followed by NAP (0.1, 0.5, 1, 2 and 10 μM), DMSO (0.1% dimethyl sulfoxide) , N ₃ -TMPs@NAP (0.1, 0.5, 1, 2 and 10μM) or TMPs (0, 1, 5, 10, 20, 50 and 100μg/mL) treated CT26 cells for 24h, and finally used CCK-8 to detect cell survival. Rate.

CCK-8 was used to detect the cytotoxicity of NAP and N ₃ -TMPs@NAP. Different concentrations of NAP and N ₃ -TMPs@NAP were used to treat CT26 cells. As the concentrations of NAP and N3-TMPs@NAP increased, the cell activity was obvious. decreased, and the cell survival rate in the N ₃ -TMPs@NAP group was lower. (See Figure 1I). In addition, CT26 cells were co-cultured with TMPs at different concentrations (up to 100 μg/mL), and the results showed that the survival rate of cells in each group was greater than 90%, indicating that TMPs have no obvious cytotoxicity in vitro (see Figure 1J).

(3) EdU cell proliferation staining was performed using EdU kit (BeyoClickTM, EdU-488, China). Briefly, CT26 cells were seeded in 12-well plates at 2 × 10 cells ( ^per well) and cultured at 37°C overnight. Then CT26 cells were divided into different groups, namely Control group (0.1% dimethyl sulfoxide), NAP group (0.1 μM), TMPs group (1 μg/mL), and N ₃ -TMPs@NAP (NAP is 0.1 μM). Use EdU kit to detect cell proliferation ability.

The EdU detection results (see Figure 1K) were consistent with the CCK8 detection results. The EdU-positive proportion of cells in the N ₃ -TMPs@NAP group was the lowest. In addition, the fluorescence signal of the TMPs group was similar to that of the control group. Therefore, it can be concluded that TMPs have basically no anti-tumor effect in vitro, but they can enhance the anti-tumor effect by increasing the uptake of NAP by tumor cells, confirming that N ₃ -TMPs@NAP has a good anti-tumor effect.

Example 5

This example examines the tumor targeting and anti-tumor effects of N ₃ -TMPs@NAP in vivo. The steps are as follows:

(1) Tumor-bearing mouse model

The mouse experiments were approved by the Animal Care Committee of Tongji Medical College, University of Science and Technology of China, China. CT26 cells (approximately 1 × 10 ⁶ cells) resuspended in 100 μL PBS were injected subcutaneously into the right upper limb of BALB/C mice (female, 6 weeks old, Beijing Huafukang Biotechnology Co., Ltd., China). Testing was performed after tumors reached approximately 5 mm. The colon cancer liver metastasis model was prepared as follows: the spleens of BALB/C mice were exposed through laparotomy, CT26 cells (5×10 ⁶ ) suspended in 50 μL PBS were injected into their spleens and quickly compressed for 2 min, and then the spleens were gently returned. abdominal cavity and suture the wound in layers. Mice were sacrificed after 2 weeks and livers and spleens were collected.

(2) Living animal PET/CT imaging and biodistribution analysis

N ₃ -TMPs@NAP was injected into CT26 tumor-bearing mice (200 μg) at a predetermined time point (24 h) using different administration methods (tail vein injection or oral administration). ⁶⁸ Ga-L-NETA-DBCO (3.7MBq) was then injected into the mice via the tail vein. Mice were anesthetized with 2% isoflurane, and small animal PET/CT static imaging was performed 2 hours after injection of ⁶⁸ Ga-L-NETA-DBCO. Mice (n=3) were sacrificed after PET/CT imaging. The mouse brain, heart, lung, liver, spleen, kidney, stomach, small intestine, large intestine, muscle, bone and tumor tissues were collected and weighed, and the radioactivity of each tissue was detected using a gamma counter. Radioactivity in organs and tissues was calculated as percent of injected dose per gram of tissue (%ID/g), corrected for radioactive decay.

N ₃ -TMPs@NAP and Cy5-NHS were incubated at 37°C for 30 min to form Cy5/N3-TMPs@NAP. Cy5/N3-TMPs@NAP (100 μg) was injected into CT26 tumor-bearing mice through different administration methods (tail vein injection or oral administration), and pathological sections of tumor tissue were detected by fluorescence imaging to evaluate the presence of Cy5/N ₃ -TMPs@NAP at the tumor site. accumulation.

According to a previous study, PET/CT imaging was performed based on different imaging time points (1h and 2h) and predetermined time points (20h). By comparing different administration methods of N ₃ -TMPs@NAP (tail vein injection group and oral administration group), the maximum uptake of tumor tissue was observed in the tail vein injection group 2 hours after injection of ⁶⁸ Ga-L-NETA-DBCO ( Figures 2A and 2B). The biodistribution results showed that the uptake of tumor tissue in the tail vein injection group was higher than that of the oral administration group, and the uptake of the heart, lung, kidney, liver, and spleen tissues of the model in the tail vein injection group was also higher than that of the oral administration group (Figure 2C and 2D). In addition, the tumor-to-muscle uptake ratio and the fluorescence signal at the tumor site in the tail vein injection group were higher than those in the oral administration group (Figures 2E and 2F). Therefore, N ₃ -TMPs@NAP accumulates more in tumor tissues after injection through the tail vein and shows good bioavailability.

(3) Evaluation of anti-tumor effects in vivo

CT26 tumor-bearing mice were randomly divided into 4 groups (5 mice in each group) and treated with NS (0.1% DMSO), NAP (20 mg/kg), TMPs (10 mg/kg) and N3-TMPs@NAP (NAP was 20 mg/kg). kg) treatment. After treatment, tumor size and mouse body weight were measured every 2 days. On day 14, all mice were sacrificed, and tumor tissues from each group were collected, weighed, photographed, and stored for further histological examination. Immunohistochemistry was used to detect the expression level of Ki67 protein in tumor tissues.

The treatment plan and grouping of tumor-bearing mice are shown in Figure 3A. The tumor size of tumor-bearing mice was observed after treatment. According to the tumor growth curve (Figure 3B), the average tumor volume of the NS group continued to increase, reaching 1345.24±157.10mm ³ on day 14. The tumor volume in the NAP, TMPs and N ₃ -TMPs@NAP groups was significantly reduced, with the N ₃ -TMPs@NAP group having the smallest average tumor volume of 270.55±107.59mm ³ . Similarly, the average tumor weight of the N ₃ -TMPs@NAP group was also the lightest, 0.30 ± 0.12 grams (Figure 3C). The gross images of tumors in each group after treatment are shown in the figure (Figure 3D). Ki67 immunohistochemical staining was used to evaluate the proliferation ability of tumor cells (Figure 3E). The Ki67 positive rate in N3-TMPs@NAP tumor tissues was also the lowest. The data show that N ₃ -TMPs@NAP has good anti-tumor ability in vivo.

The treatment plan and grouping of the colon cancer liver metastasis model are shown in Figure 4A. After 14 days, PET/CT imaging was performed according to the selected optimal imaging conditions. The morphology and radioactivity uptake of livers in each group can be observed on PET/CT images (Figures 4B-4E). As expected, the lowest number of liver metastases was detected in the N ₃ -TMPs@NAP group, as representative images are shown (Fig. 4F). The average liver and spleen weight was lowest in the N3-TMPs@NAP group (Figures 4G and 4H). In addition, H&E staining and corresponding quantitative analysis also showed that the N3-TMPs@NAP group had the smallest area of liver metastasis (Figures 4I and 4J). These results further prove that N ₃ -TMPs@NAP has good anti-tumor ability and can inhibit liver metastasis of colon cancer.

Example 6

This example detects the in vivo toxicity of N ₃ -TMPs@NAP. The steps are as follows:

After treatment, the blood and major organs (liver, spleen, kidney, heart, and lungs) of the mice were collected and functional indicators of the liver and kidneys, such as alanine aminotransferase (ALT) and aspartate aminotransferase, were detected. (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine (CRE) were measured using a blood biochemical automatic analyzer (Chemray 240, China Leto Life Analytical Sciences Co., Ltd.). Major organs (heart, liver, spleen, lung, and kidney) were stained with hematoxylin and eosin (H&E) and examined under a light microscope (Olympus IX73, Japan).

In all groups, no significant changes in mouse body weight were observed, indicating that the treatment had no obvious toxicity (Fig. 5A). In addition, liver and kidney function indicators, including ALT, AST, ALP, BUN, and CRE, showed no obvious liver or kidney toxicity (Figures 5B-5D). H&E staining also showed no pathological evidence of major organ damage (Fig. 5E). The results show that N ₃ -TMPs@NAP has no obvious toxicity to normal tissues.

Example 7

This example detects the in vivo immune activation of N ₃ -TMPs@NAP. The steps are as follows:

After treatment, the tumor tissues of mice in each group were fixed with paraformaldehyde, and T cell subsets in each group were detected by tissue flow cytometry and immunofluorescence, including CD3 ⁺ CD4 ⁺ T cells and CD3 ⁺ CD8 ⁺ T cells.

Flow cytometry and immunofluorescence results showed that the N ₃ -TMPs@NAP group had more CD3 ⁺ CD4 ⁺ and CD3 ⁺ CD8 ⁺ T cells (6A-6B). The immunofluorescence results of tumor tissue were consistent with flow cytometry (6C-6D). TMPs and NAP can synergistically stimulate anti-tumor immune responses, and the N ₃ -TMPs@NAP probe can effectively activate anti-tumor immune responses.

In this study, an integrated nanoprobe for tumor diagnosis and treatment was prepared using TMPs as nanocarriers. Through this nanoprobe, PET/CT imaging was successfully used for the diagnosis of colon cancer. Thanks to the function of NAP and the function of TMPs to activate innate immunity, nanoprobes can inhibit colon cancer by inhibiting cancer stem cells and triggering immune responses. In addition, the nanoprobe can activate anti-tumor immune responses more effectively. Overall, the strategy described here is multifaceted and not limited to colon cancer but also holds great promise in the clinical treatment of various tumors.

Comparative example 1

In the evaluation of the anti-tumor effect in vivo in Example 5(3), in the colon cancer stem cell inhibitor treatment group (NAP treatment group), this comparative example used a mixed solution of NAP and PBS. The reason was that the water solubility of NAP was poor. , cannot be dissolved in PBS and just forms a suspension.

Solution: Dissolve NAP in DMSO and then dilute it with PBS. The DMSO ratio is 2%.

Significance: TMPs@NAP has good water solubility, and no mice in this experimental group died. The loading of TMPs can solve the problem of poor water solubility of NAP.

The above-described embodiments only describe the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. All deformations and improvements shall fall within the protection scope determined by the claims of the present invention.

Claims (9)

1. A tumor-targeted diagnosis and treatment probe, characterized in that the probe preparation process is: adding 4% (v/v) dimethyl sulfoxide to PBS and encapsulating Napabucasin, and adding tumor-derived microparticles Mix with Napabucasin to prepare TMPs@NAP, and then mix 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido (polyvinyl ethanol)-2000] with TMPs@NAP at 35-38℃ The N ₃ -TMPs@NAP probe can be prepared by stirring. 2. The tumor-targeted diagnosis and treatment probe according to claim 1, wherein the diameter of the probe ranges from 40 to 970 nm, and the average diameter is 220.13±4.52 nm. 3. The tumor-targeted diagnosis and treatment probe according to claim 2, characterized in that the potential of the probe is –36.92±1.01 mV. 4. A method for preparing a tumor-targeted diagnostic and therapeutic probe, characterized in that the method includes the following steps: adding 4% (v/v) dimethyl sulfoxide to PBS and mixing tumor-derived microparticles and Napabucasin. Napabucasin was encapsulated in the above solution to prepare TMPs@NAP, and then 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[azido (polyvinyl ethanol)-2000] and TMPs@NAP were The N ₃ -TMPs@NAP probe can be prepared by stirring at 35-38°C. 5. The preparation method according to claim 4, characterized in that the encapsulation process of Napabucasin is: adding 4% (v/v) dimethyl sulfoxide in PBS, mixing tumor-derived microparticles and Napabucasin in This solution was reacted at 37°C and then purified by dialysis in a 12,000 Da permeation bag. 6. The preparation method according to claim 5, wherein the reaction time between the tumor-derived microparticles and Napabuca sin is 12 h. 7. The preparation method according to claim 6, wherein the mass ratio of the tumor-derived microparticles to Napabuca sin is 1:1. 8. The preparation method according to claim 7, characterized in that the 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[azido (polyvinyl ethanol)-2000] and The mass ratio of TMPs@NAP is 1:5. 9. Application of the tumor-targeted diagnostic and therapeutic probe according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.