A prostate cancer-specific extracellular vesicle fluorescent probe and a preparation method thereof

Abstract

The application belongs to the technical field of biomedical materials, and discloses a prostate cancer specific extracellular vesicle fluorescent probe and a preparation method thereof.The fluorescent probe comprises an extracellular vesicle with a targeting STEAP1 single-chain antibody on the outer surface and a near-infrared fluorescent molecule S0456 loaded in the extracellular vesicle.The extracellular vesicle realizes precise delivery of a target organ through targeting STEAP1, and realizes precise imaging of a prostate cancer tissue through loading of the near-infrared fluorescent molecule, so that the fluorescent probe can be applied to fluorescent navigation surgery of the prostate cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a prostate cancer-specific extracellular vesicle fluorescent probe and its preparation method. Background Technology

[0002] Prostate cancer is the most common malignant tumor of the urinary system, and its incidence is increasing rapidly year by year, placing a huge burden on my country's national economy and public health. For localized prostate cancer, active monitoring, local radiotherapy, and endocrine therapy are all treatment options. However, radical prostatectomy with pelvic lymph node dissection is currently the only method that can cure high-risk early-stage localized prostate cancer. But traditional surgical treatment of prostate cancer still faces challenges such as difficulty in determining the tumor boundary during surgery, difficulty in determining whether to perform lymph node dissection, and difficulty in determining the extent of lymph node dissection.

[0003] Fluorescent navigation surgery is a new technique that uses fluorescently labeled tumor cells to guide surgical tumor resection in real time. It relies on the accumulation of fluorescent markers in tumor tissue, which produce fluorescence of a specific wavelength upon absorbing excitation light. With the aid of a fluorescence camera, it can effectively mark tumor boundaries, lymph nodes, and distant metastases, thereby reducing the positive margin rate and detecting minute metastases. Therefore, it is an ideal solution to many challenges in radical prostatectomy. Near-infrared fluorescence is favored due to its advantages such as low tissue autofluorescence, high signal-to-noise ratio, and high tissue penetration, making it the most suitable fluorescence choice for fluorescent navigation surgery. The da Vinci Xi robotic system, commonly used in radical prostatectomy, has a near-infrared fluorescence imaging mode, providing the hardware foundation for precise navigation of the surgical resection area. Indocyanine green (ICB-1) is a non-specific, water-soluble fluorescent contrast agent and the only near-infrared probe approved by the U.S. Food and Drug Administration (FDA) for use in radical prostatectomy. However, as a non-tumor-targeting near-infrared fluorescent probe, ICB-1 has limitations such as low tumor affinity, sensitivity, specificity, poor tumor background ratio, and interference from uptake by the digestive system. Currently, there is still a lack of tumor-specific near-infrared fluorescent probes with high safety and specificity in the field of fluorescent navigation surgery for prostate cancer. Therefore, developing prostate cancer-specific fluorescent probes with high tumor background ratio and high safety is crucial to achieving the goal of precise surgery.

[0004] Extracellular vesicles are membrane-like structures enclosed by a double membrane. Almost all eukaryotic and prokaryotic cells can secrete extracellular vesicles, and they are present in almost all body fluids. As an excellent carrier, extracellular vesicles possess many characteristics and advantages: they can cross various biological barriers, such as the blood-brain barrier; they can protect their contents from degradation by various enzymes in the body; they are very stable in the blood and can, to some extent, avoid phagocytosis by the mononuclear phagocyte system. Compared with synthetic liposomes, vesicles have better biocompatibility and safety, rarely causing immunosuppression or allergic reactions, and are easy to prepare and store, thus they are currently widely used as carriers for in vivo drug delivery. Furthermore, researchers can directly modify vesicles or engineer the cells that produce them, thereby endowing vesicles with various properties, such as targeting. However, currently, no research has used targeting vesicles as carriers to construct tumor-specific fluorescent probes for fluorescently guided surgery. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a prostate cancer-specific extracellular vesicle fluorescent probe and its preparation method. The extracellular vesicles achieve precise delivery to the target organ by targeting STEAP1, and achieve precise imaging of prostate cancer tissue by loading near-infrared fluorescent molecules, making it applicable to fluorescence-guided surgery for prostate cancer.

[0006] To achieve the above objectives, this application adopts the following technical solution:

[0007] In a first aspect, the present invention provides a prostate cancer-specific extracellular vesicle fluorescent probe, comprising an extracellular vesicle whose outer surface is modified with a single-chain antibody targeting STEAP1 and a near-infrared fluorescent molecule S0456 loaded within the extracellular vesicle.

[0008] In the above technical solution, the method for constructing the extracellular vesicles is as follows: by constructing plasmids, packaging lentiviruses, and infecting target cells, a single-chain antibody targeting STEAP1 is expressed on the surface of target cells HEK293T, thereby constructing engineered HEK293T cells with a single-chain antibody targeting STEAP1 expressed on their surface, and extracting the extracellular vesicles from the engineered HEK293T cells.

[0009] In the above technical solution, the nucleotide sequence encoding the single-chain antibody targeting STEAP1 includes a heavy chain and a light chain, the nucleotide sequence of the heavy chain is shown in SEQ ID NO.1, and the nucleotide sequence of the light chain is shown in SEQ ID NO.2.

[0010] In the above technical solution, the N-terminus and C-terminus of the sequence encoding the single-chain antibody targeting STEAP1 are respectively connected to the Igκ chain guide sequence and the transmembrane domain sequence of platelet-derived growth factor receptor β. The nucleotide sequence of the Igκ chain guide sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the transmembrane domain sequence is shown in SEQ ID NO.4.

[0011] In the above technical solution, the loading efficiency of the near-infrared fluorescent molecule SO456 is 0.1%-7%, for example, it can be 0.5%, 1% or 7%. The loading amount is 0.05-0.4 μmol per milligram vesicle, for example, it can be 0.05 μmol, 0.1 μmol, 0.2 μmol, 0.3 μmol or 0.4 μmol.

[0012] In the above technical solution, the structure of the fluorescent probe is a bilayer phospholipid vesicle.

[0013] In the above technical solutions, the particle size of the fluorescent probe is 30-300 nm, for example, it can be 30 nm, 50 nm, 80 nm, 100 nm, 120 nm, 150 nm, 160 nm, 180 nm, or 200 nm. The zeta potential is -5 to -120 mV, for example, it can be -5 mV, -10 mV, -15 mV, -20 mV, -25 mV, -30 mV, -35 mV, or -40 mV.

[0014] In the above technical solution, the extracellular vesicles are derived from HEK293T cells.

[0015] Secondly, the present invention provides a method for preparing a prostate cancer-specific extracellular vesicle fluorescent probe, characterized by comprising the following steps:

[0016] S1: The engineered HEK293T cells were cultured to a confluence of 80%-90%, the complete culture medium was removed, serum-free culture medium was added, and the cells were irradiated under a UV lamp and then placed in a cell culture incubator for 12-16 hours. The culture medium was then collected.

[0017] S2: The culture medium obtained in S1 was centrifuged to obtain extracellular vesicles;

[0018] S3: The fluorescent probe is obtained by loading the S0456 molecule into the extracellular vesicles obtained in S2 through electroporation.

[0019] In the above technical solutions, in S1, when the confluence is 80%-90%, the amount of HEK293T cells per 1 mL of culture medium is 1×10⁻⁶. 6 -5×10 7Cells; complete culture medium was DMEM medium with 10% fetal bovine serum and 1% penicillin-dextrose antibody added.

[0020] In the above technical solution, the specific steps of centrifugation in S2 are as follows: centrifuge at 1500 rpm / min for 6 min and collect the supernatant; centrifuge at 5000 rpm / min for 15 min and collect the supernatant; centrifuge at 18000g for 1 h and collect the precipitate to obtain the extracellular vesicles.

[0021] In the above technical solution, in S2, the obtained extracellular vesicles need to be resuspended in sterile PBS buffer and stored at -80℃; the mass ratio of extracellular vesicles to sterile PBS buffer is 1:(1-5).

[0022] In the above technical solution, the specific steps of electroporation in step S3 are as follows: 200 μg of extracellular vesicles are mixed with 100 μL of a certain concentration of SO456 solution and added to a disposable electroporation cuvette. Electroporation is performed three times at 580 V and a pulse time of 120 ms, with a 1-minute interval between each electroporation. After electroporation, a large number of bubbles are observed rising from the bottom of the cuvette to the top, and the current curve shows a broad single peak, indicating good electroporation effect. After electroporation, the mixture is incubated at 37℃ for 1 hour to allow the vesicle membrane to repair. Then, the mixture is washed twice with PBS to thoroughly remove any SO456 fluorescent molecules that were not transfected into the vesicles. Finally, the precipitate is resuspended in an appropriate amount of PBS to obtain the fluorescent probe.

[0023] The concentration of the SO456 solution is 0.1-10 μM, for example, it can be 0.1 μM, 1 μM or 10 μM.

[0024] The beneficial effects of this invention are as follows:

[0025] This invention targets STEAP1, a prostate cancer-specific high-expression antibody. Anti-STEAP1 single-chain antibodies were expressed on the cell membrane of HEK293T cells via plasmid construction and lentiviral infection, and stably expressing cell lines were selected. Extracellular vesicles generated by engineered HEK293T cells were extracted using UV irradiation and high-speed centrifugation. S0456 was then loaded into these extracellular vesicles via electroporation, successfully constructing a prostate cancer-specific extracellular vesicle fluorescent probe. Compared to the traditional near-infrared probe indocyanine green used in prostate cancer fluorescence-guided surgery, this fluorescent probe exhibits good safety and stronger prostate cancer targeting ability, and its preparation process is relatively simple. In animal surgery, it significantly reduces the positive margin rate and improves postoperative survival. Attached Figure Description

[0026] Figure 1This is a graph showing the binding ability of various single-chain antibodies targeting STEAP1 at different concentrations to the STEAP1 antigen.

[0027] Figure 2 This is a schematic diagram of the structure of the constructed STEAP1 single-chain antibody expression plasmid.

[0028] Figure 3 This is a statistical diagram of the particle size distribution of extracellular vesicles. From left to right, they are extracellular vesicles produced by uninfected HEK293T cells, HEK293T cells infected with empty vector virus, and HEK293T cells infected with lentivirus expressing STEAP1 single-chain antibody.

[0029] Figure 4 This is a transmission electron microscope image of extracellular vesicles, with a scale bar of 100 nm. From left to right, the images show extracellular vesicles produced by uninfected HEK293T cells, HEK293T cells infected with empty vector virus, and HEK293T cells infected with lentivirus expressing STEAP1 single-chain antibody.

[0030] Figure 5 This is a statistical diagram of the zeta potential of extracellular vesicles. From left to right, they are extracellular vesicles produced by HEK293T cells infected with empty virus and HEK293T cells infected with lentivirus expressing STEAP1 single-chain antibody.

[0031] Figure 6 This describes the targeting ability of AS-EVs on LNCaP cell lines. (A) Immunofluorescence observation of the uptake of AS-EVs and Ctrl-EVs by LNCaP cells at different time points (3 hours and 6 hours). (B) Average red fluorescence intensity of each group. (C) Flow cytometry detection of the uptake of AS-EVs and Ctrl-EVs by LNCaP cells at different time points (3 hours and 6 hours). (D) Proportion of cells taking up vesicles in each group. (E) Average red fluorescence intensity of each group. (**: P < 0.01; ****: P < 0.0001).

[0032] Figure 7 This describes the targeting ability of AS-EVs on the C4-2 cell line. (A) Immunofluorescence observation of the uptake of AS-EVs and Ctrl-EVs by C4-2 cells at different time points (3 hours and 6 hours). (B) Average red fluorescence intensity of each group. (C) Flow cytometry detection of the uptake of AS-EVs and Ctrl-EVs by C4-2 cells at different time points (3 hours and 6 hours). (D) Proportion of cells taking up vesicles in each group. (E) Average red fluorescence intensity of each group. (ns: P≥0.05; ****: P<0.0001).

[0033] Figure 8 This describes the targeting ability of AS-EVs on the 22Rv1 cell line. (A) Immunofluorescence observation of the uptake of AS-EVs and Ctrl-EVs by 22Rv1 cells at different time points (3 hours and 6 hours). (B) Average red fluorescence intensity of each group. (C) Flow cytometry detection of the uptake of AS-EVs and Ctrl-EVs by 22Rv1 cells at different time points (3 hours and 6 hours). (D) Proportion of cells taking up vesicles in each group. (E) Average red fluorescence intensity of each group. (*: P < 0.05; ***: P < 0.001; ****: P < 0.0001).

[0034] Figure 9 This study analyzes the loading efficiency of AS-EVs loaded with S0456 and examines the basic characteristics of AS-EVs@S0456. (A) Standard curve of S0456 concentration-fluorescence intensity. (B) Detection of S0456 content in AS-EVs after electroporation incubation with different concentrations (0, 0.01, 0.1, 1, 10 μM) of S0456. (C) Detection of S0456 loading efficiency in AS-EVs after electroporation incubation with different concentrations (0, 0.01, 0.1, 1, 10 μM). (D) Observation of the morphology of AS-EVs@S0456 under transmission electron microscopy. (E) NTA analysis of particle size and concentration of AS-EVs@S0456. (F) Detection of the change in Zeta potential of AS-EVs@S0456 after 7 days at 4℃.

[0035] Figure 10 This study validated the safety of AS-EVs@S0456 in vitro. (A) Growth curves of RWPE1 cells after treatment with AS-EVs@S0456 at different concentration gradients (PBS, 10⁹ / mL, 10¹⁰ / mL, 10¹¹ / mL, 10¹² / mL). (B) The proportion of apoptotic cells was determined by flow cytometry after 24 hours of treatment with AS-EVs@S0456 at different concentration gradients (0 / mL, 10⁹ / mL, 10¹⁰ / mL, 10¹¹ / mL, 10¹² / mL). The bar chart represents the proportion of total apoptotic cells. (ns: P≥0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001).

[0036] Figure 11This section describes the changes in liver and kidney function in nude mice 3 days before and 1 week after AS-EVs@S0456 administration. (A) Changes in blood biochemical indicators reflecting liver function: alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) before and after AS-EVs@S0456 administration. (B) Changes in the AST / ALT ratio before and after AS-EVs@S0456 administration. (C) Changes in blood urea nitrogen (BUN) before and after AS-EVs@S0456 administration. (D) Changes in serum creatinine (CREA) before and after AS-EVs@S0456 administration.

[0037] Figure 12 These are HE-stained images of heart, liver, spleen, lung, and kidney tissue sections from nude mice in the AS-EVs@S0456 treatment group and the saline control group one week after treatment. From top to bottom, the images show HE-stained sections of heart, liver, spleen, lung, kidney, and brain tissue sections. From left to right, they represent saline group 1 and 2, and AS-EVs@S0456 group 1 and 2, respectively. The magnification is 400×.

[0038] Figure 13 This study investigates the imaging effects of AS-EVs@S0456 on tumor tissue after intracardiac injection in a nude mouse prostate orthotopic xenograft model. (A) Imaging effects of AS-EVs@S0456 on a nude mouse prostate cancer muscle xenograft model at different time points. From left to right, the images show bioluminescence, fluorescence at 3 hours, 24 hours, 48 ​​hours, and 72 hours post-injection. (B) Bioluminescence (left) and fluorescence (right) images of mice after laparotomy 72 hours following intracardiac injection of AS-EVs@S0456. (C) Bioluminescence (left) and fluorescence (right) images of mouse organs 72 hours after intracardiac injection of AS-EVs@S0456. From 12 o'clock clockwise, the organs are liver, spleen, kidney, gastrointestinal tract, lung, and heart, with the mouse's urogenital system (bladder, testes, seminal vesicles, and prostate) in the center.

[0039] Figure 14 These are surgical views of prostate muscle implantation tumor resection under white light and white light + fluorescence navigation. From top to bottom, they are the surgical fields under fluorescence and white light. From left to right, they are the surgical fields before surgery, during surgery, during tumor resection, suturing the incision, and after surgery.

[0040] Figure 15These are bioluminescence images before and after surgery. (A) Bioluminescence images before and after surgery under white light. The top row shows images before surgery, and the bottom row shows images after surgery. The results indicate that all nude mice in this group had residual tumors at the surgical margins after surgery. (B) Bioluminescence images before and after surgery under white light + fluorescence navigation. The top row shows images before surgery, and the bottom row shows images after surgery. The results indicate that all nude mice in this group had no residual tumors at the surgical margins after surgery. Figure 16 The results show the survival and surgical margin positivity of nude mice in each group after surgery. (A) Kaplan-Meier analysis showed a significant difference in survival rate between the fluorescence-guided surgery group (n=5) and the white light surgery group (n=5) (Log-rank test, P=0.0342). (B) Immunohistochemical staining confirmed high expression of STEAP1 protein in the resected tumor. (C) HE staining results showed that the surgical margins of nude mice in the fluorescence-guided surgery group were negative after surgery. (D) HE staining results showed that a large number of tumor cells remained at the surgical margins of nude mice in the white light surgery group after surgery. Detailed Implementation

[0041] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0042] I. Construction and screening of HEK293T cell lines with stable high expression of anti-STEAP1 single-chain antibody on cell membrane surface

[0043] 1. Constructing plasmids and lentiviruses overexpressing anti-STEAP1 single-chain antibodies

[0044] The anti-STEAP1 single-chain antibody sequence was obtained through phage display technology screening, and its binding ability to the STEAP1 antigen was verified by cell ELISA. The specific nucleic acid sequence encoding this anti-STEAP1 single-chain antibody is shown in Table 1. To express the anti-STEAP1 single-chain antibody on the cell membrane, the anti-STEAP1 single-chain antibody was combined with the transmembrane domain of PDGFRβ to construct an anti-STEAP1 single-chain antibody-PDGFRβ™ domain fusion protein. Two tag proteins, HA (YPYDVPDYA) and MYC (EEKLISEEDL), were inserted at both ends of the anti-STEAP1 single-chain antibody for easy detection. All fragments are linked by a (G4S)3 structure. The specific fragment structures are shown in Table 1. Figure 2 Finally, the fragment was inserted into the pCDH-EF1α-MCS-T2A-copGFP-Puro plasmid and packaged into lentivirus.

[0045] Table 1. Nucleic acid sequences encoding anti-STEAP1 single-chain antibodies.

[0046]

[0047] 2. Lentiviral infection of target cells and pressure screening

[0048] The target cells, HEK293T, were digested the night before lentiviral infection. The target cells were seeded into six-well plates at a density of 300,000-500,000 cells / well, ensuring that the cell density was 30%-50% of the plate the next day. Two wells were seeded in total: one well was used for lentiviral infection, and the other well was used for empty lentiviral infection as a control.

[0049] The next day, the cells were observed under a microscope. Lentiviral infection could begin when all cells adhered and were in good growth condition. The amount of lentivirus used was calculated based on the lentivirus titer, the number of cells per well, and the optimal multiplicity of infection (MOI) for the cell line. Based on literature review, the optimal MOI for HEK293T cells was set to 2, using the following formula:

[0050] MOI 0.7 × Viral concentration titer × Viral volume and cell count

[0051] Calculate the required volume of lentivirus. Then, prepare the lentivirus infection system according to the proportions in Table 2 below:

[0052] Table 2 Proportions of Lentiviral Infection Systems

[0053] Element Dosage DMEM complete medium containing 10% FBS 1mL Target lentiviral fluid or empty lentiviral fluid Calculate usage Transfection reagent: Polybrene (10 mg / mL) 0.1μL

[0054] Discard the cell culture medium in the six-well plate and wash once with PBS solution. Prepare the lentivirus infection system according to the above ratio and add it to the wells of the experimental group and control group, respectively. Then, put the six-well plate back into the incubator and continue culturing for 12-24 hours. During the culture period, observe the cell status under a microscope regularly. When cells show vacuolation, obvious shrinkage, or a large number of cell debris appear in the culture medium, discard the lentivirus-containing culture medium in time and replace it with 2 mL of fresh culture medium, and put it back into the incubator to continue culturing.

[0055] After culturing for 24–48 hours, observe the expression of green fluorescent protein (GFP) in the target cells under a fluorescence microscope. If the number of cells expressing GFP is low or the fluorescence intensity is weak, continue observation for another 72 hours. Forty-eight hours after lentiviral infection, puromycin-based selection can begin based on GFP expression. For HEK293T cells, fresh complete medium containing 3 μg / mL puromycin is generally used for selection. The puromycin-containing medium is changed every 2–3 days, and cell passages are performed as needed based on cell growth. One week after selection, observe GFP expression again under a fluorescence microscope. When GFP expression is relatively stable, extract total RNA and total protein from the cells to verify the expression of anti-STEAP1 single-chain antibody. After successful verification, continue passage and cell expansion, and promptly cryopreserve the relevant cell lines for subsequent research.

[0056] II. Synthesis of Vesicular Fluorescent Probes

[0057] 1. Extraction of extracellular vesicles (EVs) derived from HEK293T

[0058] 1) HEK293T cells infected with lentivirus were transferred to 15cm culture dishes. When the cell density reached 80%–90% of the dish's volume, the original culture medium was removed and replaced with fresh serum-free culture medium. The cells were then irradiated under UV light for 1 hour, followed by incubation for 24 hours. HEK293T cells produced a large number of vesicles under starvation and UV stimulation.

[0059] 2) Centrifugation to collect vesicles

[0060] (1) Collect the culture medium in the 15cm culture dish into a 50mL centrifuge tube.

[0061] (2) First, centrifuge at 1500 rpm / min for 6 min to remove cells and large cell debris, then centrifuge at 5000 rpm / min for 15 min to remove small cell debris and other impurities.

[0062] (3) Transfer the supernatant after centrifugation to a high-speed centrifuge tube and centrifuge at 18000×g for 1 hour. Remove the supernatant, and the precipitate is EVs. Wash with PBS once and resuspend. Take a small portion to measure the protein concentration using the BCA method, and store the rest at -80℃ for later use.

[0063] 2. Loading S0456 in AS-EVs using the electro-electric transfer method

[0064] 1) All of the following operations should be performed under light-protected conditions.

[0065] 2) Prepare SO456 solutions of different concentration gradients (0, 0.1, 1, 10 μM) using sterile PBS solution.

[0066] 3) After centrifuging 200 μg of freshly extracted vesicles and removing the supernatant, resuspend the vesicles in SO456 solutions of different concentrations, and add 100 μL to a disposable electroporation cup (volume 100 μL). Note that air bubbles should be avoided during the addition process to prevent them from affecting the subsequent electroporation effect.

[0067] 4) Place the electro-rotating cup into the electro-rotator and rotate it three times under conditions of 580V and a pulse time of 120ms, with a 1-minute interval between each rotation. After rotation, a large number of bubbles can be seen rising from the bottom of the cup to the lid, and the current curve shows a broad single peak, proving that the electro-rotation effect is good.

[0068] 5) After electroporation, incubate the electroporated mixture at 37°C for 1 hour to allow the vesicle membranes to repair. Then wash twice with PBS to thoroughly remove any untransfected S0456 fluorescent molecules. Finally, resuspend the precipitate in an appropriate amount of PBS to obtain the vesicle fluorescent probe.

[0069] III. Identification of Basic Properties of Vesicular Fluorescent Probes

[0070] 1. NTA detection of vesicle size and concentration

[0071] 1) Dilute the extracellular vesicles to be tested to an appropriate concentration using PBS.

[0072] 2) Turn on the nanoparticle size tracking analyzer (NTA) and install the detection plate. Use clean ddH2O to check if the instrument is in good working order. After no more bubbles are generated in the detection plate, open the NTA software, rinse with sterile PBS, and set the parameters.

[0073] 3) Use a syringe to draw up the vesicle to be tested, connect it to the injection port, and be careful not to mix in air bubbles when connecting. After injecting about 200 μL, start automatic detection.

[0074] 4) After the test is completed, the software will automatically analyze the particle size and concentration.

[0075] like Figure 3 The image shows the statistical results of the particle size distribution of extracellular vesicles. From left to right, the images show extracellular vesicles produced by uninfected HEK293T cells, HEK293T cells infected with empty vector virus, and HEK293T cells infected with lentivirus expressing STEAP1 single-chain antibody. This indicates that engineering HEK293T cells did not significantly alter the particle size of the extracellular vesicles they produced.

[0076] 2. Observation of vesicle morphology using transmission electron microscopy (TEM)

[0077] 1) Adsorption: Take 20 μL of the diluted vesicle suspension and drop it onto the copper grid. After natural adsorption for 5 to 10 minutes, use filter paper strips to remove excess droplets and let it air dry naturally.

[0078] 2) Staining: Take 20 μL of 2% phosphotungstic acid solution and drop it onto the copper grid, then let it stand for 3-5 minutes.

[0079] 3) Drying: Use filter paper strips to absorb the remaining droplets and air dry under an incandescent lamp.

[0080] 4) Observation and photography: Observe and photograph under a transmission electron microscope.

[0081] like Figure 4 The image shows a transmission electron microscope image of extracellular vesicles. The scale bar is 100 nm. From left to right, the images show extracellular vesicles produced by uninfected HEK293T cells, HEK293T cells infected with empty vector virus, and HEK293T cells infected with lentivirus expressing STEAP1 single-chain antibody.

[0082] 3. Measure the ζ-potential of extracellular vesicles

[0083] 1) Prepare the vesicles to be tested in advance and dilute them to the appropriate concentration with PBS.

[0084] 2) Turn on the Partical Metrix (ZetaVIEW) instrument and its corresponding software, and aspirate the vesicle to be tested into the instrument for detection. After the test is completed, the software will automatically analyze the zeta potential of the vesicle.

[0085] like Figure 5 The figure shows the statistical results of the ζ-potential of extracellular vesicles. From left to right, they are extracellular vesicles produced by HEK293T cells infected with empty virus and HEK293T cells infected with lentivirus expressing STEAP1 single-chain antibody.

[0086] like Figure 9 The figure shows the experimental conditions for constructing vesicular fluorescent probes using electroporation and the basic properties of the vesicular fluorescent probes. As can be seen from the figure, the loading efficiency was highest when S0456 was 0.1 μM. Compared with the extracellular vesicles before electroporation, the particle size and stability of the vesicular fluorescent probes did not change significantly.

[0087] IV. Safety Testing of Vesicle Fluorescent Probes

[0088] 1. Flow cytometry to detect cell apoptosis

[0089] 1) RWPE1 cells were digested to prepare a cell suspension, and the cell density was measured using an automated cell counter. The cells were then seeded into 6-well plates at a density of 100,000 cells / well. The seeded 6-well plates were placed in an incubator, and after 12 hours of complete cell adhesion, the old cell culture medium was discarded. 2 mL of freshly prepared culture medium containing different concentrations of vesicles (0 vesicles / mL, 10 vesicles / mL, etc.) was added. 9 cells / mL, 10 10 cells / mL, 10 11 cells / mL, 10 12 The cells were cultured in a medium containing cells per mL and then returned to the incubator for 24 hours.

[0090] 2) Remove the six-well plate and collect the cell culture medium from each group into a centrifuge tube. Wash the cells twice with PBS, and collect the PBS used for washing each group into the corresponding centrifuge tube. Digest the cells with trypsin without EDTA, and collect the resulting cell suspension into the corresponding centrifuge tube. Centrifuge at 2000 rpm / min for 5 minutes at 4°C, and discard the supernatant.

[0091] 3) Wash the cells twice with PBS, and centrifuge at 2000 rpm / min for 5 minutes after each wash.

[0092] 4) Add 500 μL of Binding Buffer to each group of cell pellets and gently pipette to prepare a single-cell suspension.

[0093] 5) Add 5 μL of Annexin V-APC or 5 μL of Propidium Iodide to the Annexin V-APC single-label group and the PI single-label group, respectively. For the remaining groups, except for the blank control group, add 5 μL of Annexin V-APC and 5 μL of Propidium Iodide simultaneously. After adding the dye, gently pipette to mix the cell suspension and incubate at room temperature in the dark for 5–10 minutes.

[0094] 6) After incubation, filter the cell suspension in the centrifuge tube through a 400-mesh filter and transfer it to a flow cytometer for flow cytometry analysis.

[0095] 7) Export the file as FCS format and analyze it using Flowjo software (version 10.8.1). Use cells from the control group and single-label group to perform fluorescence compensation adjustment to remove spectral overlap and set the position of the cross gate.

[0096] like Figure 10 Figure B shows the results after passing through different concentration gradients (0 cells / mL, 10...). 9 cells / mL, 10 10 cells / mL, 10 11 cells / mL, 10 12After treatment with a vesicle fluorescent probe (number of cells / mL) for 24 hours, the proportion of apoptotic cells was detected by flow cytometry. As shown in the figure, 10 9 Vesicle fluorescent probes at concentrations of 1 / mL or lower did not significantly increase the proportion of late apoptotic cells or the total proportion of apoptotic cells in RWPE1 cells.

[0097] 2. CCK-8 cell proliferation experiment

[0098] 1) After digesting RWPE1 cells, prepare a cell suspension, measure the cell concentration using an automated cell counter, and dilute the cell concentration to 2000 cells / 100μL with complete culture medium.

[0099] 2) Set four detection time points (24 hours, 48 ​​hours, 72 hours, and 96 hours), and simultaneously set five vesicle concentration gradient groups (0 vesicles / mL, 10...). 9 cells / mL, 10 10 cells / mL, 10 11 cells / mL, 10 12 The cell suspension was prepared in a 96-well plate (cells / mL), with six sub-wells for each group at each time point as replicates. 200 μL of PBS solution was added to each well along the outermost ring to reduce culture medium evaporation. The prepared cell suspension was carefully mixed by pipetting, and 100 μL of cell suspension was slowly added dropwise along the well wall. After spreading, the four walls of the 96-well plate were gently tapped to ensure even distribution of cells at the bottom. The 96-well plate was then placed in an incubator. After 12 hours of culture, once all cells had adhered to the wells, the original cell culture medium was discarded, and 200 μL of freshly prepared complete culture medium containing different concentrations of vesicles was added. The 96-well plate with the changed medium was then returned to the incubator, and the time was set to 0.

[0100] 3) Two hours before each detection time point, prepare the CCK-8 medium mixture for the measurement under light-protected conditions at a ratio of CCK-8 solution: incomplete medium = 1:9. Take out the 96-well plate, discard the original medium, add 100 μL of freshly prepared CCK-8 medium mixture to each well, and then put it back into the incubator for 2 hours.

[0101] 4) After incubation, remove the 96-well plate and use a microplate reader to measure the OD value of each well at a wavelength of 450 nm, i.e., OD450.

[0102] 5) After all four time points have been measured, growth curves are plotted based on the OD450 values ​​of each group.

[0103] like Figure 10Figure A shows the proliferation of RWPE1 cells after co-incubation with different concentrations of vesicular fluorescent probes for different times, as detected by the CCK-8 assay. As can be seen from the figure, compared with the PBS control group, 10 9 cells / mL and 10 10 AS-EVs@S0456 at a concentration of cells / mL did not significantly inhibit the growth of RWPE1 cells.

[0104] 3. Determination of liver and kidney function in mice

[0105] 1) Prepare a 1% heparin sodium saline solution and moisten a 1.5 mL EP tube with the prepared heparin sodium solution for later use.

[0106] 2) Disinfect the mouse's tail with an alcohol swab, quickly cut off the tip of the mouse's tail with sterile surgical scissors, collect the blood flowing from the tip of the mouse's tail with a 1.5mL EP tube moistened with sodium heparin, and gently massage the mouse's heart with your fingertips while collecting the blood. When no more blood flows from the mouse's tail, press the wound with a sterile cotton ball to stop the bleeding, and at the same time quickly place the collected blood in an ice box to prevent hemolysis.

[0107] 3) Place the collected blood in an EP tube and centrifuge at 3000 rpm for 15 minutes at 4°C. Collect the supernatant after centrifugation and repeat the above operation once to thoroughly remove blood cells. Dilute the collected supernatant according to a certain ratio and place it in a fully automated blood biochemistry analyzer to detect alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine (CREA), and blood urea nitrogen (BUN).

[0108] 4) After the initial blood collection, 200 μL of a 10% concentration of [unspecified substance] was injected into the mice via tail vein injection. 10 Seven days after injection, blood was collected from the tail tip of the mice to measure liver and kidney function. The mice were injected with vesicle fluorescent probes at a concentration of 1 / mL.

[0109] like Figure 11 Figure AD shows the changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), AST / ALT ratio, blood urea nitrogen (BUN), and serum creatinine (CREA) in mice before and 7 days after injection of the vesicular fluorescent probe. The figure shows that AS-EVs@S0456 did not significantly damage liver and kidney function in nude mice.

[0110] 4. Paraffin embedding and sectioning

[0111] 1) Modification and tissue sampling: Heart, liver, spleen, lung, and kidney tissues and surgically removed tumor tissues from mice fixed for more than 24 hours were extracted using 4% paraformaldehyde and cut into appropriate sizes. The modified tissue blocks were then placed in a dehydration box for later use.

[0112] 2) Dehydration and Clearing: The fixed and modified tissue blocks were subjected to gradient dehydration and clearing treatment in the following order: 75% ethanol for 4 hours – 85% ethanol for 2 hours – 90% ethanol for 2 hours – 95% ethanol for 1 hour – anhydrous ethanol for 30 minutes – anhydrous ethanol for 30 minutes. Then, the tissue blocks were soaked in xylene I for 8 minutes and xylene II for 8 minutes. Finally, the tissue blocks were immersed in paraffin at 65°C for 2 hours.

[0113] 3) Paraffin embedding: After paraffin infiltration, remove the tissue blocks, heat them to melt the paraffin into a liquid state, and then place them into an embedding frame. Then, place all tissue blocks in a -20°C freezer to cool. After the paraffin blocks solidify, remove them and trim the edges of the paraffin blocks neatly.

[0114] 4) Tissue sectioning: The embedded paraffin blocks were placed in a paraffin microtome for serial sectioning, with each section being 4 μm thick. The sections were then flattened in warm water and carefully retrieved using a glass slide, taking care not to mix sections from different groups. After grouping, the sections were placed in a 60°C oven to dry the water and melt the wax. The sections were then stored at room temperature for later use. In this study, the largest possible cross-section was removed to obtain the largest possible sections of the heart, liver, spleen, lung, kidney, and tumor tissue, in order to assess the toxic effects of the vesicular fluorescent probe on the organs and the positivity of the surgical margins.

[0115] like Figure 12 The image shows HE staining images of heart, liver, spleen, lung, and kidney tissue sections from nude mice in the AS-EVs@S0456 treatment group and the saline control group one week after treatment. From top to bottom, the images show HE staining images of heart, liver, spleen, lung, kidney, and brain tissue sections. From left to right, these are saline group 1 and 2, and AS-EVs@S0456 group 1 and 2, respectively. As can be seen from the images, compared with the control group, no significant substantial damage was found in any of the vital organs in the AS-EVs@S0456 group.

[0116] like Figure 16 Figure AD shows the postoperative survival and resection margin positivity of nude mice in each group. As can be seen from the figure, AS-EVs@S0456 performed well in fluorescence-guided surgery. Compared with white light surgery, fluorescence-guided surgery using AS-EVs@S0456 had a lower resection margin positivity rate and better postoperative survival. V. Detection of the effectiveness of vesicle fluorescent probes

[0117] 1. Flow cytometry analysis of the uptake of extracellular vesicles by prostate cancer cells

[0118] 1) Prostate cancer cell lines (22Rv1, LNCaP, and C4-2) were seeded at a density of 50,000 cells / well in six-well plates, with three groups: targeted vesicle group (AS-EVs), control vesicle group (Ctrl-EVs), and unstained vesicle group (Blank-EVs). Each group had two time points: 3 hours and 6 hours. Each time point in each group corresponded to one well in the six-well plate. The seeded six-well plates were incubated for 12 hours to allow complete cell adhesion.

[0119] 2) PKH26 labeling of vesicles: Vesicles were labeled using the Sigma Aldrich PKH26 membrane staining kit. EVs resuspended in PBS were centrifuged at 18000×g for 1 hour at 4°C. EVs were resuspended in 1 mL of Dilution Buffer, and 4 μL of PKH26 dye was added to another 1 mL of Dilution Buffer and mixed well. The Dilution Buffer containing PKH26 dye was then mixed with the Dilution Buffer containing resuspended EVs and incubated at room temperature in the dark for 5 minutes. The reaction was then terminated with 20 mL of complete culture medium or 2 mL of serum. Centrifuged at 18000×g for 1 hour at 4°C, the supernatant was removed, and the vesicles were washed 1–2 times with PBS to thoroughly remove any unbound free PKH26 dye.

[0120] 3) Add 30 μg of PKH26-labeled and unlabeled EVs to the corresponding wells of a six-well plate. Place the treated six-well plates in an incubator for further culture. At the set time points, remove the six-well plates, discard the culture medium containing EVs, wash twice with PBS solution, digest the cells, and centrifuge. After centrifugation, wash the cell pellet twice more with PBS solution, resuspend the cell pellet in 200 μL of PBS solution to obtain a cell suspension, filter through a 400-mesh filter, and transfer to flow cytometry tubes.

[0121] 4) Flow cytometer is used to detect the intensity of red fluorescence in cells.

[0122] like Figure 6 Figure CE shows the flow cytometry results and corresponding statistical results of the targeting ability of extracellular vesicles to the prostate cancer cell line LNCaP at different incubation times (3h and 6h). The figure shows that the targeting ability of AS-EVs to the prostate cancer cell line LNCaP is significantly higher than that of the control group vesicles.

[0123] like Figure 7Figure CE shows the flow cytometry results and corresponding statistical results of the targeting ability of extracellular vesicles against the prostate cancer cell line C4-2 at different incubation times (3h and 6h). The figure shows that the targeting ability of AS-EVs against the prostate cancer cell line C4-2 is significantly higher than that of the control group vesicles.

[0124] like Figure 8 Figure CE shows the flow cytometry results and corresponding statistical results of the targeting ability of extracellular vesicles to the prostate cancer cell line 22Rv1 at different incubation times (3h and 6h). The figure shows that the targeting ability of AS-EVs to the prostate cancer cell line 22Rv1 is significantly higher than that of the control group vesicles.

[0125] 2. Immunofluorescence detection of the uptake of extracellular vesicles by prostate cancer cells

[0126] 1) Cell Preparation: 1×10⁴ prostate cancer cells (LNCaP, C4-2, and 22Rv1) were seeded in confocal culture dishes and cultured for 12 hours. Then, 30 μg of PKH26-labeled vesicles were added to the experimental and control groups, respectively. After co-incubation with the vesicles for 3 and 6 hours, the confocal dishes at the corresponding time points were removed, the old culture medium was discarded, and the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 25 minutes.

[0127] 2) Staining

[0128] (1) Closure of membrane rupture: Same as 3.2.14.1.

[0129] (2) Staining cell nuclei: Discard the blocking and perforation solution, add PBS and wash 3 times on a shaker for 5 minutes each time. Add 100 μL DIPI (1:1000 dilution) and incubate on a horizontal shaker at room temperature in the dark for 5-10 minutes.

[0130] (3) Mounting: Discard DAPI, add PBS and wash three times on a shaker for 5 minutes each time. Slowly add a few drops of anti-fluorescence quenching mounting medium, ensuring the mounting medium evenly covers the bottom of the confocal dish before mounting. Care should be taken to avoid generating air bubbles during the mounting process.

[0131] (4) Photography: Images were taken using an inverted single-photon laser confocal microscope. The average fluorescence intensity of the red fluorescence was analyzed using ImageJ.

[0132] like Figure 6 Figures A and B show immunofluorescence images of extracellular vesicles targeting the LNCaP prostate cancer cell line at different incubation times (3 h and 6 h), along with corresponding statistical results. The figures demonstrate that the targeting ability of AS-EVs to target the LNCaP prostate cancer cell line was significantly higher than that of the control group vesicles.

[0133] like Figure 7 Figures A and B show immunofluorescence images and statistical results of the targeting ability of extracellular vesicles against the prostate cancer cell line C4-2 at different incubation times (3 h and 6 h). The figures show that the targeting ability of AS-EVs against the prostate cancer cell line C4-2 is significantly higher than that of the control group vesicles.

[0134] like Figure 8 Figures A and B show immunofluorescence images and statistical results of the targeting ability of extracellular vesicles against the prostate cancer cell line 22Rv1 at different incubation times (3 h and 6 h). The figures show that the targeting ability of AS-EVs against the prostate cancer cell line 22Rv1 is significantly higher than that of the control group vesicles.

[0135] 3. Small animal live imaging

[0136] Fifteen minutes before the designated time, nude mice were anesthetized with isoflurane and placed on the imaging platform of an IVISSpectrum small animal live imaging system. The fluorescence intensity of the emitted light at a wavelength of 820 nm under excitation with 745 nm light was measured. The acquired images were processed and analyzed using LivingImage software.

[0137] like Figure 13 Figures AC show the imaging effect of AS-EVs@S0456 on tumor tissue in a nude mouse orthotopic prostate xenograft model after intracardiac injection. As can be seen from the figure, AS-EVs@S0456 has a good imaging effect on prostate xenografts in nude mice.

[0138] 4. Fluorescent navigation surgery

[0139] 1) Fluorescence-guided tumor resection surgery was performed on a nude mouse prostate cancer muscle implantation model using the near-infrared fluorescence imaging surgical platform of Shanghai Hengguang Zhiying Medical Technology Co., Ltd.

[0140] 2) The tumor growth in nude mice two weeks after intramuscular injection of tumor cells was verified by bioluminescence imaging. Ten nude mice with successful tumor modeling and similar tumor size were randomly divided into a fluorescence-guided surgery group and a white light surgery group, with 5 nude mice in each group.

[0141] 3) 24 hours before the operation, nude mice in the fluorescent navigation surgery group and the white light surgery group were injected with 200 μL of AS-EVs@S0456 at a concentration of 1 μg / μL.

[0142] 4) Nude mice were anesthetized with isoflurane. Before the operation, 15 mg / mL of D-luciferin potassium salt solution was injected into the right leg muscle of the nude mice, and the presence of the tumor was confirmed again by bioluminescence imaging.

[0143] 5) Fix the anesthetized nude mouse in a lateral recumbent position on the imaging stage, turn on the software camera, wait for the camera temperature to drop to the operating temperature of -50℃, set the exposure time to 200 milliseconds, cover the camera, capture the background, remove the camera cover, adjust the lens to a suitable focal length, insert the 1050 filter, open the 808 nm laser channel, set the laser power to 4500 mA, set the contrast to automatic mode, set the file name and save path, adjust the number of shots to 6000, and click Start Shooting.

[0144] 6) After disinfecting the surgical field with iodine, for the fluorescence-guided surgery group, the surgeon, combining the real-time fluorescence image of the tumor in the nude mouse's leg on the display screen with the white light image under the naked eye, and their own judgment, completed the tumor resection. For the white light surgery group, the surgeon completed the tumor resection solely based on the white light image under the naked eye and their own judgment. After tumor resection, the cut margin tissue was fixed and stained with hematoxylin and eosin (HE). Then, the muscle and skin were sutured layer by layer, and the incision was disinfected with iodine. After the surgery, the resected tumor tissue and the postoperative nude mouse were photographed together.

[0145] 7) Postoperatively, bioluminescence imaging was used again to confirm the presence of residual tumor tissue. The nude mice were then placed on a heating pad to await awakening, and then placed back into the culture cage.

[0146] 8) Monitor the survival and tumor recurrence of nude mice daily and plot the survival curves of nude mice.

[0147] like Figure 14 The images show the resection of a prostate muscle implant under white light and white light + fluorescence navigation. From top to bottom, the surgical field of view is shown under fluorescence and white light. From left to right, the surgical field of view is shown before surgery, during surgery, during tumor resection, after suturing the incision, and after surgery. As can be seen from the images, under fluorescence surgical vision, the boundaries of the tumor tissue are more clearly visible and easier to distinguish from the surrounding normal tissue, thus allowing for complete removal of the tumor tissue.

[0148] VI. Construction of Animal Models

[0149] 1. Construction and bioluminescence imaging of 22Rv1-luc cells

[0150] 1) Construction of the 22Rv1 cell line expressing luciferase (22Rv1-luc)

[0151] (1) The 22Rv1 cell line (22Rv1-luc) stably expressing luciferase was constructed using PGMLV-CMV-luc-Neo Lentivirus purchased from Jimman Gene Technology Co., Ltd.

[0152] (2) The specific steps for lentivirus infection are the same as those in Part 3.2.4 of this article.

[0153] (3) Screening was performed using fresh culture medium containing 800 μg / mL G-418, and the culture medium was changed every 3 days.

[0154] (4) Detection of luciferase expression: D-luciferin potassium was diluted with sterile PBS solution (without Mg) 2+ and Ca 2+ Prepare a 30 mg / mL stock solution (200×). Use immediately after mixing or aliquot and store at -20°C. Seed 10,000 22Rv1 cells (selected by G-418 one week prior) into 96-well microplates with black borders and white backgrounds. After 8 hours, once the cells have fully adhered, detection can begin. Dilute the D-luciferin potassium stock solution 1:200 with preheated (37°C) complete culture medium to prepare a D-luciferin potassium working solution (150 μg / mL). Remove the original culture medium from the cells, add the prepared working solution, and incubate at 37°C for 1–2 minutes before detection. Place the microplate in a microplate reader and use the bioluminescence detection mode. Compare the detection results with the fluorescence intensity of the negative control group (cells not expressing luciferase) and the positive control group (cells expressing luciferase) to determine if the 22Rv1-luc has been successfully constructed.

[0155] 2) Bioluminescence imaging

[0156] (1) Substrate preparation: Use sterile PBS solution (Mg-free) 2+ and Ca 2+ Dissolve potassium D-luciferin to prepare a 15 mg / mL potassium D-luciferin salt solution. Filter sterilize using a 0.2 μm filter membrane, aliquot, and store at -20°C or -80°C, avoiding repeated freeze-thaw cycles. Protect from light during use.

[0157] (2) Substrate injection: Luciferin potassium salt solution was injected into mice via intraperitoneal injection at a concentration of 10 μL / g body weight. Imaging analysis was performed 10–20 minutes after injection, once the fluorescence signal reached its strongest stable plateau.

[0158] (3) Imaging analysis: Bioluminescence detection mode of IVIS Spectrum or VILBER FUSION FX7.EDGE in vivo imaging system was used for detection. Mice were anesthetized with isoflurane and placed in the center of the imaging platform. Biochemiluminescence images of the mice were captured using the automatic parameter mode and analyzed using the software provided with the instrument.

[0159] like Figure 15Figures A and B show bioluminescence images before and after surgery. (A) shows bioluminescence images before and after surgery under white light. The top row shows images before surgery, and the bottom row shows images after surgery. The figures show that all nude mice in this group had residual tumor at the surgical margins after surgery. (B) shows bioluminescence images before and after surgery under white light + fluorescence navigation. The top row shows images before surgery, and the bottom row shows images after surgery. The figures show that all nude mice in this group had no residual tumor at the surgical margins after surgery.

[0160] 2. Construction of a nude mouse tumor model

[0161] 1) Nude mouse muscle tumor implantation model

[0162] (1) Digest 22Rv1-luc cells and mix them with ABW matrix gel at a 1:1 ratio in equal volumes to prepare a final cell concentration of 2×10⁻⁶. 7 Cell matrix gel mixture of cells / mL.

[0163] (2) After stabilizing the nude mouse with the left hand, wipe the skin on the right thigh of the nude mouse with an alcohol swab, and use a sterile syringe to draw 50 μL of the cell matrix gel mixture, i.e., 1×10 6 To inject cells, insert a needle parallel to the femur above the mouse's heel and slowly inject the cell matrix mixture into the thigh muscle of the nude mouse. After injection, quickly withdraw the needle and press the injection site with an alcohol swab for about 10 seconds to prevent cells from leaking out of the needle tract.

[0164] (3) Two weeks later, the tumor formation and size of nude mice were monitored by bioluminescence imaging.

[0165] 2) Nude mouse orthotopic prostate tumor model

[0166] (1) Digest 22Rv1-luc cells and mix them with ABW matrix gel at a 1:1 ratio in equal volumes to prepare a final cell concentration of 2×10⁻⁶. 7 Cell matrix gel mixture of cells / mL.

[0167] (2) After anesthetizing the nude mouse with isoflurane, fix it in a supine position on a sterile surgical drape. Disinfect the abdominal skin of the nude mouse with alcohol swabs and iodine. Make a 1 cm long incision about 1 cm from the penis of the nude mouse with a sterile scalpel, and then cut the skin and abdominal wall muscles with surgical scissors in turn.

[0168] (3) Locate the bladder and seminal vesicles on both sides of the nude mouse, and turn the bladder and seminal vesicles out of the incision one by one. Gently press the bladder towards the tail of the nude mouse with a sterile cotton swab to expose the bladder neck. The part where the bladder neck connects to the seminal vesicle is the prostate of the mouse.

[0169] (4) Draw 50 μL of the cell matrix gel mixture using a sterile insulin needle, i.e., 1 × 10⁻⁶ μL. 6Insert the needle parallel to the rectum and slowly inject the cell matrix gel mixture into the lateral lobe of the prostate. After injection, a bulge will be visible at the prostate site. Quickly withdraw the needle after injection and press the injection site with a cotton swab for about 10 seconds to prevent cells from leaking out of the needle tract.

[0170] (5) Carefully reposition the organ, avoiding twisting. Suture the abdominal wall muscles and skin sequentially with absorbable sutures, and disinfect the surgical incision again with povidone-iodine after suturing.

[0171] (6) Two weeks later, the tumor formation and size of nude mice were monitored by bioluminescence imaging.

[0172] like Figure 13 Image A shows a bioluminescence imaging image of an orthotopic prostate xenograft in nude mice. As shown in the image, an orthotopic prostate xenograft model in nude mice has been functionally constructed for subsequent imaging experiments.

[0173] In summary, this invention has successfully constructed a novel fluorescent probe for targeting prostate cancer specific vesicles and verified its effectiveness and safety, which is expected to be applied in fluorescently guided surgery for prostate cancer in the future.

[0174] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A fluorescent probe for prostate cancer-specific extracellular vesicles, characterized in that: It includes extracellular vesicles with an outer surface modified with a single-chain antibody targeting STEAP1 and a near-infrared fluorescent molecule S0456 loaded within the extracellular vesicles; The method for constructing the extracellular vesicles is as follows: by constructing plasmids, packaging lentiviruses, and infecting target cells, a single-chain antibody targeting STEAP1 is expressed on the surface of target cells HEK293T, thereby constructing engineered HEK293T cells with a single-chain antibody targeting STEAP1 expressed on their surface, and extracting the extracellular vesicles from the engineered HEK293T cells. The nucleotide sequence encoding the single-chain antibody targeting STEAP1 includes a heavy chain and a light chain, the nucleotide sequence of the heavy chain being shown in SEQ ID NO.1 and the nucleotide sequence of the light chain being shown in SEQ ID NO.

2.

2. The fluorescent probe according to claim 1, characterized in that: The N-terminus and C-terminus of the sequence encoding the single-chain antibody targeting STEAP1 are respectively connected to an Igκ chain guide sequence and a transmembrane domain sequence of platelet-derived growth factor receptor β. The nucleotide sequence of the Igκ chain guide sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the transmembrane domain sequence is shown in SEQ ID NO.

4.

3. The fluorescent probe according to claim 1, characterized in that: The loading efficiency of the near-infrared fluorescent molecule SO456 is 0.1%-7%, and the loading amount is 0.05-0.4 μmol per milligram vesicle.

4. The fluorescent probe according to claim 1, characterized in that: The fluorescent probe has a structure of a bilayer phospholipid vesicle.

5. The fluorescent probe according to claim 1, characterized in that: The fluorescent probe has a particle size of 30-300 nm and a ζ-potential of -5 to -120 mV.

6. The method for preparing the prostate cancer-specific extracellular vesicle fluorescent probe according to any one of claims 1-5, characterized in that: Includes the following steps: S1: The engineered HEK293T cells were cultured to a confluence of 80%-90%, the complete culture medium was removed, serum-free culture medium was added, and the cells were irradiated under a UV lamp and then placed in a cell culture incubator for 12-16 hours. The culture medium was then collected. S2: The culture medium obtained in S1 was centrifuged to obtain extracellular vesicles; S3: The fluorescent probe is obtained by loading the S0456 molecule into the extracellular vesicles obtained in S2 through electroporation.

7. The preparation method according to claim 6, characterized in that: In S1, when the confluence was 80%-90%, the amount of HEK293T cells per 1 mL of culture medium was 1 × 10⁻⁶. 6 -5×10 7 Cells; complete culture medium was DMEM medium with 10% fetal bovine serum and 1% penicillin-dextrose antibody added.

8. The preparation method according to claim 6, characterized in that: In S2, the specific steps of centrifugation are as follows: centrifuge at 1500 rpm / min for 6 min and collect the supernatant; centrifuge at 5000 rpm / min for 15 min and collect the supernatant; centrifuge at 18000g for 1 h and collect the precipitate to obtain the extracellular vesicles.

Images