Prostate cancer specific extracellular vesicle fluorescent probe and preparation method thereof

Abstract

The invention belongs to the technical field of biomedical materials, and discloses a prostatic cancer specific extracellular vesicle fluorescent probe and a preparation method thereof, the fluorescent probe comprises an extracellular vesicle with the outer surface modified with a targeted STEAP1 single-chain antibody and a near-infrared fluorescent molecule S0456 loaded in the extracellular vesicle. The extracellular vesicles realize accurate delivery of target organs through targeting STEAP1, and realize accurate imaging of prostatic cancer tissues by loading near-infrared fluorescent molecules, so that the extracellular vesicles can be applied to fluorescent navigation surgery of prostatic cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a prostate cancer-specific extracellular vesicle fluorescent probe and a preparation method thereof. Background Art

[0002] Prostate cancer is the most common malignant tumor of the urinary system, and the incidence of prostate cancer is increasing rapidly year by year, which has brought a huge burden to 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 way to cure high-risk early localized prostate cancer. However, traditional surgical treatment of prostate cancer still has problems such as difficulty in judging tumor boundaries during surgery, difficulty in determining whether to perform lymph node dissection, and the scope of lymph node dissection.

[0003] Fluorescence navigation surgery is a new technology that uses fluorescently labeled tumor cells to guide surgical tumor resection in real time. It relies on the accumulation of fluorescent markers in tumor tissues and produces fluorescence of a certain wavelength when absorbing excitation light of a specific wavelength. With the help of a fluorescent camera, it can well mark the tumor boundaries, lymph nodes and distant metastatic lesions, thereby reducing the positive rate of resection margins and discovering tiny metastatic lesions. Therefore, it is an ideal solution to many problems in radical prostatectomy. Near-infrared fluorescence is highly favored due to its advantages such as low tissue autofluorescence, high signal-to-noise ratio, and high tissue penetration. It is the most suitable fluorescence choice for fluorescence navigation surgery. The da Vinci robot Xi system commonly used in radical prostatectomy has a near-infrared fluorescence imaging mode, which provides a hardware foundation for precise navigation of the surgical resection range. Indocyanine green is a non-specific water-soluble fluorescent contrast agent and the only near-infrared probe approved by the US Food and Drug Administration for radical prostatectomy. However, as a non-tumor-targeted near-infrared fluorescent probe, indocyanine green has limitations such as low tumor affinity, sensitivity, specificity, poor tumor-to-background ratio, and interference caused by its 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 prostate cancer fluorescence navigation surgery. Therefore, it is crucial to develop prostate cancer-specific fluorescent probes with high tumor-to-background ratio and high safety to achieve the goal of precision surgery.

[0004] Extracellular vesicles are membranous structures wrapped by a double membrane. Almost all eukaryotic and prokaryotic cells can secrete extracellular vesicles, which are also present in almost all body fluids. As an excellent carrier, extracellular vesicles have many characteristics and advantages: they can pass through various biological barriers, such as the blood-brain barrier; they can protect the contents from being degraded by various enzymes in the body; they are very stable in the blood and can avoid being engulfed by the mononuclear macrophage system in the body to a certain extent. Compared with artificially synthesized liposomes, vesicles have good biocompatibility and high safety, rarely cause immunosuppression and allergic reactions, and are easy to prepare and preserve. Therefore, they are currently widely used as carriers for in vivo drug delivery. In addition, researchers can directly modify vesicles or engineer cells that produce vesicles to give vesicles various properties, such as targeting. However, there is currently no research using targeted vesicles as carriers to construct tumor-specific fluorescent probes for fluorescence navigation surgery. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a prostate cancer-specific extracellular vesicle fluorescent probe and a preparation method thereof. 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, so that it can be applied to fluorescence navigation surgery for prostate cancer.

[0006] In order to achieve the above purpose, this application adopts the following technical solutions:

[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 in the extracellular vesicle.

[0008] In the above technical scheme, the method for constructing the extracellular vesicles is: by constructing a plasmid, packaging a lentivirus and infecting the target cells, the single-chain antibody targeting STEAP1 is expressed on the surface of the target cell HEK293T, thereby constructing an engineered HEK293T cell expressing the single-chain antibody targeting STEAP1 on the surface, and extracting the extracellular vesicles from the engineered HEK293T cell.

[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 STEAP1-targeting single-chain antibody 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 S0456 is 0.1%-7%, for example, 0.5%, 1% or 7%, etc. The loading amount is 0.05-0.4 μmol per mg of vesicles, for example, 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 double-layer phospholipid vesicle.

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

[0014] In the above technical solution, the source of the extracellular vesicles is HEK293T cells.

[0015] In a second aspect, the present invention provides a method for preparing a prostate cancer-specific extracellular vesicle fluorescent probe, characterized in that it comprises 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, the cells were irradiated under UV light and placed in a cell culture incubator for 12-16 hours, and the culture medium was collected;

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

[0018] S3: By means of electroporation, the S0456 molecule is loaded into the extracellular vesicles obtained in S2 to obtain the fluorescent probe.

[0019] In the above technical scheme, in S1, the amount of HEK293T cells per 1 mL of culture medium was 1×10 6 -5×10 7cells; the complete culture medium was DMEM medium, and 10% fetal bovine serum and 1% double antibody were added.

[0020] In the above technical scheme, in S2, the specific steps of centrifugation are: centrifugation at a speed of 1500 rpm / min for 6 minutes, collecting the supernatant; centrifugation at a speed of 5000 rpm / min for 15 minutes, collecting the supernatant; centrifugation at a centrifugal force of 18000g for 1 hour, collecting the precipitate, and obtaining the extracellular vesicles.

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

[0022] In the above technical scheme, in S3, the specific steps of the electroporation operation are: 200 μg of extracellular vesicles are mixed with 100 μL of a certain concentration of S0456 solution and added to a disposable electroporation cup. Under the conditions of 580V and a pulse time of 120ms, electroporation is performed 3 times, with an interval of 1 minute each time. After electroporation, a large number of bubbles can be seen rising from the bottom of the cup to the cup cover, and the current curve is a wide single peak, which proves that the electroporation effect is good; after the electroporation is completed, the electroporated mixture is placed in a 37°C incubator and incubated for 1 hour to repair the membrane of the vesicle. After that, wash twice with PBS to fully remove the S0456 fluorescent molecules that have not been transferred into the vesicles. Finally, resuspend the precipitate with an appropriate amount of PBS to obtain the fluorescent probe.

[0023] The concentration of the S0456 solution is 0.1-10 μM, for example, 0.1 μM, 1 μM or 10 μM.

[0024] The beneficial effects of the present invention are:

[0025] The present invention takes STEAP1, which is highly expressed specifically in prostate cancer, as a target, expresses the anti-STEAP1 single-chain antibody on the cell membrane surface of the target cell HEK293T by constructing plasmids and lentiviral infection, and screens to obtain a stable expression cell line, extracts the extracellular vesicles produced by the engineered HEK293T by ultraviolet irradiation and high-speed centrifugation, and loads S0456 into the extracellular vesicles by electroporation to successfully construct a prostate cancer-specific extracellular vesicle fluorescent probe. Compared with the traditional near-infrared probe indocyanine green used in prostate cancer fluorescence navigation surgery, this fluorescent probe shows good safety and stronger prostate cancer targeting ability, and the preparation process is relatively simple. It can significantly reduce the positive rate of resection margins and improve postoperative survival in animal surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1It is a curve diagram showing the binding ability of various single-chain antibodies targeting STEAP1 with STEAP1 antigen at different concentrations.

[0027] Figure 2 It 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 the extracellular vesicles produced by HEK293T cells that are not infected with the virus, HEK293T cells that are infected with an empty virus, and HEK293T cells that are infected with a lentivirus expressing a STEAP1 single-chain antibody.

[0029] Figure 4 This is a transmission electron micrograph of extracellular vesicles. The scale bar is 100 nm. From left to right, they are HEK293T cells that are not infected with the virus, HEK293T cells that are infected with an empty virus, and HEK293T cells that are infected with a lentivirus expressing a STEAP1 single-chain antibody.

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

[0031] Figure 6 is the targeting ability of AS-EVs to LNCaP cell line. (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) The average fluorescence intensity of red fluorescence in 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) The proportion of cells that took up vesicles in each group. (E) The average fluorescence intensity of red fluorescence in each group. (**: P < 0.01; ****: P < 0.0001).

[0032] Figure 7 is the targeting ability of AS-EVs to 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 fluorescence intensity of red fluorescence in 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) The proportion of cells that took up vesicles in each group. (E) Average fluorescence intensity of red fluorescence in each group. (ns: P≥0.05; ****: P<0.0001).

[0033] Figure 8 is the targeting ability of AS-EVs to 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) The average fluorescence intensity of red fluorescence in 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) The proportion of cells that took up vesicles in each group. (E) The average fluorescence intensity of red fluorescence in each group. (*: P < 0.05; ***: P < 0.001; ****: P < 0.0001).

[0034] Fig. 9 The loading efficiency analysis of AS-EVs loaded with S0456 and the basic characteristics of AS-EVs@S0456. (A) Standard curve of S0456 concentration-fluorescence intensity. (B) After incubation of AS-EVs with S0456 at different concentrations (0, 0.01, 0.1, 1, 10 μM) by electroporation, the content of S0456 loaded in AS-EVs was detected. (C) After incubation of AS-EVs with S0456 at different concentrations (0, 0.01, 0.1, 1, 10 μM) by electroporation, the loading efficiency of S0456 loaded in AS-EVs was detected. (D) The morphology of AS-EVs@S0456 was observed under transmission electron microscopy. (E) NTA analysis of the particle size and concentration of AS-EVs@S0456. (F) Detection of the change in the Zeta potential of AS-EVs@S0456 after being placed at 4°C for 7 days.

[0035] Fig.10 The safety of AS-EVs@S0456 was verified in vitro; (A) Growth curve of RWPE1 cells after treatment with AS-EVs@S0456 at different concentration gradients (PBS, 109 cells / mL, 1010 cells / mL, 1011 cells / mL, 1012 cells / mL). (B) The proportion of apoptotic cells was detected by apoptosis flow cytometry after treatment with AS-EVs@S0456 at different concentration gradients (0 cells / mL, 109 cells / mL, 1010 cells / mL, 1011 cells / mL, 1012 cells / mL) for 24 hours. The bar graph shows the proportion of total apoptotic cells. (ns: P≥0.05; **: P<0.01; ***: P<0.001****: P<0.0001).

[0036] Fig.11The following are the changes in liver and kidney function of nude mice 3 days before and one week after the use of AS-EVs@S0456. (A) Changes in the blood biochemical indicators of liver function, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) before and after the use of AS-EVs@S0456. (B) Changes in the AST / ALT ratio before and after the use of AS-EVs@S0456. (C) Changes in blood urea nitrogen (BUN) before and after the use of AS-EVs@S0456. (D) Changes in blood creatinine (CREA) before and after the use of AS-EVs@S0456.

[0037] Fig.12 HE staining of heart, liver, spleen, lung and kidney tissue sections of nude mice in the AS-EVs@S0456 treatment group and the saline control group after one week of treatment. From top to bottom, HE staining of heart, liver, spleen, lung, kidney and brain tissue sections, from left to right are saline group 1, 2 and AS-EVs@S0456 group 1, 2. Magnification is 400×.

[0038] Fig.13 This is a nude mouse prostate orthotopic transplant tumor model to explore the imaging effect of AS-EVs@S0456 on tumor tissue in vivo after intracardiac injection. (A) The imaging effect of AS-EVs@S0456 in the nude mouse prostate cancer muscle implant tumor model at different time points. From left to right are the bioluminescence image, the fluorescence image 3 hours after injection, the fluorescence image 24 hours after injection, the fluorescence image 48 hours after injection, and the fluorescence image 72 hours after injection. (B) Bioluminescence (left) and fluorescence (right) images of mice opened 72 hours after 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. Clockwise from 12 o'clock are the liver, spleen, kidney, gastrointestinal tract, lung and heart, and in the middle is the mouse's urogenital system (bladder, testicle, seminal vesicle and prostate).

[0039] Fig.14 The images are of prostate muscle implant 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, tumor resection, suture of incision, and after surgery.

[0040] Fig.15These are bioluminescent images before and after surgery. (A) Bioluminescent images before and after surgery under white light. The upper row is the image before surgery, and the lower row is the image after surgery. The results show that all nude mice in this group had residual tumors at the surgical margin after surgery. (B) Bioluminescent images before and after surgery under white light + fluorescence navigation. The upper row is the image before surgery, and the lower row is the image after surgery. The results show that all nude mice in this group had no residual tumors at the surgical margin after surgery. Fig.16 The survival and positive resection margin of nude mice in each group after surgery. (A) Kaplan-Meier analysis showed that there was a significant difference in survival rate between the fluorescence navigation surgery group (5 mice) and the white light surgery group (5 mice) (Log-rank test P = 0.0342). (B) Immunohistochemical staining proved that the resected tumor had high expression of STEAP1 protein. (C) HE staining results showed that the postoperative resection margin of nude mice in the fluorescence navigation surgery group was negative. (D) HE staining results showed that there were a large number of tumor cells remaining at the resection margin of nude mice in the white light surgery group. DETAILED DESCRIPTION

[0041] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0042] 1. Construction and screening of HEK293T cell lines that stably and highly express anti-STEAP1 single-chain antibody on the cell membrane surface

[0043] 1. Construction of plasmids and lentiviruses overexpressing anti-STEAP1 single-chain antibody

[0044] The anti-STEAP1 single-chain antibody sequence was obtained by phage display technology screening, and its binding ability to the STEAP1 antigen was verified by cell Elisa. The specific nucleic acid sequence encoding the anti-STEAP1 single-chain antibody is shown in Table 1. In order 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-PDGFRβTM domain fusion protein, and two tag proteins HA (YPYDVPDYA) and MYC (EEKLISEEDL) were inserted at both ends of the anti-STEAP1 single-chain antibody to facilitate detection. The fragments are connected by a (G4S)3 structure. The specific fragment structure is shown in Figure 2 , and 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. Lentivirus infection of target cells and pressure screening

[0048] Digest the target HEK293T cells the night before lentivirus infection, and plate the target cells in a six-well plate at a density of 300,000-500,000 cells / well, ensuring that the cell density is 30%-50% of the plate the next day. Plate two wells in total, one for infection with the target lentivirus, and the other for infection with an empty lentivirus as a control.

[0049] Observe the cells under a microscope the next day. When all cells are attached and growing well, you can start lentiviral infection. Calculate the amount of lentivirus based on the titer of the lentivirus, the number of cells per well, and the optimal multiplicity of infection (MOI) of the cell line. By consulting the literature, the optimal MOI value for HEK293T cells was set to 2, according to the following formula:

[0050] MOI 0.7 × virus concentration titer × virus volume cell number

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

[0052] Table 2 Proportion of lentiviral infection system

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

[0054] Discard the cell culture medium in the six-well plate, wash once with PBS solution, prepare the lentiviral infection system according to the above ratio, add it to the wells of the experimental group and the control group respectively, and then return the six-well plate to the incubator for further culture for 12 to 24 hours. During the culture period, observe the cell state under a microscope regularly. When the cells show vacuoles, obvious shrinkage, or a large amount of cell debris appears in the culture medium, discard the culture medium containing lentivirus in time and replace it with 2mL of fresh culture medium, and return it to the incubator for further culture.

[0055] After 24 to 48 hours of continuous culture, observe the expression of green fluorescent protein in the target cells under a fluorescence microscope. If there are few cells expressing green fluorescent protein or the fluorescence intensity is weak, continue to observe until 72 hours. 48 hours after lentiviral infection, puromycin pressure screening can be started according to the expression of green fluorescent protein. For HEK293T cells, fresh complete medium containing 3μg / mL puromycin is generally used for screening. The medium containing puromycin is replaced every 2 to 3 days, and the cells are passaged as appropriate according to the growth of the cells. After one week of screening, the cells are observed again under a fluorescence microscope for the expression of green fluorescent protein. When the cells express green fluorescent protein more stably, extract the total RNA and total protein of the cells to verify the expression of anti-STEAP1 single-chain antibody in the cells. After successful verification, continue to passage and expand the cells, and freeze the relevant cell lines in time for subsequent research.

[0056] 2. Synthesis of vesicle fluorescent probes

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

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

[0059] 2) Collect vesicles by centrifugation

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

[0061] (2) Centrifuge at 1500 rpm / min for 6 min to remove cells and large cell debris, and 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 18,000 × g for 1 hour. Remove the supernatant and the precipitate is EVs. Wash it once with PBS and resuspend it. Take a small portion to measure the protein concentration using the BCA method, and store the rest at -80°C for later use.

[0063] 2. Loading S0456 in AS-EVs by electroporation

[0064] 1) The following operations should be completed under light-proof conditions.

[0065] 2) Prepare S0456 solutions with different concentration gradients (0, 0.1, 1, 10 μM) using sterile PBS solution.

[0066] 3) Take 200 μg of freshly extracted vesicles, centrifuge and remove the supernatant, resuspend with S0456 solution of different concentrations, take 100 μL and add it to a disposable electroporation cup (volume is 100 μL). Be careful to avoid generating bubbles during the addition process to avoid affecting the effect of subsequent electroporation.

[0067] 4) Place the electroporation cup in the electroporator and perform electroporation 3 times at 580V and 120ms pulse time, with a 1-minute interval between each time. After electroporation, a large number of bubbles can be seen rising from the bottom of the cup to the cup cover, and the current curve shows a wide single peak, indicating that the electroporation effect is good.

[0068] 5) After electroporation, the mixture was placed in a 37°C incubator for 1 hour to repair the vesicle membrane. Then, it was washed twice with PBS to fully remove the S0456 fluorescent molecules that were not transferred into the vesicles. Finally, the precipitate was resuspended with an appropriate amount of PBS to obtain the vesicle fluorescent probe.

[0069] 3. Identification of the basic properties of vesicle 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) Open the nanoparticle size tracking analysis (NTA) instrument and install the detection plate. Use clean ddH2O to check whether the instrument is intact. After no 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 absorb the vesicles to be tested and connect it to the injection port. Be careful not to mix in bubbles during the connection. Start automatic detection after boosting about 200 μL.

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

[0075] like Figure 3 The figure shows the statistical results of the particle size distribution of extracellular vesicles. From left to right, HEK293T cells that are not infected with viruses, HEK293T cells infected with empty virus, and HEK293T cells infected with lentivirus expressing STEAP1 single-chain antibody produce extracellular vesicles. It can be seen that the engineering of HEK293T cells did not significantly change the particle size of the extracellular vesicles they produced.

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

[0077] 1) Adsorption: Take 20 μL of the diluted vesicle suspension and drop it onto the copper mesh. After natural adsorption for 5 to 10 minutes, use a filter paper strip to absorb the excess droplets and wait for it to dry naturally.

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

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

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

[0081] like Figure 4 Shown are transmission electron micrographs of extracellular vesicles, with a scale bar of 100 nm. From left to right, they are HEK293T cells that are not infected with the virus, HEK293T cells that are infected with an empty virus, and HEK293T cells that are infected with a lentivirus expressing a STEAP1 single-chain antibody and produce extracellular vesicles.

[0082] 3. Measuring the zeta potential of extracellular vesicles

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

[0084] 2) Open the instrument PARTICLE METRIX (ZetaVIEW) and the corresponding software, and draw the vesicles to be tested into the instrument for testing. After the test is completed, the software will automatically analyze the zeta potential of the vesicles.

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

[0086] like Fig. 9 The figure shows the exploration of experimental conditions for constructing vesicle fluorescent probes by electroporation and the basic properties of vesicle fluorescent probes. It can be seen from the figure that the loading efficiency is highest when S0456 is 0.1μM, and compared with the extracellular vesicles before electroporation, the particle size and stability of the vesicle fluorescent probe have not changed significantly.

[0087] IV. Safety Testing of Vesicle Fluorescent Probes

[0088] 1. Detection of cell apoptosis by flow cytometry

[0089] 1) RWPE1 cells were digested and prepared into cell suspension, and the cell density was measured by an automatic counter, and 100,000 cells / well were plated on a 6-well plate. The plated 6-well plate was placed in an incubator, and after 12 hours of complete cell adhesion, the old cell culture medium was discarded, and 2 mL of freshly prepared vesicles containing different concentrations (0 cells / mL, 1 ... 9 / mL, 10 10 / mL, 10 11 / mL, 10 12 The cells were then added with 1% 4% ethanol / mL (cell culture medium) and returned to the incubator for 24 h.

[0090] 2) Take out the six-well plate, collect the cell culture fluid of each group into a centrifuge tube, wash twice with PBS, and collect the PBS used for washing in each group into the corresponding centrifuge tube. After digesting the cells with EDTA-free trypsin, collect the cell suspension obtained by digestion 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 the cell pellet of each group 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-labeled group and the PI single-labeled group, respectively. Except for the blank control group, all other groups were added with 5 μL of Annexin V-APC and 5 μL of Propidium Iodide at the same time. After adding the dye, gently blow and mix the cell suspension, and incubate at room temperature in the dark for 5 to 10 minutes.

[0094] 6) After incubation, the cell suspension in the centrifuge tube was filtered through a 400-mesh filter and transferred to a flow cytometry tube for flow cytometry detection.

[0095] 7) The files were exported to FCS format and analyzed using Flowjo software (version 10.8.1). Fluorescence compensation was adjusted using cells from the control group and single-label group to remove spectral overlap and set the position of the cross gate.

[0096] like Fig.10 B shows the concentration gradient (0 / mL, 10 9 / mL, 10 10 / mL, 10 11 / mL, 10 12After 24 hours of treatment with vesicle fluorescent probes containing 100 μg / mL of vesicles, apoptotic cells were detected by flow cytometry. 9 The vesicle fluorescent probe at a concentration of 100 / mL or below did not significantly increase the proportion of late apoptotic cells and the total proportion of apoptotic cells in RWPE1 cells.

[0097] 2. CCK-8 cell proliferation assay

[0098] 1) RWPE1 cells were digested to prepare a cell suspension, the cell concentration was measured using an automatic cell counter, and the cell concentration was diluted to 2000 cells / 100 μL using complete culture medium.

[0099] 2) Set 4 detection time points (24 hours, 48 ​​hours, 72 hours, 96 hours) and set 5 vesicle concentration gradient groups (0 cells / mL, 10 9 / mL, 10 10 / mL, 10 11 / mL, 10 12 / mL), and set six replica wells for each group at each time point as replicates. Add 200μL PBS solution to each well in the outermost circle of the 96-well plate to reduce the volatilization of the culture medium. Carefully blow and mix the prepared cell suspension, and slowly add 100μL of cell suspension to each well along the wall of the well. After paving, gently tap the four walls of the 96-well plate in turn to make the cells evenly distributed at the bottom of the 96-well plate. After paving, place the 96-well plate in an incubator for culture. After 12 hours of culture, when all cells are attached to the wall, discard the original cell culture medium, add 200μL of freshly prepared complete culture medium containing vesicles of different concentrations, put the 96-well plate with the changed liquid back into the incubator for culture, and set this time as 0.

[0100] 3) 2 hours before each test time point, prepare the CCK-8 culture medium mixture used for measurement in a ratio of CCK-8 solution: incomplete culture medium = 1:9 under light-proof conditions. Take out the 96-well plate, discard the original culture medium, add 100 μL of the freshly prepared CCK-8 culture medium mixture to each well, and then return it to the incubator for 2 hours.

[0101] 4) After the incubation, the 96-well plate was taken out and the OD value of each well at a wavelength of 450 nanometers, i.e., OD450, was measured using an ELISA reader.

[0102] 5) After all four time points are measured, a growth curve is drawn according to the OD450 value of each group.

[0103] like Fig.10A shows the proliferation of RWPE1 cells after incubation with different concentrations of vesicle fluorescent probes for different time periods using CCK-8 method. 9 / mL and 10 10 AS-EVs@S0456 at a concentration of pc / mL did not significantly inhibit the growth of RWPE1 cells.

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

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

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

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

[0108] 4) After the first blood draw, 200 μL of 10 10 The mice were injected with vesicle fluorescent probes containing cells / mL. Blood was collected from the tail tip 7 days after injection to measure the liver and kidney function of the mice.

[0109] like Fig.11 AD shows the changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), AST / ALT ratio, blood urea nitrogen (BUN) and blood creatinine (CREA) of mice before and 7 days after injection of vesicle fluorescent probe. It can be seen from the figure that AS-EVs@S0456 has no obvious damage to the liver and kidney function of nude mice.

[0110] 4. Paraffin embedding and sectioning

[0111] 1) Modification and sampling: Use 4% paraformaldehyde to remove the mouse heart, liver, spleen, lung, kidney tissues and surgically removed tumor tissues that have been fixed for more than 24 hours, and cut the tissue blocks into appropriate sizes. Put the modified tissue blocks into a dehydration box for later use.

[0112] 2) Dehydration and transparency: The fixed and modified tissue blocks were dehydrated and transparentized in the following order: 75% ethanol soaked for 4 hours - 85% ethanol soaked for 2 hours - 90% ethanol soaked for 2 hours - 95% ethanol soaked for 1 hour - anhydrous ethanol soaked for 30 minutes - anhydrous ethanol soaked for 30 minutes. Then, use xylene I to soak for 8 minutes, and xylene II to soak for 8 minutes. Then, immerse in wax at 65°C for 2 hours.

[0113] 3) Wax embedding: After the wax immersion is completed, the tissue block is taken out, heated to melt the wax into liquid, and then placed in the embedding frame. Then all tissue blocks are placed in a -20℃ refrigerator to cool, and the wax blocks are taken out after solidification and the edges of the wax blocks are trimmed neatly.

[0114] 4) Tissue sectioning: Place the embedded tissue wax block into a paraffin slicer for continuous sectioning, with each slice being 4 μm thick. Then flatten the slices in warm water, carefully fish out the slices with a glass slide, and be careful not to mix up the slices of each group during this process. After the slices are fished out and divided into groups, they are placed in a 60°C oven to bake the slices. After the water on the slices is dried and the wax is melted, take out the slices and store them at room temperature for later use. In this study, resections were performed along the largest cross section to obtain the largest section of the heart, liver, spleen, lung, kidney and tumor tissue to determine the toxic and side effects of the vesicle fluorescent probe on the organs and the positive resection margin after surgery.

[0115] like Fig.12 The following are HE staining images of heart, liver, spleen, lung and kidney tissue sections of nude mice in the AS-EVs@S0456 treatment group and the saline control group after one week of treatment. From top to bottom, they are HE staining images of heart, liver, spleen, lung, kidney and brain tissue sections, and from left to right they are saline group 1, 2 and AS-EVs@S0456 group 1, 2. It can be seen from the figure that compared with the control group, no obvious substantial damage was found in the important organs of the AS-EVs@S0456 group.

[0116] like Fig.16 AD shows the survival and positive margin of each group of nude mice after surgery. It can be seen from the figure that AS-EVs@S0456 has a good effect in fluorescence navigation surgery. Compared with white light surgery, fluorescence navigation surgery using AS-EVs@S0456 has a lower positive margin rate and better postoperative survival. 5. Detection of the effectiveness of vesicle fluorescent probes

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

[0118] 1) Prostate cancer cell lines (22Rv1, LNCaP and C4-2) were plated in a six-well plate at a density of 50,000 cells / well. Three groups were set up: targeted vesicle group (AS-EVs), control vesicle group (Ctrl-EVs) and unstained vesicle group (Blank-EVs). Two time points were set for each group: 3 hours and 6 hours. Each time point of each group corresponded to one well of the six-well plate. The plated six-well plate was placed in an incubator and cultured for 12 hours to allow the cells to fully adhere to the wall.

[0119] 2) Labeling vesicles with PKH26: Use Sigma Aldrich's PKH26 membrane staining kit to label vesicles. Centrifuge EVs resuspended in PBS at 18,000 × g for 1 hour at 4°C. Resuspend EVs with 1 mL of Dilution Buffer, add 4 μL of PKH26 dye to another 1 mL of Dilution Buffer and mix well. Mix the Dilution Buffer with PKH26 dye and the Dilution Buffer with resuspended EVs, incubate at room temperature in the dark for 5 minutes, and then terminate the reaction with 20 mL of complete culture medium or 2 mL of serum. Centrifuge at 18,000 × g for 1 hour at 4°C, remove the supernatant, and then wash 1 to 2 times with PBS solution to fully remove free PKH26 dye that is not bound to EVs.

[0120] 3) Add EVs labeled with PKH26 and unlabeled EVs to the corresponding wells of the six-well plate, add 30 μg EVs to each well, and place the treated six-well plate in the incubator for further culture. Take out the six-well plate at the set time point, discard the culture medium containing EVs, wash twice with PBS solution, digest the cells and centrifuge, wash the cell pellet with PBS solution twice, resuspend the cell pellet with 200 μL PBS solution to obtain a cell suspension, filter through a 400-mesh filter and transfer to a flow tube.

[0121] 4) Use flow cytometer to detect the red fluorescence intensity in cells.

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

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

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

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

[0126] 1) Cell preparation 1×10 4 prostate cancer cells (LNCaP, C4-2 and 22Rv1) were plated in a confocal culture dish and cultured in an incubator for 12 hours. Then, 30 μg of PKH26-labeled vesicles of the experimental group and the control group were added. After incubation with the vesicles for 3 and 6 hours, the confocal dish at the corresponding time point was taken out, the old culture medium was discarded, and the dish was washed twice with PBS and fixed with 4% paraformaldehyde for 25 minutes.

[0127] 2) Dyeing

[0128] (1) Closed membrane rupture: Same as the closed membrane rupture in 3.2.14.1.

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

[0130] (3) Sealing: Discard DAPI, add PBS and wash on a shaker for 3 times, 5 minutes each time. Slowly add a few drops of anti-fluorescence quenching sealing agent to evenly cover the bottom of the confocal dish for sealing. Be careful not to create bubbles during the sealing process.

[0131] (4) Photography: An inverted single-photon laser confocal microscope was used to take pictures, and the average fluorescence intensity of red fluorescence was analyzed using ImageJ.

[0132] like Figure 6 AB shows the immunofluorescence images of extracellular vesicles on the prostate cancer cell line LNCaP at different incubation times (3h and 6h) and the corresponding statistical results. It can be seen from the figure that the targeting ability of the targeted vesicles AS-EVs on the prostate cancer cell line LNCaP is significantly higher than that of the control group vesicles.

[0133] like Figure 7 AB shows the immunofluorescence images of the targeting ability of extracellular vesicles to the prostate cancer cell line C4-2 at different incubation times (3h and 6h) and the corresponding statistical results. It can be seen from the figure that the targeting ability of the targeted vesicles AS-EVs to the prostate cancer cell line C4-2 is significantly higher than that of the control group vesicles.

[0134] like Figure 8 AB shows the immunofluorescence images of the targeting ability of extracellular vesicles to the prostate cancer cell line 22Rv1 at different incubation times (3h and 6h) and the corresponding statistical results. It can be seen from the figure that the targeting ability of the targeted vesicles AS-EVs to the prostate cancer cell line 22Rv1 is significantly higher than that of the control group vesicles.

[0135] 3. Small Animal In Vivo Imaging

[0136] 15 minutes before the specified time point, nude mice were anesthetized with isoflurane and placed on the imaging platform of the IVISSpectrum small animal living imager. The fluorescence intensity of the emission light with a wavelength of 820 nanometers under the excitation light of 745 nanometers was detected. The acquired images were processed and analyzed using LivingImage software.

[0137] like Fig.13 AC shows a nude mouse prostate orthotopic transplant tumor model to explore the imaging effect of AS-EVs@S0456 on tumor tissue in vivo after intracardiac injection. It can be seen from the figure that AS-EVs@S0456 has a good imaging effect on nude mouse prostate transplant tumors.

[0138] 4. Fluorescence navigation surgery

[0139] 1) The near-infrared fluorescence imaging surgery platform of Shanghai Hengguang Zhiying Medical Technology Co., Ltd. was used to perform fluorescence-guided tumor resection surgery on a nude mouse prostate cancer muscle implant tumor model.

[0140] 2) Bioluminescence imaging was used to verify the tumor growth of nude mice two weeks after intramuscular injection of tumor cells. Ten nude mice with successful modeling and similar tumor sizes were randomly divided into a fluorescence navigation surgery group and a white light surgery group, with 5 nude mice in each group.

[0141] 3) 24 hours before surgery, the nude mice in the fluorescence 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) The nude mice were anesthetized with isoflurane, and 15 mg / mL D-luciferin potassium salt solution was injected into the right leg muscle of the nude mice before surgery, and the presence of the tumor was confirmed again by bioluminescence imaging.

[0143] 5) Fix the anesthetized nude mouse in lateral position on the imaging table, turn on the software camera, wait for the camera temperature to drop to the working temperature of -50℃, set the exposure time to 200 milliseconds, cover the camera cover, remove the camera cover after collecting the background, adjust the lens to the appropriate focal length, insert the 1050 filter, turn on the 808 nm laser channel, set the laser power to 4500 mA, set the contrast to automatic mode, set the save file name and save path, adjust the number of shots to 6000, and click to start shooting.

[0144] 6) After disinfecting the visual field with iodine, for the fluorescence navigation surgery group, the surgeon combined the real-time fluorescence image of the nude mouse leg tumor on the display screen and the white light image under the naked eye, and combined his own judgment to complete the tumor resection; for the white light surgery group, the surgeon only completed the tumor resection based on the white light image under the naked eye and his own judgment. After the tumor was resected, the cut edge tissue was fixed and HE staining was performed, and then the muscles and skin were sutured layer by layer, and the incision was disinfected with iodine. After the operation was completed, the resected tumor tissue and the postoperative nude mouse were photographed.

[0145] 7) After surgery, bioluminescence imaging was used to confirm whether there was residual tumor tissue. The nude mice were then placed on a heating pad and waited to wake up. After waking up, they were placed in a culture cage.

[0146] 8) Monitor the survival and tumor recurrence of nude mice on a daily basis and draw a survival curve for nude mice.

[0147] like Fig.14 The figure shows the removal of prostate muscle implant tumors under white light and white light + fluorescence navigation. From top to bottom, the surgical field of view under fluorescence and white light. From left to right, the surgical field of view before surgery, during surgery, tumor removal, suture incision and after surgery. It can be seen from the figure that under the fluorescent surgical field of view, the boundary of the tumor tissue is more clearly visible and easier to distinguish from the surrounding normal tissue, so the tumor tissue can be completely removed.

[0148] VI. Construction of Animal Model

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

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

[0151] (1) PGMLV-CMV-luc-Neo Lentivirus purchased from Jiman Gene Technology Co., Ltd. was used to construct a 22Rv1 cell line (22Rv1-luc) that stably expresses luciferase.

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

[0153] (3) Use fresh culture medium containing 800 μg / mL G-418 for screening, and replace the culture medium every 3 days.

[0154] (4) Detection of luciferase expression: D-luciferin potassium was diluted with sterile PBS solution (without Mg 2+ and Ca 2+ ) was prepared into a 30 mg / mL stock solution (200×). After mixing, use immediately or store in a -20°C refrigerator after aliquoting. Ten thousand 22Rv1 cells that had been screened by G-418 for one week were seeded into a 96-well ELISA plate with a black border and a white bottom. After 8 hours, the detection can be started after the cells are completely attached to the wall. D-luciferin potassium stock solution was diluted 1:200 with preheated (37°C) complete culture medium to prepare D-luciferin potassium working solution (150μg / mL). Remove the original culture medium of the cells, add the prepared working solution, and place it in a 37°C incubator for 1 to 2 minutes before testing on the machine. Place the ELISA plate in an ELISA reader and use the bioluminescence detection mode for detection. Compare the detection results with the fluorescence intensity of the negative control group (cells that do not express luciferase) and the positive control group (cells that express luciferase) to determine whether 22Rv1-luc is successfully constructed.

[0155] 2) Bioluminescence imaging

[0156] (1) Substrate preparation: Use sterile PBS solution (without Mg 2+ and Ca 2+ ) Dissolve D-luciferin potassium to prepare a 15 mg / mL D-luciferin potassium salt solution, filter and sterilize using a 0.2 μm filter membrane, and store in a -20°C or -80°C refrigerator after aliquoting to avoid repeated freezing and thawing. Keep away from light during use.

[0157] (2) Injection of substrate: Inject luciferin potassium salt solution into mice via intraperitoneal injection at a concentration of 10 μL / g body weight. After 10 to 20 minutes of injection, wait until the fluorescence signal reaches the strongest stable plateau, and then use the instrument for imaging analysis.

[0158] (3) Imaging analysis: The bioluminescence detection mode of IVIS Spectrum or VILBER FUSION FX7.EDGE in vivo imager was used for detection. The mouse was anesthetized with isoflurane and placed in the center of the imaging platform. The biochemiluminescence image of the mouse was taken using the automatic parameter mode and analyzed using the software provided with the instrument.

[0159] like Fig.15AB shows the bioluminescent images before and after the operation. (A) is the bioluminescent image before and after the operation under white light. The upper row is the image before the operation, and the lower row is the image after the operation. It can be seen from the figure that all nude mice in this group have residual tumors at the resection margin after surgery. (B) is the bioluminescent image before and after the operation under white light + fluorescence navigation. The upper row is the image before the operation, and the lower row is the image after the operation. It can be seen from the figure that all nude mice in this group have no residual tumors at the resection margin after surgery.

[0160] 2. Construction of nude mouse tumor model

[0161] 1) Nude mouse muscle tumor model

[0162] (1) After digestion, 22Rv1-luc cells were mixed with ABW matrix gel at a ratio of 1:1 to obtain a final cell concentration of 2×10 7 cells / mL of cell matrix gel mixture.

[0163] (2) After fixing the nude mouse with the left hand, wipe the skin on the right thigh of the nude mouse with an alcohol cotton ball and use a sterile syringe to extract 50 μL of the cell matrix glue mixture, i.e., 1×10 6 cells, insert the needle from the top of the mouse's heel parallel to the femur, and slowly inject the cell matrix mixture into the thigh muscle of the nude mouse. After the injection, quickly withdraw the needle and press the needle port with an alcohol cotton ball for about 10 seconds to prevent the cells from overflowing from the needle channel.

[0164] (3) Two weeks later, bioluminescence imaging was used to monitor the tumor formation and size of nude mice.

[0165] 2) Nude mouse prostate orthotopic transplant tumor model

[0166] (1) After digestion, 22Rv1-luc cells were mixed with ABW matrix gel at a ratio of 1:1 to obtain a final cell concentration of 2×10 7 cells / mL of cell matrix gel mixture.

[0167] (2) After anesthetizing the nude mouse with isoflurane, fix it in a supine position on a sterile surgical pad. Disinfect the abdominal skin of the nude mouse with alcohol cotton balls and iodine. Use a sterile scalpel to make an incision about 1 cm long on the penis of the nude mouse. Use surgical scissors to cut the skin and abdominal wall muscles in turn.

[0168] (3) Find the bladder and the seminal vesicles on both sides of the bladder of the nude mouse, and turn the bladder and seminal vesicles out of the incision one by one. Use a sterile cotton swab to gently press the bladder toward the tail of the nude mouse to expose the bladder neck. The part where the bladder neck and the seminal vesicle are connected is the prostate of the mouse.

[0169] (4) Use a sterile insulin needle to extract 50 μL of the cell matrix gel mixture, i.e., 1×10 6cells, insert the needle parallel to the rectum, and slowly inject the cell matrix gel mixture into the lateral lobe of the prostate. After the injection is completed, a bulge can be seen in the prostate. After the injection is completed, quickly withdraw the needle and press the needle port with a cotton swab for about 10 seconds to prevent cells from overflowing from the needle channel.

[0170] (5) Carefully reposition the organ to avoid twisting. Use absorbable sutures to suture the abdominal wall muscles and skin in sequence. After suturing, disinfect the surgical incision again with iodine.

[0171] (6) Two weeks later, bioluminescence imaging was used to monitor the tumor formation and size of nude mice.

[0172] like Fig.13 A shows the bioluminescent imaging image of the nude mouse prostate orthotopic transplanted tumor. It can be seen from the figure that the nude mouse prostate orthotopic transplanted tumor model has been functionally constructed for subsequent imaging experiments.

[0173] In summary, the present invention successfully constructed a new type of prostate cancer-specific targeted vesicle fluorescent probe and verified its effectiveness and safety, which is expected to be used in fluorescence navigation surgery for prostate cancer in the future.

[0174] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A prostate cancer-specific extracellular vesicle fluorescent probe, characterized in that: The invention comprises an extracellular vesicle whose outer surface is modified with a single-chain antibody targeting STEAP1 and a near-infrared fluorescent molecule S0456 loaded in the extracellular vesicle.

2. The fluorescent probe according to claim 1, characterized in that: The method for constructing the extracellular vesicles is as follows: a single-chain antibody targeting STEAP1 is expressed on the surface of a target cell HEK293T by constructing a plasmid, packaging a lentivirus, and infecting the target cell, thereby constructing an engineered HEK293T cell expressing a single-chain antibody targeting STEAP1 on the surface, and extracting the extracellular vesicles from the engineered HEK293T cell.

3. The fluorescent probe according to claim 1, characterized in that: The nucleotide sequence encoding the STEAP1-targeting single-chain antibody 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.

4. The fluorescent probe according to claim 1, characterized in that: The N-terminus and C-terminus of the sequence encoding the STEAP1-targeting single-chain antibody 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.

5. The fluorescent probe according to claim 1, characterized in that: The loading efficiency of the near-infrared fluorescent molecule S0456 is 0.1%-7%, and the loading amount is 0.05-0.4 μmol per mg of vesicle.

6. The fluorescent probe according to claim 1, characterized in that: The structure of the fluorescent probe is a double-layer phospholipid vesicle.

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

8. A method for preparing a prostate cancer-specific extracellular vesicle fluorescent probe, characterized in that: The following steps are involved: 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, the cells were irradiated under UV light and placed in a cell culture incubator for 12-16 hours, and the culture medium was collected; S2: The culture medium obtained in S1 is centrifuged to obtain extracellular vesicles; S3: By means of electroporation, the S0456 molecule is loaded into the extracellular vesicles obtained in S2 to obtain the fluorescent probe.

9. The preparation method according to claim 8, characterized in that: In S1, the amount of HEK293T cells per 1 mL of culture medium was 1 × 10 6 -5×10 7 cells; the complete culture medium was DMEM medium, and 10% fetal bovine serum and 1% double antibody were added.

10. The preparation method according to claim 8, characterized in that: In S2, the specific steps of the centrifugation treatment are: centrifugation at a speed of 1500 rpm / min for 6 minutes, collecting the supernatant; centrifugation at a speed of 5000 rpm / min for 15 minutes, collecting the supernatant; centrifugation at a centrifugal force of 18000g for 1 hour, collecting the precipitate, and obtaining the extracellular vesicles.

Images