Antibody-exosome, preparation method thereof and application of antibody-exosome in preparation of ricin detoxification medicine

Abstract

The invention provides an antibody-exosome, a preparation method thereof and application of the antibody-exosome in preparation of ricin detoxification drugs, and belongs to the technical field of drug delivery and intracellular therapy. The antibody-exosome disclosed by the invention comprises a milk-derived exosome (mExo) and a single-domain antibody V9E1 of an anti-ricin A chain; the amino acid sequence of the V9E1 is as shown in SEQ ID NO. 1. V9E1 is loaded into a milk exosome by using a saponin perforation method to obtain a V9E1 antibody-exosome, and the antibody-exosome shows reliable biological safety in both cells and animal bodies; the cell survival rate can be obviously improved in a Vero cell acute poisoning model; after mice are poisoned by ricin for 2 hours and 6 hours, the survival rate of the mice can be remarkably improved; through continuous administration, all mice injected with ricin with a complete lethal dose can survive, liver and spleen tissues can be protected, and the method has the potential of replacing a traditional ricin antibody therapy.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery and intracellular therapy technology, and particularly relates to an antibody-exosome, its preparation method, and its application in the preparation of ricin toxin antidote. Background Technology

[0002] Ricin (RT) is a type II ribosome-inactivating protein extracted from castor seeds. It consists of a heterodimer formed by the A chain (RTA) and B chain (RTB) linked by disulfide bonds. It is extremely toxic; the median lethal dose (LD50) in a mouse intraperitoneal injection model is [not specified]. 50 The concentration of RT (reactive protein) was 6.6605 μg / kg, which can lead to apoptosis and tissue damage by inhibiting cellular protein synthesis. Since RT spreads within 2 hours of entering the body, and traditional antibodies have difficulty entering cells to neutralize the ingested toxins, the treatment window after poisoning is short, and the consequences are severe once symptoms appear. Therefore, developing effective prevention and treatment methods for RT poisoning is of great significance.

[0003] Currently, neutralizing antibodies are the traditional primary treatment for RT poisoning. Although various anti-RT antibodies, such as V5E1, have been studied, traditional antibodies, as large protein molecules, have difficulty penetrating the cell membrane and entering the cytoplasm, thus failing to neutralize intracellular toxic RTA and limiting their effectiveness in post-exposure therapy. V9E1's CDR3 region can directly occupy the ribosomal P-stalk binding pocket of RTA, physically blocking the binding of RTA to the ribosome. Due to its unique binding site, it cannot neutralize intact RT extracellularly and requires a delivery system to enter the cell and exert its effect.

[0004] In recent years, exosomes, as natural nanoscale membrane vesicles, have shown great potential in the field of drug delivery. They possess good biocompatibility, low immunogenicity, intrinsic stability, and the ability to cross biological barriers, protecting the loaded drugs from degradation and enabling them to be effectively taken up by recipient cells, releasing their contents into the cytoplasm.

[0005] In the current technology, no mature scheme has been found to use exosomes to target and deliver toxin antibodies into cells to achieve intracellular treatment after toxin poisoning. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an antibody-exosome, a method for preparing the same, and its application in the preparation of an antidote for ricin toxin.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides an antibody-exosome comprising milk exosomes and nanobody V9E1, wherein the amino acid sequence of nanobody V9E1 is shown in SEQ ID NO.1 and the nucleotide sequence encoding nanobody V9E1 is shown in SEQ ID NO.2.

[0008] The present invention also provides a method for preparing the aforementioned antibody-exosomes, comprising the following steps: 1) Milk exosomes were isolated and purified from fresh skim milk using ultracentrifugation; 2) The nucleotide sequence encoding V9E1 was constructed into the pET22b vector to obtain the pET22b-V9E1-His recombinant vector. The pET22b-V9E1-His recombinant vector was introduced into Escherichia coli BL21(DE3) competent cells and purified by nickel column affinity chromatography to obtain high-purity recombinant protein V9E1. 3) Mix milk exosomes and V9E1 protein solution, add saponins and incubate on a shaker, ultrafilter, centrifuge and wash, and collect antibody-exosomes.

[0009] Preferably, the rotation speed of the ultracentrifugation method in step 1) is 100,000~140,000×g, and the ultracentrifugation time is 70~80min.

[0010] Preferably, in step 3), the mass ratio of milk exosomes to V9E1 protein solution is 1:1, and the final concentration of saponin is 1 mg / mL.

[0011] Preferably, the temperature of the shaker incubation in step 3) is 35~40℃, and the incubation time is 10~30min; the conditions for ultrafiltration centrifugation washing are centrifugation at 4,000~6,000 r / min for 3~8min, repeated 2~4 times.

[0012] The present invention also provides the application of the antibody-exosomes described herein or the antibody-exosomes prepared by the described preparation method in the preparation of ricin toxin antidote drugs.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The Exo-V9E1 antibody-exosome of this invention is formed by encapsulating a V9E1 antibody that specifically binds to RTA in milk exosomes (mExo) with targeted delivery capability. The constructed antibody-exosome (Exo-V9E1) can effectively deliver V9E1 into cells and neutralize RTA proteins. Exo-V9E1, prepared using the saponin perforation method, exhibits reliable biosafety in both cells and animals. In an acute Vero cell poisoning model, it can significantly improve cell survival. Administration at 2 h and 6 h after RT poisoning in mice can significantly improve their survival rate. Continuous administration can ensure the survival of all mice injected with a completely lethal dose of RT. It can also target and accumulate in the liver and protect the liver and spleen tissues from damage, showing great potential to replace traditional ricin antibody therapy. Attached Figure Description

[0014] Figure 1 Electrophoresis diagram of V9E1 protein low-dose induced expression (lane 1 is the protein molecular weight standard, lane 2 is the supernatant of recombinant bacterial culture after induction with 0.1mM IPTG at 16℃, lane 3 is the precipitate of recombinant bacterial culture after induction with 0.1mM IPTG at 16℃, lane 4 is the supernatant of recombinant bacterial culture after induction with 0.4mM IPTG at 16℃, lane 5 is the precipitate of recombinant bacterial culture after induction with 0.4mM IPTG at 16℃, lane 6 is the supernatant of recombinant bacterial culture after induction with 1mM IPTG at 16℃, and lane 7 is the precipitate of recombinant bacterial culture after induction with 1mM IPTG at 16℃). Figure 2 This is an SDS-PAGE electrophoresis image of purified V9E1 (lane 1 is the protein molecular weight standard, lanes 2-6 are the purified protein solutions). Figure 3 To observe the structure of Exo under a transmission electron microscope; Figure 4 The results of Zeta potential and particle size analysis for Exo; Figure 5 Simple Western blotting results for Exo; Figure 6 For the cell safety evaluation of Exo; Figure 7 Survival rate and weight change of mice injected with Exo-V9E1 (left figure shows the survival rate of mice (n=5), right figure shows the change in the proportion of the mouse's weight to its original weight). Figure 8 The physiological indicators of mice after injection of Exo-V9E1 were compared (the percentage of multiple physiological indicators and the average normal physiological indicators were compared between the PBS group and the Exo group). Figure 9 An acute ricin poisoning model of Vero cells50 Measurement; Figure 10 For in vitro neutralization and cytotoxicity experiments (n=3); Figure 11 A mouse model of intraperitoneal poisoning (n=4); Figure 12 The therapeutic effects of injecting Exo-V9E1 at different time points on mice poisoned with different concentrations of ricin (n=4). Figure 13 The therapeutic effect of daily injection of Exo-V9E1 on mice poisoned with different concentrations of ricin (n=4). Detailed Implementation

[0015] This invention provides an antibody-exosome, comprising milk exosomes and nanobody V9E1. The amino acid sequence of the nanobody V9E1 is shown in SEQ ID NO.1, specifically QVQLVETGGLVQPGGSLRLSCAASGLTLDYYNIGWFRQAPGKEREWVSSISSSDGRKYYVNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADRDRLPSAITYEYNYWGQGTQVTVSSEPKTPKPQHHHHHH. The nucleotide sequence encoding the nanobody V9E1 is shown in SEQ ID NO.1. As shown in NO.2, it is specifically CATATGCAGGTTCAACTAGTAGAAACAGGTGGATTGGTTCAGCGGGCGGTTCTTTGCGCCTGAGCTGTGCCGCGAGCGGTCTGACTCTGGATTATTACAACATCGGCTGGTTTCGTCAAGCACCGGGCAAAGAGCGCGAATGGGTTTCGTCCATTAGCTCTAGCGATGGTCGTAAGTACTACGTGAATTCAGTAAAAGGCCGTTTCA CCATCAGCCGTGACAACGCTAAAAACACCGTGTACCTGCAAATGAATTCCCTGAAACCAGAAGACACGGCTGTGTACTACTGCGCGGCGGATCGTGACCGCTTACCGAGCGCGATTACCTATGAGTATAACTATTGGGGTCAAGGTACGCAGGTTACCGTCAGCTCCGAGCCGAAGACCCCGAAGCCGCAGCATCATCACCATCACCACTAACTCGAG.

[0016] The present invention also provides a method for preparing the aforementioned antibody-exosomes, comprising the following steps: 1) Milk exosomes were isolated and purified from fresh skim milk using ultracentrifugation; 2) The nucleotide sequence encoding V9E1 was constructed into the pET22b vector to obtain the pET22b-V9E1-His recombinant vector. The pET22b-V9E1-His recombinant vector was introduced into Escherichia coli BL21(DE3) competent cells and purified by nickel column affinity chromatography to obtain high-purity recombinant protein V9E1. 3) Mix milk exosomes and V9E1 protein solution, add saponins and incubate on a shaker, ultrafilter, centrifuge and wash, and collect antibody-exosomes.

[0017] In this invention, milk exosomes are separated and purified from fresh skim milk using ultracentrifugation. The preferred rotation speed for ultracentrifugation is 100,000–140,000 × g, more preferably 110,000–130,000 × g, and even more preferably 120,000 × g; the preferred ultracentrifugation time is 70–80 min, more preferably 72–78 min, and even more preferably 75 min.

[0018] In this invention, the nucleotide sequence encoding V9E1 is constructed into the pET22b vector to obtain the pET22b-V9E1-His recombinant vector. The pET22b-V9E1-His recombinant vector is introduced into Escherichia coli BL21(DE3) competent cells and purified by nickel column affinity chromatography to obtain high-purity recombinant protein V9E1.

[0019] In this invention, milk exosomes and V9E1 protein solution are mixed, saponins are added, and the mixture is incubated on a shaker. After ultrafiltration and centrifugation, the mixture is washed to collect the antibody-exosomes. The preferred mass ratio of milk exosomes to V9E1 protein solution is 1:1; the preferred final concentration of saponins is 1 mg / mL; the preferred temperature for shaker incubation is 35-40°C, more preferably 36-39°C, and even more preferably 37°C; the preferred incubation time is 10-30 min, more preferably 15-25 min, and even more preferably 20 min; the preferred conditions for ultrafiltration and centrifugation are centrifugation at 4,000-6,000 r / min for 3-8 min, repeated 2-4 times, more preferably centrifugation at 5,000 r / min for 5 min, repeated 3 times.

[0020] The present invention also provides the application of the antibody-exosomes described herein or the antibody-exosomes prepared by the described preparation method in the preparation of ricin toxin antidote drugs.

[0021] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0022] The experimental materials used in the embodiments of this invention are as follows: Trypsin was purchased from Gibco, USA; Escherichia coli BL21 (DE3) was purchased from TransGen Biotech Ltd., China; PBS buffer was purchased from Solarbio Science & Technology Co., Ltd., China; Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Solarbio Science & Technology Co., Ltd., China; Yeast Extract was purchased from Oxoid, UK; Ampicillin (AMP) was purchased from Solarbio Science & Technology Co., Ltd., China; Kanamycin (KANA) was purchased from Abcam, China; BCA protein quantification kit was purchased from Biomed Biotechnology, China; Disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4) were purchased from Sinopharm Chemical Reagent Co., Ltd., China; His Trap™ HP chromatography column was purchased from GE Healthcare, Sweden; DMEM culture medium was purchased from Gibco, USA; ELISA reagents (coating solution, Tween 20, 5% BSA, kit, TMB chromogenic solution) were purchased from Solarbio Science & Technology Co., Ltd., China; CD9 rabbit monoclonal antibody and TSG101 rabbit monoclonal antibody were purchased from Abcam, China; goat anti-rabbit IgG was purchased from Solarbio Science & Technology Co., Ltd., China; PVDF membrane was purchased from GE Healthcare, Sweden; ricin and Vero cells were preserved by the Unknown Pathogen Analysis Laboratory of the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, China; fetal bovine serum (FBS) was purchased from Gibco, USA; saponins were purchased from Sigma-Aldrich, USA; MTS was purchased from Abcam, China.

[0023] The instruments used in the embodiments of this invention are as follows: high-speed refrigerated centrifuge and refrigerated ultracentrifuge were purchased from Beckman, Germany; a Class II biosafety cabinet was purchased from NUAIR, USA; a constant temperature incubator was purchased from Tianjin Zhonghuan Experimental Furnace Co., Ltd., China; an enzyme-linked immunosorbent assay (ELISA) analyzer and a full-wavelength scanning reader were purchased from Thermor Fisher, USA; a flow nanoanalyzer (FNA) was purchased from Fuliu Biotechnology Co., Ltd., China; an ultrasonic disruptor was purchased from Qsonica LLC, USA; a transmission electron microscope was purchased from FEI, USA; an autoclave was purchased from SAKURA, Japan; an electric thermostatic water bath was purchased from Beijing Liuyi Instrument Factory, China; a constant temperature shaking incubator was purchased from Jiangsu Taicang Experimental Equipment Factory, China; a gel image analysis system was purchased from Biometra, USA; an AKTA protein purification system was purchased from GE Healthcare, USA; a protein electrophoresis system was purchased from Bio-Rad, USA; and a laser particle size analyzer was also used. The analyzer (LPS) was purchased from Malvernpanalytical, UK; the electronic analytical balance from Sartorius, Germany; the benchtop high-speed centrifuge from SAKURA, Japan; the pure water system from Millipore, USA; and the small animal 3D optical in vivo imaging system from PerkinElmer, USA.

[0024] The experimental animals used in the embodiments of this invention were SPF-grade female BALB / c mice purchased from Spiford (Beijing) Biotechnology Co., Ltd., China. The mice were 6-8 weeks old and weighed 18-22 g each. All experimental procedures were strictly in accordance with experimental animal ethics and operating procedures.

[0025] The preparation of the reagents used in the embodiments of the present invention: His-tagged protein binding buffer: Na₂HPO₄ 5.54 g, NaH₂PO₄ 0.7 g, NaCl 29.22 g, imidazole 1.36 g, deionized water 1000 mL, pH 7.4; His-tagged protein washing buffer: Na₂HPO₄ 5.54 g, NaH₂PO₄ 0.7 g, NaCl 29.22 g, imidazole 34.04 g, deionized water 1000 mL, pH 7.4; LB liquid medium: tryptone 10 g, yeast extract 5 g, NaCl 10 g, deionized water 1000 mL; LB solid medium: tryptone 10 g, yeast extract 5 g, NaCl 10 g, deionized water 1000 mL, agar powder 15 g; 1 N hydrochloric acid: 83.3 mL concentrated hydrochloric acid, deionized water 1000 mL; PBST: 500 mL PBS solution, 250 μL Tween-20.

[0026] Example 1

[0027] V9E1 protein amino acid sequence design

[0028] The single-domain antibody V9E1 sequence, which specifically binds to the ricin A chain (RTA), was selected. Its CDR3 region directly occupies the ribosomal P-stalk binding pocket of RTA, blocking RTA binding to the ribosome through steric hindrance. This sequence is relatively stable and easy to express due to the lack of atypical disulfide bonds. A 6×His tag was introduced at the 3' end of V9E1 to facilitate purification by nickel column affinity chromatography. Based on the restriction enzyme sites of the pET-22b vector, an Nde I (CATATG) restriction site was added to the 5' end of the sequence, and an Xho I (CTCGAG) restriction site was added to the 3' end to construct the pET22b-V9E1 expression vector. Synonymous substitutions were performed according to the codon preference of *E. coli*, removing rare codons to ensure protein activity while improving its expression efficiency and soluble expression level in the host bacteria. The amino acid sequence of the V9E1 protein is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0029] Example 2

[0030] Low-dose induction of V9E1 protein expression

[0031] One BL21(DE3) competent cell was removed from a -80℃ freezer and thawed on ice. 10 μL of the pET22b-V9E1-His recombinant vector was added to each competent cell, and the mixture was gently mixed by tapping the bottom of the tube with a finger. The cells were then incubated on ice for 30 min, followed by a heat shock at 42℃ for 45 s, and immediately returned to the ice bath for 3 min. 500 μL of antibiotic-free LB medium was added, and the cells were incubated at 37℃ with shaking at 180 rpm for 60 min. After centrifugation at 2000 rpm for 4 min, most of the supernatant was discarded, leaving 100 μL to resuspend the cells. The cells were then evenly spread onto LB solid medium containing ampicillin (AMP) using a sterile L-shaped spreader and incubated overnight at 37℃ with inverted incubator. A single colony was picked and inoculated into 5 mL of LB liquid medium containing AMP, and incubated at 37℃ with shaking at 180 rpm for 5 h. 1 mL of the bacterial culture was then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0032] Take 50 μL of correctly sequenced V9E1-His bacterial culture and inoculate it into two 5 mL LB agar plates containing AMP. Incubate at 37°C with shaking until... A 600To reach a concentration of 0.6, IPTG was added to each tube to achieve final concentrations of 0.1 mM, 0.4 mM, and 1 mM, respectively, and the cells were induced overnight at 16°C. After induction, the cells were centrifuged at 8,000 × g for 5 min, the supernatant was discarded, and the cells were resuspended in 2 mL of PBS. The cells were then sonicated for 5 min at 75% power and a pulse mode of 3 s / 2 s intervals on ice until the bacterial culture was clear. The cells were then centrifuged at 10,000 × g for 10 min to separate the supernatant from the precipitate, and the precipitate was resuspended in 1 mL of PBS. 24 μL of each supernatant and precipitate sample was mixed with 8 μL of 4× non-denaturing loading buffer, boiled at 95°C for 5 min, loaded onto a 4–20% gradient precast gel, and subjected to SDS-PAGE analysis at 160 V for 45 min.

[0033] Experimental results: such as Figure 1 As shown, the theoretical molecular weight of V9E1 is approximately 15 kDa. Under the conditions of 16℃ and 1mM IPTG induction, the target protein was significantly expressed in the supernatant in a secretory form, with the best expression effect. The protein was then purified under these conditions.

[0034] Example 3

[0035] Purification of V9E1 protein

[0036] According to V9E1 Ni 2+ The 6×His tag at the end of the protein can specifically bind to nickel ions, allowing for the purification of V9E1 protein using affinity chromatography. When the elution buffer for the target protein reaches 20%, A 280 A significant increase in UV absorption peak was observed. The eluent was collected until the UV absorption peak completely returned to near the baseline. The collected eluent was then identified by SDS-PAGE.

[0037] Experimental results: such as Figure 2 As shown. Contaminating proteins were removed by ultrafiltration, and the buffer system was replaced with PBS. Protein concentration was determined by the BCA method.

[0038] Example 4

[0039] Extraction of exosomes

[0040] Take fresh skim milk from Sanyuan brand, slowly add 1N hydrochloric acid at 4℃ and stir until a milky white precipitate appears. Adjust the pH to 4.6 and let stand. Centrifuge at 8,000 r / min for 15 min at 4℃, discard the precipitate and keep the supernatant. Filter the supernatant through a 0.22 μm filter to remove impurities and store at 4℃ for later use. Aliquot the whey into ultracentrifuge tubes, balance the weight (error ≤0.01 g), and place them symmetrically on the rotor. Centrifuge at 120,000×g for 75 min, discard the supernatant, and rinse the tube walls and bottom of the tube with pre-cooled PBS three times. Resuspend the precipitate in 1 mL PBS and freeze at -80℃.

[0041] Example 5

[0042] Characterization of exosomes

[0043] The purified exosome samples were taken and sent to Beijing Zhongke Baice Technology Service Co., Ltd. for transmission electron microscopy analysis.

[0044] Experimental results: such as Figure 3 As shown, the obtained nanoparticles exhibit a typical saucer-like or concave hemispherical structure, consistent with the morphological characteristics of exosomes.

[0045] The Beijing Zhongke Baice Technology Service Co., Ltd. was commissioned to conduct laser particle size analysis: after diluting the exosome samples with ultrapure water, their particle size distribution and zeta potential were determined.

[0046] Experimental results: such as Figure 4 As shown, the average particle size of the exosomes was 138.8 nm, and the zeta potential was approximately -16.8 mV, both consistent with the typical biological characteristics of exosomes.

[0047] Total exosome protein was extracted and tested using Simple Western blotting technology by Beijing Liebaike Technology Co., Ltd., and exosome marker protein antibodies (CD9, TSG101, CD63) were selected.

[0048] Experimental results: such as Figure 5 As shown, positive expression of CD9, TSG101, and CD63 can be detected in milk-derived exosomes (mExo).

[0049] Protein concentration determination: The BCA method was used. Exosomes were lysed on ice for 30 min with RIPA lysis buffer. A standard curve was plotted using standard proteins, and the concentration was detected. A 562 The protein concentration was calculated using the value. The mExo protein concentration was 1.78 mg / mL.

[0050] Example 6

[0051] Exosome drug delivery

[0052] Take 500 μg of purified milk exosomes and mix them with 500 μg of anti-RTA single-domain antibody V9E1 protein solution. Add saponins to a final concentration of 1 mg / mL and control the total solution volume to 500 μL. Incubate the mixture in a shaker at 37℃ for 20 min. Then transfer it to a 100 kDa ultrafiltration tube, centrifuge at 5,000 r / min for 5 min, discard the filtrate, and wash three times with PBS to remove uncoated V9E1 protein and residual saponins. Finally, collect the liquid in the ultrafiltration tube and dilute it to 200 μL to obtain Exo-V9E1.

[0053] Example 7

[0054] Safety assessment

[0055] The safety of exosome-based drug delivery systems is a core prerequisite for their therapeutic application. The in vitro safety of Exo-V9E1 prepared in Example 6 was evaluated using in vitro cytotoxicity experiments. Well-grown Vero cells were collected, the culture medium was discarded in a biosafety cabinet, 5 mL of pre-chilled PBS solution was added, and the mixture was gently mixed. The supernatant was discarded, and this step was repeated twice. 5 mL of trypsin was added, and the mixture was incubated at 37°C in a CO2 incubator for 5 min. Digestion was terminated by adding 3 mL of DMEM medium containing 10% fetal bovine serum. The digested adherent cells were gently aspirated using a 3 mL Pasteur pipette, and the cell suspension was transferred to a 15 mL centrifuge tube. The cells were centrifuged at 4°C, 1000×g for 5 min, and the supernatant was discarded. The cells were resuspended in 2 mL of DMEM medium. 10 μL of the cell suspension was gently mixed with 10 μL of 2% trypan blue and added to a cell counting chamber for cell counting. The Vero cell suspension was diluted to 1×10⁻⁶. 5 Cells / mL. Add 100 μL of cell suspension to each well of a 96-well plate and incubate at 37°C in a CO2 incubator for 24 h. Using the total protein content of coated V9E1 as a standard, dilute Exo-V9E1, Exo, and V9E1 to 2 mg / mL using DMEM medium, and then perform multiple down-dilutes of this concentration. Remove the 96-well plate, discard the culture medium in the wells, add 100 μL of pre-chilled PBS solution to each well, mix gently, and discard the supernatant. Repeat twice. Add 100 μL of each diluted sample solution, with 6 replicates for each gradient; add 100 μL of DMEM medium as a negative control, with 6 replicates; and use wells containing a completely lethal dose of ricin as a positive control. Incubate at 37°C in a CO2 incubator for 24 h. DMEM medium and MTS chromogenic solution were mixed at a ratio of 100:20. 120 μL of the chromogenic solution was added to each well, and the mixture was incubated at 37°C in a CO2 incubator for 3 hours. The absorbance of each well was then measured. A 492 Calculate cell survival rate (Survival Percent = (Experimental Group) / (Experimental Group)). A 492 - Positive control group A 492 (Negative control group) A 492 - Positive control group A 492 (×100%)

[0056] Experimental results: such as Figure 6As shown, at an Exo protein concentration as high as 2 mg / mL, the survival rate of Vero cells was close to 100%, indicating that Exo-V9E1 does not harm Vero cells and has good cell safety.

[0057] The in vivo safety of Exo-V9E1 was assessed by detecting changes in mouse body weight and blood physiological parameters. Six- to eight-week-old SPF-grade female BALB / c mice were selected and divided into an Exo-V9E1 group and a PBS control group, with four mice in each group. Mice in the Exo-V9E1 group were injected with 100 μL of Exo-V9E1 solution via the tail vein, while the PBS control group received an equal volume of PBS solution. Following injection, the mice's condition and body weight were monitored daily. Blood samples were collected 24 hours later via ocular sampling to detect blood parameters (total protein, albumin, total bilirubin, urea, and gamma-glutamyl transferase, etc.).

[0058] Experimental results: such as Figure 7 As shown in the figure, the body weight of mice in the Exo-V9E1 group showed a steady increasing trend, with no significant difference from the PBS group; the results of blood total protein, albumin and other indicators are as follows. Figure 8 As shown, all values ​​were within the normal range, with no abnormal fluctuations. The mice did not exhibit symptoms such as ruffled fur or weight loss, indicating that Exo-V9E1 has good in vivo safety.

[0059] Example 8

[0060] Construction of an acute cell poisoning model

[0061] The acute cellular poisoning model was constructed to simulate the rapid uptake of toxins by cells and to evaluate the neutralizing effect of therapeutic agents on intracellular toxins. Using Vero cells as the model cells, well-grown Vero cell culture flasks were first prepared and the following procedures were performed in a biosafety cabinet: the culture medium was discarded, 5 mL of pre-chilled PBS solution (pH 7.4) was added, the cells were gently shaken, and the supernatant was slowly poured off. This was repeated twice to remove residual culture medium. 5 mL of trypsin digestion solution was added, and the cells were incubated at 37°C with 5% CO2 for 5 min. When the cells were observed to become rounded and detach under a microscope, 3 mL of DMEM medium containing 10% FBS was immediately added to terminate the digestion. The cells were repeatedly pipetted using a Pasteur pipette to form a homogeneous suspension. The cell suspension was transferred to 15 mL centrifuge tubes and centrifuged at 4°C, 1000×g for 5 min. The supernatant was discarded, and the cells were resuspended in 2 mL of DMEM medium. 10 μL of the cell suspension was mixed with 10 μL of 2% trypan blue and added to a cell counting chamber for counting. The cell concentration was adjusted to 1×10⁶ cells / mL. 5 cells / mL. Add 100 μL of cell suspension (approximately 5 × 10⁶ cells / mL) to each well of a 96-well plate. 4Cells / well were incubated at 37 ℃ in a 5% CO2 incubator for 24 h until the cell density reached 70-80%. Then, RT was serially diluted with DMEM medium (containing 10% FBS) to remove the original medium from the 96-well plate. 100 μL of different concentrations of RT solution was added to each well, and the plate was incubated for 2 h. After incubation, the supernatant containing toxins was discarded, and the cells were gently washed three times with pre-cooled PBS to remove unabsorbed toxins. After 24 h, DMEM medium and MTS chromogenic solution were mixed at a ratio of 100:20, and 120 μL of the chromogenic solution was added to each well. The plate was incubated at 37 ℃ in a CO2 incubator for 3 h. The absorbance of each well was measured. A 492 Calculate cell survival rate (Survival Percent = (Experimental Group) / (Experimental Group)). A 492 - Positive control group A 492 (Negative control group) A 492 - Positive control group A 492 (×100%)

[0062] Experimental results: such as Figure 9 As shown.

[0063] Example 9

[0064] In vitro therapeutic experiments

[0065] To evaluate the therapeutic effect of Exo-V9E1 prepared in Example 6 on acute ricin poisoning cells, an in vitro treatment experiment was conducted based on the constructed Vero cell acute poisoning model.

[0066] After Vero cells in 96-well plates were exposed to ricin for 2 hours and free toxin was eluted, therapeutic intervention was initiated. Exo-V9E1 was serially diluted with DMEM medium containing 10% FBS according to the coating concentration of V9E1 (0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, etc.). Free V9E1, conventional antibody V9E1, and toxin control groups (medium only) were established, with 6 replicates per group. 100 μL of the corresponding treatment solution was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. After incubation, the liquid in the wells was discarded, and 120 μL of a 100:20 mixture of DMEM medium and MTS chromogenic solution was added to each well, and the plates were incubated for another 3 hours. The absorbance of each well was measured using a microplate reader. A 492 Calculate cell viability using the formula (cell viability = (experimental group)). A492 - Positive control group A 492 (Negative control group) A 492 - Positive control group A 492 (×100%)

[0067] Experimental results: such as Figure 10 As shown, when the concentration of V9E1 in Exo-V9E1 is ≥0.1 mg / mL, it has a significant therapeutic effect on acutely poisoned cells with RT concentration <1800 ng / mL, and the cell survival rate is significantly higher than that of the free V9E1 group and the V5E1 group. However, there is no statistically significant difference in cell survival rate between the free V9E1 group and the toxin group, indicating that V9E1 needs to be delivered into the cell via exosomes to neutralize RTA, and that Exo-V9E1 has a stronger ability to clear toxins that have been taken up by the cell.

[0068] Example 10

[0069] Establishment of a mouse model of intraperitoneal poisoning

[0070] The construction of the intraperitoneal poisoning model aims to simulate the poisoning process of ricin (RT) after it enters the body via the intraperitoneal route, providing a stable model for evaluating the in vivo efficacy of therapeutic agents.

[0071] Six- to eight-week-old SPF-grade female BALB / c mice, weighing 18–22 g, were selected and acclimatized for 3 days before the experiment. RT was diluted with sterile PBS to different concentrations (6, 7, 10, 20, 40 μg / mL), aliquoted, and stored at 4 °C for later use. During the experiment, the left thumb and forefinger held the skin on the back of the mouse's neck, while the ring and little fingers stabilized the tail, ensuring full exposure of the mouse's abdomen. The right hand held a 1 mL syringe (31G needle) to draw the corresponding concentration of RT solution, expelled air bubbles, and inserted the needle into the mouse's abdominal cavity at a 45° angle (to a depth of approximately 1–2 cm). The needle core was slowly advanced to complete the injection. The injection volume per mouse was adjusted according to body weight (usually 0.1–0.2 mL). After injection, the needle was slowly withdrawn, and the injection site was gently pressed with a dry cotton ball for a moment. The mouse was then returned to its cage and observed for 15 minutes to confirm no leakage or abnormal reaction. Mice were housed separately at different RT concentrations, and their mental state, activity level, and time of death were observed daily for 14 consecutive days. The median lethal dose (LD50) of intraperitoneal RT was calculated using a probability unit method, with mortality as the indicator. 50 Ultimately, 10 μg / kg and 20 μg / kg were determined as completely lethal concentrations for subsequent treatment trials.

[0072] Experimental results: such as Figure 11As shown, after intraperitoneal injection of RT in mice, the mortality time shortened with increasing concentration. All mice in the high-concentration group (40 μg / kg) died within 24-48 h after injection, while some mice in the low-concentration group (6 μg / kg) survived for more than 7 days. The model showed good stability and can be used to evaluate the in vivo therapeutic effect of Exo-V9E1.

[0073] Example 11

[0074] Therapeutic effects in mice

[0075] To evaluate the in vivo therapeutic effect of Exo-V9E1 prepared in Example 6 on mice with intraperitoneal ricin (RT) poisoning, an experiment was conducted based on a previously established mouse intraperitoneal poisoning model. Six- to eight-week-old SPF-grade female BALB / c mice were randomly divided into different treatment groups, with four mice in each group. An acute poisoning model was established by intraperitoneal injection of a completely lethal dose of RT (10 μg / kg or 20 μg / kg, diluted with PBS).

[0076] Treatment interventions are divided into two methods: single injection and multiple injections. Single injection: At 2 h, 6 h, 12 h and 24 h after poisoning, mice were injected with different concentrations of Exo-V9E1 (V9E1 concentration of 0.1 mg / mL) via the tail vein, and a control group was set up at the same time.

[0077] Multiple injections: Exo-V9E1 (V9E1 concentration of 0.1 mg / mL) was first injected via the tail vein at 2 h and 6 h after poisoning in mice, followed by daily supplemental injections. A corresponding control group was also set up. The survival status, activity level, and time of death of the mice were observed daily after injection for 14 consecutive days, and survival curves were plotted.

[0078] Results of a single injection Figure 12 As shown, the survival rate of mice injected with Exo-V9E1 2 h after poisoning was significantly higher than that of the V5E1 group and the PBS group, with Exo-V9E1 at a concentration of 0.1 mg / mL showing better therapeutic effect; even after injection of Exo-V9E1 at 12 h and 24 h after poisoning, some mice still survived.

[0079] Results of multiple injections Figure 13 As shown, in the groups that received initial injections of Exo-V9E1 at 2 h and 6 h post-poisoning, followed by daily supplemental injections, all mice injected with 10 μg / kg RT survived, and the survival rate in the 20 μg / kg RT group was also significantly improved. This indicates that continuous administration can accumulate therapeutic effects and completely block the lethal effect of RT within a certain toxin concentration. This result confirms that Exo-V9E1 has excellent therapeutic effects on RT poisoning in vivo and is significantly superior to traditional antibody therapy.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antibody-exosome, characterized in that, The invention includes milk exosomes and nanobody V9E1, the amino acid sequence of which is shown in SEQ ID NO.1 and the nucleotide sequence encoding which is shown in SEQ ID NO.

2.

2. The method for preparing antibody-exosomes according to claim 1, characterized in that, Includes the following steps: 1) Milk exosomes were isolated and purified from fresh skim milk using ultracentrifugation; 2) The nucleotide sequence encoding V9E1 was constructed into the pET22b vector to obtain the pET22b-V9E1-His recombinant vector. The pET22b-V9E1-His recombinant vector was introduced into Escherichia coli BL21(DE3) competent cells and purified by nickel column affinity chromatography to obtain high-purity recombinant protein V9E1. 3) Mix milk exosomes and V9E1 protein solution, add saponins and incubate on a shaker, wash by ultrafiltration and centrifugation, and collect antibody-exosomes.

3. The preparation method according to claim 2, characterized in that, Step 1) The rotation speed of the ultracentrifugation method is 100,000~140,000×g, and the ultracentrifugation time is 70~80min.

4. The preparation method according to claim 2, characterized in that, In step 3), the mass ratio of milk exosomes to V9E1 protein solution is 1:1, and the final concentration of saponin is 1 mg / mL.

5. The preparation method according to claim 2, characterized in that, Step 3) The temperature of the shaker incubation is 35~40℃, and the incubation time is 10~30min; the conditions for ultrafiltration centrifugation washing are centrifugation at 4,000~6,000 r / min for 3~8min, repeated 2~4 times.

6. The use of the drug-loaded antibody exosomes according to claim 1 or the antibody-exosomes prepared by the preparation method according to any one of claims 2 to 6 in the preparation of ricin toxin antidote.

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