CN115093459A - Delivery compound capable of carrying out axon targeted modification on surface of exosome and modification method of exosome by delivery compound - Google Patents
Delivery compound capable of carrying out axon targeted modification on surface of exosome and modification method of exosome by delivery compound Download PDFInfo
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- CN115093459A CN115093459A CN202210582830.4A CN202210582830A CN115093459A CN 115093459 A CN115093459 A CN 115093459A CN 202210582830 A CN202210582830 A CN 202210582830A CN 115093459 A CN115093459 A CN 115093459A
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- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
- C08G65/32—Polymers modified by chemical after-treatment
- C08G65/329—Polymers modified by chemical after-treatment with organic compounds
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- C08G65/33331—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing imide group
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Abstract
The invention provides a delivery compound capable of carrying out axon targeting modification on the surface of an exosome and a modification method of the exosome, wherein the delivery compound is prepared from a TAxI polypeptide and DSPE-PEG 2000-NHS; the TAxI polypeptide consists of SEQ ID NO: 1 is added with a trilysine block at each end and forms a disulfide bond between two cysteines; the TAxI polypeptide is connected with Biotin at the C-terminal lysine, and the TAxI polypeptide is grafted with DSPE-PEG2000-NHS at the N-terminal lysine. The axon-targeting exosome is formed by surface modification of a stem cell exosome by the delivery complex; the delivery complex is prepared into DSPE-PEG2000-TAxI-Biotin micelle in advance. The axon-targeting exosome has good blood stability, stealth property and targeting property, and can effectively identify axon tissues of a central nervous system.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a delivery compound capable of carrying out axon targeting modification on the surface of an exosome, a preparation method thereof and a method for modifying the exosome by the delivery compound.
Background
Neurodegenerative diseases (NDD) are caused by loss of neuronal and nerve myelin, which worsen over time and cause dysfunction, including brain atrophy, alzheimer's Disease, parkinson's syndrome, multiple sclerosis and amyotrophic lateral sclerosis. As the aging population increases, the impact of the disease on the longevity and quality of life of patients also increases.
The Targeted axon introduction (TAxI) polypeptide has been proved by scientists through phage display technology to have good axon targeting in the central nervous system, but the stability in blood is extremely low, and the solubility in axon needs to be improved. DSPE-PEG2000 (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000) is a highly biocompatible, biodegradable amphiphilic material, can be used for the functionalization of various biological macromolecules (such as functional polypeptides and proteins) to realize specific functions, and is an activated lipid-phospholipid-polyethylene glycol conjugate widely used in drug delivery system applications.
The exosome has the nanometer particle size of about 150nm, has the capability of passing through a blood brain barrier and a good immunoregulation function, and a plurality of articles show that the stem cell exosome has better curative effect than the traditional medicine when being used for treating neurodegenerative system diseases. Exosomes are suitable for intravenous delivery, but the main reason for the barrier of clinical transformation process is that the bioavailability of exosomes is too low, and most exosomes are degraded and absorbed by organs such as liver and spleen due to permeability and retention (EPR) effect of vasculature after entering into the body.
In view of this, it is urgently needed to solve the targeting problem of exosomes, improve the bioavailability of exosomes, and make them applied to the treatment of nervous system diseases, which is especially important for solving clinical nervous system degenerative diseases.
Disclosure of Invention
Aiming at the defects of the prior art, the inventor optimizes the pro-sequence fragment of the TAxI polypeptide to ensure that the TAxI polypeptide has better solubility, blood stability and in-vivo tracing convenience, and the pro-sequence fragment is jointed together by substitution reaction of amino DSPE-PEG2000-NHS of lysine on the optimized sequence to obtain a delivery compound targeting axons, and then the delivery compound targeting axons is integrated on the surface of an exosome membrane by controlling temperature and ultrasonic frequency to ensure that the modified exosomes have an axon targeting function. The technical scheme used for realizing the purpose of the invention is as follows:
a delivery complex for axon-targeting modification at the surface of exosomes, the delivery complex made of a TAxI polypeptide and DSPE-PEG 2000-NHS; the TAxI polypeptide consists of SEQ ID NO: 1 is added with a trilysine block at each end and forms a disulfide bond between two cysteines; the TAxI polypeptide is connected with Biotin at the lysine of the C terminal, and the TAxI polypeptide is grafted with DSPE-PEG2000-NHS at the lysine of the N terminal.
The original sequence of the TAxI polypeptide fragment of the targeted axon is (SACQSQSQMRCGGG) (SEQ ID NO: 1), in order to ensure the solubility of the fragment, trilysine blocks are respectively added at two ends of the sequence, meanwhile, the amino group on the N-terminal lysine also takes the grafting point of the TAxI polypeptide as the amide reaction with NHS active ester of DSPE-PEG2000-NHS to complete grafting, and the C-terminal lysine is connected with Biotin (Biotin) for tracing; in addition, in order to ensure the blood stability of the polypeptide, a disulfide bond is formed between the sulfhydryl groups of two cysteines in the polypeptide sequence so that the polypeptide forms a cyclized structure. Finally, the desalted polypeptide solution was concentrated and purified by reverse phase high performance liquid chromatography (RP-HPLC) to obtain (KKKSACQSQSQMRCGGGKKK(Biotin) -OH disulfide bond 6-14) (SEQ ID NO: 2) polypeptide fragments, which were identified by High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS).
The dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol-succinimidyl ester (DSPE-PEG 2000-NHS) has the molecular formula of C139H271N4O57P, and the molecular weight of PEG is as follows: 2000, overall molecular weight: 2900. the structural formula is as follows:
DSPE-PEG2000-NHS is a linear heterobifunctional pegylation reagent comprising DSPE phospholipid and activated NHS carboxylate. The reagent is a self-assembly reagent, is used for preparing polyethylene glycol liposome or micelle, and simultaneously provides NHS group for conjugation with amine-containing molecules, can provide stealth, prolong circulation half-life and reduce the combination of modified exosome and nonspecific protein or cell adhesion. The amino group on the N-terminal lysine of the TAxI polypeptide fragment is used as a grafting point, an amido bond is generated through the covalent conjugation between the amino group on the lysine and an NHS group on DSPE-PEG2000-NHS, and finally, the DSPE-PEG2000-TAxI-biotin linear targeting axon delivery compound is synthesized and is used for membrane surface targeting modification of exosomes.
The invention also provides a method for modifying exosome by the axon targeted delivery compound, which is formed by modifying the surface of the stem cell exosome by using the delivery compound; the delivery compound is prepared into DSPE-PEG2000-TAxI-Biotin micelle in advance; the mass ratio of the DSPE-PEG2000-TAxI-Biotin micelle to the exosome is 1:1 (in microgram protein: microgram protein).
Preferably, the exosome is any one of human umbilical cord mesenchymal stem cell exosome, blood exosome, saliva exosome or urine exosome.
The DSPE-PEG2000-TAxI insertion repair process of the surface of an exosome membrane is carried out by carrying out co-incubation of the DSPE-PEG2000-TAxI-biotin and exosome under a series of temperature changes and ultrasonic oscillation conditions. In addition, membrane labeling of the exosome modified by the axon targeting delivery complex is carried out by a DiR dye, and DSPE-PEG2000-TAxI-Biotin can also be labeled by coupling a dye-labeled (such as HRP, FITC and the like) streptavidin protein chimera with Biotin in DSPE-PEG2000-TAxI-Biotin to form a biological reaction amplification system.
Preferably, the specific preparation steps are as follows:
(1) synthesizing a TAxI polypeptide;
(2) joining AxI polypeptide and DSPE-PEG2000-NHS to obtain delivery composite DSPE-PEG 2000-TAxI-Biotin;
(3) preparing the delivery compound synthesized in the step (2) into a micelle, and inserting the micelle into an exosome lipid bilayer membrane.
Preferably, the step of synthesizing the tax polypeptide is converting SEQ ID NO: the carboxyl of the C-terminal amino acid of the sequence 1 is connected with insoluble polymer resin in a covalent bond form, then the amino of the amino acid is taken as the starting point of polypeptide synthesis, the amino reacts with activated carboxyl of other amino acids to generate peptide bond, the process is continuously repeated to obtain polypeptide, then disulfide bond is formed between sulfhydryl groups of two cysteine of polypeptide fragments for cyclization modification, and finally, the TAxI polypeptide is obtained by purifying with high performance liquid chromatography.
Preferably, in step (2), the mass ratio of DSPE-PEG2000-NHS to TAxI polypeptide is 2: 1.
preferably, in step (3), the delivery complex is pre-micelle-formed at 60 ℃ and then cooled to room temperature, and then inserted into the lipid bilayer membrane of exosome by co-incubation with exosome.
The invention has the beneficial effects that:
compared with a natural exosome, the exosome modified by the DSPE-PEG2000-TAxI-biotin has good blood stability, stealth property and targeting property, and can effectively identify axon tissues of a central nervous system to achieve a good treatment effect on neurodegenerative diseases.
Drawings
FIG. 1 is a high performance liquid chromatogram of the TAxI polypeptide of example 1 of the present invention, wherein the target polypeptide is collected at 8min of sample collection, and the purity is 96.06%.
FIG. 2 is a mass spectrum of TAxI polypeptide of example 1 of the present invention, which shows that the polypeptide has a peak value of [ M +2H ]2+ at a mass/charge ratio (M/z) 941.649, and the relative molecular mass is 2453.0.
Figure 3 is a nuclear magnetic resonance hydrogen spectrum of the delivery complex of example 2 of the invention.
Figure 4 is an infrared spectrum of a delivery complex of example 2 of the invention.
FIG. 5 is a diagram of the process of modifying the surface of exosome membranes by the axon-targeted delivery complex of example 3 of the present invention.
FIG. 6 is transmission electron microscopy results of HT7700 for exosomes modified by axon-targeted delivery complexes of example 3 of the invention, with scale of 200 nm.
FIG. 7 is ZetaView particle size analyzer results for the axon-targeted delivery complex modified exosomes of example 3 of the present invention, with the particle size of the modified exosomes increased by about 25nm compared to the native exosomes.
FIG. 8 shows the tracing result of the modified exosome of the axon-targeted delivery complex of example 4 in the small animal, and the administered group has good axon targeting property compared with the control group.
FIG. 9 is an observation of the modified exosomes of the axon-targeted delivery complex of example 5 of the present invention incubated with BV2 cells, and the modified exosome group had good cell binding and retention capacity compared to the control group.
Detailed Description
In order to make the present invention clearer and more complete, the following embodiments are further detailed descriptions of the present invention, and it is obvious that the described embodiments are only selected parts of the present invention, but not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive work or reagent modifications based on the embodiments of the present invention, are within the scope of protection of the present invention.
The technical solutions and the step sequences of the actual operations in the embodiments may be adjusted according to actual situations, but it is necessary to be based on the realization of those skilled in the art, and when the combination of the adjustment and the technical solution of the present invention contradicts each other or a detection result corresponding to the actual operation cannot be realized, the adjustment and the combination of the technical solution should be considered to be absent, that is, the present invention is not within the protection scope of the present invention.
It should be noted that the results shown in the figures of the present invention are only the results of the crude product obtained in a certain step under the given apparatus and parameter conditions, and if one of the apparatus and parameter conditions is changed, the result is also changed correspondingly.
EXAMPLE 1 Synthesis of TAxI polypeptide
In this example, the C-terminal amino acid carboxyl group of the desired target polypeptide (SACQSQSQMRCGGG) (SEQ ID NO: 1) was covalently bonded to an insoluble polymer resin, and then peptide bonds were formed between the amino group of this amino acid and the already activated carboxyl groups of other amino acids by using the amino group of this amino acid as the starting point of the polypeptide synthesis, and the sequence of the target polypeptide was (KKKSACQSQSQMRCGGGKKK(biotin) -OH disulfide bond 6-14) (SEQ ID NO: 2) by purification using HPLC by repeating this procedure.
In actual practice, according to the Fmoc chemical synthesis procedure,
firstly, swelling Resin, weighing 0.6g of 2-Chlorotrityl Chloride Resin (2-CTC Resin) with the substitution degree of 0.4mmol/g, putting the Resin into a reaction tube, adding 15ml (15ml/g) of dichloromethane, and oscillating for 30 min;
secondly, connecting the first amino acid, filtering off the solvent through sand core, adding 3 times molar excess of Fmoc-Lys (biotin) -OH amino acid, adding 10 times molar excess of N, N-Diisopropylethylamine (DIPEA), finally adding a small amount of DMF to dissolve, oscillating for 1h, and alternately cleaning for 6 times by using N, N-Dimethylformamide (DMF) and Dichloromethane (DCM);
thirdly, deprotection is carried out, 15ml of 20% piperidine DMF solution (15ml/g) is added for 5min, 15ml of 20% piperidine DMF solution (15ml/g) is removed, and 15min is carried out;
and fourthly, detecting, washing 6 times by using DMF, and taking a small amount of resin (about 10 resin) for color reaction. Taking out DMF solution, taking a small amount of resin, washing with ethanol for three times, performing qualitative color reaction, namely ninhydrin color development (Kaiser method), adding 6% ninhydrin ethanol solution, 2% 0.001M KCN pyridine solution and 80% phenol ethanol solution, heating for 5min at 105-110 ℃, and turning dark blue to obtain positive reaction;
replacing the solvent, twice with DMF (10ml/g), twice with methanol (10ml/g) and twice with DMF (10 ml/g);
sixthly, condensing, namely performing triple excess on protected amino acid (Fmoc-L-Gly-OH), performing triple excess on benzotriazole-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU), dissolving with a small amount of DMF, adding into a reaction tube, immediately adding N-methylmorpholine (NMM) for ten times of excess, reacting for 30min, and repeating the step five;
seventhly, repeating the steps from two to six, and sequentially connecting the amino acids in the sequence from right to left;
eighthly, deprotection is carried out, the resin is washed after the last amino acid is connected, DMF (10ml/g) is carried out twice, methanol (10ml/g) is carried out twice, DMF (10ml/g) is carried out twice, DCM (10ml/g) is carried out twice, and the resin is drained for 10 min;
ninthly, preparing a cutting fluid (10ml/g) by cutting the polypeptide from the resin: TFA 94.5%, water 2.5%, EDT 2.5%, and TIS 1%, charging the dried resin into a flask, and shaking the resin and the cutting fluid at a constant temperature for 2h according to a ratio of 10 ml/g;
filtering, precipitating and washing, drying the cutting fluid by using nitrogen as much as possible, uniformly mixing the cutting fluid with filtrate 6:1 by using ethyl acetate, washing for three times at 3000rpm/min for 10min, and performing vacuum pumping drying to obtain a crude polypeptide which can be stored at the temperature of-20 ℃;
eleventh, purifying the crude polypeptide by HPLC.
The synthesized polypeptide fragment forms a disulfide bond between thiol groups of two cysteines for cyclization modification.
In practical operation, the deprotected free cysteine sulfhydryl and the reduced cyclized TAxI-biotin polypeptide are dissolved in 20% dimethyl sulfoxide (DMSO) aqueous solution, and the two cysteine sulfur atoms in the polypeptide fragment are dehydrooxidized to form disulfide bond to cyclize the linear polypeptide, and the process is carried out at pH 8 for 3-4 hours.
Sufficient cyclized targeting polypeptide fragments were obtained and purified using high performance liquid chromatography, as shown in figure 1. Meanwhile, the molecular weight is identified by mass spectrometry, and the result is shown in figure 2.
Example 2 Synthesis of delivery complexes
In this example, the cyclized polypeptide fragment synthesized in example 1 was loaded with DSPE-PEG2000-NHS and the final axon-targeting delivery complex was synthesized.
In practice, DSPE-PEG2000-NHS dissolved in a suitable amount of water is added to the cyclized TAxI polypeptide dissolved in PBS (0.1M phosphate, pH 7.4) in an amount of 2: 1, pH 8-8.5, stirring overnight at room temperature. Then neutralizing with acid to pH7.0, dialyzing with distilled water, and concentrating and purifying the desalted polypeptide solution by reverse phase high performance liquid chromatography (RP-HPLC).
Then, for characterization detection, FITC and NH2-PEG2000-DSPE were dissolved in DMF for 2h, and FITC-PEG2000-DSPE was synthesized by covalent conjugation. No free amino group was present as determined by ninhydrin test, indicating that DSPE-PEG2000-NH2 is linked to FITC. The mixture was dialyzed against distilled water (MWCO 3500Da) to remove excess cyclized TAxI polypeptide and reagents, and the solution was lyophilized under vacuum and the functional material was characterized by 1HNMR (Varian 400MHz, Palo Alto, USA).
As shown in FIG. 3, in the nuclear magnetic hydrogen spectrum of DSPE-PEG2000-NHS, a peak appeared at 3.7ppm for NHS group, while in the hydrogen spectrum of DSPE-PEG2000-TAxI-Biotin, the peak disappeared completely, indicating that NHS group reacted with amino group on TAxI lysine. In addition, in the infrared spectrum of DSPE-PEG2000-TAxI-Biotin, strong absorption peak is generated in 1640-1820cm < -1 > region to prove the existence of carbonyl, and medium absorption peak is generated near 3500 cm < -1 > to prove the existence of amide bond, as shown in figure 4.
Example 3 synthetic axon-targeting delivery Complex-modified exosomes
As shown in the attached figure 5, the DSPE-PEG2000-TAxI-Biotin synthesized in the embodiment 2 and a natural exosome are incubated together under a series of conditions, and then the modification of the exosome by the axon targeted delivery compound can be completed, wherein the exosome is the human umbilical cord mesenchymal stem cell exosome.
In practical operation, DSPE-PEG2000-TAxI-Biotin is dissolved in 100 mu M HEPES buffer solution (pH 7.4) at 60 ℃ for 15min to form micelles, and then the solution is cooled to room temperature after completion. The exosomes and the DSPE-PEG2000-TAxI-Biotin micelles are mixed according to the ratio of 1:1 (mug protein: mug protein), the total volume is 100 and 150 mug, and the mixture is incubated for 2 hours at 40 ℃ in a PCR instrument. After the incubation was completed, the mixture was cooled to 4 ℃ and the modified exosomes were immediately purified from the free micelles by exclusion chromatography. And (3) observing the exosome modified by the targeting delivery compound under a transmission electron microscope and analyzing the particle size.
As shown in figure 6, the dark area of the exosome membrane surface modified by the targeted delivery complex is darkened by observation under an electron microscope, which proves that the targeted delivery complex is effectively modified on the exosome membrane surface. The analytical results in fig. 7 prove that the electron microscope results in fig. 6 prove that the exosome modified by the targeting complex has obviously increased particle size, and the successful modification is proved.
Example 4 animal experiments with axon-targeted delivery of Complex-modified exosomes
The exosome modified by the axon targeting delivery complex is subjected to surface labeling by using a DiR dye, and a C57BL/6 mouse is used as an in-vivo tracing experimental animal to perform a small animal in-vivo imaging experiment of the DiR dye labeled exosome.
In actual operation, 2 mu M DiR working solution is prepared, the target modification exosome is added into the working solution, the final concentration of the target modification exosome in the DiR working solution is 1x10^ 8-1 x10^9 particles/ml, water bath at 37 ℃ is carried out for 20min, a 100KDa ultrafiltration tube is used for replacing a solvent to be PBS buffer solution, and the final concentration is 10 mu g/mu l. C57BL/6 mice were selected as in vivo tracer experimental animals, and 100 μ g of DiR-stained targeted modified exosomes were intravenously administered to each group of mice and detected under 720nm excitation light of an IVIS Spectrum small animal in vivo imager, as shown in FIG. 8.
The results show that compared with the natural exosome, the exosome modified by the targeting delivery compound has better blood stability and can effectively reside in the brain for 48 hours.
Example 5 cell binding and Retention Capacity assays for exosomes modified by axon-Targeted delivery complexes
In this example, BV2 cells (a mouse glial cell line) were used as the subject for cell binding and retention of the targeted delivery complex. The exosomes modified by the axon targeting delivery complex were surface labeled with DiR dye, BV2 cells were labeled with CFSE dye, and finally observed under laser confocal microscope.
In practice, the DiR working solution labeling of exosomes was performed as described above, 5 μ M CFSE working solution was prepared, resuspended BV2 cells were added to the working solution to a final concentration of 1x10^ 6/ml, water bath was 30min at 37 ℃, 5 volumes of cold medium was added to stop staining and incubate for 5min, cells were pelleted by centrifugation at 1000rpm/min ^ 5min and washed three times with medium, and finally cells were collected and re-seeded into dishes to incubate for 24h with DiR stained exosomes modified with targeting delivery complex at a concentration of 1x10^8 particles/ml, and observed with 488nm excitation light and 750nm excitation light under confocal laser microscopy, as shown in FIG. 9.
The results show that exosomes modified with targeted delivery complexes have better binding and retention capacity to BV2 cells than native exosomes.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Sequence listing
<110> Guangxi university
<120> a delivery complex capable of carrying out axon targeting modification on exosome surface and its modification method for exosome
<141> 2022-05-26
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Claims (10)
1. A delivery complex for axon-targeted modification of an exosome surface, the delivery complex
Is prepared from TAxI polypeptide and DSPE-PEG 2000-NHS;
the TAxI polypeptide consists of SEQ ID NO: 1 is prepared by adding a trilysine block at each end of the sequence shown in 1;
the TAxI polypeptide is connected with Biotin at the C-terminal lysine, and the TAxI polypeptide is grafted with DSPE-PEG2000-NHS at the N-terminal lysine.
2. The delivery complex for axonal targeting modification at an exosome surface according to claim 1, wherein the sequence of the TAxI polypeptide is as set forth in SEQ ID NO: 2, respectively.
3. The delivery complex for axon-targeting modification at an exosome surface according to claim 1 or 2, wherein disulfide bond formation between two cysteines of the TAxI polypeptide cyclizes the polypeptide.
4. The delivery complex for axon-targeting modification at an exosome surface according to claim 1, wherein the DSPE-PEG2000-NHS has the structural formula:
5. the delivery complex for axon-targeting modification of an exosome surface according to claim 4, wherein the DSPE-PEG2000-NHS has a molecular formula of C139H271N4O57P and a molecular weight of 2900.
6. A method for modifying exosomes by an axon-targeted delivery complex, which is characterized in that a stem cell exosome is subjected to surface modification by the delivery complex of claim 1; the delivery compound is prepared into DSPE-PEG2000-TAxI-Biotin micelle in advance;
the mass ratio of the DSPE-PEG2000-TAxI-Biotin micelle to the exosome is 1:1 (in microgram protein: microgram protein).
7. The method for modifying an exosome according to claim 6, wherein the exosome is any one of a human umbilical cord mesenchymal stem cell exosome, a blood exosome, a saliva exosome or a urine exosome.
8. The method for modifying exosomes by axon-targeted delivery complex according to claim 6, which is characterized by comprising the following specific preparation steps:
(1) synthesizing a TAxI polypeptide;
(2) coupling AxI polypeptide with DSPE-PEG2000-NHS to obtain a delivery complex;
(3) preparing the delivery complex synthesized in the step (2) into a micelle, and inserting the micelle into an exosome lipid bilayer membrane.
9. The method for modifying exosomes according to claim 8, wherein the step of synthesizing a TAxI polypeptide is converting SEQ ID NO: the carboxyl of the C-terminal amino acid of the sequence 1 is connected with insoluble polymer resin in a covalent bond form, then the amino of the amino acid is taken as the starting point of polypeptide synthesis, the amino reacts with activated carboxyl of other amino acids to generate peptide bond, the process is continuously repeated to obtain polypeptide, then disulfide bond is formed between sulfhydryl groups of two cysteine of polypeptide fragments for cyclization modification, and finally, the TAxI polypeptide is obtained by purifying with high performance liquid chromatography.
10. The method for modifying exosomes according to claim 8, wherein in step (2), the mass ratio of DSPE-PEG2000-NHS to TAxI polypeptide is 2: 1.
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