CN115364243A - Construction and application of visual regulation probe for T cell immunocompetence - Google Patents
Construction and application of visual regulation probe for T cell immunocompetence Download PDFInfo
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- CN115364243A CN115364243A CN202211043903.9A CN202211043903A CN115364243A CN 115364243 A CN115364243 A CN 115364243A CN 202211043903 A CN202211043903 A CN 202211043903A CN 115364243 A CN115364243 A CN 115364243A
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
T cell immune activityThe visual regulation probe comprises an active oxygen scavenging group R 1 T cell targeting antibody fragment R 2 And fusogenic liposome units, wherein the fusogenic liposome unit is comprised of DOPE, DOPE-PEG 2000‑ MAL and positively charged lipid DOTAP; the visual regulation probe utilizes amphipathic molecules to form a spherical liposome vesicle structure through self-assembly, and R is 1 、R 2 Respectively with DOPE-PEG 2000‑ The hydrophilic end of the MAL is covalently coupled. The preparation is carried out by a hydration film method, the dispersibility is good, the target modification range is wide, and the regulation and control performance on the T cell immunocompetence is excellent. The reactive oxygen scavenging groups are displayed on the surface of the T cell membrane and act as reactive oxygen decoys to resist inactivation of T cells and simultaneously provide protons T for MRI quantification of T cell immune activity activation 1 The obvious change of the relaxation time is used for the visual regulation and the early curative effect evaluation of diseases related to the T cell immune activity.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to construction and application of a visual regulation probe for T cell immunocompetence.
Background
T cells in a healthy body have stable metabolic function and differentiation capacity, and further maintain the normal immune defense function of the body. When the metabolic function and the number of T cells in the body are abnormally changed, a series of immune metabolic disorders can appear in the body, and further pathological changes of the body are triggered. Clinical studies have shown that the occurrence and development of tumors, acute/chronic infections, metabolic syndrome, immunodeficiency diseases, etc. are all associated with abnormal changes in T cell immune activity. Therefore, studies based on T cell immune activity are of great importance to understand the pathogenesis of the disease, control the development of the disease, and guide clinical treatment. However, there is a multifactorial depletion of cells, which in turn leads to the failure of the number of T cells in the pathological microenvironment to accurately reflect their immune activity. Therefore, the visual regulation and control of the immune activity of the T cells have important significance on the precise regulation and control of curative effect and prognostic evaluation of diseases related to abnormal immune activity of the T cells.
Clinical pathological tissue biopsy is a gold standard for monitoring cell activity, but is limited by factors such as the material drawing position, and the like, so that false positive or false negative results are easily caused, and great interference is caused to the correct judgment of a clinician. Compared with the traditional invasive tissue biopsy method, the molecular imaging technology can provide anatomical structure and functional imaging information with rapidness, accuracy, time resolution and space resolution, and provides a more effective tool for drug research and development, treatment scheme optimization and the like. At present, living body imaging aiming at T cells is mainly based on a biological binding targeting strategy of a cell-specific marker, the result mainly reflects the number and receptor distribution of the T cells, and the immune activity of the T cells cannot be accurately revealed.
Several studies have shown that: the proliferation, activation and maintenance of homeostasis of different types of T cells is closely related to the microenvironment they are in. The level of reactive oxygen species in the environment also plays a critical role in the function and activity of T cells. An imbalance in reactive oxygen species production and clearance can cause severe T cell damage and potential cell death. For example: in a tumor microenvironment, a large amount of inflammatory reactive oxygen species can cause the rise of oxidative stress level in the tumor, so that T cells are reduced or lost, and immune tolerance is expressed; in autoimmune diseases such as rheumatoid arthritis, high doses of inflammatory reactive oxygen species can lead to reduced or lost regulatory T cell (Treg) activity, which can exacerbate the disease process by immune imbalance. Further studies have shown that the immunological activity of T cells is directly proportional to the number of free thiol groups (-SH, reduced state) of the membrane surface reducing proteins (e.g., trx) and inversely proportional to the number of disulfide bonds (S-S, oxidized state). In the active oxygen microenvironment, the reductive proteins on the surface of T cell membrane are easily oxidized (-SH to S-S) to lose their proliferation ability and immunological activity. Based on the mutual relation between the redox state (-balance of SH and S-S) of the surface of the T cell membrane and the immunological activity of the T cell in an active oxygen environment, the balance state of the-SH and S-S on the surface of the target T cell membrane is expected to realize the diagnosis and treatment regulation of the immunological activity of the T cell and provide a new idea for the treatment effect regulation and prognosis evaluation of tumor radiotherapy. However, there are challenges in the targeted regulation based on the redox state of the T cell membrane surface, and there are few reports on the visual regulation of T cell immune activity and the research on its diagnostic and therapeutic applications.
Nitroxide-based active oxygen scavengers are a class of reagents widely used for the detection and quantification of active oxygen species, including 2, 6-Tetramethylpiperidine (TEMP), 5-dimethyl-1-pyrroline-N-oxide (DMPO), bocMPO, and the like. The magnetic capture agent can not only realize the anti-oxidation effect, but also oxidize molecules of the diamagnetic capture agent to form paramagnetic stable free radicals in the active oxygen capture process, thereby realizing the amplification of signals in magnetic resonance imaging. Unlike the molecular marker imaging method of radionuclides (Always ON), magnetic resonance imaging in this mode can achieve imaging in response to activation (switching), signal amplification, and multiple modes, with particular advantages in contrast imaging of small molecules with biochemical activity. Therefore, the introduction of the nitro-oxygen active oxygen capture group is expected to realize the diagnosis and treatment regulation of T cell immune activity in an oxidation microenvironment. However, how to display the active oxygen scavenger on the cell is also a big difficulty.
Disclosure of Invention
The invention aims to solve the problems in the prior art, provides the construction and application of a visual regulation probe of T cell immunocompetence, and particularly provides multifunctional fused liposome T-Fulips which consists of a nitro-oxygen active oxygen trapping group with active oxygen scavenging capacity and modified by a T cell targeting antibody, is fixed in a liposome, has the functions of targeting fused T cells and helping to resist oxidative inactivation, has the magnetic resonance activation imaging capacity, and can be used for visual regulation of T cell immunocompetence-related diseases (such as tumors, rheumatoid arthritis and the like) and early evaluation of treatment efficacy.
In order to achieve the purpose, the invention adopts the following technical scheme:
a probe for visual regulation of T cell immune activity, comprising a reactive oxygen species scavenging group R 1 T cell targeting antibody fragment R 2 And a fusogenic liposome unit, wherein the fusogenic liposome unit is composed of DOPE (dioleoylphosphatidylethanolamine), DOPE-PEG 2000- MAL (dioleoylphosphatidylethanolamine-polyethylene glycol-maleimide) and a positively charged lipid DOTAP (trimethyl-2, 3-dioleoyloxypropylammonium chloride); the visual regulation and control probe utilizes amphipathic molecules to form a spherical liposome vesicle structure through self-assembly, and R is 1 、R 2 Respectively with DOPE-PEG 2000- The hydrophilic end of the MAL is covalently coupled.
The active oxygen scavenging group R 1 Nitro-oxygen active oxygen trapping agent is adopted.
The active oxygen scavenging group R 1 Comprises at least one of 2, 6-Tetramethylpiperidine (TEMP), 5-dimethyl-1-pyrroline-N-oxide (DMPO) and BocMPO.
The T cell targeting antibody fragment R 2 Comprises at least one of anti-CD3, anti-CD4, anti-CD8, anti-CD25 and anti-TIM-3.
The preparation method of the visual regulation probe for the T cell immunocompetence comprises the following steps:
1) Synthesis of active oxygen scavenging Unit Structure DOPE-PEG 2000 -R 1 ；
2) Mixing DOPE, DOPE-PEG 2000 -MAL、DOPE-PEG 2000 -R 1 Uniformly mixing the positive charge lipid DOTAP in a solvent, evaporating the solvent to dryness, adding phosphate buffer solution PBS for dispersion, then vortexing to generate a multi-layer liposome vesicle, and then ultrasonically homogenizing to obtain a unilamellar fusion liposome vesicle;
3) Targeting of antibody fragment R by ligation of T cells 2 Preparing a visual regulation probe for the immunological activity of the T cells.
The preparation of step 1) of the invention is as follows: adding DOPE-PEG 2000 MAL and thiolated R 1 Mixing in phosphate buffer solution PBS, reacting under nitrogen protection, removing unreacted raw material compound by dialysis bag, lyophilizing dialyzed solution to obtain solid product DOPE-PEG 2000 -R 1 。
The invention, step 2) involves the components DOPE, DOPE-PEG 2000 -MAL、DOPE-PEG 2000 -R 1 DOTAP in molar ratio DOPE: DOPE-PEG 2000 -MAL：DOPE-PEG 2000 -R 1 : DOTAP =1: (0.1-0.5): (0.1-0.5): 1, the liposome prepared by the material with the proportioning has high target cell fusion efficiency and high yield, and is an ideal carrier for displaying active oxygen capture on cell membranes.
The preparation of step 3) of the invention is as follows: reduction of T cell targeting antibody fragment R with Dithiothreitol (DTT) 2 Then using desalting column to remove free DTT, and finally using the stepChemically linking the unilamellar fusogenic liposome vesicles obtained in step 2).
The chemical connection is carried out under the protection of nitrogen, and then the reaction is carried out spin-washing and purification in phosphate buffer solution PBS solution, thus obtaining the visual regulation and control probe for the T cell immunocompetence, wherein the particle size is 10-500 nm.
The application of the visual regulation probe of the T cell immunocompetence is used for the visual regulation of the diseases related to the T cell immunocompetence and the early evaluation of the treatment effect.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
1. the probe is a liposome nanoparticle, has the functions of target fusion of T cells and assistance in resisting oxidative inactivation, and has the magnetic resonance activation imaging capability;
2. the probe can be prepared by a hydration film method, the steps are simple and convenient to operate, the obtained nanoparticles have uniform particle size, good dispersibility, wide target modification range and excellent regulation and control performance on T cell immunocompetence;
3. the lipid proportion is optimized in the invention, so that the high-efficiency membrane display of the functional group TEMP is realized, and the bioavailability is improved;
4. in the present invention, the active oxygen scavenging group R is used 1 The active oxygen scavenging property and the imaging characteristic of the compound realize the diagnosis and treatment regulation of the immune activity of T cells, R 1 Including but not limited to nitro-oxygen active oxygen scavengers such as TEMP, DMPO and BocMPO;
5. preferred T cell targeting antibody fragments R of the invention 2 Solves the problem of poor specificity of non-target nano-drugs, reduces the off-target effect of the fusogenic liposome complex by utilizing the target function, greatly improves the synergistic effect and the imaging effect of the fusogenic liposome complex and the antioxidant functional group, and R 2 T cell targeting antibodies or antibody fragments including, but not limited to, anti-CD3, anti-CD4, anti-CD8, anti-CD25, and anti-TIM-3;
6. preferred T cell targeted R-containing of the invention 1 The antioxidant fusogenic liposome complex can be used for treating diseases (such as tumor and tumor-like) caused by abnormal T cell immunocompetenceRheumatic arthritis, etc.) has great advantages compared with the clinical method that the curative effect needs to be known for a plurality of weeks after the treatment.
Drawings
FIG. 1 is a transmission electron micrograph and particle size distribution of T-Fulips;
FIG. 2 is a targeted fusion assay of T-Fulips;
FIG. 3 shows the proliferation and activation of T cells in an oxidative environment promoted by T-Fulips;
FIG. 4 shows the MRI monitoring effect of T-Fulips injected intravenously after radiotherapy in a mouse breast cancer model;
FIG. 5 is a tumor growth curve following intravenous injection of T-Fulips following radiation therapy in a mouse breast cancer model;
FIG. 6 evaluation of the application of T-Fulips in tumor stratification.
Detailed Description
The invention will be further illustrated with reference to specific examples, in which TEMP is selected as R 1 anti-CD 3F (ab') 2 is R 2 The application in the radiotherapy model of mouse breast cancer is taken as an example to enable a person skilled in the art to better understand the present invention and to implement the present invention, but the present invention is not limited by the examples.
Example 1 Synthesis of T-Fulips
1. Active oxygen scavenging unit structure DOPE-PEG in T-Fulips 2000 The main process of the chemical synthesis route of TEMP is divided into two steps:
(1) The synthesis of sulfhydrylated TEMP, namely the synthesis of 3-sulfydryl-N- (2, 6-tetramethyl piperidine) -4-propionamide;
(2) Compound DOPE-PEG 2000 -synthesis of TEMP;
synthesis of the Compound 3-mercapto-N- (2, 6-tetramethylpiperidine) -4-propionamide: n-hydroxysuccinimide (12mmol, 1.38g) was stirred in anhydrous tetrahydrofuran (30 mL) for 30min, followed by addition of 3-mercaptopropionic acid (10mmol, 1.064g) to give a transparent solution. 1, 3-dicyclohexylcarbodiimide (12mmol, 2.488g) was dissolved in tetrahydrofuran (50 mL) and added dropwise to the reaction mixture, ice-cooled for 60min, and stirred at room temperature for 24h. Filtering the precipitate, and reducing pressureRemoving solvent by rotary evaporation to obtain yellow oily product, purifying with flash chromatography column 1 H-NMR characterization (solvent CDCl) 3 ) Obtaining the purified 3-mercaptopropionic acid-N-hydroxysuccinimide. 3-mercaptopropionic acid-N-hydroxysuccinimide (1mmol, 0.25g), N-hydroxysuccinimide (1mmol, 0.25g) and triethylamine (50. Mu.L) were mixed well in a dichloromethane (25 mL) solvent, and 4-amino-2, 6-tetramethylpiperidine (TEMP-NH) dissolved in anhydrous tetrahydrofuran (5 mL) was added dropwise thereto 2 1mmol, 0.156g), ice-cooled for 60min, and stirred at room temperature for 24h. The dichloromethane solution was extracted with distilled water (2X 100 mL) to collect the water layer, and lyophilized to obtain a yellow waxy solid, namely thiolated TEMP, 3-mercapto-N- (2, 6-tetramethylpiperidine) -4-propionamide. Product adoption 1 H-NMR characterization (solvent is MeOD).
The compound DOPE-PEG 2000 Synthesis of TEMP: adding DOPE-PEG 2000 -MAL (0.017mmol, 50mg) and 3-mercapto-N- (2, 6-tetramethylpiperidine) -4-propionamide (0.2mmol, 48.8mg) were mixed in phosphate buffered saline PBS (pH = 6.8), reacted for 12h at room temperature under nitrogen protection, and then dialyzed for 24h in distilled water using dialysis bag (MWCO 1000 Da) to remove unreacted starting compounds. Freeze-drying the dialyzed solution to obtain a yellow solid product, namely DOPE-PEG 2000 -TEMP。
2. DOPE and DOPE-PEG 2000 -MAL、DOPE-PEG 2000 the-TEMP and the positive charge lipid DOTAP are evenly mixed in a chloroform solvent, the chloroform solvent is evaporated by rotation under the vacuum condition, and a proper volume of PBS is added to disperse the concentration of 2mg/mL. The solution was vortexed for 2min to generate multilamellar liposome vesicles, followed by sonication for 20min at 4 ℃ to give unilamellar fusogenic liposome vesicles.
3.T cell targeted antioxidant fusogenic liposome T-Fulips samples were prepared by ligation of Anti-CD 3F (ab') 2 fragments: reducing the antibody fragment with 2mM Dithiothreitol (DTT) at 25 deg.C for 90min to obtain free sulfhydryl, removing free DTT with 7kDa desalting column, and chemically linking with unilamellar fusogenic liposome vesicle; reacting for 12h at 25 ℃ under the protection of nitrogen, and then performing rotary washing for 2 times in PBS solution at 70000g for 60min for purification to obtain the T cell targeted antioxidant fusogenic liposome T-Fulips.
T-Fulips were mixed in phosphate buffered saline PBS (pH = 6.8), the particle size distribution of the synthesized T-Fulips was measured at room temperature under nitrogen using a particle size analyzer, and the morphology of the T-Fulips was observed using a transmission electron microscope, as shown in FIG. 1. As can be seen from FIG. 1A, the obtained T-Fulips has uniform particle size, spherical shape and smooth surface; as can be seen from FIG. 1B, the particle size distribution of T-Fulips was 81.6. + -. 16.7nm.
Example 2T-Fulips Targeted fusion of T cells
Using DiR fluorescent dye-labeled liposomes and 30000 CD3 at 37 deg.C + T cells are incubated for 30min, then washed 3-5 times by PBS, and then the targeted fusion efficiency of the fusogenic liposome is evaluated by detecting the DiR fluorescence by a laser confocal microscope. The results are shown in FIG. 2. As can be seen from FIG. 2 (A), the DiR fluorescent species are uniformly distributed on the T cell membrane, suggesting that T-Fulips have good T cell targeting and fusion performance. The quantitative result is shown in FIG. 2 (B), and can reach 84.3%.
Example 3T-Fulips promote proliferation and activation of T cells in an oxidative environment
T cells were pretreated with T-Fulips followed by CFSE staining, which was performed on H 2 O 2 In the environment, the activation factor conA is given for incubation for 48h, and the proliferation activation is monitored by flow cytometry and ELISA. The results are shown in FIG. 3. The results in FIG. 3A show: even in the presence of conA, H 2 O 2 It also greatly attenuated T cell proliferation, in contrast to T cell CFSE in the T-Fulips-pretreated group low The percentage of components increases significantly. This result is also consistent with the secretion of IFN-. Gamma.from T cells treated with T-Fulips and the significant increase in the number of surface thiols (FIG. 3B, FIG. 3C). In conclusion, T-Fulips can block active oxygen-induced oxidation of-SH on the surface of T cells so as to maintain favorable reducing environment of the T cells, and can reverse the inactivation of the T cells caused by the oxidizing environment.
Example 4 in vivo MRI evaluation of T-Fulips modulation and quantification of T cell Activity
BALB/c mice were used to inoculate 1X 10 subcutaneous implants on the right thigh 6 4T1 breast cancer tumor cells. When tumor is presentThe volume of the water-saving filter reaches 30-40 mm 3 On the left and right, the groups were randomly divided into 6 groups (6 in each group). Mice of different groups were treated with PBS control, X-ray radiation + Iso-Fulips, and X-ray radiation + T-Fulips, respectively. Iso-Fulips is a T-Fulips isotype control group. By T 1 Relaxation time series and multilayer T 1 The weighted sequences were MRI pre-scanned separately for different groups of mice. By intravenous injection of T-Fulips and Iso-Fulips probes (injection dose 10mg/kg mouse body weight), 24h later by the same T 1 Relaxation time series and multilayer T 1 The weighted sequence was subjected to an MRI scan and a comparative analysis. The results are shown in FIG. 4: t1 relaxation time changes were significantly higher in tumors of mice treated with T-Fulips 48h after RT than in the Iso-Fulips group (248.3. + -. 64.6ms vs.61.8. + -. 34.2ms,. Times.P<0.0018). These results indicate that diamagnetic TEMP can be oxidized to paramagnetic TEMPO radicals in RT-treated tumors.
Example 5 modulation of immune response in tumors by T-Fulips to improve the efficacy of RT therapy
Tumor size was recorded every two days for each mouse in example 4 (tumor volume =1/2 × length × width) 2 ). As shown in fig. 5 results: mice receiving T-Fulips and X-ray treatment showed significant inhibition of tumor growth compared to the Iso-Fulips group ([ P ])<0.05 The tumor inhibition rate is greatly improved. The results also show that the T-Fulips are used for regulating the immune response in the tumor and have great application prospect in the aspect of improving the treatment effect of radiotherapy
Example 6 evaluation of the use of T-Fulips in tumor stratification
For T based on T-Fulips 1 The relaxation time change is analyzed in relation to T cell immune activity and tumor inhibition rate. First, the correlation between the-SH density of T cells and the number of cytotoxic T cells in a mouse tumor at day 12 in different treatment groups was analyzed, and the Pearson's correlation coefficient R was 0.8281 (R) 2 = 0.6857). The positive correlation between the density of the T cell membrane-SH and the immune activity of T cells is shown. More importantly, as shown in FIG. 6A, X-ray + T-Fulips/Iso-Fulips mice had tumor area T before and after treatment 1 Changes in relaxation time and the amount of cytotoxic T cell infiltration in the tumor at day 12The Pearson coefficient R between the two is respectively as high as 0.9230 (R) 2 = 0.8519). The results indicate T 1 Changes in relaxation time may to some extent reflect changes in T cell immune activity within the tumor. In addition, tumor T was obtained in mice of different treatment groups as shown in FIG. 6B 1 The analysis result of the correlation between the change of relaxation time and the tumor inhibition rate shows that: mice in the X-ray + T-Fulips treatment group had tumor area T before and after treatment 1 The Pearson coefficient R between the change of relaxation time and the tumor growth inhibition rate at 32 days is respectively as high as 0.9599 (R) 2 = 0.9214); in contrast, mice in the Iso-Fulips treatment group (Iso-Fulips + X-ray, X-ray + Iso-Fulips) had tumor regions T before and after treatment 1 The Pearson's coefficient R between the change in relaxation time and the tumor growth inhibition rate at day 32 was only 0.2282 (R), respectively (R) 2 = 0.0521). These results highlight the promise of strategies based on T-Fulips quantitative MRI for early stratification of the therapeutic effect of RT.
In conclusion, the invention constructs the multifunctional fusogenic liposome T-Fulips which consists of the nitrooxygen active oxygen trapping groups which are modified by a T cell targeting antibody and fixed in the liposome and have the active oxygen scavenging capacity, can specifically identify and fuse T cells, displays the active oxygen trapping groups on T cell membranes, prevents the T cells from activity reduction induced by oxidation, regulates the immune activity of the T cells and realizes magnetic resonance imaging monitoring at the same time.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
- A visual regulatory probe for T cell immune activity, characterized by: comprising an active oxygen scavenging group R 1 T cell targeting antibody fragment R 2 And fusogenic liposome units, wherein the fusogenic liposome unit is comprised of DOPE, DOPE-PEG 2000- MAL and positive charge lipid DOTAP; the visual regulation and control probe utilizes amphiphilic molecules to form through self assemblySpherical liposome vesicle structure, and R 1 、R 2 Respectively react with DOPE-PEG 2000- The hydrophilic end of the MAL is covalently coupled.
- 2. The visual modulation probe of T cell immune activity of claim 1, characterized by: the active oxygen scavenging group R 1 Nitro-oxygen active oxygen trapping agent is adopted.
- 3. The visual modulation probe of T cell immune activity of claim 2, wherein: the active oxygen scavenging group R 1 Comprises at least one of 2, 6-Tetramethylpiperidine (TEMP), 5-dimethyl-1-pyrroline-N-oxide (DMPO) and BocMPO.
- 4. The visual modulation probe of T cell immune activity of claim 1, characterized by: the T cell targeting antibody fragment R 2 Comprises at least one of anti-CD3, anti-CD4, anti-CD8, anti-CD25 and anti-TIM-3.
- 5. The method for preparing a probe for visually regulating T cell immune activity according to any one of claims 1 to 4, characterized by comprising the steps of:1) Synthesis of active oxygen scavenging Unit Structure DOPE-PEG 2000 -R 1 ；2) DOPE and DOPE-PEG 2000 -MAL、DOPE-PEG 2000 -R 1 Uniformly mixing the positive charge lipid DOTAP in a solvent, evaporating the solvent to dryness, adding phosphate buffer solution PBS for dispersion, then vortexing to generate a multi-layer liposome vesicle, and then ultrasonically homogenizing to obtain a unilamellar fusion liposome vesicle;3) Targeting of antibody fragment R by ligation of T cells 2 Preparing a visual regulation probe for the immunological activity of the T cells.
- 6. The method according to claim 5, wherein the step 1) is performed by: adding DOPE-PEG 2000 MAL and thiolated R 1 Mixing in phosphate buffer PBIn S, reacting under the protection of nitrogen, removing unreacted raw material compounds by a dialysis bag, and freeze-drying the dialyzed solution to obtain a solid product, namely DOPE-PEG 2000 -R 1 。
- 7. The method of claim 5, wherein: the components DOPE and DOPE-PEG are involved in the step 2) 2000 -MAL、DOPE-PEG 2000 -R 1 DOTAP is DOPE: DOPE-PEG 2000 -MAL：DOPE-PEG 2000 -R 1 ：DOTAP＝1：(0.1～0.5)：(0.1～0.5)：1。
- 8. The method according to claim 5, wherein the step 3) is performed by: reduction of T cell-targeting antibody fragment R with Dithiothreitol (DTT) 2 And then removing free DTT by using a desalting column, and finally carrying out chemical connection with the unilamellar fusogenic liposome vesicle obtained in the step 2).
- 9. The method of claim 8, wherein: the chemical connection is carried out under the protection of nitrogen, and then the reaction is carried out spin-washing and purification in phosphate buffer solution PBS, thus obtaining the visual regulation and control probe for the T cell immunocompetence.
- The application of the visual regulation probe for the T cell immune activity is characterized in that: the method is used for the visual regulation of the diseases related to the T cell immune activity and the early evaluation of the treatment effect.
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