阅读说明：本技术 基于中性粒细胞的活细胞探针构建方法 (Live cell probe construction method based on neutrophils ) 是由 冯峰 邱钱赛 温亚 于 2019-09-27 设计创作，主要内容包括：本发明涉及生物医学检测技术领域,公开了一种基于中性粒细胞的活细胞探针构建方法,包括A、BSA还原；B、合成Gd负载BSA纳米颗粒；C、Bodipy标记Gd负载BSA纳米颗粒；以及D、中性粒细胞探针构建,首先以PBS为介质,将DTNB加入至步骤C的产物溶液中,并在室温下相互作用一定时间,完成BSA纳米颗粒活化；超滤纯化后,将活化的BSA纳米颗粒用无FBS的RPMI 1640培养基稀释至0.1mg mL<Sup>-1</Sup>,然后在室温下与1.25×10<Sup>6</Sup>cells.mL<Sup>-1</Sup>中性粒细胞一起孵育；随后用冰冷的PBS洗涤,获得中性粒细胞探针。(The invention relates to the technical field of biomedical detection, and discloses a live cell probe construction method based on neutrophils, which comprises A, BSA reduction; B. synthesizing Gd-loaded BSA nanoparticles; C. bodipy labeled Gd loaded BSA nanoparticles; d, constructing a neutrophil probe, namely adding DTNB into the product solution obtained in the step C by taking PBS as a medium, and interacting for a certain time at room temperature to complete BSA nanoparticle activation; after purification by ultrafiltration, activated BSA nanoparticles were diluted to 0.1mg mL with FBS-free RPMI1640 medium ‑1 Then reacted with 1.25X 10 at room temperature 6 cells.mL ‑1 Incubating the neutrophils together; followed by washing with ice-cold PBS to obtain a neutrophil probe.)

1. A live cell probe construction method based on neutrophils is characterized by comprising the following steps:

A. BSA reduction

Introducing a reducing agent and 2% SDS into the marked BSA protein solution, and carrying out strip reaction at 90-100 DEG CContinuously stirring under the condition, fully exposing free sulfydryl, and then diluting by adopting MES solution to prepare the product with the concentration of 1mg^-1The working solution of (1);

B. synthesis of Gd-loaded BSA nanoparticles

The working solution is incubated at 35-40 ℃ with shaking, and then GdCl with the concentration of 2mmol/L is added₃Solution, the working solution and the GdCl₃The volume ratio of the solution is 30-40: 1; after incubation for 20-30 min, adding 0.1mmol/L NaOH solution, continuing to react for 40-60 min, and then performing ultrafiltration purification to obtain the Gd-loaded BSA nano-particles;

C. bodipy-labeled Gd-loaded BSA nanoparticles

The Gd-loaded BSA nanoparticle is in NaHCO₃Stirring the solution with bodipy solution at room temperature, followed by purification by ultrafiltration to obtain the product;

D. neutrophil probe construction

Firstly, adding DTNB into the product solution obtained in the step C by taking PBS as a medium, and interacting for a certain time at room temperature to complete the BSA nanoparticle activation; after purification by ultrafiltration, activated BSA nanoparticles were diluted to 0.1mg mL with FBS-free RPMI1640 medium^-1Then reacted with 1.25X 10 at room temperature⁶cells.mL^-1Incubating the neutrophils together; the neutrophil probe was then obtained by washing with ice-cold PBS.

2. The method of claim 1, wherein the method comprises the steps of:

in the step A, the marked BSA protein is a BSA protein molecule marked by FITC, and the marking method is as follows:

will dissolve in 10mg.mL^-1NaHCO of₃Mixing BSA protein and FITC in a solution according to a mass ratio of 1:40, and stirring at a constant speed for at least 5 hours to fully react; followed by purification by ultrafiltration to give FITC-BSA.

3. The method of claim 2, wherein the method comprises the steps of:

wherein, when BSA is reduced, the FITC-BSA protein solution and a reducing agent DTT are mixed according to the molar ratio of 1:16, and are continuously stirred with 2% SDS at the temperature of 90 ℃ for 2 hours to carry out reduction reaction, and free sulfhydryl groups are fully exposed; then the concentration is 0.1mg.mL^-1And MES solution of pH 4.4.

4. The method of claim 1, wherein the method comprises the steps of:

wherein, in step B, the working solution is incubated at 37 ℃ for 5 hours with shaking at 130rpm, and then GdCl having a concentration of 2mmol/L is added₃Solution, the working solution and the GdCl₃The volume ratio of the solution is 33: 1; after 20min of incubation, adding NaOH solution with the volume of 25-30% of that of the working solution and the concentration of 0.1mmol/L, continuing to react for 400min, and then performing ultrafiltration purification to obtain the Gd-loaded BSA nano-particles.

5. The method of claim 1, wherein the method comprises the steps of:

wherein, in the step C, the Gd-loaded BSA nano-particle is added into NaHCO at 0.1mmol/L₃In solution with 10mg.mL^{- 1}The Bodipy solution was stirred at room temperature for 1 hour, followed by ultrafiltration purification to obtain Bodipy-labeled Gd-loaded BSA nanoparticles,

the molar ratio of the Gd-loaded BSA nanoparticles to bodipy was 1: 3.

6. The method of claim 3, wherein the method comprises the steps of:

in the step D, the mass ratio of BSA to DTNB is 1:0.42, and the activation time of BSA nanoparticles is 1 hour; the diluted BSA nanoparticles were incubated with neutrophils for 20min at room temperature, and then washed with ice-cold PBS to obtain the neutrophil probe.

7. The method of claim 1, wherein the method comprises the steps of:

wherein the particle size of the Gd-loaded BSA nanoparticle is 42 nm.

Technical Field

The invention relates to the technical field of biomedical detection, relates to construction of a living cell probe, and particularly relates to a living cell probe construction method based on neutrophils.

Background

The timely and accurate identification of tumor lesions is an important prerequisite for clinical cancer intervention and is also the target of tumor imaging diagnosis. Advances in nanoparticle-based molecular imaging have greatly improved the sensitivity and specificity of tumor detection. For exampleInspired by biomineralization, early studies successfully constructed chelated gadolinium ion (Gd)³⁺) The Bovine Serum Albumin (BSA) nanoparticles have obvious advantages in the aspect of detecting tumors by Magnetic Resonance Imaging (MRI) compared with the commercialized gadolinium contrast agent (DTPA). Some reports have made further studies on Gd-loaded nanoparticles, in particular modifying the functionality of overexpressed cancer cell receptors to confer active tumor recognition, with encouraging results also at the animal level.

However, few nanoparticle-based drug delivery systems are used for clinical tumor diagnosis or treatment due to rapid clearance of blood circulation, limited tumor targeting ability, and off-target effects in vivo. Therefore, the development of an imaging agent that can be "free of examination" in the blood circulation and that can effectively identify the Tumor Microenvironment (TME) remains a clinical urgent need.

During the development of tumors, various cytokines and chemokines can recruit a large number of immune cells to fight the disease, inducing the formation of inflammatory TME. Inspired by the natural chemotactic response of immune cells to inflammation, leukocyte-based cell carriers are of increasing interest as they are thought to potentially increase the poor tumor delivery efficiency of nanoparticles. Among the many cell vectors studied, neutrophils are considered one of the most potential choices for the following reasons: neutrophils are the most abundant leukocytes in circulation in the body (50% -70%) and are readily available; various cytokines secreted by cancer cells and chemokines secreted by tumor-associated granulocytes can cause rapid migration of circulating neutrophils to TME in response to inflammation; compared with the nano-carrier with relatively single structure and function, the neutrophil has multiple physiological mechanisms to escape.

The immune system attacks, overcoming various physiological barriers, enabling it to specifically target tumor regions. Neutrophils have been studied and selected as a delivery platform for internalized drug-loaded nanoparticles and have shown excellent efficacy in tumor therapy applications. Delivery of contrast agents (e.g., Gd) by internalization may be limited because Gd3⁺Is not easy to exchange with free water in cellsThereby affecting the Gd chelated on the protein nanoparticles³⁺High T1 relaxation rate. In addition, intracellular delivery of imaging agents may potentially affect the activity and biological properties of neutrophils, leading to impaired specific tumor delivery capabilities.

Disclosure of Invention

The invention aims to successfully construct a live cell probe based on the neutrophil on the basis of the research background, and the live cell probe is used for accurate imaging diagnosis of tumors.

The inventor researches a large amount of recent biomaterial literature reports, and proposes an idea of an intelligent probe for accurate tumor diagnosis based on deep research on autoimmunity, tumor microenvironment and nanotechnology, and the main idea is as follows:

imaging agent (Gd and bodipy) loaded BSA nanoparticles expose multiple free thiol groups by reduction of DTT, which are then activated by 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB), i.e. disulfide bond substitution of 5-thio-2-nitrobenzoate (TNB) in DTNB; the activated BSA nanoparticles form disulfide bond cross-links with thiol groups on the surface of neutrophils through substitution reaction again, and finally the living cell probe (NEs probe) with imaging function is obtained.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides a method for constructing a live cell probe based on neutrophils, which comprises the following steps:

A. BSA reduction

Introducing a reducing agent and 2% SDS (sodium dodecyl sulfate) into a marked BSA (bovine serum albumin) protein solution, continuously stirring at 90-100 ℃, fully exposing free sulfydryl, and then diluting by adopting an MES (MES) solution to prepare the BSA protein solution with the concentration of 1mg^-1The working solution of (1);

B. synthesis of Gd-loaded BSA nanoparticles

The working solution is incubated at 35-40 ℃ with shaking, and then GdCl with the concentration of 2mmol/L is added₃Solutions, working solutions and GdCl₃The volume ratio of the solution is 30-40: 1; after incubation for 20-30 min, adding 0.1mmol/L NaOH solution, continuing to react for 40-60 min, and then carrying out ultrafiltrationFiltering and purifying to obtain Gd loaded BSA nano particles;

C. bodipy-labeled Gd-loaded BSA nanoparticles

Gd loaded BSA nanoparticle in NaHCO₃Stirring the solution with bodipy solution at room temperature, followed by purification by ultrafiltration to obtain the product;

D. neutrophil probe construction

Firstly, adding DTNB into the product solution obtained in the step C by taking PBS as a medium, and interacting for a certain time at room temperature to complete the BSA nanoparticle activation; after purification by ultrafiltration, activated BSA nanoparticles were diluted to 0.1mg mL with FBS-free RPMI1640 medium^-1Then reacted with 1.25X 10 at room temperature⁶cells.mL^-1Incubating the neutrophils together; followed by washing with ice-cold PBS to obtain a neutrophil probe.

Preferably, in step a, the labeled BSA protein is a BSA protein molecule labeled with FITC, and the labeling method is as follows: will dissolve in 10mg.mL^-1NaHCO of₃Mixing BSA protein and FITC in a solution according to a mass ratio of 1:40, and stirring at a constant speed for at least 5 hours to fully react; followed by purification by ultrafiltration to give FITC-BSA.

When BSA is reduced, mixing a FITC-BSA protein solution and a reducing agent DTT in a molar ratio of 1:16, and continuously stirring the mixture and 2% SDS at 90 ℃ for 2 hours to perform a reduction reaction, so as to fully expose free sulfhydryl; then the concentration is 0.1mg.mL^-1And MES solution of pH 4.4.

Preferably, in step B, the working solution is incubated at 37 ℃ for 5 hours with shaking at 130rpm, followed by addition of GdCl at a concentration of 2mmol/L₃Solutions, working solutions and GdCl₃The volume ratio of the solution is 33: 1; after 20min of incubation, adding NaOH solution with the volume of 25-30 percent of that of the working solution and the concentration of 0.1mmol/L, continuing to react for 400min, and then performing ultrafiltration purification to obtain Gd-loaded BSA nanoparticles, wherein the particle size of the BAS nanoparticles is 42 nm.

Preferably, in step C, the Gd-loaded BSA nanoparticles are loaded with NaHCO at 0.1mmol/L₃In solution with 10mg.mL^{- 1}The bodipy solution was stirred at room temperature for 1 hourFollowed by ultrafiltration purification to obtain Bodipy-labeled Gd-loaded BSA nanoparticles. Wherein the molar ratio of Gd-loaded BSA nanoparticles to bodipy is 1: 3.

Preferably, in the step D, the mass ratio of BSA to DTNB is 1:0.42, and the activation time of BSA nanoparticles is 1 hour; the diluted BSA nanoparticles were incubated with neutrophils for 20min at room temperature, and then washed with ice-cold PBS to obtain neutrophil probes.

In the invention, the neutrophilic granulocyte is collected in the abdominal cavity of a mouse, and a sterile thioglycollate culture medium is injected into the abdominal cavity of the mouse to simulate an abdominal cavity inflammation environment; after a period of "recruitment", the abdominal cavity was perfused 3-4 times with ice-cold PBS, and the erythrocytes in the lavage were lysed according to the instructions, centrifuged, and resuspended in RPMI1640 medium to obtain mouse leukocytes; the cell suspension was then allowed to stand at 37 ℃ in 5% CO₂Incubators for 30 minutes to remove adherent macrophages; the supernatant was collected and centrifuged (850g) for 5 minutes to obtain mouse neutrophils.

The preliminary basic research only provides and verifies the concept of an intelligent probe that utilizes the natural tumor chemotactic ability of immune cells to improve diagnostic efficacy in animal models. In clinical transformation, the imaging probe can be constructed in vivo by utilizing the animal neutrophil granulocytes and tumor specific imaging can be realized, and the mode can reduce trauma and avoid potential self rejection, thereby increasing the practicability and the popularization.

After the probe is prepared, the inventor firstly researches the influence of the probe on the biological characteristics such as the activity, the morphology, the membrane protein labeling and the chemotactic capacity in a mouse of the neutrophil. The results show that the anchoring of the nanoparticles has little effect on neutrophil function, and that the NEs probe retains the physiological function of neutrophils and can migrate across the vascular barrier from the circulation to the "inflamed area" of the abdominal cavity.

Subsequently, the inventor evaluates the tumor-specific targeting ability of the NEs probe through a mouse subcutaneous lung cancer model, and the result shows that neutrophils as a carrier can efficiently deliver the imaging agent to a tumor region, so that the NEs probe can effectively migrate to the tumor region by identifying complex biological signals in vivo, thereby generating a T1WI signal with diagnostic application value, and the imaging effect is remarkably superior to that of the current nanoparticle-based imaging agent.

The invention has the following beneficial guarantee and effects:

first, neutrophils are the most abundant leukocytes in the circulation in the body (50% -70%) and are readily available; various cytokines secreted by cancer cells and chemokines secreted by tumor-associated granulocytes can cause circulating neutrophils to rapidly migrate to the tumor microenvironment (TEM) in response to inflammation, and therefore, neutrophils are selected as a cell carrier for the probe in the present invention. Compared with the nano-carrier with relatively single structure and function, the neutrophil has multiple physiological mechanisms, can escape the attack of an immune system, overcomes various physiological barriers, can specifically target a tumor region, further efficiently delivers the imaging agent to the tumor region, has more efficient imaging agent delivery efficiency, and thus improves the detection sensitivity of the tumor.

Secondly, disulfide bonds in the fetal Bovine Serum Albumin (BSA) are reduced by DTT to expose a plurality of free sulfydryl groups, and the sulfydryl groups can form disulfide bonds again under certain conditions to serve as main acting forces in the protein nano-skeleton. The nano construction method is simple, convenient, green, efficient and stable, and the loading of the imaging agents (Gd and bodipy) is based on early-stage research, the synthesis technology is mature and convenient, and excessive investment is not needed, so that the preparation cost of the probe is reduced, the industrial production of the probe is favorably realized, and the method is clinically popularized and applied.

Thirdly, the neutrophil and BSA nano-particles are realized by using DTNB and undergoing two times of sulfydryl-disulfide bond exchange, the cell surface engineering method has strong operability and small influence on the physiological function of cells, and can ensure the high relaxation rate (14.53 s) of Gd^-1mM) to ensure its ultimate imaging effect in the target area.

In conclusion, the invention utilizes the inflammation recognition and tumor homing capacity of neutrophils to construct a highly sensitive living cell probe. The probe can realize faster, stronger and more durable signal enrichment of a tumor region, and is favorable for accurate tumor diagnosis.

Drawings

FIG. 1 is a schematic diagram of the construction of a live cell probe and the imaging verification process in a mouse according to the present invention;

FIG. 2 is a graph showing the results of characterization of Bodipy-labeled Gd-loaded BSA nanoparticles (Gd @ BSA-FITC/Bodily NPs) in accordance with the present invention;

FIG. 3 is a diagram showing the preparation and results of a neutrophil probe (Gd @ BSA-FITC NPs/NE) according to the present invention;

FIG. 4 shows the effect of the probe of the present invention on the performance of neutrophils;

FIG. 5 shows the results of in vivo chemotaxis studies of the probe of the present invention;

FIG. 6 shows the results of fluorescence imaging of a mouse with the probe of the present invention;

FIG. 7 shows the result of the probe of the present invention used in MR imaging of a mouse in vivo.

Detailed Description

The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The reagents and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. The examples do not include detailed descriptions of conventional methods, experimental methods without specifying specific conditions, and are generally performed according to conventional conditions, experimental methods on a technical tool book, or conditions recommended by the manufacturer. Percentages and parts are by volume unless otherwise indicated.

FIG. 1 illustrates the working procedure of the present invention, first constructing a neutrophil probe and observing the probe under a fluorescence microscope; then the probe is injected into a mouse body through tail vein, the probe passes through endothelial cells to be enriched at the tumor position, and the value of the NEs probe in the tumor specific targeting and diagnosis application is researched through living fluorescence imaging and magnetic resonance imaging. See the following examples for specific experimental procedures: