A flexible indwelling needle device for capturing tumor cells in vivo and a capturing method thereof

Abstract

The present application provides a flexible indwelling needle device for in vivo capture of tumor cells and a capture method using the same, which comprises a flexible indwelling needle shell and a hard inner core; the surface of the flexible indwelling needle shell is connected with a specific antibody. The flexible indwelling needle device of the present application combines tumor cells with antibodies on the surface of the flexible indwelling needle shell that stay in the vein, thereby achieving high-sensitivity capture of tumor cells while minimizing the pain of the test subject.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical engineering, and in particular relates to a flexible indwelling needle device for capturing tumor cells in vivo and a capturing method. Background Art

[0002] As an important biomarker for liquid biopsy, tumor cells free in peripheral blood, i.e., circulating tumor cells, have broad application prospects in assisting clinical diagnosis, early cancer screening, and targeted drug development. However, the extremely low abundance in peripheral blood determines the necessity of efficiently enriching them from peripheral blood for further clinical diagnosis and treatment.

[0003] At present, the enrichment detection technology of circulating tumor cells is almost all concentrated on in vitro detection, that is, by extracting a certain volume of peripheral blood, using physical or biological methods to enrich and purify tumor cells, and finally identifying them by immunofluorescence staining (IF), fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), etc. For example, the CellSearch system launched by Johnson & Johnson, which is the only one that has passed the US Food and Drug Administration (FDA), is to separate tumor cells from blood cells under the action of a magnetic field after co-culturing the patient's peripheral blood with immunomagnetic beads. However, although later researchers have done a lot of work on improving the efficiency of tumor cell separation, this in vitro detection method is fundamentally limited by the volume of each blood collection and the cell loss caused by centrifugation, filtration and other operations required for sample processing. At the same time, due to the uneven distribution of tumor cells in peripheral blood, simply extracting a part of the volume of peripheral blood for detection will also lead to the problem of false negative test results. In contrast, the in vivo detection technology of circulating tumor cells can capture the number of tumor cells in the patient's entire peripheral blood, greatly improving the sensitivity of the detection. Summary of the invention

[0004] Based on the above problems of cell loss and false negative in in vitro tumor cell detection and the advantages of high sensitivity in in vivo detection, the present invention proposes a flexible indwelling needle device for in vivo tumor cell detection and provides an operating method for in vivo capture using the device.

[0005] To achieve this object, the present invention provides the following technical solutions:

[0006] A flexible indwelling needle device for capturing tumor cells in vivo comprises a flexible indwelling needle shell and a hard inner core; the surface of the flexible indwelling needle shell is connected with specific antibodies.

[0007] The flexible indwelling needle shell is used for in vivo capture, the hard inner core is used to assist the flexible indwelling needle shell in being placed into a blood vessel, and the specific antibody is used to specifically bind to tumor cells; when the indwelling needle is in use, the device retained in the patient's body for in vivo capture of tumor cells in the blood is only the flexible indwelling needle shell.

[0008] Furthermore, the material of the outer shell of the flexible indwelling needle is fluorinated ethylene propylene copolymer, and the material of the hard inner core is stainless steel.

[0009] Furthermore, the specific antibodies are coupled to the surface of the flexible shell step by step through a layer-by-layer self-assembly method with sulfur-gold bonds as connection base points.

[0010] Furthermore, the step of connecting the specific antibody to the surface of the flexible indwelling needle shell includes:

[0011] (1) immersing the indwelling needle in a silanization reagent for silanization treatment to amino-modify its surface;

[0012] (2) placing the indwelling needle treated in step (1) in a colloidal gold solution to obtain a flexible indwelling needle shell surface modified with nano-gold particles; preferably, the nano-gold particles have a particle size of 5 nm;

[0013] (3) placing the indwelling needle treated in step (1) in an anhydrous ethanol solution containing 6-thio-1-hexanol and 11-mercaptoundecanoic acid to obtain a thiol-modified flexible indwelling needle shell surface;

[0014] (4) placing the indwelling needle treated in step (1) in deionized water dissolved with N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to obtain a carboxyl-activated flexible indwelling needle shell surface;

[0015] (5) placing the indwelling needle treated in step (4) in a buffer solution containing a specific antibody to obtain a modified indwelling needle.

[0016] Furthermore, the silanization agent in step (1) is a solution of 3-aminopropyltrimethoxysilane, deionized water and methanol in a volume ratio of 1:1:3.

[0017] Furthermore, in the anhydrous ethanol solution containing 6-mercapto-1-hexanol and 11-mercapto-undecanoic acid dissolved in step (3), the mass ratio of 6-mercapto-1-hexanol to 11-mercapto-undecanoic acid is 5:4.

[0018] Furthermore, in step (4), the mass ratio of N-hydroxysuccinimide to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in deionized water is 0.93:1.

[0019] Furthermore, the buffer solution containing the specific antibody in step (5) is a 1xPBS buffer solution with an anti-EpCAM antibody concentration of 2 μg / mL.

[0020] On the other hand, the present application provides a method for in vivo capture of tumor cells using the above-mentioned flexible indwelling needle device.

[0021] Furthermore, the capture method includes simultaneously inserting the flexible indwelling needle shell and the hard inner core of the indwelling needle into the patient's blood vessel, pulling out the hard inner core, retaining the flexible indwelling needle shell in the blood vessel for 1 hour and then slowly pulling it out; washing the pulled out flexible indwelling needle shell in a buffer solution and then placing it in an eluent to elute the captured tumor cells.

[0022] A person skilled in the art can select a suitable specific antibody according to the type of tumor cell to be detected and his knowledge of the specific antigen thereon; the specific antibody can be a polyclonal or monoclonal antibody; for a person skilled in the art who has a known antigen, the person skilled in the art can obtain the specific antibody by purchasing or making it himself.

[0023] The indwelling needle or capture method of the present application is preferably used in a venous blood vessel.

[0024] The capture method of the present application can be used as an intermediate sample / information acquisition step in the diagnostic process, and can also be used for non-diagnostic purposes such as scientific research.

[0025] All materials known in the art for indwelling needles can be used for the indwelling needles of the present application, and commercially available finished indwelling needles can also be directly used to modify and transform the indwelling needles of the present application.

[0026] Those skilled in the art can determine the size characteristics of the indwelling needle according to the type and tolerance of the user.

[0027] The advantage of the present invention is that by designing an indwelling needle device comprising a flexible outer shell and a hard inner core and having a surface modified with specific antibodies, tumor cells in the patient's body can be captured in vivo, and the flexible indwelling needle material can also minimize the patient's pain. Compared with the currently commonly used in vitro detection, the present invention has the following advantages:

[0028] (1) It can overcome the problem of false negative detection caused by small amount of blood sample and uneven distribution of tumor cells in vitro;

[0029] (2) It can avoid the physical and mental harm to patients caused by repeated peripheral blood extraction;

[0030] (3) It can improve the sensitivity of tumor cell detection;

[0031] (4) It can reduce the problem of cell loss caused by basic operations such as centrifugation that are necessary during in vitro testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a real picture of the indwelling needle used for in vivo tumor cell capture after the surface is modified layer by layer according to the present invention.

[0033] Figure 2 The figure is a schematic diagram of the capture process of the indwelling needle used for in vivo capture of tumor cells according to the present invention.

[0034] Figure 3 This is a diagram showing the effect of capturing cells using the indwelling needle used for in vivo capture of tumor cells according to the present invention.

[0035] Figure 4 This is a data diagram comparing the effects of the present invention on in vivo capture and in vitro detection of tumor cells under the same conditions. DETAILED DESCRIPTION

[0036] The following examples are provided to facilitate a better understanding of the present invention, but are not limited thereto. These examples are only used for illustrative purposes and in no way limit the scope of protection of the present invention.

[0037] Example 1 Silanization treatment method and selection of gold nanoparticles

[0038] Try various methods to silanize the surface of the catheter:

[0039] (1) Using 3-aminopropyltrimethoxysilane stock solution, the reagent has a good silanization effect. The surface of the indwelling needle shell after silanization treatment was characterized. However, it was rapidly hydrolyzed during the modification of nano-gold colloid. The heat released caused the nano-gold particles to agglomerate and become ineffective.

[0040] (2) Silanization was performed using a mixture of 3-aminopropyltrimethoxysilane stock solution and water. Mixtures of 3-aminopropyltrimethoxysilane stock solution and water in ratios of 0.5:1, 1:1, 1.5:1, and 2:1 were tested. The experimental results showed that when the ratio of silane to water was less than 1:1, the silane was not fully hydrolyzed, and secondary heat would occur during the modification of the nanogold layer, causing the nanogold particles to agglomerate. When the ratio was higher than 1:1, heat was released severely during the silane modification process, the film was thick, and the nanogold modification effect was not obvious.

[0041] Based on the above experiments, the inventors tried to add methanol, an organic solvent, to the hydrolysis equilibrium of silane and water to maintain the balance of the system, and explored solution systems with ratios of 3-aminopropyltrimethoxysilane stock solution, deionized water, and methanol of 1:1:1; 1:1:2; 1:1:3; 1:1:4; and 1:1:5, respectively. By characterizing the surface of the indwelling needle during the silanization modification process, it was concluded that the silanization modification effect was optimal and the nano-gold layer was the densest when the ratio was 1:1:3.

[0042] Selection of gold nanoparticle diameter and concentration:

[0043] In the experiment, the gold nanoparticles were selected to be 5 nm in diameter. This diameter is determined by the cell diameter, the distance between the antibody recognition sites on the cell surface, and the length of the self-assembly chain. Using 5 nm gold nanoparticles can achieve the best modification effect.

[0044] The applicant tested different nanogold concentrations, including 0.1 mg / mL; 0.05 mg / mL; 0.025 mg / mL; 0.0125 mg / mL. The results of the surface characterization experiment of the indwelling needle showed that when the concentration was 0.1 mg / mL, the nanogold concentration on the surface of the indwelling needle was too high, and a large number of gold particles were enriched on the surface of the shell, which was not conducive to the formation of self-organized chains; when the concentration was 0.05 mg / mL, the nanogold self-organized membrane structure was dense and had good uniformity; when the concentration was further reduced, the gold particles on the surface of the shell were loosely arranged, and there was a lot of blank space under the electron microscope, which was not conducive to the subsequent modification of the self-assembled chain. Therefore, the concentration of nanogold particles in subsequent experiments was determined to be 0.05 mg / mL.

[0045] Mass ratio of MUA to MCH:

[0046] During the modification process, the mass ratio of MUA to MCH was optimized to be 4:5 after experiments. The role of MUA is to provide carboxyl groups for the layer-by-layer self-assembly chains, and the role of MCH is to separate the carboxyl groups to prevent them from connecting to each other, so that they can be connected to subsequent branch chains.

[0047] Example 2 Preparation process of indwelling needle

[0048] The base of the indwelling needle used in the present invention adopts a conventional fluorinated ethylene propylene copolymer shell and a stainless steel hard inner core. The specifications and models can be selected according to the experimental subjects (patients, nude mice, rabbits, etc.). The original indwelling needle is as follows Figure 1 -I. The surface of the flexible shell is modified with specific anti-EpCAM antibodies by layer-by-layer self-assembly, and the specific process is as follows:

[0049] (1) Immerse the catheter in a solution of silanization reagent 3-aminopropyltrimethoxysilane, deionized water, and methanol in a volume ratio of 1:1:3 to perform silanization treatment to amino-modify its surface, such as Figure 1 -II;

[0050] (2) Place the surface amino-modified indwelling needle into 5 nm nano-gold colloid to allow the gold particles to adsorb on the surface of the indwelling needle. Figure 1 -III;

[0051] (3) 6-mercapto-1-hexanol (MCH) and 11-mercaptoundecanoic acid (MUA) were dissolved in anhydrous ethanol at a mass ratio of 5:4, and the gold particle-functionalized indwelling needle was placed therein to fix the thiol through the sulfur-gold bond between the thiol and gold;

[0052] (4) dissolving N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in deionized water at a mass ratio of 0.93:1, and immersing the indwelling needle with the thiol molecules fixed therein to activate the carboxyl groups on the shell surface;

[0053] (5) Immerse the activated indwelling needle in 1xPBS buffer containing 2 μg / mL anti-EpCAM antibody to complete the modification of the shell surface antibody, such as Figure 1 -IV.

[0054] Example 3 Indwelling needle capture process

[0055] This example uses nude mice as experimental subjects to introduce the process of indwelling needle capture in vivo. Figure 2 .

[0056] The capture process is to insert the flexible outer shell and stainless steel inner core of the indwelling needle into the tail vein of the nude mouse at the same time, remove the stainless steel inner core, keep the flexible outer shell in the vein for 1 hour and then slowly remove it. The removed indwelling needle is washed in 1xPBS buffer and placed in the elution solution to elute the captured tumor cells for subsequent downstream analysis.

[0057] Example 4 Capture effect verification

[0058] In order to clearly observe the MCF-7 cells captured on the removed indwelling needle, the removed indwelling needle was thoroughly washed several times in 1xPBS buffer to wash away the red blood cells, white blood cells and other blood cells adsorbed on the surface of the indwelling needle during the capture process. The washed indwelling needle was placed under a microscope for observation, and different viewing angles were adjusted to observe the capture status of cells at different positions.

[0059] Example 5 Comparison of in vivo and in vitro capture effects using healthy nude mouse model and nude mouse tumor model

[0060] To verify the effects of in vivo capture and in vitro detection, this example constructed a xenogeneic orthotopic breast cancer nude mouse model and a healthy nude mouse model as a blank control. After modeling, peripheral whole blood of nude mice was collected in conventional anticoagulant EP tubes at 7, 14, 21, and 28 days after inoculation, and cell counting was performed by in vitro screening; at the same time, the modified indwelling needle was implanted in the tail vein of the nude mouse for 1 hour, and the indwelling needle was removed and fully washed with buffer and eluted with eluent, and then counted and data compared. The statistical results are shown in Figure 4 .from Figure 4 It can be seen that compared with in vitro detection, the in vivo capture method can observe tumor cells 7 days after tumor modeling, and as the cancer progresses, more tumor cells are captured at each stage than in vitro detection. It can be seen that this method greatly improves the sensitivity of tumor cell detection, overcomes the false negative problem caused by the small amount of in vitro detection samples, and also avoids problems such as cell loss in in vitro detection.

Claims

1. A flexible indwelling needle for in vivo capture of tumor cells, characterized in that: The flexible indwelling needle comprises a flexible indwelling needle shell and a hard inner core; the surface of the flexible indwelling needle shell is connected with a specific antibody; the material of the flexible indwelling needle shell is a fluorinated ethylene propylene copolymer, and the material of the hard inner core is stainless steel; the specific antibody is coupled to the surface of the flexible indwelling needle shell step by step through a layer-by-layer self-assembly method, with sulfur-gold bonds as the connection base points; The step of connecting the specific antibody to the surface of the flexible indwelling needle shell comprises: (1) immersing the flexible indwelling needle in a silanization agent for silanization treatment to aminize its surface; the silanization agent is a solution of 3-aminopropyltrimethoxysilane, deionized water, and methanol in a volume ratio of 1:1:3; (2) placing the flexible indwelling needle treated in step (1) in a colloidal gold solution, and modifying the surface of the flexible indwelling needle shell with nano-gold particles; (3) placing the flexible indwelling needle treated in step (2) in an anhydrous ethanol solution containing 6-thio-1-hexanol and 11-mercaptoundecanoic acid, thereby modifying the surface of the flexible indwelling needle shell with thiol; (4) placing the flexible indwelling needle treated in step (3) in deionized water dissolved with N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to activate the carboxyl groups on the surface of the flexible indwelling needle shell; (5) placing the flexible indwelling needle treated in step (4) in a buffer solution containing a specific antibody to obtain a modified flexible indwelling needle; the buffer solution containing the specific antibody is a 1xPBS buffer solution with an anti-EpCAM antibody concentration of 2 μg / mL.

2. The flexible indwelling needle according to claim 1, wherein the nano-gold particles used in step (2) have a particle size of 5 nm.

3. The flexible indwelling needle according to claim 1, wherein the mass ratio of 6-mercapto-1-hexanol to 11-mercapto-undecanoic acid in the anhydrous ethanol solution containing 6-mercapto-1-hexanol and 11-mercapto-undecanoic acid in step (3) is 5:

4.

4. The flexible indwelling needle according to claim 1, wherein in the deionized water in which N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are dissolved, the mass ratio of N-hydroxysuccinimide to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.93:1.

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