CN108392232B - In vivo cell capturing device using functional protein silk thread as carrier - Google Patents

Abstract

The present invention provides an in vivo cell trap using functionalized protein filaments as a carrier, including an in vivo cell capturing tube using functionalized protein filaments as a carrier. The in vivo cell capturing tube includes a fixed tube, several functionalized protein filaments and cell capture ligands fixed on the functionalized protein filaments. One end of the functionalized protein filaments is fixed in the fixed tube, and a guide pin is sheathed on the outside of the fixed tube. The other end of the functionalized protein thread is located in the cavity, and the fixed tube is also connected with a guide wire rod, which is located in the guide pin and can slide relative to the guide pin. The capture device of the invention has good biocompatibility, is easy to contact with target cells, and improves the capture efficiency of characteristic cells in vivo.

Description

In vivo cell traps based on functionalized protein filaments

technical field

The invention relates to the field of medical devices, in particular to an in vivo cell trap with functionalized protein silk as a carrier.

Background technique

The disease-related characteristic cells in human blood have important clinical application value, especially circulating tumor cells (CTC). However, the number of characteristic cells (especially CTC) in blood is extremely rare, and they are rare cells. Usually, the detection method includes the process of cell capture and enrichment. The capture method is divided into in vitro method and in vivo method. The former is prone to false negatives due to the small amount of blood in the test sample. It is for this reason that several in vivo cell capture methods are now developed. Whether in vivo or in vitro, the specific capture ligands of target cells (such as epithelial cell adhesion molecule antibodies, nucleic acid aptamers, and polypeptide aptamers) are usually immobilized on a carrier to construct a cell trap, and then the trap is placed in blood vessels to capture high-speed flowing target cells.

Currently, the carriers used in in vivo cell traps include chemical fiber filaments, organic polymer wires, medical indwelling needles, medical stainless steel wires, and venous catheters. However, in vivo cell traps constructed with these vectors have obvious deficiencies in four aspects. First, in order to resist the high-speed impact of venous blood, a larger diameter (above 500 microns) is often required to avoid the silk thread from being broken and placed in the blood vessel; second, the large-diameter material is more rigid, and the probability of contact with the high-speed flowing target cells in the flexible blood vessel will be greatly reduced; The specific surface area, which will also greatly reduce the contact probability between the high-speed flowing target cells and the capture device; fourth, although these carrier materials have good biocompatibility and low toxicity, they are not biodegradable, and once broken and placed in blood vessels, it will definitely cause serious medical problems.

Contents of the invention

In order to solve the above-mentioned technical problems, the object of the present invention is to provide an in vivo cell trap using functionalized protein filaments as a carrier, which not only has good biocompatibility, but also can be biodegraded, easily contacts with target cells, and improves the capture efficiency of characteristic cells in vivo.

The present invention provides an in vivo cell trap using functionalized protein filaments as a carrier, including an in vivo cell capturing tube using functionalized protein filaments as a carrier. The in vivo cell capturing tube includes a fixed tube, several flexible functionalized protein filaments and cell capture ligands fixed on the functionalized protein filaments. The plug surrounds the outer side of the fixing tube, the sealing plug, the fixing tube and the guide pin form a cavity with an opening at one end, the other end of the functionalized protein thread is located in the cavity, the fixing tube is also connected with a guide wire rod, the guide wire rod is located in the guide pin and can slide relative to the guide pin.

Further, the number of functionalized protein filaments is 2-60. Immobilizing multiple functionalized protein filaments in the cell capture tube at the same time greatly increases the specific surface area of the capture device, and significantly increases the probability of contact with the cells to be captured in the blood, which significantly increases the capture rate of cells and realizes efficient cell capture.

Further, the functionalized protein filaments include protein filaments and functional substances fixedly connected to the surface of the protein filaments, and the functional substances are connected to cell capture ligands.

Further, the protein silk thread is mulberry silk, tussah silk or spider silk, the functional substance is a compound containing functional groups, or a compound containing functional groups and functionalized nanoparticles, and the cell capture ligand is one or more of specific antibodies (such as epithelial cell adhesion molecule antibodies), specific nucleic acid aptamers and polypeptide aptamers.

Further, the compound containing functional groups is one or more of compounds containing epoxy groups, compounds containing carboxyl groups and compounds containing amino groups.

Further, the epoxy group-containing compound is diglycidyl ether, such as polyethylene glycol diglycidyl ether and/or 1,4-butanediol diglycidyl ether.

Further, the carboxyl-containing compound is polyacrylic acid (PAA), a copolymer of acrylic acid and methyl acrylate, a copolymer of acrylic acid and ethyl acrylate, or a partially acemidated polymer of polyacrylic acid.

Further, the amino-containing compound is a polyamine compound, such as one or more of ethylenediamine, propylenediamine, butylenediamine and polyetherimide (PEI).

Further, the functionalized nanoparticles are one of silica nanoparticles and titanium dioxide nanoparticles.

Further, the preparation method of the functionalized protein thread immobilized with the cell-trapping ligand comprises the following steps:

The protein filaments were reacted with functional group-containing compounds or functional group-containing compounds and functionalized nanoparticles at 60°C for 6h, then reacted with the cell-trapping ligand under the action of a cross-linking agent at 37°C for 3h, and then added alkylamines for 4h.

Further, the crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

Further, one end of the functionalized protein thread is fixed in the fixed tube through hydrogel. The hydrogel is a cross-linked polyacrylamide gel.

Further, the fixed pipe is a hose made of high molecular polymer, such as polytetrafluoroethylene, PVC, fluorinated ethylene propylene copolymer (FEP) or polyurethane.

Further, the cells are circulating tumor cells (CTCs) or fetal-derived cells in maternal blood.

Further, the end of the guide pin away from the guide wire rod is detachably connected to a liquid storage unit, the liquid storage unit includes a liquid storage sleeve, a sealing cover and a preservation solution located in the storage sleeve, the sealing cover is provided with a through hole, the guide needle is penetrated in the through hole and communicated with the storage sleeve, and the cavity is filled with the storage solution. The preservation solution is filled in the cavity where the storage solution sleeve and the functionalized protein silk thread are located, so that the capture ligand on the functionalized protein silk thread will not be inactivated during preservation.

Further, a plurality of guide plugs are fixedly connected to the inner wall of the guide needle.

Further, there are two guide plugs, one of which surrounds the outside of the fixing tube, and the other surrounds the outside of the guide wire rod.

Further, a sheet-shaped guide wire handle is fixedly connected to the outside of the guide needle, a sheet-shaped guide wire handle is connected to the end of the guide wire rod away from the fixing tube, and a positioning line is connected between the guide wire handle and the guide wire handle.

Further, small holes are opened on the guide needle handle and the guide wire handle, and the two ends of the positioning line are passed through the small holes and fixedly connected with the guide needle handle and the guide wire handle respectively. The setting of the positioning line can prevent one end of the guide wire rod from slipping out of the guide pin when it slides relative to the guide pin.

Further, the guide needle handle and guide wire handle are made of elastic material. Convenient to hold on to the skin while in use.

Furthermore, several anti-slip bumps are provided on the handle of the guide needle and the handle of the guide wire.

By means of the above solution, the present invention has at least the following advantages:

The in vivo cell capture tube in the in vivo cell trap of the present invention uses functionalized protein filaments as a carrier, and the protein filaments have extremely strong tensile strength and good toughness, which can fully resist the high-speed impact of venous blood. Moreover, the diameter of protein filaments is very small, usually about 10-15 μm, which is only 1/30, sometimes even 1/50 of that of conventional capture filaments. The diameter of the natural protein filament is equivalent to the diameter of the cell to be captured, which is beneficial to reduce the steric hindrance of the capture carrier. At the same time, the ultra-thin protein filaments also show obvious softness macroscopically. In the high-speed flowing blood, these soft filaments will swing at high speed to make them fully contact with the cells to be captured, thus overcoming the difficulty of contacting the rigid carrier with the target cells. Due to the extremely thin protein filaments, several capture filaments can be fixed in the cell capture tube at the same time, and the capture device placed in the venous blood vessel will not significantly affect the normal flow of blood. The capture device thus formed greatly increases the specific surface area of the capture device, and the probability of contact with the cells to be captured in the blood is also significantly increased, so that the capture rate of cells is significantly improved, and efficient cell capture is achieved. Since the raw materials of the functionalized protein silk thread are all natural proteins, they have excellent biocompatibility and good biodegradability. In case of breakage, they can be taken out by clinical surgery like the current vascular indwelling materials such as indwelling needles and indwelling catheters, or they can be self-biodegradable, which greatly reduces the risk of the capture thread breakage to the human body.

The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly and implement it according to the contents of the description, the preferred embodiments of the present invention and accompanying drawings are described in detail below.

Description of drawings

Fig. 1 is a schematic structural view of the cell trap in vivo of the present invention;

Figure 2 is an enlarged view of circle A in Figure 1;

Fig. 3 is the selective result of CTC capture by the cell trap of the present invention and the control group;

Figure 4 is a photomicrograph of CTCs captured by the control group;

Figure 5 is a photomicrograph of CTCs captured by the cell trap of the present invention;

Fig. 6 is a test result diagram of the relationship between different numbers of functionalized protein filaments fixed in the cell trap of the present invention and the number of CTCs captured;

Figure 7 is a scanning electron micrograph of the immobilized functionalized protein filaments in the cell trap of the present invention;

Fig. 8 is a scanning electron micrograph of silicon nanoparticles immobilized on the surface of the functionalized protein filament immobilized in the cell trap of the present invention;

Fig. 9 is the toxicity test result of different substances to cells;

Fig. 10 is the hemolysis test result of different substances;

Fig. 11 is the coagulation test result of the functionalized protein silk thread and the medical stainless steel wire in the cell trap of the present invention;

Explanation of reference signs:

1-Reservoir sleeve; 2-Guide needle; 3-Protein thread; 4-Sealing plug; 5-Guide plug; 6-Guide needle handle; 7-Positioning line; 8-Capturing ligand; 9-Fixing tube; 10-Hydrogel;

Detailed ways

The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1

Referring to Figures 1-2, an in vivo CTC cell trap using functionalized protein filaments as a carrier in a preferred embodiment of this embodiment includes an in vivo CTC cell capture tube, and the in vivo cell CTC capture tube includes a fixed tube 9 and several functionalized protein filaments, preferably 40. The fixed pipe 9 is a hose, and its material is a high molecular polymer, such as polytetrafluoroethylene, PVC, FEP or polyurethane. One end of the functionalized protein thread is fixedly connected in the fixed tube 9 through the hydrogel 10. The functionalized protein thread includes a protein thread 3 and a functional substance fixedly connected to the surface of the protein thread 3. The functional substance is connected with a capture ligand 8 for capturing cells. The protein thread 3 can be mulberry silk, tussah silk or spider silk.

A guide pin 2 is sheathed on the outside of the fixing tube 9, and a sealing plug 4 is fixedly connected to the inside of the guide pin 2. The sealing plug 4 surrounds the outer side of the upper end of the fixing tube 9. The sealing plug 4, the fixing tube 9 and the guide pin 2 form a cavity with an opening at one end. The other end of the functionalized protein silk thread is located in the cavity and can move freely relative to the fixing tube 9.

The needle of the guide needle 2 is detachably connected with a liquid storage unit. The liquid storage unit includes a liquid storage sleeve 1, a sealing cover 14 and a preservation solution 13 located in the storage sleeve 1. A through hole is opened on the sealing cover 14. The needle head of the guide needle 2 is penetrated in the through hole and communicated with the storage sleeve 1. The storage solution 13 is filled in the storage sleeve 1 and the cavity. The preservation solution 13 prevents the capture ligand 8 on the functionalized protein silk from being inactivated during preservation.

The lower end of the fixed tube 9 is fixedly connected with a guide wire rod 11 , and the guide wire rod 11 is located in the guide pin 2 and can slide relative to the guide pin 2 . One end of the guide wire rod 11 is located outside the guide needle 2 . The inner wall of the guide needle 2 is also fixedly connected with two guide plugs 5 , one of which surrounds the outer side of the lower end of the fixed tube 9 , and the other surrounds the outer side of the guide wire rod 11 .

The outside of the guide needle 2 is fixedly connected with a flaky guide needle handle 6, the end of the guide wire rod 11 is connected with a flaky guide wire handle 12, the guide needle handle 6 and the guide wire handle 12 are provided with a number of non-slip bumps 15, the material of the guide needle handle 6 and the guide wire handle 12 is elastic material, which is convenient to be fixed on the skin during use. Small holes are provided on the guide needle handle 6 and the guide wire handle 12, and a positioning line 7 is fixedly connected in the small holes. The setting of the positioning line 7 can prevent one end of the guide wire rod 11 from slipping out of the guide needle 2 when it slides relative to the guide needle 2 .

The method for immobilizing the functionalized protein filament with capture ligand 8 and its fixed connection in the fixation tube 9 (i.e. the preparation of CTC cell capture tube in vivo) is as follows:

(1) Put the extracted protein silk (including mulberry silk, tussah silk or spider silk) into the fixing tube, inject hydrogel to fix the protein silk, and then put the protein silk into the degumming solution for degumming. The degummed protein filaments were washed several times with triple distilled water to obtain fixed protein filaments.

(2) Put the fixed protein (including a fixed pipe) into a round bottle, and use two-shrimply gly gly ether (including polyethylene glycol two-shrimply gly gly gly gly gly gly glycerin ether or 1,4-butanol dilatein gly gly glycerin ether) and PAA. Aunar compounds (ethimine, propyramine, butyramine, or PEI) and PAA responded in a 60 ° C water bath for 6 hours. After that, wash with distilled water several times to obtain surface-functionalized immobilized protein filaments.

(3) Put the surface-functionalized immobilized protein filaments into a round bottom flask, add EDC and NHS solutions, shake, and react in a water bath at 37° C. for 3 hours. After the reaction, wash with distilled water several times. Then put it into a round bottom flask, add CTC capture ligand (epithelial cell adhesion molecule antibody, nucleic acid aptamer or polypeptide aptamer), shake, and react in a water bath at 37°C for 3 hours. After the reaction, wash with distilled water several times. Then put it into a round bottom bottle, add alkylamine, shake, and react in a water bath at 25°C for 4 hours. After the reaction, wash with distilled water several times to obtain the in vivo CTC cell capture tube.

When the target capture cells are other rare cells, different capture ligands 8 can be connected to the protein filament 3 through functional substances as needed.

According to the above structure, the in vivo CTC cell capture tube and other components are assembled into an in vivo CTC cell trap. After the assembly is completed, the liquid storage set 1 is opened, and the preservation solution 13 is filled into the cavity where the functionalized protein filament is located through the end of the guide pin 2, and the preservation solution 12 is also added to the liquid storage set 1, and the liquid storage set 1 is sealed, sterilized, and set aside.

The steps of using the in vivo CTC cell trap of the present invention are as follows:

When in use, the guide needle 2 is pulled out from the fluid storage set 1, and then the needle is inserted into the blood vessel by about 0.5-1.0cm. Then the guide wire rod 11 is pushed about 2 cm, and the guide wire rod 11 pushes the fixing tube 9 and the functionalized protein thread connected thereto to move relative to the guide needle 2 and emerge from the guide needle 2, so that the fixing tube 9 is completely exposed in the blood vessel. When the blood flows at high speed, a large number of functionalized protein filaments fixed on the fixed tube 9 swing at a high speed in the flowing blood, fully contacting the CTC, so that the CTC capture ligand immobilized on it interacts with the receptor on the CTC, so that the CTC is immobilized on the protein filament 3 . During the capture process, the wire guide handle 12 and the guide needle handle 6 can be fixed on the skin with adhesive tape, without affecting the patient's activities. After capturing for a period of time, pull the guide wire rod 11 to drive the capture tube to enter the guide needle 2 again, and then pull the guide wire handle 12 to pull the guide needle 2 out of the blood vessel, which completes the CTC cell capture process in vivo.

In order to verify the effect of the CTC cell trap in vivo of the present invention, the capture tube was pushed out from the guide needle, and the functionalized protein filaments in it were completely exposed, and washed with PBS for 2 minutes. Digest the cleaned capture tube in trypsin for 3 minutes, add culture medium to stop the digestion, and take out the capture tube. Finally, the captured CTCs in the culture medium are counted and analyzed. In order to serve as a control, protein filaments without any treatment were selected to make cell traps, and the same method was used to capture CTCs in vivo. The selectivity results of the two groups of experiments on CTC capture are shown in Figure 3. It can be seen from Figure 3 that the cell capture efficiency of the CTC cell trap of the present invention is significantly higher than that of the control experiment. Figures 4-5 are photomicrographs of CTCs captured by two groups of cell traps. The arrows in the figure indicate CTC cells, and the CTCs captured by the traps of the present invention are significantly more than those of the control group.

Changing the number of functionalized protein filaments fixed in the cell trap changes the number of CTCs captured accordingly. Figure 6 shows the relationship between the number of functionalized protein filaments and the number of CTCs captured. It can be seen from the figure that as the number of functionalized protein filaments increases, the number of CTCs captured also increases. This further proves that the capture efficiency of the trap constructed with multiple small-diameter protein filaments can be significantly increased, which is significantly higher than that of the trap constructed with a single carrier material.

In addition, in order to change the CTC capture efficiency, nanoparticles can also be immobilized in the functionalized protein filaments. FIG. 7 is a scanning electron micrograph of the functionalized protein filaments immobilized in the cell trap of the present invention; FIG. 8a is a scanning electron micrograph of silicon nanoparticles immobilized, and FIG.

Figures 9-10 show the test results of the toxicity of different substances to cells and the test results of hemolysis experiments. Figures 9-10 show that the functionalized protein filaments of the present invention are less toxic to cells and have minimal hemolysis, which is almost equivalent to negative samples. The silk-ligand in the figure is the functionalized protein silk used in the present invention. Figure 11 shows the coagulation test results of the functionalized protein silk thread and the medical stainless steel wire, which shows that the functionalized protein silk thread of the present invention is similar to the commercially available medical stainless steel wire in coagulation property, and can be applied to the human body.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (10)

1. An in vivo cell capturer taking a functionalized protein wire as a carrier is characterized in that: the in-vivo cell capturing tube comprises a fixing tube, a plurality of flexible functional protein wires and a cell capturing ligand fixed on the functional protein wires, wherein the cell capturing ligand is used for capturing cells, one end of the functional protein wires is fixed in the fixing tube, the other end of the functional protein wires can freely move relative to the fixing tube, a guide pin is sleeved outside the fixing tube, a sealing plug is fixedly connected in the guide pin, the sealing plug surrounds the outer side of the fixing tube, a cavity with an opening at one end is formed by the sealing plug, the fixing tube and the guide pin, the other end of the functional protein wires is positioned in the cavity, and the fixing tube is also connected with a guide rod which is positioned in the guide pin and can slide relative to the guide pin.

2. The in vivo cell harvester of claim 1, wherein: the functional protein silk thread comprises a protein silk thread and a functional substance fixedly connected with the surface of the protein silk thread, wherein the functional substance is connected with the cell capturing ligand.

3. The in vivo cell harvester of claim 2, wherein: the protein silk thread is mulberry silk, tussah silk or spider silk, the functional substance is a compound containing a functional group, or a compound containing a functional group and functionalized nanoparticles, and the cell capturing ligand is one or more of a specific antibody, a specific nucleic acid aptamer and a polypeptide aptamer.

4. An in vivo cell harvester according to claim 3, wherein: the compound containing functional groups is one or more of epoxy group-containing compounds, carboxyl group-containing compounds and amino group-containing compounds.

5. An in vivo cell harvester according to claim 3, wherein: the functionalized nanoparticle is one of a silicon dioxide nanoparticle and a titanium dioxide nanoparticle.

6. The in vivo cell harvester of claim 1, wherein: one end of the functionalized protein wire is fixed in the fixing tube, and the fixation is realized through hydrogel.

7. The in vivo cell harvester of claim 1, wherein: the cells are circulating tumor cells or fetal cells in maternal blood.

8. The in vivo cell harvester of claim 1, wherein: the guide pin is far away from the one end detachable of guide screw is connected with the stock solution unit, the stock solution unit includes stock solution cover, sealed lid and is located stock solution in the stock solution cover, set up the through-hole on the sealed lid, the guide pin wear to locate in the through-hole and with stock solution cover intercommunication, the cavity intussuseption is filled with stock solution.

9. The in vivo cell harvester of claim 1, wherein: the inner wall of guide pin still fixedly connected with a plurality of guide plugs.

10. The in vivo cell harvester of claim 1, wherein: the guide needle is characterized in that a flaky guide needle handle is fixedly connected to the outside of the guide needle, one end, away from the fixed tube, of the guide screw rod is connected with a flaky guide wire handle, and a positioning wire is connected between the guide needle handle and the guide wire handle.