CN108392232B

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

Info

Publication number: CN108392232B
Application number: CN201810321288.0A
Authority: CN
Inventors: 谢洪平
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2018-04-11
Filing date: 2018-04-11
Publication date: 2023-07-25
Anticipated expiration: 2038-04-11
Also published as: CN108392232A

Abstract

The invention provides an in-vivo cell capturing device taking a functionalized protein wire as a carrier, which comprises an in-vivo cell capturing tube taking the functionalized protein wire as the carrier, wherein the in-vivo cell capturing tube comprises a fixing tube, a plurality of functionalized protein wires and a cell capturing ligand fixed on the functionalized protein wire, one end of the functionalized protein wire is fixed in the fixing tube, a guide needle is sleeved outside the fixing tube, a sealing plug is fixedly connected in the guide needle, 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 needle, the other end of the functionalized protein wire is positioned in the cavity, the fixing tube is also connected with a guide screw, and the guide screw is positioned in the guide needle and can slide relative to the guide needle. The trap has good biocompatibility, is easy to contact with target cells, and improves the trapping efficiency of characteristic cells in vivo.

Description

In vivo cell capturing device using functional protein silk thread as carrier

Technical Field

The invention relates to the field of medical equipment, in particular to an in-vivo cell catcher taking a functionalized protein wire as a carrier.

Background

Characteristic cells related to diseases in human blood have important clinical application value, in particular to Circulating Tumor Cells (CTCs). However, the number of characteristic cells (especially CTCs) in the blood is extremely rare, and the usual detection methods involve the process of cell capture and enrichment. The capturing method is classified into an in vitro method and an in vivo method, the former method is liable to have false negative due to a small blood volume of a test sample. For this reason, several in vivo cell capture methods are now being developed. Whether in vivo or in vitro, specific capture ligands for target cells (e.g., such as cell adhesion molecule antibodies, nucleic acid aptamers, polypeptide aptamers) are typically immobilized on a carrier to construct a cell capture device, which is then placed in a blood vessel to capture high-velocity flowing target cells.

Currently, carriers used for in vivo cell traps include chemical fiber filaments, organic polymer wires, medical indwelling needles, medical stainless steel wires, and intravenous catheters. However, in vivo cell arresters constructed with these vectors clearly suffer from four disadvantages. First, to combat the high-speed impact of venous blood, a larger (500 micron or more) diameter is often required to avoid wire breakage and lodging in the vessel; secondly, the rigidity of the large-diameter material is enhanced, and the contact probability with high-speed flowing target cells in the flexible blood vessel is greatly reduced; thirdly, on the premise of not influencing the normal function of the blood vessel, the large-diameter carrier material cannot be used for placing the trap containing a plurality of carrier wires (or tubes) in the narrow venous blood vessel, and in fact, the trap is constructed by taking a single wire (or tube) as the carrier material, and has extremely small specific surface area, so that the contact probability of target cells flowing at high speed and the trap is greatly reduced; fourth, although these carrier materials are biocompatible and have little toxicity, they are not biodegradable and, once broken, they remain in the blood vessel, which can lead to serious medical problems.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide an in-vivo cell capturer taking a functionalized protein wire as a carrier, which has good biocompatibility, can be biodegraded, is easy to contact with target cells, and improves the capture efficiency of characteristic cells in vivo.

The invention provides an in-vivo cell capturing device taking a functional protein wire as a carrier, which comprises an in-vivo cell capturing tube taking the functional protein wire as the carrier, wherein the in-vivo cell capturing tube comprises a fixed tube, a plurality of flexible functional protein wires and a cell capturing ligand fixed on the functional protein wire, the cell capturing ligand is used for capturing cells, one end of the functional protein wire is fixed in the fixed tube, the other end of the functional protein wire can freely move relative to the fixed tube, a guide pin is sleeved outside the fixed tube, a sealing plug is fixedly connected in the guide pin, the sealing plug surrounds the outer side of the fixed tube, the sealing plug, the fixed tube and the guide pin form a cavity with an opening at one end, the other end of the functional protein wire is positioned in the cavity, the fixed tube is also connected with a guide rod, and the guide rod is positioned in the guide pin and can slide relative to the guide pin.

Further, the number of the functionalized protein wires is 2-60. The cell capturing tube is internally provided with a plurality of functional protein wires, so that the specific surface area of the capturing device is greatly increased, the probability of contacting with cells to be captured in blood is also obviously increased, the capturing rate of the cells is obviously improved, and the efficient cell capturing is realized.

Further, the functionalized protein thread comprises a protein thread and a functional substance fixedly connected with the surface of the protein thread, wherein the functional substance is connected with a cell capturing ligand.

Further, the protein silk 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 (such as an epithelial cell adhesion molecule antibody), a specific nucleic acid aptamer and a polypeptide aptamer.

Further, the compound containing a functional group is one or more of an epoxy group-containing compound, a carboxyl group-containing compound and an amino group-containing compound.

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

Further, the carboxyl group-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 imidized 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 nanoparticle is one of a silica nanoparticle and a titania nanoparticle.

Further, the preparation method of the functionalized protein wire immobilized with the cell capturing ligand comprises the following steps:

the protein silk thread is reacted with the compound containing functional group or the compound containing functional group and the functionalized nano particle for 6 hours at 60 ℃, then reacted with the cell capturing ligand for 3 hours at 37 ℃ under the action of the cross-linking agent, and then reacted for 4 hours after adding the alkylamine.

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

Further, one end of the functionalized protein wire is fixed in the fixed tube through hydrogel. The hydrogel is a crosslinked polyacrylamide gel.

Further, the fixing tube is a hose, and the material of the fixing tube is high molecular polymer, such as polytetrafluoroethylene, PVC, fluorinated ethylene propylene copolymer (FEP) or polyurethane.

Further, the cells are Circulating Tumor Cells (CTCs) or fetal cells in maternal blood.

Further, the guide pin is far away from the detachable stock solution unit that is connected with of one end of guide screw, and the stock solution unit includes stock solution cover, sealed lid and is located the preservation solution of stock solution cover, has seted up the through-hole on the sealed lid, and the guide pin is worn to locate in the through-hole and communicate with the stock solution cover, and the cavity intussuseption is filled with preservation solution. The preservation liquid is filled in the cavity where the liquid storage sleeve and the functionalized protein silk thread are located, so that the capture ligand on the functionalized protein silk thread cannot be inactivated in the preservation process.

Further, the inner wall of the guide pin is also fixedly connected with a plurality of guide plugs.

Further, the number of the guide plugs is two, one of the guide plugs surrounds the outer side of the fixed pipe, and the other guide plug surrounds the outer side of the guide screw rod.

Further, 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.

Further, small holes are formed in the guide needle handle and the guide wire handle, and two ends of the positioning wire penetrate through the small holes and are fixedly connected with the guide needle handle and the guide wire handle respectively. The setting of positioning line can make the lead screw when sliding with the guide pin relatively, and its one end can not follow the guide pin and slide out.

Further, the guide needle handle and the guide wire handle are made of elastic materials. Is convenient to be fixed on the skin when in use.

Further, a plurality of anti-skid protruding points are arranged on the guide needle handle and the guide wire handle.

By means of the scheme, the invention has at least the following advantages:

the in-vivo cell capturing tube in the in-vivo cell capturing device takes the functionalized protein silk thread as a carrier, has extremely strong tensile strength, has better toughness, and can completely resist the high-speed impact of venous blood. Also, the diameter of the protein thread is very small, typically around 10-15 μm, only 1/30, sometimes even 1/50 of that of a conventional capture thread. The diameter of the natural protein silk thread is equivalent to the diameter of the cells to be captured, which is beneficial to reducing the steric hindrance of the capture carrier. At the same time, the superfine protein wires macroscopically show obvious softness, and in the blood flowing at high speed, the soft wires swing at high speed to make the wires fully contact with the cells to be captured, so that the problem that the rigid carrier is difficult to contact with target cells is solved. Because the protein silk threads are extremely fine, a plurality of capturing silk threads can be fixed in the cell capturing tube at the same time, the capturing device is placed in a vein, and the normal flow of blood is not obviously influenced, so that the specific surface area of the capturing device is greatly increased, the probability of contacting cells to be captured in the blood is obviously increased, the capturing rate of the cells is obviously improved, and the efficient cell capturing is realized. As the raw materials of the functional protein silk thread are natural proteins, the functional protein silk thread has excellent biocompatibility and good biodegradability, and if fracture occurs, the functional protein silk thread can be taken out by clinical operation like the current vascular indwelling materials such as indwelling needles and indwelling catheters, and can also be self-biodegraded, thus greatly reducing the risk of fracture of the capturing thread on human body.

The foregoing description is only an overview of the present invention, and is intended to provide a better understanding of the present invention, as it is embodied in the following description, with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 is a schematic diagram of the structure of an in vivo cell harvester according to the present invention;

FIG. 2 is an enlarged view of FIG. 1 at circle A;

FIG. 3 is a graph showing the selective results of the cell arrestor of the invention versus the control group for CTC capture;

FIG. 4 is a photomicrograph of CTC captured by the control group;

FIG. 5 is a photomicrograph of CTC captured by the cell harvester of the present invention;

FIG. 6 is a graph of test results of the relationship between the number of immobilized functionalized protein threads and the number of CTC captures in the cell harvester according to the invention;

FIG. 7 is a scanning electron micrograph of immobilized functionalized protein strands in a cell harvester according to the invention;

FIG. 8 is a scanning electron microscope photograph of the surface of the immobilized functionalized protein wire in the cell catcher of the present invention immobilized with silicon nanoparticles;

FIG. 9 shows the results of toxicity tests of different substances on cells;

FIG. 10 shows the results of hemolysis experiments of different substances;

FIG. 11 is a clotting assay test result of a functionalized protein wire and a medical stainless steel wire in a cell harvester of the invention;

reference numerals illustrate:

1-a liquid storage sleeve; 2-a guide pin; 3-protein silk thread; 4-sealing plugs; 5-a guide plug; 6-a guide pin handle; 7-positioning lines; 8-capture ligand; 9-fixing the tube; 10-hydrogel; 11-a guide screw; 12-a guidewire handle; 13-preserving fluid; 14-sealing the cover; 15-anti-skid convex points.

Detailed Description

The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

Example 1

Referring to fig. 1-2, an in vivo CTC cell trap using functionalized protein wires as carriers in the preferred embodiment of the present invention includes an in vivo CTC cell trap tube, which includes a fixing tube 9 and a plurality of functionalized protein wires, preferably 40. The fixing tube 9 is a hose made of high molecular polymer such as polytetrafluoroethylene, PVC, FEP or polyurethane. One end of the functionalized protein silk thread is fixedly connected in the fixed tube 9 through the hydrogel 10, the functionalized protein silk thread comprises a protein silk thread 3 and a functional substance fixedly connected with the surface of the protein silk thread 3, the functional substance is connected with a capturing ligand 8 for capturing cells, and the protein silk thread 3 can be mulberry silk, tussah silk or spider silk.

The guide pin 2 is sleeved outside the fixed tube 9, the sealing plug 4 is fixedly connected in the guide pin 2, the sealing plug 4 surrounds the outer side of the upper end of the fixed tube 9, the sealing plug 4, the fixed tube 9 and the guide pin 2 enclose a cavity with one end provided with an opening, and the other end of the functional protein silk thread is positioned in the cavity and can move freely relative to the fixed tube 9.

The detachable stock solution unit that is connected with in syringe needle department of guide pin 2, stock solution unit include stock solution cover 1, sealed lid 14 and be located stock solution 13 of stock solution cover 1, have seted up the through-hole on the sealed lid 14, and the syringe needle of guide pin 2 wears to locate in the through-hole and communicates with stock solution cover 1, and stock solution cover 1 is interior and above-mentioned cavity intussuseption is filled with stock solution 13. The preservation solution 13 prevents the capture ligand 8 on the functionalized protein wire from being inactivated during preservation.

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

The outside fixedly connected with flaky guide pin handle 6 of guide pin 2, the end of guide screw 11 and be connected with flaky seal wire handle 12 all are equipped with a plurality of anti-skidding bump 15 on guide pin handle 6 and the seal wire handle 12, and the material of guide pin handle 6 and seal wire handle 12 is elastic material, conveniently fixes on skin when using. The guide needle handle 6 and the guide wire handle 12 are provided with small holes, and the small holes are fixedly connected with positioning wires 7. The positioning wire 7 is arranged so that one end of the guide screw 11 does not slide out of the guide needle 2 when the guide screw 11 slides relative to the guide needle 2.

The method of immobilizing the functionalized protein wire with capture ligand 8 and its immobilization within the immobilization tube 9 (i.e., preparation of the in vivo CTC cell capture tube) is as follows:

(1) Placing the extracted protein silk (including mulberry silk, tussah silk or spider silk) into a fixing tube, injecting hydrogel to fix the protein silk, and then placing the protein silk into degumming liquid for degumming. The degummed protein silk is washed for a plurality of times by three distilled water, and the fixed protein silk is obtained.

(2) The immobilized protein filaments (including the immobilization tube) were placed in a round bottom flask and reacted with diglycidyl ether (including polyethylene glycol diglycidyl ether or 1, 4-butanediol diglycidyl ether) and PAA, respectively, or with diglycidyl ether (including polyethylene glycol diglycidyl ether or 1, 4-butanediol diglycidyl ether), polyamine compounds (ethylenediamine, propylenediamine, butylenediamine, or PEI), and PAA, respectively, in a water bath at 60 ℃ for 6 hours. And then washing the surface-functionalized immobilized protein yarn for a plurality of times by using distilled water to obtain the immobilized protein yarn with the surface functionalized surface.

(3) The surface functionalized immobilized protein filaments were placed in a round bottom flask, EDC and NHS solution were added, shaken, and reacted in a water bath at 37 ℃ for 3 hours. After the reaction was completed, the reaction mixture was washed with distilled water several times. Then put it into round bottom bottle, add capture ligand (epithelial cell adhesion molecule antibody, nucleic acid aptamer or polypeptide aptamer) of CTC, shake, react in water bath at 37 ℃ for 3 hours. After the reaction was completed, the reaction mixture was washed with distilled water several times. Then put it into round bottom bottle, add alkylamine, shake, react in water bath at 25 deg.C for 4 hours. And after the reaction is finished, washing the mixture for multiple times by using distilled water to obtain the in-vivo CTC cell capturing tube.

When the target capturing cell is other rare cell, different capturing ligands 8 may be attached to the protein thread 3 via functional substances, as required.

According to the structure, the in-vivo CTC cell capturing tube and other parts are assembled into the in-vivo CTC cell capturing device, after the assembly is completed, the liquid storage sleeve 1 is opened, the cavity where the functionalized protein silk thread is positioned is filled with the preservation liquid 13 through the tail end of the guide pin 2, the preservation liquid 12 is also added into the liquid storage sleeve 1, the liquid storage sleeve 1 is sealed, and the liquid storage sleeve is sterilized for later use.

The use steps of the in vivo CTC cell catcher are as follows:

in use, the lead 2 is withdrawn from the fluid reservoir 1 and the needle is then inserted into the blood vessel at about 0.5 cm to about 1.0cm. The guide screw 11 is then pushed about 2cm, and the guide screw 11 pushes the fixing tube 9 and the connected functional protein thread 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. During high-speed blood flow, a plurality of functionalized protein threads immobilized on the immobilization tube 9 swing at high speed in flowing blood, and are fully contacted with CTC, so that CTC capture ligand immobilized on the CTC interacts with a receptor on the CTC, thereby immobilizing the CTC on the protein threads 3. During the capturing process, the guide wire handle 12 and the guide needle handle 6 can be fixed on the skin by using the adhesive tape, so that the movement of the patient is not affected. After capturing for a period of time, the guide wire rod 11 is pulled to drive the capturing tube to enter the guide needle 2 again, and then the guide wire handle 12 is pulled to pull the guide needle 2 out of the blood vessel, so that the in-vivo CTC cell capturing process is completed.

In order to verify the effect of the CTC cell trap in the body of the present invention, the trap tube was pushed out of the guide needle with the functionalized protein thread completely exposed, washed with PBS for 2min. The washed trapping tube was put into pancreatin for digestion for 3min, and the digestion was stopped by adding medium, and the trap was taken out. Finally, the captured CTCs in the medium are counted and analyzed. For control purposes, cell arresters were made from protein threads without any treatment and CTCs were captured in vivo using the same procedure. The results of selectivity of CTC capture for both sets of experiments are shown in fig. 3, and it can be seen from fig. 3 that the cell capture efficiency of the CTC cell capturer of the present invention is significantly higher than that of the control experiment. Figures 4-5 are photomicrographs of CTCs captured by two sets of cell arresters, indicated by the arrows in the figures as CTC cells, with significantly more CTCs captured by the arresters of the invention than the control.

The number of immobilized functionalized protein wires in the cell harvester was changed, and thus the number of CTC captures was also changed, and fig. 6 is a graph showing the relationship between the number of functionalized protein wires and the number of CTC captures, and it can be seen from the graph that the number of CTC captures was increased as the number of functionalized protein wires was increased. This also further demonstrates that the capture efficiency of a trap constructed with multiple small diameter protein threads as carriers can be significantly increased, i.e. significantly higher than a trap constructed with a single carrier material.

In addition, in order to change CTC capturing efficiency, nanoparticles may be immobilized in the functionalized protein wire, and fig. 7 is a scanning electron microscope photograph of the functionalized protein wire immobilized in the cell harvester of the present invention; fig. 8a is a scanning electron microscope photograph in which silicon nanoparticles are immobilized, and fig. 8b is an enlarged view of the silicon nanoparticles in fig. 8 a.

FIGS. 9-10 show the toxicity test results and the hemolysis test results of different substances on cells, and FIGS. 9-10 show that the functionalized protein wire has smaller cytotoxicity, extremely small hemolysis and almost equivalent to a negative sample. The silk thread-ligand in the figure is the functional protein silk thread used in the invention. Fig. 11 is a test result of the coagulation test of the functionalized protein wire and the medical stainless steel wire, which shows that the functionalized protein wire of the invention has similar coagulation property with the commercial medical stainless steel wire widely used currently and can be applied to human bodies.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and it should be noted that it is possible for those skilled in the art to make several improvements and modifications without departing from the technical principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

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.