CN111632202A

CN111632202A - Tumor cell adhesion material and preparation method and application thereof

Info

Publication number: CN111632202A
Application number: CN202010607320.9A
Authority: CN
Inventors: 欧来良
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2020-06-29
Filing date: 2020-06-29
Publication date: 2020-09-08

Abstract

The invention provides a tumor cell adhesion material and a preparation method and application thereof, and relates to the technical field of medicines. In addition, the material is simple to prepare, low in cost and easy to operate in use, and has important clinical value and economic and social benefits.

Description

Tumor cell adhesion material and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a tumor cell adhesion material and a preparation method and application thereof.

Background

Cancer diseases are continuously high, cancer becomes the first cause of death in cities, the death rate of cancer patients is not reduced by the improvement of science and technology, and the death rate of cancer patients is shown by investigation, and 90 percent of cancer patients are caused by recurrent metastasis of cancer. Therefore, how to prevent and reduce the metastasis of tumor becomes a significant problem.

How to effectively reduce the metastasis of the tumor is an important factor for improving the prognosis of the tumor. However, there are many types of tumors, and metastasis has already occurred when the tumor is found, which is also one of the reasons for poor prognosis of tumor treatment. However, it is generally considered that the metastasis of tumor is mainly based on several ways, one of which is blood metastasis, and the metastasis accounts for more than 80% of tumor metastasis; the second is lymphatic metastasis; the third is wound spreading, such as local shedding of tumor cells caused by operation. Therefore, how to block the metastasis of tumor cells will be an important issue for the tumor treatment.

Among the factors causing tumor metastasis, Circulating Tumor Cells (CTCs) refer to tumor cells released into peripheral blood circulation from solid tumors or metastases during diagnosis and treatment operations, and the local scattering and scattering in the operation area account for more than 90% of tumor metastasis. Therefore, if circulating tumor cells or tumor cells scattered in the operation area can be effectively eliminated, the risk of tumor metastasis can also be effectively reduced.

The rapid adhesion technology of tumor cells is reported in the detection of circulating tumor cells both at home and abroad, and most of cell adhesion materials for tumor cell detection adopt methods of antigen-antibody, exosome, DNA, RNA and the like for identification, for example, the technology for capturing CTC based on the CTC surface marker principle includes: CTC-chip (CTC-chip), iFISH-CTC (immunofluorescence staining in combination with FISH technology), and the like. The inventor also bases on the surface grafting of multiple antibodies on the anti-fouling material for capturing and detecting tumor cells (patent application number: 201910485658.9), and the technology based on biomolecule recognition has a wide detection prospect, has very few researches on clearing CTC, and is rarely reported for clearing scattered tumor cells in an operation area. Because the adhesion materials based on biomacromolecules (antibodies, exosomes, DNA and the like) as ligands have acceptable cost when being used for trace detection, but have no feasibility in aspects of manufacturing process, cost and the like when being used for removing a large number of tumor cells.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The main object of the present invention is to provide a tumor cell adhesion material to alleviate at least one of the technical problems of the prior art.

The second object of the present invention is to provide a method for preparing the tumor cell adhesion material.

The third purpose of the invention is to provide the application of the tumor cell adhesion material.

The fourth purpose of the invention is to provide a medical appliance comprising the tumor cell adhesion material provided by the invention.

The invention provides a tumor cell adhesion material, which comprises a carrier, an anti-fouling polymer, a connector and a small molecule adhesion ligand;

the anti-fouling polymer is coated on the surface of the carrier;

the connector is arranged on the surface of the carrier and is used for coupling the small molecule adhesion ligand.

Further, the carrier includes a magnetic carrier or a non-magnetic carrier.

Further, the magnetic carrier includes magnetic iron oxide;

optionally, the non-magnetic carrier comprises polyvinyl alcohol, chitosan, cellulose, agarose, polymethacrylates, or polystyrene.

Further, the anti-fouling polymer comprises polyvinyl alcohol, polycaprolactone, polyethylene glycol or polyacrylate.

Further, the linker comprises a molecular arm or a cross-linking agent;

preferably, the molecular arm bears a bifunctional group;

preferably, the molecular arm carries one or more of an amine group, a carboxyl group, an aldehyde group or a hydroxyl group.

Further, the small molecule adhesion ligand comprises one or more of a small molecule pectin, a polypeptide, a folate, or a polyamine compound.

Further, the shape of the tumor cell adhesion material comprises a tube shape, a sheet shape, a flocculent shape, a block shape, a powder shape or a spherical shape.

The invention also provides a preparation method of the tumor cell adhesion material, which comprises the steps of treating the carrier by the anti-fouling polymer, activating by the connector, and coupling the small molecule adhesion ligand to obtain the tumor cell adhesion material.

The invention also provides the application of the tumor cell adhesion material in removing tumor cells; and/or the presence of a gas in the gas,

the application in preparing products for preventing and treating tumor metastasis.

In addition, the invention also provides a medical appliance, which comprises the tumor cell adhesion material.

Compared with the prior art, the invention has the following beneficial effects:

the tumor cell adhesion material provided by the invention comprises a carrier, an anti-fouling polymer coated on the surface of the carrier, a molecular arm arranged on the surface of the carrier and a coupled small molecular adhesion ligand, wherein the anti-fouling polymer can effectively improve the blood compatibility of the tumor cell adhesion material, and the tumor affinity small molecular adhesion ligand is coupled to the carrier through a connector, so that the tumor cell adhesion material provided by the invention has strong adhesion capacity to tumor cells and can specifically adhere to the tumor cells in blood, body fluid or solution in an operation area, thereby reducing the occurrence of metastatic cancer caused by blood metastasis or surgical wound dissemination of primary cancer, and providing an auxiliary treatment method for cancer treatment. In addition, the material is simple to prepare, low in cost and easy to operate in use, and has important clinical value and economic and social benefits.

The preparation method of the tumor cell adhesion material provided by the invention is simple in process, convenient to operate and suitable for popularization and application.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention aims to solve the problems of tumor metastasis and the like caused by local scattering of tumor cells caused by clinical circulating tumor cells and operation and the like, and provides a tumor cell adhesion material which is simple to operate, short in treatment time, reliable in result, moderate in price and capable of effectively removing scattered tumor cells in blood and operation areas and a preparation method thereof.

According to one aspect of the present invention, there is provided a tumor cell adhesion material comprising a carrier, an anti-fouling polymer, a linker and a small molecule adhesion ligand;

the anti-fouling polymer is coated on the surface of the carrier;

In the tumor cell adhesion material provided by the invention, the anti-fouling polymer can effectively improve the blood compatibility of the tumor cell adhesion material, and the tumor affinity small molecule adhesion ligand is coupled to the carrier through the connector, so that the tumor cell adhesion material provided by the invention has strong adhesion capability to tumor cells, and can specifically adhere to the tumor cells in blood, body fluid or solution in an operation area, thereby reducing the occurrence of metastatic cancer caused by blood metastasis or surgical wound dissemination of primary cancer, and providing an auxiliary treatment method for cancer treatment. In addition, the material is simple to prepare, low in cost and easy to operate in use, and has important clinical value and economic and social benefits.

In the present invention, the carrier may include a magnetic carrier or a non-magnetic carrier.

According to the actual adsorption requirement, different materials can be selected as carriers to prepare the tumor cell adhesion material. For example, non-magnetic carriers can be used to remove tumor cells directly from blood, body fluids, or surgical field solutions; the magnetic carrier can be dispersed in blood, body fluid or solution, and then the magnetic carrier is adsorbed by the magnet, thereby eliminating tumor cells in the blood, body fluid or solution in an operation area.

An alternative magnetic carrier comprises magnetic iron oxide.

Alternative non-magnetic carriers include polyvinyl alcohol, chitosan, cellulose, agarose, polymethacrylates, or polystyrene.

In the invention, after the carrier material is subjected to surface anti-fouling treatment by the anti-fouling polymer, the required material with excellent blood compatibility can be obtained.

The specific choice of the antifouling polymer is not limited, and any high molecular substance capable of increasing the blood compatibility of the carrier can be used. Preferably, the anti-fouling polymer comprises polyvinyl alcohol, polycaprolactone, polyethylene glycol or polyacrylate, chitosan, polyamino acid and the like according to different uses of the tumor cell adhesion material. For example, when the anti-fouling polymer is used for removing tumor cells from blood, materials with excellent blood compatibility are required, and polyvinyl alcohol, polyethylene glycol, polycaprolactone, polyacrylate and the like can be selected as the anti-fouling polymer; for the removal of tumor cells in non-blood, such as tissue fluid or surgical solution, materials with excellent tissue compatibility, including chitosan, polyamino acids, polyacrylic acids, etc., can be used as the anti-fouling polymer of the present invention.

In the invention, the linker is used for activating the surface of the carrier, so that the activated surface of the carrier can be coupled with a small molecule adhesion ligand having affinity to tumor cells.

Typical linkers may be molecular arms or cross-linkers.

The type of the molecular arm functional group is generally determined according to the functional group of the ligand to be grafted, so as to ensure that the two functional groups can chemically react, and in the present invention, the molecular arm preferably has a bifunctional group, such as, but not limited to, one or more of an amine group, a carboxyl group, an aldehyde group, or a hydroxyl group.

It should be noted that the meaning of "one or more" means that the specific selection of the bifunctional group can be one of an amine group, a carboxyl group, an aldehyde group or a hydroxyl group, or two, three or four of them, according to different applications. The invention is not limited in this regard.

In the invention, the micromolecule adhesion ligand has high affinity to the tumor cells, and the effective adsorption to the tumor cells can be realized by coupling the micromolecule adhesion ligand. The small molecule adhesion ligand comprises one or more of a small molecule pectin, a polypeptide, a folate, or a polyamine compound, depending on the use of the tumor cell adhesion material. For example, when the polypeptide, the pectin and the folic acid are used for removing tumor cells in blood, the polypeptide, the pectin and the folic acid are generally used as the small molecule adhesion ligands in the invention, and the compatibility with blood is better; when the polypeptide is used for removing tissue fluid or liquid tumor cells in an operation area, besides the aglycone, polyamine aglycone can be used.

Likewise, "one or more" means that the particular choice of small molecule adhesion ligand may be one, or two, three or four, of small molecule pectin, polypeptide, folate or polyamine compounds, depending on the application. The invention is not limited in this regard.

Among them, the polypeptide is preferably a polypeptide having 18 or less amino acids, for example, RKEM, RHCLS, RHCLSGGGRKEM, etc.

In the present invention, the carrier may have any shape, and for example, but not limited to, a tube shape, a sheet shape, a flocculent shape, a block shape, a powder shape, or a spherical shape.

When the carrier is tubular, spherical or blocky, the carrier can be more suitable for removing tumor cells in circulating blood; when the carrier is in the form of powder, sheet, flocculent or sphere, the carrier can be more suitable for removing tumor cells in body fluid or operation area solution.

According to a second aspect of the present invention, the present invention further provides a preparation method of the tumor cell adhesion material, comprising the steps of treating the carrier with the anti-fouling polymer, activating the carrier with the linker, and coupling the small molecule adhesion ligand to obtain the tumor cell adhesion material.

Based on the beneficial effects of the tumor cell adhesion material provided by the invention, the invention also provides the application of the tumor cell adhesion material in removing tumor cells. And the application of the tumor cell adhesion material in preparing products for preventing and treating tumor metastasis.

The tumor cell adhesion material provided by the invention can adsorb all types of tumor cells and can adhere to the tumor cells in blood, body fluid and operating area exudate. It can be used for preventing tumor cell metastasis before or after operation.

By the specific adhesion effect of the tumor cell adhesion material on tumor cells, the tumor cell adsorption material can be applied to the existing medical instruments, is convenient for clinical operation or use, and can be used as a coating material of extracorporeal circulation or venous circulation system pipelines during operation; also can be used for the membrane surface coating of a dialyzer or hemofilter; and the like, in combination with the above medical instruments, and the present invention is not limited thereto.

The invention is further illustrated by the following examples. The materials in the examples are prepared according to known methods or are directly commercially available, unless otherwise specified.

Example 1 preparation and application of tumor cell adhesion microspheres

1. Preparation of tumor cell adhesion microsphere carrier

Synthesizing a polyvinyl alcohol (PVA) spherical support by a suspension polymerization method:

1000mL of a 1.5% (w/v) aqueous polyvinyl alcohol solution was prepared.

② 67.6mL of monomeric vinyl acetate, 27g of crosslinking agent triallyl isocyanurate (TAIC), 45mL each of liquid pore-forming agents ethyl acetate and n-heptane were added into a 1000mL three-necked flask and stirred at 40 ℃ until completely dissolved (about 30 min). 2g of Azobisisobutyronitrile (ABIN) as an initiator was added to the reaction system, and stirred until completely dissolved.

③ adding 500mL of water phase (1.5 percent (w/v) of polyvinyl alcohol aqueous solution) prepared in advance, adjusting the stirring speed until the system is dispersed into even small oil droplets, gradually raising the temperature to 75 ℃, and reacting for 3 hours to fully react.

Fourthly, washing the obtained microspheres with hot water to slightly wash away the oil, screening and washing the microspheres with a 200-mesh sieve until the microspheres are odorless, and airing the microspheres. And then fully extracting the microspheres for about 48 hours at 90 ℃ by using methanol to remove the pore-forming agent and redundant organic matters (monomer and pore-forming agent) in the microspheres.

Fifthly, adding the microspheres into 3 percent (w/v) NaOH/methanol solution, performing ester exchange for 18 hours at 40 ℃, and replacing the solution once during the reaction. Washing the microspheres to neutrality with water, filtering with methanol, and air drying. And finally, extracting methanol for 24 hours to fully remove impurities and the like in the microspheres, and drying at low temperature to obtain the required spherical carrier.

2. Activation of spherical carriers coupled with ligands

Taking 10g of the microspheres, adding the microspheres into 100ml of 10% (w/v) epoxy chloropropane aqueous solution, adjusting the pH value to 11 by NaOH, reacting for 4 hours at 50 ℃, and then fully cleaning by using 200ml of deionized water. And adding the activated microspheres into a 1% (w/v) micromolecular pectin solution, reacting for 1.5 hours under the condition of pH 10, filtering out reaction liquid, and washing with deionized water to obtain the required tumor cell adhesion microspheres.

EXAMPLE 2 preparation of tumor cell adhesive sheet

1. Preparation of adhesive sheet Carrier

Taking 10g of chitosan, putting into 500ml of 1% acetic acid aqueous solution, stirring and dissolving for 4 hours at room temperature, pouring into a container with a certain shape, then slowly adding 1% NaOH aqueous solution to solidify the solution into a film, washing with a large amount of water, freezing in a refrigerator at-70 ℃, and drying for 24 hours by a freeze dryer after freezing is finished for later use.

2. Grafted molecular arm

Taking lyophilized adhesive sheet carrier 10cm²Adding NH with the concentration of 1mg/ml prepared in advance in excess₂And (3) reacting at room temperature for 30 minutes, absorbing and removing reaction liquid, and washing with PBS for 3 times, wherein each time is 5 minutes, so as to obtain the crosslinked adhesive sheet carrier with ligand bonding activity.

3. Coupled affinity ligands

Mixing the above 10cm after activation²Adhering the sheet, putting the sheet into 10ml of 10mg/ml folic acid aqueous solution prepared in advance, and reacting for 2 hours at 45 ℃ to obtain the tumor cell adhering and adsorbing sheet.

EXAMPLE 3 preparation of tumor cell-adhering Cotton

1. Activation of adhered cotton

Taking 10g of dry medical cotton, adding 100ml of dimethyl sulfoxide and 80ml of epoxy chloropropane, swelling for 30min at 40 ℃, adding 10ml of 3mol/l NaOH solution, carrying out oscillation reaction for 5h at 40 ℃, removing reaction liquid, leaching with deionized water to be neutral, storing at 4 ℃ for later use, and obtaining the activated cotton ball with the epoxy value of 150 umol/g.

2. Coupling of adhesion Cotton ligands

And (3) weighing 10g of activated wet cotton, adding the wet cotton into an aqueous solution containing 50mg of polypeptide RKEM, oscillating and reacting for 24h at 37 ℃, filtering, and leaching with deionized water to be neutral to obtain the cotton ball for tumor cell adhesion.

EXAMPLE 4 preparation of magnetic tumor cell adhesion microspheres

1. Preparation of coated magnetic microspheres

Taking 5g of ferroferric oxide microspheres with the particle size of 10-15nm, putting the ferroferric oxide microspheres into a beaker, adding 10ml of a 1% acetic acid aqueous solution of 1% chitosan prepared in advance into the beaker, quickly stirring the mixture (30-50 rpm) for 30 minutes, and gradually heating the mixture (1-2 ℃/minute) to 60 ℃ until the microspheres are fully dried. Then slowly dripping 0.1% sodium hydroxide water solution to solidify the coating agent for later use.

2. Anti-fouling arm ligand coupling of magnetic microspheres

5g of the prepared film magnetic microspheres are taken and put into a 50ml flask, and then 2 percent NH is added into the flask₂10ml of PEG-NH2 solution, then slowly adding 1% glutaraldehyde solution dropwise to coat the chitosan and make active NH on the chitosan₂Radical with NH₂PEG-NH2 coupling or cross-linking to obtain magnetic microsphere with great amount of amine groups.

Experimental example 1 tumor cell adhesion test

Examination of tumor cell adhesion effects of the tumor cell adsorbents prepared in examples 1, 2, 3, and 4.

1.5 ml of PBS is taken respectively, a certain amount of MCF7-pEGFP cells are added, the mixture is stirred and mixed evenly, 0.25g of the tumor cell adhesion material prepared in the above embodiment is added respectively, and the mixture is incubated and adhered for 30min at 37 ℃ by shaking. Each sample was tested in duplicate 3 times.

2. The incubated suspensions of examples 1, 2 and 3 were added to a filtration apparatus, filtered to allow the liquid to slowly pass through the filter and filter (0.22 μm pore size), and 5ml of buffer was added to flush the walls of the loading tube and the filter. The incubated suspension of example 4 was passed through a magnet ring and then through a filter unit, the liquid was filtered slowly through the filter and filter (0.22 micron pore size) and 5ml of buffer was added to wash the walls of the loading tube and the filter.

3. The filters were removed, fixed on glass slides, and tumor cells were identified and counted under a fluorescence microscope.

4. The results show that the adhesion rate of the material of the invention to various tumor cells in PBS is above 90%.

Experimental example 2

The kits prepared in examples 1, 2, 3, 4 were used to detect in vitro mock circulating tumor cells.

1. 10000 tumor cells (HeLa cells) pre-labeled with red fluorescent DiI were added to the peripheral blood of 7.5ml healthy volunteers to simulate the peripheral blood specimen of tumor patients. 0.5g of the tumor cell adhesion material was added, and incubated at 37 ℃ for 30 min.

2. The suspension after incubation in examples 1, 2 and 3 was added to a filtration apparatus, the filtration process was performed to allow the liquid to slowly pass through the filter and the filter membrane, and 5ml of buffer was added to wash the walls of the loading tube and the filter membrane. The incubated suspension of example 4 was passed through a magnet ring and then through a filter unit, the liquid was filtered slowly through the filter and filter (0.22 micron pore size) and 5ml of buffer was added to wash the walls of the loading tube and the filter.

3. And taking out the filter membrane, fixing the filter membrane on a glass slide, and identifying and counting the tumor cells according to a judgment standard under a fluorescence microscope.

4. The results show that the adhesion rate of the adhesion material to the in vitro simulated blood tumor cells is over 85 percent.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tumor cell adhesion material, which is characterized by comprising a carrier, an anti-fouling polymer, a connector and a small molecule adhesion ligand;

the anti-fouling polymer is coated on the surface of the carrier;

2. The tumor cell adhesion material of claim 1, wherein the carrier comprises a magnetic carrier or a non-magnetic carrier.

3. The tumor cell adhesion material of claim 2, wherein the magnetic carrier comprises magnetic iron oxide;

4. The tumor cell adhesion material of claim 1, wherein the anti-fouling polymer comprises polyvinyl alcohol, polycaprolactone, polyethylene glycol, or polyacrylate.

5. The tumor cell adhesion material of claim 1, wherein the linker comprises a molecular arm or a cross-linking agent;

preferably, the molecular arm bears a bifunctional group;

6. The tumor cell adhesion material of claim 1, wherein the small molecule adhesion ligand comprises one or more of a small molecule pectin, a polypeptide, a folate, or a polyamine compound.

7. The tumor cell adhesion material of any one of claims 1-6, wherein the shape of the tumor cell adhesion material comprises a tube, a sheet, a flocculent, a block, a powder or a sphere.

8. The method for preparing the tumor cell adhesion material of any one of claims 1 to 7, which comprises treating a carrier with an anti-fouling polymer, activating the carrier with a linker, and coupling small molecule adhesion ligands to obtain the tumor cell adhesion material.

9. Use of a tumor cell adhesion material according to any one of claims 1 to 7 for the elimination of tumor cells; and/or the presence of a gas in the gas,

10. A medical device comprising the tumor cell adhesion material of any one of claims 1 to 7.