CN111840658A - Intravenous indwelling needle catheter and preparation method and application thereof - Google Patents
Intravenous indwelling needle catheter and preparation method and application thereof Download PDFInfo
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- A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
- A61M5/14—Infusion devices, e.g. infusing by gravity; Blood infusion; Accessories therefor
- A61M5/158—Needles for infusions; Accessories therefor, e.g. for inserting infusion needles, or for holding them on the body
- A61M2005/1587—Needles for infusions; Accessories therefor, e.g. for inserting infusion needles, or for holding them on the body suitable for being connected to an infusion line after insertion into a patient
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Abstract
The invention provides a vein indwelling needle catheter and a preparation method and application thereof. The outer wall of the venous indwelling needle catheter is modified with circulating tumor cell capture molecules, and the inner wall of the venous indwelling needle catheter is modified with carboxylated photosensitizer. The circulating tumor cell capturing molecules can be combined with CTCs surface proteins and modified on a catheter material, so that the enrichment of CTCs in blood in vivo can be realized, the detection of CTCs in blood of patients with early cancer or relapse is facilitated, the detection result is accurate and reliable, the biocompatibility of the venous indwelling needle catheter is good, the venous indwelling needle catheter can be easily inserted into and taken out from superficial blood vessels, CTCs in whole peripheral blood are captured, and the CTCs can be eluted and cracked for downstream analysis; meanwhile, the vein indwelling needle catheter can kill and kill the enriched CTCs under the action of near infrared light to realize targeted clearing, so the vein indwelling needle catheter has the double functions of enriching and killing the CTCs.
Description
Technical Field
The invention belongs to the technical field of separation and capture of circulating tumor cells, particularly relates to an enrichment method of circulating tumor cells, and particularly relates to a venous indwelling needle catheter and a preparation method and application thereof.
Background
Circulating Tumor Cells (CTCs) are currently defined as a class of tumor cells that are released into the peripheral circulation from solid tumors or metastases, either spontaneously or as a result of a diagnostic procedure. Detection of CTCs in peripheral blood is predictive of the potential for tumor metastasis. The number of CTCs in the periphery is too small, and thus the detection of CTCs must be enriched. The CTCs separation and capture technology mainly comprises a biochemical characteristic enrichment method (affinity enrichment method) and a physical characteristic enrichment method, and is respectively based on CTCs specific surface markers and physical properties.
CN110133294A discloses a method for capturing and detecting peripheral blood CTCs, which relates to the technical field of in vitro diagnosis and is characterized in that erythrocytes are removed firstly, the interference of the erythrocytes is reduced, then an antibody is easier to combine with the CTCs by adopting a scheme of preferentially combining a first affinity substance and the CTCs, and then a magnetic substance is adsorbed on the CTCs by utilizing the super-strong affinity of the first affinity substance and a second affinity substance, so that the CTCs are magnetic, and the CTCs are separated from the non-magnetic cells under the action of a magnetic field. Through the simultaneous incubation of various antibodies, the capture omission caused by the heterogeneity of CTCs can be avoided; and the magnetic separation is adopted, so that the mechanical damage to the CTCs is effectively reduced.
However, in vitro enrichment methods typically only use 5-10mL of blood for isolation, 5-10mL of blood represents only 0.1-0.2% of the total amount of human blood, contains very small amounts of CTCs, may have false negative results in patients with early cancer or relapse, and cannot be targeted at enrichment of CTCs in whole peripheral blood. Hemodialysis can treat several liters of blood, but is at risk for acute and distant complications and is not suitable for the isolation of CTCs in peripheral blood of patients with early stage tumors.
Therefore, there is a need to optimize the existing liquid biopsy techniques to achieve safe and convenient separation of CTCs from large volumes of blood.
Studies have shown that in cancer patients, such as metastatic breast and colorectal cancer patients, the number of CTCs has become an important predictor of progression-free survival and overall survival, with the greater the number of CTCs, the shorter the survival of the patients. Therefore, clearance of CTCs is crucial to reducing the metastatic potential of tumors, and if clearance of CTCs is performed early in a tumor, it may even prevent the occurrence of metastasis, ultimately improving patient prognosis.
However, surgical resection or chemoradiotherapy cannot help CTCs in peripheral blood, but rather reduces the immunity of the organism and accelerates the metastatic spread of CTCs. Moreover, therapies with novel tumors that target metastatic characteristics of the tumor, such as angiogenesis, lymphangiogenesis, specific signaling pathways or biomarkers, etc., have been reported to achieve desirable clinical results, but they are generally ineffective for CTCs.
Photodynamic therapy (PDT) for removing CTCs is carried out by intravenous injection of photosensitizer, and the photosensitizer accumulated in tumor cells is activated under 660nm laser irradiation and in the presence of oxygen to generate singlet oxygen to destroy the tumor cells. Current PDT applications are mainly limited to localized areas of disease, such as skin, head, neck, etc. Near-infrared (NIR) light has a lower tissue absorption rate than visible light and thus has a stronger tissue penetration ability. Novel two-dimensional nanomaterials with near-infrared light response characteristics, such as graphene, MXenes (two-dimensional transition metal carbides, nitrides or carbonitrides), transition metal sulfides, and Black Phosphorus (BP), have been widely used in photothermal therapy (PTT) of tumors.
Therefore, how to achieve the target elimination of CTCs and improve the prognosis of tumors by using the novel therapeutic methods is another key problem to be solved urgently in the field.
Disclosure of Invention
In view of the problems in the prior art, the invention provides a Venous indwelling needle (Venous) catheter and a preparation method and application thereof. The vein indwelling needle catheter has the functions of enriching and killing CTCs, and can be used for preparing a vein indwelling needle, the enrichment of CTCs in blood in vivo can be realized simultaneously when the outline function of the vein indwelling needle is not influenced, after the enrichment is finished, the CTCs enriched on the surface of the catheter can be further killed through the near infrared effect, and a new thought is provided for the separation and targeted removal of CTCs in large-volume blood.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a venous indwelling needle catheter, wherein the outer wall of the venous indwelling needle catheter is modified with a circulating tumor cell capturing molecule, and the inner wall of the venous indwelling needle catheter is modified with a carboxylated photosensitizer.
The material of the vein indwelling needle catheter is modified, so that the outer wall of the vein indwelling needle catheter is combined with CTCs capture molecules, the inner wall of the vein indwelling needle catheter is combined with a photosensitizer, the CTCs capture molecules can be combined with CTCs surface protein, the catheter is small in diameter, flexible and good in biocompatibility, can exist in a human body for a long time, can realize the enrichment of CTCs in blood in the human body, is beneficial to the detection of CTCs in blood of patients with early cancer or relapse, and enables the detection result to be accurate and reliable, the vein indwelling needle catheter can be easily inserted into and taken out of superficial blood vessels, so that CTCs in the whole peripheral blood are captured, CTCs can be eluted and cracked for downstream analysis, the enriched CTCs can be collected after the vein indwelling needle is taken out, and the gene detection and cell typing of the CTCs are realized; meanwhile, the photosensitizer is combined on the inner wall of the catheter, so that the enriched CTCs can be killed or killed under the action of near infrared light, the side effect of the targeted clearing method is small, the metastasis can be prevented, and the prognosis of a patient is finally improved.
In a preferred embodiment of the present invention, the venous indwelling needle catheter material contains a free amino group, and CTCs capture molecules are immobilized on the outer wall of the venous indwelling needle catheter by an amino-carboxyl condensation reaction.
Preferably, the material of the intravenous indwelling needle catheter contains free amino, and the carboxylated photosensitizer is fixed on the inner wall of the intravenous indwelling needle catheter through amino carboxyl condensation reaction.
In the invention, the catheter material of the venous indwelling needle left in the blood vessel contains free amino functional groups, and the venous indwelling needle and the catheter material can be fixed on the surface of the hose in a covalent coupling mode.
In a preferred embodiment of the present invention, the intravenous indwelling needle catheter is made of a polyurethane material.
In the invention, the chemical component of the catheter of the vein indwelling needle left in the blood vessel is polyurethane, and the chemical component is macromolecular compound with a main chain containing repeated urethane groups. Since the polyurethane contains free amino functional groups, carboxyl groups can be activated by carbodiimide, and antibodies for capturing CTCs can be fixed on the surface of the hose by means of covalent coupling.
Preferably, the circulating tumor cell capture molecule comprises: any one or a combination of at least two of an antibody, a polypeptide, or an aptamer that specifically binds to a surface antigen of CTCs.
Preferably, the circulating tumor cell capture molecule comprises an EpCAM antibody.
Epithelial cell adhesion molecules (EpCAM) are highly expressed in almost all Epithelial and highly invasive tumor cells, such as human liver cancer cells HepG2, but are not present in blood cells, which is beneficial to high-efficiency specific enrichment of CTCs. In the present invention, the surface of the intravenous indwelling catheter is coated with the EpCAM antibody, and CTC can be captured. Meanwhile, the CTC capture molecule may also be other molecules capable of binding CTC, such as polypeptide or aptamer.
As a preferable technical scheme of the invention, the photosensitizer has near infrared light response characteristics. The photosensitizer can realize local temperature rise of the catheter under the action of near infrared light, so that cells enriched on the catheter are killed.
Preferably, the photosensitizer comprises a photosensitive small molecule drug and/or a photosensitive nanomaterial.
Preferably, the coating concentration of the photosensitizer on the inner wall of the intravenous indwelling needle catheter is 5-10mg/mL, and may be, for example, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL or the like.
If the coating concentration of the photosensitizer on the inner wall exceeds 10mg/mL, the temperature in the intravenous indwelling needle catheter is too high, and the photosensitizer can influence the survival of normal cells and generate side effects besides killing CTCs.
Preferably, the photosensitizer comprises any one of graphene, MXenes, transition metal sulfides or black phosphorus or a combination of at least two of the same, preferably black phosphorus.
In a second aspect, the present invention provides a method for manufacturing an intravenous indwelling needle catheter according to the first aspect, comprising the steps of:
(1) injecting a mixed solution containing a carboxylated photosensitizer, an activating agent and a buffer solution into the catheter of the venous indwelling needle, reacting, cleaning, and then sealing unreacted activated carboxyl to obtain a carboxylated photosensitizer modified venous indwelling needle catheter;
(2) putting the vein indwelling needle catheter obtained in the step (1) into a mixed solution containing a circulating tumor cell capture molecule, an activating agent and a buffer solution, reacting, cleaning, and then sealing unreacted activated carboxyl to obtain the vein indwelling needle catheter;
alternatively, the preparation method comprises the following steps:
(1') putting the vein indwelling needle catheter into a mixed solution containing circulating tumor cell capture molecules, an activating agent and a buffer solution, reacting, cleaning, and then sealing unreacted activated carboxyl to obtain the vein indwelling needle catheter modified by the circulating tumor cell capture molecules;
(2 ') injecting a mixed solution containing a carboxylated photosensitizer, an activating agent and a buffer solution into the catheter of the venous indwelling needle obtained in the step (1'), reacting, cleaning, and then blocking unreacted activated carboxyl to obtain the venous indwelling needle catheter.
In the present invention, the order of modification of the carboxylated photosensitizer and the capture molecule of circulating tumor cells is not limited. As a preferred embodiment of the present invention, the activating agent in the step (1) and the step (2') comprises EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride).
Preferably, the mixed solution of step (1) and step (2') comprises: EDC solution at a mass concentration of 15-25mg/mL (e.g., 15mg/mL, 18mg/mL, 20mg/mL, 21mg/mL, 22mg/mL, 24mg/mL, 25mg/mL, etc.), a carboxylated photosensitizer at 15-25 μ g/mL (e.g., 15 μ g/mL, 18 μ g/mL, 20 μ g/mL, 22 μ g/mL, 23 μ g/mL, 24 μ g/mL, 25 μ g/mL, etc.), and PBS buffer.
Preferably, the operation of withdrawing the needle head of the venous indwelling needle before injecting the mixed solution into the catheter in the step (1) and the step (2') is further included.
Preferably, the method further comprises the step of returning the needle to the original position after the step (1) and the step (2') are finished.
As a preferred technical scheme of the invention, the activating agent in the step (2) and the step (1') comprises EDC.
Preferably, the mixed solution of step (2) and step (1') includes: 15-25mg/mL (e.g., 15mg/mL, 18mg/mL, 20mg/mL, 21mg/mL, 22mg/mL, 24mg/mL, 25mg/mL, etc.) EDC solution, 3-5 μ g/mL (e.g., 3 μ g/mL, 3.2 μ g/mL, 3.5 μ g/mL, 3.8 μ g/mL, 4 μ g/mL, 4.5 μ g/mL, or 5 μ g/mL, etc.) circulating tumor cell capture molecule, and PBS buffer;
in a preferred embodiment of the present invention, the method for blocking the activated carboxyl group comprises:
glycine is added at a molar concentration of 0.8-1.2mM (e.g., 0.8mM, 0.85mM, 0.9mM, 1mM, 1.05mM, 1.1mM, 1.15mM, or 1.2 mM) and shaken for 20-40min, e.g., 20min, 22min, 25min, 30min, 32min, 35min, or 40 min.
Preferably, the reaction temperature in step (1) and step (2') is 20-30 deg.C, such as 20 deg.C, 22 deg.C, 24 deg.C, 25 deg.C, 27 deg.C, 28 deg.C or 30 deg.C, and the time is 20-40min, such as 20min, 22min, 25min, 30min, 32min, 35min or 40 min.
Preferably, the reaction temperature in step (2) and step (1') is 20-30 deg.C, such as 20 deg.C, 22 deg.C, 24 deg.C, 25 deg.C, 27 deg.C, 28 deg.C or 30 deg.C, and the time is 20-40min, such as 20min, 22min, 25min, 30min, 32min, 35min or 40 min.
As a preferred technical scheme of the invention, the preparation method comprises the following steps:
(1) extracting a needle head of the venous indwelling needle by 1-2cm under the aseptic condition, injecting a mixed solution containing 15-25mg/mL of EDC solution, 15-25 mu g/mL of carboxylated photosensitizer and PBS buffer solution into the catheter of the venous indwelling needle, carrying out oscillation reaction at 20-30 ℃ for 20-40min, washing, adding 0.8-1.2mM of glycine, oscillating for 20-40min, sealing unreacted activated carboxyl, washing again, and restoring the needle head to the original position to obtain the carboxylated photosensitizer modified venous indwelling needle catheter;
(2) inserting the venous indwelling needle catheter obtained in the step (1) into a mixed solution containing 3-5 mug/mL of EpCAM antibody, 15-25mg/mL of EDC solution and buffer, carrying out shake reaction at 20-30 ℃ for 20-40min, cleaning, adding 0.8-1.2mM of glycine, and carrying out shake reaction for 20-40min to seal unreacted activated carboxyl, thus obtaining the venous indwelling needle catheter;
alternatively, the preparation method comprises the following steps:
(1') inserting the venous indwelling needle catheter into a mixed solution containing 3-5 mu g/mL of EpCAM antibody, 15-25mg/mL of EDC solution and buffer solution, carrying out oscillation reaction at 20-30 ℃ for 20-40min, washing, adding glycine with the molar concentration of 0.8-1.2mM, oscillating for 20-40min, and sealing unreacted activated carboxyl to obtain the circulating tumor cell capture molecule modified venous indwelling needle catheter;
(2 ') extracting the needle head of the venous indwelling needle by 1-2cm under the aseptic condition, injecting a mixed solution containing EDC solution with the mass concentration of 15-25mg/mL, carboxylated photosensitizer with the mass concentration of 15-25 mu g/mL and PBS buffer solution into the catheter of the venous indwelling needle in the step (1'), carrying out shake reaction at 20-30 ℃ for 20-40min, washing, adding glycine with the molar concentration of 0.8-1.2mM, shaking for 20-40min, sealing unreacted activated carboxyl, washing again, and restoring the needle head to the original position to obtain the venous indwelling needle catheter.
In the invention, the bifunctional venous indwelling needle catheter can be prepared by the following preparation method:
(1) modification of carboxylated photosensitizer on inner wall of catheter of intravenous indwelling needle
20mg EDC was weighed out and dissolved in pre-cooled 1mL PBS and ready to use. 980 mu.L of PBS, 2 mu.L of EDC solution and 20 mu g of carboxylated photosensitizer (the carboxylated photosensitizer comprises photosensitive micromolecular drug or nano material and the like) are added into an EP tube and mixed evenly. Extracting the needle head of a venous indwelling needle (24G multiplied by 0.75) by 1.5cm upwards under the aseptic condition, injecting the mixed solution into a hose, slightly shaking for reaction for 30min at room temperature, washing with PBS for 3 times, adding 1mM glycine, slightly shaking for 30min, sealing activated carboxyl, finally washing with double distilled water, and restoring the needle head to the original position.
(2) Modification of CTC capture molecule on outer wall of venous indwelling needle catheter
10mg EDC was weighed out and dissolved in pre-cooled 1mL PBS and ready to use. 980. mu.L of PBS, 1. mu.L of EDC solution and 4. mu.g of antibody were added to the EP tube, and the venous indwelling needle was inserted into the EP tube. Shaking to react for 30min at room temperature, washing with PBS for 3 times, adding 1mM glycine, shaking for 30min, and sealing activated carboxyl; adding 0.5% BSA, shaking for 30min, and blocking the non-specific sites on the surface of the catheter to obtain the venous indwelling needle catheter.
To further verify whether the venous indwelling needle catheter is bound to a CTC capture molecule, a secondary antibody may also be modified on the outer wall of the catheter, namely: adding secondary antibody labeled by HRP (horse radish peroxidase), oscillating at room temperature for 30min, washing with PBS for 4 times, 5min each time, adding substrate, developing for 5min, and stopping reaction with stop solution. The solution in the tube was transferred to a 96-well plate and the absorbance of the solution was measured at 450nm to determine whether CTC capture molecules were bound by absorbance.
Meanwhile, in the present invention, after the intravenous indwelling needle catheter is prepared, multiple verifications of cell level, in vitro and in vivo are also performed. The method specifically comprises the following steps:
(1) selection and validation of EpCAM positive and negative expression cell models
The expression level of EpCAM in different cells is searched in a website https:// www.proteinatlas.org/ENSG00000119888-EPCAM/cell, and finally HepG2 and HeLa cells are selected as cell models for positive expression and negative expression of EpCAM. The expression of EpCAM on the surface of both cells was further verified by flow cytometry, immunofluorescence and western blot.
(2) The functions of enriching and killing CTCs of the intravenous indwelling needle catheter are researched in a peripheral blood closed circulation simulation device (namely an in-vitro device) and a rabbit (New Zealand rabbit) auricular vein model, and meanwhile, the influence of the liquid flow rate on the near-infrared thermal effect of the photosensitizer in the catheter is researched in the peripheral blood closed circulation simulation device.
In a third aspect, the present invention provides a venous indwelling needle prepared using the catheter for an intravenous indwelling needle according to the first aspect.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the invention has at least the following beneficial effects:
the invention provides a vein indwelling needle catheter, which can realize enrichment by combining CTC capture molecules and a photosensitizer through the CTC capture molecules and CTC, and remove the enriched CTCs in vivo through the near infrared photothermal effect of the photosensitizer on the inner wall of the catheter in a targeted manner, so the vein indwelling needle catheter has the dual functions of enrichment and killing of the CTCs;
Meanwhile, the catheter has good biocompatibility, can be kept in a blood vessel for about 3 days, and can be easily inserted into and taken out of a superficial blood vessel, so that CTCs in the whole peripheral blood are captured, the purity of the enriched CTCs is high, the CTCs can be eluted and cracked for downstream analysis, the enriched CTCs can be collected after the venous indwelling needle is taken out, and the gene detection and typing of the CTCs are realized; the kit has important research significance and biomedical application prospect for realizing early diagnosis of tumor patients, reducing the number of circulating tumor cells in peripheral blood of postoperative tumor patients, reducing the probability of blood circulation metastasis and prolonging the life cycle of patients.
Drawings
Fig. 1 is a schematic view of the working principle and the using flow of the venous indwelling needle catheter provided by the invention.
Figure 2 is a schematic representation of the binding process of the EpCAM antibody to the polyurethane of the catheter material under EDC activation in example 1.
FIG. 3(a) is a standard graph used to calculate the number of HepG2 cells.
FIG. 3(b) is a standard graph for calculating the number of HeLa cells.
FIG. 4(a) is a graph of the capturing effect of the venous indwelling needle catheter on HepG2 and HeLa cells in both resting and flowing conditions (scale 200 microns) in example 2.
FIG. 4(b) is a histogram showing the capturing efficiency of HepG2 and HeLa cells in the static and flowing states of the catheter of the intravenous indwelling needle in example 2.
FIG. 5 is a graph showing the temperature of the irradiated portion of the catheter with the inner wall coated with black phosphorus of different concentrations under laser irradiation as a function of irradiation time in example 2.
FIG. 6(a) is a graph showing the effect of HepG2 cells on the outer wall of the catheter in example 2 on a fluorescence microscope (scale 100 μm) before and after near-infrared illumination, when stained with visible light, AO and PI.
FIG. 6(b) is a graph showing the effect of HeLa cells on the outer wall of the catheter in example 2 under a fluorescence microscope before and after near-infrared illumination, when stained with visible light, AO and PI (scale 100 μm).
FIG. 7 is a histogram showing the trapping efficiency of HepG2 and HeLa cells in rabbit ear vein model of the catheter of intravenous indwelling needle of example 3.
FIG. 8 is a graph showing the effect of HepG2 and HeLa cells on the outer wall of the catheter in example 3 under a fluorescent microscope (scale 100 μm) after near-infrared illumination when stained with visible light, AO and PI.
Detailed Description
The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.
In the following examples, consumables and reagents used were all commercially available from conventional manufacturers.
Firstly, the working principle of the vein indwelling needle catheter provided by the invention is described in detail with reference to fig. 1: modifying an antibody capable of capturing CTCs on the surface of a catheter of an intravenous indwelling needle, wherein the binding principle of CTCs capturing molecules and the catheter is shown in figure 2, and free carboxyl on the capturing molecules and free amino on the catheter material are subjected to dehydration condensation reaction under the activation of EDC and finally bound on the outer wall of the catheter; meanwhile, the inner wall of the conduit is decorated with photosensitive materials, and the principle of the photosensitive materials is the same as that shown in figure 2;
then the blood vessel is placed into the vein of a human body, and the normal body temperature of the human body is 37 ℃; after the outer wall of the catheter is combined with the CTCs, 808nm light irradiation is provided, the local temperature of the photosensitive material part is raised to 55 ℃, and the combined CTCs are killed; or taking out the venous indwelling needle, eluting the CTCs attached to the venous indwelling needle, and subsequently performing cell culture or high-throughput sequencing on the eluted CTCs so as to perform the next research.
Example 1
The embodiment provides an EpCAM antibody/black phosphorus bifunctional indwelling catheter, and the preparation method comprises the following steps:
(1) preparation and carboxylation modification of black phosphorus nanosheet
a) Liquid phase stripping of black phosphorus crystals: and stripping the block black phosphorus by adopting a non-contact probe ultrasonic liquid stripping method to obtain the few-layer black phosphorus nanosheet.
Firstly, grinding the block black phosphorus into powder, and dispersing the powder in an isopropanol solvent; carrying out probe ice bath ultrasonic treatment on the dispersion liquid, and stripping to obtain a few-layer black phosphorus nanosheet; and carrying out gradient centrifugation treatment on the black phosphorus dispersion liquid after ultrasonic treatment to obtain black phosphorus nanosheet solutions with different thicknesses and layer number distribution.
b) Preparation of carboxyl-modified black phosphorus nanosheet
20mg of DSPE-PEG-COOH (dipalmitoylphosphatidylethanolamine-polyethylene glycol-carboxyl) is suspended in 10mL of black phosphorus nanosheet (two-dimensional black phosphorus) solution, and the final concentration of black phosphorus is adjusted to be 10 mg/mL.
And (3) carrying out ultrasonic treatment on the room temperature probe for 30min, then slowly shaking for 4h, centrifuging at 12000rpm for 5min at 4 ℃, repeatedly washing twice by using PBS, and removing excessive DSPE-PEG-COOH.
And (4) suspending the purified carboxylated black phosphorus nanosheets in PBS, and storing at 4 ℃ for later use.
(2) Preparation of EpCAM antibody/black phosphorus bifunctional indwelling needle catheter
a) Carboxylated black phosphorus modified on outer wall of catheter and verification
20mg EDC was weighed out and dissolved in pre-cooled 1mL PBS buffer and ready to use. Add 980. mu.L of LPBS buffer, 2. mu.L of EDC solution and 20. mu.g of carboxylated black phosphorus to the EP tube and mix well. Extracting the needle head of a venous indwelling needle (24G multiplied by 0.75) by 1.5cm upwards under the aseptic condition, injecting the mixed solution outside a hose, slightly shaking for reaction at room temperature for 30min, washing with PBS for 3 times, adding 1mM glycine, slightly shaking for 30min, sealing activated carboxyl, finally washing with double distilled water, and restoring the needle head to the original position.
After coating, 808nm laser was used to irradiate the coated hose part with a power density of 1.0W/cm2And observing the change of the temperature of the irradiated part by a thermal imager to verify whether the black phosphorus nano material is coated successfully.
b) Catheter surface modification antibody and validation
10mg EDC was weighed out and dissolved in pre-cooled 1mL PBS and ready to use. 980. mu.L of PBS buffer, 1. mu.L of EDC solution and 4. mu.g of EpCAM antibody were added to the EP tube, and the venous indwelling needle was inserted into the EP tube. Shaking for 30min at room temperature, activating free carboxyl on the EpCAM antibody by EDC, and then performing condensation reaction with free amino on polyurethane, wherein the EpCAM antibody is combined with the polyurethane and fixed on the outer wall of the catheter.
Subsequently, washing with PBS buffer solution for 3 times, adding 1mM glycine, shaking for 30min, and blocking the activated carboxyl; and adding 0.5% BSA, shaking for 30min, and blocking non-specific sites on the surface of the catheter.
Adding HRP-labeled secondary antibody, oscillating at room temperature for 30min, washing with PBS for 4 times, each time for 5min, adding substrate, developing for 5min, and stopping reaction with stop solution. The solution in the tube was transferred to a 96-well plate and the absorbance of the solution was measured at 450nm, after which it was known that the surface of the catheter had bound to the EpCAM antibody.
Example 2
This example investigated the ability of the indwelling needle catheter provided in example 1 to enrich and kill CTCs by means of a peripheral blood closed circulation simulator.
(1) Selection and validation of EpCAM positive and negative expression cell models
The expression level of EpCAM in different cells is searched in a website https:// www.proteinatlas.org/ENSG00000119888-EPCAM/cell, and finally HepG2 and HeLa cells are selected as cell models for positive expression and negative expression of EpCAM.
(2) Construction of peripheral blood circulation simulation device
A peripheral blood circulation simulation device was constructed using a peristaltic pump and a flexible tube with an inner diameter of 2.5 mm. The liquid inlet pipe is 30cm long, and the liquid outlet pipe is 15cm long. The tubes were treated with 1% BSA for 30min to block non-specific binding sites. In the experiment, a liquid inlet pipe and a liquid outlet pipe are simultaneously inserted into a 50mL test tube, the test tube contains 40mL of single cell suspension preheated at 37 ℃, and the single cell suspension is circulated at the speed of 10 mL/min.
(3) Study of the Functions of the bifunctional cathether-enriched CTCs in an in vitro device
The double-functional catheter is inserted into a liquid inlet pipe and fixed, 10mL of DMEM culture solution containing HepG2 or HeLa single-cell suspension preheated at 37 ℃ is added into the test tube, the flow rate of the liquid is adjusted to be 10mL/min, after circulation for 15min, the catheter is carefully drawn out, and PBS is washed for 3 times.
Adding Trizol to lyse the cells, extracting RNA, detecting the expression level of EpCAM gene or GAPDH gene by RT-qPCR, and using cells with gradient concentration (1X 10)4，1×103，1×102) A standard curve was prepared, and the obtained standard curve was shown in FIG. 3(a) and FIG. 3 (b).
Wherein, fig. 3(a) is a standard curve of HepG2, specifically, y ═ 3.353x +33.273, R20.99942, fig. 3(b) shows a standard curve for HeLa, specifically y-3.325 x +28.746, R2The number of captured HepG2 and HeLa cells was calculated according to the standard curve, 0.99999.
FIGS. 4(a) and 4(b) show fluorescence microscopy patterns and capture efficiencies of HepG2 and HeLa cells captured by a venous indwelling needle catheter in a test tube (static) and a closed circulation simulator for peripheral blood (flow), respectively, with 10 additions to the test tube and the closed circulation simulator for peripheral blood in the static and flow states5Capturing HepG2 or HeLa cells for 30min at room temperature, digesting the captured cells by using 0.01% trypsin, extracting RNA in the cells, detecting by RT-qPCR to obtain Ct values of EpCAM and GAPDH of the HepG2 or HeLa cells respectively, and calculating by using a standard curve, wherein the capturing quantity of the HepG2 cells in a static state and a flowing state is 8100 and 2210, and the capturing efficiency is 8.1% and 2.21% respectively; the number of captured HeLa cells was 350 and 175, respectively, and the capturing efficiency was 0.35% and 0.175%, respectively.
(4) Research on influence of liquid flow velocity on near-infrared photothermal effect of black phosphorus in catheter in vitro device
Inserting catheters with inner walls coated with black phosphorus (0, 1, 5, 10mg/mL) with different concentrations into a liquid inlet pipe and then fixing, adding 40mL of DMEM culture solution without FBS (Fetal calf serum) into a test tube, irradiating the catheter indwelling part with 808nm laser when the liquid flow rates are 0 and 10mL/min respectively, and recording the change of the temperature of the irradiated part along with the irradiation time by using a thermal imaging instrument.
The graph of the temperature change with time is shown in fig. 5, the irradiation time is used as the abscissa, the temperature is used as the ordinate, when the black phosphorus is not coated, namely the concentration is 0mg/mL, the temperature inside and outside the catheter is consistent, the temperature of the catheter is increased along with the increase of the concentration of the black phosphorus under the irradiation of the near-infrared laser, the temperature of the catheter is increased along with the increase of the irradiation time, the local temperature is at most 10mg/mL, and the temperature can reach 65 ℃ after the irradiation is carried out for 140 min.
(4) Study of the Functions of the bifunctional ductal killer CTCs in an in vitro device
Inserting the dual-functional catheter into a liquid inlet pipe and fixing, adding 40mL of DMEM culture solution containing HepG2 and HeLa single cell suspension preheated at 37 ℃ into a test tube, adjusting the flow rate of the liquid to be 10mL/min, circulating for 15min, irradiating the catheter part of the indwelling needle by 808nm laser, observing the change of the temperature of the irradiated part by using a thermal imaging instrument, and adjusting the power density of the laser at any time to control the temperature to be 55 ℃ and keeping for 5 min.
After the light irradiation is finished, the catheter is carefully withdrawn, and AO (Acridine Orange)/PI (Propidium Iodide) stains the cells enriched on the surface of the catheter and observes by using a fluorescence microscope, wherein the living cells are green fluorescence, and the dead cells are red fluorescence. FIGS. 6(a) and 6(b) show the enlarged area before and after illumination, respectively, in FIG. 6(a), the HepG2 on the outer wall of the catheter under visible light, AO and PI and the HeLa cell under a fluorescence microscope, and the cell killing rate is 100%.
Example 3
This example investigates the ability of the indwelling needle catheter provided in example 1 to enrich and kill CTCs by a rabbit auricular vein model.
(1) Study of the function of bifunctional duct-enriched CTCs in rabbit ear vein model
The experimental rabbits were fasted overnight before the operation; before operation, the ear edge vein is anesthetized with l% sodium pentobarbital (3mL/kg) and fixed on the rabbit table in a supine position.
The proximal end of the ear vein of the experimental rabbit is inserted with 1 piece of bifunctional indwelling needle catheter, and the distal end is inserted with 1 piece of untreated indwelling needle catheter. The vascular clamp clamps the auricle proximal vein of the experimental rabbit to reduce the collateral blood flow. HepG2 and HeLa cells (1mL, 1X 10)5mL) after 1min of manual injection through the distal catheter, the catheter was flushed with sodium heparin (1mL, 10U/mL) for 1 min.
The bifunctional catheters were carefully withdrawn and washed 3 times with PBS. Adding Trizol to crack the cell, extracting RNA, and detecting the expression quantity of the EpCAM gene by RT-qPCR. The number of the captured HepG2 and HeLa cells was respectively 2130 and 100 according to the standard curve, and the capturing efficiency of HepG2 and HeLa cells in the rabbit ear vein model of the intravenous indwelling needle catheter was calculated, as shown in fig. 7, the capturing efficiency of HepG2 cells was about 2.13% and the capturing efficiency of HeLa cells was 0.01%.
(2) Research on function of dual-functionalized intraductal black phosphorus photothermal killing CTCs in rabbit ear vein model
The experimental rabbits were fasted overnight before the operation; before operation, the ear edge vein is anesthetized with l% sodium pentobarbital (3mL/kg) and fixed on the rabbit table in a supine position.
The proximal end of the ear vein of the experimental rabbit is inserted with 1 piece of bifunctional indwelling needle catheter, and the distal end is inserted with 1 piece of untreated indwelling needle catheter. The vascular clamp clamps the auricle proximal vein of the experimental rabbit to reduce the collateral blood flow. HepG2 and HeLa cells (1mL, 1X 10)5mL) after 1min of manual injection through the distal catheter, the catheter was flushed with sodium heparin (1mL, 10U/mL) for 1 min.
Irradiating the indwelling needle catheter part with 808nm laser, observing the change of the temperature of the irradiated part by using a thermal imaging instrument, and adjusting the laser power density at any time to control the temperature to be 55 ℃ and keep for 5 min. After the illumination is finished, the catheter is carefully drawn out, AO/PI stains the cells enriched on the surface of the catheter, the cell viability is observed under a fluorescence microscope, and FIG. 8 shows the effect graphs of HepG2 and HeLa cells on the outer walls of the catheter under visible light, AO and PI respectively under the fluorescence microscope, wherein the viable cells are green fluorescence, the dead cells are red fluorescence, and the cell killing rate is 100%.
Example 4
The embodiment provides an EpCAM antibody/graphene oxide bifunctional indwelling catheter, and the preparation method comprises the following steps:
(1) injecting a mixed solution containing carboxylated graphene oxide, an activating agent and a buffer solution into the catheter of the venous indwelling needle, reacting, cleaning, and then sealing unreacted activated carboxyl to obtain a carboxylated photosensitizer modified venous indwelling needle catheter;
(2) and (2) putting the vein indwelling needle catheter obtained in the step (1) into a mixed solution containing a circulating tumor cell capture molecule, an activating agent and a buffer solution, reacting, cleaning, and sealing activated carboxyl to obtain the vein indwelling needle catheter.
In conclusion, the venous indwelling needle catheter provided by the invention has the functions of enriching and killing CTCs, and has good biocompatibility and realizes separation and targeted removal of CTCs in large-volume blood when the prepared venous indwelling needle has no influence on the general functions of the venous indwelling needle.
The applicant declares that the present invention illustrates the detailed structural features of the present invention through the above embodiments, but the present invention is not limited to the above detailed structural features, that is, it does not mean that the present invention must be implemented depending on the above detailed structural features. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of selected components of the present invention, additions of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. The venous indwelling needle catheter is characterized in that the outer wall of the venous indwelling needle catheter is modified with circulating tumor cell capture molecules, and the inner wall of the venous indwelling needle catheter is modified with carboxylated photosensitizer.
2. The intravenous indwelling needle catheter according to claim 1, wherein the intravenous indwelling needle catheter material contains free amino groups, and the circulating tumor cell capturing molecule is fixed on the outer wall of the intravenous indwelling needle catheter by amino carboxyl condensation reaction;
preferably, the material of the intravenous indwelling needle catheter contains free amino, and the carboxylated photosensitizer is fixed on the inner wall of the intravenous indwelling needle catheter through amino carboxyl condensation reaction.
3. The intravenous indwelling needle catheter of claim 1 or claim 2, wherein the intravenous indwelling needle catheter is a polyurethane material;
preferably, the circulating tumor cell capture molecule comprises: any one or a combination of at least two of an antibody, a polypeptide, or an aptamer that specifically binds to a circulating tumor cell surface antigen;
preferably, the circulating tumor cell capture molecule comprises an EpCAM antibody.
4. The intravenous indwelling needle catheter of any of claims 1-3, wherein the photosensitizer has near infrared light response characteristics;
Preferably, the photosensitizer comprises a photosensitive small molecule drug and/or a photosensitive nanomaterial;
preferably, the coating concentration of the photosensitizer on the inner wall of the intravenous indwelling needle catheter is 5-10 mg/mL;
preferably, the photosensitizer comprises any one of graphene, MXenes, transition metal sulfides or black phosphorus or a combination of at least two of the same, preferably black phosphorus.
5. A method of making a venous indwelling needle catheter, as claimed in any one of claims 1 to 4, comprising the steps of:
(1) injecting a mixed solution containing a carboxylated photosensitizer, an activating agent and a buffer solution into the catheter of the venous indwelling needle, reacting, cleaning, and then sealing unreacted activated carboxyl to obtain a carboxylated photosensitizer modified venous indwelling needle catheter;
(2) putting the vein indwelling needle catheter obtained in the step (1) into a mixed solution containing a circulating tumor cell capture molecule, an activating agent and a buffer solution, reacting, cleaning, and then sealing unreacted activated carboxyl to obtain the vein indwelling needle catheter;
alternatively, the preparation method comprises the following steps:
(1') putting the vein indwelling needle catheter into a mixed solution containing circulating tumor cell capture molecules, an activating agent and a buffer solution, reacting, cleaning, and then sealing unreacted activated carboxyl to obtain the vein indwelling needle catheter modified by the circulating tumor cell capture molecules;
(2 ') injecting a mixed solution containing a carboxylated photosensitizer, an activating agent and a buffer solution into the catheter of the venous indwelling needle obtained in the step (1'), reacting, cleaning, and then blocking unreacted activated carboxyl to obtain the venous indwelling needle catheter.
6. The process of claim 5, wherein the activator of steps (1) and (2') comprises EDC;
preferably, the mixed solution of step (1) and step (2') comprises: EDC solution with mass concentration of 15-25mg/mL, carboxylated photosensitizer with mass concentration of 15-25 μ g/mL and PBS buffer solution;
preferably, before injecting the mixed solution into the catheter in the steps (1) and (2'), the operation of withdrawing the needle head of the venous indwelling needle is further included;
preferably, the method further comprises the step of returning the needle to the original position after the step (1) and the step (2') are finished.
7. The method according to claim 5 or 6, wherein the activator of step (2) and step (1') comprises EDC;
preferably, the mixed solution of step (2) and step (1') includes: EDC solution with mass concentration of 15-25mg/mL, circulating tumor cell capture molecules with mass concentration of 3-5 mu g/mL and PBS buffer solution.
8. The production method according to any one of claims 5 to 7, wherein the blocking of the unreacted activated carboxyl group is carried out by: adding glycine with molar concentration of 0.8-1.2mM, and shaking for 20-40 min;
Preferably, the temperature of the reaction in the step (1) and the step (2') is 20-30 ℃ and the time is 20-40 min;
preferably, the reaction of step (2) and step (1') is carried out at a temperature of 20 to 30 ℃ for a time of 20 to 40 min.
9. The method according to any one of claims 5 to 8, characterized by comprising the steps of:
(1) extracting a needle head of the venous indwelling needle by 1-2cm under the aseptic condition, injecting a mixed solution containing 15-25mg/mL of EDC solution, 15-25 mu g/mL of carboxylated photosensitizer and PBS buffer solution into the catheter of the venous indwelling needle, carrying out oscillation reaction at 20-30 ℃ for 20-40min, washing, then adding 0.8-1.2mM of glycine, oscillating for 20-40min to seal unreacted activated carboxyl, washing again, and then restoring the needle head to the original position to obtain the carboxylated photosensitizer modified venous indwelling needle catheter;
(2) inserting the venous indwelling needle catheter obtained in the step (1) into a mixed solution containing 3-5 mug/mL of EpCAM antibody, 15-25mg/mL of EDC solution and buffer solution, carrying out shake reaction at 20-30 ℃ for 20-40min, washing, adding glycine with the molar concentration of 0.8-1.2mM, and carrying out shake reaction for 20-40min to seal unreacted activated carboxyl, thus obtaining the venous indwelling needle catheter;
Alternatively, the preparation method comprises the following steps:
(1') inserting the venous indwelling needle catheter into a mixed solution containing 3-5 mu g/mL of EpCAM antibody, 15-25mg/mL of EDC solution and buffer solution, carrying out oscillation reaction at 20-30 ℃ for 20-40min, washing, adding glycine with the molar concentration of 0.8-1.2mM, oscillating for 20-40min, and sealing unreacted activated carboxyl to obtain the circulating tumor cell capture molecule modified venous indwelling needle catheter;
(2 ') extracting the needle head of the venous indwelling needle by 1-2cm under the aseptic condition, injecting a mixed solution containing EDC solution with the mass concentration of 15-25mg/mL, carboxylated photosensitizer with the mass concentration of 15-25 mu g/mL and PBS buffer solution into the catheter of the venous indwelling needle obtained in the step (1'), carrying out shake reaction at 20-30 ℃ for 20-40min, washing, adding glycine with the molar concentration of 0.8-1.2mM, shaking for 20-40min, sealing unreacted activated carboxyl, washing again, and restoring the needle head to the original position to obtain the catheter of the venous indwelling needle.
10. A venous indwelling needle prepared using a venous indwelling needle catheter as claimed in any one of claims 1 to 4.
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