A bionic three-dimensional polymer fiber network structure and its preparation method and application

Abstract

This invention discloses a biomimetic three-dimensional polymer fiber network structure, its preparation method, and its application, belonging to the field of cancer diagnostic materials technology. It solves the problems of insufficient space utilization, cumbersome preparation, and damage to cell viability in the capture of circulating tumor cells using traditional fiber materials. The invention includes the following steps: preparing a 3D-L fiber network using liquid receiving electrospinning technology; placing the prepared fibers into a foaming liquid for gas foaming to obtain a 3D-G fiber network; immersing the 3D-G fiber network in an aqueous solution containing TA and PEG, washing it, and then immersing it again in a solution containing FeCl3 to obtain a 3D-G@FTP fiber network. This material can achieve efficient separation and non-destructive release of tumor cells, and its preparation is simple and inexpensive, making it widely applicable in the preparation of biomaterials.

Description

Technical Field

[0001] The present invention belongs to the technical field of cancer diagnosis materials, and in particular relates to a bionic three-dimensional polymer fiber network structure and a preparation method and application thereof. Background Art

[0002] Circulating Tumor Cells (CTCs) refer to tumor cells that break away from the original tumor lesions and enter the human peripheral circulatory system, enter other tissues and organs through the blood circulation, and grow into new tumor tissues. As an important link in tumor metastasis, circulating tumor cells not only carry all the genetic information of the original tumor lesions, but also exist in the peripheral blood circulation system at the early stage of cancer. They are considered to be important biomarkers for early diagnosis and prognosis prediction of cancer. However, due to their rarity and fragility, it is still challenging to efficiently capture and gently release CTCs from complex blood components.

[0003] At present, electrospun fibers are widely used in the detection and separation of CTCs due to their high specific surface area, adjustable pore size and porosity. However, the recognition ligands (antibodies, aptamers, etc.) used in this method have the disadvantages of cumbersome grafting, high price, easy inactivation, etc., and most fiber materials are two-dimensional structures with tight stacking and low space utilization, making the capture of CTCs challenging. In addition, the current methods of releasing CTCs based on enzymatic hydrolysis, electrical stimulation, ultraviolet light irradiation, etc. are invasive and will destroy cell activity. Summary of the invention

[0004] In view of the problems in the prior art of cumbersome grafting, high cost, easy inactivation, tight stacking and destruction of cell activity, the present invention provides a bionic three-dimensional polymer fiber network structure and a preparation method and application thereof, the purpose of which is to achieve efficient detection and separation of tumor cells.

[0005] The technical solution adopted by the present invention is as follows:

[0006] Application of a bionic three-dimensional polymer fiber network structure in the efficient capture and lossless release of circulating tumor cells.

[0007] A bionic three-dimensional polymer fiber network structure for efficiently capturing and non-destructively releasing circulating tumor cells, comprising a 3D-G fiber network, wherein a Fe III -TA-PEG coating.

[0008] Using the above scheme, the tumor microenvironment (TME) is an important ecosystem for the survival and metastasis of tumor cells. The extracellular matrix (ECM) is the main component of the TME. It is a three-dimensional (3D) fiber network structure composed of a variety of biomacromolecules such as complex dynamic structural proteins, glycosaminoglycans and signaling proteins. In addition, since tumor cell metabolism is more vigorous than normal cells, it will produce more reactive oxygen species during the metabolic process. In order to avoid these reactive oxygen species from causing oxidative damage to tumor cells, tumor cells produce a large amount of glutathione (GSH) to promptly remove excess reactive oxygen species. Therefore, inspired by the 3D fiber network structure of the tumor microenvironment and the high abundance of GSH, the 3D fiber network with GSH responsiveness helps to efficiently capture and friendly release CTCs to maintain their high cell viability.

[0009] The three-dimensional structure of the 3D-G fiber network has a high porosity, which allows circulating tumor cells to enter the 3D-G fiber network, increasing the contact probability between the material and the cells. Tumor cells entering the 3D-G fiber network are Fe III -TA-PEG coating recognition and capture. This is because TA has poor affinity for some tightly wound small and dense proteins, but has a strong affinity for extended proteins and peptides containing a high proportion of proline residues in the sequence, such as sialic acid, gelatin, etc. The main driving force for binding is the "hydrophobic effect", which can form hydrophobic stacking with the pyrrolidine ring on proline through the phenol ring, and the "hydrogen bond" between the phenolic hydroxyl group and the carbonyl group of proline on the protein will enhance the complexation. Therefore, circulating tumor cells with a layer of glycocalyx structures rich in salivary proteins, gelatin, etc. on the surface are easily recognized and bound by TA, allowing TA to capture a wide range of circulating tumor cells. Under the action of glutathione, Fe III -Fe in TA-PEG coating III Reduction to Fe II , making the insoluble Fe III -TA network becomes soluble Fe II -TA network, thereby promoting Fe III -TA-PEG coating degradation achieves the purpose of releasing captured CTCs. At the same time, glutathione is an endogenous substance in cells, has good cell compatibility, and can maintain the activity of released cells. PEG is used to prevent leukocyte adhesion.

[0010] Preferably, the porosity of the 3D-G fiber network is 30%-70% and the pore size is 20-60 μm.

[0011] After adopting this technical solution, since the size of cancer cells is about 10-20um, if the pore size is too small, the cells cannot enter the pores, and the purpose of improving the capture efficiency cannot be achieved. If the pore size is too large, the mechanical properties of the material will be poor and the pores will easily collapse.

[0012] A bionic three-dimensional polymer fiber network structure and a preparation method thereof, comprising the following steps:

[0013] Step A: preparing 3D-L fiber network;

[0014] Step B: increasing the porosity and pore size of the 3D-L fiber network obtained in step A to form a 3D-G fiber network;

[0015] Step C: Setting up FeIII-TA-PEG coating on 3D-G fiber network.

[0016] Preferably, in step A, the 3D-L fiber network is prepared by liquid receiving electrospinning technology.

[0017] Preferably, the needle in the liquid receiving electrospinning technology is a 20-23 gauge needle, the voltage is 11-16 kV, the flow rate is 0.1-2.0 mL / h, the receiving device is a conductive metal basin connected to a conductive receiving substrate, the receiving distance is 10-20 cm, the ambient temperature is 20-30°C, and the humidity is below 50%.

[0018] Preferably, the raw material for preparing the 3D-L fiber network in step A is PCL, and the specific steps of step A include:

[0019] Step A1: Completely dissolving PCL particles;

[0020] Step A2: preparing the dissolved PCL particles into a 3D-L fiber network through a liquid receiving electrospinning technique, wherein the liquid in the liquid receiving electrospinning technique is ethanol;

[0021] Step A3: Soak the 3D-L fiber network obtained in step A2 in RO water to displace the ethanol in the 3D-L fiber network, and freeze-dry to obtain a dry 3D-L fiber network.

[0022] Preferably, in step B, the 3D-G fiber network is prepared by bubble foaming technology, and the foaming liquid of the bubble foaming technology is NaBH4 with a concentration of 1-10 mg / mL.

[0023] Preferably, the Fe III -TA-PEG coating is adsorbed on the surface of the 3D-G fiber network through self-assembly chelation.

[0024] Preferably, step C specifically comprises the following steps:

[0025] Step C1: preparing a mixed aqueous solution of 0.5-2.0 mg / mL TA and 0.05-0.2 mg / mL PEG and a 0.5-2.0 mg / mL FeCl3·6H2O aqueous solution;

[0026] Step C2: The 3D-G fiber network obtained in step B is placed in a mixed aqueous solution containing TA and PEG and immersed for 10-30 minutes and then taken out;

[0027] Step C3: washing the 3D-G fiber network obtained in step C2 to remove the TA and PEG that are not completely adsorbed in the fiber gaps of the 3D-G fiber network;

[0028] Step C4: soak the 3D-G fiber network obtained in step C3 in a FeCl3·6H2O aqueous solution for 5-15 minutes and then take it out;

[0029] Step C5: Clean the 3D-G fiber network obtained in step C2 to remove excess FeCl3 on the surface.

[0030] The above scheme was adopted to receive electrospinning technology through ethanol liquid, sodium borohydride gas foaming technology and Fe III -TA-PEG coating self-assembly technology prepared PCL three-dimensional fiber network has a large porosity and pore size, Fe III The introduction of -TA-PEG coating can efficiently capture a variety of cancer cells, and has the characteristics of glutathione response, and can release the captured circulating tumor cells with high activity under glutathione response. This material is simple to prepare and low in price, and can be widely used in the process of biomaterial preparation.

[0031] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0032] 1. Through liquid receiving electrospinning technology and gas foaming technology, the originally tightly stacked two-dimensional fiber structure is expanded into a loose and porous three-dimensional structure, which increases the pore size and porosity of the material. The porosity and pore size can be adjusted by changing the gas foaming time. After the pore size and porosity of the material are increased, cells can enter the pores of the fiber material, further increasing the contact probability between cells and materials, thereby improving the capture efficiency;

[0033] 2.Fe III The introduction of -TA-PEG coating can efficiently and broadly capture a variety of cancer cells, and has the characteristics of glutathione response, and can release the captured circulating tumor cells with high activity under glutathione response. This material is simple to prepare and low in price, and can be widely used in the process of biomaterial preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention will now be described by way of example with reference to the accompanying drawings, in which:

[0035] Figure 1 The following are: a Fourier transform infrared spectrum of a material before and after coating according to an embodiment of the present invention (a) and an element distribution diagram of the material after coating (b);

[0036] Figure 2 Yes; Scanning electron microscope image of HeLa cells captured by a material according to one embodiment of the present invention;

[0037] Figure 3 is: a capture efficiency diagram of four types of cancer cells that are EpCAM positive and negative according to one embodiment of the present invention;

[0038] Figure 4 It is: a comparison diagram of the capture efficiency of a 2D@FTP two-dimensional fiber scaffold and a 3D-G@FTP three-dimensional fiber network according to an embodiment of the present invention;

[0039] Figure 5 is: a graph of release efficiency after capturing cells according to one embodiment of the present invention;

[0040] Figure 6 Yes: Cell activity diagram of released cells according to one embodiment of the present invention. DETAILED DESCRIPTION

[0041] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0042] Combine the following Figure 1-Figure 6 The present invention is described in detail.

[0043] Embodiment 1:

[0044] A bionic three-dimensional polymer fiber network structure for efficiently capturing and non-destructively releasing circulating tumor cells comprises a 3D-G fiber network, wherein the 3D-G fiber network is provided with Fe III -TA-PEG coating, after inspection, the porosity of the 3D-G fiber network was 64.9% and the pore size was 58.6 μm.

[0045] A bionic three-dimensional polymer fiber network structure and a preparation method thereof, comprising the following steps:

[0046] Step A: preparing a 3D-L fiber network; the raw material for preparing the 3D-L fiber network is PCL, and the specific steps of step A include:

[0047] Step A1: Dissolve 2 g of PCL particles in 8 mL of dichloromethane and 2 mL of N,N-dimethylformamide and stir overnight until the PCL particles are completely dissolved;

[0048] Step A2: The dissolved PCL particles are prepared into a 3D-L fiber network by liquid receiving electrospinning technology, wherein the needle in the liquid receiving electrospinning technology is a 22-gauge needle, the flow rate of the microfluidic pump is 1.0 mL / h, the voltage is set to 11.5 kv, the distance between the needle and the receiving device is controlled to be 10 cm, the receiving device includes a conductive metal basin connected to a conductive receiving substrate, the liquid in the liquid receiving electrospinning technology is ethanol set in a conductive metal basin, the ambient temperature is 25° C., and the humidity is 50%;

[0049] Step A3: Soak the 3D-L fiber network obtained in step A2 in RO water to displace the ethanol in the 3D-L fiber network, and freeze-dry to obtain a dry 3D-L fiber network.

[0050] Step B: increasing the porosity and pore size of the 3D-L fiber network obtained in step A to form a 3D-G fiber network;

[0051] In step B, a 3D-G fiber network is prepared by a bubble foaming technique, and the specific steps include: placing the 3D-L fiber network in a 1 mg / mL NaBH4 solution for 20 minutes for foaming to obtain a 3D-G fiber network, and after the foaming is completed, the 3D-G fiber network is placed in a beaker containing RO water for washing to remove residual NaBH4, and then freeze-dried to shape it.

[0052] Step C: Setting Fe on 3D-G fiber network III -TA-PEG coating, the Fe III -TA-PEG coating is adsorbed on the surface of the 3D-G fiber network through self-assembly chelation, and step C specifically comprises the following steps:

[0053] Step C1: 50 mg TA and 5 mg PEG were dissolved in two beakers containing 100 mL RO water, respectively, and 50 mg FeCl3·6H2O was dissolved in another beaker containing 100 mL RO water to prepare a 0.5 mg / mL TA aqueous solution, a 0.05 mg / mL PEG aqueous solution, and a 0.5 mg / mL FeCl3·6H2O aqueous solution;

[0054] Step C2: The 3D-G fiber network obtained in step B is placed in a TA aqueous solution and a PEG aqueous solution and immersed for 10 minutes respectively, and then taken out;

[0055] Step C3: placing the 3D-G fiber network obtained in step C2 in RO water for ultrasonic cleaning for 30 seconds to remove the TA and PEG that are not completely adsorbed in the fiber gaps of the 3D-G fiber network;

[0056] Step C4: soak the 3D-G fiber network obtained in step C3 in a FeCl3·6H2O aqueous solution for 5 minutes and then take it out;

[0057] Step C5: The 3D-G fiber network obtained in step C2 is ultrasonically cleaned for 30 seconds to remove excess FeCl3 on the surface. III -TA-PEG coated 3D-G@FTP PCL fiber network.

[0058] The chemical composition of 3D-G@FTP PCL can be characterized and analyzed by Fourier transform infrared spectroscopy, and the element distribution can be characterized and analyzed by energy dispersive spectrometer. Figure 1 .

[0059] Depend on Figure 1 a It can be seen that compared with PCL fiber, new absorption peaks appeared in the FTIR spectrum of PCL@FTP, which can be attributed to the CC stretching vibration of the benzene ring of TA (1610, 1535 and 1443 cm-1) and the -COH shear vibration of PEG (947 cm-1). Figure 1 In b, Fe element can be observed in the energy spectrum of 3D-G@FTP fiber network compared with PCL, which indicates the successful coating.

[0060] The prepared 3D-G@FTP PCL fiber network was co-incubated with HeLa cells and its scanning images were taken to investigate the affinity of the material to the cells. The results are shown in Figure 2 .

[0061] Depend on Figure 2 It can be seen that HeLa cells can enter the fiber pores, and the three-dimensional fiber network has a good affinity for HeLa cells. After combining with the material, HeLa cells can extend more filopodia.

[0062] The samples obtained in the example were applied to capture four types of heterogeneous CTCs. The cells after passage were counted using a blood cell counting plate and diluted to 1×10 with PBS buffer. 5cells / mL. The cells were added to a 48-well plate containing 2D@FTP fiber scaffolds and 3D-G@FTP PCL fiber networks and incubated on a shaker for 30 minutes. The supernatant containing uncaptured cells was aspirated, and then gently washed three times with PBS buffer and the supernatant was aspirated. The aspirated supernatant was collected and the number of uncaptured cells was counted under a microscope using a hemocytometer. The capture efficiency was defined as: (total number of cells - number of uncaptured cells in the supernatant) / total number of cells; where the total number of cells is 1×10 5 The statistical results are as follows Figure 3 and Figure 4 shown.

[0063] Depend on Figure 3 It can be seen that the capture efficiency of 3D-G@FTP three-dimensional fiber network for HeLa cells, HepG2 cells, A549 cells and HCT116 cells is 90.4±2.4%, 92.0±2.2%, 81.1±1.6% and 87.7±1.4%, respectively. This shows that the three-dimensional fiber network coated with TA has a broad-spectrum capture performance, which has great potential for practical clinical applications in the future.

[0064] Depend on Figure 4 It can be seen that the 3D-G@FTP three-dimensional fiber network can achieve higher capture efficiency (90.4±2.4% vs 78.5±3.1%) in a shorter time (30min vs 90min).

[0065] Finally, the sample obtained in the example was applied to the release of CTCs. The captured cells were transferred to DMEM-high glucose medium containing 25mM glutathione, placed in a shaker and continued to be incubated for 1h, 2h, 4h, 6h, 8h, and 10h, then washed with PBS buffer, and the cells released in the supernatant were collected and counted with a hemocytometer under an inverted phase contrast microscope. The release efficiency is defined as the number of cells released in the supernatant / (total number of cells-number of cells not captured); where the total number of cells is 1×10 5 The statistical results are as follows Figure 5 shown.

[0066] Depend on Figure 5 It can be seen that after the captured cells were placed in the culture medium and cultured for another 1 hour, the release efficiency was only 19.4±1.3%; as the time was extended and cultured for another 8 hours, the release efficiency could reach 60.4±3.2%; and after another 10 hours of culture and release, the release efficiency was 62.5±1.6%, which was not much different from the release efficiency of another 8 hours of culture. This shows that the CTCs adhered to the three-dimensional fiber network are gradually separated from the fiber network as the coating degrades, thereby realizing the release of CTCs.

[0067] Depend on Figure 6 It can be seen that the released cells have good cell activity. After being released and then cultured for 0h, 6h, 24h, and 48h, the cells have good cell activity, and the cell survival rates are all higher than 90%.

[0068] In summary, the three-dimensional fiber network and preparation method for capturing and non-destructively releasing circulating tumor cells provided by the present invention use PCL as the base material, and greatly improve the porosity and pore size of the fiber through liquid receiving electrospinning technology and gas foaming technology, so that circulating tumor cells can enter the fiber interior, thereby increasing the contact probability between the material and the cells; III -TA-PEG coating, TA can effectively capture a variety of circulating tumor cells, PEG and TA can also achieve the effect of anti-leukocyte adhesion, PCL substrate and Fe III -TA-PEG coating combination produces a three-dimensional fiber network that can efficiently capture and non-destructively release circulating tumor cells.

[0069] It should be noted that TA mentioned in the present invention is tannic acid, which is a component abbreviation well known to those skilled in the art and will not be further elaborated in the present invention.

[0070] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A bionic three-dimensional polymer fiber network structure, characterized in that: The 3D-G fiber network comprises a Fe III - Tannic acid-PEG coating; The method for preparing the bionic three-dimensional polymer fiber network structure comprises the following steps: Step A: preparing a 3D-L fiber network by liquid receiving electrospinning technology, wherein the raw material for preparing the 3D-L fiber network is PCL; Step B: increasing the porosity and pore size of the 3D-L fiber network obtained in step A by bubble foaming technology to form a 3D-G fiber network, wherein the foaming liquid of the bubble foaming technology is a NaBH4 solution with a concentration of 1-10 mg / mL; Step C: Fe chelation via self-assembly III -Tannic acid-PEG coating is adsorbed on the surface of the 3D-G fiber network.

2. The bionic three-dimensional polymer fiber network structure according to claim 1, characterized in that: The porosity of the 3D-G fiber network is 30%-70%, and the pore size is 20-60 μm.

3. A method for preparing a biomimetic three-dimensional polymer fiber network structure according to any one of claims 1 to 2, characterized in that: The following steps are involved: Step A: preparing a 3D-L fiber network by liquid receiving electrospinning technology, wherein the raw material for preparing the 3D-L fiber network is PCL; Step B: increasing the porosity and pore size of the 3D-L fiber network obtained in step A by bubble foaming technology to form a 3D-G fiber network, wherein the foaming liquid of the bubble foaming technology is a NaBH4 solution with a concentration of 1-10 mg / mL; Step C: Fe chelation via self-assembly III -Tannic acid-PEG coating is adsorbed on the surface of the 3D-G fiber network.

4. The method for preparing a biomimetic three-dimensional polymer fiber network structure according to claim 3, characterized in that: The needle in the liquid receiving electrospinning technology is a 20-23 gauge needle, the voltage is 11-16 kV, the flow rate is 0.1-2.0 mL / h, the receiving device is a conductive metal basin connected to a conductive receiving substrate, the receiving distance is 10-20 cm, the ambient temperature is 20-30° C., and the humidity is below 50%.

5. The method for preparing a biomimetic three-dimensional polymer fiber network structure according to any one of claims 3 to 4, characterized in that: The specific steps of step A include: Step A1: obtaining a PCL solution; Step A2: preparing the PCL solution into a 3D-L fiber network by liquid receiving electrospinning technology, wherein the liquid in the liquid receiving electrospinning technology is ethanol; Step A3: Soak the 3D-L fiber network obtained in step A2 in RO water to displace the ethanol in the 3D-L fiber network, and freeze-dry to obtain a dry 3D-L fiber network.

6. The method for preparing a biomimetic three-dimensional polymer fiber network structure according to any one of claims 3 to 4, characterized in that: Step C specifically includes the following steps: Step C1: preparing a mixed aqueous solution of 0.5-2.0 mg / mL tannic acid and 0.05-0.2 mg / mL PEG and a 0.5-2.0 mg / mL FeCl3·6H2O aqueous solution; Step C2: The 3D-G fiber network obtained in step B is placed in a mixed aqueous solution containing tannic acid and PEG for 10-30 minutes and then taken out; Step C3: washing the 3D-G fiber network obtained in step C2 to remove the tannic acid and PEG that are not completely adsorbed in the fiber gaps of the 3D-G fiber network; Step C4: soak the 3D-G fiber network obtained in step C3 in a FeCl3·6H2O aqueous solution for 5-15 minutes and then take it out; Step C5: Clean the 3D-G fiber network obtained in step C2 to remove excess FeCl3 on the surface.

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