A polypeptide-functionalized micro-nano interface material for separating and enriching circulating tumor cells, and its preparation method and application

Abstract

The present invention discloses a polypeptide-functionalized micro-nano interface material for separating and enriching circulating tumor cells, and a preparation method and application thereof. The polypeptide-functionalized micro-nano interface material includes a tumor-specific targeting polypeptide, nanofibers, and a disulfide bond connecting the polypeptide and the nanofibers. This polypeptide-functionalized micro-nano interface material can achieve efficient capture of tumor cells (>90%), and can simultaneously separate and capture EpCAM-positive and EpCAM-negative CTCs, realizing the detection of CTCs in the body of liver cancer patients and during the treatment process and their changes, and has application prospects in early diagnosis, metastasis prediction, and prognosis.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomaterials and clinical detection, and particularly relates to a polypeptide-functionalized micro-nano interface material for separating and enriching circulating tumor cells, a preparation method thereof, and an application thereof. Background Art

[0002] Circulating tumor cells (CTCs) are cancer cells existing in the blood system and originating from primary tumors. They are closely related to tumor metastasis, recurrence, etc., and are important markers for cancer research. As a new means of liquid biopsy, the detection of CTCs plays an important role in guiding the treatment process, predicting disease development, and evaluating survival rate. In addition, the separation of CTCs and subsequent molecular profiling can provide important information for personalized treatment and understanding the biological mechanism of metastasis. However, the content of CTCs in the blood is extremely low, and only a few to dozens of CTCs exist in 10 9 blood cells, which brings great challenges to the separation and detection of CTCs.

[0003] So far, many techniques for identifying CTCs have been developed based on the physical and biochemical properties of cancer cells. Some nanomaterials and devices can distinguish the size or surface roughness between CTCs and blood cells and have been widely used in the capture and separation of CTCs. To target CTCs at the molecular level, antibodies, aptamers, polypeptides, etc. have also been used as capture elements for CTCs. Epithelial cell markers, such as epithelial cell adhesion molecule (EpCAM), are the most common target proteins in CTC analysis. Currently, the development of strategies that combine size selection and molecular recognition has greatly improved the sensitivity and selectivity of CTC separation. However, CTCs are heterogeneous and prone to losing some common target proteins during the epithelial-mesenchymal transition (EMT) process, which limits the accuracy of CTC detection in actual samples. For tumor cells with high metastatic potential, the expression of epithelial markers (such as EpCAM) will be downregulated, making these CTC subsets undetectable by methods based on epithelial markers. In addition, the expression levels of epithelial markers in liver cancer cells and soft tissue cancer cells are also very low, so it is difficult to apply them to general CTC separation methods. For example, more than 80% of liver cancer patients are EpCAM-negative, making it difficult to separate CTCs by EpCAM-based methods. At the same time, maintaining the biological activity of CTCs during the separation process is crucial for subsequent molecular profiling. Therefore, the development of a new type of CTC separation and enrichment material and method to obtain different subtypes of circulating tumor cells is crucial for early tumor screening, precision treatment, and molecular mechanism research. Summary of the Invention

[0004] To solve the problem in the prior art that mainly EpCAM-positive CTCs are captured and EpCAM-negative CTCs are easily lost, resulting in inaccurate detection results, the present invention provides a polypeptide-functionalized material with a micro-nano surface topography, which can achieve efficient capture, separation, and enrichment of different subtypes of circulating tumor cells in blood samples.

[0005] In the first aspect of the present invention, there is provided a polypeptide-functionalized micro-nano interface material for separating and enriching circulating tumor cells, which comprises three components:

[0006] A) Tumor-specific targeting polypeptide; B) nanofibers; C) disulfide bond connecting the polypeptide and the nanofibers;

[0007] Preferably, the tumor-specific targeting polypeptide uses the marker protein lysosome-associated protein transmembrane 4 beta (LAPTM4B) carried on the surface of the tumor cells as a specific target; specifically, the tumor-specific targeting polypeptide is the AP2H polypeptide, and its amino acid sequence is shown as follows: IHGHHIISVG (SEQ ID NO:1).

[0008] Preferably, the nanofibers are poly (D,L-lactide-co-glycolide) (PLGA) nanofibers (with a molecular weight of 38,000 - 54,000); specifically, the nanofibers are prepared by electrospinning.

[0009] Furthermore, the fiber diameter of the poly (D,L-lactide-co-glycolide) (PLGA) nanofibers is preferably 100 - 1500 nm.

[0010] Preferably, the disulfide bond of the linking polypeptide and the nanofibers has the structure shown in the following formula:

[0011]

[0012] Among them, R1 and R2 are selected from the alkyl groups of straight-chain alkanes with C1 - C5, and preferably both R1 and R2 are (CH2)2.

[0013] The second aspect of the present invention provides a preparation method of a polypeptide-functionalized micro-nano interface material for separating and enriching circulating tumor cells, and the method includes the following steps:

[0014] 1) Prepare the tumor-specific targeting polypeptide by solid-phase polypeptide synthesis;

[0015] 2) Prepare the nanofibers by electrospinning;

[0016] 3) Couple the tumor-specific targeting polypeptide and the nanofibers through a disulfide bond to obtain the polypeptide-functionalized micro-nano interface material.

[0017] In the preferred technical solution, the tumor-specific targeting polypeptide in step 1) is the AP2H polypeptide, and the specific preparation method includes:

[0018] a1) Using Fmoc-Gly-Wang resin as the starting material, 20% piperidine / N,N'-dimethylformamide (DMF) solution as the Fmoc group deprotecting agent, and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) as the carboxyl activating reagent, AP2H with the sequence IHGHHIISVG was synthesized; wherein, the glycine bonding amount in the Fmoc-Gly-Wang resin was 0.354 mmol / g;

[0019] b1) Deprotecting the Fmoc protecting group of the AP2H polypeptide sequence and cleaving AP2H from the resin using a cleavage solution; wherein the cleavage solution consisted of 95% (v / v) trifluoroacetic acid (TFA), 2.5% water, and 2.5% triisopropylsilane (TIS).

[0020] In the preferred technical solution, the nanofibers in step 2) were PLGA nanofibers, and the specific preparation method included:

[0021] a2) Dissolving PLGA in hexafluoroisopropanol to obtain a polymer solution;

[0022] b2) During the electrospinning process, placing a silicon wafer (1 cm × 1 cm) in the center of an aluminum-covered receiver, injecting the PLGA solution through a syringe pump, and setting the distance and voltage between the needle tip and the receiver;

[0023] c2) After electrospinning for 30 - 90 min, PLGA nanofibers were collected on the surface of the silicon wafer. The obtained PLGA nanofibers were vacuum dried at room temperature to obtain the substrate material Si@PLGA-NF.

[0024] Further, in the a2), the PLGA concentration was 3 wt% - 15 wt%, specifically such as: 3 wt%, 5 wt%, 7 wt%, 10 wt%, 13 wt% or 15 wt%.

[0025] Further, in the b2), the syringe pump flow rate was 0.1 - 0.5 mL h -1 , specifically such as: 0.1, 0.3 or 0.5 mL h -1 , the distance between the needle tip and the receiver was 15 cm, and the voltage therebetween was 10 kV.

[0026] In a preferred technical solution, the specific method for the connection mode of the disulfide bond between the tumor-specific targeting polypeptide and the nanofiber in step 3) specifically includes:

[0027] a3) Place the PLGA nanofibers Si@PLGA-NF in a six-well plate, and then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (N-(3-dimethylaminopropyl)-N’-ethylcarbodiimidehydrochloride, EDC) and N-hydroxysuccinimide (N-hydroxysuccinimide, NHS) for an activation reaction to obtain carboxyl-activated Si@PLGA-NF;

[0028] b3) In phosphate buffered saline (PBS), successively couple the carboxyl-activated Si@PLGA-NF with cysteamine, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and AP2H polypeptide to obtain the polypeptide-functionalized micro-nano interface material Si@PLGA-NF@AP2H.

[0029] Further, in a3), the molar ratio of EDC to NHS is 10:1, and the conditions for the activation reaction are shaking reaction at room temperature for 20 - 30 min.

[0030] Further, in b3), the concentration of cysteamine is 1 - 2 mg / mL -1 , the concentration of SPDP is 0.45 - 0.6 mg / mL -1 , and the concentration of AP2H polypeptide is 0.5 - 1 mg / mL -1 . The conditions for the coupling reaction are shaking reaction at room temperature for 10 - 12 h.

[0031] The third aspect of the present invention provides the application of the polypeptide-functionalized micro-nano interface material (specifically such as Si@PLGA-NF@AP2H) in the efficient separation and enrichment of circulating tumor cells.

[0032] The tumor cells are cancer cells (including drug-resistant cancer cells); the cancer cells are liver cancer cells, breast cancer cells, lung cancer cells, human glioma cells, melanoma cells, glioblastoma cells, cervical cancer cells, nasopharyngeal cancer cells, brain cancer cells, pancreatic cancer cells, ovarian cancer cells, uterine cancer cells, testicular cancer cells, skin cancer cells, gastric cancer cells, colon cancer cells, bladder cancer cells, or rectal cancer cells.

[0033] The liver cancer cells may specifically be HepG2, HuH-7 or Hep3B; the human glioma cells may specifically be human glioma cell U251.

[0034] In the fourth aspect of the present invention, there is provided an application of the polypeptide-functionalized micro-nano interface material (specifically, Si@PLGA-NF@AP2H) in the preparation of a reagent for highly efficient separation and enrichment of circulating tumor cells.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] (1) The present invention uses LAPTM4B protein as a new target. The high expression of LAPTM4B protein on CTCs and during the EMT process enables a new method for CTC capture based on polypeptide AP2H to be independent of epithelial markers. This new strategy can simultaneously capture different subtypes of CTCs, and can not only separate EpCAM-positive CTCs, but also separate and capture EpCAM-negative CTCs, which can provide accurate and comprehensive information for cancer diagnosis and treatment;

[0037] (2) The nanofibers are formed by electrospinning of PLGA. There are abundant carboxyl groups on PLGA, so multiple polypeptide ligands can be bound to the fiber surface, forming a multivalent affinity effect on CTCs. The nanofibers stacked on the silicon wafer surface simulate the morphological structure of the tumor cell extracellular matrix, thereby enhancing the adhesion of CTCs and reducing the non-specific adsorption of blood cells. Through the combination of molecular recognition of polypeptide-protein and morphological recognition of nanofibers, efficient and high-purity enrichment of CTCs in whole blood of patients is achieved;

[0038] (3) A reversible disulfide bond responsive to oxidation-reduction is introduced in the polypeptide modification to achieve reversible release of tumor cells. The released tumor cells have high viability and are suitable for subsequent molecular profiling and gene analysis of CTCs;

[0039] (4) Separation and capture of CTCs in actual sample whole blood can be achieved with low sample consumption; at the same time, EpCAM-positive and EpCAM-negative CTCs are separated; changes in CTCs during the treatment process are monitored, and it has application prospects in early diagnosis and prognosis prediction. Description of the Drawings

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0041] Figure 1Schematic diagram of the working principle of the polypeptide-functionalized micro-nano interface material Si@PLGA-NF@AP2H of the present invention;

[0042] Figure 2 Flow chart of the preparation of the material Si@PLGA-NF@AP2H in the embodiment of the present invention;

[0043] Figure 3 Electron microscopy characterization diagrams of the diameters of PLGA-NF prepared with different parameters and Si@PLGA-NF with different fiber diameters in the embodiment of the present invention;

[0044] Figure 4 Electron microscopy characterization diagram of the material Si@PLGA-NF@AP2H in the embodiment of the present invention;

[0045] Figure 5 Result data diagram of the influence of different fiber diameters on the tumor cell capture efficiency in the embodiment of the present invention;

[0046] Figure 6 Result data diagram of the influence of substrates with different structures on the tumor cell capture efficiency in the embodiment of the present invention;

[0047] Figure 7 Result data diagram of the capture efficiency of the material Si@PLGA-NF@AP2H for different phenotypic tumor cells in the embodiment of the present invention;

[0048] Figure 8 Result data diagram of the capture efficiency of the material Si@PLGA-NF@AP2H for tumor cells in simulated samples in the embodiment of the present invention;

[0049] Figure 9 Result data diagram of the release rate of the captured tumor cells by the material Si@PLGA-NF@AP2H and the detection of the activity of the tumor cells after release in the embodiment of the present invention;

[0050] Figure 10 Result data diagram of the capture and phenotypic analysis of CTCs in clinical samples by the material Si@PLGA-NF@AP2H in the embodiment of the present invention;

[0051] Figure 11 Monitoring result diagram of CTCs in the whole blood of liver cancer patients before and after treatment by the material Si@PLGA-NF@AP2H in the embodiment of the present invention. Detailed implementation manners

[0052] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work belong to the scope of protection of the present invention. The materials and reagents used in the present invention are commercially available unless otherwise specified.

[0053] The present invention will be further described below in conjunction with the specification drawings and embodiments, but it does not limit the present invention.

[0054] Example 1 Synthesis of Polypeptide-Functionalized Micro-Nano Interface Material Si@PLGA-NF@AP2H

[0055] According to Figure 2 the reaction flow chart for preparation.

[0056] 1. Synthesis of Polypeptide AP2H

[0057] It was manually synthesized using the Fmoc solid-phase synthesis strategy. Fmoc-Gly-Wang resin was used as the starting material (the glycine bonding amount was 0.354 mmol / g), 20% piperidine / DMF solution was used as the Fmoc group deprotecting agent, and HBTU was used as the carboxyl activating reagent.

[0058] The specific synthesis steps of the AP2H polypeptide sequence were as follows: The weighed Fmoc-Gly-Wang resin was loaded into a solid-phase polypeptide synthesis tube, swollen with DMF for 30 minutes, and then washed three times with DMF. 20% (volume fraction) piperidine / DMF solution was added at a ratio of 30 mL / g resin, magnetically stirred for 10 minutes and then drained, and repeated twice to remove the Fmoc protecting group of the amino acid. The resin was washed six times with DMF, stirred for 1 minute each time, and drained for 10 seconds. 4-fold amounts of Fmoc-AA-OH, 4-fold amounts of HBTU, and 4-fold amounts of 1-Hydroxybenzotriazole (HOBt) monohydrate were weighed and dissolved in 0.4 mol / L N-methylmorpholine / DMF solution. The above reaction solution was added to the resin in the solid-phase polypeptide synthesis tube, magnetically stirred and reacted for 2 hours, and then Kaiser detection was carried out. The resin not turning blue-violet indicated that the reaction was complete, otherwise the reaction time was extended, or the coupling reaction was repeated once, and then the resin was washed six times with DMF. The above deprotection and coupling steps were repeated to complete the extension of the polypeptide chain. After the synthesis of the AP2H polypeptide sequence (IHGHHIISVG) was completed, the Fmoc protecting group at the amino terminus of the peptide chain was removed, and the resin was washed 6 times with DMF. Then, the AP2H was cleaved from the resin using freshly prepared cleavage solution (95% TFA, 2.5% H2O, 2.5% TIS, v / v / v), and the crude product was purified by Shimadzu semi-preparative liquid chromatography.

[0059] 2. Synthesis of PLGA nanofibers Si@PLGA-NF

[0060] Dissolve PLGA (with a molecular weight of 38000 - 54000) in hexafluoroisopropanol to obtain polymer solutions with concentrations of 3 wt%, 5 wt%, 7 wt%, 10 wt%, 13 wt% and 15 wt% respectively. During the electrospinning process, place a silicon wafer (1 cm × 1 cm) in the center of an aluminum-covered receiver. The PLGA solution is injected through a syringe pump at a flow rate of 0.1 - 0.5 mL h -1 . The distance between the needle tip and the receiver is 15 cm, and the voltage between them is 10 kV. After electrospinning for 60 min, PLGA nanofibers are collected on the surface of the silicon wafer. The obtained PLGA nanofibers are dried in vacuum at room temperature to obtain the substrate material Si@PLGA-NF. The greater the concentration of the polymer solution, the larger the fiber diameter; at the same time, the greater the flow rate during electrospinning, the larger the fiber diameter. Si@PLGA-NF with different fiber diameters are prepared from polymer solutions with different concentrations and different flow rates, and the microstructure of Si@PLGA-NF with different fiber diameters is characterized by scanning electron microscopy (SEM), and the results are shown in Figure 3 .

[0061] 3. Synthesis of polypeptide-functionalized micro-nano interface material Si@PLGA-NF@AP2H

[0062] Place Si@PLGA-NF in a six-well plate, drop 1 mL of 0.5 M EDC and 0.05 M NHS phosphate buffer solution on the surface, and react at room temperature for 20 min to activate the carboxyl group. After washing with water, add 1 mg mL -1 cysteamine / PBS solution and react at room temperature for 12 h. Then wash with water, add 0.45 mg mL -1 SPDP solution (the solvent is DMSO:PBS = 3:7, v / v) and react at room temperature for 12 h. Finally, after washing with water, add 0.5 mg mL -1 AP2H / PBS solution and react at room temperature for 12 h to obtain the polypeptide-functionalized micro-nano interface material Si@PLGA-NF@AP2H. The microstructure of Si@PLGA-NF@AP2H is characterized by SEM, and the results are shown in Figure 4 . Figure 4 The Si@PLGA-NF used in the product is prepared from 10 wt% PLGA at a flow rate of 0.5 mL h -1 . Figure 1 is the working principle diagram of Si@PLGA-NF@AP2H.

[0063] Performance Investigation of the Polypeptide-Functionalized Micro-Nano Interface Material Si@PLGA-NF@AP2H in Example 2

[0064] 1. Selection of the Optimal Fiber Diameter

[0065] (1) Experimental Procedure: Prepare Si@PLGA-NF@AP2H with different PLGA fiber diameters (100 - 1500 nm) respectively, place them in a six-well plate, add 1 mL of human hepatoma cell (HepG2) suspension or rat whole blood, and incubate at 37 °C and 5% CO2 for 1 h. After incubation, wash 5 times with PBS, add 1 mL of Hoechst 33342 and incubate for 20 min. After washing with PBS, image and count using a laser confocal microscope.

[0066] (2) Result Analysis: Refer to Figure 5 a, as the fiber diameter increases from 100 nm to 1000 nm, the number of captured HepG2 cells gradually increases; further increasing the fiber diameter from 1000 nm to 1500 nm, the capture rate gradually decreases. Therefore, fibers with an average diameter of 1 μm exhibit the best capture performance on Si@PLGA-NF@AP2H. On the other hand, see Figure 5 b, when the diameter of PLGA nanofibers on the surface of Si@PLGA-NF@AP2H is 1 μm, the number of adsorbed white blood cells (WBCs) is less than 200, showing the least non-specific adsorption.

[0067] 2. Influence of Substrates with Different Structures on the Capture Efficiency of Tumor Cells

[0068] (1) Experimental Procedure: Place Si, Si@PLGA-NF, and Si@PLGA-NF@AP2H (each 1 cm × 1 cm) in a six-well plate respectively, add 1 mL of HepG2 cell suspension, and incubate at 37 °C and 5% CO2 for 1 h. After incubation, wash 5 times with PBS, add 1 mL of Hoechst 33342 and incubate for 20 min. After washing with PBS, image and count using a laser confocal microscope.

[0069] (2) Result Analysis: Refer to Figure 6 , the bare silicon wafer can hardly capture cells, and the capture efficiency is as low as 10%. In contrast, the number of HepG2 captured by Si@PLGA-NF increases, and the capture rate is 34%. In the presence of both nanofibers and targeting polypeptides, Si@PLGA-NF@AP2H captures the largest number of HepG2 cells, and the capture rate is as high as 96%.

[0070] 3. Specific capture of Si@PLGA-NF@AP2H on tumor cells with different phenotypes

[0071] (1) Experimental procedure: Place Si@PLGA-NF@AP2H (1 cm × 1 cm) in a six-well plate, and add 1 mL of suspension of human embryonic kidney cells (HEK293) with low expression of LAPTM4B protein and four kinds of tumor cells with high expression of LAPTM4B protein (human glioma cell U251, human hepatoma cell HepG2, HuH-7, and Hep3B) respectively. Incubate at 37 °C and 5% CO2 for 1 h. After incubation, wash 5 times with PBS, add 1 mL of Hoechst 33342 and incubate for 20 min. After washing with PBS, image and count using a laser confocal microscope.

[0072] (2) Result analysis: Refer to Figure 7 , the capture rates of Si@PLGA-NF@AP2H on HepG2, HuH-7, Hep3B, and U251 cells are all higher than 90%, while normal cells are hardly captured, indicating that Si@PLGA-NF@AP2H can selectively recognize and capture tumor cells. It is worth noting that among the four kinds of tumor cells, the expression level of EpCAM on Hep3B cells is the highest, and the content of EpCAM on HuH-7, HepG2, and U251 cells is greatly reduced. The efficient capture of the four kinds of tumor cells shows that Si@PLGA-NF@AP2H can not only capture EpCAM-positive tumor cells but also capture EpCAM-negative tumor cells.

[0073] 4. High-sensitivity capture of Si@PLGA-NF@AP2H on tumor cells in simulated samples

[0074] (1) Experimental procedure: The whole blood simulated sample is obtained by adding tumor cells to healthy human whole blood. HepG2 cells are pre-stained with calcein-AM. Use a flow cytometer (BD FACSAriall, USA) to count the pre-stained cells, and then add the sorted tumor cells to 1 mL of healthy human whole blood to obtain CTC simulated samples with different spiking densities (5 - 100 mL -1 ). Add Si@PLGA-NF@AP2H to a 24-well plate, add 1 mL of CTC simulated sample, and incubate at 37 °C and 5% CO2 for 1 h. After incubation, wash 10 times with PBS, add 1 mL of Hoechst 33342 and incubate for 20 min. After washing with PBS, image and count using a laser confocal microscope. Calcein-AM + / Hoechst 33342 + positive ones are HepG2 cells, Calcein-AM- / Hoechst 33342 + Those that did were non-specifically adsorbed WBCs.

[0075] (2) Result analysis: Refer to Figure 8 , directly incubate 1 mL of the unpretreated CTC mock sample with Si@PLGA-NF@AP2H, and the capture rate is 65%-93%. It is worth noting that when the spiked density of HepG2 cells is 5 mL -1 , the average capture rate is as high as 93%. In contrast, the capture rate of WBCs is only 0.01%. Si@PLGA-NF@AP2H can directly capture rare tumor cells from whole blood and exclude the interference of blood cells, and is expected to be used for the analysis of CTCs in clinical samples.

[0076] 5. Release of tumor cells by Si@PLGA-NF@AP2H

[0077] (1) Experimental procedure: In the cell release experiment, add 3×10 4 HepG2 cells to PBS and rat whole blood respectively. Incubate the above cells with Si@PLGA-NF@AP2H at 37 °C and 5% CO2 for 1 h. Then wash with PBS, add 1 mL of glutathione (GSH) / PBS solution (1 mg / mL) and react at room temperature for 30 min to release the captured cells. The remaining cells on the material surface after release are imaged and counted using a confocal laser scanning microscope to calculate the release rate. At the same time, the cells released from the material surface are seeded in a culture dish and cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% double antibody for 72 h. The control group is to directly seed the untreated HepG2 cells into the culture dish at the same density. After culturing for 24, 48, and 72 h, image the cells using an inverted microscope.

[0078] (2) Result analysis: Refer to Figure 9 (a, b), after treatment with GSH (1 mg mL -1 ), more than 80% of the HepG2 cells are released from the material surface. Culture the released HepG2 cells in the medium for 3 days, see Figure 9 c, and observe the adhesion and proliferation of the cells in the culture dish, indicating that this release process retains the proliferative activity of the cells and is suitable for subsequent molecular profiling and gene analysis of CTCs.

[0079] Example 3 Capture and analysis of CTCs in clinical samples by Si@PLGA-NF@AP2H

[0080] (1) Experimental procedures: Whole blood samples were collected in vacuum blood collection tubes (BD EDTA vacuum blood collection tubes). Si@PLGA-NF@AP2H was added to a 24-well plate, and 0.7 mL of whole blood samples were added. The samples were incubated at 37 °C under 5% CO2 for 1 h. After incubation, they were washed 10 times with PBS, and triple-color immunostaining was used to distinguish and identify CTCs. First, 1 mL of 2.5% glutaraldehyde / PBS was added for fixation for 20 min, and then washed 2 times with PBS (5 min each time). Then, 1 mL of Triton X-100 was added for permeabilization for 20 min, and washed 2 times with PBS (5 min each time). Next, 1 mL of 1% bovine serum albumin (BSA) was used as the blocking solution for blocking for 30 min. Then, 0.2 mL of Alexa Fluor 488-labeled CD45 antibody (Abcam, 1:100) and Alexa Fluor 647-labeled CK antibody (Abcam, 1:50) diluted with PBS were added and incubated together at 25 °C for 1 h. After antibody incubation, they were washed 2 times with PBS (5 min each time), and finally 1 mL of DAPI nuclear dye was added for incubation for 10 min. After washing with PBS, observation was carried out using a laser confocal microscope. The expression of EpCAM protein and LAPTM4B protein on CTCs was analyzed by immunofluorescence.

[0081] (2) Result analysis: Refer to Figure 10 (a, b), cells that were DAPI positive / CK positive / CD45 negative (DAPI + / CK + / CD45 - ) were counted as CTCs, and cells that were DAPI + / CK - / CD45 + were WBCs. It can be seen that different numbers of CTCs were detected in the whole blood of liver cancer patients. The number of CTCs was 6 - 55 mL -1 (the median was 30 mL -1 , and the average value was 29 ± 14 mL -1 ), and no CTCs were detected in the whole blood of healthy people. According to the BCLC (Barcelona Clinic Liver Cancer) staging system, among 20 liver cancer patients, 2 patients were clinically diagnosed with stage A liver cancer. Based on the Si@PLGA-NF@AP2H method, CTCs in the whole blood of these stage A patients were effectively enriched and detected, and 6 mL -1 and 12 mL -1 were detected respectively. It should be noted that in addition to capturing single CTCs, Si@PLGA-NF@AP2H also captured CTC clusters from the whole blood of liver cancer patients. The immunofluorescence results of EpCAM protein and LAPTM4B protein on CTCs are shown inFigure 10 (c, d) indicated that Si@PLGA-NF@AP2H could not only separate EpCAM-positive CTCs, but also capture EpCAM-negative CTCs; among the captured CTCs, LAPTM4B protein was highly expressed and stably present.

[0082] Based on the above analysis results of clinical samples, Si@PLGA-NF@AP2H was further applied to monitor the treatment process of liver cancer patients. See Figure 11 Before treatment, the number of CTCs in the whole blood of patient A with clinical stage B was 16 / mL -1 and the number of CTCs in patient B with clinical stage C was 30 / mL -1 For patient A, two days after receiving transarterial chemoembolization (TACE), the number of CTCs in the whole blood of patient A decreased to 10 / mL -1 ; 30 days after treatment, the number of CTCs further decreased to 8 / mL -1 , which was half of that before treatment. After clinical diagnosis, it was found that 30 days after treatment, the tumor cells of patient A had necrosis, and the gradual decrease in the number of CTCs was consistent with this clinical feature. For patient B, patient B received TACE and radiofrequency ablation treatment. Two days after treatment, the number of CTCs decreased sharply to 2 / mL -1 ; however, 30 days after treatment, the number of CTCs increased to 16 / mL -1 , and at the same time, new tumor lesions were also found in the corresponding clinical diagnosis.

[0083] The above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention. Sequence Listing <110> Institute of Chemistry, Chinese Academy of Sciences <120> Polypeptide-functionalized micro-nano interface material for separating and enriching circulating tumor cells, its preparation method and application <130> 211404 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial sequence <400> 1 Ile His Gly His His Ile Ile Ser Val Gly 1 5 10

Claims

1. A polypeptide-functionalized micro-nano interface material, characterized in that: The material includes three components: a tumor-specific targeting polypeptide, nanofibers, and a disulfide bond connecting the polypeptide to the nanofibers; The tumor-specific targeting polypeptide uses the marker protein lysosome-associated transmembrane protein 4B (LAPTM4B) carried on the surface of tumor cells as a specific target; The tumor-specific targeting polypeptide is the AP2H polypeptide, and its amino acid sequence is as follows: IHGHHIISVG; The nanofibers are poly(lactic-co-glycolic acid) (PLGA) nanofibers; The poly(lactic-co-glycolic acid) (PLGA) nanofibers are prepared by electrospinning, and their fiber diameter is 1000 nm; The disulfide bond connecting the polypeptide to the nanofibers has the structure shown by the following formula: Wherein, both R1 and R2 are (CH2)2.

2. A preparation method of the polypeptide-functionalized micro-nano interface material according to claim 1, characterized in that, It includes the following steps: 1) Prepare the tumor-specific targeting polypeptide by solid-phase peptide synthesis; 2) Prepare nanofibers by electrospinning; 3) Couple the tumor-specific targeting polypeptide to the nanofibers through a disulfide bond to obtain the polypeptide-functionalized micro-nano interface material.

3. According to the preparation method described in claim 2, characterized in that: In step 1), the tumor-specific targeting polypeptide is the AP2H polypeptide, and the specific method includes: a1) Using Fmoc-Gly-Wang resin as the starting material, 20% piperidine / N,N-dimethylformamide solution as the Fmoc group deprotecting agent, and 2-(3'-oxo-benzotriazol)-1,1',3,3'-tetramethyluronium hexafluorophosphate as the carboxyl activating reagent, AP2H with the sequence IHGHHIISVG was synthesized; wherein, the glycine bonding amount in the Fmoc-Gly-Wang resin is 0.354 mmol / g; N, N ’ -dimethylformamide solution as the Fmoc group deprotecting agent, 2-(3'- N -oxo-benzotriazol)-1,1',3,3'-tetramethyluronium hexafluorophosphate as the carboxyl activating reagent, AP2H with the sequence IHGHHIISVG was synthesized; wherein, the glycine bonding amount in the Fmoc-Gly-Wang resin is 0.354 mmol / g; b1) Remove the Fmoc protecting group of the AP2H polypeptide sequence, and cleave AP2H from the resin using a cleavage solution, thus obtaining it; wherein the cleavage solution is composed of 95% (v / v) trifluoroacetic acid, 2.5% water, and 2.5% triisopropylsilane.

4. According to the preparation method described in claim 2 or 3, characterized in that: In step 2), the nanofibers are PLGA nanofibers, and the specific method includes: a2) Dissolve PLGA in hexafluoroisopropanol to obtain a PLGA solution; b2) During electrospinning, place a silicon wafer in the center of an aluminum-covered receiver, and inject the PLGA solution through a syringe pump; set the distance and voltage between the needle tip and the receiver; c2) After electrospinning for 60 min, collect PLGA nanofibers on the surface of the silicon wafer, and vacuum-dry the obtained PLGA nanofibers at room temperature to obtain the substrate material Si@PLGA-NF; In a2), the PLGA concentration is 3 wt% - 15 wt%; In the above b2), the flow rate of the syringe pump is 0.1 - 0.5 mL∙h -1 ; In b2), the distance between the needle tip and the receiver is 15 cm, and the voltage therebetween is 10 kV.

5. According to the preparation method described in claim 2 or 3, characterized in that: In step 3), the specific method for the connection mode of the disulfide bond between the tumor-specific targeting polypeptide and the nanofibers includes: a3) Place the PLGA nanofibers Si@PLGA-NF in a six-well plate, then add 0.5 M EDC and 0.05 M NHS for an activation reaction to obtain carboxyl-activated Si@PLGA-NF; b3) In phosphate buffer, the carboxyl-activated Si@PLGA-NF was successively coupled with 1 mg∙mL -1 cysteamine, 0.45 mg∙mL -1 SPDP, 0.5 mg∙mL -1 AP2H polypeptide to obtain the polypeptide-functionalized micro-nano interface material Si@PLGA-NF@AP2H.

6. Application of the polypeptide-functionalized micro-nano interface material according to claim 1 in the preparation of a circulating tumor cell separation and enrichment reagent.

7. According to the application described in claim 6, characterized in that: The tumor cells are cancer cells; the cancer cells include liver cancer cells, breast cancer cells, lung cancer cells, human glioma cells, melanoma cells, glioblastoma cells, cervical cancer cells, nasopharyngeal cancer cells, brain cancer cells, pancreatic cancer cells, ovarian cancer cells, uterine cancer cells, testicular cancer cells, skin cancer cells, gastric cancer cells, colon cancer cells, bladder cancer cells, or rectal cancer cells.

Images