Construction of 3D cell-matrix interaction model and application of 3D cell-matrix interaction model in drug screening
By constructing a 3D cell-matrix interaction model, using RGD functionalized spiral polypeptides to simulate the mechanical properties and fibrous structure of extracellular matrix, the problem of difficulty in comprehensively revealing the mechanism of cell-matrix interaction in the prior art is solved, and a more accurate drug screening effect is achieved.
Patent Information
- Application Number
- CN202510062152.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-04-18
AI Technical Summary
Existing drug screening platforms mainly focus on biochemical cell-matrix interactions, and it is difficult to fully reveal the mechanisms of cell-matrix interactions, especially in simulating the biophysical microenvironment, which affects the effectiveness of drug screening.
A 3D cell-matrix interaction model is used to mix RGD functionalized spiral polypeptides with cell suspension to form a single-cell 3D culture system, simulating the complex mechanical properties and fibrous network structure of natural extracellular matrix, and achieving dynamic bidirectional interaction between cells and matrix.
This model can more accurately simulate cell-matrix interactions in the tumor microenvironment, providing a drug screening platform closer to real physiological conditions, improving the effectiveness and reliability of drug screening.
Smart Images
Figure CN119842622A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of drug screening model development, and specifically relates to the construction of a 3D cell-matrix interaction model and its application in drug screening. Background Art
[0002] Tumor cell invasion is a core step in the cancer metastasis cascade, and is crucial both in penetrating the extracellular matrix (ECM) and crossing the vascular endothelium. In this complex process, especially in the tumor microenvironment (TME), cell-matrix interactions mainly coordinately regulate cell behavior through biomechanics (such as cell traction) and biochemical signals (such as cytokine secretion). With the deepening of research, people have become increasingly aware of the importance of this interaction to tumor cell invasion. However, traditional experimental designs, whether in vitro Transwell models or in vivo mouse models, have limitations in controlling ECM parameters, making it difficult to fully reveal the mechanism of cell-matrix interactions.
[0003] During the tumor invasion process, matrix metalloproteinases (MMPs) belong to the zinc-dependent endopeptidase family and play a key role in multiple aspects of cancer progression, such as invasion, metastasis and tumor angiogenesis. These proteases can also be shed from the matrix into the plasma and are therefore considered as potential biomarkers of disease progression or the efficacy of related therapeutic interventions. Given their importance, the pharmaceutical industry has invested a lot of resources in the research and development of MMP inhibitors (MMPIs) aimed at providing oral treatment for cancer patients. However, some MMPs, such as the broad-spectrum inhibitor Marimastat, have not shown good results in clinical trials alone or in combination with gemcitabine for the treatment of patients with unresectable pancreatic cancer; another MMP3, MMP9 and MMP 13 The inhibitor Tanomastat (BAY-12-9566) combined with gemcitabine also showed poor efficacy in a phase III trial in pancreatic cancer patients, indicating that under real physiological conditions, inhibition of MMPs alone cannot effectively prevent cancer cell invasion. Although these inhibitors showed good anti-invasion effects in the Matrigel invasion experiment, they failed in the clinical phase III. It is worth noting that the Matrigel-based invasion experiment may only simulate the biochemical microenvironment, while ignoring the important effects of the biophysical microenvironment on tumor invasion and drug efficacy.
[0004] An ideal physical microenvironment platform (a platform that simulates the biophysical environment) should have the following characteristics: ① It can simulate the complex mechanical properties of natural ECM. Natural ECM is not a simple linear material. It exhibits complex nonlinear mechanical properties, such as stress stiffening effect, time-dependent viscoelasticity and mechanical plasticity; ② It has a fibrous network structure similar to natural ECM; ③ It can respond to cell feedback, thereby forming a dynamic bidirectional cell-matrix interaction with cells, thereby affecting cell proliferation, migration and other cell behaviors. At present, most of the reported hydrogels do not have a biomechanical response, which limits the exploration of multiple factors of cancer cell invasion and further affects the effectiveness of clinical drugs. Therefore, it is of great significance to study new biomimetic hydrogels that play a role in the physical microenvironment in the drug screening and application of tumor invasion. Summary of the invention
[0005] In view of the technical defect that the current drug screening platform only focuses on biochemical cell-matrix interactions, the present invention provides a 3D model for reconstructing complex cell-matrix physical interactions in vitro and a construction method thereof, and applies it to drug screening evaluation.
[0006] The cell-matrix interaction 3D model provided by the present invention is prepared by a method comprising the following steps:
[0007] 1) Preparation of helical polypeptides functionalized with cell adhesion peptide RGD
[0008] The helical polypeptide with azide was subjected to click chemistry reaction with DBCO-GRGDS to obtain the RGD-functionalized helical polypeptide;
[0009] 2) Construction of 3D model of cell-matrix interaction
[0010] The RGD-functionalized helical polypeptide was dissolved in PBS, mixed evenly with the cell suspension to form a gel, and then single-cell 3D culture was performed to obtain a cell-matrix interaction 3D model to observe the interaction between cells and the matrix.
[0011] In step 1) of the above method, the ratio of DBCO-RGD to the helical polypeptide with azide is: 1-2 mg: 100-350 mg, specifically 1.8 mg: 300 mg;
[0012] The click chemistry reaction is carried out at room temperature for 18-28 hours.
[0013] In step 2) of the above method, the RGD functionalized helical polypeptide is mixed with PBS, placed in a refrigerator at 4°C overnight, and then evenly blown on an ice box to fully dissolve it;
[0014] The cells may be tumor cells and / or cancer cells, specifically 231 (MBA-MB-231) triple-negative human breast cancer cells;
[0015] The single cell 3D culture operation is as follows: mixing the cell suspension with the RGD functionalized helical polypeptide, inoculating it in a cell culture plate, placing it in a 37°C incubator for gelation, and culturing it;
[0016] The volume ratio of cells to RGD-functionalized helical polypeptide is 1:1; the cell concentration (referring to the concentration of cells in the cell suspension) can be: 1×10 5 / mL; the concentration of the RGD functionalized helical polypeptide may be 10mg / mL-20mg / mL, specifically 10mg / mL;
[0017] The gelling time may be 15-30 minutes, specifically 20 minutes;
[0018] The culture time may be 6-9 days, specifically 7 days;
[0019] During the culture process, the medium was changed every two days and the cell activity was measured.
[0020] The cell-matrix interaction 3D model prepared by the above method also belongs to the protection scope of the present invention.
[0021] The application of the above-mentioned cell-matrix interaction 3D model as a platform for determining the invasive ability of cells also falls within the protection scope of the present invention.
[0022] In the application, the cell may be a tumor cell and / or a cancer cell, specifically 231 (MBA-MB-231) triple-negative human breast cancer cell.
[0023] The application of the above-mentioned cell adhesion polypeptide RGD functionalized helical polypeptide or cell-matrix interaction 3D model in the drug screening platform for preparing anti-tumor drugs also falls within the protection scope of the present invention.
[0024] The present invention also provides a method for screening and evaluating drugs using the above cell-matrix interaction 3D model.
[0025] The method for screening and evaluating drugs using the above-mentioned cell-matrix interaction 3D model comprises the following steps: dissolving the RGD-functionalized helical polypeptide in PBS, culturing with tumor cells and / or cancer cells, adding drugs and serum-free DMEM culture medium to the upper chamber, adding serum-containing DMEM culture medium to the lower chamber, 3D culturing for 3-4 days (specifically 3 days), and measuring the invasive ability of tumor cells and / or cancer cells in the presence of drugs, thereby judging the inhibitory effect of the drug on the migration of tumor cells and / or cancer cells.
[0026] Wherein, the concentration of the RGD functionalized helical polypeptide may be: 10 mg / mL-20 mg / mL;
[0027] The tumor cells and / or cancer cells may specifically be 231 (MBA-MB-231) triple-negative human breast cancer cells;
[0028] The concentration of tumor cells and / or cancer cells can be 1×10 5 Pieces / mL;
[0029] The drug may specifically be a MMP inhibitor.
[0030] The invention also provides a drug screening system.
[0031] The drug screening system provided by the present invention contains the above-mentioned cell adhesion polypeptide RGD functionalized helical polypeptide or contains the above-mentioned cell-matrix interaction 3D model.
[0032] As a highly biomimetic reversible temperature-sensitive biomaterial, helical polypeptide hydrogel can accurately simulate the fiber structure and mechanical properties of the natural extracellular matrix. Its unique responsive biomimetic properties create an excellent environment for cell growth, while showing excellent resistance to biological enzymes such as matrix metalloproteinases (MMPs), ensuring that the growth of cells in helical polypeptide hydrogels is not disturbed by enzymatic hydrolysis, and mainly regulating the cell state through biophysical mechanisms. Helical polypeptide also has good temperature sensitivity. When the temperature rises to the gel point, a stable gel network structure is formed. Through the "click chemistry" strategy, we accurately attached the cell adhesion peptide RGD to the side chain of the helical polypeptide hydrogel, providing adhesion sites for cells, which greatly promoted the stable attachment and growth of cells on the gel matrix. With its unique properties, the RGD-functionalized helical polypeptide hydrogel has been successfully applied to a variety of cell lines and organoid 3D culture systems, providing cells with a highly biomimetic growth environment, which helps to deeply explore the behavioral characteristics of cells under complex physiological conditions. In addition, the mechanical properties of helical polypeptide hydrogels are adjustable. By precisely controlling its hardness, elasticity and other parameters, it can directly affect biological processes such as cell proliferation, differentiation, and migration, providing a powerful tool for cell biology research. Therefore, helical polypeptide hydrogels have shown great application potential and broad prospects in biomedical fields such as tumor drug screening platforms due to their unique temperature sensitivity, bionic fiber structure, adjustable mechanical properties and good biocompatibility.
[0033] The present invention uses RGD functionalized helical polypeptide to explore the interaction between cells and matrix in the tumor microenvironment. 231 cells were cultured with helical polypeptide alone, and only in RGD did 231 cells form compact spheres. In the fluorescence experiment, it was clearly proved that 231 cells can generate tension to reshape the extracellular matrix. In the drug screening system, after adding MMP inhibitors, a large number of cells still invaded in the helical polypeptide model. This three-dimensional tumor model is a promising method for future tumor drug discovery.
[0034] In order to more comprehensively evaluate the efficacy of anti-tumor drugs, the present invention designs a platform capable of simulating a biophysical environment. Through this platform, the actual effect of drugs in a complex biological environment can be more accurately evaluated, providing a more reliable basis for drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Figure 1 The morphology and size of helical polypeptide-encapsulated 231 cells cultured for 7 days were observed by fluorescence microscopy in Example 2 of the present invention.
[0036] Figure 2 It is the survival rate of 231 cells in the hydrogel measured in Example 3 of the present invention.
[0037] Figure 3 The matrix remodeling after 3 days of encapsulating 231 cells in hydrogel in Example 4 of the present invention was photographed by fluorescence microscopy.
[0038] Figure 4 and Figure 5 The results of cell invasion and quantitative analysis after the helical polypeptide hydrogel in the present invention was treated with Marimastat as a platform.
[0039] Figure 6 and Figure 7 The helical polypeptide hydrogel in the present invention is used as a platform to treat the cell invasion and quantitative analysis results with Tanomastat. DETAILED DESCRIPTION
[0040] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0041] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.
[0042] The DBCO-RGD used in the following examples was purchased from Shanghai Ambio Biotech Co., Ltd.
[0043] Example 1. Preparation of RGD-functionalized helical polypeptide
[0044] Connect DBCO-RGD to the helical polypeptide hydrogel. Take 300 mg of the helical polypeptide and dissolve it in 14 mL of acetonitrile. Dissolve 1.8 mg of DBCO-RGD in 8 μL of DMSO until it is completely dissolved, then add it to the helical polypeptide solution and stir to react. The stirring time is about 24 hours to ensure that the reaction is fully carried out. After the reaction is completed, precipitate with isopropyl ether. Collect the precipitate by centrifugation and place it in a dry environment for air drying. The air drying time is about 24 hours to ensure that the precipitate is completely dry.
[0045] The structural formula of the helical polypeptide is as follows:
[0046]
[0047] n represents the degree of polymerization, n=50-50000.
[0048] Example 2: 231 Single Cell Culture
[0049] The 231 cells were digested, centrifuged for 10 min, and the cell concentration was diluted to 2×10 5 / mL; the RGD functionalized helical polypolypeptide prepared in Example 1 was mixed with PBS, placed in a refrigerator at 4°C overnight, and then evenly blown on an ice box to fully dissolve it, with a concentration of 20 mg / mL, and mixed evenly with the cell suspension at a ratio of 1:1. A certain amount of the mixture was added to a cell culture plate and placed in an incubator for 20 minutes. After that, an appropriate amount of DMEM culture medium was added for culture, and the morphology and size of the helical polypolypeptide-encapsulated 231 cells after culture were observed by fluorescence microscopy after 7 days.
[0050] Figure 1 The morphology and size of 231 cells are shown. The detected size is about 50 μm and the cells form compact tumor spheres.
[0051] Example 3: Cytotoxicity of the prepared hydrogel was measured by 231 cell survival rate to explore the biocompatibility of the helical polypeptide hydrogel
[0052] 231 cells were cultured at 2×10 5A cell density of 100 μL / mL was mixed with the hydrogel (20 mg / mL) prepared in Example 1 at an equal volume at 4°C. Subsequently, the mixed solution was placed in a cell culture plate for culture, 100 μL of the mixed solution was added to each well, and each group of experiments was set to contain three parallel samples. After the hydrogel solidified, 200 μL of DMEM culture medium was added to each well. After culturing for 1 day, 3 days, 5 days, and 7 days, 20 μL of CCK-8 was added to each well, and the absorbance at 450 nm was recorded with a microplate reader after 2 hours. Relative cell survival rate (%) = absorbance measured in the experimental group / absorbance measured in the control group × 100%.
[0053] Figure 2 It shows that the hydrogel provided by the present invention has no obvious cytotoxicity to 231 cells and has good biocompatibility.
[0054] Example 4: Matrix displacement and remodeling induced by 231 single cell force
[0055] The cells were incubated with fluorescent dye for 45 minutes for live cell staining; the helical polypolypeptide prepared in Example 1 was incubated with Cy5 for 20 minutes. The previously stained cells were treated and the cell concentration was diluted to 6×10 4 The cells were encapsulated in the hydrogel and left to stand for 3 days before being photographed with a fluorescence microscope. Through the above steps, we performed live cell staining and fluorescence photography of 231 cells.
[0056] Figure 3 Fluorescence microscopy clearly shows that the blue fluorescence is 231 cells and the red fluorescence is hydrogel. It can be found that the red fluorescence around the cells has become significantly brighter, indicating that the cells have undergone significant matrix remodeling.
[0057] Example 5. Evaluation of Helical Polypeptide Hydrogel as an In Vitro Tumor Drug Platform
[0058] In this experiment, we used Corning's Matrigel as a control experiment. Its main components are laminin, collagen, heparin sulfate proteoglycans and several soluble factors, which are derived from the secreted basement membrane extract of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. The invasion experiment of Matrigel may only simulate the biochemical microenvironment, while ignoring the important effects of the biophysical microenvironment on tumor invasion and drug efficacy.
[0059] First, 231 cells were digested and the cell density was counted using a cell counting plate to 2×10 5 / mL. The cells and the 20mg / mL helical polypolypeptide prepared in Example 1 were placed in a chamber for invasion experiment. The control group was treated with 231 single cells in the same manner with a 44% concentration of matrix gel. After treatment with MMP inhibitors (Marimastat, Tanomastat), the cells were cultured under specific conditions. In the following 3 days, the invasion behavior of the cells was observed. During this process, the invasion ability of the cells in the Transwell system will be recorded in detail to evaluate the effect of MMP inhibitors on cell activity.
[0060] The specific operation is as follows: remove the culture medium in the chamber, fix the cells with the fixative for 15 minutes, and then incubate with crystal violet for 30 minutes. Take out the Transwell chamber and gently wipe the matrix gel and cells in the chamber with a cotton swab or cotton soaked in PBS. Add 4% paraformaldehyde fixative to the clean wells of the 24-well plate and place the chamber in the fixation for 30 minutes. Discard the fixative and wash the inside and outside of the chamber with PBS once. Add crystal violet staining solution to the clean wells of the 24-well plate and place the chamber in the staining for 10 minutes. Take out the chamber and wash the inside and outside of the chamber with PBS 3 times. After proper air drying, observe and take pictures under a microscope, and use ImageJ to take the average value for quantitative research.
[0061] Figure 4 and Figure 5 After Marimastat treatment, the number of cells invading in the matrix gel group and the helical polypeptide group and the number of cells invading were counted separately. The helical polypeptide group was significantly higher than the matrix gel group.
[0062] Figure 6 and Figure 7 After Tanomastat treatment, the number of cells invading in the matrix gel group and the helical polypeptide group and the number of cells that invaded were counted separately. The helical polypeptide group was significantly higher than the matrix gel group.
[0063] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.
Claims
1. A method for preparing a 3D cell-matrix interaction model, comprising the following steps: 1) Preparation of helical polypeptides functionalized with cell adhesion peptide RGD The helical polypeptide with azide was subjected to click chemistry reaction with DBCO-GRGDS to obtain the RGD-functionalized helical polypeptide; 2) Construction of 3D model of cell-matrix interaction The RGD-functionalized helical polypeptide was dissolved in PBS, mixed evenly with the cell suspension to form a gel, and then 3D culture was performed to obtain a 3D model of cell-matrix interaction and observe the interaction between cells and the matrix.
2. The method according to claim 1, characterized in that: In step 1), the ratio of DBCO-RGD to the helical polypeptide with azide is: 1-2 mg: 100-350 mg; The click chemistry reaction is carried out at room temperature for 18-28 hours.
3. The method according to claim 1, characterized in that The cells are tumor cells and / or cancer cells; The single cell 3D culture operation is as follows: mixing the cell suspension with the RGD functionalized helical polypeptide, inoculating it in a cell culture plate, placing it in a 37°C incubator for gelation, and culturing it; The volume ratio of cells to RGD-functionalized helical polypeptide was 1:1; cell concentration: 1×10 5 / mL; the concentration of RGD functionalized helical polypeptide is 10mg / mL-20mg / mL; The gelling time is 15-30min; The culture time is 6-9 days.
4. A 3D cell-matrix interaction model prepared by the method according to any one of claims 1 to 3.
5. Use of the cell-matrix interaction 3D model according to claim 4 as a platform for determining the invasive ability of cells.
6. The use according to claim 5, characterized in that: The cells are tumor cells and / or cancer cells.
7. Use of the cell adhesion polypeptide RGD functionalized helical polypeptide of claim 1 or the cell-matrix interaction 3D model of claim 4 in a drug screening platform for preparing anti-tumor drugs.
8. A method for screening and evaluating drugs using the cell-matrix interaction 3D model of claim 4, comprising the following steps: dissolving the RGD-functionalized helical polypeptide in PBS, culturing with tumor cells and / or cancer cells, adding drugs and serum-free DMEM culture medium to the upper chamber, adding serum-containing DMEM culture medium to the lower chamber, 3D culturing for 3-4 days, and measuring the invasive ability of tumor cells and / or cancer cells in the presence of drugs, thereby determining the inhibitory effect of the drug on the migration of tumor cells and / or cancer cells.
9. A drug screening system, comprising the cell adhesion polypeptide RGD functionalized helical polypeptide of claim 1 or the cell-matrix interaction 3D model of claim 4.