Construction of 3D cell-matrix interaction model and use thereof in drug screening
By constructing a 3D cell-matrix interaction model and using RGD-functionalized helical polypeptides to simulate the natural extracellular matrix, the problem of neglecting the biophysical microenvironment in existing technologies is solved, and more accurate drug screening results are achieved.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- HEBEI UNIV OF TECH
- Filing Date
- 2026-01-15
- Publication Date
- 2026-07-23
Smart Images
Figure CN2026072920_23072026_PF_FP_ABST
Abstract
Description
Construction of a 3D Cell-Matrix Interaction Model and Its Application in Drug Screening Technical Field
[0001] This invention belongs to the field of drug screening model development, specifically involving the construction of a 3D cell-matrix interaction model and its application in drug screening. Background Technology
[0002] Tumor cell invasion is a core step in the cancer metastasis cascade, crucial for both penetrating the extracellular matrix (ECM) and crossing vascular endothelium. In this complex process, cell-matrix interactions within the tumor microenvironment (TME), in particular, synergistically regulate cellular behavior through both biomechanical mechanisms (such as cellular traction) and biochemical signaling (such as cytokine secretion). As research progresses, the critical role of these interactions in tumor cell invasion becomes increasingly apparent. However, traditional experimental designs, whether in vitro using Transwell models or in vivo using mouse models, have limitations in controlling ECM parameters, making it difficult to fully elucidate the mechanisms of cell-matrix interactions.
[0003] Matrix metalloproteinases (MMPs), belonging to the zinc-dependent endopeptidase family, play crucial roles in multiple stages of cancer progression, such as invasion, metastasis, and tumor angiogenesis. These proteases can also detach from the matrix and enter the plasma, thus being considered potential biomarkers for disease progression or the effectiveness of related treatment interventions. Given their importance, the pharmaceutical industry has invested heavily in the development of MMP inhibitors (MMPIs) to provide oral treatment for cancer patients. However, some MMPs, such as the broad-spectrum inhibitor Marimastat, have not shown good efficacy in clinical trials, either alone or in combination with gemcitabine, for the treatment of unresectable pancreatic cancer patients; another type of MMP targeting MMP3, MMP9, and MMP2 has also failed to demonstrate efficacy. 13 The inhibitor Tanomastat (BAY-12-9566), when used in combination with gemcitabine, also showed poor efficacy in a phase III trial in pancreatic cancer patients, indicating that under real physiological conditions, inhibiting MMPs alone is not effective in preventing cancer cell invasion. Although these inhibitors all showed good anti-invasive effects in the Matrigel invasion assay, they failed in phase III clinical trials. It is worth noting that Matrigel-based invasion assays may only simulate the biochemical microenvironment, neglecting the important influence of the biophysical microenvironment on tumor invasion and drug efficacy.
[0004] An ideal physical microenvironment platform (a platform that simulates the biophysical environment) should possess the following characteristics: ① It should be able to simulate the complex mechanical properties of the natural ECM. The natural ECM is not a simple linear material; it exhibits complex nonlinear mechanical properties, such as stress stiffening effects, time-dependent viscoelasticity, and mechanical plasticity. ② It should possess a fibrous network structure similar to the natural ECM. ③ It should be able to respond to cellular feedback, thereby forming a dynamic bidirectional cell-matrix interaction with cells, which in turn affects cell proliferation, migration, and other cellular behaviors. Currently, most reported hydrogels lack biomechanical response, limiting the exploration of multiple factors contributing to cancer cell invasion and further affecting the effectiveness of clinical drugs. Therefore, researching novel biomimetic hydrogels that leverage the role of the physical microenvironment in drug screening for tumor invasion is of great significance. Summary of the Invention
[0005] This invention addresses the technical shortcomings of current drug screening platforms that only focus on biochemical cell-matrix interactions by providing a 3D model of complex cell-matrix physical interactions reconstructed in vitro and its construction method, and applying it to drug screening and evaluation.
[0006] The cell-matrix interaction 3D model provided by this invention is prepared by a method comprising the following steps:
[0007] 1) Preparation of helical polypeptides functionalized with cell adhesion peptide RGD
[0008] Azid-containing helical polypeptides were reacted with DBCO-GRGDS via click chemistry to obtain RGD-functionalized helical polypeptides.
[0009] 2) Construction of a 3D model of cell-matrix interaction
[0010] RGD-functionalized helical polypeptides were dissolved in PBS, mixed evenly with cell suspension to form a gel, and then single-cell 3D culture was performed to obtain a cell-matrix interaction 3D model, and the interaction between cells and matrix was observed.
[0011] In step 1) of the above method, the ratio of DBCO-RGD to the azide-containing helical polypeptide is 1-2 mg: 100-350 mg, specifically 1.8 mg: 300 mg;
[0012] The click chemical reaction was carried out at room temperature for 18-28 hours.
[0013] Step 2 of the above method involves mixing the RGD-functionalized helical polypeptide with PBS, placing it in a refrigerator at 4°C overnight, and then gently blowing it onto an ice pack to ensure complete dissolution.
[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 procedure is as follows: the cell suspension is mixed with RGD-functionalized helical polypeptide, seeded in a cell culture plate, and placed in a 37°C incubator for gelation and culture.
[0016] The volume ratio of cells to RGD-functionalized helical polypeptides is 1:1; the cell concentration (referring to the concentration of cells in the cell suspension) can be 1×10⁻⁶. 5 The concentration of RGD-functionalized helical polypeptides can be 10 mg / mL to 20 mg / mL, specifically 10 mg / mL.
[0017] The gelation time can be 15-30 min, specifically 20 min;
[0018] The culture time can be 6-9 days, specifically 7 days;
[0019] The culture medium was changed every two days during the culture process, and cell viability was measured.
[0020] The cell-matrix interaction 3D model prepared by the above method is also within the scope of protection of this invention.
[0021] The application of the aforementioned cell-matrix interaction 3D model as a platform in determining the invasive ability of cells also falls within the scope of protection of this invention.
[0022] In the application described, the cells may be tumor cells and / or cancer cells, specifically 231 (MBA-MB-231) triple-negative human breast cancer cells.
[0023] The application of the above-mentioned cell adhesion peptide RGD-functionalized helical polypeptide or cell-matrix interaction 3D model in the drug screening platform for the preparation of antitumor drugs is also within the scope of protection of this invention.
[0024] The present invention also provides a method for screening and evaluating drugs using the above-mentioned cell-matrix interaction 3D model.
[0025] The method for screening and evaluating drugs using the above-mentioned cell-matrix interaction 3D model includes the following steps: dissolving RGD-functionalized helical polypeptides in PBS, culturing them with tumor cells and / or cancer cells, adding the drug and serum-free DMEM medium to the upper chamber, adding serum-containing DMEM medium to the lower chamber, culturing for 3-4 days (specifically 3 days), measuring the invasive ability of tumor cells and / or cancer cells in the presence of the drug, thereby determining the inhibitory effect of the drug on the migration of tumor cells and / or cancer cells.
[0026] The concentration of the RGD-functionalized helical polypeptide can be 10 mg / mL to 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 1×10⁻⁶. 5 cells / mL;
[0029] The drug may specifically be an MMP inhibitor.
[0030] This invention also provides a drug screening system.
[0031] The drug screening system provided by this invention contains either the above-mentioned cell adhesion polypeptide RGD-functionalized helical polypeptide or the above-mentioned cell-matrix interaction 3D model.
[0032] Helical polypeptide hydrogels, as highly biomimetic reversible thermosensitive biomaterials, can precisely mimic the fibrous structure and mechanical properties of the natural extracellular matrix. Their unique responsive biomimetic properties create a superior environment for cell growth, while exhibiting excellent resistance to matrix metalloproteinases (MMPs) and other enzymes, ensuring that cell growth in the helical polypeptide hydrogel is not interfered with by enzymatic degradation, primarily regulating cell state through biophysical mechanisms. Helical polypeptides also possess good thermosensitivity; when the temperature rises to the gelation point, a stable gel network structure is formed. Using a "click chemistry" strategy, we precisely attached the cell adhesion peptide RGD to the side chains of the helical polypeptide hydrogel, providing adhesion sites for cells and greatly promoting stable cell attachment and growth on the gel matrix. RGD-functionalized helical polypeptide hydrogels, with their unique properties, have been successfully applied to various cell lines and organoid 3D culture systems, providing cells with a highly biomimetic growth environment and contributing to a deeper exploration of cellular behavior under complex physiological conditions. Furthermore, the mechanical properties of helical polypeptide hydrogels are tunable. By precisely controlling parameters such as hardness and elasticity, they can directly influence biological processes such as cell proliferation, differentiation, and migration, providing a powerful tool for cell biology research. Therefore, helical polypeptide hydrogels, with their unique temperature sensitivity, biomimetic fiber structure, tunable mechanical properties, and good biocompatibility, demonstrate enormous application potential and broad prospects in biomedical fields such as tumor drug screening platforms.
[0033] This invention utilizes RGD-functionalized helical polypeptides to explore cell-matrix interactions in the tumor microenvironment. When 231 cells were cultured alone with the helical polypeptides, they formed compact spherical structures only within the RGD model. Fluorescence experiments clearly demonstrated that 231 cells could generate tension that remodeled the extracellular matrix. In drug screening systems, even with the addition of MMP inhibitors, a large number of cells still invaded in the helical polypeptide model. This three-dimensional tumor model represents a promising method for future tumor drug discovery.
[0034] To more comprehensively evaluate the efficacy of anti-tumor drugs, this invention designs a platform capable of simulating a biophysical environment. This platform allows for more accurate assessment of the actual effects of drugs in complex biological environments, providing a more reliable basis for drug development. Attached Figure Description
[0035] Figure 1 shows the morphology and size of 231 cells encapsulated by helical polypeptide and cultured for 7 days using a fluorescence microscope in Example 2 of this invention.
[0036] Figure 2 shows the survival rate of 231 cells in the hydrogel as measured in Example 3 of this invention.
[0037] Figure 3 shows the matrix remodeling of 231 cells encapsulated in hydrogel after 3 days, as captured by a fluorescence microscope in Example 4 of this invention.
[0038] Figures 4 and 5 show the results of cell invasion and quantitative analysis after treatment with Marimastat using the helical polypeptide hydrogel as a platform in this invention.
[0039] Figures 6 and 7 show the results of cell invasion and quantitative analysis after treatment with Tanomastat using the helical polypeptide hydrogel as a platform in this invention. Detailed Implementation
[0040] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0042] The DBCO-RGD used in the following examples was purchased from Shanghai Angbo Biotechnology Co., Ltd.
[0043] Example 1: Preparation of RGD-functionalized helical polypeptides
[0044] DBCO-RGD was attached to a helical polypeptide hydrogel. 300 mg of the helical polypeptide was dissolved in 14 mL of acetonitrile. 1.8 mg of DBCO-RGD was dissolved in 8 μL of DMSO until completely dissolved, and then added to the helical polypeptide solution. The mixture was stirred for approximately 24 h to ensure complete reaction. After the reaction was complete, precipitation was performed using isopropyl ether. The precipitate was collected by centrifugation and air-dried in a dry environment for approximately 24 h to ensure complete drying.
[0045] The structural formula of the helical polypeptide is shown below:
[0046]
[0047] n represents the degree of aggregation, n = 50 to 50000.
[0048] Example 2, 231 Single-cell culture
[0049] Cells were digested, centrifuged for 10 min, and diluted to a concentration of 2 × 10⁻⁶. 5 Cells / mL; The RGD-functionalized helical polypeptide prepared in Example 1 was mixed with PBS, placed in a refrigerator at 4°C overnight, and then evenly pipetted on an ice box to ensure complete dissolution, achieving a concentration of 20 mg / mL. This was then mixed thoroughly with the cell suspension at a 1:1 ratio. A certain amount of the mixture was added to a cell culture plate and incubated for 20 min. Afterward, an appropriate amount of DMEM medium was added for culturing. Seven days later, the morphology and size of the 231 cells encapsulated by the helical polypeptide were observed using a fluorescence microscope.
[0050] Figure 1 shows the morphology and size of 231 cells, with a detected size of approximately 50 μm and cells forming compact tumor spheres.
[0051] Example 3: The cytotoxicity of the prepared hydrogel was investigated by measuring the viability of 231 cells, thus exploring the biocompatibility of the helical polypeptide hydrogel.
[0052] Cell 231 was fed at a rate of 2 × 10⁻⁶. 5Cells at a density of [number] cells / mL were mixed in an equal volume with the hydrogel prepared in Example 1 (20 mg / mL) at 4°C. The mixture was then placed in cell culture plates, with 100 µL of the mixture added to each well, and each experiment included three replicates. After the hydrogel solidified, 200 µL of DMEM medium was added to each well. After 1, 3, 5, and 7 days of culture, 20 µL of CCK-8 was added to each well, and the absorbance at 450 nm was recorded using a microplate reader after 2 h. Relative cell viability (%) = (Absorbance of experimental group / Absorbance of control group) × 100%.
[0053] Figure 2 shows that the hydrogel provided by the present invention has no obvious cytotoxicity to 231 cells and has good biocompatibility.
[0054] Example 4, 231 Single-cell force-induced matrix displacement and remodeling
[0055] Live cell staining was performed by incubating cells with a fluorescent dye for 45 min; the helical polypeptide prepared in Example 1 was incubated with Cy5 for 20 min. The previously stained cells were then treated by diluting the cell concentration to 6 × 10⁻⁶. 4 Cells were encapsulated in hydrogels at a concentration of cells / mL and incubated for 3 days before fluorescence microscopy imaging. Following these steps, we performed live-cell staining and fluorescence imaging on 231 cells.
[0056] Figure 3, taken using a fluorescence microscope, clearly shows that the blue fluorescence represents cells 231, and the red fluorescence represents the hydrogel. The red fluorescence around the cells is noticeably brighter, indicating significant matrix remodeling within the cells.
[0057] Example 5: Evaluation of helical polypeptide hydrogels as an in vitro tumor drug platform
[0058] In this experiment, we used Corning's Matrigel as a control. Its main components are laminin, collagen, heparin sulfate proteoglycans, and several soluble factors, derived from the secretory basement membrane extract of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. The Matrigel invasion assay may only simulate the biochemical microenvironment, neglecting the significant influence of the biophysical microenvironment on tumor invasion and drug efficacy.
[0059] First, the 231 cells were digested and the cell density was counted using a cell counting chamber to be 2 × 10⁻⁶. 5Cells / mL. Cells and the 20 mg / mL helical polypeptide prepared in Example 1 were placed in a chamber for invasion experiments. The control group was treated with 44% matrix gel and 231 single cells under the same procedure. After treatment with MMP inhibitors (Marimastat, Tanomastat), cells were cultured under specific conditions. Cell invasion behavior was observed over the following 3 days. During this process, the invasive ability of cells in the Transwell system was recorded in detail to assess the effect of MMP inhibitors on cell activity.
[0060] The specific procedure is as follows: Remove the culture medium from the chamber, fix the cells with fixative for 15 min, and then incubate with crystal violet for 30 min. Remove the Transwell chamber and gently wipe the matrix gel and cells inside with a cotton swab or cotton ball moistened with PBS. Add 4% paraformaldehyde fixative to a clean well of a 24-well plate and place the chamber in for fixation for 30 min. Discard the fixative and wash the chamber inside and outside once with PBS. Add crystal violet staining solution to a clean well of a 24-well plate and stain the chamber for 10 min. Remove the chamber and wash the chamber inside and outside three times with PBS. After appropriate air drying, observe and photograph under a microscope, and then use ImageJ to calculate the average value for quantitative analysis.
[0061] Figures 4 and 5 show the number of cells that invaded the matrix gel group and the number of cells that survived the invasion after Marimastat treatment, and the number of cells that survived the invasion was significantly higher in the helical polypeptide group than in the matrix gel group.
[0062] Figures 6 and 7 show the number of cells that invaded the matrix gel group and the number of cells that survived the invasion after Tanomastat treatment, and the number of cells that invasive in the matrix gel group was significantly higher than that in the matrix gel group.
[0063] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. A method for preparing a 3D model of cell-matrix interaction, comprising the following steps: 1) Preparation of helical polypeptides functionalized with cell adhesion peptide RGD Azid-containing helical polypeptides were reacted with DBCO-GRGDS via click chemistry to obtain RGD-functionalized helical polypeptides. 2) Construction of a 3D model of cell-matrix interaction RGD-functionalized helical polypeptides were dissolved in PBS, mixed evenly with cell suspension to form a gel, and then cultured in 3D to obtain a 3D model of cell-matrix interaction, allowing observation of the interaction between cells and matrix.
2. The method according to claim 1, characterized in that, In step 1), the ratio of DBCO-RGD to the azide-containing helical polypeptide is 1-2 mg: 100-350 mg; The click chemical reaction was 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 procedure is as follows: the cell suspension is mixed with RGD-functionalized helical polypeptide, seeded in a cell culture plate, and placed in a 37°C incubator for gelation and culture. The volume ratio of cells to RGD-functionalized helical polypeptides was 1:1; cell concentration: 1×10⁻⁶. 5 The concentration of RGD-functionalized helical polypeptides is 10 mg / mL to 20 mg / mL. The gelation time is 15-30 min; The culture time is 6-9 days.
4. A cell-matrix interaction 3D model prepared by the method described in any one of claims 1-3.
5. The application of the cell-matrix interaction 3D model of claim 4 as a platform for determining the invasive ability of cells.
6. The application according to claim 5, characterized in that, The cells are tumor cells and / or cancer cells.
7. The application of the helical polypeptide functionalized with the cell adhesion polypeptide RGD as described in claim 1 or the cell-matrix interaction 3D model as described in claim 4 in a drug screening platform for the preparation of antitumor drugs.
8. A method for screening and evaluating drugs using the cell-matrix interaction 3D model as described in claim 4, comprising the following steps: dissolving RGD-functionalized helical polypeptides in PBS, culturing them with tumor cells and / or cancer cells, adding the drug and serum-free DMEM medium to the upper chamber, adding serum-containing DMEM medium to the lower chamber, culturing for 3-4 days, and measuring the invasive ability of tumor cells and / or cancer cells in the presence of the drug, thereby determining the inhibitory effect of the drug on the migration of tumor cells and / or cancer cells.
9. A drug screening system comprising a helical polypeptide functionalized with the cell adhesion polypeptide RGD as described in claim 1 or a cell-matrix interaction 3D model as described in claim 4.