Application of hydrogen bond organic framework nano material PFC-73 in cell culture and cell proliferation
The hydrogen-bonded organic framework nanomaterial PFC-73, when used in cell culture media, solves the problems of biosafety and standardization of nanomaterials in cell culture, promotes cell proliferation and migration, and has significant biological activity and application potential.
Patent Information
- Application Number
- CN202411183943.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-27
- Publication Date
- 2026-03-06
AI Technical Summary
Existing nanomaterials face challenges in terms of biosafety, standardization, and cost in cell culture media, limiting their widespread adoption in large-scale production and clinical applications.
The hydrogen-bonded organic framework nanomaterial PFC-73, through self-assembly to form a stable framework structure, was used in cell culture medium at a concentration of 50 μg/mL to promote cell proliferation, migration, and vascular endothelial growth factor expression.
It significantly promotes cell proliferation and migration, optimizes cell growth conditions, and enhances the expression of vascular endothelial growth factor and proliferation factor, showing broad application prospects and potential.
Smart Images
Figure CN121610440A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to the application of hydrogen-bonded organic framework nanomaterial PFC-73 in cell culture and cell proliferation. Background Technology
[0002] Nanomaterials, such as metal-organic frameworks, covalent metal-organic frameworks, and quantum dots, are often used as additives in cell culture media due to their excellent biocompatibility and tunable surface properties. These nanoparticles can interact with biomolecules in the culture medium through electrostatic adsorption, covalent binding, and other mechanisms, thereby influencing the cell growth environment. Experiments have shown that adding appropriate amounts of nanoparticles can significantly improve cell proliferation rate and survival rate, while also promoting cell differentiation.
[0003] Hydrogen-bonded organic frameworks (HOFs), as a novel class of porous crystalline materials, have shown broad application prospects in the biomedical field due to their unique structure and properties. HOFs are composed of organic or metal-organic building blocks linked by intermolecular forces such as hydrogen bonds, and possess characteristics such as high specific surface area, designable structure, and tunable pores. These properties give HOFs significant advantages in drug delivery, gas adsorption and separation, and fluorescence sensing. Particularly in drug delivery, HOFs, with their good biocompatibility and low cytotoxicity, hold promise as candidate materials for novel drug carriers.
[0004] However, nanomaterials face several potential challenges when applied to culture media, including: 1. Biosafety: The long-term biosafety of nanomaterials requires further research to ensure their safety in clinical applications. 2. Standardization: Currently, there is a lack of unified standards and specifications for the preparation and application of nanomaterials, necessitating the establishment of corresponding quality control systems. 3. Cost: The high cost of preparing high-performance nanomaterials limits their application in large-scale production. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides the application of the hydrogen-bonded organic framework nanomaterial PFC-73 in cell culture and cell proliferation, migration, and drug preparation. Details are as follows:
[0006] Applications of PFC-73, a hydrogen-bonded organic framework nanomaterial, in cell culture, especially in the preparation of cell culture reagents.
[0007] Furthermore, the hydrogen-bonded organic framework nanomaterial PFC-73, as a hydrogen-bonded organic framework material, is mainly composed of the self-assembly of the organic ligand tetracarboxylate porphyrin and the metal ion (nickel). The ligand and the metal ion are connected together through hydrogen bonds and other non-covalent interactions to form a stable framework structure. Currently, most research on PFC-73 focuses on photoelectrocatalysis, gas adsorption and separation, fluorescence sensing, and other fields; this is its first application in the field of cell culture.
[0008] Furthermore, the PFC-73 has a particle size of 5-6 nm.
[0009] Furthermore, the cell culture involves adding hydrogen-bonded organic framework nanomaterial PFC-73 to the culture medium before cell culture. Preferably, the concentration of PFC-73 in the culture medium is 50 μg / mL.
[0010] A cell culture medium containing the hydrogen-bonded organic framework nanomaterial PFC-73 includes a basal culture medium and the hydrogen-bonded organic framework nanomaterial PFC-73. Furthermore, the concentration of PFC-73 in the culture medium is 50 μg / mL.
[0011] The hydrogen-bonded organic framework nanomaterial PFC-73 exhibits remarkable bioactivity, particularly in promoting cell proliferation. It can also be applied to promote cell proliferation, cell migration, and the expression of vascular endothelial growth factor (VEGF) and proliferative factors. It can be formulated into corresponding reagents, such as those for promoting cell proliferation, cell migration, and the expression of VEGF and proliferative factors. Furthermore, it shows great potential in promoting angiogenesis and cell proliferation, making it suitable for drug development, especially for vascular-related drugs.
[0012] The beneficial effects of this invention are:
[0013] (1) This application discloses the remarkable bioactivity exhibited by the hydrogen-bonded organic framework nanomaterial PFC-73, particularly in promoting cell proliferation. Experimental results from this application show that treatment of cells with different concentrations of PFC-73 significantly enhanced cell proliferation, with this effect peaking at a PFC-73 concentration of 50 μg / mL, indicating that this concentration is the optimal condition for cell growth. Particularly noteworthy is that treatment with PFC-73 (50 μg / mL) significantly upregulated the expression of vascular endothelial growth factor (Vegf) and the key proliferation factor (Ki67), revealing not only the molecular-level mechanism of action of PFC-73 but also its significant potential in promoting angiogenesis and cell proliferation.
[0014] (2) Treatment with the hydrogen-bonded organic framework nanomaterial PFC-73 also significantly promoted the migration ability of vascular endothelial cells. This discovery is of great significance for understanding angiogenesis, wound healing, and various pathophysiological processes related to cell migration. In summary, PFC-73 of this invention, with its unique biological activity and broad application prospects, provides new research ideas and treatment strategies for cell biology, regenerative medicine, and drug development.
[0015] (3) This application provides a culture medium for hydrogen-bonded organic framework nanomaterials, comprising a basal culture medium, 10% serum, and PFC-73 nanomaterials. The concentration of PFC-73 nanomaterials is 0-200 μg / mL, with an optimal concentration of 50 μg / mL for promoting cell proliferation. This invention has the following advantages: treatment of cells with different concentrations of PFC-73 significantly promotes cell proliferation, with the highest proliferation rate observed at a PFC-73 concentration of 50 μg / mL. Specifically, PFC-73 (50 μg / mL) treatment significantly upregulated the expression of vascular endothelial growth factor Vegf and proliferation factor Ki67. Furthermore, PFC-73 treatment significantly promoted the migration of vascular endothelial cells. Attached Figure Description
[0016] Figure 1 The image shows the cell viability of HUVECs incubated with different concentrations of PFC-73 for 24 hours.
[0017] Figure 2 The image shows the cell viability of HUVECs incubated with different concentrations of PFC-73 for 48 hours.
[0018] Figure 3 The results of cell proliferation assays after incubation of HUVECs with PFC-73 (50 μg / mL) for 1, 3, and 5 days are shown in the figure.
[0019] Figure 4 Image showing the results of live / dead cell detection by Calcein-AM / PI fluorescence staining after HUVECs were treated with PFC-73 (50 μg / mL) for 72 hours. Scale bar: 100 μm.
[0020] Figure 5 Wound healing images were acquired under a microscope after co-culturing PFC-73 (50 μg / mL) with HUVEC for 24 h and 48 h. Scale bar: 200 μm.
[0021] Figure 6 This is a graph showing the quantitative results of the healing rate.
[0022] Figure 7The image shows the results of qPCR detection of Ki67 mRNA expression level after PFC-73 (50 μg / mL) was co-cultured with HUVEC for 24 h.
[0023] Figure 8 The image shows the results of qPCR detection of Vegf mRNA expression level after co-culturing PFC-73 (50 μg / mL) with HUVEC for 24 h.
[0024] Figure 9 The image shows the results of immunofluorescence detection of Ki67 expression level after PFC-73 (50 μg / mL) was co-cultured with HUVEC for 24 h. Detailed Implementation
[0025] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.
[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0027] All materials and reagents used in the following examples are commercially available unless otherwise specified.
[0028] Example 1: A culture medium for hydrogen-bonded organic framework nanomaterials
[0029] The culture medium includes DMEM high-glucose basal medium, serum, antibiotics, mycoplasma scavenger, and PFC-73 nanomaterials.
[0030] Example 2 Cell Viability and Proliferation Experiment
[0031] Cytotoxicity test
[0032] The cytotoxicity of PFC-73 against HUVECs was determined using the CCK8 assay kit. First, HUVECs (5 × 10⁻⁶) were... 3 Cells were seeded at 1000 cells / well in 96-well plates and treated with different concentrations of PFC-73 (0-200 μg / mL) for 24 and 48 hours. After washing with PBS, 100 μL of 10% CCK-8 working solution (Life-iLab, China) was added, and the plates were incubated at 37°C for 1.5 hours. OD was measured at 450 nm using a microplate reader.
[0033] Calcein-AM and PI staining:
[0034] HUVECs were seeded in 24-well plates overnight and treated with PFC-73 (50 μg / mL) for 24 hours. After washing three times with PBS, 250 μL of Calcein-AM / PI working solution (0.25 μL Calcein-AM + 0.25 μL PI) (Beyotime, Shanghai, China) was added, and the cells were incubated at 37°C in the dark for 30 minutes. Cells were washed three times with PBS, and cell proliferation was observed under a fluorescence microscope.
[0035] HUVECs were seeded in 96-well plates (2 × 10⁻⁶). 3 Cells were divided into wells and treated with PFC-73 (50 μg / mL). Cell proliferation was measured at 1, 3, and 5 days using CCK-8 working solution (Life-iLab, China).
[0036] Compared with the control group, different concentrations of PFC-73 showed good biocompatibility with HUVECs after co-culturing for 24 h. Furthermore, the cell viability of HUVECs increased significantly by approximately 1.5-fold after 48 hours. Calcein-AM and PI staining were used to detect the cell status of HUVECs co-cultured with PFC-73 for 72 h. Compared with the control group, cell death (PI-positive cells, red fluorescence) was minimal after treatment with PFC-73 (50 μg / mL). HUVECs maintained good viability and proliferation rate for 5 days after incubation with PFC-73, confirming that PFC-73 significantly enhanced the proliferation of HUVECs. Figures 1-4 ).
[0037] Example 3 Scratch Test
[0038] To further investigate the effect of PFC-73 on the migration ability of human umbilical vein endothelial cells (HUVECs), we designed a scratch assay.
[0039] To evaluate the effect of PFC-73 on scratch healing of HUVECs, cells were seeded at a certain density into 6-well plates. Scratches of equal width were made using a 200 μL pipette tip, and cell debris was washed away with PBS. Subsequently, the cells were exposed to PFC-73 (50 μg / mL) for 24 hours, and the scratch closure rate was determined by microscopic image analysis.
[0040] After 12 hours of co-culture, the results showed that the wound healing rate in the PFC-73-treated group was significantly higher than that in the untreated control group. This preliminary result suggests that PFC-73 may have the potential to promote cell migration. Further extending the observation period to 24 hours, we observed a more significant migration enhancement effect, conclusively verifying the positive promoting effect of PFC-73 on the migration ability of HUVECs. Figure 5 and Figure 6 ).
[0041] Example 4 Real-time Quantitative PCR Experiment
[0042] Total RNA was extracted using FreeZol reagent (Vazyme, Nanjing, China), and then reverse transcribed into cDNA via strand cDNA synthesis (Vazyme, Nanjing, China). Real-time quantitative PCR was performed using SYBR Green qPCR Master Mix (Abclonal, Wuhan, China).
[0043] To further investigate the mechanism by which PFC-73 promotes cell proliferation, we co-cultured HUVECs with PFC-73 for 24 hours and then examined the expression of key cell proliferation markers. The results showed that the PFC-73 treatment group significantly upregulated the mRNA expression levels of the proliferation markers Ki67 and vascular endothelial growth factor Vegf. This finding strongly demonstrates the positive promoting effect of PFC-73 on the proliferation activity of HUVECs. Figure 7 and Figure 8 ).
[0044] Example 5 Immunofluorescence Experiment
[0045] Cells were seeded in culture dishes pre-placed with treated coverslips. Once the cells nearly formed a monolayer, the coverslips were removed, and the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min. The samples were treated with 0.5% Triton X-100 for 5-15 minutes, followed by washing with PBS. Blocking with 10% goat serum was typically for 30 minutes. Primary antibody was incubated overnight at 4°C. Incubation was then carried out at room temperature in the dark for 1 hour. After incubation, the cells were washed with PBS to remove unbound secondary antibody. Cell morphology and localization were observed under a fluorescence microscope after DAPI staining.
[0046] Further validation using immunofluorescence assays revealed a significant increase in Ki67 nuclear expression in PFC-73-treated HUVECs. This finding not only reinforces previous conclusions regarding PFC-73's promotion of cell proliferation but also elucidates the specific mechanism by which it enhances cell proliferation at the molecular level. Figure 9 ).
[0047] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.
Claims
1. Use of hydrogen-bonded organic framework nanomaterial PFC-73 in cell culture.
2. Use according to claim 1, characterized in that, The hydrogen-bonded organic framework nanomaterial PFC-73. PFC-73 is a kind of hydrogen-bonded organic framework material, the main components of which include self-assembly of organic ligand tetracarboxylic porphyrin and metal ions (nickel). The ligand and metal ions are connected together by hydrogen bonds and other non-covalent interactions to form a stable framework structure.
3. Use according to claim 1, characterized in that, The PFC-73 has a particle size of 5-6 nm.
4. The use according to claim 1, characterized in that, The cell culture is used in cell culture after adding the hydrogen-bonded organic framework nanomaterial PFC-73 in the culture medium.
5. Use according to claim 4, characterized in that, After adding the hydrogen-bonded organic framework nanomaterial PFC-73 in the culture medium, the concentration of PFC-73 therein is 50 μg / mL.
6. A cell culture medium comprising a hydrogen-bonded organic framework nanomaterial PFC-73, characterized in that, The culture medium comprises a basic medium and the hydrogen-bonded organic framework nanomaterial PFC-73.
7. Use of hydrogen-bonded organic framework nanomaterial PFC-73 in promoting cell proliferation.
8. Use of hydrogen-bonded organic framework nanomaterial PFC-73 in promoting cell migration.
9. Use of hydrogen-bonded organic framework nanomaterial PFC-73 in promoting expression of vascular endothelial growth factor and proliferation factor.
10. Use of hydrogen-bonded organic framework nanomaterial PFC-73 in preparing a drug.