Integrated micro-fluidic platform for preclinical evaluation of radiotherapy nano-material and evaluation method of integrated micro-fluidic platform

Abstract

The invention discloses an integrated micro-fluidic platform for preclinical evaluation of a radiotherapy nano material and an evaluation method thereof, the platform comprises a G chip and a T chip which are cascaded, the G chip generates uniform matrigel liquid drops for encapsulating tumor cells through a flow focusing orifice, and tumor spheres with uniform size and high activity are formed through solidification and fluid perfusion culture; the T chip anchors a tumor sphere through an array type fixing structure, and forms a concentration gradient of a radiotherapy nanometer material through the regulation and control of a double-fluid channel, thereby achieving the precise pretreatment of the tumor sphere. The platform analyzes the activity and proliferation condition of tumor spheres through fluorescence imaging and immunofluorescence staining, and can quickly and reliably evaluate the curative effect of the radiotherapy nano material. The problems that a traditional 2D cell culture model is low in fidelity and an animal model is high in cost are solved, the method has the advantages of being easy and convenient to operate, low in cost and high in fidelity of simulated in-vivo tumor microenvironment, and a new scheme is provided for preclinical evaluation of radiotherapy nanometer materials.

Description

Technical Field

[0001] This invention belongs to the fields of microfluidics, tumor biology and radiotherapy, and particularly relates to an integrated microfluidic platform and its evaluation method for preclinical evaluation of radiotherapy nanomaterials. Background Technology

[0002] Radiotherapy nanomaterials offer a new direction for traditional radiotherapy by enhancing therapeutic response and eliminating side effects. However, current preclinical evaluation faces two major challenges: first, 2D cell culture models lack the three-dimensional structure and microenvironment gradient of in vivo tumors, resulting in low evaluation fidelity; second, animal models are costly, time-consuming, and raise ethical concerns. While tumor spheroids, as three-dimensional cell aggregates, can simulate the structure and characteristics of solid tumors in vivo, traditional generation methods (such as bioreactors and pendant drop methods) struggle to control the microenvironment of individual spheroids, leading to inconsistent spheroid sizes, poor data reproducibility, and cumbersome manual processing, thus limiting the accurate evaluation of radiotherapy nanomaterials.

[0003] Microfluidics has shown advantages in cell manipulation and analysis due to its ability to precisely manipulate microscale fluids. However, current technologies have not yet achieved full integration of the entire process of tumor spheroid generation, culture, pretreatment and radiotherapy assessment, which makes it difficult to meet the needs of high-throughput and high-fidelity assessment of nanomaterials. Summary of the Invention

[0004] The purpose of this invention is to provide an integrated microfluidic platform and its evaluation method for preclinical evaluation of radiotherapy nanomaterials, enabling rapid generation, long-term culture, and precise pretreatment of uniform tumor spheres, solving the problems of low fidelity and high cost of existing evaluation models, and providing an efficient solution for preclinical evaluation of radiotherapy nanomaterials.

[0005] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides an integrated microfluidic platform for preclinical evaluation of radiotherapy nanomaterials, comprising two core chips, namely... G-chip: Used to generate and culture uniform tumor spheres, comprising a first microchannel, a second microchannel, a third microchannel, and a fourth microchannel, each having a first inlet, a second inlet, a third inlet, and a first liquid outlet. The first, second, and third microchannels converge at a flow focusing orifice. Substances flowing into the first and second microchannels mix at the flow focusing orifice and then flow into the third microchannel. The third microchannel is connected to a first microchamber, and the first microchamber is connected to the fourth microchannel. T-chip: Used for anchoring tumor spheres and pretreatment of radiotherapy nanomaterials, comprising a fifth, sixth, seventh, and eighth microchannel, each with a fourth, fifth, and sixth inlet and a second liquid outlet. The fifth, sixth, and seventh microchannels are all connected to a second microchamber. The second microchamber contains multiple fixing structures, each consisting of two symmetrical right-angled trapezoids. The hypotenuses of the two trapezoids form a figure-eight structure, and the upper bases of the two trapezoids form parallel channels. The second microchamber is connected to the eighth microchannel.

[0006] In the above technical solution, the width of the first microchannel and the second microchannel is 100μm, and the width of the third microchannel is 400μm.

[0007] In the above technical solution, the upper base of the right trapezoid is 375μm, the lower base is 750μm, and the height is 360μm; the width of the parallel channel is 80μm; and the spacing between adjacent fixed structures is 80μm.

[0008] In the above technical solution, the method for generating tumor spheroids using the G chip is as follows: tumor cells are mixed with matrix gel to form a dispersed phase, which is injected into the second microchannel through the second inlet. Fluorinated oil containing 2.5% surfactant is injected into the first microchannel through the first inlet as a continuous phase. Uniform droplets are generated through the flow focusing orifice at 4°C and flow into the first microchamber. After the first microchamber is filled with droplets, it is transferred to a 37°C incubator. After solidification, culture medium is injected into the first microchamber through the third inlet. After a period of incubation, tumor spheroids with a diameter of 180-240 μm are formed.

[0009] In the above technical solution, the method of the T chip to achieve nanomaterial concentration gradient pretreatment is as follows: tumor spheres are injected into the second microchamber through the fourth inlet, and radiotherapy nanomaterial solution and culture medium are injected into the second microchamber through the fifth inlet and the sixth inlet, respectively. By adjusting the flow rate and flow rate ratio of the two, a concentration gradient is formed in the second microchamber, and the tumor spheres anchored by the fixed structure are simultaneously treated with nanomaterials of different concentrations.

[0010] In the above technical solutions, the flow rate of both is 60-100μL / h, and the flow rate ratio is 2:1 to 1:2.

[0011] Secondly, this invention provides the application of the above-mentioned integrated microfluidic platform in the preclinical evaluation of radiotherapy nanomaterials.

[0012] Thirdly, the present invention provides a method for evaluating radiotherapy nanomaterials based on the above-mentioned integrated microfluidic platform, characterized by comprising the following steps: S1, generating and culturing uniform tumor spheroids in the G chip; S2, the tumor spheres are transferred to the T-chip and anchored by a fixation structure; S3, forming a concentration gradient of radiotherapy nanomaterials in the T chip to pre-treat tumor spheres; S4, T-chip was subjected to different doses of radiotherapy, and the viability and proliferation of tumor spheroids were analyzed by FDA / PI fluorescence staining and Ki-67 immunofluorescence staining to evaluate the radiotherapy effect of nanomaterials.

[0013] In the above technical solutions, the tumor spheroids include spheroids formed by MCF-7 human breast cancer cells, CT26 mouse colon cancer cells, or 4T1-GFP mouse breast cancer cells, with a viability greater than 90%.

[0014] In the above technical solution, the radiotherapy nanomaterial is a membrane-coated FeS2 nanozyme, and its killing effect on tumor spheroids is evaluated by controlling the concentration gradient in combination with a radiotherapy dose of 2-6 Gy.

[0015] The beneficial effects of this invention are as follows: (1) High-fidelity simulation of in vivo tumors: The generated tumor spheres have necrotic cores, oxygen and nutrient gradients, and are highly similar to the structure of solid tumors. The evaluation results are consistent with those of animal models.

[0016] (2) Uniformity and reproducibility: G chip achieves narrow distribution of tumor spheroid size (coefficient of variation <10%), and T chip has high precision in concentration gradient control to ensure the reliability of experimental data.

[0017] (3) High efficiency and low cost: Compared with animal models, the cost is reduced by more than 80%, the evaluation cycle is shortened to 7 days, and no complicated animal operations are required.

[0018] (4) Integrated process: The entire process from sphere generation to radiotherapy evaluation is integrated into the microfluidic chip, reducing manual operation errors and making it suitable for high-throughput screening. Attached Figure Description

[0019] Figure 1 Schematic diagram of the structure of the G chip and T chip integrating the microfluidic platform.

[0020] Figure 2 :Schematic diagram of the fixed structure 20 on the T chip.

[0021] Figure 3Uniform tumor spheroids for evaluating radiotherapy nanomaterials were prepared using an integrated microfluidic platform. Uniform tumor spheroids were formed and cultured using a droplet microfluidic chip (G-chip), then sequentially collected and transferred to another pretreated microfluidic chip (T-chip) for incubation under precisely controlled radiotherapy nanomaterial environment conditions. Finally, the T-chip was subjected to radiotherapy to evaluate the tumor-killing performance of the radiotherapy nanomaterials.

[0022] Figure 4 (A) Photograph of G-chip; (B) Micrograph of matrix gel droplet formation containing tumor cells; (C) Micrograph of solidified matrix gel microbeads containing tumor cells; (D) Effect of DP flow rate and cell count on droplet diameter when CP flow rate is set to 480 μL / h; (E) Effect of CP flow rate and cell count on droplet diameter when DP flow rate is set to 10 μL / h; (F) Effect of cell concentration in DP on droplet diameter; (G) and (H) Statistical analysis and micrographs show the proliferation of tumor spheroids cultured in culture dishes; (I) and (J) Statistical analysis and micrographs show the proliferation of tumor spheroids cultured in G-chip.

[0023] Figure 5 (A) Photomicrographs of tumor spheroids formed by MCF-7 and CT26 cells, and corresponding fluorescence images with FDA / PI staining; (B) Photomicrographs and corresponding fluorescence images showing tumor spheroids of GFP-transfected 4T1 cells cultured in G-chips and harvested on days 1, 2, and 4, respectively; (C) Viability analysis of tumor spheroids formed by MCF-7 and CT26 cells; (D) Viability analysis of 4T1 tumor spheroids cultured in G-chips for 108 hours; (E) Size distribution of 4T1 tumor spheroids cultured in G-chips on days 1, 2, and 4, respectively.

[0024] Figure 6 (A) Photograph of the T-chip; (B) Microscopic image and corresponding fluorescence image showing 4T1-GFP tumor spheres immobilized by fixators (as shown in the blue dashed box in A); (C) Numerical simulation showing the concentration gradient of the therapeutic agent in the T-chip when used to treat tumor spheres immobilized in different fixators; (D) Changes in the concentration gradient in the T-chip by adjusting the flow rate and the ratio of therapeutic agent to culture medium; (E) GFP fluorescence intensity, showing the viability of 4T1-GFP tumor spheres immobilized in fixators 1, 3, 5, 7, 9, and 11 under cisplatin concentration gradients (as shown by the dashed line in C); (F) GFP fluorescence intensity analysis of the 4T1-GFP tumor spheres in E. The yellow dashed line indicates the outline of the fixator.

[0025] Figure 7(A) Schematic diagram showing radiotherapy assessment based on a T-chip containing uniform tumor spheres; (B) GFP fluorescence image showing the viability of 4T1-GFP tumor spheres after radiotherapy CF pretreatment with or without radiation, with the yellow dashed line representing the outline of the fixator; (C) Analysis of B; (D) GFP fluorescence intensity of 4T1-GFP tumor spheres in GFP; (E) Immunofluorescence showing the post-radiotherapy viability and proliferation of 4T1-GFP tumor spheres with or without radiotherapy CF pretreatment on the T-chip; (F) GFP fluorescence intensity analysis of 4T1-GFP tumor spheres in D; (G) Ki-67 fluorescence intensity analysis of 4T1-GFP tumor spheres in E.

[0026] Wherein: 1. First microchannel; 2. Second microchannel; 3. Third microchannel; 4. Fourth microchannel; 5. First inlet; 6. Second inlet; 7. Third inlet; 8. First liquid outlet; 9. Flow focusing orifice; 10. First microchamber; 11. Fifth microchannel; 12. Sixth microchannel; 13. Seventh microchannel; 14. Eighth microchannel; 15. Fourth inlet; 16. Fifth inlet; 17. Sixth inlet; 18. Second liquid outlet; 19. Second microchamber; 20. Fixed structure. Detailed Implementation

[0027] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0028] This invention provides an integrated microfluidic platform and its evaluation method for preclinical evaluation of nanomaterials used in radiotherapy, such as... Figure 1-2 As shown, this integrated microfluidic platform includes two core chips, namely... G-chip: Used to generate and culture uniform tumor spheres, comprising a first microchannel 1, a second microchannel 2, a third microchannel 3, and a fourth microchannel 4, each with a first inlet 5, a second inlet 6, a third inlet 7, and a first liquid outlet 8. The first microchannel 1, the second microchannel 2, and the third microchannel 3 converge at a flow focusing orifice 9. Substances flowing into the first microchannel 1 and the second microchannel 2 are mixed at the flow focusing orifice 9 and then flow into the third microchannel 3. The third microchannel 3 is connected to the first microchamber 10, and the first microchamber 10 is connected to the fourth microchannel 4.

[0029] Preferably, the shape of the first microchamber 10 can be a square, rectangle, hexagon, etc. For example, the shape of the first microchamber 10 is a square with an area of ​​600 mm².

[0030] Preferably, the width of the first microchannel 1 and the second microchannel 2 is 100 μm, and the width of the third microchannel 3 is 400 μm. This differentiated width design is to facilitate the generation of droplets.

[0031] Preferably, the upper base of the right trapezoid is 375 μm, the lower base is 750 μm, and the height is 360 μm. The width of the parallel channel is 80 μm, and the spacing between adjacent fixing structures 20 is 80 μm. The dimensions of the right trapezoid are designed based on the dimensions of the tumor sphere, which facilitates its anchoring of the tumor sphere and simultaneous treatment with different concentrations of nanomaterials.

[0032] Preferably, the method for generating tumor spheroids using the G-chip is as follows: tumor cells are mixed with matrix gel to form a dispersed phase, which is injected into the second microchannel 2 through the second inlet 6. Fluorinated oil containing 2.5% surfactant is injected into the first microchannel 1 as a continuous phase through the first inlet 5. Uniform droplets are generated through the flow focusing orifice 9 at 4°C and flow into the first microchamber 10. After the first microchamber 10 is filled with droplets, it is transferred to a 37°C incubator. After solidification, culture medium is injected into the first microchamber 10 through the third inlet 7. After a period of culture, tumor spheroids with a diameter of 180-240 μm are formed.

[0033] T-chip: Used for anchoring tumor spheres and pretreatment of radiotherapy nanomaterials, comprising a fifth microchannel 11, a sixth microchannel 12, a seventh microchannel 13, and an eighth microchannel 14, each with a fourth inlet 15, a fifth inlet 16, a sixth inlet 17, and a second liquid outlet 18. The fifth microchannel 11, the sixth microchannel 12, and the seventh microchannel 13 are all connected to a second microchamber 19. The second microchamber 19 contains multiple fixing structures 20, each consisting of two symmetrical right-angled trapezoids. The hypotenuses of the two right-angled trapezoids form a figure-eight structure, and the upper bases of the two right-angled trapezoids form parallel channels. The second microchamber 19 is connected to the eighth microchannel 14.

[0034] Preferably, the widths of the fifth microchannel 11, the sixth microchannel 12, and the seventh microchannel 13 are 400 μm.

[0035] Preferably, the fixed structure 20 is arrayed within the second microchamber 19, for example: 7×11, 7×12, 7×13, 7×14, 7×15, 8×11, 8×12, 8×13, 8×14, 8×15, etc.

[0036] Preferably, the method for T-chip to achieve nanomaterial concentration gradient pretreatment is as follows: tumor spheres are injected into the second microchamber 19 through the fourth inlet 15, and radiotherapy nanomaterial solution and culture medium are injected into the second microchamber 19 through the fifth inlet 16 and the sixth inlet 17, respectively. By adjusting the flow rate and flow rate ratio of the two, a concentration gradient is formed in the second microchamber 19, and the tumor spheres anchored by the fixed structure 20 are simultaneously treated with nanomaterials of different concentrations.

[0037] Preferably, both have a flow rate of 60-100 μL / h and a flow rate ratio of 2:1 to 1:2.

[0038] This invention also provides the application of the above-mentioned integrated microfluidic platform in the preclinical evaluation of radiotherapy nanomaterials, such as... Figure 3 As shown, the specific evaluation method includes the following steps: S1, generating and culturing uniform tumor spheroids in the G chip; S2, the tumor spheres are transferred to the T-chip and anchored by a fixation structure; S3, forming a concentration gradient of radiotherapy nanomaterials in the T chip to pre-treat tumor spheres; S4, T-chip was subjected to different doses of radiotherapy, and the viability and proliferation of tumor spheroids were analyzed by FDA / PI fluorescence staining and Ki-67 immunofluorescence staining to evaluate the radiotherapy effect of nanomaterials.

[0039] Preferably, the tumor spheroids are spheroids formed from MCF-7 human breast cancer cells, CT26 mouse colon cancer cells, or 4T1-GFP mouse breast cancer cells, with a viability greater than 90%.

[0040] Preferably, the radiotherapy nanomaterial is a membrane-coated FeS2 nanozyme, and its killing effect on tumor spheroids is evaluated by controlling the concentration gradient in combination with a radiotherapy dose of 2-6 Gy.

[0041] The G and T chips of this invention are made of polydimethylsiloxane (PDMS) and are fabricated using SU-8 photoresist molds and soft photolithography. The chip height is 450 μm. The specific fabrication process includes the following steps: A mold was prepared on a silicon wafer using SU-8 photoresist. After casting with PDMS (precursor: crosslinking agent = 10:1), it was cured at 120℃ for 15 minutes, and then plasma bonded to a glass substrate after drilling.

[0042] Before use, the chip was sealed overnight at 4°C with a 3% bovine serum albumin (BSA) solution to prevent non-specific adsorption.

[0043] As one embodiment, the G-chip of the present invention is used for the generation and culture of tumor spheroids, mainly including cell preparation, G-chip operation, and spheroid harvesting, as detailed below: (1) Cell preparation: MCF-7, CT26 or 4T1-GFP cells were cultured in DMEM containing 10% fetal bovine serum, digested with 0.25% trypsin, washed with PBS and resuspended, and counted with a hemocytometer.

[0044] (2) G chip operation: Cells and matrix gel were mixed at a concentration of 1000-20000 cells / μL as the dispersion phase, and HFE7500 fluorinated oil containing 2.5% PicoSurf was used as the continuous phase. The mixture was injected into the G chip at a flow rate of 10 μL / h for DP and 480 μL / h for CP using a peristaltic pump. Droplets were generated at 4℃. After the microchamber was filled with droplets, it was transferred to a 37℃ incubator. After solidification, the culture medium was injected into the microchannel at a flow rate of 50 μL / h and cultured for 4 days.

[0045] (3) Sphere harvesting: Inject cell recovery solution to degrade matrix gel, collect spheres from the outlet, wash with PBS, detect viability by FDA / PI staining (live cells show green fluorescence, dead cells show red), and observe under a confocal microscope.

[0046] As one embodiment, the T-chip of the present invention is used for the pretreatment and radiotherapy evaluation of tumor spheroids, mainly including T-chip anchoring, radiotherapy implementation, and effect analysis. Details are as follows: (1) T-chip anchoring: The harvested tumor spheres are resuspended in the culture medium and injected into the T-chip for anchoring through a fixed structure; a nanomaterial solution (such as 200 μg / mL FeS2 nanozyme) and culture medium are injected from the microchannel, and the flow rate ratio is adjusted to 1:1 (total flow rate 80 μL / h) to form a concentration gradient.

[0047] (2) Radiotherapy implementation: The T-chips were divided into four groups, which were respectively treated with PBS, 2Gy radiotherapy, 6Gy radiotherapy, and 2Gy radiotherapy + nanomaterials, with 3 chips in each group.

[0048] (3) Effect analysis: After radiotherapy, the tumor was fixed by injection of 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked with 5% BSA, stained with Ki-67 kit, stained with DAPI nucleus, observed GFP fluorescence and immunofluorescence under an inverted microscope, and the fluorescence intensity was analyzed by Image-Pro Plus software to evaluate the tumor spheroid viability and proliferation inhibition rate.

[0049] Experimental verification like Figure 4-7As shown, taking 4T1-GFP tumor spheroids as an example, after 4 days of culture on the G chip, the spheroid diameter was 180±15μm, and the viability was 95.13%. In the T chip, when the cisplatin concentration gradient was 10μg / mL, the GFP fluorescence intensity of the spheroids in the high-concentration region (Retainer 1) decreased to 73% of the control group, while the fluorescence intensity in the low-concentration region (Retainers 7-11) was similar to that of the control group (95%). When FeS2 nanozyme was combined with 2Gy radiotherapy, the tumor spheroid viability inhibition rate was increased by 40% compared with radiotherapy alone, and the number of Ki-67 positive cells was reduced by 50%. The results were consistent with the mouse model, verifying the high-fidelity assessment capability of the platform.

[0050] The integrated microfluidic platform and evaluation method provided by this invention can be implemented under routine conditions in biomedical laboratories. It is applicable to the preclinical screening and optimization of various radiotherapy nanomaterials and has significant industrial application value and market prospects.

[0051] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An integrated microfluidic platform for preclinical evaluation of nanomaterials for radiotherapy, characterized in that: It includes two core chips, namely G-chip: Used to generate and culture uniform tumor spheres, comprising a first microchannel (1), a second microchannel (2), a third microchannel (3) and a fourth microchannel (4), respectively provided with a first inlet (5), a second inlet (6), a third inlet (7) and a first liquid outlet (8). The first microchannel (1), the second microchannel (2) and the third microchannel (3) converge at the flow focusing orifice (9). The substances flowing into the first microchannel (1) and the second microchannel (2) are mixed at the flow focusing orifice (9) and then flow into the third microchannel (3). The third microchannel (3) is connected to the first microchamber (10), and the first microchamber (10) is connected to the fourth microchannel (4). T-chip: used for anchoring tumor spheres and pretreatment of radiotherapy nanomaterials, comprising a fifth microchannel (11), a sixth microchannel (12), a seventh microchannel (13), and an eighth microchannel (14), which are respectively provided with a fourth inlet (15), a fifth inlet (16), a sixth inlet (17), and a second liquid outlet (18). The fifth microchannel (11), the sixth microchannel (12), and the seventh microchannel (13) are all connected to a second microchamber (19). The second microchamber (19) is provided with multiple fixing structures (20). The fixing structure (20) is composed of two symmetrical right trapezoids. The hypotenuses of the two right trapezoids form a figure-eight structure, and the upper bases of the two right trapezoids form a parallel channel. The second microchamber (19) is connected to the eighth microchannel (14).

2. The integrated microfluidic platform according to claim 1, characterized in that: The width of the first microchannel (1) and the second microchannel (2) is 100 μm, and the width of the third microchannel (3) is 400 μm.

3. The integrated microfluidic platform according to claim 1, characterized in that: The upper base of the right trapezoid is 375μm, the lower base is 750μm, and the height is 360μm. The width of the parallel channel is 80μm, and the spacing between adjacent fixed structures (20) is 80μm.

4. The integrated microfluidic platform according to claim 1, characterized in that: The method for generating tumor spheres using the G chip is as follows: tumor cells are mixed with matrix gel to form a dispersed phase and injected into the second microchannel (2) through the second inlet (6). Fluorinated oil containing 2.5% surfactant is injected into the first microchannel (1) through the first inlet (5) as a continuous phase. Uniform droplets are generated through the flow focusing orifice (9) at 4°C and flow into the first microchamber (10). After the first microchamber (10) is filled with droplets, it is transferred to a 37°C incubator. After solidification, culture medium is injected into the first microchamber (10) through the third inlet (7). After a period of culture, tumor spheres with a diameter of 180-240 μm are formed.

5. The integrated microfluidic platform according to claim 1, characterized in that: The method for achieving nanomaterial concentration gradient pretreatment using the T-chip is as follows: tumor spheres are injected into the second microchamber (19) through the fourth inlet (15), and radiotherapy nanomaterial solution and culture medium are injected into the second microchamber (19) through the fifth inlet (16) and the sixth inlet (17) respectively. By adjusting the flow rate and flow rate ratio of the two, a concentration gradient is formed in the second microchamber (19), and the tumor spheres anchored by the fixed structure (20) are simultaneously treated with nanomaterials of different concentrations.

6. The integrated microfluidic platform according to claim 5, characterized in that: Both had a flow rate of 60-100 μL / h and a flow rate ratio of 2:1 to 1:

2.

7. The application of the integrated microfluidic platform according to any one of claims 1-6 in the preclinical evaluation of radiotherapy nanomaterials.

8. A method for evaluating radiotherapy nanomaterials based on the integrated microfluidic platform according to any one of claims 1-6, characterized in that: Includes the following steps: S1, generating and culturing uniform tumor spheroids in the G chip; S2, the tumor spheres are transferred to the T-chip and anchored by a fixation structure; S3, forming a concentration gradient of radiotherapy nanomaterials in the T chip to pre-treat tumor spheres; S4, T-chip was subjected to different doses of radiotherapy, and the viability and proliferation of tumor spheroids were analyzed by FDA / PI fluorescence staining and Ki-67 immunofluorescence staining to evaluate the radiotherapy effect of nanomaterials.

9. The evaluation method according to claim 8, characterized in that: The tumor spheroids comprise spheroids formed from MCF-7 human breast cancer cells, CT26 mouse colon cancer cells, or 4T1-GFP mouse breast cancer cells, with a viability greater than 90%.

10. The evaluation method according to claim 8, characterized in that: The radiotherapy nanomaterial is a membrane-coated FeS2 nanozyme. Its killing effect on tumor spheroids was evaluated by controlling the concentration gradient in combination with a radiotherapy dose of 2-6 Gy.

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