EpCAM aptamer modified nanomaterial and preparation method and application thereof

Abstract

The application relates to the technical field of nanomaterials, in particular to an EpCAM aptamer modified nanomaterial and a preparation method and application thereof, wherein the nanomaterial takes carboxylated mesoporous silica nanoparticles as a carrier, and an EpCAM specific DNA aptamer is covalently coupled to the surface of the nanomaterial; the sequence of the EpCAM specific DNA aptamer is shown as SEQ ID No. 1. The nanomaterial can specifically capture tumor-derived exosomes (EVs) in blood, and can realize sensitive detection of EVs in the 7-day (early stage) and 21-day (late stage) stages of an HT29 colon cancer model and the 7-day stage of an A549 / H460 lung cancer model, thereby providing a minimally invasive liquid biopsy new tool for early diagnosis of colon cancer and lung cancer, and solving the inherent limitation problems of sensitivity and specificity of existing diagnosis means in cancer detection.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, specifically to an EpCAM aptamer-modified nanomaterial, its preparation method, and its application. Background Technology

[0002] Existing diagnostic methods have inherent limitations in sensitivity and specificity for cancer detection (Baghban and Mojra 2018; Madani and Mojra 2017), especially in the early stages of carcinoma in situ where clinical features are not yet apparent. Notably, tumor cells continuously release tumor-derived exosomes (EVs) in their early stages. Analyzing these exosomes opens up promising new avenues for early cancer detection. Recent evidence suggests that detecting tumor-derived exosomes and their molecular payloads holds promise for overcoming the limitations of traditional diagnostic methods, as these nanoscale vesicles carry tumor-specific biomarkers reflecting the biological state of the source cells (Ma et al. 2024; Ma et al. 2023).

[0003] Therefore, there is a need to develop a nanomaterial that can specifically recognize tumor-derived exosomes in blood samples. Summary of the Invention

[0004] The purpose of this invention is to provide a nanomaterial capable of specifically recognizing tumor-derived exosomes in blood samples.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention proposes an EpCAM aptamer-modified nanomaterial, wherein the nanomaterial comprises: carboxylated mesoporous silica nanoparticles as a carrier, with EpCAM-specific DNA aptamers covalently coupled to the surface, the sequence of which is shown in SEQ ID No. 1; the carboxylated mesoporous silica nanoparticles have a particle size of 112.4±15.6 nm, a zeta potential of -23.5±1.5 mV, and an aptamer loading of ≥10 pmol / mg.

[0006] This invention also includes a method for preparing the aforementioned nanomaterials, comprising the following steps: S1, CTAC and TEA were reacted, then TEOS was added to continue the reaction, centrifuged, washed, and MSN-OH was obtained; S2, MSN-OH is added to a hydrochloric acid-ethanol solution, heated, APTES is added, and stirred to obtain aminated MSN; S3, amino-modified MSN was reacted with succinic anhydride to prepare carboxylated mesoporous silica nanoparticles; S4. Carboxylated mesoporous silica nanoparticles were coupled with EpCAM-specific DNA aptamers at room temperature to prepare nanomaterials.

[0007] Furthermore, in step S1, ethanol is used for washing, and the washing is performed three times.

[0008] Furthermore, in step S2, the heating temperature is 78°C.

[0009] Further, step S4 involves mixing carboxylated mesoporous silica nanoparticles with EDC, NHS, and EpCAM-specific DNA aptamers, stirring, centrifuging, and washing to obtain the nanomaterials.

[0010] The present invention also includes the application of the nanomaterials as biomarkers for detecting tumor-derived exosomes.

[0011] Compared with existing technologies, the nanomaterials of this invention can specifically capture tumor-derived exosomes (EVs) in the blood. Sensitive detection of EVs can be achieved in the 7-day (early) and 21-day (late) stages of the HT29 colon cancer model and the 7-day stage of the A549 / H460 lung cancer model. This provides a new minimally invasive liquid biopsy tool for the early diagnosis of colon cancer and lung cancer, and solves the inherent limitations of existing diagnostic methods in terms of sensitivity and specificity in cancer detection. Attached Figure Description

[0012] Figure 1 Dynamic light scattering characterization of (A) carboxylated MSNs and (B) EpCAM-MSNs; Figure 2 Transmission electron microscopy shows the EV capture effect of the HT29 model at (A) 7 days and (B) 21 days; Figure 3 A comparison of EV signals in the early (7 days) vs. late (21 days) stages detected by flow cytometry; Figure 4 Validation of the 7-day detection performance of the A549 / H460 model. Detailed Implementation

[0013] The technical solution of the present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0014] Prepare nanomaterials according to the following steps: S1, CTAC and TEA were reacted, then TEOS was added to continue the reaction, centrifuged, washed, and MSN-OH was obtained; S2, MSN-OH is added to a hydrochloric acid-ethanol solution, heated, APTES is added, and stirred to obtain aminated MSN; S3, amino-modified MSN is reacted with succinic anhydride to prepare carboxylated mesoporous silica nanoparticles (hereinafter referred to as carboxylated MSN). S4. The carboxylated MSN was coupled with the EpCAM-specific DNA aptamer at room temperature to prepare nanomaterials (EpCAM-MSNs). The carboxylated MSN has a particle size of 112.4±15.6 nm, a zeta potential of -23.5±1.5 mV, and an aptamer loading of ≥10 pmol / mg. Example 1:

[0015] Prepare nanomaterials according to the following steps: S1, firstly, 10 g of hexadecyltrimethylammonium chloride (CTAC) and 0.5 g of triethanolamine (TEA) were dissolved in 100 mL of distilled water, heated to 95 °C under vigorous stirring and maintained for 1 hour, then 8 mL of tetraethoxysilane (TEOS) was added dropwise to the solution, and after reacting for 1 hour, the product was separated by centrifugation and purified by washing three times with ethanol to obtain MSN-OH; S2, then MSN-OH was dissolved in a 10% (v / v) hydrochloric acid-ethanol solution and heated at 78°C to remove CTAC; 150 mL of (3-aminopropyl)triethoxysilane (APTES) was added to 150 mL of distilled water containing 2 mg / mL (after CTAC removal) MSN-OH, and the mixture was stirred at 80°C for 18 hours to obtain aminated MSN; S3, then the amino-modified MSN was reacted with succinic anhydride at room temperature for 12 hours to achieve carboxyl modification, and carboxylated MSN was successfully prepared. After centrifugation, carboxylated MSN was obtained.

[0016] S4. Finally, 100 mg of carboxylated MSN, 75 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 75 mg of N-hydroxysuccinimide (NHS), and 2.8 nmol of EpCAM-specific DNA aptamer (FITC-5'-CACTAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG-3'-NH2, Shanghai Sanger Biotechnology Co., Ltd.) (Xie et al. 2016) were dissolved in 10 mL of distilled water and reacted at room temperature for 24 hours with continuous stirring. Afterward, the mixture was centrifuged at 15,000 g for 15 minutes and washed twice with distilled water to obtain nanomaterials (EpCAM-MSNs), which were stored at -4 °C for subsequent experiments. Example 2:

[0017] The EpCAM-MSNs prepared in Example 1 were subjected to the following tests: The particle size distribution and zeta potential of EpCAM-MSNs were analyzed using a Zetasizer HS III (Malvin Instruments, Malvin, UK). The coupling of aptamers on EpCAM-MSNs was assessed by polyacrylamide gel electrophoresis (PAGE). For PAGE analysis, 6 μL of each sample was mixed with 7 μL of loading buffer and 2.5 μL of SYBR Green dye. After incubation for 15 minutes, the samples were separated by polyacrylamide gel electrophoresis and developed using a Bio-Rad ChemiDoc MP imaging system. Fluorescence standard curves were established using a TECAN Infinite F200 microplate reader to determine the number of EpCAM-specific DNA aptamers bound. In summary, a series of dilutions (0.02, 0.05, 0.1, 0.2, 0.5, 1 μM) of fluorescently labeled aptamers were prepared to generate a standard calibration curve relating concentration to fluorescence intensity. Subsequently, the concentration of aptamers bound to EpCAM-MSNs was measured, and the difference in aptamer concentration between the initial incubation solution and the supernatant after centrifugation following carboxylated MSN functionalization was calculated.

[0018] Dynamic light scattering (DLS) results are as follows Figure 1 As shown, Figure 1 A and B represent the particle size distributions of carboxylated MSN and EpCAM-MSNs, respectively. The average particle size of carboxylated MSN is 112.4 nm (PDI=0.149), and the average particle size of EpCAM-MSNs is 125.4 nm (PDI=0.221).

[0019] Figure 1 C represents polyacrylamide gel electrophoresis (PAGE) showing the coupling between carboxylated MSN and aptamers. The coupling between carboxylated MSN and EpCAM-specific DNA aptamers was verified by PAGE experiments: under voltage, free aptamers dissociate to the lower end of the gel, while the aptamers modified with carboxylated MSN remain at the upper end of the gel, indicating that the aptamers have been successfully anchored to the surface of carboxylated MSN.

[0020] Aptamer standard concentration curves were used to quantitatively analyze aptamers on EpCAM-MSNs. The aptamer content on the MSN surface was determined by fluorescence quantification and a pre-set standard curve. The results are as follows: Figure 1 D showed that 10.66 pmol of aptamers were bound to the surface of each milligram of EpCAM-MSNs. Example 3:

[0021] Early cancer diagnosis remains a crucial pathway to reducing patient mortality. To evaluate the diagnostic efficacy of EpCAM-MSNs prepared in Step 1 at different stages of tumor progression, this study established an HT29 colon cancer model in female Balb / c nude mice (7 weeks old). The ability of EpCAM-MSNs to capture tumor-derived exosomes from mouse blood at different tumor stages was assessed using flow cytometry (FACS Celesta). Blood samples were collected from HT29 colon cancer model mice at different disease stages, placed in EDTA anticoagulant tubes, and centrifuged at 1200g for 10 minutes to remove cellular components. The resulting supernatant was incubated with EpCAM-MSNs for 30 minutes under gentle shaking at 100 rpm. After incubation, the mixture was centrifuged at 15,000 × g for 10 minutes. The precipitate was collected and washed with PBS containing 10% bovine serum albumin. The isolated tumor-derived exosomes were detected by transmission electron microscopy or by incubation with 10 μL of CD63 detection microspheres (Invitrogen, catalog number 10606D) for 30 minutes. The samples were finally washed with PBS and analyzed by flow cytometry.

[0022] Figure 2 Transmission electron microscopy (TEM) images of tumor exosomes captured by EpCAM-MSNs in blood samples from HT29 lung cancer mouse models at different tumor stages (7 days and 21 days) provide a direct visual demonstration that EpCAM-MSNs can capture tumor exosomes in the blood of HT29 tumor-bearing mice.

[0023] Figure 3 A represents the treatment plan and testing procedures; Figure 3 B shows the detection capability of EpCAM-MSNs in blood samples from colon tumor-bearing mice on days 7 and 21 by flow cytometry, demonstrating that EpCAM-MSNs can sensitively detect EVs in HT29 tumor-bearing mice at both the 7-day (early) and 21-day (late) stages.

[0024] To further evaluate the early diagnostic potential of EpCAM-MSNs in a broader spectrum of cancers, a tumor xenograft model was established in nude mice using A549 and H460 cell lines. Blood samples were collected from A549 and H460 cancer model mice on day 7 post-transplantation, and cellular components were removed by centrifugation at 1200g for 10 minutes. The supernatant was incubated with EpCAM-MSNs under gentle shaking at 100 rpm for 30 minutes, followed by centrifugation at 15000×g for 10 minutes. The precipitate was collected and washed with PBS containing 10% bovine serum albumin. The isolated tumor-derived exosomes were then incubated with 10 μL of CD63 detection microspheres. Specifically, 10 μL of FITC-labeled anti-CD63 detection microspheres were added to the EVs-EpCAM-MSNs solution, and the mixture was incubated by continuous rotation at room temperature for 30 minutes. The mixture was then diluted with 600 μL of ice-cold PBS and incubated overnight at 4°C. After adding 400 μL of 1M glycine (pH 8.0) solution, the mixture was incubated at room temperature for 1 hour by rotation, followed by centrifugation at 12,000 × g for 1 minute. The precipitate was resuspended in 100 μL of 10% BSA (w / v) in PBS, incubated at room temperature by rotation for 45 minutes, then centrifuged (12,000 × g, 1 minute, room temperature) and the supernatant was completely aspirated. The final precipitate (microspheres with the EVs-EpCAM-MSNs complex attached) was resuspended in 200 μL of PBS buffer containing 2% bovine serum albumin and analyzed by flow cytometry with a 488 nm laser. Single microsphere events were gated using FSC-H and FSC-A parameters. The negative control group consisted of EpCAM-MSNs incubated only with CD63 microspheres. Blood samples from healthy mice were centrifuged and then co-incubated with EpCAM-MSNs, followed by the addition of CD63-labeled magnetic beads as a control group.

[0025] Figure 4 To investigate the detection capability of EpCAM-MSNs in blood samples from A549 and H460 tumor-bearing mice on day 7 using flow cytometry, the results showed that EpCAM-MSNs did not show a positive signal in the control group blood samples. However, at the 7-day time point, it was able to effectively detect tumor-derived EVs in blood samples from A549 and H460 xenograft models, demonstrating its ability to specifically capture tumor-derived exosomes (EVs) in the blood and showcasing its potential for application in early diagnosis.

[0026] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the invention. Modifications and variations made by those skilled in the art in accordance with the spirit of the invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A nanomaterial modified with EpCAM aptamer, characterized in that: The nanomaterial is: a carrier of carboxylated mesoporous silica nanoparticles, with EpCAM-specific DNA aptamers covalently coupled to the surface, and the sequence of the EpCAM-specific DNA aptamer is as shown in SEQ ID No.

1.

2. The method for preparing nanomaterials as described in claim 1, characterized in that: Includes the following steps: S1, CTAC and TEA were reacted, then TEOS was added to continue the reaction, centrifuged, washed, and MSN-OH was obtained; S2, MSN-OH is added to a hydrochloric acid-ethanol solution, heated, APTES is added, and stirred to obtain aminated MSN; S3, amino-modified MSN was reacted with succinic anhydride to prepare carboxylated mesoporous silica nanoparticles; S4. Carboxylated mesoporous silica nanoparticles were coupled with EpCAM-specific DNA aptamers at room temperature to prepare nanomaterials.

3. The method for preparing nanomaterials according to claim 2, characterized in that: In step S1, ethanol is used for washing, and the washing is performed three times.

4. The method for preparing nanomaterials according to claim 2, characterized in that: In step S2, the heating temperature is 78°C.

5. The method for preparing nanomaterials according to claim 2, characterized in that: Step S4 is as follows: carboxylated mesoporous silica nanoparticles are mixed with EDC, NHS and EpCAM specific DNA aptamers, stirred, centrifuged and washed to obtain the nanomaterials.

6. The use of the nanomaterial as described in claim 1 as a biomarker for detecting tumor-derived exosomes.

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