Application of nucleolin as a target in preparation of double-targeted therapeutic drugs for colorectal cancer

Abstract

The application provides application of nucleolin as a drug target in preparation of a double-targeted treatment drug for colorectal cancer, and application of nucleolin as a double-targeted drug target in preparation of a drug for double-targeted killing of colorectal cancer tumor cells and tumor-related fibroblasts, the drug for double-targeted killing of colorectal cancer tumor cells and tumor-related fibroblasts, a pharmaceutical composition containing the drug and application thereof. The application uses nucleolin as a common drug target of colorectal cancer tumor cells and tumor-related fibroblasts, applies a double-targeted killing drug with nucleolin as a target, simultaneously kills colorectal cancer tumor cells and tumor-related fibroblasts, removes tumor cells while destroying tumor-related fibroblast-mediated tumor supporting microenvironment, and synergistically enhances the overall treatment effect of colorectal cancer.

Description

[0001] This application claims priority to Chinese Patent Application No. 202610119440.1, filed on January 28, 2026, entitled "Application of nucleolin as a target in the preparation of a dual-targeted therapy for colorectal cancer", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of pharmaceutical technology, and in particular to the application of nucleolin as a drug target in the preparation of a dual-targeted therapy for colorectal cancer. Background Technology

[0003] Colorectal cancer (CRC) is one of the most prevalent and deadliest malignant tumors worldwide. Its development and progression are closely related to the complex regulation of the tumor microenvironment, among which cancer-associated fibroblasts (CAFs) are one of the most abundant and functionally important stromal cell types in the CRC tumor microenvironment. Numerous studies have shown that CAFs can form a tight functional interaction network with tumor cells through various mechanisms, including paracrine signaling, metabolic reprogramming, and extracellular matrix remodeling, playing a crucial role in tumor progression. On the one hand, cancer cell fibroblasts (CAFs) can secrete various growth factors and cytokines, such as transforming growth factor-β (TGF-β) and hepatocyte growth factor (HGF), promoting tumor cell proliferation, angiogenesis, and invasion. On the other hand, CAFs form physical barriers and immunosuppressive microenvironments within tumor tissues by remodeling the extracellular matrix structure, increasing interstitial fluid pressure, and secreting immunosuppressive factors (such as interleukin-6 (IL-6) and programmed death-ligand 1 (PD-L1)). Against this backdrop, both traditional chemotherapeutic drugs lacking cell specificity and monoclonal antibodies or nanomedicines with single-target efficacy are susceptible to the CAF-mediated microenvironment barrier, leading to a significant reduction in drug penetration efficiency in tumor tissues and thus limiting their therapeutic effects. However, due to the diversity of CAF origins, functional heterogeneity, and high plasticity of CAFs, specific targeted therapy strategies targeting CAFs alone still face challenges such as unstable efficacy and potential side effects in practical applications.

[0004] Currently, commonly used tumor-targeting biomarkers in colorectal cancer treatment mainly focus on molecules on the surface of cancer cells, such as epithelial cell adhesion molecule (EpCAM) and mucin 1 (MUC1). Research on cancer cell adhesion fibroblasts (CAFs) primarily focuses on fibroblast-specific molecules such as alpha-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP). However, these targets can only act on tumor cells or CAFs separately, making it difficult to simultaneously intervene in both tumor cells and CAFs within the colorectal cancer tumor microenvironment. Therefore, there are still significant limitations in disrupting the overall structure and function of the tumor microenvironment. Thus, there is an urgent need to discover a common target that is highly expressed in both colorectal cancer tumor cells and CAFs, and to construct a therapeutic strategy based on this target that can synergistically act on both types of cells. This would allow for the direct killing of tumor cells while simultaneously disrupting the CAF-mediated tumor-supporting microenvironment, thereby improving the overall therapeutic effect of colorectal cancer.

[0005] Existing research suggests that nucleolin, a multifunctional nucleolar protein, is abnormally expressed in various malignant tumors and can be localized on the cell membrane surface, offering the potential for its use as a tumor-targeting molecule. However, systematic and effective research is still lacking on the expression characteristics of nucleolin in colorectal cancer cell arteriovenous fibroblasts (CAFs) and on technical solutions that utilize nucleolin as a dual target to achieve simultaneous targeting intervention of tumor cells and CAFs. Summary of the Invention

[0006] The purpose of this application is to provide an application of nucleolin as a drug target in the preparation of a dual-targeted therapeutic drug for colorectal cancer. This drug, by simultaneously targeting nucleolin in colorectal cancer tumor cells and cancer cell support fibroblasts (CAFs), achieves synergistic killing of both tumor cells and CAFs, clearing tumor cells while disrupting the CAF-mediated tumor support microenvironment, thereby improving the therapeutic effect of colorectal cancer. The specific technical solution is as follows:

[0007] The first aspect of this application provides the use of nucleolin as a drug target in the preparation of a drug for the dual-target killing of colorectal cancer tumor cells and CAFs.

[0008] In one embodiment, the drug achieves synergistic killing of the colorectal cancer tumor cells and the CAFs by simultaneously targeting nucleolin in both colorectal cancer tumor cells and CAFs.

[0009] The second aspect of this application provides the use of nucleolin as a drug target in the preparation of a dual-targeted therapeutic agent for colorectal cancer.

[0010] In one embodiment, the drug achieves synergistic killing of the colorectal cancer tumor cells and the CAFs by simultaneously targeting nucleolin in both colorectal cancer tumor cells and CAFs.

[0011] A third aspect of this application provides a drug for dual-targeted killing of colorectal cancer tumor cells and CAFs, the drug comprising a nucleolin-targeting molecule selected from at least one of nucleic acid aptamers, CD44, F3 peptide, NLS peptide, HB-19 monoclonal antibody, MS-3 monoclonal antibody, and 4LB5 monoclonal antibody; and / or,

[0012] The drug comprises magnetic nanomaterials selected from at least one of Fe3O4, FexC, Cu@Fe3O4, and Cu@FexC.

[0013] In one embodiment, the nucleic acid aptamer is selected from at least one of AS1411, T40214, and T30695.

[0014] The fourth aspect of this application provides a pharmaceutical composition comprising the medicament provided in the third aspect of this application and a pharmaceutically acceptable carrier.

[0015] In one embodiment, the carrier comprises the magnetic nanomaterial; the pharmaceutical composition comprises the magnetic nanomaterial modified with the nucleolin-targeting molecule.

[0016] In one embodiment, the magnetic nanomaterial is selected from at least one of Fe3O4, FexC, Cu@Fe3O4, and Cu@FexC.

[0017] The fifth aspect of this application provides the use of the pharmaceutical composition provided in the fourth aspect of this application in the preparation of a medicament for dual-target killing of colorectal cancer tumor cells and CAFs.

[0018] In one embodiment, the pharmaceutical composition achieves synergistic killing of colorectal cancer tumor cells and CAFs by simultaneously targeting nucleolin in both colorectal cancer tumor cells and CAFs.

[0019] The sixth aspect of this application provides the use of the pharmaceutical composition provided in the fourth aspect of this application in the preparation of a dual-targeted therapeutic agent for colorectal cancer.

[0020] The beneficial effects of this application are:

[0021] This application provides the use of nucleolin as a drug target in the preparation of a dual-targeted therapeutic drug for colorectal cancer, and also provides the use of nucleolin as a dual-targeted drug target in the preparation of a drug for dual-targeted killing of colorectal cancer tumor cells and CAFs, a drug for dual-targeted killing of colorectal cancer tumor cells and CAFs, and a pharmaceutical composition containing the drug and its application. This application uses nucleolin as a common drug target for colorectal cancer tumor cells and CAFs, and applies a dual-targeted killing drug with nucleolin as the target, simultaneously killing colorectal cancer tumor cells and CAFs. This eliminates tumor cells while disrupting the CAF-mediated tumor-supporting microenvironment, thereby improving the overall therapeutic effect of colorectal cancer.

[0022] Of course, implementing any product or method of this application does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other embodiments can be obtained based on these accompanying drawings.

[0024] Figure 1A The results of Example 1 show the analysis of the expression characteristics of marker genes of common major cell types, the annotation of each cell cluster, and the identification of multiple cell types; and the results of extracting normal fibroblasts (NFs) and CAFs, and analyzing them again using dimensionality reduction clustering to obtain the NFs and CAFs subpopulations.

[0025] Figure 1B Example 1: Bubble heatmap of expression of major marker genes in NFs and CAFs in fibroblast subsets;

[0026] Figure 1C Example 1: Bubble heatmap of the expression of 20 reported colorectal cancer biomarkers in NFs and CAFs in fibroblast subsets;

[0027] Figure 2A For Example 2, standardization, dimensionality reduction, and cluster analysis were performed on all epithelial cell subpopulations extracted, including tumor epithelial cells and normal epithelial cells, to obtain the results of 7 different epithelial cell subpopulations.

[0028] Figure 2B This is a graph showing the results of copy number change analysis of epithelial cells at the transcriptome level in Example 2;

[0029] Figure 2CExample 2: Bubble heatmap of the expression of 20 reported colorectal cancer biomarkers in epithelial cell subsets in colorectal cancer tumor cells and normal intestinal epithelial cells;

[0030] Figure 2D This is a graph showing the expression levels of nucleolin (NCL) mRNA in colorectal cancer tissue and adjacent normal tissue in Example 2.

[0031] Figure 3A Microscopic field photographs of fibroblasts migrating from normal colonic epithelial tissue and fibroblasts migrating from CRC tissue after 14 days and 90 days of culture, as shown in Example 3.

[0032] Figure 3B The graph shows the protein expression levels of FAP, α-SMA, and Vimentin in CAFs and NFs cells in Example 3.

[0033] Figure 4A The graph shows the protein expression levels of nucleolin in CAFs and NFs cells in Example 3.

[0034] Figure 4B This is a graph showing the protein expression levels of nucleolin in colorectal cancer tumor tissue and adjacent normal epithelial tissue in Example 3.

[0035] Figure 5 The image shown in Example 4 illustrates the membrane localization and expression levels of nucleolin protein in CAFs, NFs, HCT116 cells, and NCM460 cells, obtained by live-cell immunofluorescence staining combined with laser confocal imaging.

[0036] Figure 6A This is a diagram illustrating the operational steps for obtaining a co-culture model of primary tumor cells and CAFs derived from a patient-derived xenograft (PDX) model in vitro, as shown in Example 5.

[0037] Figure 6B The image shows the results of Example 5, which uses immunofluorescence technology to stain specific protein markers of tumor epithelial cells and CAFs to identify the successful establishment of an in vitro co-culture model of primary tumor cells derived from PDX and CAFs.

[0038] Figure 7A This is a diagram illustrating the grouping and treatment steps in Example 5 of a co-culture model of PDX-derived tumor cells and CAFs, with no drugs, with Cu@Fe3O4, and with Cu@Fe3O4-AS1411.

[0039] Figure 7BThe following are fluorescence micrographs of cells viable in the PDX-derived tumor cell and CAF co-culture model in Example 5, after the addition of no drug, the addition of Cu@Fe3O4, and the addition of Cu@Fe3O4-AS1411, as determined by Calcein / PI staining.

[0040] Figure 8 Example 5: Fluorescence micrographs of cells viable in a colorectal cancer PDO model with no drugs, with Cu@Fe3O4, and with Cu@Fe3O4-AS1411, as determined by Calcein / PI staining.

[0041] Figure 9A This is a diagram illustrating the grouping and treatment steps of mice in a subcutaneous xenograft model containing tumor cells and fibroblasts, with and without drugs, with Cu@Fe3O4, and with Cu@Fe3O4-AS1411.

[0042] Figure 9B The image shows the tumor volume of mice in each group in Example 6 at 5, 8, 11, 14 (drug injection), 17 (drug injection), 20 (drug injection), and 23 days after mixed inoculation of tumor cells and fibroblasts.

[0043] Figure 9C The graph shows the changes in tumor volume in each mouse in each group of Example 6 after mixed inoculation with tumor cells and fibroblasts.

[0044] Figure 10 The figure shows the body weight of mice in each group after different days following inoculation with tumor cells and fibroblasts in Example 6. Detailed Implementation

[0045] The technical solutions of this application will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0046] The inventors discovered that simultaneously targeting tumor cells and their dependent CAFs microenvironment, thereby directly eliminating tumor cells while disrupting the "soil" that provides them with physical support and functional assistance, could provide a potentially advantageous new approach to improving the treatment effect of colorectal cancer. Furthermore, research has shown that nucleolin can be used as a dual-target drug in the preparation of drugs for the dual-target killing of colorectal cancer tumor cells and tumor-associated fibroblasts, and this application was completed based on this discovery.

[0047] The first aspect of this application provides the use of nucleolin as a drug target in the preparation of a drug for the dual-target killing of colorectal cancer tumor cells and CAFs.

[0048] The inventors discovered that nucleolin is significantly highly expressed on the surface of tumor cells and CAFs in colorectal cancer, while its expression level is low on the surface of normal intestinal epithelial cells and NFs. Based on this, this application uses nucleolin as a common target for colorectal cancer tumor cells and CAFs, and constructs a dual-targeting drug that simultaneously targets these two cell types, effectively killing colorectal cancer tumor cells and CAFs, thereby improving the specificity and efficacy of targeted therapy for colorectal cancer.

[0049] In one embodiment, the drug achieves synergistic killing of both colorectal cancer tumor cells and CAFs by simultaneously targeting nucleolin in both. The drug of this application achieves synergistic killing of both cell types by simultaneously targeting nucleolin highly expressed in both colorectal cancer tumor cells and CAFs.

[0050] The second aspect of this application provides the use of nucleolin as a drug target in the preparation of a dual-targeted therapeutic agent for colorectal cancer.

[0051] The dual-targeted therapy drug described in this application can act on both colorectal cancer tumor cells and CAFs simultaneously. Using the nucleolin-targeted dual-targeted therapy drug of this application, it can act on the common target of both colorectal cancer tumor cells and CAFs, overcoming the limitation of existing technologies that can only target tumor cells or CAFs individually. It achieves synergistic intervention on tumor cells and CAFs, which helps to enhance the enrichment and penetration of drugs in colorectal cancer tumor tissue, thereby significantly improving the treatment effect of colorectal cancer.

[0052] In one embodiment, the drug achieves synergistic killing of the colorectal cancer tumor cells and the CAFs by simultaneously targeting nucleolin in both colorectal cancer tumor cells and CAFs.

[0053] In one embodiment, the colorectal cancer is colonic adenocarcinoma and / or rectal adenocarcinoma.

[0054] A third aspect of this application provides a drug for dual-targeted killing of colorectal cancer tumor cells and CAFs, the drug comprising a nucleolin-targeting molecule selected from at least one of nucleic acid aptamers, CD44, F3 peptide, NLS peptide, HB-19 monoclonal antibody, MS-3 monoclonal antibody, and 4LB5 monoclonal antibody; and / or,

[0055] The drug comprises magnetic nanomaterials selected from at least one of Fe3O4, FexC, Cu@Fe3O4, and Cu@FexC.

[0056] In one embodiment, the nucleic acid aptamer is selected from at least one of AS1411, T40214, and T30695.

[0057] A fourth aspect of this application provides a medicament for dual-targeted therapy of colorectal cancer, the medicament comprising a nucleolin-targeting molecule selected from at least one of nucleic acid aptamers, CD44, F3 peptide, NLS peptide, HB-19 monoclonal antibody, MS-3 monoclonal antibody, and 4LB5 monoclonal antibody; and / or,

[0058] The drug comprises magnetic nanomaterials selected from at least one of Fe3O4, FexC, Cu@Fe3O4, and Cu@FexC.

[0059] In one embodiment, the nucleic acid aptamer is selected from at least one of AS1411, T40214, and T30695.

[0060] The fifth aspect of this application provides a pharmaceutical composition comprising a drug provided in the third aspect of this application or a drug provided in the fourth aspect of this application, and a pharmaceutically acceptable carrier.

[0061] In one embodiment, the carrier comprises a nanomaterial; in another embodiment, the nanomaterial comprises the magnetic nanomaterial; in yet another embodiment, the pharmaceutical composition comprises the magnetic nanomaterial modified with the nucleolin-targeting molecule. The magnetic nanomaterial modified with the nucleolin-targeting molecule described in this application refers to the magnetic nanomaterial having the nucleolin-targeting molecule modified on its surface.

[0062] In one embodiment, the magnetic nanomaterial modified with the nucleolin-targeting molecule includes, but is not limited to, magnetic nanomaterials modified with AS1411, T40214, or T30695.

[0063] In one embodiment, the magnetic nanomaterial includes, but is not limited to, at least one of Fe3O4, FexC, Cu@Fe3O4, and Cu@FexC.

[0064] In this application, x can be 2-5 in FexC and Cu@FexC.

[0065] In one embodiment, the Cu@Fe3O4 can be prepared by the following method:

[0066] Copper acetylacetonate (Cu(acac)2), octadecene (ODE), oleic acid, and oleylamine (OAm) were mixed, and the solution was heated to 100-130°C under a nitrogen atmosphere and held for 0.5-2 hours to remove organic impurities. The mixture was then heated to 150-250°C, and iron pentacarbonyl (Fe(CO)5) was added to the reaction system and the temperature was maintained for 5-30 minutes. The solution was then heated to 260-300°C and held for 0.5-5 hours. After cooling to room temperature, Cu@Fe3O4 was obtained; wherein the molar ratio of copper acetylacetonate, octadecene, oleic acid, oleylamine, and iron pentacarbonyl can be 1:(40-50):(1.5-2.5):(8-15):(3-7).

[0067] In one embodiment, the nucleolin-targeted molecule-modified magnetic nanomaterial can be AS1411-modified magnetic nanoparticles Cu@Fe3O4 (Cu@Fe3O4-AS1411); the Cu@Fe3O4-AS1411 can be prepared by the following method:

[0068] The Cu@Fe3O4 was modified with distearate-phosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG-NH2). 1-Ethyl-3-(3-(dimethylamino)propyl)urea hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were mixed with the DSPE-PEG-NH2-modified Cu@Fe3O4 nanoparticles in an aqueous solution for 4-8 hours. AS1411 was then added to the reaction system and mixing continued for 12-48 hours to obtain Cu@Fe3O4-AS1411; wherein the mass-to-volume ratio of 1-ethyl-3-(3-(dimethylamino)propyl)urea hydrochloride, N-hydroxysuccinimide, DSPE-PEG-NH2-modified Cu@Fe3O4 nanoparticles, AS1411, and water could be 1 mg:(1-1.5) mg:(8-15) mg:(25-35) μg:(2-20) mL.

[0069] The sixth aspect of this application provides the use of the pharmaceutical composition provided in the fifth aspect of this application in the preparation of a medicament for dual-target killing of colorectal cancer tumor cells and CAFs.

[0070] In one embodiment, the pharmaceutical composition achieves synergistic killing of colorectal cancer tumor cells and CAFs by simultaneously targeting nucleolin in both colorectal cancer tumor cells and CAFs.

[0071] The seventh aspect of this application provides the use of the pharmaceutical composition provided in the fifth aspect of this application in the preparation of a dual-targeted therapeutic agent for colorectal cancer.

[0072] In one embodiment, the pharmaceutical composition achieves synergistic killing of colorectal cancer tumor cells and tumor CAFs by simultaneously targeting nucleolin in both colorectal cancer tumor cells and CAFs.

[0073] The eighth aspect of this application provides a treatment method for colorectal cancer, comprising administering to a subject a therapeutically effective amount of the drug provided in the third aspect of this application, or the drug provided in the fourth aspect of this application, or the pharmaceutical composition provided in the fifth aspect of this application.

[0074] In this application, the term "therapeutic effective amount" refers to the amount of a drug, when used on a subject, sufficient to affect the treatment of a disease, condition, or symptom. Therapeutic effective amount can vary depending on the drug, the symptoms of the disease or condition, the severity of the symptoms, etc. Wherever possible, a suitable dose may be obvious to those skilled in the art or may be determined by routine experiments.

[0075] The terms “subject” or “patient” refer to humans and non-human mammals, including but not limited to primates, rabbits, pigs, horses, dogs, cats, sheep, and cattle.

[0076] Example

[0077] The following examples illustrate the implementation of this application in more detail. Various tests and evaluations were conducted according to the methods described below. Furthermore, unless otherwise specified, "parts" and "%" are quality standards.

[0078] Example 1: Differential expression analysis of colorectal cancer biomarkers in NFs and CAFs

[0079] 1.1 Acquisition and preprocessing of single-cell RNA sequencing data:

[0080] The publicly available colorectal cancer single-cell RNA sequencing dataset E-MTAB-8107 was selected as the analysis object. This dataset, sourced from a public database, contains single-cell transcriptome data and corresponding cell annotation information for various cell types in colorectal cancer patient tumor tissues. The raw single-cell RNA sequencing data underwent quality control and preprocessing, including: removing low-quality cells with excessively low gene expression levels or abnormal mitochondrial gene ratios; normalizing the retained high-quality cells to eliminate the influence of sequencing depth differences on the analysis results; screening for representative highly variable genes for subsequent dimensionality reduction and cluster analysis; linearly scaling the data; and regressing covariates irrelevant to the analysis. After these processing steps, a high-quality single-cell expression matrix was obtained for downstream analysis.

[0081] 1.2 Data dimensionality reduction, clustering, and fibroblast subpopulation annotation:

[0082] Subsequent data analysis was performed using the Seurat package in R Studio. Based on the preprocessed single-cell data, the "RunPCA" and "RunUMAP" functions were used for principal component analysis (PCA) and nonlinear dimensionality reduction. Following this, cell clustering analysis was performed using the "FindNeighbors" and "FindClusters" functions. Each cell cluster was annotated by analyzing the expression characteristics of known cell type marker genes in different clusters. The annotated cell types were visualized using the "DimPlot" function. Figure 1A As shown, it identifies various cell types, including cancer cells, epithelial cells, fibroblasts, immune cells (myeloid cells), and endothelial cells, as well as B cells, enteric glial cells, mast cells, and T cells. UMAP1 and UMAP2 are two-dimensional coordinate axes generated by UMAP dimensionality reduction technology.

[0083] The "Subset" function was used to further extract fibroblast subpopulations from the above results, and these subpopulations were then subjected to further standardization, dimensionality reduction, and cluster analysis. By comparing the expression characteristics of typical marker genes in each fibroblast subpopulation, the fibroblasts were clearly distinguished into: normal fibroblasts (NFs) and tumor-associated fibroblasts (CAFs). Subsequently, the "DotPlot" function was used to visualize the expression characteristics of marker genes in NFs and CAFs, as shown below. Figure 1B As shown. Among them, the marker genes of CAFs include ACTA2, TAGLN, and INHBA, and the marker genes of NFs include DCN, IGFBP6, and MFAP5.

[0084] 1.3 Expression of 20 common colorectal cancer biomarkers in NFs and CAFs:

[0085] Subsequently, gene expression data of 20 reported colorectal cancer biomarkers were analyzed in NFs and CAFs, including: NCL, CD44, TFRC, CXCR4, MUC1, EPCAM, PROM1, SELP, CEACAM5, EGFR, LRP1, NR3C1, FOLR2, NRP1, MRC1, TMEM97, SSTR1, FOLR1, CHEK2, and LYVE1. Logcount data for these genes were scaled, and their expression in NFs and CAFs and their proportion in corresponding cell types were ranked. The mRNA expression levels of each biomarker and the proportion of positively expressed cells in different fibroblast cell types were statistically analyzed. The mRNA expression results of these 20 genes in NFs and CAFs were visualized using the DotPlot function. The results are shown below. Figure 1C As shown, compared with NFs, nucleolin (NCL) had the highest average mRNA expression level and the highest proportion of positive expression cells in CAFs. By analyzing the expression differences of various colorectal cancer-related biomarkers in NFs and CAFs, nucleolin was screened as a target biomarker for colorectal cancer CAFs.

[0086] This embodiment reveals the differential expression characteristics of various colorectal cancer biomarkers in NFs and CAFs through systematic analysis of single-cell RNA sequencing data. Among them, nucleolin showed significantly high expression in CAFs, indicating that it can serve as an important potential target for CAFs in the colorectal cancer tumor microenvironment, providing a theoretical basis for the design of subsequent dual-targeted therapy strategies.

[0087] Example 2: Expression analysis of colorectal cancer biomarkers in tumor epithelial cells and normal intestinal epithelial cells

[0088] 2.1 Identification of tumor epithelial cells and analysis of biomarker expression based on single-cell RNA sequencing data

[0089] Based on the single-cell RNA sequencing data from Example 1, the "subset" function was used to further extract all epithelial cell (epi) subsets, including tumor epithelial cells and normal epithelial cells. The obtained epithelial cell subsets were then standardized, dimensionality reduced, and clustered, further dividing the epithelial cells into seven distinct subsets (epithelial cell-1 to epithelial cell-7). The results are as follows: Figure 2A As shown. Based on the origin information of each subpopulation of epithelial cells in the database (normal tissue, tumor marginal tissue, tumor tissue), it was found that one of the seven subpopulations of epithelial cells originated from normal tissue (epithelial cell 1), and six epithelial cell subpopulations originated from tumor or tumor marginal tissue (epithelial cell-2 to epithelial cell-7).

[0090] To further analyze whether all epithelial cells-1 derived from normal tissues are normal epithelial cells without genomic variations, this embodiment continues to use inferring copy number variation (inferCNV) analysis for verification. For the entire copy number variation analysis process, firstly, a subset of cells from the epithelial cell-1 subpopulation was selected as reference cells, and an infercnv object was created using the "CreateInfercnvObject" function. Subsequently, the "infercnv::run" function was used to perform transcriptomic-level copy number variation analysis on the seven epithelial cell subpopulations to distinguish between colorectal cancer tumor epithelial cells and normal intestinal epithelial cells. Next, the "infercnv::apply_median_filtering" function was used to perform median filtering on the copy number variation matrix to reduce single-cell noise. Finally, the "infercnv::plot_cnv" function was used to visualize the filtered copy number variation results. The inferring copy number variation analysis results are as follows: Figure 2B As shown, the expression distribution represents gene expression distribution, the corrected expression level is the standardized gene expression value (around 1 is a reference value for copy number variation, greater than 1 indicates copy number amplification, and less than 1 indicates copy number loss), and the count is the observed value within the expression interval. The target (cell) is the result of copy number variation analysis and clustering for each cell, and the genomic region is the gene sequence on the chromosome (Chr). The results showed that epithelial cells-1 from normal tissues had no significant copy number variation and no abnormalities in genomic information, while epithelial cells 2-7 from tumor and tumor margin tissues showed significant copy number variation. Based on the above cell classification results, the average expression levels and the proportion of positively expressing cells of the 20 reported colorectal cancer biomarkers in Example 1 were compared and analyzed in colorectal cancer tumor cells and normal intestinal epithelial cells. The expression results of these 20 genes in tumor and normal intestinal epithelial cells were visualized using the "DotPlot" function, and the results are shown below. Figure 2C As shown, compared with normal intestinal epithelial cells, the mRNA expression level of nucleolin (NCL) in colorectal cancer tumor cells is significantly increased, and the proportion of cells expressing NCL positive expression is the highest among the analyzed biomarkers. By analyzing the expression differences of various colorectal cancer-related biomarkers in normal intestinal epithelial cells and colorectal cancer tumor epithelial cells, nucleolin was screened as a targeted biomarker for colorectal cancer tumor cells.

[0091] 2.2 Nucleolin Expression Validation Based on TCGA Database

[0092] To further validate the expression characteristics of nucleolin in colorectal cancer tissues, transcriptomic data from colorectal cancer patients (COAD and READ) in the TCGA database were analyzed. The expression levels of nucleolin (NCL) mRNA in colorectal cancer tissues and adjacent normal colorectal epithelial tissues were standardized, and nonparametric statistical methods were used to compare the expression differences between the two groups. The results are as follows: Figure 2D As shown ( (P < 0.001) The expression level of NCL mRNA is expressed as Log2(FPKM+1). It can be seen that compared with normal colorectal epithelial tissue, the expression level of nucleolin (NCL) mRNA in colorectal cancer tissue is significantly increased, and the difference is statistically significant.

[0093] This embodiment combines single-cell RNA sequencing data with TCGA transcription datasets to systematically verify the high expression characteristics of nucleolin transcripts in colorectal cancer tumor epithelial cells at both the single-cell and tissue levels. This result is consistent with the high expression characteristics of nucleolin in CAFs described in Example 1, indicating that nucleolin can serve as a common target for both tumor cells and CAFs in the colorectal cancer tumor microenvironment, providing important experimental evidence for subsequent nucleolin-based dual-targeted diagnostic and therapeutic technologies.

[0094] Example 3: Detection of nucleolin protein expression in primary NFs and CAFs, normal intestinal epithelial tissue, and colorectal cancer tissue from colorectal cancer patients.

[0095] 3.1 Isolation and Culture of Primary Fibroblasts

[0096] Following ethical approval, samples were obtained from fresh colorectal cancer tumor tissue and adjacent normal colonic epithelial tissue surgically removed from colorectal cancer patients. These samples were placed in tissue preservation solution and rapidly transferred to the laboratory. Under aseptic conditions, the tissues were repeatedly washed with pre-cooled phosphate buffered saline (PBS, Solarbio, catalog number P1020) to remove blood and impurities, and visible necrotic tissue was removed. The tissues were then cut into small pieces suitable for adherent culture, evenly distributed at the bottom of the culture dish, and allowed to stand. High-glucose DMEM medium (Hyclone, catalog number SH30022.01) containing 15% fetal bovine serum (Gibco, catalog number 16000) and 1% penicillin-streptomycin (Solarbio, catalog number P1400) was added, and the culture was carried out at 37°C and 5% carbon dioxide. The culture medium was changed regularly during the culture process, and the migration and growth of cells from the tissue blocks were observed under an inverted optical microscope (manufacturer: Leica, model: DMi1). Micrographs of NFs migrating from normal colon tissue and CAFs migrating from CRC tissue were obtained at 14 and 90 days of culture. Figure 3A As shown, cells are indicated by arrows. Once the cells reached an appropriate density, the old culture medium was discarded, and the cells were washed with PBS. An appropriate amount of 0.25% trypsin-EDTA digestion solution (Gibco, catalog number 25200) was added, and the cells were incubated at 37°C. Close observation was maintained under a microscope. When the intercellular spaces increased, the cells became more rounded, and some cells began to detach, serum-containing complete culture medium was immediately added to terminate the reaction. The cells were gently pipetted to completely disperse them into a single-cell suspension. After centrifugation, the suspension was resuspended and passaged.

[0097] 3.2 Identification of fibroblasts and detection of nucleolin expression

[0098] Western blot (WB) was used to identify NFs and CAFs and to detect the expression of nucleolin in NFs and CAFs. Specifically, primary NFs and CAFs were collected, and total cell protein was extracted using RIPA lysis buffer (Solarbio, catalog number R0020) containing a protease inhibitor (Roche, model number 4693116001). The protein concentration was determined using a Brandford protein quantification kit (Biorad, catalog number 5000505). Equal volumes of protein samples were subjected to SDS-PAGE gel electrophoresis and membrane transfer. Fibroblast-associated marker protein antibodies FAP (Cell Signaling Technology, model: E1V9V), α-SMA (STARTER, model: S0B2103), and vimentin (Cell Signaling Technology, model: 5741s) were then detected, with β-actin (Sigma-Aldrich, model: A1978) used as an internal control. Western blotting results are shown below. Figure 3B As shown, the normal fibroblast groups 1-3 (NF1-NF3) were three normal fibroblast cell lines obtained from primary culture, and the tumor-associated fibroblast groups 1-3 (CAF1-CAF3) were three patient-derived tumor-associated fibroblast cell lines obtained from primary culture. The results showed that the markers of CAFs, FAP, α-SMA and Vimentin, were highly expressed in CAF1-CAF3, while their expression was significantly reduced in NF1-NF3, indicating that the obtained NFs and CAFs populations had good purity and representativeness.

[0099] Western blot analysis (WB) was used to detect the expression levels of nucleolin (manufacturer: Abcam, model: ab129200) in the aforementioned NFs and CAFs, with β-actin used as an internal reference protein. The WB results are as follows: Figure 4AAs shown, nucleolin was significantly highly expressed in tumor-associated fibroblasts (NFs) groups 1–3, while its expression level was low or absent in normal fibroblasts (NFs) groups 1–3. These results indicate that nucleolin can serve as an effective molecular marker for distinguishing between colorectal cancer NFs and CAFs.

[0100] 3.3 Detection of nucleolin protein expression levels in tumor tissues of colorectal cancer patients and normal control tissues.

[0101] Fresh tumor tissue and adjacent normal colorectal epithelial tissue from colorectal cancer patients who underwent aseptic resection during ethically approved surgery were collected and rinsed thoroughly with pre-chilled PBS. An appropriate amount of lysis buffer containing protease inhibitors was added, and the tissue was homogenized thoroughly using a tissue homogenizer. The homogenate was then lysed on ice and centrifuged to obtain the total protein extract. Protein concentration was determined using protein quantification methods. Equal volumes of protein samples were subjected to gel electrophoresis and membrane transfer to detect the expression of nucleolin protein in colorectal cancer tumor tissue and normal control tissue, using β-actin as an internal control protein. Western blotting results of nucleolin protein expression are shown below. Figure 4B As shown, the results of nucleolin protein expression in normal colorectal epithelial tissues (groups 1-6) and tumor tissues (groups 1-6) are the results of nucleolin protein expression in tumor tissues. It can be seen that in the six pairs of colorectal cancer and adjacent normal epithelial tissues, the expression of nucleolin protein in cancer tissues is significantly higher than that in the paired normal tissues.

[0102] This embodiment first successfully isolated and identified primary nucleoli and coronary artery fibroids (NFs) from colorectal cancer patients. Subsequent experiments verified that the protein expression level of nucleolin in CAFs was significantly higher than that in NFs, confirming the reliability of nucleolin as a specific biomarker for CAFs. Further analysis of nucleolin protein in patient tumor tissue samples strongly demonstrated that the expression of this protein in colorectal cancer tissue was significantly higher than in normal colorectal epithelial tissue, providing important evidence for a dual-target therapy strategy for colorectal cancer using nucleolin.

[0103] Example 4: Detection of nucleolin expression on the surface of normal colorectal epithelial cells, tumor cells, and NFs and CAFs membranes.

[0104] 4.1 Cell Culture

[0105] Colorectal cancer cell line HCT116, normal colorectal epithelial cell line NCM460, and patient-derived colorectal cancer CAFs and NFs were cultured in DMEM high-glucose complete medium (manufacturer: Hyclone, catalog number: SH30022.01) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. All cells were cultured in a cell culture incubator at 37°C and 5% CO2 until they reached the logarithmic growth phase, at which point they were used for subsequent experiments.

[0106] 4.2 Detection of nucleolin's cell membrane localization expression by live-cell immunofluorescence staining

[0107] To clarify the expression characteristics of nucleolin proteins on the cell membrane surface of different cell types, live-cell immunofluorescence staining was used to detect HCT116 and NCM460 cell lines, as well as primary cultured CAFs and NFs. During the experiment, without disrupting cell membrane integrity, live cells in the logarithmic growth phase were immunofluorescently labeled with the green fluorescent antibody CoraLite® Plus 488-conjugated Nucleolin (manufacturer: Proteintech, model: CL488-83380) to detect the distribution of nucleolin on the cell membrane surface. Simultaneously, the red fluorescent antibody CoraLite® 594-conjugated Nucleolin was used as a positive control marker for cell membrane localization to verify the accuracy of cell membrane staining. Hoechst 33342 (a blue nuclear fluorescent dye) was used to stain the nuclei of live cells to indicate nuclear location and aid in cell structure identification. Images were observed and acquired using a laser confocal microscope (manufacturer: Zeiss, model: LSM 880), and the cell membrane localization fluorescence signals of Nucleolin in different cell types were compared and analyzed. Figure 5 Laser confocal microscopy images show the expression of nucleolin in CAFs, NFs, HCT116 cells, and NCM460 cells, and its localization on the cell membrane. The results show that nucleolin exhibits significant fluorescence signals on the cell membranes of HCT116 and CAFs, while the fluorescence signals on the cell membranes of normal colorectal epithelial cell lines NCM460 and NFs are weak or indistinct. These results indicate that nucleolin has a specific high expression characteristic on the cell membranes of colorectal cancer tumor cells and CAFs, demonstrating that nucleolin expression on the cell membranes of colorectal cancer cells is higher than that of normal intestinal epithelial cells, and its expression on the cell membranes of CAFs is higher than that of NFs.

[0108] This embodiment further verified the feasibility of nucleolin as a common target of colorectal cancer tumor cells and CAFs from the cell membrane localization level through live cell immunofluorescence staining combined with laser confocal imaging experiments, providing direct experimental evidence for its application in dual-targeted therapy.

[0109] Example 5: Killing effect of nucleolin-targeting nucleic acid aptamer-modified magnetic nanomaterials on colorectal cancer tumor cells and CAFs.

[0110] 5.1 Construction of a co-culture model of PDX-derived tumor cells and CAFs

[0111] Tumor tissue from primary tumor cells (PDX) of colorectal cancer patients was aseptically isolated in a biosafety cabinet. The tissue was thoroughly washed with pre-cooled PBS to remove blood and necrotic tissue. The tumor tissue was then minced and enzymatically digested (digestion and dissociation). After digestion was terminated, the tissue was filtered and centrifuged to obtain a mixed cell population. The resulting cells were seeded into culture plates and cultured in DMEM high-glucose complete medium (manufacturer: Hyclone, catalog number: SH30022.01) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin to construct an in vitro co-culture model containing primary tumor cells and cancer cells (CAFs). The experimental procedures are as follows: Figure 6A As shown. The co-culture model was identified by immunofluorescence staining. Carcinoembryonic antigen (CEA) and Ki67 were used as tumor epithelial cell markers, with anti-CEA monoclonal antibody (Huabio, catalog number: ET1705-52) and anti-Ki67 monoclonal antibody (Abcam, catalog number: ab16667) as primary antibodies, and red fluorescent CoraLite594–conjugated goat anti-rabbit IgG (H+L) antibody (Proteintech, catalog number: SA00013-4) as the secondary antibody. α-SMA and Vimentin were used as fibroblast markers, with anti-α-SMA monoclonal antibody (STARTER, model number: S0B2103) and anti-Vimentin monoclonal antibody (Cell Signaling) as secondary antibodies. The primary antibody was Proteintech (model: 5741s), and the secondary antibody was a green fluorescent goat anti-rabbit IgG (H+L) antibody (manufacturer: Proteintech, catalog number: SA00003-2). Cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI, blue fluorescent dye). Immunofluorescence staining images of the primary tumor cells and CAFs co-culture model were obtained using an inverted fluorescence microscope (manufacturer: Leica, model: DMi8). Figure 6B As shown, the results confirm that the co-culture model contains both tumor cells and CAFs, indicating that the PDX co-culture model was successfully constructed.

[0112] 5.2 Construction of Tumor Organoid Models

[0113] With ethical approval and informed consent obtained from patients, fresh tumor tissue specimens from biopsies or intraoperative resections of colorectal cancer patients were collected and transported in organoid tissue preservation solution. After cleaning and mincing, the tissue samples were dissociated using a tumor tissue digestion solution (manufacturer: BioGenous, model: K601003). Digestion was terminated, and the samples were filtered and washed to obtain a tumor cell population. The cells were resuspended in matrix gel (manufacturer: BioGenous, model: M315077) and seeded in culture plates. They were then cultured in three dimensions in colorectal cancer organoid culture medium (manufacturer: BioGenous, catalog number: K-2605) to form a patient-derived organoid (PDO) model with glandular structural features. This model effectively simulates the biological characteristics of the patient's primary tumor at both structural and functional levels.

[0114] 5.3 Nanomedicine Preparation

[0115] 5.3.1 Preparation of unmodified magnetic nanoparticles Cu@Fe3O4:

[0116] Copper acetylacetonate (Cu(acac)2) (1 mmol, J&K brand), octadecene (ODE) (46.875 mmol, Alabaster brand), oleic acid (2 mmol, Alabaster brand), and oleylamine (OAm) (10 mmol, J&K brand) were mixed in a four-necked flask. The solution was heated to 120°C under a mild nitrogen atmosphere and held for 1 hour to remove organic impurities. The mixture was then heated to 180°C, and ferric pentacarbonyl (Fe(CO)5) (5 mmol) was added to the reaction system and held for 10 minutes. The solution was then heated to 280°C and held for 2 hours. After cooling to room temperature, the solution was washed three times with acetone and hexane to obtain magnetic nanoparticles Cu@Fe3O4, which were stored in chloroform.

[0117] 5.3.2 Preparation of Cu@Fe3O4-AS1411 magnetic nanoparticles modified with the nucleolin-targeting nucleic acid aptamer AS1411

[0118] Cu@Fe3O4 nanoparticles were modified with distearate-phosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG-NH2). 1-Ethyl-3-(3-(dimethylamino)propyl)urea hydrochloride (EDC) (1.9 mg, Aldrich, 99% purity) and N-hydroxysuccinimide (NHS) (2.2 mg, Aldrich, 98% purity) were mixed with the above Cu@Fe3O4 nanoparticles (20 mg) in 10 mL of aqueous solution for 6 hours. Then, AS1411 (60 μg, CAS No.: 301636-59-9) was added to the reaction system and mixing continued for 24 hours to obtain AS1411-modified magnetic nanoparticles Cu@Fe3O4-AS1411. The product was washed three times with deionized water and then stored in aqueous solution.

[0119] 5.4 Calcein / PI staining to detect the killing effect of nucleolin-targeted nucleic acid aptamer-modified magnetic nanomaterials on tumor cells and CAFs

[0120] In the co-culture models of PDX-derived tumor cells and CAFs and the PDO model, the NC group without added materials was used as a control. The killing effects of magnetic nanomaterials without added targeting probes (Cu@Fe3O4) and magnetic nanomaterials modified with nucleolin-targeting nucleic acid aptamer AS1411 (Cu@Fe3O4-AS1411) on CRC tumor cells and CAFs in the co-culture model and on PDO derived from colorectal cancer patients were compared. In an in vitro co-culture model of primary tumor cells derived from colorectal cancer PDX and CAFs, the dosage of magnetic nanomaterials added was the drug concentration that could induce half-life death in CAFs. Specifically: No drug added group (NC): 100 μl of PBS was added; Cu@Fe3O4 added group (NPs): 100 μl of PBS containing 2.63 μM Cu@Fe3O4 was added; Cu@Fe3O4-AS1411 added group (NPs-AS1411): 100 μl of PBS containing 2.63 μM Cu@Fe3O4-AS1411 was added. In the colorectal cancer organoid model, the dosage of magnetic nanomaterials added was the drug concentration that could induce half-life death in colorectal cancer organoids. Specifically: No drug added group (NC): 100 μl of PBS was added; Cu@Fe3O4 added group (NPs): 100 μl of PBS containing 7.11 μM Cu@Fe3O4 was added; Cu@Fe3O4-AS1411 added group (NPs-AS1411): 100 μl of PBS containing 7.11 μM Cu@Fe3O4-AS1411 was added.

[0121] After 24 hours of co-incubation, cell viability was assessed using the Calcein / PI double staining method. Calcein staining labeled surviving cells, emitting green fluorescence; PI staining labeled dead cells, emitting red fluorescence. Cell survival and death in different treatment groups were compared and analyzed using fluorescence microscopy (manufacturer: Leica, model: DMi8). In the in vitro co-culture model of primary tumor cells derived from colorectal cancer PDX and CAFs, the grouping treatment steps were as follows: no drug, Cu@Fe3O4 added, and Cu@Fe3O4-AS1411 added. Figure 7A As shown, the micrographs of each group of Calcein / PI staining were obtained as follows: Figure 7B As shown. Micrographs of Calcein / PI staining in the PDO model with and without the drug, with the addition of Cu@Fe3O4, and with the addition of Cu@Fe3O4-AS1411 are shown below. Figure 8 As shown.

[0122] Figure 7B The results showed that in the PDX-derived tumor cell and CAF co-culture model, compared with the unmodified magnetic nanomaterials (NPs), the AS1411-modified magnetic nanomaterials (NPs-AS411) significantly enhanced the combined killing effect on colorectal cancer tumor cells and CAFs, as evidenced by a significant decrease in the proportion of surviving cells and a significant increase in the proportion of dead cells. Figure 8 The results showed that, in the PDO model, the magnetic nanomaterials modified with AS1411 (NPs-AS411) significantly enhanced the killing effect on colorectal cancer PDO compared with the unmodified group (NPs).

[0123] This embodiment, using PDX co-culture and PDO models, verified the significant killing advantage of nucleolin-targeting dual-targeting nanomaterials in a colorectal cancer model from two-dimensional and three-dimensional perspectives. It demonstrates that nucleolin-targeting nucleic acid aptamer-modified magnetic nanomaterials can more effectively improve the efficacy against tumor cells and CAFs, achieving synergistic killing of both cell types. Furthermore, it proves that nucleolin-targeting nucleic acid aptamer-modified magnetic nanomaterials can be well applied to complex tumor microenvironments, not only effectively killing tumor cells but also simultaneously disrupting the CAF-mediated tumor support microenvironment, improving the stability of therapeutic efficacy, reducing the risk of side effects, and enhancing the therapeutic effect of drugs on colorectal cancer.

[0124] Example 6: In vivo efficacy verification of nucleolin-targeting nucleic acid aptamer-modified magnetic nanomaterials in a mouse subcutaneous xenograft model of colorectal cancer cells and fibroblasts co-transplanted.

[0125] 6.1 Cell Culture

[0126] Mouse colon cancer cell line CT26 and mouse embryonic fibroblasts BALB / 3T3 were cultured in DMEM high-glucose complete medium containing 10% FBS and 1% penicillin-streptomycin, and then cultured in a 37°C, 5% CO2 cell culture incubator. Subsequent experiments were conducted after the cells reached the logarithmic growth phase.

[0127] 6.2 Nanomedicine Preparation

[0128] The preparation of unmodified magnetic nanoparticles Cu@Fe3O4 and the preparation of magnetic nanoparticles Cu@Fe3O4-AS1411 modified with nucleolin-targeting nucleic acid aptamer AS1411 are the same as in Example 5.

[0129] 6.3 Construction of a subcutaneous xenograft model by co-injection of CT26 cells and BALB / 3T3 cells

[0130] This animal experiment was conducted in accordance with relevant animal ethics guidelines. Six- to eight-week-old BALB / c mice (weighing 20g) were selected and housed in a barrier environment. CT26 cells and BALB / 3T3 cells in the logarithmic growth phase were collected. CT26 cells (1.95 × 10⁻⁶) were... 6 (5 × 10 cells) and BALB / 3T3 cells (5 × 10) 4 (100 cells) were mixed in 0.1 mL of PBS and then subcutaneously injected into the right hind leg root of BALB / c mice to construct a subcutaneous xenograft model containing tumor cells and fibroblasts. Tumor growth was monitored regularly after inoculation, and drug treatment was initiated when the tumor volume reached a predetermined range.

[0131] 6.4 Grouping and Dosing Regimen

[0132] Experimental animals were randomly divided into three groups, each containing the same number of mice: ① Control group (NC group, no drug): PBS (200 μl) was injected via tail vein; ② Unmodified magnetic nanoparticle group (NPs group, with Cu@Fe3O4): PBS containing Cu@Fe3O4 magnetic nanoparticles (20 mg / kg, 200 μl) was injected via tail vein; ③ AS1411 modified magnetic nanoparticle group (NPs-AS1411 group, with Cu@Fe3O4-AS1411): PBS containing Cu@Fe3O4-AS1411 magnetic nanoparticles (20 mg / kg, 200 μl) was injected via tail vein. Each group was administered the drug multiple times at predetermined doses and time intervals.

[0133] The grouping and treatment procedures for each group of mice are as follows: Figure 9AAs shown, on day 14 after cell seeding, mice were intravenously injected via tail vein into PBS containing Cu@Fe3O4 magnetic nanoparticles (20 mg / kg, 200 μl) or Cu@Fe3O4-AS1411 magnetic nanoparticles (20 mg / kg, 200 μl). The injections were repeated on days 17 and 20 after cell seeding.

[0134] 6.5 Tumor growth and safety monitoring

[0135] During the drug administration period, the mouse's body weight and general activity status were recorded regularly. The long and short diameters of the tumor were measured using calipers. The tumor volume was calculated and recorded according to the formula: Volume = Long Diameter × Short Diameter × Short Diameter / 2, to assess tumor growth. The tumor volumes of each group of mice at 5, 8, 11, 14 (drug injection), 17 (drug injection), 20 (drug injection), and 23 days after tumor cell inoculation are shown below. Figure 9B As shown, the tumor volume of each mouse in each group at 8 days, 14 days (drug injection), and 20 days (drug injection) after tumor cell inoculation is as follows: Figure 9C As shown in the figure (n=6), the experimental results showed that compared with the NC group, the tumor volume of the NPs group was significantly smaller than that of the NC group, and the tumor volume of the NPs-AS1411 group was significantly smaller than that of the NPs group. The differences between the groups were significant, indicating that the dual-targeting strategy of this application can effectively kill tumor cells and CAFs, and has a significant tumor-suppressing effect. The body weight of mice in each group at different days after tumor cell inoculation is shown in the figure. Figure 10 As shown, there was no difference in body weight among the groups of mice during the treatment period, indicating that the treatment regimen of this application is safe.

[0136] This embodiment demonstrates that magnetic nanomaterials modified with nucleic acid aptamers targeting nucleolin have good feasibility for application under in vivo conditions. Targeting nucleolin can promote the effect of magnetic nanomaterials in tumor microenvironments rich in tumor cells and fibroblasts, providing experimental support for the in vivo application of dual-targeted therapy for colorectal cancer.

[0137] In summary, this application, through analysis of reported colorectal cancer cell surface markers using single-cell RNA sequencing databases, combined with clinical sample detection and live-cell immunofluorescence analysis, found that nucleolin exhibits significantly high expression on the cell membranes of both colorectal cancer tumor cells and CAFs, while its expression level is low on the cell membranes of normal intestinal epithelial cells and NFs. Based on this, this application uses nucleolin as a common target for both colorectal cancer tumor cells and CAFs, and constructs a dual-targeting therapeutic drug that simultaneously targets these two cell types, which is beneficial for improving the specificity and efficacy of targeted therapy for colorectal cancer. The dual-targeting therapeutic drug targeting nucleolin, as described in this application, was applied in in vitro models of tumor cells and CAFs coexisting, as well as in tumor organoid models. It can significantly and efficiently kill colorectal cancer tumor cells and CAFs, achieving a synergistic killing effect on both types of cells and disrupting the tumor-supporting microenvironment formed by CAFs. Furthermore, when applied in a mouse model of tumor cell and fibroblast co-transplantation, it can enhance the accumulation and uptake of the drug in colorectal cancer tumor tissue, exhibiting excellent tumor growth inhibition effects, thereby significantly improving the therapeutic effect of colorectal cancer.

[0138] It should be noted that, in this document, the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, or article apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, or article.

[0139] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. Application of nucleolin as a drug target in the preparation of drugs for dual-target killing of colorectal cancer tumor cells and tumor-associated fibroblasts.

2. Application of nucleolin as a drug target in the preparation of dual-targeted therapeutic drugs for colorectal cancer.

3. The application according to claim 1 or 2, characterized in that, The drug achieves synergistic killing of colorectal cancer tumor cells and tumor-associated fibroblasts by simultaneously targeting nucleolin in both colorectal cancer tumor cells and tumor-associated fibroblasts.

4. A drug for the dual-targeted killing of colorectal cancer tumor cells and tumor-associated fibroblasts, characterized in that, The drug comprises a nucleolin-targeting molecule, wherein the nucleolin-targeting molecule is selected from at least one of nucleic acid aptamers, CD44, F3 peptide, NLS peptide, HB-19 monoclonal antibody, MS-3 monoclonal antibody, and 4LB5 monoclonal antibody; and / or, The drug comprises magnetic nanomaterials selected from at least one of Fe3O4, FexC, Cu@Fe3O4, and Cu@FexC.

5. The drug according to claim 4, characterized in that, The nucleic acid aptamer is selected from at least one of AS1411, T40214, and T30695.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the drug of claim 4 or 5, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The carrier includes the magnetic nanomaterial; the pharmaceutical composition includes the magnetic nanomaterial modified with the nucleolin-targeting molecule.

8. Use of the pharmaceutical composition of any one of claims 6-7 in the preparation of a medicament for the dual-targeted killing of colorectal cancer tumor cells and tumor-associated fibroblasts.

9. The application according to claim 8, characterized in that, The pharmaceutical composition achieves synergistic killing of colorectal cancer tumor cells and tumor-associated fibroblasts by simultaneously targeting nucleolin in both colorectal cancer tumor cells and tumor-associated fibroblasts.

10. Use of the pharmaceutical composition of any one of claims 6-7 in the preparation of a dual-targeted therapeutic agent for colorectal cancer.

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