Nano-drug for oral cancer as well as preparation method and application of nano-drug

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a nano-drug for oral cancer as well as a preparation method and application of the nano-drug. The nano-drug provided by the invention is a drug-loaded mesoporous polydopamine nano-particle wrapped by a lipidosome modified by RGD peptide; wherein the load components of the mesoporous polydopamine nanoparticles comprise a cis-platinum drug and an RBMX gene expression inhibitor. The molecular RBMX related to regulation and control of oral cancer progress and cis-platinum drug resistance is screened, a nano biomimetic material drug loading system is constructed to load si-RBMX and cis-platinum to synergistically resist oral cancer treatment, and a novel nano drug with strong targeting property, good biological safety and excellent tumor lethality is obtained. The invention also provides a preparation method and application of the nano-drug.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nanomedicine for oral cancer, its preparation method, and its application. Background Technology

[0002] Oral cancer, one of the most common malignant tumors of the head and neck, refers to all malignant tumors occurring in the oral cavity. Its incidence rate is increasing globally, with over 260,000 new cases diagnosed annually worldwide, according to the World Health Organization (WHO). The occurrence of oral cancer is closely related to various factors, including poor lifestyle habits, oral hygiene, nutrition, environment, and genetics. Early symptoms of oral cancer are easily overlooked by patients, but the treatment outcome worsens as the disease progresses. If oral cancer is detected early and treated scientifically and appropriately, the survival rate can exceed 80%. Conversely, if neglect or delay leads to advanced stages, patients often face more severe treatment challenges and poorer prognoses. Therefore, emphasizing early detection, diagnosis, and treatment strategies for oral cancer is of immeasurable importance in improving patients' quality of life and extending their survival time.

[0003] Oral cancer is primarily treated with a combination of methods, including surgery, radiotherapy, and chemotherapy. Improving the long-term survival rate of patients with intermediate to advanced oral cancer remains challenging, with the five-year survival rate hovering between 50% and 60%. This is mainly due to the difficulty in effectively controlling tumor recurrence, including recurrence at the primary site and regional or distant lymph node metastasis. Cisplatin is a commonly used chemotherapy drug in oral cancer treatment. Cisplatin is typically administered intravenously, a passive delivery method that has drawbacks such as poor targeting, significant systemic toxicity, and a high risk of developing drug resistance. Furthermore, cisplatin resistance is a key reason for treatment failure in oral cancer. Currently, the key molecules influencing cisplatin resistance in oral cancer are unclear, and targeted treatment methods are lacking.

[0004] RNA-binding motif protein X-linked (RBMX) has been identified as one of the diverse nuclear proteomes that binds to polyadenylated RNA and is crucial for RNA processing, metabolism, and maintaining genome stability (including DNA splicing, damage repair, and transcription). RBMX plays the role of a readout protein in m6A modification of RNA. As a key regulator, RBMX is closely linked to various cancer drivers, suggesting its important role in tumorigenesis, development, and treatment resistance. However, the role of RBMX in oral cancer remains unclear.

[0005] Therefore, in-depth research into the role of RBMX in oral cancer progression and cisplatin resistance, and the exploration and development of innovative RBMX-based treatment strategies, are of paramount clinical significance in fundamentally improving the prognosis of oral cancer patients and significantly enhancing their survival rate and quality of life through precision medicine and personalized treatment. To this end, there is an urgent need to develop novel treatment strategies for oral cancer, improve the targeting of cisplatin drugs, and enhance the therapeutic efficacy for oral cancer. Summary of the Invention

[0006] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention provides a nanomedicine for oral cancer. This invention screens and regulates RBMX molecules associated with oral cancer progression and cisplatin resistance, constructs a nanobiomimetic drug delivery system to load si-RBMX and cisplatin for synergistic anti-oral cancer treatment, aiming to obtain a novel nanomedicine with strong targeting, good biocompatibility, and excellent tumor-killing efficacy. Specifically, this invention utilizes MDPA to load si-RBMX and cisplatin to solve the problem of inefficient in vivo siRNA delivery, sensitizing tumor cells to cisplatin, improving the precise targeting of the drug to tumors, and further enhancing the bioavailability of the drug.

[0007] In a first aspect, the present invention provides a nanomedicine, characterized in that the nanomedicine is drug-loaded mesoporous polydopamine nanoparticles encapsulated in RGD peptide-modified liposomes.

[0008] The loading components of the mesoporous polydopamine nanoparticles include cisplatin and RBMX gene expression inhibitors.

[0009] According to some embodiments of the present invention, the RBMX gene expression inhibitor comprises siRNA of the RBMX gene.

[0010] According to some embodiments of the present invention, the nucleotide sequence of the sense strand of the RBMX gene siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.2.

[0011] According to some embodiments of the present invention, the loading component of the mesoporous polydopamine nanoparticles further includes a fluorescent dye.

[0012] A second aspect of the present invention provides a method for preparing nanomedicines as described in the first aspect of the present invention, comprising the following steps:

[0013] S1. F127, dopamine hydrochloride and tetramethylbenzidine were added to an ethanol solution, and the mixture was sonicated to form an emulsion. Ammonia was added to react the emulsion, and the particles generated by the reaction were separated, washed and dried to obtain mesoporous polydopamine nanoparticles.

[0014] S2. The solution of cisplatin and RBMX gene expression inhibitor was added to anhydrous ethanol and mixed. Then, a dispersion of mesoporous polydopamine powder was added, stirred, and separated to obtain drug-loaded mesoporous polydopamine nanoparticles.

[0015] S3. Lecithin and DSPE-PEG are added to an organic solvent, evaporated to form a film to obtain liposomes, DSPE-PEG-RGD is added and mixed, impurities are removed by dialyzing, drug-loaded mesoporous polydopamine nanoparticles are added, hydrated and sonicated, and then DSPE-PEG-RGD is added and stirred to obtain the nanomedicine.

[0016] According to some embodiments of the present invention, in step S1, the temperature of the reaction of adding ammonia water is 50℃~70℃ and the time is 2~4h; the drying temperature is 50℃~70℃ and the time is 10~14h.

[0017] According to some embodiments of the present invention, in step S2, the mass ratio of the cisplatin drug to the mesoporous polydopamine powder is (10~15):1.

[0018] A third aspect of the invention provides the use of nanomedicines as described in the first aspect of the invention in the preparation of pharmaceutical formulations for treating oral cancer.

[0019] According to some embodiments of the present invention, the oral cancer includes cisplatin-resistant oral cancer.

[0020] According to some embodiments of the present invention, the oral cancer is oral squamous cell carcinoma.

[0021] A fourth aspect of the present invention provides the use of nanomedicines as described in the first aspect of the present invention in the preparation of imaging materials for oral cancer lesions.

[0022] A fifth aspect of the present invention provides a pharmaceutical composition comprising the nanomedicine described in the first aspect of the present invention.

[0023] The beneficial effects of this invention are:

[0024] This invention, through the analysis of the TCGA database and the collection of oral cancer (OSCC) patient tissue samples, revealed a significant upregulation trend of RBMX expression in OSCC tissues. It also found that RBMX expression levels are closely related to tumor grade, with OSCC patients exhibiting high RBMX expression showing poor prognosis. Furthermore, RBMX upregulation promotes the proliferation, migration, and invasion of OSCC cells and enhances their cisplatin resistance. This invention further validates that reducing RBMX expression can significantly weaken OSCC cell resistance to cisplatin, thereby making them more sensitive to chemotherapeutic drugs.

[0025] This invention utilizes MDPA to carry si-RBMX and cisplatin, which not only avoids the problem of inefficient in vivo siRNA delivery but also sensitizes tumor cells to cisplatin, improving the precise targeting of the drug to tumors and further enhancing drug bioavailability. This invention validates the anti-tumor effect of the provided composite nanomedicine using a nude mouse orthotopic xenograft model, providing new ideas and directions for developing novel clinical treatment strategies for oral cancer, and is of great significance for the treatment of patients with refractory and cisplatin-resistant oral cancer.

[0026] The composite nanomedicine of this invention utilizes mesoporous polydopamine, which possesses a rich pore structure, providing a good structural basis for drug delivery. Furthermore, the chemical composition of dopamine remains stable at neutral pH, allowing for degradation and release of the target drug in the acidic tumor microenvironment. This property can significantly reduce the adverse effects of tumor drugs on healthy physiological tissues. This invention further organically combines liposomes with mesoporous dopamine, improving the material's targeting and delivery effects. Combined with the inherent safety, biocompatibility, and targeting ability of RGD peptides, it achieves better targeted binding to tumor tissues, resulting in a superior anti-tumor effect.

[0027] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0029] Figure 1 Figure A shows the results of the study on m6A levels in oral cancer tissue in Example 1 of the present invention; Figure A is a histogram of m6A levels in oral cancer tissue (OC) and adjacent normal tissue (NC); Figure B is a heatmap showing significant differences in m6A-related genes between oral cancer and normal oral tissue.

[0030] Figure 2 Figure 1 shows the results of the study on RBMX regulation of m6A levels in oral cancer cells in Example 1 of this invention. Figure A is an m6A subtyping diagram; Figure B is a PCA analysis showing the expression of m6A-related genes in two subtypes; Figure C is a differential m6A analysis result; Figure D is a random forest tree model based on the oral cancer group and the control group, with red representing the error in the oral cancer group, green representing the error in the control group, and black representing the error in all samples; Figure E is a gene importance score analysis result; and Figure F is the effect of RBMX knockdown on m6A methylation levels in oral cancer cells.

[0031] Figure 3The following figures illustrate the results of the study in Example 2 of this invention, exploring the impact of RBMX on the grading and prognosis of oral cancer. Figure A shows the histogram of RBMX differences between oral cancer tissues and normal tissues in the TCGA database; Figure B shows the paired RBMX differences between oral cancer tissues and adjacent normal tissues in the TCGA database; Figure C shows the results of a Western blot (WB) experiment comparing RBMX proteins in five pairs of oral cancer tissues and adjacent normal tissues; Figure D shows a heatmap of the clinical relevance of RBMX high and low expression groups in oral cancer patients; Figure E shows a box plot of the relationship between RBMX expression levels and oral cancer patients at different grades; Figure F shows the survival curves of RBMX high and low expression groups in oral cancer patients; and Figure G shows a forest plot of univariate regression analysis of RBMX and different clinical traits.

[0032] Figure 4 Figure A shows the GO and KEGG analysis results of RBMX in Example 3 of the present invention; where Figure A is the GO analysis diagram of RBMX-related genes in oral cancer; and Figure B is the KEGG analysis diagram of RBMX-related genes in oral cancer.

[0033] Figure 5 Figure A shows the GESA analysis results of RBMX in Example 3 of the present invention; where Figure A is the GSEA-GO analysis of RBMX-related genes in oral cancer; and Figure B is the GSEA-KEGG analysis of RBMX-related genes in oral cancer.

[0034] Figure 6 Figure 4 shows the experimental results of the effect of RBMX knockdown on the activity of oral cancer cells in Example 4 of the present invention; Figure A is the experimental histogram of cck-8 in SCC-4 cells with RBMX knockdown; Figure B is the experimental histogram of cck-8 in CAL-27 cells with RBMX knockdown; Figure C is the experimental image (left) and statistical graph (right) of the effect of RBMX knockdown on cell colony formation in SCC-4 and CAL-27 cells.

[0035] Figure 7 Figure 4 shows the experimental results of the effect of RBMX knockdown on the migration ability of oral cancer cells in Example 4 of the present invention; Figure A shows the scratch test image (left) and statistical graph (right) of RBMX knockdown on SCC-4 cells; Figure B shows the scratch test image (left) and statistical graph (right) of RBMX knockdown on CAL-27 cells.

[0036] Figure 8 Representative images (left) and statistical graphs (right) of the Transwell experiment on SCC-4 and CAL-27 cells by knocking down RBMX in Example 4 of this invention;

[0037] Figure 9 This describes the effect of RBMX knockdown on the sensitivity of SCC-4 and CAL-27 cells to cisplatin in Example 5 of this invention.

[0038] Figure 10 The following are the characterization results of the nanomedicine prepared in Example 6 of the present invention; wherein, Figure A is a scanning electron microscope image of G-LMDR nanoparticles; Figure B is a particle size analysis diagram of MPDA nanoparticles (blue) and G-LMDR nanomedicine (orange); Figure C is a zeta potential analysis diagram of MPDA nanoparticles and G-LMDR nanomedicine.

[0039] Figure 11 The results of the hemolytic activity test of the nanomedicine prepared in Example 6 of this invention;

[0040] Figure 12 The results of the drug distribution and tumor targeting experiments of G-LMDRs in Example 7 of the present invention are shown. Among them, Figure A is a schematic diagram of the process of in vivo treatment of oral cancer in nude mice with G-LMDRs; Figure B is a live fluorescence imaging image of oral cancer nude mice at different time points in vivo treatment with G-LMDR nanotherapy drugs.

[0041] Figure 13 The following are the tumor detection results of nude mice treated with G-LMDR in vivo for oral cancer in Example 8 of the present invention; wherein, Figure A is a visual diagram of the tumor size of nude mice treated with G-LMDR in vivo for oral cancer; Figure B is a statistical diagram of the tumor volume of nude mice treated with G-LMDR in vivo for oral cancer; and Figure C is a statistical diagram of the weight change of nude mice treated with G-LMDR in vivo for oral cancer.

[0042] Figure 14 This is a graph showing the results of immunofluorescence detection of the effect of G-LMDR on the expression level of NEK2 protein in oral cancer tissue in Example 9 of the present invention;

[0043] Figure 15 The figure shows the H&E detection results of the effects of G-LMDR on the major organs of nude mice treated with oral cancer in vivo in Example 10 of the present invention. Detailed Implementation

[0044] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0045] Unless otherwise specified in the examples, standard conditions or manufacturer-recommended conditions were followed. Reagents or instruments whose manufacturers are not specified are all commercially available products. The main sources of reagents are shown in Table 1.

[0046] Unless otherwise specified, in the result graphs of the embodiments of the present invention, * indicates a significance level of p<0.05, ** indicates a significance level of p<0.01, *** indicates a significance level of p<0.001, and n=3.

[0047] All animal experiments in this invention were conducted in accordance with the guidelines of the Animal Care and Use Committee of the Experimental Animal Institutions of Hunan Normal University and Central South University. Unless otherwise specified, the immunology, biochemistry, chemistry, molecular biology, cell biology, genomics, and recombinant DNA techniques used in this invention are standard skills in the field. The statistical methods used in this invention are as follows: independent samples t-tests were used to compare differences between two independent samples, and paired t-tests were used to compare differences between matched samples. The statistical significance level was set at p < 0.05, and statistical analysis and graph creation were performed using Graph Pad Prism (8.0.2).

[0048] Example 1

[0049] This embodiment investigated the relationship between the m6A methylation modifier RBMX and oral cancer.

[0050] m6A modification and dysregulation of m6A-related genes may influence the biological behavior of oral cancer cells. Investigating the role of m6A modification in oral squamous cell carcinoma could potentially provide a better foundation and basis for personalized treatment of oral cancer.

[0051] 1. m6A detection in oral cancer tissues and cells:

[0052] 1) Collection of clinical specimens: This study was approved by the Medical Ethics Committee of Xiangya Stomatological Hospital, Central South University, Hunan Province, with ethics approval number 2024060605. Informed consent was obtained from all patients before the surgery, and informed consent forms were signed.

[0053] Inclusion criteria: (1) Age 18-60 years; (2) Patients with newly diagnosed oral cancer confirmed by surgery and pathological tissue diagnosis; (3) No other tumors or systemic diseases; (4) Complete pathological and medical history data.

[0054] Exclusion criteria: (1) Age <18 years or >60 years; (2) Comorbid tumors or other systemic diseases; (3) Missing pathological or medical history data.

[0055] We collected oral squamous cell carcinoma patients who visited the Department of Stomatology, Xiangya Hospital, Central South University between June 2020 and December 2021. All samples obtained in this study were oral cancer tissue and adjacent normal oral mucosa tissue removed during surgery, with a size of approximately 1cm*1cm*0.5cm. After removal, the tissue was immediately placed at 4℃ and aliquoted into liquid nitrogen within 30 minutes for subsequent experiments.

[0056] 2) Use TRIzol reagent to extract total RNA from fresh clinical tissue specimens or cells.

[0057] 3) Quantitative detection of m6A was performed using an m6A methylation detection kit.

[0058] Test results as follows Figure 1 As shown in Figure A, the m6A level in the three pairs of OSCC tumors collected in this embodiment was significantly higher than that in adjacent normal tissues (P<0.05).

[0059] 2. Screening for oral cancer m6A-related genes:

[0060] Raw data collection and processing: Gene expression data (dataset) of oral cancer patients were screened and downloaded from the GEO database. Expression data of m6A-related genes were retrieved from normal oral tissues (n=71) and oral cancer tissues (n=130), and the expression levels of m6A-related genes in normal and oral cancer tissues were analyzed using the R package "Limma". Differential gene expression heatmaps were created using the "pheatmap" package.

[0061] The results of exploring m6A-related differentially expressed genes using standard datasets related to oral cancer in the GEO database are as follows: Figure 1 As shown in Figure B, several m6A-related genes were significantly altered in OSCC, including RBMX, RBM15, CBLL1, YTHDC2, and RBM15B.

[0062] Based on the expression level of the m6A regulatory factor and combined with the similarity analysis of the fuzzy clustering measure proportion, the best cluster stability was observed when the number of clusters k was set to 2. 320 OSCC patients were divided into subtypes 1 and 2, with subtype 1 containing 52 patients and subtype 2 containing 168 patients (e.g., ...). Figure 2 (As shown in Figure A); PCA analysis results are as follows: Figure 2 As shown in Figure B, these samples can be divided into two subtypes, A and B, based on the expression levels of m6A-related genes. Furthermore, there are significant differences in m6A scores between these two subtypes (e.g., ...). Figure 2 (As shown in Figure C). The dynamic relationship between random forest prediction error and the number of random trees is as follows: Figure 2As shown in Figure D, the error is basically stable when the number of random trees is 200. A schematic diagram of the random forest tree model constructed based on the oral cancer group and the control group is provided, identifying the point with the smallest error.

[0063] To identify key molecules regulating m6A modification levels in OSCC, importance scoring analysis was performed on differentially expressed genes including RBMX, IGF2BP3, RBM15B, LRPPRC, IGFBP3, YTHDC2, YTHDC1, RBM15, ELAVL1, IGFBP2, and CBLL1. The results are as follows: Figure 2 As shown in Figure E, RBMX is the key molecule among these genes that has the most significant impact on m6A methylation levels. Furthermore, after successfully knocking down RBMX expression in the OSCC cell lines SCC-4 and CAL-27 using siRNA technology, the m6A methylation levels in the cells also decreased significantly (e.g., ...). Figure 2 (As shown in Figure F in the figure). This result confirms that RBMX has a significant effect on the m6A modification level in OSCC.

[0064] Example 2

[0065] This embodiment investigates the impact of RBMX on the grading and prognosis of oral cancer.

[0066] First, the difference in RBMX gene expression levels between oral cancer tissues and normal oral mucosa tissues in the TCGA database was analyzed. The results are shown in Figure A in 3. The expression level of RBMX in OSCC tissues was significantly higher than that in normal mucosa tissues.

[0067] Focusing on paired OSCC tumor samples and their adjacent non-tumor tissues (adjacent tissues) within the TCGA database, a comparative analysis of RBMX expression levels was performed. The results are as follows: Figure 3 As shown in Figure B, this result indicates that even in more stringent paired sample contrast, RBMX expression in OSCC tissues remains significantly upregulated.

[0068] To verify the reliability of the bioinformatics analysis results, this embodiment further collected cancer tissue and paired adjacent normal tissue samples from 5 pairs of OSCC patients. Proteins were extracted from the tissues, and the expression of RBMX was verified using Western blot technology. The results are as follows: Figure 3 As shown in Figure C, the expression level of RBMX protein in the cancerous tissue of OSCC patients was significantly increased compared with its corresponding adjacent normal tissue, which is highly consistent with the results of the bioinformatics analysis in this experiment.

[0069] To further investigate the differences in RBMX gene expression and its potential significance among various clinical characteristics (including age, sex, tumor grade, and TNM stage) in patients with oral squamous cell carcinoma (OSCC), this study performed a correlation analysis on clinical data of OSCC patients from the TCGA database. The results showed that RBMX expression levels did not exhibit a significant correlation with clinical factors such as age, sex, clinical stage, primary tumor size, and lymph node involvement in OSCC patients. However, RBMX expression showed a significant correlation with tumor grade (e.g., ...). Figure 3 As shown in Figure D). Further subgroup analysis also revealed the specific expression trend of RBMX in tumor grading: as the tumor grade gradually increases, the expression level of RBMX also shows an increasing trend (as shown in Figure D). Figure 3 (As shown in Figure E). Although RBMX expression levels did not show a significant difference compared to other groups in grade 4 tumors, this is likely due to the smaller sample size at this grade, resulting in statistical error due to sample size limitations.

[0070] Furthermore, survival analysis of the OSCC patient dataset revealed that the overall survival rate of the RBMX high-expression group was significantly lower than that of the RBMX low-expression group, showing a poorer prognostic trend (P=0.08). Figure 3 (As shown in Figure F). To eliminate the interference of other potential clinical confounding factors and determine the impact of RBMX on patient prognosis, this embodiment further performed univariate regression analysis on RBMX and other relevant clinical characteristics, and the results are as follows. Figure 3 As shown in Figure G, RBMX is an independent risk factor affecting the prognosis of OSCC patients (P=0.029), which means that high expression of RBMX is closely related to poor prognosis in OSCC patients.

[0071] Example 3

[0072] This embodiment performs GO, KEGG, and GESA analyses on RBMX.

[0073] By deeply analyzing the dataset, a gene population closely related to RBMX expression was further screened, and then GO and KEGG enrichment analyses were performed on these genes. The results showed that these related genes are mainly involved in key biological processes such as meiosis, the meiotic cell cycle, and epithelial cell differentiation, which are crucial for cell proliferation and the transmission of genetic information. In terms of cellular localization, these genes are mainly enriched in structures such as synaptic membranes, transmembrane complexes, and condensed chromosomes. From a molecular functional perspective, the functions of these genes are mainly concentrated in transport activities and single-stranded DNA helicase activities (e.g., ...). Figure 4(As shown in Figure A). The RBMX gene is involved in signaling pathways including the cell cycle, DNA replication, cAMP signaling pathway, and drug metabolism (e.g., Figure 4 (As shown in Figure B).

[0074] After in-depth functional annotation of the RBMX gene population using GSEA enrichment analysis, the study confirmed a significant negative correlation between RBMX and keratinocyte differentiation, biological processes, cellular composition of immunoglobulin complexes, and the function of antigen-binding molecules (e.g., Figure 5 (As shown in Figure A). Furthermore, the results also revealed a positive correlation between RBMX and signaling pathways such as drug-metabolizing enzymes and retinol metabolism (e.g., ...). Figure 5 (As shown in Figure B).

[0075] These results suggest that upregulation of RBMX expression may inhibit the maturation and differentiation of keratinocytes through specific drug metabolism pathways.

[0076] Example 4

[0077] This embodiment explores the effect of RBMX knockdown on oral cancer cells. The RBMX siRNA sequences used in this embodiment are: Sense (5'-3'): UUCAUCAAGAGUACUUCCATT (SEQ ID NO.1), Antiense (5'-3'): UGGAAGUACUCUUGAUGAATT (SEQ ID NO.2).

[0078] 1. Effect of RBMX knockdown on OSCC cell viability:

[0079] OSCC cell lines SCC-4 and CAL-27 were successfully transfected using siRNA, which successfully reduced the expression level of RBMX in the cells. The effect on cell viability and proliferation was evaluated using the CCK-8 assay. Results are as follows: Figure 6 As shown in Figures A and B, the cell viability of SCC-4 and CAL-27 cells decreased significantly after RBMX expression was inhibited compared to the control group.

[0080] In addition, to more comprehensively evaluate the effect of RBMX expression on cell proliferation and colony formation ability, this embodiment further conducted a plate colony formation experiment, and the results are as follows: Figure 6 As shown in Figure C, after RBMX knockdown, the number of cell colonies formed by SCC-4 / CAL-27 cells was significantly reduced compared to the control group.

[0081] These results indicate that inhibiting RBMX expression in OSCC cells significantly suppresses the cell's clonogenic ability.

[0082] 2. Effect of RBMX knockdown on OSCC cell migration ability:

[0083] To investigate the changes in the migration function of OSCC cells after RBMX expression was suppressed, appropriate amounts of SCC-4 and CAL-27 cells were seeded in 6-well plates and cultured using standard methods. Then, these cells were transfected with siRNA to specifically reduce RBMX expression levels. After the cells reached confluence, a scratch assay was performed, and cell migration was recorded under a microscope.

[0084] Experimental results are as follows Figure 7 As shown, during the 24-hour observation period, compared with the si-NC control group, the si-RBMX group had lower levels of SCC-4 (e.g., ...). Figure 7 Figure A in the diagram) and CAL-27 (as shown in Figure A) Figure 7 (Figure B in the image) The intercellular spaces formed by the cells after the scratch are significantly increased.

[0085] The results indicate that after RBMX expression was effectively inhibited by siRNA, the migration speed of both types of OSCC cells was significantly reduced, showing a marked weakening of their migration ability.

[0086] 3. Effect of RBMX knockdown on the invasive ability of OSCC cells:

[0087] To assess the effect of reduced RBMX expression on cell invasion ability, SCC-4 and CAL-27 cells treated with si-NC and si-RBMX, respectively, were seeded in the upper chamber of a Transwell. After 48 hours of cell culture, the number of SCC-4 and CAL-27 cells that crossed the upper chamber and entered the lower chamber of the Transwell was counted using crystal violet staining. The results are as follows: Figure 8 As shown, compared with the si-NC control group, the number of transmembrane cells was significantly reduced in both SCC-4 and CAL-27 cells after si-RBMX knockdown.

[0088] This result indicates that RBMX knockdown can weaken the invasive ability of OSCC cells.

[0089] Example 5

[0090] This embodiment explores the effect of RBMX expression on chemotherapy resistance in oral cancer cells.

[0091] The CCK-8 assay was used to detect the cell survival rates of SCC-4 and CAL-27 cells after RBMX expression knockdown under different concentrations of cisplatin treatment. The results are as follows: Figure 9As shown, with increasing cisplatin concentration, the number of SCC-4 and CAL-27 cells was significantly reduced compared to the untreated control group, and cell viability exhibited a clear dose-dependent relationship with cisplatin concentration. Notably, after RBMX expression was knocked down, the survival rate of both SCC-4 and CAL-27 cell lines was significantly lower than that of the control group at the same cisplatin concentration. Further analysis showed that the half-maximal inhibitory concentration (IC50) of cisplatin in the si-RBMX group was much lower than that in the si-NC group. Specifically, for the SCC-4 cell line, the IC50 value after RBMX knockdown (1.192 μM) was significantly lower than that in the control group (3.264 μM); similarly, in the CAL-27 cell line, the IC50 value after RBMX knockdown (1.108 μM) was also much lower than that in the control group (2.247 μM).

[0092] These results indicate that reducing RBMX expression can significantly reduce OSCC cell resistance to cisplatin, thereby making them more sensitive to chemotherapeutic drugs.

[0093] Example 6

[0094] Based on the aforementioned experimental findings on the effects of RBMX on oral cancer cells, this embodiment designs and prepares gene therapy nanomedicines targeting RBMX. The specific preparation steps are as follows:

[0095] 1) Preparation of mesoporous polydopamine (MPDA): Mix 25 mL of water and 25 mL of ethanol; add 0.5 g of F127, 0.5 g of dopamine hydrochloride and 0.8 mL of TMB to the mixture; sonicate in a water bath for 2 min to form an emulsion; add 2 mL of ammonia to the emulsion, react at 60 °C for 3 h, centrifuge and wash, redisperse in deionized water to obtain stable MPDA particles; wash the MPDA particles and dry in a vacuum drying oven at 60 °C for 12 h to obtain purified MPDA powder;

[0096] 2) Preparation of drug-loaded (cisplatin + si-RBMX) mesoporous polydopamine (MDR): 29.5 mg of cisplatin (DDP) was dissolved in 100 μL of deionized water. After complete dissolution, 9.9 mL of anhydrous ethanol was added, and the mixture was gently stirred until homogeneous. 400 μL of a solution containing si-RBMX was added, along with 0.1 mg of the fluorescent dye Cy-5.5. 2 mg of pre-prepared MPDA dispersion was added to the above solution to ensure uniform distribution of MPDA particles. The mixture was stirred at room temperature in the dark for 24 h. Particles were collected by ultracentrifugation and washed multiple times with deionized water or PBS to remove unloaded free drug and unreacted substances. The supernatant was collected after centrifugation, and the content of unloaded si-RBMX and DDP in the supernatant was detected to calculate the encapsulation efficiency and drug loading. The potential and particle size were also measured.

[0097] 3) Preparation of RGD peptide-modified liposome-encapsulated drug-loaded mesoporous polydopamine (G-LMDR) (cisplatin + si-RBMX): 17.4 mg of lecithin and 1.7 mg of... DSPE-PEG was dissolved in chloroform, and the dissolved lipid material solution was evaporated at 40℃~50℃ using a rotary evaporator to form a uniform film. DSPE-PEG-RGD was mixed with the liposome film at a 1:1 mass ratio and stirred at 37℃ for 6 h. Unbound free RGD peptides, DSPE-PEG-RGD, and other small molecule impurities were removed by dialysis at 4℃. MDR dispersion was added to the RGD-modified lipid film, and the mixture was subjected to short-term hydration sonication (1~3 s) to help the lipid film hydrate and reform the liposome structure, while simultaneously encapsulating the drug-loaded MDR particles. 2 mg of DSPE-PEG-RGD was further added, and the mixture was stirred for 2 h to ensure uniform RGD modification. Potential and particle size were measured. When the particle size was stable and the potential was close to that of the liposome, the G-LMDR was successfully prepared.

[0098] Comparative Example 1

[0099] The specific steps for preparing liposome-encapsulated mesoporous polydopamine (LMDR) (cisplatin + si-RBMX) in this comparative example are as follows:

[0100] 17.4 mg of lecithin and 1.7 mg of DSPE-PEG were dissolved in chloroform, and the dissolved lipid material solution was evaporated to dryness using a rotary evaporator at 40°C~50°C to form a uniform film. The dispersion of MDR prepared in Example 6 was added to the evaporated lipid film, and the mixture was subjected to short-term hydration sonication (1~3 s) to help the lipid film hydrate and reform the liposome structure, while simultaneously encapsulating the drug-loaded MDR particles. Potential and particle size were measured. When the particle size was stable and the potential was close to that of the liposome, it indicated that the LMDR was successfully prepared.

[0101] The nanomedicine prepared in this invention was tested:

[0102] 1. Physical property characterization:

[0103] The morphology of G-LMDR nanomedicines was observed using SEM, such as... Figure 10 As shown in Figure A, it can be observed that the nanoparticles are uniformly distributed spherical particles with smooth surfaces and diameters ranging from 100 to 200 nm. A small number of nanoparticles form clusters, but most particles are uniformly distributed, and the shape and size of individual particles are consistent, with no obvious morphological defects observed.

[0104] Particle size analysis results are as follows Figure 10As shown in Figure B, the particle size distribution of MPDA (blue) is concentrated between 150 and 300 nm, while that of G-LMDR (orange) is concentrated between 200 and 400 nm. The particle size of MPDA nanoparticles is approximately 245.20 nm with a polydispersity index (PDI) of 0.026, while the particle size of G-LMDR nanomedicine is 264.28 nm with a PDI of 0.136. The particle size distribution of G-LMDR is significantly wider, with larger and more widely distributed particles, while the particle size of MPDA is more concentrated, exhibiting a narrower distribution range.

[0105] Zeta potential test, such as Figure 10 As shown in Figure C, the zeta potential of MPDA is -5mV, while that of G-LMDR is -10mV. It can be seen that G-LMDR has a larger surface negative charge, indicating that it may have stronger colloidal stability.

[0106] 2. To further verify the biosafety of the composite nanomedicine and the feasibility of intravenous injection, this invention tested the hemolysis rate of the synthesized MDR, LMDR, and G-LMDR nanoparticle materials. The test results are as follows: Figure 11 As shown:

[0107] Gross observation revealed that the water control group exhibited obvious red hemolysis, serving as a positive control. However, no visible red hemolysis was observed in any of the other nanomaterial groups, indicating low hemolysis rates.

[0108] The hemolysis rate results calculated from OD values ​​showed that the hemolysis rate increased for all samples with increasing concentration. However, the hemolysis rate remained low for all sample groups across the concentration gradient. Overall, these three nanomedicines demonstrated good safety for erythrocytes.

[0109] Among them, at concentrations of 20 μg / mL and 40 μg / mL, there was no significant difference in the hemolysis rate among the three groups of MDRs, LMDRs and G-LMDRs.

[0110] At a concentration of 80 μg / mL, the hemolysis rate of the MDRs group was significantly higher than that of the LMDRs and G-LMDRs groups (P<0.05), while the hemolysis rates of LMDRs and G-LMDRs were both lower and the differences were not significant (ns).

[0111] At a concentration of 160 μg / mL, the hemolysis rate of the MDRs group was significantly higher than that of the LMDRs and G-LMDRs groups (P<0.001), while the hemolysis rates of LMDRs and G-LMDRs remained low and the differences were not significant (ns).

[0112] In summary, the experimental results show that all three nanomedicines exhibited good hemocompatibility at gradient concentrations, but at high concentrations (160 μg / mL), especially for MDRs, the hemolysis rate increased significantly. In contrast, the hemolysis rate of G-LMDRs and LMDRs increased less. These results indicate that encapsulation with liposomes can effectively reduce the hemolysis of mesoporous polydopamine particles, improving their biosafety and biocompatibility.

[0113] Example 7

[0114] This embodiment explores the drug distribution and tumor targeting of G-LMDRs.

[0115] To further verify the targeting and therapeutic effects of composite nanomaterials on tumor cells, this embodiment constructed an oral cancer xenograft model in nude mice. The nude mice were first injected with CAL-27 tumor cells. After 14 days, once tumor cell survival and tumor formation were confirmed, the tumor-bearing mice were treated with different materials via tail vein administration every other day for 10 days. A schematic diagram of the treatment process is shown below. Figure 12 As shown in Figure A, tumor-bearing mice were divided into four groups based on the treatment they received: PBS (control), MDR, LMDR, and G-LMDR.

[0116] In vivo fluorescence imaging was used to track the distribution of two different nanomedicines (LMDR and G-LMDR) in nude mice at different time points (6h, 24h, and 48h) after the initial injection, observing the distribution in various major tissues. A color bar (EPI-FL) displays the relative values ​​of fluorescence intensity, with red representing the highest intensity and blue representing the lowest. Figure 12 As shown in Figure B:

[0117] In the LMDR group, the drug was mainly concentrated in the well-circulating region at 6 hours, with cross-targeting of the tumor area. After 24 hours, it diffused to a wider area in the body, and the overall fluorescence intensity decreased after 48 hours. In the G-LMDR group, the fluorescence was concentrated in the subcutaneous tissue near the tumor in the thigh of the mouse at 6 hours. After 24 hours, the fluorescence intensity in this area increased, and a significant fluorescence signal was still maintained at 48 hours, indicating that G-LMDR accumulated more persistently at the tumor site and had better targeting ability.

[0118] Therefore, G-LMDR nanomedicines showed better enrichment at tumor sites than LMDR, and the fluorescence intensity of G-LMDR at tumor sites was significantly higher than that of LMDR, suggesting that G-LMDR may have better targeted delivery capabilities.

[0119] Example 8

[0120] This embodiment investigates the oral cancer inhibitory effect of G-LMDRs.

[0121] To further verify the inhibitory effect of various composite nanomaterials on oral cancer cells, in this embodiment, tumor tissue was isolated after the completion of the tumor treatment course (24 days), and gross observation of the isolated tumor tissue was performed (e.g. Figure 13 (As shown in Figure A). Compared with the control group (PBS), the tumor volume in the G-LMDR group was significantly reduced, while the tumor volume in the MDR and LMDR groups was reduced, but the effect was significantly less than that in the G-LMDR group. The above experimental results indicate that G-LMDR has a significant inhibitory effect on tumors in vivo.

[0122] In this embodiment, the relative size of tumors was recorded simultaneously during drug treatment of nude mice. The tumor size on day 1 of drug treatment (day 14 after tumor transplantation) was used as the baseline value. Changes in tumor volume in each group were used to quantify the tumor-suppressing effect of different treatment groups (e.g., ...). Figure 13 (As shown in Figure B). The curve trend shows that the tumor volume in the PBS control group increased rapidly over time, while the tumor volume in all drug treatment groups decreased over time. Among them, the tumor volume reduction over time in the G-LMDR group was more significant than that in other groups (MDR, LMDR), and there was a significant difference compared with other groups on day 10 of the experiment (P<0.01), indicating that G-LMDR has a stronger anti-tumor effect.

[0123] In this embodiment, when performing nanomedicine treatment on nude mice, the weight changes of the nude mice were recorded simultaneously (e.g., ...). Figure 13 As shown in Figure C, during the experiment, there was no significant difference in body weight among the groups of nude mice, indicating that MDR, LMDR, and G-LMDR all have good safety profiles. They did not cause significant weight fluctuations or toxic reactions when inhibiting tumor growth.

[0124] Example 9

[0125] This embodiment investigates the effect of G-LMDRs on NEK2 expression in tumor tissue.

[0126] The tumor-causing gene NEK2 (NIMA-related kinase 2) has attracted much attention due to its aberrant expression and function in various cancers. Our research group's studies have shown a regulatory and regulated relationship between RBMX and NEK2. This invention develops a novel nanomedicine, G-LMDR, for oral cancer, which can precisely target oral cancer tissue and achieve highly effective anti-tumor therapy by loading siRBMX and cisplatin. To further explore the mechanism of action and efficacy of this novel nanomedicine G-LMDR in treating oral cancer, the expression level of NEK2 protein in nude mouse models of oral cancer xenografts was detected using immunofluorescence technology. The experimental results are as follows: Figure 14 As shown.

[0127] The experimental results show that NEK2 expression was high and fluorescence signal was strong in the tumor tissue of the PBS treatment group, indicating active tumor cell proliferation. NEK2 expression was significantly reduced in the tumor tissue of the G-LMDR treatment group, indicating that G-LMDR can effectively inhibit NEK2 expression in oral cancer tissue in in vivo anti-tumor experiments. This may be mainly due to the regulatory role of si-RBMX loaded on G-LMDR, which inhibits tumor growth by suppressing NEK2 levels in oral cancer tissue. These experimental results verify that RBMX can affect the development of oral cancer in vivo by regulating NEK2 expression.

[0128] Example 10

[0129] This embodiment further evaluates the safety of G-LMDR through H&E staining.

[0130] The histological structures of major organs (heart, liver, spleen, lungs, and kidneys) in nude mice with oral cancer xenografts were analyzed by H&E staining to assess the potential toxicity of G-LMDR to the organism.

[0131] Experimental results are as follows Figure 15 As shown, H&E sections of the heart, liver, spleen, lungs, and kidneys reveal the organ and tissue structures of the PBS and G-LMDR groups compared to the PBS treatment group. No significant pathological changes were observed in the major organs and tissues of the G-LMDR treatment group. The fibrous structure of the heart was clear, liver cells were regularly arranged, the follicular structure of the spleen was normal, the alveolar structure of the lungs was undamaged, and the glomeruli and tubules of the kidneys were intact. These results indicate that G-LMDR has no significant toxicity to the major organs at the experimental dose.

[0132] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A nanomedicine, characterized in that, The nanomedicine is drug-loaded mesoporous polydopamine nanoparticles encapsulated in RGD peptide-modified liposomes. The loading components of the mesoporous polydopamine nanoparticles include cisplatin and RBMX gene expression inhibitors.

2. The nanomedicine according to claim 1, characterized in that, The RBMX gene expression inhibitor includes siRNA of the RBMX gene.

3. The nanomedicine according to claim 2, characterized in that, The nucleotide sequence of the sense strand of the siRNA of the RBMX gene is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand of the siRNA is shown in SEQ ID NO.

2.

4. The nanomedicine according to any one of claims 1 to 3, characterized in that, The loading components of the mesoporous polydopamine nanoparticles also include fluorescent dyes.

5. The method for preparing nanomedicine according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1. F127, dopamine hydrochloride and tetramethylbenzidine were added to an ethanol solution, and the mixture was sonicated to form an emulsion. Ammonia was added to react the emulsion, and the particles generated by the reaction were separated, washed and dried to obtain mesoporous polydopamine nanoparticles. S2. The solution of cisplatin and RBMX gene expression inhibitor was added to anhydrous ethanol and mixed. Then, a dispersion of mesoporous polydopamine powder was added, stirred, and separated to obtain drug-loaded mesoporous polydopamine nanoparticles. S3. Add lecithin and DSPE-PEG to an organic solvent, evaporate to dryness to form a film to obtain liposomes, add DSPE-PEG-RGD to mix, dialyze to remove impurities, add drug-loaded mesoporous polydopamine nanoparticles, hydrate and sonicate, then add DSPE-PEG-RGD and stir to obtain the nanomedicine.

6. The preparation method according to claim 5, characterized in that, In step S1, the temperature for adding ammonia water is 50℃~70℃ and the time is 2~4h; the drying temperature is 50℃~70℃ and the time is 10~14h; in step S2, the mass ratio of cisplatin drug to mesoporous polydopamine powder is (10~15):

1.

7. The use of the nanomedicine as described in any one of claims 1 to 4 in the preparation of a pharmaceutical formulation for treating oral cancer.

8. The application according to claim 7, characterized in that, The oral cancer in question is cisplatin-resistant oral cancer.

9. The application of the nanomedicine as described in claim 4 in the preparation of imaging materials for oral cancer lesions.

10. A pharmaceutical composition, characterized in that, Including the nanomedicines described in any one of claims 1 to 4.

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