Preparation of cationic radical-based gene delivery system and its application in treatment of pancreatic cancer

Abstract

The application discloses a preparation of a gene delivery system based on cationic free radicals and application thereof in treatment of pancreatic cancer. A series of pyrrole compounds are synthesized, and structural formulae of the pyrrole compounds are shown in formula I or formula III; then, the pyrrole compounds are screened, and it is found that 2,5-dimethyl-1-phenyl-1H-pyrrole derivatives are rapidly converted into red-near infrared emitting pyrrole radical cations (P·+) in an acidic environment, which indicates that the pyrrole radical molecules have excellent bioimaging capability. The pyrrole radical cation P6·+ is further used as a mitochondrion-targeting functional gene carrier, is combined with KRAS siRNA through electrostatic adsorption, and is self-assembled into P6@siKRAS bifunctional nanoparticles, which can effectively induce simultaneous degradation of mutp53 and oncogenic KRAS proteins in pancreatic cancer cells, and provides a new treatment strategy for pancreatic cancer.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedicine, and specifically relates to the preparation of a gene delivery system based on cationic free radicals and its application in the treatment of pancreatic cancer. Background Technology

[0002] Pancreatic cancer is a highly malignant digestive system tumor. Early diagnosis is difficult, and once it develops, it progresses rapidly. The overall surgical resection rate is low, and the postoperative recurrence or metastasis rate is high, resulting in an extremely poor prognosis. The overall 5-year survival rate is only 4.1%, earning it the title of "King of Cancers." Traditional nucleoside analog chemotherapy drugs based on gemcitabine are the mainstay of pancreatic cancer treatment, but their effectiveness is limited.

[0003] In recent years, with the deepening research on tumor molecular (EGFR, IGF, VEGF, and other signal transduction pathway-related molecules) targeted drugs, although initial benefits have been seen in adjuvant therapy and treatment of advanced pancreatic cancer, the improvement in survival has only been 6 months. The reasons for this are twofold. First, pancreatic cancer is a genetically challenging and complex disease, with an average of 63 different gene mutations per tumor cell. Genetic heterogeneity and the uncertainty of target molecules make pancreatic cancer extremely difficult to treat, hindering the successful development of many new drugs. Currently, the effective application of immunotherapy in many solid tumors is changing the landscape of cancer treatment and bringing hope to pancreatic cancer patients. However, the unique tumor microenvironment leads to pancreatic cancer exhibiting lower antigen specificity, poor antigen exposure, lack of effector T cell infiltration, and suppression of immune cells by various cells and cytokines compared to other solid tumors, ultimately resulting in poor efficacy of immunotherapy for pancreatic cancer.

[0004] Besides its high heterogeneity and abundant stromal tissue, the complex gene mutations in pancreatic cancer pose a significant challenge to drug development. Studies show that frequently mutated genes in pancreatic cancer patients include KRAS (83.2%), TP53 (70.6%), CDKN2A (28.8%), SMAD4 (23.0%), ARID1A (12.8%), and CDKN2B (8.9%). The majority of patients carry mutations in the RTK / Ras / MAPK signaling pathway (90.7%). Previous research has demonstrated that KRAS oncogenic mutations play a crucial genetic driving role in the initiation, progression, and maintenance of tumorigenic growth in pancreatic cancer. Activating mutations in KRAS lead to the loss of GTPase activity, resulting in the sustained activation of downstream pro-proliferative and pro-survival pathways, including RAF / MEK / ERK and PI3K / AKT. Despite 40 years of research into targeted therapies against KRAS oncoproteins, the covalent inhibitor AMG510 against the KRASG12C mutation only recently received FDA approval in May 2021. Nevertheless, the presence of various codon mutations in the KRAS oncogene leads to multiple forms of mutant KRAS proteins, posing a significant challenge to effectively targeting other KRAS mutations. TP53 mutations are the second most common genetic alteration in PDAC. Studies have shown that approximately 70% of PDAC cases exhibit TP53 hotspot missense mutations, resulting in the loss of TP53's tumor-suppressive function. Furthermore, mice carrying p53 hotspot mutations exhibit greater aggression than heterozygous or unmutated p53 mice, suggesting that mutant p53 (mutp53) performs a gain-of-function function. Existing literature indicates a close link between mutant p53 and the mutant KRAS oncogenic signaling axis, jointly promoting pancreatic cancer growth, metastasis, and immune rejection. Therefore, combination therapies targeting multiple key driver oncogene factors may offer hope for pancreatic cancer treatment. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a pyrrole free radical molecule.

[0006] Another object of the present invention is to provide a method for preparing the pyrrole radical molecule.

[0007] Another object of the present invention is to provide the application of the pyrrole radical molecule.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A pyrrole radical molecule, the structural formula of which is shown in Formula I or Formula III:

[0010]

[0011] Where R1 is -H or -OCH3; R2 is -H or -OCH3; R3 is -H, -OCH3, -Br or -F.

[0012] The pyrrole radical molecule is preferably at least one of P-3OCH3, P-OCH3, P6-Br, P6-F, and P6, and its structural formula is shown in Formulas IV to VIII:

[0013]

[0014] The method for preparing the pyrrole radical molecule includes the following steps:

[0015] S1. Aniline compounds and 2,5-hexanedione were dissolved in tetrahydrofuran, iodine was added as a catalyst, the mixture was stirred at room temperature, and then an excess of thiosulfate solution was added and the mixture was stirred again. After the reaction was completed, the mixture was washed with saturated brine, the organic phase was dried with anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the residue was purified by column chromatography to obtain pyrrole radical molecules as shown in Formula I.

[0016] S2. Oxaloyl chloride was added dropwise to N,N-dimethylformamide under ice bath conditions, followed by the white solid produced by dichloromethane dissolution, then the pyrrole radical molecules obtained in step S1 and excess sodium hydroxide aqueous solution were added. The mixture was stirred at room temperature. After the reaction was completed, the mixture was washed with saturated brine, the organic phase was dried with anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the residue was purified by column chromatography to obtain the intermediate product shown in Formula II.

[0017] S3. Dissolve the intermediate product obtained in S2 in methanol, add sodium borohydride under ice bath conditions, stir the reaction, remove methanol by rotary evaporation after the reaction is complete, wash, collect the insoluble solid and freeze dry to obtain pyrrole radical molecules as shown in Formula III.

[0018] The aniline compound mentioned in step S1 is preferably at least one of 3,4,5-trimethoxyaniline, 4-methoxyaniline, 4-bromoaniline, and 4-fluoroaniline.

[0019] The molar ratio of aniline compounds, 2,5-hexanedione, iodine and thiosulfate in step S1 is 5:6 to 10:0.5:10 to 20; preferably 5:6:0.5:10.

[0020] The amount of tetrahydrofuran used in step S1 is calculated as 10-20 ml of tetrahydrofuran per millimole of aniline compound; preferably, it is calculated as 10 ml of tetrahydrofuran per millimole of aniline compound.

[0021] The room temperature stirring reaction time in step S1 is 36 to 48 hours; preferably 36 hours.

[0022] The concentration of the thiosulfate solution mentioned in step S1 is preferably 1 mmol / mL.

[0023] The stirring reaction time described in step S1 is 1 to 2 hours; preferably 1 hour.

[0024] The washing with saturated saline solution described in steps S1 and S2 is performed more than 3 times.

[0025] The column chromatography described in step S1 is 200-300 mesh silica gel column chromatography, and the eluent used is obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 8:1.

[0026] The molar ratio of oxalyl chloride, N,N-dimethylformamide, pyrrole radical molecules and sodium hydroxide in step S2 is 4:4:4:5 to 10.

[0027] The amount of dichloromethane used in step S2 is calculated as 5 ml of dichloromethane per millimole of oxalyl chloride.

[0028] The stirring reaction at room temperature described in step S2 takes 1 to 2 hours.

[0029] The column chromatography described in step S2 is 200-300 mesh silica gel column chromatography, and the eluent used is obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 4:1.

[0030] The intermediate product mentioned in step S3 is preferably 2,5-dimethyl-1-phenyl-1H-pyrrole-3-carboxaldehyde.

[0031] The molar ratio of the intermediate product to sodium borohydride in step S3 is 1:2 to 4; preferably 1:2.

[0032] The amount of methanol used in step S3 is calculated based on 4 to 15 ml of methanol per millimol of intermediate product.

[0033] The washing described in step S3 involves using ultrapure water to remove residual inorganic salts.

[0034] The stirring reaction at room temperature in step S3 is carried out for 2 to 3 hours; preferably 2 hours.

[0035] A cationic free radical molecule is obtained by adding the above-mentioned pyrrole free radical molecule to a hydrochloric acid solution, followed by ultrasonic treatment and freeze drying.

[0036] The hydrochloric acid solution is a hydrochloric acid solution with pH = 1 ± 0.1; preferably, it is a hydrochloric acid solution with pH = 1.

[0037] The amount of hydrochloric acid solution used is calculated based on 0.08 mL of hydrochloric acid solution per milligram of pyrrole free radical molecule.

[0038] The power of the ultrasonic treatment is 40 kHz.

[0039] The duration of the ultrasonic treatment is 1 to 1.5 hours; preferably 1 hour.

[0040] A nanoparticle containing pyrrole radical molecules was obtained by adding the above-mentioned pyrrole radical molecules and cucurbita[7]urea to water and then subjecting it to water bath ultrasound.

[0041] The mass ratio of the pyrrole radical molecule to cucurbit[7]urea is 20.1:38.7.

[0042] The water is preferably DEPC water.

[0043] The ultrasonic treatment time is 0.5 to 1 hour; preferably 0.5 hours.

[0044] A self-assembled bifunctional nanoparticle was obtained by adding KRAS siRNA and the aforementioned nanoparticles containing pyrrole radical molecules to water, incubating at room temperature, and then collecting by centrifugation; wherein the nucleotide sequence of the KRAS siRNA is as follows:

[0045] siKRAS justice chain sequence: 5'-GAUCCGACAAUACAGAUUGTT-3' (SEQ ID NO.1);

[0046] siKRAS antisense sequence: 5'-CAAUCUGUAUUGUCGGAUCTT-3' (SEQ ID NO.2).

[0047] The molar ratio of the concentration of pyrrole radical molecules in the nanoparticles containing pyrrole radical molecules to the base concentration in KRASsiRNA is 2 to 10:1; preferably 10:1.

[0048] The room temperature incubation time is 1 to 1.5 hours; preferably 1 hour.

[0049] The centrifugation conditions are: 15,000 rpm for 100 minutes.

[0050] The use of at least one of the pyrrole radical molecules, cationic radical molecules, nanoparticles containing pyrrole radical molecules, and self-assembled bifunctional nanoparticles in the preparation of bioimaging agents (fluorescence imaging, cell imaging), gene delivery vectors, and / or drugs for treating pancreatic cancer.

[0051] The pancreatic cancer mentioned refers to gene-mutant pancreatic cancer, including p53-mutant pancreatic cancer, KRAS-mutant pancreatic cancer, and p53 and KRAS double-mutant pancreatic cancer.

[0052] The pancreatic cancer mentioned includes pancreatic ductal adenocarcinoma.

[0053] The use of at least one of the pyrrole radical molecules, cationic radical molecules, nanoparticles containing pyrrole radical molecules, and self-assembled bifunctional nanoparticles in the preparation of products that degrade mutant p53 protein, degrade KRAS protein, and / or downregulate the expression level of KRAS gene.

[0054] The aforementioned degradation mutant p53 protein is degraded via the proteasome pathway after p53 ubiquitination.

[0055] The mutant p53 protein is the p53 protein mutant R248W.

[0056] The application of at least one of the pyrrole radical molecules, cationic radical molecules, nanoparticles containing pyrrole radical molecules, and self-assembled bifunctional nanoparticles in the preparation of chemotherapeutic drug sensitizers.

[0057] The preferred chemotherapy drug is gemcitabine.

[0058] The present invention has the following advantages and effects compared with the prior art:

[0059] 1. This invention synthesizes a series of pyrrole compounds and, through screening these compounds, reveals that 2,5-dimethyl-1-phenyl-1H-pyrrole derivatives rapidly transform into red-near-infrared pyrrole radical cations (P·+) under acidic conditions, indicating that pyrrole radical molecules possess excellent bioimaging capabilities. This invention also reveals that P·+ effectively triggers the degradation of mutant p53 in a K48 ubiquitinated proteasome-dependent manner.

[0060] 2. This invention reports for the first time a functionalized gene vector that targets and degrades mitochondria mutp53. Specifically, P6·+, a pyrrole compound with the best mutp53 degradation ability, was selected as the mitochondrial-targeting functionalized gene vector. P6·+ was then bound to KRAS siRNA via electrostatic adsorption to form P6@siKRAS bifunctional nanoparticles. The results showed that P6@siKRAS nanoparticles can effectively induce the simultaneous degradation of mutp53 and oncogenic KRAS proteins in pancreatic cancer cells. Therefore, this therapeutic strategy eliminates the gain-of-function effect of mutp53 and inhibits downstream signaling pathways regulated by RAS, providing a new approach for the development of combined therapy for pancreatic ductal adenocarcinoma.

[0061] 3. The P6@siKRAS nanoparticles in this invention have shown significant therapeutic effects in both the Mia PaCa-2 cell line and the KPC p53-KRAS double-mutant pancreatic tumor model. Furthermore, P6@siKRAS can eliminate the mutant p53-KRAS, thereby inhibiting tumor growth, invasion, metastasis, and drug resistance. At the same time, it can reshape the tumor microenvironment by promoting the infiltration of anti-tumor immune cells, thus providing a new therapeutic strategy for p53-KRAS double-mutant pancreatic cancer.

[0062] 4. The P6@siKRAS nanoparticles of this invention, when treated in combination with the chemotherapy drug gemcitabine, exhibit a better killing effect on cancer cells, indicating that P6@siKRAS nanoparticles can be used as a sensitizer for chemotherapy drugs. Attached Figure Description

[0063] Figure 1 This is the hydrogen NMR spectrum of P-3OCH3.

[0064] Figure 2 This is the hydrogen NMR spectrum of P-OCH3.

[0065] Figure 3 This is the hydrogen NMR spectrum of P6-Br.

[0066] Figure 4 This is the hydrogen NMR spectrum of P6-F.

[0067] Figure 5 This is the hydrogen NMR spectrum of P6.

[0068] Figure 6 This is the carbon NMR spectrum of P6.

[0069] Figure 7 This is a high-resolution mass spectrum of P6.

[0070] Figure 8 These are the electronic paramagnetic resonance spectra of pyrrole radical molecules and cationic radical molecules; where a represents P-3OCH3 and P-3OCH3·+; b represents P-OCH3 and P-OCH3·+; c represents P6-Br and P6-Br·+; d represents P6-F and P6-F·+; and e represents P6 and P6·+.

[0071] Figure 9 These are cellular fluorescence images of pyrrole free radical molecules and cationic free radical molecules.

[0072] Figure 10 This is a Western blot verification result of the degradation mutant p53 induced by different cationic free radicals in PA-TU-8988S cells.

[0073] Figure 11The figures are Western blot verification results of the degradation mutant p53 induced by P6·+ nanoparticles in different pancreatic cancer cells; where a is the dose-response plot and b is the time-response plot.

[0074] Figure 12 This is an immunofluorescence image of P6·+ nanoparticles degrading mutant p53 in PA-TU-8988S cells and MIA PaCa-2 cells.

[0075] Figure 13 This is a Western blot result of p53 protein induced by P6·+ nanoparticles in wild-type p53.

[0076] Figure 14 The graph shows the statistical results of real-time PCR of the TP53 gene from P6·+ nanoparticles and the Western blot results of p53 protein in PA-TU-8988S cells after CHX treatment; where a is the result of real-time PCR and b is the result of Western blot.

[0077] Figure 15 The images show the results of treating PA-TU-8988S cells with P6·+ nanoparticles, including the ubiquitin-proteasome pathway. Image a shows the Western blot results of p53 protein after treatment with CQ and MG-132; image b shows the Western blot results of p53 protein ubiquitination after immunoprecipitation following P6·+ nanoparticle treatment; and image c shows the Western blot results of P6·+ nanoparticles treated with PYR-41 and PR619.

[0078] Figure 16 This is a Western blot diagram of p53 degradation in PA-TU-8988S cells after treatment with the endocytosis inhibitors Genistein and Cyto D using P6·+ nanoparticles.

[0079] Figure 17 Fluorescence verification images of mitochondrial localization, increased reactive oxygen species (ROS), and mitochondrial destruction of P6·+ nanoparticles in PA-TU-8988S cells, and Western blot analysis of p53 degradation in cells after co-treatment with P6·+ nanoparticles and ROS inhibitors; where a represents mitochondrial localization results; b represents increased ROS levels; c represents mitochondrial destruction; and d represents p53 degradation in cells as detected by Western blot.

[0080] Figure 18 This is a DNA gel electrophoresis image from the supernatant.

[0081] Figure 19Characterization diagrams of P6·+ nanoparticles and P6@siKRAS nanoparticles are shown below; where a is a transmission electron microscope image of the two nanoparticles; b is a particle size diagram of the two nanoparticles; c is a surface potential diagram of the two nanoparticles; and d is a diagram showing the 24-hour stability test results of the two nanoparticles.

[0082] Figure 20 These are the UV absorption and fluorescence spectra of P6·+ nanoparticles and P6@siKRAS nanoparticles; where a is the UV absorption spectrum of the nanoparticles and b is the fluorescence spectrum of the nanoparticles.

[0083] Figure 21 These are the EPR spectra of P6·+ nanoparticles and P6@siKRAS nanoparticles.

[0084] Figure 22 These are cellular fluorescence images of P6·+ nanoparticles and P6@siKRAS nanoparticles.

[0085] Figure 23 This is a fluorescence verification image of cellular uptake and lysosomal colocalization of P6@siKRAS nanoparticles; where a represents cellular uptake and b represents lysosomal colocalization fluorescence results.

[0086] Figure 24 The figure shows the RT-PCR results verifying the effect of P6@siKRAS nanoparticles on knocking down the KRAS gene; where a represents the downregulation of KRAS gene expression level by P6@siKRAS nanoparticles; b represents the downregulation of KRAS gene expression level by P6@siKRAS nanoparticles in PA-TU-8988S cells; and c represents the downregulation of KRAS gene expression level by P6@siKRAS nanoparticles in MIA PaCa-2 cells.

[0087] Figure 25 This is a Western blot image showing the expression levels of key proteins in the downstream signaling pathway after the KRAS gene was knocked down by P6@siKRAS nanoparticles; where a represents MIAPaCa-2 cells and b represents PA-TU-8988S cells.

[0088] Figure 26 The image shows the cell viability results of each cell type after treatment with P6@siKRAS nanoparticles for double mutant cell lines and normal cells, as determined by the MTT assay. In the image, a represents the double mutant cell line, and b represents normal cells.

[0089] Figure 27The images show the effects of P6@siKRAS nanoparticles on double mutant cells; where a is a photograph of the effect on cell migration; b is a photograph of cell clone formation; c is a diagram of the killing effect after treatment with gemcitabine chemotherapy in PA-TU-8988S cells; and d is a real-time PCR image of genes related to the downstream signaling pathway of the mutant p53.

[0090] Figure 28 This is an in vivo distribution map of P6@siKRAS nanoparticles in the MIAPaCa-2 tumor-bearing model; where a is an in vivo imaging map of small animals at different time points after tail vein administration; b is an imaging map of the isolated organs after sacrificing mice 24 hours after tail vein administration; and c is a statistical map of the average fluorescence of the isolated organs.

[0091] Figure 29 The images show the therapeutic effects of P6@siKRAS nanoparticles on MIAPaCa-2 tumor-bearing mice; where a is the mouse weight statistics during treatment; b is the tumor volume change during treatment; c is the image of the ex vivo tumor tissue after treatment; and d is the weight statistics of the ex vivo tumor tissue after treatment.

[0092] Figure 30 The images show the therapeutic effects of P6@siKRAS nanoparticles on a mouse model of spontaneous pancreatic cancer (KPC). Image a shows the gene sequencing of the spontaneous KPC tumor tissue; image b shows the tumor volume changes during treatment in the KPC model; image c shows an image of the ex vivo tumor tissue after treatment in the KPC model; and image d shows the weight statistics of the ex vivo tumor tissue after treatment in the KPC model.

[0093] Figure 31 This is a statistical graph analyzing the tumor immune microenvironment after P6@siKRAS nanoparticle treatment for spontaneous pancreatic cancer (KPC). In the graph, a represents the serum IFN-γ level after treatment; b represents the proportion of CD8+ T cells in the tumor microenvironment after treatment; c represents the proportion of regulatory T cells (Tregs) in the tumor microenvironment after treatment; and d represents the proportion of anti-inflammatory M2 macrophages in the tumor microenvironment after treatment.

[0094] Figure 32 This is a TUNEL staining result of tumor tissue in MIAPaCa-2 tumor-bearing mice after treatment with P6@siKRAS nanoparticles.

[0095] Figure 33 This is a graph showing the serum test results after P6@siKRAS nanoparticle treatment of MIAPaCa-2 tumor-bearing mice; where a is the concentration of ALT (alanine aminotransferase); b is the concentration of AST (aspartate aminotransferase); c is the concentration of CREA (serum creatinine); and d is the concentration of UREA (blood urea nitrogen).

[0096] Figure 34 This is a diagram showing the H&E staining results of mouse organs after treatment with P6@siKRAS nanoparticles for MIAPaCa-2 tumor-bearing mice.

[0097] Figure 35 This is a Western blot image of p53 protein in tumor tissue after treatment with P6@siKRAS nanoparticles in MIAPaCa-2 tumor-bearing mice. Detailed Implementation

[0098] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0099] Example 1: Synthesis of Pyrrole Radical Molecules

[0100] 1. Synthesis of pyrrole free radical molecules

[0101] 1.1 Synthesis Path

[0102] ① Aniline compounds and 2,5-hexanedione were dissolved in tetrahydrofuran, a small amount of iodine was added as a catalyst, and the mixture was stirred at room temperature for 36 hours. Then, an excess of thiosulfate solution was added and the mixture was stirred for another hour. The mixture was then washed three times with saturated brine, the organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the residue was purified by column chromatography to obtain Formula I.

[0103] ② Oxaloyl chloride was added dropwise to N,N-dimethylformamide under ice bath conditions, followed by the addition of the white solid produced by dichloromethane dissolution. Formula I molecules were added all at once, and the mixture was stirred at room temperature for 1 hour. Then, excess sodium hydroxide aqueous solution was added, and stirring was continued for 1 hour. The mixture was then washed three times with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was purified by column chromatography to obtain Formula II.

[0104] ③ Dissolve the molecule of formula II in methanol, add sodium borohydride under ice bath conditions and stir for 2 hours, remove methanol by rotary evaporation, wash away residual inorganic salts with ultrapure water, collect the insoluble solid, freeze dry to obtain formula III.

[0105]

[0106] Among them, R1 is selected from -H or -OCH3; R2 is selected from -H or -OCH3; R3 is selected from -H, -OCH3, -Br or -F.

[0107] 1.2 Synthesis of pyrrole free radical molecules

[0108] (1) Synthesis of P-3OCH3 molecule

[0109] 5 mmol of 3,4,5-trimethoxyaniline and 6 mmol of 2,5-hexanedione were dissolved in 50 mL of tetrahydrofuran. 0.5 mmol of iodine was added, and the mixture was stirred at room temperature for 36 hours. Then, 10 mmol of sodium thiosulfate was dissolved in 10 mL of water and added to the reaction mixture. The mixture was stirred for another hour. The mixture was then washed three times with 50 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was purified by column chromatography on 200–300 mesh silica gel (petroleum ether: ethyl acetate = 8:1, v / v) to obtain the P-3OCH3 molecule (Formula IV).

[0110] Nuclear magnetic resonance hydrogen spectrum such as Figure 1 As shown, the NMR data are as follows: 1 H NMR of P-3OCH3(400MHz,DMSO-d6,δ):6.54(s,2H),5.77(s,2H),3.79(s,6H),3.72(s,3H),2.01(s,6H).

[0111]

[0112] (2) Synthesis of P-OCH3 molecules

[0113] 5 mmol of 4-methoxyaniline and 6 mmol of 2,5-hexanedione were dissolved in 50 mL of tetrahydrofuran. 0.5 mmol of iodine was added, and the mixture was stirred at room temperature for 36 hours. Then, 10 mmol of thiosodium thiosulfate was dissolved in 10 mL of water and added to the reaction mixture. The mixture was stirred for another hour. The mixture was then washed three times with 50 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was purified by column chromatography on a 200–300 mesh silica gel column (petroleum ether: ethyl acetate = 8:1, v / v) to obtain the P-OCH3 molecule (formula V).

[0114] Nuclear magnetic resonance hydrogen spectrum such as Figure 2 As shown, the NMR data are as follows: 1 H NMR of P-OCH3(400MHz,DMSO-d6,δ):7.17(d,2H),7.05(d,2H),5.76(s,2H),3.82(s,3H),1.94(s,6H).

[0115]

[0116] (3) Synthesis of P6-Br molecules

[0117] ① Dissolve 5 mmol of 4-bromoaniline and 6 mmol of 2,5-hexanedione in 50 mL of tetrahydrofuran, add 0.5 mmol of iodine, stir at room temperature for 36 hours, then dissolve 10 mmol of thiosodium in 10 mL of water and add it to the reaction solution, continue stirring for 1 hour, then wash three times with 50 mL of saturated brine, dry the organic phase with anhydrous sodium sulfate, remove the solvent by rotary evaporation, and purify the residue by column chromatography on 200-300 mesh silica gel (petroleum ether: ethyl acetate = 8:1, v / v) to obtain intermediate I.

[0118] ② Add 10 mmol of oxalyl chloride dropwise to 10 mmol of N,N-dimethylformamide under ice bath conditions, add 50 mL of dichloromethane to dissolve the white solid, add 2.5 mmol of intermediate I to the reaction solution at once, stir at room temperature for 1 hour, dissolve 12.5 mmol of sodium hydroxide in 10 mL of water and add it to the reaction solution, continue stirring for 1 hour, wash the organic phase three times with 50 mL of saturated brine, dry with anhydrous sodium sulfate, remove the solvent by rotary evaporation, and purify the residue by 200-300 mesh silica gel column chromatography (petroleum ether: ethyl acetate = 4:1, v / v) to obtain intermediate II.

[0119] ③ Dissolve 1.5 mmol of intermediate II in 20 mL of methanol, add 3 mmol of sodium borohydride under ice bath conditions, stir in an ice bath for 2 hours, remove methanol by rotary evaporation, wash away residual inorganic salts with ultrapure water, collect the insoluble solid, and freeze-dry to obtain P6-Br (Formula VI).

[0120] Nuclear magnetic resonance hydrogen spectrum such as Figure 3 As shown, the NMR data are as follows: 1 H NMR of P6-Br(400MHz,DMSO-d6,δ):7.70(d,2H),7.21(d,2H),5.86(s,1H),4.47(t,1H),4.26(d,2H),1.94(d,6H).

[0121]

[0122] (4) Synthesis of P6-F molecules

[0123] ① Dissolve 5 mmol of 4-fluoroaniline and 6 mmol of 2,5-hexanedione in 50 mL of tetrahydrofuran, add 0.5 mmol of iodine, stir at room temperature for 36 hours, then dissolve 10 mmol of thiosodium in 10 mL of water and add it to the reaction solution, continue stirring for 1 hour, then wash three times with 50 mL of saturated saline, dry the organic phase with anhydrous sodium sulfate, remove the solvent by rotary evaporation, and purify the residue by column chromatography on 200-300 mesh silica gel (petroleum ether: ethyl acetate = 8:1, v / v) to obtain intermediate I.

[0124] ② Add 10 mmol of oxalyl chloride dropwise to 10 mmol of N,N-dimethylformamide under ice bath conditions, add 50 mL of dichloromethane to dissolve the white solid, add 2.5 mmol of intermediate I to the reaction solution at once, stir at room temperature for 1 hour, dissolve 12.5 mmol of sodium hydroxide in 10 mL of water and add it to the reaction solution, continue stirring for 1 hour, wash the organic phase three times with 50 mL of saturated brine, dry with anhydrous sodium sulfate, remove the solvent by rotary evaporation, and purify the residue by 200-300 mesh silica gel column chromatography (petroleum ether: ethyl acetate = 4:1, v / v) to obtain intermediate II.

[0125] ③ Dissolve 1.5 mmol of intermediate II in 20 mL of methanol, add 3 mmol of sodium borohydride under ice bath conditions, stir in an ice bath for 2 hours, remove methanol by rotary evaporation, wash away residual inorganic salts with ultrapure water, collect the insoluble solid, and freeze-dry to obtain P6-F (Formula VII).

[0126] Nuclear magnetic resonance hydrogen spectrum such as Figure 4 As shown, the NMR data are as follows: 1 H NMR of P6-F(400MHz,DMSO-d6,δ):7.39–7.26(m,4H),5.85(s,1H),4.46(t,1H),4.26(d,2H),1.92(d,6H).

[0127]

[0128] (5) Synthesis of P6

[0129] 10 mmol of 2,5-dimethyl-1-phenyl-1H-pyrrole-3-carboxaldehyde was dissolved in 40 mL of methanol. 20 mmol of sodium borohydride was added under ice bath conditions and the mixture was stirred for 2 hours. Methanol was removed by rotary evaporation, and residual inorganic salts were washed away with ultrapure water. The insoluble solid was collected and lyophilized to obtain P6 (formula VIII).

[0130] Nuclear magnetic resonance spectrum as shown Figure 5 and Figure 6 As shown, the NMR data are as follows: 1 H NMR of P6(400MHz,DMSO-d6,δ):7.55–7.48(m,2H),7.44(t,1H),7.23(d,2H),5.86(s,1H),4.45(s,1H),4.28(s,2H),1.93(d,6H). 13C NMR of P6(101MHz,DMSO-d6,δ):138.84,129.73,128.52,128.10,126.83,125.11,120.11,107.74,56.49,13.14,10.97.

[0131] Mass spectrum as shown Figure 7 As shown, the mass spectrometry data are as follows: HRMS(ESI) m / z: [M+Na]+calcd forC13H15NONa: 224.1052; found: 224.1046.

[0132]

[0133] 2. Preparation and Electron Paramagnetic Resonance (EPR) Spectroscopy of Cation Radicals

[0134] Take 25 mg of the pyrrole free radical molecules (P-3OCH3, P-OCH3, P6-Br, P6-F, P6) prepared above, add 2 mL of hydrochloric acid solution with pH=1, sonicate (40 kHz) for 1 hour, freeze dry to obtain cationic free radical (P-3OCH3·+, P-OCH3·+, P6-Br·+, P6-F·+, P6·+) powder, and set aside for later use.

[0135] 20 mg of cationic free radical powder and pyrrole free radical molecules were respectively added to an NMR tube, and their EPR spectra were measured. The results are as follows: Figure 8 As shown, after acid induction, all molecules exhibited EPR signals, indicating that the cationic radicals were successfully prepared.

[0136] 3. To explore the cell imaging capabilities of cationic free radical molecules.

[0137] Human pancreatic cancer PA-TU 8988S cells (purchased from ATCC) were seeded in 24-well cell culture plates at a density of approximately 3 × 10⁻⁶ cells / well. 4 ~5×10 4 Cells / wells were cultured overnight and ready for use. Before adding samples, the cells were replaced with fresh DMEM medium, and cationic and pyrrole radical molecules were added to a final concentration of 50 μM. After culturing the cells for 12 hours, fluorescence microscopy was used for detection. Results are as follows: Figure 9 As shown, red fluorescence can only be observed with pyrrole radicals generated by acid induction, while red fluorescence cannot be observed with pyrrole radical molecules, indicating that pyrrole radical molecules have excellent bioimaging capabilities.

[0138] Example 2: Testing the ability of different pyrrole radical cations to degrade mutant p53 cells.

[0139] PA-TU 8988S cells (R248W, p53 gene Arg mutated to Trp at position 248; purchased from ATCC) expressing mutant p53 protein were seeded in 24-well cell culture plates at a density of approximately 3 × 10⁻⁶ cells / well. 4 ~5×10 4 Cells / wells were cultured overnight and ready for use. Before adding samples, the cells were transferred to fresh DMEM high-glucose medium (purchased from Hyclone), and then 50 μM of pyrrole compounds and their free radical cations (various pyrrole free radical molecules prepared in Example 1, and various cationic free radical molecules induced by acid) were added. The negative control was an equal volume of PBS buffer. After 24 h of cell culture, Western blot analysis was performed. The experiment was set up in triplicate. The results are as follows: Figure 10 As shown, pyrrole radicals, after being induced by acid to become cationic radicals, can significantly induce the degradation of mutant p53 protein. Among them, the P6·+ cationic radical showed the best degradation effect and can be used for subsequent studies on degradation effects. Subsequently, we also conducted experiments in human pancreatic cancer MIA PaCa-2 cells (containing the R248Wp53 mutation site) (purchased from ATCC) using the same method, and found that P6·+ NPs showed a good degradation effect.

[0140] Simultaneously, following the same method described above, P6·+ nanoparticles (prepared in Example 6, P6·+NPs) at final concentrations of 0, 5, 10, 15, 20, and 25 μg / mL were added to PA-TU 8988S and MIA PaCa-2 cell lines, respectively, and cultured for 24 h. It was found that the degradation of mutant p53 protein by P6·+ nanoparticles exhibited a dose-dependent effect. Figure 11 a) Following the same method described above, P6·+ cation free radicals at a final concentration of 25 μg / mL were added to PA-TU 8988S and MIAPaCa-2 cell lines and cultured for 0, 2, 4, 8, 12, and 24 h. It was found that significant degradation occurred at 8 h after treatment with 25 μg / mL P6·+ nanoparticles, with the most significant degradation at 24 h. This indicates that the degradation of mutant p53 protein by P6·+ nanoparticles exhibits a time-dependent effect. Figure 11 b).

[0141] Similarly, using immunofluorescence, we further revealed that P6·+ nanoparticles significantly reduced mutant p53 protein in PA-TU 8988S and MIA PaCa-2 cell lines. Figure 12As shown. Conversely, following the same method described above, P6·+ cation radicals were added to A549 cells (ATCC) at final concentrations of 0, 5, 10, 15, 20, and 25 μg / mL and cultured for 24 h. It was found that P6·+ nanoparticles did not induce degradation of wild-type p53 protein (A549 cells). Figure 13 In summary, P6·+ nanoparticles can selectively degrade mutant p53 proteins.

[0142] Example 3: Investigating the level of p53 regulated by P6·+ nanoparticles

[0143] PA-TU 8988S cells and MIA PaCa-2 cells (both purchased from ATCC) were cultured in small dishes. When the cell density reached approximately 70%, PA-TU 8988S and MIA PaCa-2 cells were treated with P6·+ nanoparticles (prepared in Example 6, P6·+NPs) at a final concentration of 25 μg / mL for 12 h, respectively. An equal volume of PBS buffer (CON) was used as a control. RNA was extracted from the cells, and subsequent quantitative real-time PCR experiments were performed to detect p53 gene expression. The experiment was repeated three times. The results are as follows: Figure 14 As shown in figure a, there was no difference in the mRNA expression level of p53 between the P6·+ nanoparticles and the CON group, indicating that the treatment of mutant p53 cells with P6·+ nanoparticles did not regulate the transcription level of the p53 gene.

[0144] Next, the PA-TU 8988S cells were cultured using the same method as described above. When the cell density reached approximately 70%, the PA-TU 8988S cells were co-treated with the protein synthesis inhibitor cycloheximide (final concentration 20 μM; CHX) and P6·+ nanoparticles (final concentration 25 μg / mL), with cycloheximide (final concentration 20 μM) serving as a control. The experiment was performed in triplicate. The results are as follows: Figure 14 As shown in b, the level of co-treated p53 protein decreased more and more significantly with increasing time, indicating that the P6·+ nanoparticles degrade post-translational mutant p53 protein, without involving genomic-level regulation.

[0145] Example 4: Investigating the pathway of P6·+ nanoparticle degradation of mutant p53 protein and its effect on the ubiquitination level of mutant p53.

[0146] PA-TU 8988S cells were seeded in 24-well cell culture plates at a density of approximately 3 × 10⁶ cells / well. 4 ~5×10 4Cells / wells were cultured overnight and ready for use. Before adding samples, the cells were replaced with fresh DMEM medium (purchased from Hyclone), and then P6·+ nanoparticles (prepared in Example 6) and chloroquine CQ (autophagy inhibitor, purchased from MCE) were added to a final concentration of 25 μg / mL, MG-132 (proteasome inhibitor, purchased from MCE) was added to a final concentration of 20 μM, and P6·+ nanoparticles were added to a final concentration of 25 μg / mL. A negative control was prepared by adding an equal volume of PBS buffer. After culturing the cells for 24 hours, Western blot analysis was performed. The experiment was set up in triplicate. The results are as follows: Figure 15 As shown in a, the degradation of mutant p53 protein induced by P6·+ nanoparticles can be effectively inhibited by the proteasome inhibitor MG-132, while the autophagy inhibitor CQ has no such inhibitory effect, indicating that this degradation effect of mutant p53 protein is mainly through the proteasome pathway.

[0147] Meanwhile, PA-TU 8988S and MIAPaCa-2 cells were cultured using the above method, and then P6·+ nanoparticles were added to a final concentration of 25 μg / mL and cultured for 24 hours. An equal volume of PBS buffer was added as a control. The p53 protein in the cells was then precipitated using immunoprecipitation, and finally, Western blot analysis was performed. The experiment was conducted in triplicate. Results are as follows: Figure 15 As shown in b, in PA-TU 8988S and MIA PaCa-2 cells, P6·+ nanoparticles can significantly increase the ubiquitination level of p53 protein, indicating that the degradation effect of mutant p53 protein is mainly achieved by ubiquitinizing p53 and then degrading it through the proteasome pathway.

[0148] To further verify the role of the ubiquitin-proteasome pathway in the degradation of mutant p53 induced by P6·+ nanoparticles, we added the following to PA-TU 8988S cells: P6·+ nanoparticles at a final concentration of 25 μg / mL; PYR-41 (E1 ubiquitin activator, purchased from MCE) at a final concentration of 50 μM; PYR-41 at a final concentration of 25 μg / mL; P6·+ nanoparticles at a final concentration of 10 μM; and PR619 (deubiquitinating enzyme inhibitor) at a final concentration of 10 μM. An equal volume of PBS buffer was added as a negative control. After 24 hours of cell culture, Western blot analysis was performed. The experiment was conducted in triplicate. Results are shown below. Figure 15As shown in c, PYR-41 effectively inhibits the degradation of mutant p53 protein induced by P6·+ nanoparticles, while PR619 further promotes the degradation of mutant p53 protein, indicating that ubiquitination is essential for degradation.

[0149] Example 5: Investigating the mechanism by which P6·+ nanoparticles induce the degradation of mutant p53 protein.

[0150] PA-TU 8988S cells were seeded in 24-well cell culture plates at a density of approximately 3 × 10⁶ cells / well. 4 ~5×10 4 Cells / wells were cultured overnight and ready for use. Before adding samples, the cells were replaced with fresh DMEM medium, and then P6·+ nanoparticles (prepared in Example 6) and Genistein (endocytosis inhibitor, purchased from MCE) were added to a final concentration of 25 μg / mL, Cyto D (cytochalasin, purchased from MCE) to a final concentration of 50 μM, and Genistein and Cyto D to a final concentration of 10 μM were added respectively. The negative control was an equal volume of PBS buffer. After culturing the cells for 24 hours, Western blot analysis was performed. The experiment was set up in triplicate. The results are as follows. Figure 16 As shown, both the endocytosis inhibitors Genistein and cytochalasin can effectively inhibit the degradation of mutant p53 induced by P6·+ nanoparticles, indicating that cellular endocytosis is essential for the degradation induced by P6·+ nanoparticles.

[0151] Next, as Figure 17 As shown in Figure a, our fluorescence experiments revealed that P6·+ nanoparticles localized to mitochondria (Mito Traker Green, Beyotime) after entering PA-TU 8988S cells. Subsequently, we further stained PA-TU 8988S cells treated with P6·+ nanoparticles using a mitochondrial membrane potential assay kit (JC-1), and the results are shown in Figure a. Figure 17 As shown in b, after treatment with P6·+ nanoparticles, the green fluorescence of JC-1 monomers was significantly enhanced, and the mitochondrial membrane potential was significantly decreased, indicating that P6·+ nanoparticles disrupted mitochondrial homeostasis by targeting mitochondria, causing mitochondrial membrane damage. We also found that treatment of PA-TU 8988S cells with P6·+ nanoparticles significantly increased the level of intracellular reactive oxygen species (ROS). Figure 17 c).

[0152] Our previous research showed that intracellular redox levels can affect the stability of mutant p53 protein. Therefore, to further investigate the mechanism by which P6·+ nanoparticles induce the degradation of mutant p53 protein, PA-TU 8988S cells were treated with the following solutions for 24 h: P6·+ nanoparticles (prepared in Example 6) at a final concentration of 25 μg / mL; P6·+ nanoparticles at a final concentration of 25 μg / mL; NAC (antioxidant, N-acetylcysteine, Sigma) at a final concentration of 5 mM; NAC at a final concentration of 5 mM; P6·+ nanoparticles at a final concentration of 25 μg / mL; Mito Tempo (MT, mitochondrial antioxidant, MCE) at a final concentration of 1 mM; Mito Tempo at a final concentration of 1 mM; and P6·+ nanoparticles at a final concentration of 25 μg / mL + oxidase inhibitor VAS2870 (VAS, purchased from Sigma) at a final concentration of 15 μM. The blank control was an equal volume of PBS buffer. After processing, Western blot analysis was performed. The experiment was set up with three replicates. Results are as follows: Figure 17 As shown in d, both the antioxidant NAC and the mitochondrial antioxidant Mito Tempo can effectively inhibit the degradation of mutant p53 protein induced by P6·+ nanoparticles, while the oxidase inhibitor VAS2870 has no significant inhibitory effect. This indicates that reactive oxygen species originating from mitochondria induced by P6·+ nanoparticles play an important role in the degradation of mutant p53 protein.

[0153] Example 6: Synthesis and Characterization of P6@siKRAS Nanoparticles

[0154] 1. Synthesis of P6@siKRAS nanoparticles

[0155] (1) Synthesis of P6·+ nanoparticles

[0156] Take 20.1 mg of P6 and 38.7 mg of cucurbita[7]urea obtained in Example 1, add 10 mL of DEPC water, and sonicate in a water bath for 0.5 hours to obtain P6·+ nanoparticles (abbreviated as P6·+NPs).

[0157] (2) Synthesis of P6@siKRAS nanoparticles

[0158] 30 nmol of KRAS siRNA (siRNA sequences were designed and synthesized by Suzhou Beixin Biotechnology Co., Ltd., siKRAS positive strand sequence (5'-3'): GAUCCGACAAUACAGAUUGTT, siKRAS antisense strand sequence (5'-3'): CAAUCUGUAUUGUCGGAUCTT) was dissolved in 300 μL of DEPC water. Then, 1260 μL of P6·+NPs were added at once. After incubation at room temperature for 1 hour, the mixture was centrifuged at 15000 rpm for 100 minutes. The lower blue solid layer was collected, which is P6@siKRAS nanoparticles (abbreviated as P6@siRNANPs).

[0159] 2. Nanoparticle characterization

[0160] (1) siRNA binding analysis

[0161] Following the method described in step (2) above, P6@siKRAS nanoparticles were prepared by setting the molar ratio of P6 concentration in the nanoparticles to the base concentration in the siRNA to 2:1, 4:1, 6:1, 8:1, and 10:1. The supernatant after centrifugation was then analyzed by DNA gel electrophoresis. The experiment was performed in triplicate. The results are as follows: Figure 18 As shown, when the ratio of P6 concentration in the particle to the base concentration in the siRNA is 10:1, the siRNA is completely bound to the particle.

[0162] (2) Particle size and potential analysis

[0163] P6·+ nanoparticles and P6@siKRAS nanoparticles were characterized using transmission electron microscopy and laser particle size analysis. The experiment was performed in triplicate. Results are shown below. Figure 19 As shown in a, 19b, and 19c, the P6·+ nanoparticles have a particle size of 120 nm and a surface potential of +47.58 mV; the P6@siKRAS nanoparticles bound to siRNA have a particle size of 150 nm and a surface potential of -12.67 mV.

[0164] The 24-hour stability of P6·+ nanoparticles and P6@siKRAS nanoparticles was then analyzed. Both types of particles were added to DMEM medium containing 10% (v / v) fetal bovine serum at a final concentration of 25 μg / ml and allowed to stand for 24 hours. The results are as follows: Figure 19 As shown in d, in DMEM medium supplemented with 10% (v / v) fetal bovine serum, the particle size of P6·+ nanoparticles was larger than that of P6@siKRAS nanoparticles, and the fluctuation range was larger. This phenomenon indicates that the stability of P6@siKRAS nanoparticles bound to siRNA is better than that of P6·+ nanoparticles.

[0165] (3) Ultraviolet spectroscopy and fluorescence spectroscopy determination

[0166] Solutions of P6·+ nanoparticles and P6@siKRAS nanoparticles with a P6 concentration of 10 μM were prepared. Using pyrrole radical molecule P6 and cucurbitaureus (CB[7]) as controls, their UV and fluorescence spectra were scanned. The experiment was repeated three times. The results are as follows: Figure 20 As shown, the maximum absorption wavelength of both types of particles is 600 nm and the maximum emission wavelength is 670 nm. This phenomenon indicates that siRNA does not affect the photophysical properties of the particles or their imaging capabilities.

[0167] (4) EPR spectrum determination

[0168] P6·+ nanoparticles and P6@siKRAS nanoparticles, each with a P6 content of 2 mg, were weighed out, added to an NMR tube, and EPR spectra were measured. The experiment was performed in triplicate. Results are as follows: Figure 21 As shown, the EPR signal intensities of P6·+ nanoparticles and P6@siKRAS nanoparticles are the same, indicating that siRNA does not quench already generated free radicals.

[0169] (5) Investigating the cell imaging capabilities of cationic free radical molecules

[0170] PA-TU 8988S cells were seeded in 24-well cell culture plates at a density of approximately 3 × 10⁶ cells / well. 4 ~5×10 4 Cells / wells were cultured overnight and ready for use. Before adding samples, the cells were transferred to fresh DMEM medium, and P6·+ nanoparticles and P6@siKRAS nanoparticles were added to a final concentration of 25 μg / mL, respectively. After culturing the cells for 12 hours, fluorescence microscopy was performed for detection. The experiment was set up in triplicate. The results are as follows. Figure 22 As shown, we observed red fluorescence in both P6·+ nanoparticles and P6@siKRAS nanoparticles, indicating that the modification of siKRAS with P6·+ nanoparticles retains its superior molecular imaging capabilities.

[0171] Example 7: Detection of cellular uptake of P6@siKRAS nanoparticles

[0172] PA-TU 8988S cells were seeded in confocal microplates at a density of approximately 1 × 10⁻⁶ cells / mL. 5 ~2×10 5 Cells / wells were cultured overnight and ready for use. Before adding samples, the cells were transferred to fresh DMEM medium and P6@siKRAS nanoparticles (prepared in Example 6) were added to a final concentration of 25 μg / mL. Cells were cultured for 2, 4, 8, and 12 hours, and then detected using a fluorescence microscope. The experiment was repeated three times. Results are as follows: Figure 23As shown in Figure a, P6@siKRAS nanoparticles began to enter cells after 2 hours (red indicates P6·+, green indicates FAM-siKRAS). With the extension of treatment time, the uptake of P6@siKRAS nanoparticles by PA-TU 8988S cells continued to increase.

[0173] Next, we investigated the organelle localization after the P6@siKRAS nanoparticles entered the cells. PA-TU8988S cells were seeded in confocal microarrays and treated with P6@siKRAS nanoparticles for 0, 4, 8, and 12 hours. Cells were then treated with commercial Lyso-Tracker Green (a lysosomal green fluorescent probe, Beyotime) for 30 minutes, and the nuclei were stained with Hochest. The images were recorded under laser confocal microscopy. The experiment was repeated three times. The results are shown below. Figure 23 As shown in b, P6@siKRAS nanoparticles (red fluorescence) exhibit a significant co-localization effect with lysosomes (green fluorescence) at 8 h, while the co-localization phenomenon weakens at 12 h. This phenomenon indicates that P6@siKRAS nanoparticles have a good ability to escape lysosomes and can serve as a high-performance gene delivery vector.

[0174] Example 8: Detection of the efficiency of P6@siKRAS nanoparticles in silencing the KRAS gene

[0175] PA-TU 8988S and MIA PaCa-2 cells were seeded into 6-well plates at a density of approximately 1 × 10⁻⁶ cells / well. 5 ~2×10 5 Cells / wells were cultured overnight and ready for use. Before sample loading, cells were transferred to fresh DMEM medium and treated with P6@siKRAS nanoparticles (P6@KRAS siRNA) at a final concentration of 25 μg / mL for 0, 12, and 24 h, respectively. An equal volume of PBS buffer was used as a control. RNA was extracted from the cells, and subsequent quantitative real-time PCR was performed to detect KRAS gene expression. The experiment was repeated three times. Results are shown below. Figure 24 As shown in figure a, P6@siKRAS nanoparticles significantly downregulated the expression level of the KRAS gene.

[0176] Furthermore, we compared the efficiency of P6@siKRAS nanoparticles in delivering KRAS siRNA with the commercially available Lipofectamine 3000 transfection kit, using an equal volume of PBS buffer as a control. The results are as follows: Figure 24 b and Figure 24As shown in Figure c, P6@siKRAS nanoparticles exhibited superior silencing efficiency in both PA-TU 8988S and MIA PaCa-2 cell lines. This demonstrates that our designed and synthesized P6@siKRAS nanoparticles can simultaneously achieve efficient degradation of mutant p53 and serve as a gene vector for efficient delivery of KRAS siRNA, making them a high-performance bifunctional nanoparticle.

[0177] Example 9: Detection of the expression levels of proteins related to the KRAS downstream signaling pathway downregulated by P6@siKRAS nanoparticles.

[0178] PA-TU 8988S and MIA PaCa-2 cells were seeded into 24-well plates at a density of approximately 3 × 10⁻⁶ cells / well. 4 ~5×10 4 Cells / wells were cultured overnight for later use. Before adding samples, the cells were transferred to fresh DMEM medium, and different concentrations (final concentrations of 5, 10, 15, 20, and 25 μg / mL) of P6@siKRAS nanoparticles were added for 24 hours, with an equal volume of PBS buffer as a control. After treatment, Western blot was performed to detect the expression levels of KRAS and its downstream signaling pathway-related proteins. The experiment was conducted in triplicate.

[0179] The results are as follows Figure 25 As shown, P6@siKRAS nanoparticles can significantly downregulate the expression level of KRAS protein. In normal cells, KRAS protein maintains its normal biological function by cycling between an activated state bound to guanosine triphosphate (GTP) and an inactivated state bound to guanosine diphosphate (GDP) in response to extracellular signals. Activating mutations in KRAS lead to the loss of GTPase activity, resulting in the continuous activation of downstream pro-proliferation and pro-survival pathways, including RAF / MEK / ERK and PI3K / AKT. When KRAS is mutated, the KRAS mutant protein has a significantly increased affinity for GTP, forming a continuously activated GTP-binding state, and the loss of GTPase activity leads to the continuous activation of downstream pro-proliferation and pro-survival pathways, including RAF / MEK / ERK and PI3K / AKT. Our study found that treatment with P6@siKRAS nanoparticles significantly reduced the expression levels of phosphorylated AKT and phosphorylated ERK, indicating that P6@siKRAS nanoparticles downregulate KRAS expression levels and thus significantly inhibit downstream signaling pathways.

[0180] Example 10: Detection of the killing effect of P6@siKRAS nanoparticles in different cell lines

[0181] Human pancreatic cancer PA-TU-8988S and MIA PaCa-2 cells belong to the p53 and KRAS double mutant 3 cell lines, while mouse fibroblast L929 and mouse embryonic fibroblast 3T3 are normal cells. All these cells were purchased from ATCC. The above cells were seeded into 96-well cell culture plates at a density of approximately 1 × 10⁻⁶. 4 ~2×10 4 Cells / wells were cultured overnight and ready for use. Before adding samples, the cells were transferred to fresh DMEM medium, and then treated with different concentrations (final concentrations of 5, 10, 15, 20, 25, and 30 μg / mL) of P6@siKRAS nanoparticles for 24 h. The blank control was an equal volume of PBS buffer. MTT assay was then performed. The experiment was set up in triplicate.

[0182] The results are as follows Figure 26 As shown, P6@siKRAS nanoparticles do not significantly affect the cell viability of normal cells, but have a strong killing effect on double mutant cell lines. This indicates that P6@siKRAS induces the degradation of mutant p53 protein and downregulates the expression level of mutant KRAS, thereby specifically causing a severe decrease in cell viability.

[0183] Example 11: Detection of GOF (Gain of Function) in double-mutant cells treated with P6@siKRAS nanoparticles

[0184] (1) Cell migration assay: 200 μL (10,000 cells) of PA-TU-8988S and MIA PaCa-2 cell suspension (containing 1% (v / v) fetal bovine serum FBS) was added to a Transwell chamber. 600 μL of DMED medium (containing 20% ​​(v / v) FBS) was added to the lower layer. Cells were then treated with P6@siKRAS nanoparticles and P6@siNC nanoparticles (containing control siRNA, sense strand sequence (5'-3'): UUCUCCGAACGUGUCACGUTT; antisense strand sequence: ACGUGACACGUUCGGAGAATT) at a final concentration of 25 μg / mL, with an equal volume of PBS buffer as a blank control. After 24 hours of treatment, the outer membrane of the upper chamber was washed three times with PBS, and the cells were fixed with 4% paraformaldehyde for 20 minutes. The outer membrane of the chamber was then stained with 0.1% crystal violet solution for 30 minutes at room temperature. The staining solution was then removed, and the cells were gently washed several times with 1×PBS buffer to remove the background. After air-drying at room temperature, the cells were imaged using a microscope (Nikon, Ci-L). The experiment was performed in triplicate. The results are as follows: Figure 27As shown in Figure a, compared with the P6@siNC treatment group and the blank control group, P6@siKRAS nanoparticles significantly inhibited the migration of PA-TU-8988S and MIA PaCa-2 double mutant pancreatic cancer cells, indicating that the simultaneous removal of mutant p53 protein and mutant KRAS protein has a significant inhibitory effect on cancer cell migration.

[0185] (2) Cell colony formation assay: A suitable amount of PA-TU-8988S and MIA PaCa-2 single-cell suspension (1000 cells) was seeded into 6-well plates. The next day, P6@siNC and P6@siKRAS nanoparticles at a final concentration of 25 μg / mL were prepared and the cells were treated for 24 hours. An equal volume of PBS buffer was used as a blank control. After treatment, the original culture medium was aspirated, and the cell surface was gently washed twice with PBS buffer to remove the nanoparticles attached to the cell surface. Finally, complete culture medium was added, and the cells were cultured for 10–14 days, during which time the culture medium was monitored and replaced as needed. After culture, the cell surface was gently washed with PBS buffer, and 4% paraformaldehyde was added to fix the cells at room temperature for 20 minutes. The cells were then washed twice with PBS buffer. 0.1% crystal violet solution was prepared and 1 mL was added to each well. After staining at room temperature for 30 minutes, the staining solution was aspirated, and the cells were gently washed several times with PBS buffer to remove the background. After drying at room temperature, the cells were photographed and their colonies recorded. The experiment was repeated three times. The results are as follows: Figure 27 As shown in b, treatment with P6@siKRAS nanoparticles can effectively promote the proliferation and growth of cancer cells.

[0186] (3) Chemosensitization assay: MIA PaCa-2 cells were seeded in 96-well cell culture plates at a density of approximately 1×10⁻⁶. 4 ~2×10 4 Cells / wells were cultured overnight for later use. Before adding samples, cells were transferred to fresh DMEM medium, and then P6@siKRAS nanoparticles (final concentration 20 μg / mL) and Gemcitabine (final concentration 50 μM, purchased from MCE) were added. Cells were treated with Gemcitabine for 24 h. An equal volume of PBS buffer was used as a blank control. MTT assay was then performed. The experiment was conducted in triplicate. Gemcitabine is currently a first-line treatment for pancreatic cancer; however, gemcitabine chemotherapy only kills less than 10% of MIA PaCa-2 cells, indicating significant drug resistance in MIA PaCa-2 double-mutant pancreatic cancer cells. Results are as follows... Figure 27 As shown in c; after treatment with P6@siKRAS nanoparticles and combined with chemotherapy drugs, the killing effect on cancer cells reached nearly 80%, indicating that P6@siKRAS can effectively enhance the killing effect of chemotherapy drugs at the cellular level.

[0187] (4) Downstream target gene expression level calibration: MIA PaCa-2 cells were seeded in 6-well plates at a density of approximately 1×10⁻⁶. 5 ~2×10 5 Cells / wells were cultured overnight for later use. Before sample loading, cells were transferred to fresh DMEM medium and treated with 25 μg / mL P6@siKRAS nanoparticles for 24 h. An equal volume of PBS buffer was used as a control. RNA was extracted from the cells, and subsequent quantitative real-time PCR was performed to detect the expression of downstream target genes. The experiment was repeated three times. Results are shown below. Figure 27 As shown in d, P6@siKRAS nanoparticles significantly downregulated the expression levels of downstream target genes, including cell cycle regulators cyclin-dependent kinases (CDK1) and MAP2K3, MYC, and apoptosis regulator BCL2L1, by clearing the levels of mutated p53 and KRAS proteins. This evidence further demonstrates that P6@siKRAS nanoparticles reverse the GOF (Gain-of-Function) of cancer cells by clearing double-mutant proteins, effectively inhibiting the growth of cancer cells.

[0188] Example 12: Investigating the distribution of P6@siKRAS nanoparticles in animals.

[0189] The above results demonstrate that P6@siKRAS, based on its unique cationic radical properties, exhibits superior red light characteristics at a wavelength of 630 nm. To detect the distribution of P6@siKRAS in vivo and its accumulation in tumor regions, three Balb / c nude mice (approximately 20g, female, purchased from Beijing Vital River Animal Experiment Technology Co., Ltd.) were implanted with MIAPaCa-2 subcutaneous tumors, with tumor volumes ranging from 150 to 200 mm². 3 Small animal imaging experiments were conducted. 5 mg / kg of P6@siKRAS nanoparticles (prepared in Example 6) were injected via the tail vein, and small animal imaging was performed at different time points (0, 2, 4, 8, 12, and 24 hours). The results are as follows: Figure 28 As shown, after injection of P6@siKRAS, we observed a gradual increase in fluorescence intensity in the tumor region over time, reaching a maximum at 12 h. This can be attributed to the enhanced permeability and retention (EPR) properties of P6@siKRAS. Furthermore, in vitro images of tissues and organs further validated the good enrichment effect of P6@siKRAS NPs in tumor tissues of MIA PaCa-2 tumor-bearing mice.

[0190] Example 13: Investigating the in vivo antitumor activity of P6@siKRAS nanoparticles

[0191] (1) 100 μL of MIA PaCa-2 cells (1×10⁻⁶) were subcutaneously injected into the right wing of female Balb / c nude mice (weighing approximately 22 g, purchased from Vital River Pharmaceuticals, Beijing). 7 (Each tumor model was constructed. When the tumor volume reached 100 mm²...) 3 Subsequent experiments were then conducted. The obtained MIA PaCa-2 tumor-bearing mice were divided into three groups of seven mice each. Each group received a tail vein injection of 5 mg / kg P6@siNC nanoparticles (as in Example 11) or 5 mg / kg P6@siKRAS nanoparticles (prepared in Example 6), with an equal volume of PBS buffer as a control. Treatment was administered every two days. Changes in mouse weight and tumor volume were recorded during treatment. After 14 days of treatment, the mice were sacrificed, tumors were harvested, photographed, and weighed. Results are as follows: Figure 29 As shown, compared with the control group, P6@siNC nanoparticle treatment had a better therapeutic effect on MIA PaCa-2 tumors, while the P6@siKRAS nanoparticle treatment group showed the best therapeutic effect. This indicates that clearing mutant p53 protein has a certain anti-tumor effect, and simultaneously clearing mutant p53 and mutant KRAS proteins in tumor tissue can further inhibit tumor growth.

[0192] (2) LSL-Kras G12D (KI / +); LSL-Trp53 R172H (KI / +); Pdx1-Cre(TG / +)(KPC) mice (Jicui Pharmaceutical Biotechnology) are a spontaneous pancreatic cancer model mouse carrying both p53 and KRAS mutations, closely resembling key features of human pancreatic cancer. After spontaneous tumor formation, tumor tissue was dissected, and sequencing of a portion of the tumor tissue confirmed that the spontaneous pancreatic cancer tumor tissue indeed contained the double mutations of TP53 (R172H) and KRAS (G12D). A portion of the tumor tissue was washed in sterile PBS and cut into 2–3 mm pieces. 3 A 2-3 mm tissue mass was subcutaneously implanted in the right wing of female 6-8 week old C57BL / 6 mice (weighing approximately 19 g, purchased from Vital River Pharmaceuticals, Beijing). 3 Spontaneous pancreatic cancer tumor tissue, waiting for the tumor to grow to 150mm 3 At that time, tumor-bearing mice were randomly divided into 3 groups of 7 mice each. Each group received a tail vein injection of 5 mg / kg P6@siNC nanoparticles (as in Example 11) or 5 mg / kg P6@siKRAS nanoparticles (prepared in Example 6), with an equal volume of PBS buffer injected as a control. Treatment was administered every two days, and changes in mouse body weight and tumor volume were recorded during the treatment. After 14 days of treatment, the tumors of the sacrificed mice were harvested, photographed, and weighed. The results are as follows: Figure 30 As shown in the KPC model, P6@siKRAS nanoparticles also exhibit a good anti-tumor effect.

[0193] Furthermore, previous studies have shown that mutant p53 and KRAS, besides activating related signaling pathways to promote tumor development, more importantly, also create a tumor-suppressive immune microenvironment. The KPC tumor model is a mouse model with a complete immune system. To further verify the effects of clearing mutant p53 and KRAS proteins on the tumor microenvironment, we analyzed immune cells in the tumor microenvironment. First, we detected IFN-γ in mouse serum using an ELISA assay. IFN-γ is a soluble dimeric cytokine and the only member of type II interferon. It is mainly secreted by natural killer cells (NK) and natural killer T cells (NKT) and plays a role in innate immunity; in antigen-specific immunity, it is secreted by CD4 Th1 and CD8 cytotoxic T cells. The results are as follows: Figure 31 As shown, compared with the control group and P6@siNC nanoparticles, the serum IFN-γ level in mice treated with P6@siKRAS nanoparticles was significantly increased. More importantly, flow cytometry analysis of immune cells in the tumor microenvironment revealed that the infiltration of CD8+ T cells in the tumor microenvironment was significantly enhanced in the P6@siKRAS nanoparticle-treated group. Conversely, P6@siKRAS nanoparticles significantly reduced the proportion of tumor immunosuppressive cells (Treg cells) and further reduced the proportion of anti-inflammatory M2 macrophages. This evidence suggests that P6@siKRAS nanoparticles enhance tumor immunotherapy by remodeling the tumor immune microenvironment by clearing mutant p53 and mutant KRAS proteins from tumor cells.

[0194] Example 14: Detection of apoptosis levels in tumor tissues after animal-level treatment with P6@siKRAS nanoparticles

[0195] After the treatment of MIA PaCa-2 tumor-bearing mice in step (1) of Example 13 was completed, a portion of the tumor tissue from each group was taken for paraffin sectioning and TUNEL staining was performed using an apoptosis kit (purchased from Beyotime). The results are as follows: Figure 32 As shown, compared with the control group and the P6@siNC group, the tumor tissue treated with P6@siKRAS nanoparticles showed more obvious green fluorescence, indicating a higher level of apoptosis, which is consistent with the tumor weighing results after treatment.

[0196] Example 15: Detection of the effects of P6@siKRAS nanoparticles on mouse organs

[0197] At the end of treatment in MIA PaCa-2 tumor-bearing mice in step (1) of Example 13, blood was collected by enucleating the eyeballs of the mice before sacrifice, and serum was separated to detect the concentrations of AST (aspartate aminotransferase), ALT (alanine aminotransferase), BUN (blood urea nitrogen), and SCR (serum creatinine). This was repeated three times. The results are as follows: Figure 33 As shown, compared with the PBS group, there were no significant differences in liver and kidney function indicators between the P6@siNC nanoparticle and P6@siKRAS nanoparticle treatment groups and the PBS group, indicating that P6@siNC nanoparticle and P6@siKRAS nanoparticle did not have a significant impact on the organs of mice during treatment.

[0198] Furthermore, in Example 13, after the MIA PaCa-2 tumor-bearing mice completed treatment, the mice were sacrificed, and vital organs such as the heart, liver, spleen, lungs, and kidneys were harvested for sectioning and HE staining observation. The results are as follows... Figure 34 As shown, the texture of major tissues and organs (excluding tumor tissue) treated with P6@siNC nanoparticles and P6@siKRAS nanoparticles was not significantly different from that of PBS, further demonstrating that P6@siNC nanoparticles and P6@siKRAS nanoparticles do not cause significant damage to the organs in mice.

[0199] Example 16: Detection of p53 and KRAS expression levels in tumor tissues after P6@siKRAS nanoparticle treatment.

[0200] At the end of treatment in MIA PaCa-2 tumor-bearing mice in step (1) of Example 13, a portion of tumor tissue was collected from each group, and proteins were extracted from the tumor tissue using RIPA tissue lysis buffer. Western blot analysis was performed to detect the expression of p53 and KRAS proteins in the tumor tissue. The experiment was repeated three times. Results are as follows: Figure 35 As shown, in both the P6@siNC nanoparticle and P6@siKRAS treatment groups, the p53 protein level in the tumor tissue of each mouse was significantly reduced, indicating that the nanoparticles, primarily composed of P6·+ cations, possess a good ability to degrade mutant p53 protein in tumor tissue in vivo. Furthermore, a significant decrease in KRAS protein was observed in the tumor tissue of mice treated with P6@siKRAS, indicating that the P6·+ cation, as a gene delivery vector with mutation-degrading function, can exert a good KRAS siRNA delivery effect in vivo. The functional gene delivery vector invented in this study provides a new strategy for the precision treatment of double-mutant pancreatic cancer.

[0201] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A self-assembled bifunctional nanoparticle, characterized in that: KRAS siRNA was obtained by adding KRAS siRNA and nanoparticles containing pyrrole radical molecules to water, incubating at room temperature, and then collecting by centrifugation. The nucleotide sequence of the KRAS siRNA is as follows: siKRAS justice chain sequence: 5'-GAUCCGACAAUACAGAUUGTT-3'; siKRAS antisense sequence: 5'-CAAUCUGUAUUGUCGGAUCTT-3'; The molar ratio of the concentration of pyrrole radical molecules in the nanoparticles containing pyrrole radical molecules to the base concentration in KRAS siRNA is 2 to 10:

1. The nanoparticles containing pyrrole radical molecules are obtained by adding pyrrole radical molecules and cucurbita[7]urea to water and then subjecting them to ultrasonication in a water bath. The pyrrole radical molecule is selected from at least one of P6-Br, P6-F, and P6, and its structural formula is shown in Formulas VI to VIII: 。 2. The self-assembled bifunctional nanoparticles according to claim 1, characterized in that, The pyrrole radical molecule was prepared by the following method: S1. Aniline compounds and 2,5-hexanedione were dissolved in tetrahydrofuran, iodine was added as a catalyst, the mixture was stirred at room temperature, and then an excess of thiosulfate solution was added and the mixture was stirred again. After the reaction was completed, the mixture was washed with saturated brine, the organic phase was dried with anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the residue was purified by column chromatography to obtain intermediate I. S2. Oxaloyl chloride was added dropwise to N,N-dimethylformamide under ice bath conditions, followed by the white solid produced by dichloromethane dissolution, then intermediate I obtained in step S1 and excess sodium hydroxide aqueous solution were added. The mixture was stirred at room temperature. After the reaction was completed, the mixture was washed with saturated brine, dried with anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the residue was purified by column chromatography to obtain intermediate II. S3. Dissolve intermediate II or 2,5-dimethyl-1-phenyl-1H-pyrrole-3-carboxaldehyde obtained in S2 into methanol, add sodium borohydride under ice bath conditions, stir the reaction, remove methanol by rotary evaporation after the reaction is completed, wash, collect the insoluble solid and freeze dry to obtain pyrrole free radical molecules P6-Br, P6-F or P6. The aniline compound mentioned in step S1 is at least one of 4-bromoaniline and 4-fluoroaniline.

3. The self-assembled bifunctional nanoparticles according to claim 2, characterized in that: The molar ratio of aniline compounds, 2,5-hexanedione, iodine and thiosulfate in step S1 is 5:6~10:0.5:10~20; The room temperature stirring reaction time described in step S1 is 36 to 48 hours; The stirring reaction time described in step S1 is 1 to 2 hours; The column chromatography described in step S1 is 200-300 mesh silica gel column chromatography, and the eluent used is obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 8:

1. The molar ratio of oxalyl chloride, N,N-dimethylformamide, intermediate I and sodium hydroxide in step S2 is 4:4:4:5 to 10; The stirring reaction at room temperature in step S2 lasts for 1 to 2 hours; The column chromatography described in step S2 is 200-300 mesh silica gel column chromatography, and the eluent used is obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 4:

1. The molar ratio of intermediate II to sodium borohydride in step S3 is 1:2 to 4; The room temperature stirring reaction time described in step S3 is 2 to 3 hours.

4. The use of the self-assembled bifunctional nanoparticles according to any one of claims 1 to 3 in the preparation of gene delivery vectors and / or medicaments for treating pancreatic cancer.

5. The use of the self-assembled bifunctional nanoparticles according to any one of claims 1 to 3 in the preparation of products that degrade KRAS protein and / or downregulate the expression level of KRAS gene.

6. The use of the self-assembled bifunctional nanoparticles according to any one of claims 1 to 3 in the preparation of chemotherapeutic drug sensitizers.

7. The use of at least one of the pyrrole radical molecules, nanoparticles containing pyrrole radical molecules, and self-assembled bifunctional nanoparticles as described in any one of claims 1 to 3 in the preparation of products that degrade mutant p53 protein.

8. The application of cationic free radical molecules in the preparation of products that degrade mutant p53 protein, characterized in that: The aforementioned cationic free radical molecule is obtained by adding the pyrrole free radical molecule described in any one of claims 1 to 3 to a hydrochloric acid solution, followed by ultrasonic treatment and freeze drying.

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