Redox-responsive HE4-loaded siRNA nanoparticles, preparation method thereof and application of nanoparticles in treatment of cis-platinum drug-resistant ovarian cancer

Abstract

The invention provides an HE4siRNA (Hepatitis E 4 Small Interference Ribonucleic Acid) loaded nano particle with redox response. The nano particle is prepared from a tumor targeting carrier material with redox response and HE4siRNA. The tumor targeting carrier material with redox response is poly (ethyleneimine)-diselenide bond-poly (ethylene glycol) (PEI-SeSe-PEG) modified by c (RGDfK) peptide. According to the nanoparticle, a carrier material and siRNA are mixed according to the N / P ratio of 10: 1 and are self-assembled into the nanoparticle, the particle size is about 116 nm, in-vitro release has oxidation-reduction responsiveness, release of siRNA is achieved in a tumor microenvironment of high-concentration glutathione (GSH), and the nanoparticle is beneficial to play a role in a tumor site. The HE4siRNA loaded nanoparticles with redox response prepared by the invention can realize lysosome escape, have tumor targeting property, can inhibit proliferation, migration and invasion of cis-platinum drug-resistant ovarian cancer cells, and have good tumor targeting property, antitumor activity and biocompatibility in animal bodies. The nanoparticle is simple in preparation method and remarkable in anti-cancer effect, and has good application value and market prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a redox-responsive HE4 siRNA-loaded nanoparticle, a preparation method thereof and an effect in treating cisplatin-resistant ovarian cancer, and belongs to the field of medicine. BACKGROUND

[0002] Ovarian cancer (OC) is one of the malignant tumors of the female reproductive system with the highest mortality rate in the world, and its incidence is increasing year by year. It is a highly heterogeneous disease, most of which is derived from epithelial ovarian cancer, and a small part is derived from germ cells or sex cord stromal tissue. In recent years, the incidence in Northern Europe and North America has decreased, while the incidence in Eastern Europe and some parts of Asia is increasing year by year. According to statistics, there were 19680 new cases and 12740 deaths worldwide in 2024, with a mortality rate as high as 65%. Such a high mortality rate is largely due to late discovery, as most (about 66%) patients are directly diagnosed as advanced disease stage III or IV by the International Gynecological Oncology Alliance, with 5-year survival rates of 41% and 20%, respectively. If patients can be treated in the early stages of cancer, their 5-year survival rate can be significantly improved, and the likelihood of cure can be greatly increased. Therefore, early detection and treatment of OC is of great significance to improve the prognosis of patients and increase the happiness of patients.

[0003] Human epididymis protein 4 (HE4) as a new tumor marker has high accuracy and specificity in the differential diagnosis of benign and malignant ovarian masses. HE4 belongs to the whey acidic four-disulfide bond core (WFDC) protein family and was initially identified in the epithelium of the distal epididymis. More and more evidence is showing that HE4 is highly expressed in OC. Based on extensive empirical research, HE4 has been approved by the US Food and Drug Administration as a diagnostic and prognostic marker for monitoring ovarian epithelial cancer alone or in combination with CA125. Therefore, HE4 may be a potential gene target for treating OC.

[0004] RNA interference (RNAi) is an accurate and effective post-transcriptional gene silencing technique triggered by small interfering RNA (siRNA). As the core component of the RNAi complex, siRNA is a sequence-specific gene silencing method, usually a double-stranded RNA molecule composed of 19-25 base pairs, which can down-regulate the expression of specific genes and achieve therapeutic effect by selectively silencing pathogenic genes. Previous studies have found that the overexpression of HE4 in OC is related to the drug resistance of cancer cells, and HE4 interacts with epidermal growth factor receptor, insulin growth factor receptor, hypoxia-inducible factor-1α, etc., affecting the tumor microenvironment, thereby enhancing tumor cell proliferation and inducing cisplatin resistance. In the previous experiment, it was found that after the cisplatin-resistant ovarian cancer cells OVCAR-3 / DDP were transfected with Lipofectamine 2000 to transfect HE4 siRNA, the proliferation, migration and invasion functions of the cells were significantly reduced. However, naked siRNA is easily affected by various factors in the blood, resulting in its instability in the cell and poor tumor targeting. Therefore, in order to improve the stability and tumor targeting of siRNA, the development of an efficient targeted drug delivery system is the main problem to be solved in current research, which will provide a new strategy for the treatment of cisplatin-resistant ovarian cancer (OVCAR-3 / DDP). SUMMARY

[0005] In order to solve the problems in the prior art, the application provides an oxidation-reduction response HE4 siRNA-loaded nanoparticle, a preparation method thereof and an application in treating cisplatin-resistant ovarian cancer.

[0006] The technical scheme of the application is as follows: The application provides an oxidation-reduction response HE4 siRNA-loaded nanoparticle, which is composed of an oxidation-reduction response tumor-targeting carrier material and HE4 siRNA. The oxidation-reduction response tumor-targeting carrier material is a c(RGDfK) peptide modified poly(ethyleneimine)-diselenium bond-poly(ethylene glycol) (PEI-SeSe-PEG). The forward primer sequence of the HE4 siRNA is shown as SEQ ID NO. 1, and the reverse primer sequence is shown as SEQ ID NO. 2.

[0007] The sequence of SEQ ID NO. 1 is GUCCUGUGUCACUCCCAAUTT. The sequence of SEQ ID NO. 2 is AUUGGGAGUGACACAGGACTT. The application further provides a preparation method of the oxidation-reduction response HE4 siRNA-loaded nanoparticle, comprising the following steps: (1) mixed and reacted cross-linking agent SMCC with PEI-SeSe-PEG at a molar ratio of 1:1, stirred at room temperature in the dark for 2 h, and the obtained product was dialyzed in pure water for 48 h (molecular weight cut-off: 10 KD) to obtain a mixed solution.

[0008] (2) c(RGDfK) was added to the above mixed solution (molar ratio of 1:1 with SMCC), stirred at room temperature in the dark for 4 h, and freeze-dried for 48 h to obtain c(RGDfk)-PEI-SeSe-PEG freeze-dried powder.

[0009] (3) The powder was dissolved in DEPC water, and HE4 siRNA solution was added at N / P = 10, vortexed for 30 s and left standing for 25 min, and self-assembled to obtain c(RGDfK) modified PEI-SeSe-PEG loaded HE4 siRNA nanoparticles.

[0010] The application also provides an application of the HE4 siRNA-loaded nanoparticle with redox response in treating cisplatin-resistant ovarian cancer.

[0011] Compared with the prior art, the application has the following advantages and beneficial effects: The nano-drug delivery system constructed in the application has redox response, can realize release of siRNA in the tumor microenvironment with high concentration of glutathione (GSH), and is beneficial to exerting effects at the tumor site.

[0012] The nano-drug delivery system constructed in the application can improve the transfection efficiency of siRNA, realize lysosome escape, has tumor targeting, improves the anti-tumor effect, and reduces side effects.

[0013] The HE4 siRNA nanoparticle constructed in the application can inhibit the proliferation, migration and invasion of cisplatin-resistant ovarian cancer cells, and has good tumor targeting, excellent anti-tumor activity and biocompatibility in the animal body. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Transmission electron microscopy of the HE4 siRNA-loaded nanoparticle with redox response.

[0015] Figure 2 In vitro release of the HE4 siRNA-loaded nanoparticle with redox response.

[0016] Figure 3 Cellular uptake of the HE4 siRNA-loaded nanoparticle with redox response (A: representative image; B: quantitative results). Compared with the naked FAM-siRNA group, # p <0.05; compared with the lipo / FAM-siRNA group,& p <0.05; compared with NPs / FAM-siRNA group, * p <0.05 Figure 4 Redox-responsive HE4 siRNA-loaded nanoparticles escape from lysosomes.

[0017] Figure 5 Effect of redox-responsive HE4 siRNA-loaded nanoparticles on the viability of OVCAR-3 / DDP cells. Compared with Blank group, # p <0.05; compared with Negative group, $ p <0.05; compared with DDP group, ￥ p <0.05; compared with R-NPs / HE4 siRNA group, & p <0.05; compared with NPs / HE4 siRNA + DDP group, * p <0.05.

[0018] Figure 6 Effect of redox-responsive HE4 siRNA-loaded nanoparticles on the migration and invasion of OVCAR-3 / DDP cells (A: representative images; B: quantitative results). Compared with Blank group, # p <0.05; compared with Negative group, $ p <0.05; compared with DDP group, ￥ p <0.05; compared with R-NPs / HE4 siRNA group, & p <0.05; compared with NPs / HE4 siRNA + DDP group, * p <0.05. (C: representative images; D: quantitative results). Compared with Blank group, # p <0.05; compared with Negative group, $ p <0.05; compared with DDP group, ￥ p <0.05; compared with R-NPs / HE4 siRNA group, & p<0.05; compared with the NPs / HE4siRNA + DDP group, * p <0.05.

[0019] Figure 7 In vivo distribution of redox-responsive HE4-loaded siRNA nanoparticles in OVCAR-3 / DDP cell xenograft mouse model (A: representative fluorescence signal image; B: in vitro imaging of major organs; C: quantitative analysis of in vitro organs). Compared with the Cy5-siRNA group, # p <0.05; compared with the NPs / Cy5-siRNA group; $ p <0.05 Figure 8 Antitumor activity of redox-responsive HE4-loaded siRNA nanoparticles against OVCAR-3 / DDP cell xenograft tumor model mice (A: representative image; B: quantitative analysis; C: representative image). Compared with the Blank group, # p <0.05; compared to Negative, $ p < 0.05; compared with the DDP group, ￥ p <0.05; compared with the R-NPs / HE4 siRNA group, & p <0.05; compared with the NPs / HE4 siRNA + DDP group, * p <0.05. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained commercially.

[0021] The reagents and materials used in the following examples are as follows: PEI-SeSe-PEG was synthesized by Xi'an Ruixi Biosynthesis Co., Ltd.; OVCAR-3 ovarian cancer cells were purchased from Beyotime Biotechnology Co., Ltd. Fetal bovine serum was purchased from Shanghai Sangon Biotech Co., Ltd.; cell culture reagents and consumables were purchased from Beijing Solarbio Biotechnology Co., Ltd.; HE4 siRNA was designed and synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd., and its sequence is as follows: HE4 siRNA positive strand (5'-3'): GUCCUGUGUCACUCCCAAUTT (SEQ ID NO.1) HE4 siRNA antisense strand (5'-3'): AUGGGAGUGACACAGGACTT (SEQ ID NO.2) Example 1

[0022] Preparation and characterization of redox-responsive HE4-loaded siRNA nanoparticles The redox carrier PEI-SeSe-PEG was mixed with the cross-linking agent SMCC (dissolved in DMSO) (molar ratio 1:1) and stirred at room temperature in the dark for 2 h. Unbound SMCC molecules were removed by dialysis (molecular weight cutoff: 10 kDa). After dialysis, the targeting peptide c(RGDfK) was added to the mixture (molar ratio to SMCC 1:1) and stirred at room temperature in the dark for 4 h. After the reaction, the sample was placed in an ultrafiltration centrifuge tube to remove unreacted targeting peptides (molecular weight cutoff: 10 kDa). The resulting solution was then freeze-dried to obtain c(RGDfk)-PEI-SeSe-PEG lyophilized powder. The lyophilized powder can be dissolved in DEPC water for use. HE4 siRNA and the redox carrier c(RGDfk)-PEI-SeSe-PEG (N / P = 10) were mixed, vortexed for 30 s and allowed to stand for 25 min. The two self-assembled into siRNA-loaded nanoparticles R-NPs / HE4 siRNA through positive and negative charge attraction.

[0023] The R-NPs / HE4 siRNA solution was centrifuged at 18,000 rpm for 30 min at 4 °C. The concentration of free siRNA in the supernatant was measured using a microplate reader with the fluorescent dye RiboGreen, and the amount of free siRNA was calculated according to the standard curve. The encapsulation efficiency was calculated using the following formula, and the results showed that the encapsulation efficiency was 97.36 ± 2.95.

[0024] .

[0025] The particle size, polydispersity index, and zeta potential were determined using dynamic light scattering. The results showed that the nanoparticle size was approximately 116.8 ± 16.97 nm; the polydispersity index was 0.129 ± 0.022; and the zeta potential was 19.92 ± 0.32 mV. The prepared nanoparticles, observed under a transmission electron microscope, exhibited regular spherical shapes and relatively uniform dispersion. Figure 1 As shown. Example 2

[0026] In vitro release of redox-responsive HE4-loaded siRNA nanoparticles To verify the redox responsiveness of the drug delivery system, R-NPs / siRNA solutions (containing 200 ng siRNA) with an N / P ratio of 10 were diluted to 1 ml with PBS buffer containing GSH and without GSH. The complex was then transferred to a dialysis unit with a molecular weight cutoff of 20 kDa and incubated on a shaker at room temperature. Samples were taken at 0, 1, 2, 4, 8, 12, 24, 48, and 72 h and frozen at -20 °C, with the same volume of PBS added to make up the original volume. The siRNA release was determined by agarose gel electrophoresis, and the results are shown below. Figure 2 As shown, R-NPs / siRNA, after incubation for 72 h in a medium containing 10 μM glutathione (simulating a normal physiological environment), released less than 25% of the siRNA, based on grayscale values. However, after treatment for 24 h in a medium containing 10 mM glutathione (simulating a tumor microenvironment), the release rate approached 100%. The experimental results demonstrate that the nanocarrier constructed based on c(RGDfK)-PEG-SeSe-PEI exhibits excellent environmental response characteristics: it maintains structural stability under normal physiological conditions to prevent drug leakage, while triggering the breaking of diselenium bonds in the high-concentration reducing microenvironment unique to tumor tissue, thereby achieving specific and rapid release of siRNA. These results indicate that c(RGDfK)-PEG-SeSe-PEI can be an ideal carrier for tumor-targeted delivery of siRNA. Example 3

[0027] Redox-responsive HE4-loaded siRNA nanoparticles uptake in OVCAR-3 / DDP cells Take OVCAR-3 / DDP cells in logarithmic growth phase (1×10⁻⁶) 5 Cells were seeded in 24-well plates at a density of 70% (r-NPs / mL). R-NPs / FAM-siRNA or NPs / FAM-siRNA solutions were added to DMEM medium, with naked FAM-siRNA and Lipo2000-transfected FAM-siRNA (lipo / FAM-siRNA) used as controls. After 4 h of incubation, cells were washed three times and digested. For qualitative observation, cells were fixed in 4% paraformaldehyde for 20 min, and images were captured using a fluorescence microscope to observe the cellular uptake of siRNA nanoparticles. For quantitative observation, cells were suspended in PBS and seeded in 96-well plates. The fluorescence intensity of FAM was detected using a microplate reader with an excitation wavelength of 492 nm and an emission wavelength of 518 nm, reflecting the amount of cellular uptake. Results are as follows: Figure 3As shown, naked siRNA is difficult for cells to take up, and almost no green fluorescence is observed in OVCAR-3 / DDP cells. Lipo2000 is still a commonly used siRNA transfection agent, which can significantly improve the transfection efficiency of siRNA in cells, and significantly increase the intracellular green fluorescence (compared with the naked FAM-siRNA group). p <0.05]. Loading siRNA with c(RGDfK)-modified PEI-SeSe-PEG significantly improved cell transfection rate (compared to the naked FAM-siRNA group). p <0.05), and the uptake rate in OVCAR-3 / DDP cells was significantly higher than that of the group loaded with PEI-SeSe-PEG (compared to the NPs / FAM-siRNA group). p <0.05), and the intracellular fluorescence intensity was higher than that of the Lipo-2000 group (compared to the lipo / FAM-siRNA group). p <0.05), which suggests that nanoparticles made by loading HE4 siRNA onto the c(RGDfK) modified redox carrier PEI-SeSe-PEG have tumor targeting and high cellular uptake, which is beneficial for delivery in OVCAR-3 / DDP cells. Example 4

[0028] Redox-responsive HE4-loaded siRNA nanoparticles escape lysosomal in OVCAR-3 / DDP cells OVCAR-3 / DDP cells were seeded in 12-well plates at a density of 1×10⁶ cells / well. 5 / well, after culturing in DMEM medium for 24 h in a cell culture incubator, R-NPs / HE4 siRNA solution (siRNA labeled with FAM) prepared in serum-free DMEM medium was added, and incubated at 37°C for 4 h. Cells were washed with PBS solution and cultured in serum-free DMEM medium for 0, 2, and 6 h respectively. Then, Lyso Tracker Red staining for 60 min and DAPI staining for 20 min were performed, and images were taken under a fluorescence microscope. The results are as follows. Figure 4As shown, after 4 h of incubation with R-NPs / HE4 siRNA, green fluorescence appeared in the cells. A combined yellow fluorescence signal, indicating that the R-NPs / HE4 siRNA was taken up by OVCAR-3 / DDP cells, and almost all of the R-NPs / HE4 siRNA that entered the cells co-localized with lysosomes. After removal of the R-NPs / HE4 siRNA, the combined yellow fluorescence signal gradually weakened over time. By 10 h of co-incubation, only a small amount of the R-NPs / HE4 siRNA complex co-localized with lysosomes, indicating that the R-NPs / HE4 siRNA escaped from the lysosomes and diffused into the cytoplasm. Example 5

[0029] Effects of redox-responsive HE4-loaded siRNA nanoparticles on the proliferation, migration, and invasion of OVCAR-3 / DDP cells Cell viability was assessed using the CCK-8 assay. OVCAR-3 / DDP cells were seeded in 96-well plates at a density of 1 × 10⁶ cells / well. 4 / well, incubated for 24 h. Then, freshly prepared nanoparticle complexes (siRNA concentration of 100 nM) and cisplatin solution were added to the cells, respectively. An equal volume of culture medium served as a blank control (Blank group), blank nanoparticles without HE4 siRNA served as a negative control (Negative group), and the cisplatin group served as a control (DDP group). After 24 h of incubation, the optical density (OD) at 490 nm was measured using the CCK-8 assay, and cell viability was calculated using the following formula: .

[0030] The results are as follows Figure 5 As shown. In OVCAR-3 / DDP cells, compared with the Blank group, Negative group and DDP group, the cell viability of each nanoparticle complex was significantly decreased ( Figure 5 , p <0.05, and the inhibitory effect of R-NPs / HE4siRNA + DDP group modified with target peptide c (RGDfK) was significantly stronger than that of NPs / HE4siRNA + DDP group without target peptide modification. p <0.05), suggesting that R-NPs / HE4 siRNA combined with cisplatin can significantly inhibit the proliferation of OVCAR-3 / DDP cells, and that the targeting peptides enhance the antitumor effect. There was no significant difference in cell viability between the Negative group and the Blank group ( Figure 5 This indicates that the carrier material used in this patent has no obvious cytotoxic effects.

[0031] Cell migration ability was assessed using a cell scratch assay. OVCAR-3 / DDP cells (5 × 10⁶ cells) were used. 5 Cells were seeded in 6-well plates and cultured for 48 h until adherence. Then, using a 200 μL sterile pipette tip, perpendicular to the cell surface and the marked black line, a small incision was made from one end of the well to the other. Cells were divided into four groups: Blank group, Negative group, DDP group, R-NPs / HE4 siRNA group, NPs / HE4 siRNA + DDP group, and R-NPs / HE4 siRNA + DDP group. 2 mL of the solution was added to each well of each group. Cells were observed and photographed under a microscope at 0 and 72 h. The images were processed using ImageJ software to measure the scratch area, and the cell migration rate at 72 h was calculated using the following formula: .

[0032] Normalization was performed using Blank groups. The results are as follows: Figure 6 As shown. Compared with the Blank group, Negative group, and DDP group, the cell migration rate of each nanoparticle complex group was significantly decreased ( ). p <0.05), and the inhibitory effect of R-NPs / HE4 siRNA + DDP group modified with target peptide c (RGDfK) was significantly stronger than that of NPs / HE4 siRNA + DDP group without target peptide modification. p <0.05), suggesting that the combined use of R-NPs / HE4 siRNA and cisplatin can significantly inhibit the migration of OVCAR-3 / DDP cells.

[0033] Transwell assays were used to assess cell invasion ability. Chambers were placed in 24-well plates. 100 µL of serum-free DMEM medium was added to each chamber for 10 min of hydration. 100 µL of diluted Matrigel was added to each chamber, and the plates were incubated overnight at 37°C to allow gelation. The chambers were then filled with 200 µL of serum-free DMEM medium (Blank group), blank nanoparticles (Negative group), R-NPs / HE4 siRNA nanoparticle complex (R-NPs / HE4 siRNA group), NPs / HE4 siRNA nanoparticle complex + DDP (NPs / HE4 siRNA + DDP group), R-NPs / HE4 siRNA nanoparticle complex + DDP (R-NPs / HE4 siRNA + DDP group), and OVCAR-3 / DDP cells (5 × 10⁶ cells / well). 5Add 750 µL of high-concentration DMEM medium to the lower chamber and incubate for 24 h. Aspirate the medium from the upper chamber, wipe away any unmigrated cells from the upper chamber with a cotton swab, and wash the chamber twice with PBS. Fix the cells with 500 µL of 4% tissue cell fixative at room temperature for 30 min. Remove the chamber from the 4% tissue cell fixative and wash twice with PBS. Add 0.1% crystal violet and stain for 20 min in the dark. Discard the staining solution and wash twice with PBS. Observe under a fluorescence microscope, count the stained cells using ImageJ software, and normalize using a blank control group to obtain the cell invasion rate. Results are as follows: Figure 6 As shown. Compared with the Blank group, Negative group and DDP group, the cell invasion rate of each nanoparticle group was significantly reduced ( ). p <0.05), and the inhibitory effect of R-NPs / HE4 siRNA + DDP group modified with target peptide c (RGDfK) was significantly stronger than that of NPs / HE4 siRNA + DDP group without target peptide modification. p <0.05), suggesting that R-NPs / HE4 siRNA can significantly inhibit the invasion of OVCAR-3 / DDP cells. Example 6

[0034] In vivo distribution of redox-responsive HE4-loaded siRNA nanoparticles in OVCAR-3 / DDP cell xenograft mouse model OVCAR-3 / DDP cells were resuspended in 100 μL PBS solution and inoculated into the left axilla of nude mice to successfully establish a xenograft tumor model. HE4 siRNA labeled with the near-infrared fluorescent dye Cy5 was used to track the distribution of siRNA in the xenograft tumor model. The successfully established models were then randomly divided into three groups, labeled Cy5-siRNA group, NPs / Cy5-siRNA group, and R-NPs / Cy5-siRNA group, respectively. Cy5-siRNA, NPs / Cy5-siRNA, and R-NPs / Cy5-siRNA complexes (siRNA concentration 33 μg / 10 g) were injected intraperitoneally, respectively. At 0 h, 2 h, 5 h, 10 h, and 24 h after injection, the localization and distribution of Cy5-siRNA in the model at the Cy5 wavelength were monitored using an in vivo optical imaging system. Twenty-four hours later, all models were euthanized by cervical dislocation. The tumors, heart, liver, spleen, lungs, and kidneys were dissected and in vitro imaged, and standardized analysis was performed using Tanon software.

[0035] The results are as follows Figure 7As shown, the Cy5-siRNA group exhibited the strongest fluorescence signal intensity 2 hours after intraperitoneal injection, but the fluorescence intensity was mostly limited to abdominal organs, with no fluorescence observed at the tumor site. Intraperitoneal fluorescence gradually weakened after 5 hours, and the fluorescence signal was significantly weaker at 24 hours compared to the previous few hours. The NPs / Cy5-siRNA group also showed a strong fluorescence signal at the peritoneal site 2 hours after intraperitoneal injection, but weaker than the Cy5-siRNA group. The intraperitoneal signal intensity gradually weakened over time, and the signal essentially disappeared at 24 hours. Furthermore, this group showed a certain fluorescence intensity at the tumor site at the 2-hour time point, and this fluorescence did not weaken over time. This is because the redox vector PEI-SeSe-PEG delayed the degradation of siRNA, prolonged its half-life, and possessed a certain ability to target tumors. The results for the R-NPs / Cy5-siRNA group were similar to those for the Cy5-siRNA group, but the fluorescence intensity at the tumor site was significantly enhanced. This may be because c(RGDfK) enhances the tumor targeting of the nanoparticles, enabling them to better localize to tumor tissue and prolong their residence time at the tumor site. The fluorescence intensity results for ex vivo organs are as follows: Figure 7 As shown, the modification of PEI-SeSe-PEG and c(RGDfk)-PEI-SeSe-PEG can indeed improve the targeting of siRNA by observing in vitro imaging results. Compared with the Cy5-siRNA group, the fluorescence intensity of tumor sites in the other two groups was significantly enhanced (p<0.05 compared with the NPs / Cy5-siRNA group and the R-NPs / Cy5-siRNA group), while no fluorescence signal was detected in the Cy5-siRNA group. Meanwhile, the fluorescence intensity of the heart, liver, spleen, lungs, and kidneys in the Cy5-siRNA group was much higher than that in the other two groups, especially the liver and kidneys, which showed the highest intensity. This indicates that the siRNA had been gradually excreted from the body through organ metabolism at this time. In contrast, the fluorescence intensity of the organs in the other two groups with carrier modification was weaker. This also reflects that c(RGDfk)-PEI-SeSe-PEG and PEI-SeSe-PEG can significantly prolong the survival time of siRNA in cells and improve its targeting ability to tumor cells. Moreover, the effect of c(RGDfK) modified R-NPs is more significant, showing the superior targeting ability of the targeting peptide.

[0036] In summary, R-NPs / HE4 siRNA combined with cisplatin can specifically improve siRNA delivery and prolong its half-life, and exhibits excellent tumor-targeting ability. Example 7

[0037] Antitumor activity of redox-responsive HE4-loaded siRNA nanoparticles against OVCAR-3 / DDP cell xenograft tumor model mice Successfully established xenograft tumor models were randomly divided into 6 groups, with 6 animals in each group: Blank control group, Negative control group, cisplatin group (DDP), R-NPs / HE4 siRNA group (R-NPs / HE4 siRNA), NPs / HE4 siRNA + DDP group (NPs / HE4 siRNA + DDP), and R-NPs / HE4 siRNA + DDP group (R-NPs / HE4 siRNA + DDP). The Blank group received an intraperitoneal injection of an equal volume of 9% saline. The HE4 siRNA nanocomposite was administered via intraperitoneal injection every 3 days (siRNA concentration of 33 μg / animal), for a total of 6 injections. Cisplatin administration: 5 min after injection of the nanoparticle composite, it was administered via intraperitoneal injection (0.5 mg / kg, total volume 1 mL), at the same frequency as the HE4 siRNA nanocomposite. During the injection period, the tumor volume of the xenograft tumor model was measured and recorded every 3 days. The vertical diameter of the tumor was measured with calipers, and the tumor volume (V) was calculated according to the formula for a rotating ellipsoid (A is the major axis, B is the minor axis). After treatment, the model was euthanized by cervical dislocation, the tumor was dissected, and photographs were taken.

[0038] V = A × (B 2 / 2) In the OVCAR-3 / DDP xenograft tumor model, no deaths occurred in any group of nude mice. Results are as follows: Figure 8 As shown, during the first 9 days of treatment, the tumor growth rate remained consistent across all groups. After 9 days of treatment, the tumor volume in the R-NPs / HE4 siRNA + DDP group and the NPs / HE4 siRNA + DDP group was significantly smaller than that in the Blank group, Negative group, and DDP group (p<0.05 compared to the Blank group, Negative group, and DDP group). After 12 days of administration, the tumor volume in each nanoparticle complex group was smaller than that in the Blank group, Negative group, and DDP group (p<0.05 compared to the Blank group, Negative group, and DDP group), and each nanoparticle complex group exhibited an inhibitory effect on tumor growth. From day 15 to day 18 of administration, the inhibitory effect of each nanoparticle complex continued to increase. The inhibitory effect of the R-NPs / HE4 siRNA + DDP group was significantly stronger than that of the R-NPs / HE4 siRNA group and the NPs / HE4 siRNA + DDP group (p<0.05 compared with the R-NPs / HE4 siRNA group and the NPs / HE4 siRNA + DDP group).

[0039] In summary, R-NPs / HE4 siRNA nanoparticles combined with cisplatin can significantly inhibit tumor growth, and the tumor-suppressing effect is stronger than that of NPs / HE4 siRNA + DDP.

[0040] The experimental results of the above embodiments show that the c(RGDfK) peptide-modified poly(ethyleneimine)-diselement bond-poly(ethylene glycol) (PEI-SeSe-PEG) loaded HE4 siRNA nanoparticles constructed in this invention have tumor targeting and redox responsiveness. They can be taken up by tumor cells and can achieve lysosomal escape and release at the tumor site. They can significantly inhibit the proliferation, invasion and metastasis of ovarian cancer cells. Moreover, they have good tumor targeting, anti-tumor activity and biocompatibility in animals. They can effectively improve the sensitivity of cisplatin-resistant ovarian cancer to cisplatin, providing a new strategy for targeted therapy of cisplatin-resistant ovarian cancer.

Claims

1. A redox-responsive HE4-loaded siRNA nanoparticle, characterized in that, The nanoparticles are composed of a redox-responsive tumor-targeting carrier material and HE4 siRNA; wherein the redox-responsive tumor-targeting carrier material is poly(ethyleneimine)-diselement bond-poly(ethylene glycol) (PEI-SeSe-PEG) modified with the targeting peptide c(RGDfK).

2. The redox-responsive HE4-loaded siRNA nanoparticle as described in claim 1, characterized in that, The forward primer sequence of the HE4 siRNA is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.

2.

3. The method for preparing redox-responsive HE4-loaded siRNA nanoparticles as described in claim 1, characterized in that, Includes the following steps: Step 1: Mix the crosslinking agent SMCC with PEI-SeSe-PEG at a molar ratio of 1:1 and stir at room temperature in the dark for 2 hours. Dialyze the resulting product in pure water for 48 hours (molecular weight cutoff: 10KD) to obtain a mixture. Step 2: Add c(RGDfK) to the above mixture (molar ratio with SMCC is 1:1), stir at room temperature in the dark for 4 hours, and freeze dry for 48 hours to obtain c(RGDfk)-PEI-SeSe-PEG freeze-dried powder. Step 3: Dissolve the powder in DEPC water, add HE4 siRNA solution at N / P = 10, vortex for 30s and let stand for 25min to self-assemble c(RGDfK) modified PEI-SeSe-PEG loaded HE4 siRNA nanoparticles.

4. The application of the redox-responsive HE4-loaded siRNA nanoparticles as described in claim 1 in the treatment of cisplatin-resistant ovarian cancer.

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