A pH-responsive SOX10 siRNA-loaded nanoparticle, its preparation method and application in the treatment of triple-negative breast cancer and its bone metastasis

Abstract

The invention discloses a pH-responsive SOX10 siRNA-loaded nanoparticle, a preparation method thereof and application thereof in treating triple-negative breast cancer and bone metastasis thereof. The nanoparticle consists of a pH-responsive tumor-targeting carrier material and SOX10 siRNA. The pH-responsive tumor-targeting carrier material is poly(ethylene glycol)-poly(2-(dimethylamino)ethyl methacrylate) (PEG-PDMAEMA) modified with IF peptide and GALA peptide. The nanoparticles are mixed with the carrier material and siRNA at an N / P ratio of 40:1, and self-assembled into nanoparticles with a particle size of about 100 nm. The in vitro release is pH-responsive and released at pH 5.0. The pH-responsive SOX10 siRNA-loaded nanoparticles prepared by the present invention have high affinity for breast cancer cells, high cell uptake, can achieve lysosomal escape, can significantly inhibit the proliferation, migration and invasion of triple-negative breast cancer cells, and also inhibit the chemotaxis, adhesion and anchoring-independent cloning of triple-negative breast cancer cells to the bone microenvironment, and have a significant effect on treating triple-negative breast cancer and its bone metastasis. The nanoparticle preparation method is simple, the anti-cancer effect is significant, and it has good application value and market prospects.

Description

Technical Field

[0001] The invention relates to a pH-responsive SOX10 siRNA-loaded nanoparticle, a preparation method thereof and the effect of the nanoparticle in treating triple-negative breast cancer and bone metastasis thereof, and belongs to the field of medicine. Background Art

[0002] Breast cancer is the most common female cancer worldwide. It accounts for approximately 25% of all cancer types and 12% of all new cancer cases. There are several biological subtypes of breast cancer, including triple-negative breast cancer (TNBC). TNBC is characterized by the lack of expression of progesterone receptor (PR), estrogen receptor (ER), and human epidermal growth factor receptor-2 (HER-2), and accounts for approximately 15-20% of all breast cancers. Due to the aggressive biology of TNBC with early and extensive metastasis and the lack of available targeted therapies, patients have poor survival outcomes. The main metastatic sites of TNBC are bones, lungs, brain, liver, soft tissues, and adrenal glands, among which bone is the most common metastatic site for distant metastasis of TNBC. TBNC metastasis is a highly complex and poorly understood process involving multiple steps, including genetic and epigenetic alterations, angiogenesis, tumor-stroma interactions, basement membrane intravasation, circulation survival, and extravasation to distant tissues. Once TNBC metastasizes, the prognosis is very poor, with an average median overall survival (OS) of only 10-13 months. Therefore, there is an urgent need to explore new biomarkers to predict the metastasis of TNBC and develop specific treatments.

[0003] SOX10 is a member of the SRY-related high-mobility group box (SOX) family of key transcription factors. SOX10 mediates the differentiation of neural crest into oligodendrocytes, melanocytes, and glial cells, and promotes mesenchymal transition of mammary gland cells. Recent studies have found that SOX10 is expressed in primary and metastatic TNBC. SOX10 may be considered as a new biomarker for the diagnosis of TNBC. Therefore, SOX10 may be a potential gene target for the treatment of TNBC, but there is no relevant research.

[0004] In recent years, nucleic acid drugs have become potential candidate drugs for the treatment of many diseases, especially cancer. Small interfering RNA (siRNA) is a synthetic non-coding short RNA with a length of 21-22 nucleotides, which can be used as an RNA interference tool to silence target genes. Studies have found that overexpression of SOX10 in breast cancer tissues is associated with prognosis. In preliminary experiments, it was found that after TNBC cells MDA-MB-231 were transfected with SOX10 siRNA using Lipofectamine 2000, cell proliferation, migration and invasion were significantly reduced. However, siRNA is easily degraded in the blood, resulting in less accumulation in tumor tissues. Therefore, in order to improve the stability and tumor targeting of siRNA, the development of an efficient delivery targeted drug delivery system is the main problem to be solved in current research, which will provide a new strategy for the treatment of bone metastasis of triple-negative breast cancer. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a pH-responsive SOX10 siRNA-loaded nanoparticle, a preparation method thereof, and its effect in treating triple-negative breast cancer and its bone metastasis.

[0006] The technical solution of the present invention is as follows:

[0007] The invention provides a pH-responsive SOX10 siRNA-loaded nanoparticle, which is composed of a pH-responsive tumor-targeting carrier material and SOX10 siRNA. The pH-responsive tumor-targeting carrier material is poly(ethylene glycol)-poly(2-(dimethylamino)ethyl methacrylate) (PEG-PEMAEMA) modified with IF peptide and GALA peptide. The SOX10 siRNA at least comprises SOX10 siRNA1, SOX10 siRNA2 and SOX10 siRNA3, wherein the forward primer sequence of SOX10 siRNA1 is shown in SEQ ID NO.1, and the reverse primer sequence thereof is shown in SEQ ID NO.2, the forward primer sequence of SOX10 siRNA2 is shown in SEQ ID NO.3, and the reverse primer sequence thereof is shown in SEQ ID NO.4, the forward primer sequence of SOX10 siRNA3 is shown in SEQ ID NO.5, and the reverse primer sequence thereof is shown in SEQ ID NO.6.

[0008] The sequence of SEQ ID NO.1 is: GCCCUCAGGACCCUAUUAUTT;

[0009] The sequence of SEQ ID NO.2 is: AUAAUAGGGUCCUGAGGGCTT;

[0010] The sequence of SEQ ID NO.3 is: AGGCGGACGAUGACAAGUUTT;

[0011] The sequence of SEQ ID NO.4 is: AACUUGUCAUCGUCCGCCUTT;

[0012] The sequence of SEQ ID NO.5 is: AAAGACCACCCGGACUACATT;

[0013] The sequence of SEQ ID NO.6 is: UGUAGUCCGGGUGGUCUUUTT.

[0014] The present invention also provides a method for preparing pH-responsive SOX10 siRNA-loaded nanoparticles, comprising the following steps:

[0015] (1) The thiol-modified IF-7 peptide and GALA peptide were dissolved in DEPC water with MAL-PEG-PDMAEMA at a molar ratio of 1:1 and mixed evenly. The mixture was incubated at room temperature for 4 h. The obtained products were dialyzed in pure water for 48 h (molecular weight cutoff 5 KD) and freeze-dried for 48 h to obtain IF-7-modified PEG-PDMAEMA and GALA-modified PEG-PEMAEMA, respectively.

[0016] (2) IF-7 modified PEG-PDMAEMA and GALA modified PEG-PEMAEMA were mixed in a molar ratio of 1:1, dissolved in DEPC water, and SOX10 siRNA solution was added at an N / P ratio of 1:1 to obtain SOX10 siRNA nanoparticles loaded with IF-7 and GALA modified PEG-PEMAEMA by self-assembly.

[0017] The present invention also provides a pH-responsive SOX10 siRNA-loaded nanoparticle for use in treating triple-negative breast cancer and bone metastasis thereof.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] 1. The nano drug delivery system constructed by the present invention is pH responsive and can release siRNA in the acidic tumor microenvironment, which is beneficial for exerting its effect at the tumor site.

[0020] 2. The nano drug delivery system constructed by the present invention can improve the transfection efficiency of siRNA, achieve lysosomal escape, and has tumor targeting, thereby improving the anti-tumor effect and reducing side effects.

[0021] 3. The SOX10 siRNA nanoparticles constructed by the present invention can inhibit the proliferation, migration and invasion of triple-negative breast cancer cells, as well as the chemotaxis, adhesion and proliferation of triple-negative breast cancer cells to the bone microenvironment, and can be used to treat triple-negative breast cancer and its bone metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Transmission electron microscopy images of pH-responsive SOX10 siRNA-loaded nanoparticles.

[0023] Figure 2 In vitro release profile of pH-responsive SOX10 siRNA-loaded nanoparticles.

[0024] Figure 3 Cellular uptake of pH-responsive SOX10 siRNA-loaded nanoparticles (A: representative images; B: quantitative results). *P<0.05 compared with the naked siRNA group; △P<0.05 compared with MCF-10A cells.

[0025] Figure 4 Lysosomal escape of pH-responsive SOX10 siRNA-loaded nanoparticles.

[0026] Figure 5 Effects of pH-responsive SOX10 siRNA-loaded nanoparticles on the viability of MDA-MB-231 cells (A: representative images; B: quantitative results). Compared with the blank control group, *P<0.05; compared with the negative control group, #P<0.05; compared with naked siRNA, ^P<0.05.

[0027] Figure 6 Effects of pH-responsive SOX10 siRNA-loaded nanoparticles on the migration of MDA-MB-231 cells (A: representative images; B: quantitative results). Compared with the blank control group, *P<0.05; compared with the negative control group, #P<0.05; compared with naked siRNA, ^P<0.05.

[0028] Figure 7 Effects of pH-responsive SOX10 siRNA-loaded nanoparticles on the invasion of MDA-MB-231 cells (A: representative images; B: quantitative results). Compared with the blank control group, *P<0.05; compared with the negative control group, #P<0.05; compared with naked siRNA, ^P<0.05.

[0029] Figure 8Effects of pH-responsive SOX10 siRNA-loaded nanoparticles on the chemotaxis of MDA-MB-231 cells toward the bone microenvironment (A: representative images; B: quantitative results). Compared with the blank control group, *P<0.05; compared with the negative control group, #P<0.05; compared with naked siRNA, ^P<0.05.

[0030] Fig. 9 Effects of pH-responsive SOX10 siRNA-loaded nanoparticles on the adhesion of MDA-MB-231 cells to the bone microenvironment (A: representative images; B: quantitative results). Compared with the blank control group, *P<0.05; compared with the negative control group, #P<0.05; compared with naked siRNA, ^P<0.05.

[0031] Fig.10 Effects of pH-responsive SOX10 siRNA-loaded nanoparticles on the anchorage-independent clonal formation of MDA-MB-231 cells to the bone microenvironment (A: representative images; B: quantitative results). Compared with the blank control group, *P<0.05; compared with the negative control group, #P<0.05; compared with naked siRNA, ^P<0.05. DETAILED DESCRIPTION

[0032] The technical scheme of the present invention is further described below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The materials, reagents, etc. used in the embodiments of the present invention, unless otherwise specified, can all be obtained from commercial sources.

[0033] The reagents and materials used in the following examples are as follows:

[0034] PEG-PEMAEMA, IF peptide and GALA peptide were synthesized by Xi'an Ruixi Biosynthesis Co., Ltd.; TNBC cells MDA-MB-231, human mammary epithelial cells MCF-10A and osteoblast-like cells MG-63 were purchased from Shanghai Bolson Biotechnology Co., Ltd.; fetal bovine serum was purchased from Invitrogen Life Sciences, Inc.; cell culture reagents and consumables were purchased from Beijing Solebold Biotechnology Co., Ltd.; SOX10 siRNA was designed and synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd., and its sequence is as follows:

[0035] SOX10 siRNA1 positive strand (5'-3'): GCCCUCAGGACCCUAUUAUTT (SEQ ID NO.1)

[0036] SOX10 siRNA1 antisense strand (5'-3'): AUAAUAGGGUCCUGAGGGCTT (SEQ ID NO. 2)

[0037] SOX10 siRNA2 positive strand (5'-3'): AGGCGGACGAUGACAAGUUTT (SEQ ID NO. 3)

[0038] SOX10 siRNA2 antisense strand (5'-3'): AACUUGUCAUCGUCCGCCUTT (SEQ ID NO. 4)

[0039] SOX10 siRNA3 positive strand (5'-3'): AAAGACCACCCGGACUACATT (SEQ ID NO.5)

[0040] SOX10 siRNA3 antisense strand (5'-3'): UGUAGUCCGGGUGGUCUUUTT (SEQ ID NO.6)

[0041] Example 1

[0042] Preparation and characterization of pH-responsive SOX10 siRNA-loaded nanoparticles

[0043] The IF-7 peptide and GALA peptide containing thiol modification were dissolved in DEPC water with MAL-PEG-PDMAEMA at a molar ratio of 1:1 and mixed evenly, incubated at room temperature for 4 hours, and the obtained product was dialyzed in pure water for 48 hours (molecular weight cutoff 5KD), and freeze-dried for 48 hours to obtain IF-7 modified PEG-PDMAEMA and GALA modified PEG-PEMAEMA, respectively. IF-7 modified PEG-PDMAEMA and GALA modified PEG-PEMAEMA were mixed at a molar ratio of 1:1, dissolved in DEPC water, and SOX10 siRNA solution was added at an N / P ratio of 40:1, and self-assembled to obtain IF-7 and GALA modified PEG-PEMAEMA loaded SOX10 siRNA nanoparticles (IG-NP).

[0044] The IG-NP solution was centrifuged at 18000 rpm for 30 min at 4°C. The free siRNA concentration in the supernatant was measured by absorbance using a fluorescent dye RiboGreen using an enzyme marker, and the amount of free siRNA was calculated based on the standard curve. The encapsulation efficiency was calculated according to the following formula, and the results showed that the encapsulation efficiency was 92.30±2.73%.

[0045]

[0046] The particle size, polydispersity coefficient and zeta potential were determined by dynamic light scattering. The results showed that the particle size of the nanoparticles was about 103.54±3.38nm; the polydispersity coefficient was 0.254±0.019, and the zeta potential was 21.49±0.78mV. The prepared nanoparticles were observed to be regular spherical under transmission electron microscopy and were relatively evenly dispersed. Figure 1 shown.

[0047] Example 2

[0048] pH-responsive release of SOX10 siRNA-loaded nanoparticles in vitro

[0049] The prepared nanoparticles were dissolved in 2 mL of PBS buffer solutions with different pH values ​​(7.4 and 5.0), and the siRNA concentration was set to 20 nM. The solution was transferred to a dialysis bag (molecular weight cutoff 20 kDa) and dialyzed with 25 mL of PBS buffer solution at 37 °C with stirring. At 0, 2, 6, 12, 16, 20, 24, and 48 h, 1 ml of the dialysate was taken out, and the same volume of fresh solution was added. The concentration of free siRNA in the dialysate was determined using the fluorescent dye RiboGreen, and the in vitro release curve was plotted. The results are shown in Figure 2. Figure 2 As shown. In environments with pH 7.5 and 5.0, the release behavior of IG-NPs was time-dependent. As time went on, the release rate increased significantly within 24 hours, and then the release rate slowed down to 48 hours. At the same time point, the release rate at pH 5.0 was significantly higher than that at pH 7.4. After 24 hours of incubation, the amount of siRNA released at physiological pH (pH 7.4) was only about 30%, while the amount of siRNA released at pH 5.0 increased to more than 80%. pH-triggered IG-NP release is suitable for delivering siRNA to tumor sites.

[0050] Example 3

[0051] Uptake of pH-responsive SOX10 siRNA-loaded nanoparticles in MDA-MB-231 and MCF-10A cells

[0052] MDA-MB-231 cells (1×10 4 / mL) and MCF-10A (1×10 5 / mL) were inoculated in a 24-well plate. When the cell density reached 70%, IG-NP solution was added to the DMEM medium, and naked FAM-siRNA (naked siRNA) and FAM-siRNA (Lipo2000) transfected with Lipo2000 were used as controls. After incubation for 4 hours, the cells were washed 3 times and digested. For qualitative observation, the cells were fixed with 4% paraformaldehyde for 20 minutes, and images were taken with a fluorescence microscope to observe the cellular uptake of siRNA nanoparticles. For quantitative observation, the cells were suspended in PBS and inoculated in a 96-well plate. The fluorescence intensity of FAM was detected by a microplate reader with an excitation wavelength of 492nm and an emission wavelength of 518nm. The fluorescence intensity can reflect the amount of cellular uptake. The results are shown in Figure 3 As shown. Naked siRNA is difficult to be taken up by cells, and almost no green fluorescence is observed in MCF-10A and MDA-MB-231 cells. Lipo2000 is still a commonly used siRNA transfection agent, which can significantly improve the transfection efficiency of siRNA in both cells, and the intracellular green fluorescence is significantly increased (compared with the naked siRNA group, P<0.05). The cell transfection rate was improved by loading siRNA with PEG-PDMAEMA modified with IF-7 and GALA (compared with the naked siRNA group, P<0.05), and the uptake rate in MDA-MB-231 cells was significantly higher than that in MCF-10A cells (P<0.05), and the intracellular fluorescence intensity was comparable to that of the Lipo-2000 group, indicating that nanoparticles made of siRNA loaded with PEG-PDMAEMA modified with IF-7 and GALA dipeptides have tumor targeting and high cell uptake, which is conducive to delivery in MDA-MB-231 cells.

[0053] Example 4

[0054] Lysosomal escape of pH-responsive SOX10 siRNA-loaded nanoparticles in MDA-MB-231 cells

[0055] MDA-MB-231 cells were seeded in 12-well plates at a density of 1×10 4 / well, after culturing in DMEM medium in a cell culture incubator for 24 hours, IG-NP solution prepared in serum-free DMEM medium (siRNA labeled with FAM) was added and incubated at 37°C for 4 hours. The cells were washed with PBS and incubated in serum-free DMEM medium for 0, 2, and 6 hours respectively. Then, Lyso Tracker Red was used for staining for 60 minutes and DAPI was used for staining for 20 minutes. The cells were photographed under a fluorescence microscope. The results are shown in the figure. Figure 4As shown. After IG-NPs were incubated with cells for 4 hours, green fluorescence appeared in the cells, and a merged yellow fluorescence signal appeared between the green fluorescence and the red fluorescence, indicating that IG-NPs were taken up by MDA-MB-231 cells, and that almost all IG-NPs that entered the cells were co-localized with lysosomes. After the removal of GC-NPs, the merged yellow fluorescence signal gradually weakened with the extension of time. At 10 hours, only a small amount of IG-NP complexes were co-localized with lysosomes, indicating that IG-NPs escaped from lysosomes and diffused into the cytoplasm.

[0056] Example 5

[0057] Effects of pH-responsive SOX10 siRNA-loaded nanoparticles on proliferation, migration and invasion of MDA-MB-231 cells

[0058] The cell viability was detected by MTT assay. MDA-MB-231 and MCF-10A cells were seeded in 96-well plates at a density of 4 × 10 5 / well, incubated for 24h. Subsequently, freshly prepared naked SOX10 siRNA (n-siRNA, 40nM) and IG-NP solutions of different concentrations (10, 20, 40, 80, 120nM) were added to the cells. An equal amount of culture medium was used as a blank control, and blank nanoparticles without SOX10 siRNA were used as a negative control. After incubation for 24h, the optical density (OD) at 490nm was determined by the MTT method, and the cell viability was calculated as follows:

[0059]

[0060] The results are as follows Figure 5 As shown. In MDA-MB-231 cells, compared with the blank control group and naked siRNA group, the cell viability of each concentration of IG-NP group was significantly decreased ( Figure 5 A, P<0.05), and in a dose-dependent manner, indicating that IG-NP can significantly inhibit the proliferation of MDA-MB-231 cells. In MCF-10A cells, only when the concentration of IG-NP was above 40nM, its cell viability was lower than that of the blank control group and the naked siRNA group ( Figure 5 B, P<0.05), but the cell viability was above 75%, indicating that IG-NP had no obvious cytotoxic effect on normal cells. The cell viability of MDA-MB-231 cells and MCF-10A cells in the negative control group was not significantly different from that in the blank control group ( Figure 5 A and 5B), indicating that the carrier material used in this patent has no obvious cytotoxic effect. Therefore, the concentration of IG-NP was set to 80nM to observe its effect on the migration and invasion of MDA-MB-231 cells.

[0061] The cell migration ability was detected by cell scratch test. MDA-MB-231 cells (4×10 5 / well) were inoculated in a 6-well plate and cultured for 48 hours until the cells adhered to the wall. A 200 μL sterile pipette tip was used to vertically hold the cell surface and vertically to the marked black line, and scratched from one end of the well to the other end. The cells were divided into a blank control group, a negative control group, a naked siRNA group, and an IG-NP group. Each well of each group of cells was added with 2 mL of culture medium, blank nanoparticles, naked siRNA, and IG-NP solution, respectively. The cells were observed and photographed under a microscope at 0 and 48 hours, and the scratch area was measured by processing the images with Image J software. The cell migration rate at 48 hours was calculated as follows:

[0062]

[0063] The blank control group was used for normalization. Figure 6 Compared with the blank control group, negative control group and naked siRNA group, the cell migration rate in the IG-NP group was significantly decreased (P<0.05), indicating that IG-NP can significantly inhibit the migration of MDA-MB-231 cells.

[0064] Transwell assay was used to detect cell invasion ability. The chamber was placed in a 24-well plate. 100 μL of serum-free DMEM medium was added to the chamber for hydration for 10 min. 100 μL of diluted Matrigel was added to each chamber and incubated overnight in a 37°C incubator to gel. 200 μL of serum-free DMEM medium (blank control group), blank nanoparticles (negative control group), naked siRNA and IG-NP solution, and MDA-MB-231 cells (4×10 5 / mL), add 750μL high concentration DMEM culture medium to the lower chamber and culture it in an incubator for 24h. Aspirate the culture medium in the upper chamber, wipe off the cells in the upper chamber that have not migrated with a cotton swab, and wash the chamber twice in PBS. Fix the cells with 4% tissue cell fixative, 500μL per well, and fix at room temperature for 30min. Remove the chamber from the 4% tissue cell fixative and wash it twice with PBS. Add 0.1% crystal violet and stain for 20min in the dark. Discard the staining solution and wash it twice with PBS. Take pictures and observe under a fluorescence microscope, use Image J software to count the stained cells, and normalize them with the blank control group to obtain the cell invasion rate. The results are shown in Figure 7 Compared with the blank control group, negative control group and naked siRNA group, the cell invasion rate in the IG-NP group was significantly decreased (P<0.05), indicating that IG-NP can significantly inhibit the invasion of MDA-MB-231 cells.

[0065] Example 6

[0066] Effects of pH-responsive SOX10 siRNA-loaded nanoparticles on chemotaxis, adhesion, and anchorage-independent colony formation of MDA-MB-231 cells to the bone microenvironment

[0067] The cell chemotaxis assay was used to detect the chemotaxis of MDA-MB-231 cells to the in vitro bone microenvironment simulated by MG-63 cells. An appropriate amount of MG-63 cells were inoculated in a 24-well plate and cultured for 24 hours after attachment. The next day, fresh culture medium containing 10% fetal bovine serum was replaced. At the same time, MDA-MB-231 cells were transfected with culture medium (blank control group), blank nanoparticles (negative control group), naked siRNA and IG-NP solution for 24 hours, and then digested and counted under a microscope. 2.5×10 4 The cells were centrifuged at 1000rpm for 5min, the supernatant was taken, 500μL serum-free medium was added, and the cells were resuspended. The cells were added to the chemotaxis chamber and placed on the upper layer of a 24-well plate. After incubation for 12h, the chemotaxis chamber was removed and fixed with 4% paraformaldehyde for 20min at room temperature. The unpenetrated cells on the upper layer were wiped with a cotton swab, washed with PBS, and stained with crystal violet for 5min. Rinse with PBS three times, dry at room temperature, fix the cells with neutral glue, observe under a fluorescence microscope and take pictures. The stained cells are penetrated cells and are counted using Image J software. The cell penetration amount was normalized with the blank control group, and the cell chemotaxis rate was calculated. The results are shown in Figure 8 Compared with the blank control group, negative control group and naked siRNA group, the cell chemotaxis rate in the IG-NP group was significantly decreased (P<0.05), indicating that IG-NP can significantly inhibit the chemotaxis of MDA-MB-231 cells to the bone microenvironment, thereby inhibiting bone metastasis.

[0068] The cell heterogeneity adhesion assay was used to detect the adhesion ability of MDA-MB-231 cells to the in vitro bone microenvironment simulated by MG-63 cells. An appropriate amount of MG-63 cells were inoculated on a 24-well plate for an appropriate time to make the cell fusion rate reach 100%. MDA-MB-231 cells were transfected with culture medium (blank control group), blank nanoparticles (negative control group), naked siRNA and IG-NP solution for 24 hours, and then digested and counted under a microscope. MDA-MB-231 cells (1×10 5 / well) were suspended in 500 μL of culture medium and inoculated into the MG-63 cells with a fusion rate of 100%. After 20 minutes of adhesion, the non-adherent cells were removed with a pipette and gently washed with 250 μL of PBS solution. The non-adherent cells were counted, and the cell adhesion rate was calculated as follows and normalized with the blank control group:

[0069]

[0070] The MDA-MB-231 cells adhered to the MG-63 cells were fixed with 4% paraformaldehyde and photographed under bright field fluorescence microscope. Fig. 9 Compared with the blank control group, negative control group and naked siRNA group, the cell adhesion rate in the IG-NP group was significantly decreased (P<0.05), indicating that IG-NP can significantly inhibit the adhesion of MDA-MB-231 cells to the bone microenvironment, thereby inhibiting bone metastasis.

[0071] The soft agar clone formation assay was used to detect the anchorage-independent clone formation of MDA-MB-231 cells into the in vitro bone microenvironment simulated by MG-63 cells. The serum-free conditioned medium of MG-63 cells in the logarithmic growth phase was collected. 0.6% low-density agarose was prepared with serum-free complete medium, 1 mL was placed on the bottom of a 6-well plate, and cooled to solidify at 4°C. MDA-MB-231 cells transfected for 24 hours were digested and counted under a microscope. 1×10 3 The cells were mixed with 0.3% low-density agarose in complete medium containing 10% fetal bovine serum, inoculated on 0.6% low-density agarose, and incubated overnight. The next day, 500 μL of conditioned medium (MG-63: fresh medium containing 20% ​​FBS = 1:1) was added to the 6-well plate, and the conditioned medium was replaced every other day. After 14 days of culture, the 6-well plate was observed under a 100x inverted microscope, and 5 fields of view were randomly selected to count the number of clones with more than 50 cells (>0.05mm clones) and the total number of clones. The clone formation rate was calculated according to the following formula and normalized to the blank control group:

[0072]

[0073] The results are as follows Fig.10 Compared with the blank control group, negative control group and naked siRNA group, the cell clone formation rate in the IG-NP group was significantly decreased (P<0.05), indicating that IG-NP can significantly inhibit the anchorage-independent clone formation of MDA-MB-231 cells to the bone microenvironment, thereby inhibiting bone metastasis.

[0074] The experimental results of the above examples show that the IF-7 and GALA peptide-modified PEG-PDMAEMA-loaded SOX10 siRNA nanoparticles constructed by the present invention have tumor targeting and pH responsiveness, can be taken up by tumor cells and can achieve lysosomal escape, and are released at the tumor site, and can significantly inhibit the proliferation, invasion and metastasis of triple-negative breast cancer cells, inhibit the chemotaxis, adhesion and anchorage-independent cloning of triple-negative breast cancer cells to the bone microenvironment, thereby inhibiting triple-negative breast cancer and its bone metastasis, providing a new strategy for the treatment of triple-negative breast cancer and its bone metastasis.

Claims

1. A pH-responsive SOX10 siRNA-loaded nanoparticle, characterized in that: The nanoparticles are composed of a pH-responsive tumor-targeting carrier material and SOX10 siRNA; Wherein, the pH-responsive tumor targeting carrier material is PEG-PDMAEMA modified with IF-7 peptide and GALA peptide respectively; The SOX10 siRNA at least comprises SOX10 siRNA1, SOX10 siRNA2 and SOX10 siRNA3, wherein the forward primer sequence of SOX10 siRNA1 is shown as SEQ ID NO.1, and the reverse primer sequence thereof is shown as SEQ ID NO.2, the forward primer sequence of SOX10 siRNA2 is shown as SEQ ID NO.3, and the reverse primer sequence thereof is shown as SEQ ID NO.4, the forward primer sequence of SOX10 siRNA3 is shown as SEQ ID NO.5, and the reverse primer sequence thereof is shown as SEQ ID NO.

6.

2. The method for preparing a pH-responsive SOX10 siRNA-loaded nanoparticle according to claim 1, characterized in that: The following steps are involved: Step 1: Dissolve IF-7 peptide and GALA peptide containing thiol group modification with MAL-PEG-PDMAEMA in DEPC water at a molar ratio of 1:1 and mix evenly. Incubate at room temperature for 4 hours. The obtained product is dialyzed in pure water for 48 hours with a molecular weight cutoff of 5 KD. Freeze-dried for 48 hours to obtain IF-7 modified PEG-PDMAEMA and GALA modified PEG-PEMAEMA respectively. Step 2: Mix IF-7 modified PEG-PDMAEMA and GALA modified PEG-PEMAEMA in a molar ratio of 1:1, dissolve in DEPC water, add SOX10 siRNA solution at an N / P ratio of 40:1, and self-assemble to obtain IF-7 and GALA modified PEG-PEMAEMA loaded SOX10 siRNA nanoparticles.

3. Use of the pH-responsive SOX10 siRNA-loaded nanoparticles according to claim 1 in the preparation of a drug for treating triple-negative breast cancer and its bone metastasis.

Images