SOX9-loaded siRNA lipid nanoparticles, preparation method thereof and application of SOX9-loaded siRNA lipid nanoparticles in treatment of colorectal cancer

Abstract

The invention discloses a lipid nanoparticle loaded with SOX9siRNA (small interfering Ribonucleic Acid). The lipid nanoparticle is prepared from a tumor targeting carrier material and the SOX9siRNA. The tumor targeting carrier material is prepared from phospholipide-polyethylene glycol 2000 (DSPE-PEG2000-cRGDfk) modified by cRGDfK peptide, 4-(N, N-dimethylamino) butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), distearoyl phosphatidylcholine (DSPC), dimyristoyl glycerol-polyethylene glycol 2000 (DMG-PEG2000) and cholesterol, and the preparation method of the tumor targeting carrier material is characterized in that the tumor targeting carrier material is prepared from the cRGDfK peptide modified phospholipide-polyethylene glycol 2000 (DSPE-PEG2000-cRGDfk), the 4-(N, N-dimethylamino) butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA) The prepared SOX9siRNA loaded lipid nanoparticles can be taken by colorectal cancer cells, lysosome escape is achieved, and proliferation, migration and invasion of the colorectal cancer cells are inhibited. Meanwhile, the compound also has good tumor targeting property, anti-tumor activity and safety in a colorectal cancer tumor-bearing mouse body.

Description

Technical Field

[0001] The invention relates to a SOX9 siRNA-loaded lipid nanoparticle, a preparation method thereof and the effect of the nanoparticle in treating colorectal cancer, and belongs to the field of medicine. Background Art

[0002] Colorectal cancer (CRC) refers to a malignant tumor that occurs on the colorectal mucosa. The early symptoms are usually not obvious and are easily ignored, which delays the disease. According to statistics from the International Agency for Research on Cancer, colorectal cancer is the third most common cancer in the world and the second most common cause of cancer death worldwide. According to statistics, there are more than 1.9 million new cases of colorectal cancer and more than 930,000 deaths from colorectal cancer worldwide. Colorectal cancer can occur at any age, and most patients are over 50 years old, but in recent years, the incidence of colorectal cancer has shown a trend of younger age. Despite great progress in detection technology and treatment methods, most CRC patients are still at risk of recurrence, metastasis and poor prognosis. In recent years, gene therapy has promoted the advancement of cancer treatment methods, the purpose of which is to introduce exogenous genetic material into target cells for treatment without producing off-target toxicity to achieve the purpose of treatment.

[0003] SOX9 is a member of the key transcription factor SRY-related high-mobility group box (SOX) family. SOX9 is a transcription factor that is abnormally expressed in many solid tumors. A large amount of data shows that SOX9 is overexpressed in colorectal cancer and is associated with low survival rate, poor prognosis and reduced sensitivity to certain chemotherapy drugs in colorectal cancer. Downregulating SOX9 expression will affect the invasion, metastasis, and proliferation of colorectal cancer cells. Therefore, SOX9 is expected to become a valuable diagnostic, evaluation marker and therapeutic target for colorectal cancer.

[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. However, naked or unmodified siRNA has many defects such as poor stability, easy degradation by nucleases, short half-life in vivo, and low cell entry efficiency, resulting in low drugability. 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 colorectal cancer. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a SOX9 siRNA-loaded lipid nanoparticle, a preparation method thereof and its effect in treating colorectal cancer.

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

[0007] The present invention provides a lipid nanoparticle loaded with SOX9 siRNA, which is composed of a tumor-targeting carrier material and SOX9 siRNA. The tumor-targeting carrier material consists of phospholipid-polyethylene glycol 2000 (DSPE-PEG 2000 ) modified with cRGDfK peptide, methyl 4-(N,N-dimethylamino)butyrate (DLin-MC3-DMA), distearoyl phosphatidylcholine (DSPC), dimyristoyl glycerol-polyethylene glycol 2000 (DMG-PEG 2000 ) and cholesterol. The SOX9 siRNA contains at least SOX9 siRNA1 and SOX9 siRNA2. The forward primer sequence of SOX9 siRNA1 is shown in SEQ ID NO.1, and its reverse primer sequence is shown in SEQ ID NO.2. The forward primer sequence of SOX9 siRNA2 is shown in SEQ ID NO.3, and its reverse primer sequence is shown in SEQ ID NO.4.

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

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

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

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

[0012] The present invention also provides a preparation method of the lipid nanoparticle loaded with SOX9 siRNA, including the following steps:

[0013] (1) Dissolve the cRGDfK peptide modified with a thiol group and DSPE-PEG 2000 -MAL in DEPC water at a molar ratio of 3:1, mix evenly, incubate at room temperature for 4 h, dialyze the obtained product in pure water for 48 h (cut-off molecular weight 3 KD), and lyophilize for 24 h to obtain cRGDfK-modified DSPE-PEG 2000 -cRGDfK.

[0014] (2) Mix DLin-MC3-DMA, DMG-PEG, DSPC, DSPE-PEG 2000-cRGDfk and cholesterol were dissolved in chloroform and methanol solution at a molar ratio of 50:1:10:1:38, and then transferred to a round-bottom flask for rotary evaporation. The parameters of the rotary evaporator under reduced pressure were set at 42 °C and 45 rpm to volatilize the organic solvents to obtain a liposome membrane. The liposome membrane was taken out and stored at 4 °C overnight, then 2 mL of distilled water was added, and it was incubated in a water bath at 55 °C for 45 min. The hydrated liposomes were sonicated for 10 min and extruded through a 220 nm filter membrane several times to obtain blank lipid nanoparticles.

[0015] (3) According to N / P = 5, SOX9 siRNA and blank lipid nanoparticles were mixed and vortexed for 5 min, and incubated at room temperature for 30 min to obtain lipid nanoparticles loaded with SOX9 siRNA.

[0016] The present invention also provides an application of lipid nanoparticles loaded with SOX9 siRNA in the treatment of colorectal cancer.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] 1. The lipid nanodrug delivery system constructed by the present invention can improve the transfection efficiency of siRNA, achieve lysosomal escape, and has tumor targeting, improve the anti-tumor effect and reduce side effects.

[0019] 2. The SOX9 siRNA lipid nanoparticles constructed by the present invention can not only inhibit the proliferation, migration and invasion of colorectal cancer cells in vitro, but also inhibit the growth of tumor tissues in vivo, and can be used for the preparation of drugs for the treatment of colorectal cancer.

[0020] 3. The SOX9 siRNA lipid nanoparticles constructed by the present invention Brief Description of the Drawings

[0021] Figure 1 Particle size potential diagram and transmission electron microscope image of lipid nanoparticles loaded with SOX9 siRNA.

[0022] Figure 2 In vitro release curve of lipid nanoparticles loaded with SOX9 siRNA.

[0023] Figure 3 Cell uptake of lipid nanoparticles loaded with SOX9 siRNA (A: representative image; B: quantitative result). Compared with the naked FAM-siRNA group, **P < 0.01; compared with the lipo + FAM-siRNA group, # P < 0.05, ## P < 0.01; compared with the LNPs / FAM-siRNA group, && P < 0.01.

[0024] Figure 4Lysosomal escape of SOX9 siRNA-loaded lipid nanoparticles.

[0025] Figure 5 Effect of SOX9 siRNA-loaded lipid nanoparticles on the viability of HCT-116 cells. Compared with the blank control group, *P<0.05, **P<0.01; compared with the negative control group, # P<0.05, ## P<0.01; compared with naked SOX9 siRNA, & P<0.05, && P<0.01; compared with the LNPs group at the same concentration, $$ P<0.01.

[0026] Figure 6 Effect of SOX9 siRNA-loaded lipid nanoparticles on the migration of HCT-116 cells (A: representative images; B: quantitative results). Compared with the blank control group, **P<0.01; compared with the negative control group, # P<0.05, ## P<0.01; compared with the naked SOX9 siRNA group, & P<0.05, && P<0.01; compared with the LNPs group, $ P<0.05.

[0027] Figure 7 Effect of SOX9 siRNA-loaded lipid nanoparticles on the invasion of HCT-116 cells (A: representative images; B: quantitative results). Compared with the blank control group, **P<0.01; compared with the negative control group, ## P<0.01; compared with the naked SOX9 siRNA group, & P<0.05, && P<0.01; compared with the LNPs group, $ P<0.05.

[0028] Figure 8 Tissue distribution of SOX9 siRNA-loaded lipid nanoparticles in tumor-bearing mice inoculated with HCT-116 cells (A: in vivo fluorescence signal images; B: ex vivo organ fluorescence signals; C: ex vivo organ fluorescence quantitative results). Compared with the naked siRNA group, *P<0.01; compared with the LNPs group, # P<0.01.

[0029] Figure 9Inhibitory effect of SOX9 siRNA-loaded lipid nanoparticles on tumor implantation in tumor-bearing mice inoculated with HCT-116 cells and its effect on the body weight of tumor-bearing mice (A: Quantitative results; B: Body weight change; C: Representative images). Compared with the control group, *P<0.05; compared with the naked SOX9 siRNA group, # P<0.01; compared with the LNPs group, & P<0.01.

[0030] Figure 10 Safety evaluation of SOX9 siRNA-loaded lipid nanoparticles on important organs of tumor-bearing mice inoculated with HCT-116 cells (A: Representative images of HE staining; B: Organ index). Detailed implementation manners

[0031] The technical solutions of the present invention will be 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 used to limit the present invention. The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial channels without special instructions.

[0032] The reagents and materials used in the following examples are as follows:

[0033] c[RGDfK(mpa)] was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., DLin-MC3-DMA, DSPC, DMG-PEG 2000 and DSPE-PEG 2000 -MAL were synthesized by Xi'an Ruixi Biotechnology Co., Ltd., and cholesterol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Colorectal cancer cell line HCT-116 was purchased from Wuhan Zishan Biotechnology Co., Ltd.; fetal bovine serum was purchased from Shanghai Sangon Biotech; cell culture reagents and consumables were purchased from Beijing Solarbio Science & Technology Co., Ltd.; BALB / c female nude mice were purchased from Jinan Pengyue Experimental Animal Co., Ltd.; SOX9 siRNA was designed and synthesized by Shanghai GenePharma Co., Ltd., and its sequences are as follows:

[0034] SOX9 siRNA1 sense strand (5’-3’): GCGGAGGAAGUCGGUGAAGAATT (SEQ ID NO.1)

[0035] SOX9 siRNA1 antisense strand (5’-3’): UUCUUCACCGACUUCCUCCGCTT (SEQ ID NO.2)

[0036] SOX9 siRNA2 sense strand (5’-3’): ACCUUCGAUGUCAACGAGUUUTT (SEQ ID NO.3)

[0037] Antisense strand of SOX9 siRNA2 (5'-3'): AAACUCGUUGACAUCGAAGGUTT (SEQ ID NO.4)

[0038] Example 1

[0039] Preparation and Characterization of Lipid Nanoparticles Loaded with SOX9 siRNA

[0040] Dissolve the thiol-modified cRGDfK peptide and DSPE-PEG 2000 -MAL in DEPC water at a molar ratio of 3:1, mix well, incubate at room temperature for 4 h. The resulting product is dialyzed in pure water for 48 h (cut-off molecular weight 3 KD), and freeze-dried for 24 h to obtain cRGDfK-modified DSPE-PEG 2000 -cRGDfK. Dissolve DLin-MC3-DMA, DMG-PEG, DSPC, DSPE-PEG 2000 -cRGDfk and cholesterol in a chloroform and methanol solution at a molar ratio of 50:1:10:1:38, then transfer to a round-bottom flask for rotary evaporation. Set the parameters of the vacuum rotary evaporator to 42 °C and 45 rpm to volatilize the organic solvents to obtain a lipidosome membrane. Take out the lipidosome membrane, place it at 4 °C and refrigerate overnight, then add 2 mL of distilled water and incubate in a water bath at 55 °C for 45 min. Sonicate the hydrated liposomes for 10 min and extrude through a 220 nm filter membrane several times to obtain blank lipid nanoparticles. According to N / P = 5, mix SOX9 siRNA and blank lipid nanoparticles, vortex for 5 min, and incubate at room temperature for 30 min to obtain lipid nanoparticles loaded with SOX9 siRNA (R-LNPs).

[0041] Centrifuge the R-LNPs solution at 18000 rpm for 30 min at 4 °C. The concentration of free siRNA in the supernatant is measured by absorbance using the fluorescent dye RiboGreen with a microplate reader, and the amount of free siRNA is calculated according to the standard curve. Calculate the encapsulation efficiency according to the following formula. The results show that the encapsulation efficiency is 90.71 ± 1.63%.

[0042]

[0043] Measure the particle size, polydispersity index and zeta potential by dynamic light scattering. The results show that the particle size of R-LNPs is about 159.6 ± 0.93 nm; the polydispersity index is 0.207 ± 0.016( Figure 1 A), and the zeta potential is 2.74 ± 0.35 mV( Figure 1 B); the prepared R-LNPs are observed to be regular spherical shapes and are relatively uniformly dispersed under a transmission electron microscope( Figure 1 C).

[0044] Example 2

[0045] In vitro release of SOX9 siRNA-loaded lipid nanoparticles

[0046] The prepared R-LNPs and naked siRNA were separately dissolved in 1 mL of PBS buffer solution, and the siRNA concentration was set at 20 nM. The solution was transferred into a dialysis bag (molecular weight cut-off 10 kDa) and dialyzed with stirring in 100 mL of PBS buffer solution at 37 °C. At time points of 0, 2, 6, 12, 16, 24, 48 h, and 72 h, 100 μL of the dialysis solution was taken out and the same volume of fresh solution was added. The concentration of free siRNA in the dialysis solution was measured using the fluorescent dye RiboGreen, and an in vitro release curve was plotted. The results are as Figure 2 shown. The release of naked siRNA was nearly 80% at 6 h, while the release of R-LNPs reached 70% at 24 h and then slowed down until 72 h, indicating that R-LNPs can protect siRNA and control its release, thus prolonging the circulation time of siRNA.

[0047] Example 3

[0048] Uptake of SOX9 siRNA-loaded lipid nanoparticles in HCT-116 cells

[0049] HCT-116 cells in the logarithmic growth phase (1×10 5 / mL) were seeded in 12-well plates. When the cell density reached 70%, the cells were transfected with LNPs and R-LNPs prepared with FAM-siRNA respectively. Naked FAM-siRNA and FAM-siRNA transfected with Lipo8000 were used as controls. After incubation for 4 h, the cells were successively stained with 50 nM of Lyso Tracker Red for 60 min, fixed with 4% paraformaldehyde for 10 min, and stained with DAPI for 10 min. Then images were taken with a fluorescence microscope to observe cell uptake. In addition, the cells were resuspended with PBS and seeded in 96-well plates, and the fluorescence intensity of FAM was detected with a microplate reader with an excitation wavelength of 495 nm and an emission wavelength of 520 nm. This fluorescence intensity can reflect the amount of cell uptake. The results are as Figure 3 shown. Naked siRNA was difficult to be taken up by cells, and almost no green fluorescence was observed in HCT-116 cells( Figure 3 A and 3B). Lipo8000 is currently still a commonly used siRNA transfection reagent, which can significantly improve the transfection efficiency of siRNA in cells, and the intracellular green fluorescence increased significantly( Figure 3 A and 3B, compared with the naked siRNA group, P<0.01). Loading siRNA with lipid nanoparticles modified with cRGDfK peptide increased the cell transfection rate( Figure 3A and 3B. Compared with the naked siRNA group, the intracellular fluorescence intensity was comparable to that of the Lipo8000 group, and the transfection rate was also significantly increased compared with the LNPs group (P < 0.01). Figure 3 A and 3B (P < 0.01), indicating that the lipid nanoparticles modified with cRGDfK peptide have tumor targeting and high cell uptake, which is beneficial for delivery in HCT-116 cells.

[0050] Example 4

[0051] Lysosomal escape of SOX9 siRNA-loaded lipid nanoparticles in HCT-116 cells

[0052] HCT-116 cells (1×10 5 / well) were seeded in 12-well plates and cultured. When the cell density reached 70%, 1 mL of serum-free medium containing 20 nM FAM-siRNA-loaded R-LNPs was added to each well. The cells were washed with PBS and then cultured with serum-free RPMI 1640 medium for 4, 6, and 10 h respectively. Then, the cells were stained with Lyso Tracker Red for 60 min and DAPI for 10 min, and photographed under a fluorescence microscope. The results are as follows Figure 4 shown. After 4 h of transfection of HCT-116 cells with R-LNPs, the green fluorescence in the cells was almost consistent with the red fluorescence, showing orange fluorescence, indicating that R-LNPs had entered the cells and were almost co-localized with lysosomes. As time extended, the merged orange signal began to weaken at 6 h and almost completely disappeared at 10 h, indicating that R-LNPs had escaped from lysosomes. The lysosomal escape function of R-LNPs is beneficial for its gene silencing and anti-tumor effects in cells.

[0053] Example 5

[0054] Effects of SOX9 siRNA-loaded lipid nanoparticles on the proliferation, migration, and invasion of HCT-116 cells

[0055] The CCK-8 method was used to detect cell viability. HCT-116 cells (8×10 3 / well) were seeded in 96-well plates. After incubation for 24 h, freshly prepared naked SOX9 siRNA (20 nM), LNPs and R-LNPs solutions at different concentrations (10, 20, 40, 80, 120 nM) were added respectively. An equal volume of medium was used as the blank control, and blank lipid nanoparticles without SOX9 siRNA were used as the negative control. After incubation for 24 h, the optical density (OD) at 450 nm was measured using the CCK-8 method, and the cell viability was calculated according to the following formula:

[0056]

[0057] The results are as follows Figure 5 As shown. LNPs and R-LNPs with different concentrations all had inhibitory effects on the viability of HCT-116 cells to varying degrees, and the inhibitory effect increased with the increase in the concentrations of LNPs and R-LNPs. Moreover, the inhibitory effect of R-LNPs on the viability of HCT-116 cells was significantly stronger than that of LNPs at the same concentration (P<0.01). When the concentration of R-LNPs was greater than 40 nM, the cell survival rate changed little. Therefore, the concentration of R-LNPs was set at 40 nM to observe its effect on the migration and invasion of HCT-116 cells.

[0058] The cell scratch assay was used to detect cell migration ability. HCT-116 cells (5×10 5 / well) were seeded in 6-well plates. After culturing for 24 h until the cells adhered, a 200 μL sterile pipette tip was held vertically to the cell surface and perpendicular to the marked black line, and then scratched from one end of the well to the other end. The cells were divided into blank control group, negative control group, naked SOX9 siRNA group, LNPs group and R-LNPs group. 2 mL of medium, blank lipid nanoparticles, naked SOX9 siRNA, LNPs and R-LNPs solutions were added to each well of the cells in each group respectively. The cells were observed and photographed under a microscope at 0 and 48 h, and the scratched area was measured by Image J software. The cell migration rate at 48 h was calculated according to the following formula:

[0059]

[0060] The normalization method was performed using the blank control group. The results are as follows Figure 6 As shown. Compared with the blank control group, negative control group and naked siRNA group respectively, the cell migration rate in the R-LNPs group decreased significantly (P<0.05 or P<0.01). Compared with the LNPs group, the migration rate also decreased (P<0.05), indicating that R-LNPs can significantly inhibit the migration of HCT-116 cells.

[0061] The Transwell assay was used to detect cell invasion ability. The chamber was placed in a 24-well plate. 100 μL of serum-free RPMI 1640 medium was added to the chamber for hydration for 10 min. 100 μL of the diluted Matrigel (Matrigel: serum-free RPMI1640 medium = 1:8) was added to each chamber and incubated overnight in a 37 °C incubator to gel. 200 μL of serum-free RPMI 1640 medium (blank control group), blank lipid nanoparticles (negative control group), naked SOX9 siRNA, LNPs and R-LNPs solutions and HCT-116 cells (4×10 5 / mL), add 750 μL of high-concentration RPMI 1640 medium to the lower chamber, and incubate in an incubator for 48 h. Aspirate the medium in the upper chamber, wipe off the cells in the non-migrated upper chamber with a cotton swab, and wash the chamber twice in PBS. Fix the cells with 4% tissue cell fixative, 500 μL per well, and fix at room temperature for 20 min. Remove the chamber from the 4% tissue cell fixative and wash it twice with PBS; add 0.1% crystal violet and stain for 15 min in the dark. Discard the staining solution and wash twice with PBS again. Take pictures and observe under a fluorescence microscope, count the stained cells using Image J software, and obtain the cell invasion rate by normalizing with the blank control group. The results are as Figure 7 shown. Compared with the blank control group, negative control group, and naked SOX9 siRNA group respectively, the cell invasion rate of the R-LNPs group decreased significantly (P < 0.01), and there was also a significant decrease compared with the LNPs group (P < 0.05), indicating that R-LNPs can significantly inhibit the invasion of HCT-116 cells.

[0062] Example 6

[0063] Tissue distribution, antitumor effect of SOX9 siRNA-loaded lipid nanoparticles in tumor-bearing mice and safety evaluation of organs of tumor-bearing mice

[0064] Thirty-three female BALB / c nude mice were adaptively fed for 1 week; the well-grown HCT-116 cells were digested, and the cells were made into a cell suspension with a density of 2×10 7 cells / mL with PBS; 200 μL of the cell suspension was subcutaneously inoculated under the right axilla of 33 nude mice per mouse. Wait until the tumor volume grows to about 100 - 200 mm 3 and then conduct the experiment.

[0065] The tissue distribution and tumor targeting effect of lipid nanoparticles were detected using HCT-116 tumor-bearing female BALB / c nude mice. When the tumor volume grew to 100 - 200 mm 3 , nine nude mice were randomly divided into 3 groups, and Cy5-labeled siRNA, LNPs, and R-LNPs (33 μg / mouse) were injected via the tail vein respectively. Observe the distribution of Cy5-siRNA at 0, 3, 5, 10, and 24 h using an in vivo imager. Then sacrifice the nude mice, collect the tumors and various organs (heart, liver, spleen, lung, kidney, brain), and measure the fluorescence intensity using an in vivo imager. As Figure 8As shown in , the fluorescence intensity was the strongest 3 h after injection of naked siRNA and decreased with the prolongation of time. At 24 h, the fluorescence was mainly concentrated in the kidneys, indicating that naked siRNA was easily metabolized in vivo and had no specific tissue distribution. The fluorescence intensities of LNPs and R-LNPs were significantly stronger than that of naked siRNA and decreased slowly with the prolongation of time. In addition, fluorescence was visible in the tumor sites of both the LNPs group and the R-LNPs group. Importantly, the fluorescence intensity in the tumor site of the R-LNPs group was stronger, indicating that R-LNPs had good tumor targeting ability due to the modification of cRGDfK peptide. Through the detection of fluorescence intensity in ex vivo organs ( Figure 8 B and 8C), it was found that naked siRNA was mainly concentrated in the kidneys, followed by the liver, and there was almost no fluorescence in the tumor tissue. However, the fluorescence intensities in the tumor tissues of the LNPs group and the R-LNPs group were significantly higher than those of the naked siRNA group (P < 0.01), and the fluorescence intensity of the R-LNPs group was higher than that of the LNPs group (P < 0.01), suggesting that LNPs modified with cRGDfK had better tumor targeting.

[0066] The antitumor effect of lipid nanoparticles was detected using female BALB / c nude mice bearing HCT-116 tumors, and their body weight changes were also observed. Twenty-four tumor-bearing mice were randomly divided into 4 groups (n = 6): PBS group, naked SOX9 siRNA group, LNPs group, and R-LNPs group. When the tumors grew to 150 mm 3 3, the four groups of mice were respectively injected with PBS, naked SOX9 siRNA, LNPs, and R-LNPs (33 μg / mouse) via the tail vein. The administration interval was once every 3 days, and a total of 7 administrations were given. The body weight and tumor volume were recorded every 3 days. The tumor volume was calculated using the following formula: V (mm 3 ) = ab 2 / 2, where a was the tumor length and b was the tumor width. The mice were fasted on the 3rd day after the end of the administration, and then the tumors and other organs were removed. The results are as Figure 9 shown in A and B. Nine days after the tail vein injection, the tumor volumes of the LNPs group and the R-LNPs group were respectively smaller than those of the PBS group and the naked siRNA group (P < 0.05). Starting from the 15th day, the tumor volume of the R-LNPs group was significantly smaller than that of the LNPs group (P < 0.05), suggesting that R-LNPs had a stronger antitumor effect in vivo. At the same time, the body weights of the nude mice in each group fluctuated little during the administration period ( Figure 9 C).

[0067] Calculate the organ index and use hematoxylin and eosin (HE) staining to detect the organ toxicity of lipid nanoparticles in tumor-bearing female BALB / c nude mice. After the above-mentioned mice were sacrificed by cervical dislocation, the heart, liver, spleen, lungs, kidneys and brain were removed, weighed, and the organ index was calculated using the formula: Organ index (%) = Organ weight (g) / Body weight (g) × 100%. The above organs were fixed with formaldehyde, dehydrated, embedded in paraffin, and prepared into paraffin sections. The sections were spread on glass slides and baked. After dewaxing with xylene and rehydrating with gradient ethanol → Hematoxylin staining at room temperature for 10 min → Washing with water → Differentiating with differentiating solution for 5 s → Washing with water → Blueing with ammonia water for 40 s → Washing with water → Eosin staining for 1 min → Dehydrating with gradient ethanol → Clearing with xylene → Sealing with neutral resin, HE-stained sections were obtained, and the histological morphology was observed and photographed under a microscope. The results are as Figure 10 shown. There was no significant difference in the organ index between the LNP group and the R-LNP group compared with the control group ( Figure 10 B), and at the same time, the HE staining results showed no obvious damage to the important organs, indicating that R-LNPs have certain safety.

[0068] The experimental results of the above examples show that the cRGDfK peptide-modified lipid nanoparticles loaded with SOX9 siRNA constructed by the present invention have tumor targeting, can be taken up by tumor cells and achieve lysosomal escape, and are released at the tumor site, can significantly inhibit the proliferation, invasion and metastasis of colorectal cancer cells, and at the same time also have good tumor targeting, anti-tumor activity and safety in tumor-bearing mice, providing a new strategy for the treatment of colorectal cancer.

Claims

1. A SOX9 siRNA-loaded lipid nanoparticle, characterized in that: The lipid nanoparticles are composed of tumor targeting carrier materials and SOX9 siRNA; The tumor targeting carrier material is composed of phospholipid-polyethylene glycol 2000 (DSPE-PEG 2000 -cRGDfk), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG 2000 ), distearoylphosphatidylcholine (DSPC) and cholesterol; The SOX9 siRNA at least comprises SOX9 siRNA1 and SOX9 siRNA2, wherein the forward primer sequence of SOX9 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 SOX9 siRNA2 is shown as SEQ ID NO.3, and the reverse primer sequence thereof is shown as SEQ ID NO.

4.

2. The method for preparing SOX9 siRNA-loaded nanoparticles according to claim 1, characterized in that: The following steps are involved: Step 1: The cRGDfK peptide containing thiol modification was stained with phospholipid-polyethylene glycol 2000-maleimide (DSPE-PEG 2000 -MAL) was dissolved in DEPC water at a molar ratio of 3:1 and mixed evenly, incubated at room temperature for 4 h, and the obtained product was dialyzed in pure water for 48 h, with a molecular weight cutoff of 3 KD, and lyophilized for 24 h to obtain cRGDfK-modified DSPE-PEG 2000 (DSPE-PEG 2000 -cRGDfK). Step 2: DSPE-PEG 2000 -cRGDfk, DLin-MC3-DMA, DMG-PEG 2000 , DSPC and cholesterol were dissolved in chloroform and methanol solution at a molar ratio of 1:50:1:10:38, and then completely dissolved by water bath sonication. The parameters of the reduced pressure rotary evaporator were set to 42°C and 45rpm to evaporate the organic solvent to obtain the liposome membrane. After refrigeration at 4°C overnight, 2mL of distilled water was added and incubated in a water bath at 55°C for 45min. The hydrated liposomes were sonicated for 10min and squeezed through a 220nm filter membrane several times to obtain blank lipid nanoparticles. Step 3: SOX9 siRNA and blank lipid nanoparticles were mixed according to N / P = 5:1, vortexed for 5 min, and incubated at room temperature for 30 min to form SOX9 siRNA-encapsulated lipid nanoparticles (R-LNPs).

3. Use of the SOX9 siRNA-loaded lipid nanoparticles according to claim 1 in the preparation of a drug for treating colorectal cancer.

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