Double-response TGF-beta1-loaded siRNA nanoparticles, preparation method thereof and application thereof in treatment of triple-negative breast cancer and bone metastasis of triple-negative breast cancer

Abstract

The invention discloses a dual-response TGF (Transforming Growth Factor)-beta 1 (Transforming Growth Factor) loaded siRNA (Small Interference Ribonucleic Acid) nanoparticle, which is prepared from a carrier material with redox and pH (Potential of Hydrogen) dual response, namely poly (N, N-dimethylaminoethyl methacrylate)-SS-polyethylene glycol-SS-poly (N, N-dimethylaminoethyl methacrylate) (PDMAEMA-SS-PEG (Polyethylene Glycol)-SS-PDMAEMA) and TGF-beta 1 siRNA. The prepared dual-response TGF-beta1siRNA-loaded nanoparticles are released in an oxidation-reduction and pH-responsive manner, protect TGF-beta1siRNA from being degraded by RNaseA and serum, remarkably inhibit proliferation, migration and invasion of triple-negative breast cancer cells and chemotaxis and heterogeneous adhesion of the triple-negative breast cancer cells to a bone microenvironment, and have the effect of treating triple-negative breast cancer and bone metastasis thereof. The nanoparticle is simple in preparation method and remarkable in effect, and has good application value and market prospect.

Description

Technical Field

[0001] The invention relates to a dual-response TGF-β1 siRNA-loaded nanoparticle, a preparation method thereof and application thereof in treating triple-negative breast cancer and bone metastasis thereof, and belongs to the field of medicine. Background Art

[0002] Triple-negative breast cancer (TNBC) is a very biological subtype of breast cancer, accounting for approximately 15-20% of all breast cancers. It is named because it does not express progesterone receptor (PR), estrogen receptor (ER), and human epidermal growth factor receptor-2 (HER-2). Compared with other breast cancer subtypes, TNBC exhibits inherently aggressive clinical behavior and does not respond to endocrine therapy or anti-HER-2 targeted therapy. The prognosis of TNBC patients is generally poor. Chemotherapy is the main systemic treatment for TNBC. Currently, clinical targeted drugs for breast cancer include PARP inhibitors (PARPi), CDK4 / 6 inhibitors (CDK4 / 6i), PI3K inhibitors, and AKT inhibitors, which are often ineffective for TNBC. Therefore, in the future, there is an urgent need to explore the drug compatibility and potential targets of TNBC, develop new targeted preparations, and improve the prognosis of TNBC patients.

[0003] Transforming growth factor-β (TGF-β) is considered to be an important regulator of the development and progression of breast cancer. In the later stages of breast cancer progression, TGF-β enhances the migration and invasiveness of cancer cells, leading to tumor metastasis. Treatment of mice with monoclonal anti-TGF-β antibodies inhibited lung or bone metastasis in mouse breast cancer models. In addition, TGF-β plays an important role in epirubicin-induced drug resistance by regulating the stemness, epithelial-to-mesenchymal transition, and apoptosis of TNBC. In TNBC cells, the mRNA expression level of TGF-β is much higher than that of non-TNBC cells, directly or indirectly promoting EMT and increasing the migration of TNBC cells [9]. Therefore, inhibiting the TGF-β signaling pathway may be a very promising strategy for the treatment of TNBC, but there is currently a lack of systematic and in-depth research.

[0004] RNA interference technology is a technology that introduces exogenous or endogenous double-stranded RNA (dsRNA) into cells to cause degradation of target genes homologous to dsRNA, thereby inhibiting the expression of target genes. The occurrence of RNA interference depends on three types of dsRNA: short hairpin RNA (shRNA), endogenous small RNA (miRNA) and small interfering RNA (siRNA). Among them, siRNA is a type of dsRNA with a length of 21 to 23 nt, which is produced by the RNase III family member Dicer enzyme decomposing long dsRNA in the cytoplasm. siRNA has the advantages of high specificity, short development time, low production cost and long duration of efficacy in drug development, making siRNA therapy have great potential in the treatment of various tumors. siRNA exerts anti-tumor activity by precisely targeting key genes in pathways related to tumor cell proliferation, migration, apoptosis, drug tolerance, etc. However, due to the complex pathogenesis of tumors, the difficulty of targeted delivery to tumor tissues, and the low uptake rate of tumor cells, there are still many difficulties in the development of anti-tumor siRNA drugs. In previous studies, it was found that after transfection of TNBC cells MDA-MB-231 with TGF-β1 siRNA, cell proliferation, migration and invasion were significantly reduced. In order to achieve effective in vivo delivery of TGF-β1 siRNA, it is urgent to develop a drug delivery system based on the characteristics of the tumor microenvironment.

[0005] The internal environment in which tumor cells are produced and live is the tumor microenvironment (TME), which includes tumor cells and their surrounding fibroblasts, immune and inflammatory cells, glial cells, as well as intercellular matrix, microvessels, and biological molecules infiltrating therein. The extracellular microenvironment of tumors is characterized by being slightly acidic, hypoxic, and rich in tumor cell secretory enzymes; at the same time, tumor cells also have a variety of biochemical indicators that are different from normal cells, such as high concentrations of reactive oxygen species (ROS) and high concentrations of glutathione (GSH). Therefore, designing nano-drug carriers that can respond to these abnormal physiological indicators of the tumor microenvironment to achieve drug enrichment and release at the tumor site has become a hot topic in the current development of anti-tumor drugs, including siRNA drugs. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a dual-responsive TGF-β1 siRNA-loaded nanoparticle, a preparation method thereof and an application thereof in treating triple-negative breast cancer and bone metastasis thereof.

[0007] The technical solution of the present invention is as follows:

[0008] The invention provides a dual-responsive TGF-β1 siRNA-loaded nanoparticle, which is composed of a carrier material with dual redox and pH responses and TGF-β1 siRNA. The carrier material with dual redox and pH responses is poly(N,N-dimethylaminoethyl methacrylate)-SS-polyethylene glycol-SS-poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA-SS-PEG-SS-PDMAEMA). The sense sequence of TGF-β1 siRNA is GCAGAGUACACACAGCAUATT, and the antisense sequence is UAUGCUGUGUGUACUCUGCTT.

[0009] The present invention also provides a method for preparing dual-responsive TGF-β1 siRNA-loaded nanoparticles, comprising the following steps: dissolving PDMAEMA-SS-PEG-SS-PDMAEMA in DEPC water, adding TGF-β1 siRNA at an N / P ratio of 15:1, vortex mixing for 30 seconds, and standing for 30 minutes to self-assemble into nanoparticles.

[0010] The present invention also provides a dual-response TGF-β1 siRNA-loaded nanoparticle for use in treating triple-negative breast cancer and its bone metastasis.

[0011] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0012] 1. The nano drug delivery system constructed by the present invention has redox and pH dual responsiveness, and can release siRNA in a high-glutathione, slightly acidic tumor microenvironment, which is conducive to exerting its effect at the tumor site.

[0013] 2. The nano drug delivery system constructed by the present invention can effectively protect siRNA from degradation, has good stability, and prolonged circulation time. It can significantly inhibit the proliferation, migration and invasion of triple-negative breast cancer cells and their chemotaxis, adhesion and proliferation to the bone microenvironment, and can be used to treat triple-negative breast cancer and its bone metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Transmission electron microscopy image of dual-responsive TGF-β1 siRNA-loaded nanoparticles.

[0015] Figure 2 In vitro release curves of dual-responsive TGF-β1 siRNA-loaded nanoparticles (A: release in an environment with or without GSH; B: release in an environment with different pH values).

[0016] Figure 3 In vitro stability of dual-responsive TGF-β1 siRNA-loaded nanoparticles (A: stability against enzymatic degradation; B: stability against serum degradation).

[0017] Figure 4 Effects of dual-responsive TGF-β1 siRNA-loaded nanoparticles on the viability of MDA-MB-231 cells. Compared with the control group, *P<0.05;

[0018] Compared with the naked siRNA group, #P<0.05.

[0019] Figure 5 Effects of dual-responsive TGF-β1 siRNA-loaded nanoparticles on the migration (A: representative images; B: quantitative results) and invasion (C: representative images; D: quantitative results) of MDA-MB-231 cells. Compared with the control group, *P<0.05; compared with the naked siRNA group, #P<0.05.

[0020] Figure 6 Effects of dual-responsive TGF-β1 siRNA-loaded nanoparticles on chemotaxis (A: representative images; B: quantitative results) and adhesion (C: representative images; D: quantitative results) of MDA-MB-231 cells to the bone microenvironment. *P<0.05 compared with the control group; #P<0.05 compared with the naked siRNA group. DETAILED DESCRIPTION

[0021] 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 through commercial channels.

[0022] The reagents and materials used in the following examples are as follows:

[0023] PDMAEMA-SS-PEG-SS-PDMAEMA and PEG-PEMAEMA were synthesized by Xi'an Ruixi Biosynthesis Co., Ltd.; TNBC cells MDA-MB-231 and osteoblast-like cells MG-63 were purchased from Shanghai Bolson Biotechnology Co., Ltd.; fetal bovine serum was purchased from Invitrogen Life Sciences, USA; cell culture reagents and consumables were purchased from Beijing Solebogen Biotechnology Co., Ltd.; TGF-β1 siRNA was designed and synthesized by Shanghai Jima Gene, and its sense sequence is GCAGAGUACACACAGCAUATT, and its antisense sequence is UAUGCUGUGUGUACUCUGCTT.

[0024] Example 1

[0025] Preparation and characterization of dual-responsive TGF-β1 siRNA-loaded nanoparticles

[0026] PDMAEMA-SS-PEG-SS-PDMAEMA was dissolved in DEPC water, and TGF-β1 siRNA was added at a N / P ratio of 15:1. After vortex mixing for 30 seconds and standing for 30 minutes, it can self-assemble into nanoparticles (PSPD-NP).

[0027] The PSPD-NP solution was centrifuged at 18000 rpm for 30 min at 4°C, and the supernatant was taken. The amount of free siRNA in the supernatant was determined using the fluorescent dye RiboGreen method. The encapsulation efficiency was calculated according to the following formula. The results showed that the encapsulation efficiency was 94.19±0.47%.

[0028]

[0029] The particle size, polydispersity coefficient and zeta potential were determined by dynamic light scattering. The results showed that the particle size of PSPD-NP was about 122±1.69nm; the polydispersity coefficient was 0.25±0.012, and the zeta potential was 5.28±0.14mV. The prepared nanoparticles were observed to be regular spherical under transmission electron microscopy and were relatively uniformly dispersed. Figure 1 shown.

[0030] Example 2

[0031] In vitro release of dual-responsive TGF-β1 siRNA-loaded nanoparticles

[0032] The prepared PSPD-NPs were dissolved in 2 mL of PBS buffer solution with or without GSH or different pH (7.4 and 5.0), and transferred to a dialysis bag and dialyzed with 25 mL of PBS buffer solution at 37 °C with stirring. At 0, 2, 6, 12, 16, 20, 24, 48, and 72 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 drawn. The results are shown in Figure 2. Figure 2 As shown in Figure 2, the release behavior of IG-NPs in vitro was time-dependent. Figure 2 A shows that the release rate of PSPD-NP increased significantly in the presence of GSH, reaching 84.32% at 24h, while it was only 19.59% in the absence of GSH, showing the characteristics of redox-triggered drug release. Figure 2 B shows that the release rate of PSPD-NP increased significantly in the environment of pH 5.0, reaching 63.32% at 48h, while it was only 20.87% in the environment of pH 7.4, showing the characteristics of pH-triggered drug release. The release characteristics of PSPD-NP triggered by redox and pH dual responsiveness are conducive to the delivery of siRNA to the tumor microenvironment to exert its effect.

[0033] Example 3

[0034] In vitro stability of dual-responsive TGF-β1 siRNA-loaded nanoparticles

[0035] Take the freshly prepared PSPD-NP solution and the free siRNA solution of the same concentration, and divide them into two equal parts. One part is added with RNase solution (RNaseA), and the other part is added with the same volume of TE buffer. After incubation at 37℃ for 1h, add RNase inhibitor (20U / μL), incubate at room temperature for 10min to terminate the enzyme reaction, then add heparin sodium solution with a final concentration of 1mg / mL (add an equal volume of DEPC water to the free siRNA group), incubate at room temperature for 20min to displace the siRNA in the complex, and check the integrity of siRNA by agarose gel electrophoresis to evaluate the anti-enzymatic effect of PSPD-NP. Take another freshly prepared PSPD-NP solution, dilute it to a final volume of 10μL with DMEM medium containing 0, 10%, 20%, 30%, 40% and 50% fetal bovine serum, incubate at 37℃ for 1h, and mix the sample with loading buffer containing 1% SDS in a ratio of 1:1 to prepare the sample. The release of siRNA was examined by agarose gel electrophoresis to evaluate the dissociation of PSPD-NP antiserum. The results showed that after treatment with RNaseA, naked siRNA was completely degraded and no siRNA bands appeared, while the siRNA in PSPD-NP remained intact ( Figure 3 A), indicating that PSPD-NP can effectively protect siRNA from degradation by RNase. The results also showed that after PSPD-NP was co-incubated with FBS at a final concentration of 10% to 50%, obvious bands appeared at the position of naked siRNA, indicating that siRNA was displaced by SDS, indicating that PSPD-NP can resist dissociation at a concentration of up to 50% FBS and has excellent anti-serum dissociation effect. The above results show that PSPD-NP can resist degradation by RNaseA and serum, thereby ensuring good stability during in vivo delivery.

[0036] Example 4

[0037] Effects of dual-responsive TGF-β1 siRNA-loaded nanoparticles on proliferation, migration and invasion of MDA-MB-231 cells

[0038] The cell viability was detected by MTT assay. MDA-MB-231 cells were seeded in 96-well plates at a density of 4 × 10 5 / well, after incubation for 24h, freshly prepared PSPD-NP solutions of different concentrations (10, 20, 40, 80, 120nM) and naked TGF-β1 siRNA (40nM) were added, and an equal amount of culture medium was used as the control group. After incubation for 24h, the optical density (OD) at 490nm was determined by MTT method, and the cell viability was calculated as follows:

[0039]

[0040] The scratch test was used to detect the cell migration ability. 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 200μL sterile pipette tip was placed perpendicular to the cell surface and perpendicular to the marked black line to scratch from one end of the well to the other. The cells were transfected with 2mL each of naked TGF-β1 siRNA and PSPD-NP solution, and an equal amount of culture medium was used as the control group. The cells were observed and photographed under a microscope at 0 and 48 hours, and the images were processed with Image J software to measure the scratch area. The cell migration rate at 48 hours was calculated as follows and normalized with the control group.

[0041]

[0042] Transwell assay was used to detect cell invasion ability. The chamber was placed in a 24-well plate. 100 μL 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. MDA-MB-231 cells (4×10 5 / mL), and then add 200μL naked TGF-β1siRNA and PSPD-NP solution respectively, and an equal amount of serum-free DMEM medium as the control group. Add 750μL high-concentration DMEM medium to the lower chamber and place it in an incubator for 24h. Aspirate the culture medium in the upper chamber, wipe 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, and then stain with 0.1% crystal violet in the dark for 20min. After washing twice with PBS, take pictures under a fluorescence microscope, observe, count the stained cells with Image J software, and normalize the control group to obtain the cell invasion rate.

[0043] The results showed that compared with the control group and naked siRNA group, the cell viability of each concentration of PSPD-NP group was significantly decreased ( Figure 4, P<0.05), and in a dose-dependent manner, indicating that PSPD-NP can significantly inhibit the proliferation of MDA-MB-231 cells. When the concentration of PSPD-NP is 40nM, the viability of MDA-MB-231 cells is 40% ( Figure 4 ). Therefore, the concentration of PSPD-NP was set to 80nM to observe its effect on the migration and invasion of MDA-MB-231 cells. The results showed that compared with the control group and the naked siRNA group, the migration rate of cells in the PSPD-NP group ( Figure 5 A and 5B) and invasion rate ( Figure 5 C and 5D) were significantly decreased (P<0.05), indicating that PSPD-NP can significantly inhibit the migration and invasion of MDA-MB-231 cells.

[0044] Example 5

[0045] Effects of dual-responsive TGF-β1 siRNA-loaded nanoparticles on chemotaxis and heterogeneous adhesion of MDA-MB-231 cells to the bone microenvironment

[0046] The chemotaxis assay was used to observe 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 into 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 naked TGF-β1 siRNA and PSPD-NP solution for 24 hours, and an equal amount of culture medium was used as a control. The cells were digested and counted, and 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 the 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 3 times, dry at room temperature, fix the cells with neutral glue, observe and photograph under a fluorescence microscope. The stained cells are penetrated cells and are counted using Image J software. The cell penetration amount was normalized to the control group to calculate the cell chemotaxis rate.

[0047] The cell heterogeneity adhesion experiment was used to observe 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 naked TGF-β1 siRNA and PSPD-NP solution for 24 hours, and the same amount of culture medium was used as a control. The cells were digested and counted. 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 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 control group.

[0048]

[0049] The MDA-MB-231 cells to which the MG-63 cells adhered were fixed with 4% paraformaldehyde and photographed under bright field fluorescence microscope.

[0050] The results showed that compared with the control group and naked siRNA group, the chemotaxis rate of cells in the PSPD-NP group to MG-63 cells ( Figure 6 A and 6B) and adhesion rate ( Figure 6 C and 6D) were significantly decreased (P<0.05), indicating that PSPD-NP can significantly inhibit the chemotaxis and adhesion of MDA-MB-231 cells to the bone microenvironment, thereby inhibiting bone metastasis.

[0051] The experimental results of the above embodiments show that the dual-responsive TGF-β1 siRNA-loaded nanoparticles constructed by the present invention can protect siRNA from nuclease and serum degradation, and can respond to the tumor microenvironment, have redox and pH-triggered release characteristics, can release TGF-β1 siRNA at the tumor site, can significantly inhibit the proliferation, invasion and metastasis of triple-negative breast cancer cells, and at the same time inhibit the chemotaxis and adhesion of triple-negative breast cancer cells to the bone microenvironment, thereby inhibiting triple-negative breast cancer and its bone metastasis, providing a new method for the treatment of triple-negative breast cancer and its bone metastasis.

Claims

1. A dual-responsive TGF-β1 siRNA-loaded nanoparticle, characterized in that: The nanoparticles are composed of a redox and pH dual-responsive carrier material and TGF-β1 siRNA; Wherein, the redox and pH dual-responsive carrier material is poly(N,N-dimethylaminoethyl methacrylate)-SS-polyethylene glycol-SS-poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA-SS-PEG-SS-PDMAEMA). The TGF-β1 siRNA is characterized in that it is a double-stranded RNA molecule with the following nucleic acid sequence: Justice sequence: GCAGAGUACACACAGCAUATT, Antisense sequence: UAUGCUGUGUGUACUCUGCTT.

2. The method for preparing a dual-responsive TGF-β1 siRNA-loaded nanoparticle according to claim 1, characterized in that: The following steps are involved: PDMAEMA-SS-PEG-SS-PDMAEMA was dissolved in DEPC water, and TGF-β1 siRNA was added at a N / P ratio of 15:

1. After vortex mixing for 30 seconds and standing for 30 minutes, it can self-assemble into nanoparticles.

3. Use of a dual-responsive TGF-β1 siRNA-loaded nanoparticle as claimed in claim 1 in the treatment of triple-negative breast cancer and its bone metastasis.

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