Preparation method and application of LRIG1 nano microbubble in targeted activation cervical cancer HeLa cell

Abstract

The invention discloses a preparation method of saLRIG1 loaded nano microbubbles and application of the saLRIG1 loaded nano microbubbles in anti-cervical treatment. Blank Nano Bubbles (N-NBs) are prepared through a thin film hydration method and a mechanical oscillation method, and the saLRIG1-loaded nano microbubble saLRIG1-NBs is prepared through electrostatic adsorption of polylysine (PLL). The saLRIG1-NBs prepared by the invention can be used for inhibiting proliferation and migration of human cervical cancer HeLa cells and remarkably inhibiting growth of cervical cancer transplanted tumors of BALB / c nude mice.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and provides a method for preparing nano-microbubbles loaded with LRIG1 small activating RNA and use of the nano-microbubbles in preparing drugs for treating cervical cancer. Background Art

[0002] Despite the gradual promotion of early screening, the application of HPV vaccines, and the continuous improvement of diagnosis and treatment technologies, cervical cancer is still a common malignant tumor that threatens the lives and health of women worldwide. More than half of cervical cancer patients are already in the advanced stage when diagnosed. The treatment options for advanced, recurrent or metastatic cervical cancer are limited, and the 5-year survival rate is only 16.8%. Therefore, finding new and effective strategies to treat cervical cancer has important scientific significance and clinical application prospects.

[0003] LRIG1 Leucine rich repeats and immunoglobulin-like domain protein 1 (LRIG1) is a negative regulator of tyrosine kinase (RTK) receptors. It binds to the ectodomain of EGFR through its extracellular LRR and IG-like domains, triggering the degradation of EGFR, thereby inhibiting downstream signaling pathways: inhibiting cell cycle progression, inhibiting cell proliferation and invasion, and promoting cell apoptosis. LRIG1 is downregulated in bladder cancer, cervical cancer, colon cancer, rectal cancer, thyroid cancer and melanoma. The survival rate of cervical cancer patients with low expression of LRIG1 is only 1%, which is an important prognostic predictor for early cervical cancer. Overexpression of LRIG1 can inhibit the growth of prostate transplanted tumors and inhibit the growth of prostate cancer cells by downregulating the expression of c-Myc and ERBB / pERBB. It has also been found that LRIG1 overexpression can inhibit the migration and invasion of melanoma cells by inhibiting the EGFR / ERK pathway.

[0004] Small activating RNA (saRNA) is a double-stranded RNA with a length of 21nt. It can target the gene promoter region to activate the expression of the target gene. It has broad application prospects in the development of therapeutic drugs, including tumors, genetic diseases, metabolic diseases, cardiovascular and cerebrovascular diseases, etc. It has the advantages of short R&D cycle, low resistance, long-lasting effect, and high R&D success rate. The conclusion of the MTL-CEBPA clinical trial strongly confirmed the anti-tumor activity of saRNA drugs, laid the foundation for the listing of saRNA drugs, and is expected to become a new strategy for the treatment of cancer.

[0005] Ultrasound with microbubbles (USMB) is a new type of drug delivery carrier. It enhances cell permeability through cavitation and mechanical effects, delivers therapeutic drugs such as small nucleic acid drugs, chemotherapy drugs and targeted drugs to target organs, and promotes gene or drug release, thereby enhancing the efficacy of drugs. Ultrasound with microbubbles can avoid drug degradation in the blood, increase local drug concentration, reduce adverse reactions, and reduce systemic toxicity. It is a safe, stable and efficient drug delivery carrier that shows great potential in cancer treatment. So far, the preparation method of nano-microbubbles loaded with LRIG1 small activating RNA has not been reported at home and abroad. Summary of the invention

[0006] Nanobubbles loaded with saRNA can deliver drugs to target organs and release them in a controllable manner without damaging the normal functions of cells, thereby increasing local drug concentrations and reducing adverse reactions. It has the advantages of safety, stability, and efficiency.

[0007] The present invention uses thin film hydration method and mechanical oscillation method to prepare blank nano bubbles: Dipalmitoylphosphatidylcholine DPPC, polyethylene glycol 2000-distearoylphosphatidylethanolamine DSPE-PEG2000, and DC-cholesterol DC-Chol were added with anhydrous ethanol and mixed and dissolved, and then formed into a film by rotary evaporation. The obtained film was heated and dissolved again, and then filled with perfluoropropane under negative pressure and ultrasonically oscillated to obtain blank nanobubbles N-NBs.

[0008] Using the positive point property of PLL, negatively charged saLRIG1 was loaded onto blank nanobubbles by electrostatic adsorption. The specific process is as follows: N-NBs were added to poly-lysine solution, saLRIG1 was added after cold incubation, and nanobubbles loaded with saLRIG1 were obtained by cold incubation again, thus obtaining LRIG1 nanobubbles saLRIG1-NBs.

[0009] In some specific embodiments, the refrigerated incubation refers to incubation in a 4-6° C. refrigerator for 10-30 minutes.

[0010] In the step (1), the mass ratio of dipalmitoylphosphatidylcholine DPPC, polyethylene glycol 2000-distearoylphosphatidylethanolamine DSPE-PEG2000, and DC-cholesterol DC-Chol is 3-8:1-2:1-2.

[0011] In some specific embodiments, DPPC (dipalmitoylphosphatidylcholine), DSPE-PEG2000 (polyethylene glycol 2000-distearoylphosphatidylethanolamine), and DC-Chol (DC-cholesterol) are used for preparation at a mass ratio of 5:2:2.

[0012] In the step (2), the concentration of PLL is 1-5 mg / ml; the concentration of saLRIG1 is 20-50 nmol; and the concentration of N-NBs is 5 μL-30 μL.

[0013] In a preferred embodiment, the concentration of PLL is 3-4 mg / ml; the concentration of saLRIG1 is 30-40 nmol; and the concentration of N-NBs is 20-30 μL.

[0014] More preferably, the concentration of PLL is 4 mg / ml; the concentration of saLRIG1 is 30 nmol; and the concentration of N-NBs is 20 μL.

[0015] Another technical solution of the present invention is to provide a LRIG1 nanobubble prepared according to the above method, wherein saLRIG1 is loaded on the microbubble, wherein the particle size of the LRIG1 nanobubble is 500-600nm, and the concentration of the blank nanobubble is (1.0-2.0)×10 9 / mL.

[0016] The nano-microbubbles prepared by the invention are spherical, concentratedly distributed and have uniform particle sizes.

[0017] Another technical solution of the present invention is to use the LRIG1 nanobubbles prepared by the method described in the present invention in the preparation of drugs for treating cervical cancer.

[0018] In the process of realizing the application in preparing the drug for treating cervical cancer, ultrasonic treatment is performed, and the ultrasonic conditions are ultrasonic frequency 1-5 MHz and ultrasonic intensity 1.5-3.0 W / cm 2 , duty cycle 40-50%, irradiation time 1-2min.

[0019] The ultrasonic conditions are preferably an ultrasonic frequency of 1 MHz and an ultrasonic intensity of 1.5 W / cm 2 , duty cycle 50%, irradiation time 2min.

[0020] The LRIG1 nanobubbles are used in the preparation of drugs for treating cervical cancer by targeted activation of cervical cancer HeLa cells.

[0021] The LRIG1 nanobubbles are used in the preparation of drugs for treating cervical cancer by enabling saLRIG1 to enter cervical cancer HeLa cells and upregulating the LRIG1 protein content.

[0022] The LRIG1 nanobubbles are used in the preparation of drugs for treating cervical cancer by inhibiting the EGFR / AKT pathway to inhibit the proliferation of HeLa cells, and / or increasing the expression of E-cad protein and reducing the expression of N-cad protein to inhibit the migration of HeLa cells.

[0023] saLRIG1-NBs can increase the accumulation of saLRIG1 in HeLa cells under ultrasound, and significantly upregulate the protein expression level of LRIG1, thereby inhibiting the proliferation of HeLa cells by inhibiting the EGFR / AKT pathway. It also inhibits the migration of HeLa cells by downregulating the expression of N-cadherin and upregulating the expression of E-cadherin. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Characterization of saLRIG1-NBs. A: Morphological distribution of sa-LRIG1-NBs under fluorescence microscope (white light image 600×) B: Fluorescence image of sa-LRIG1-NBs under fluorescence microscope (400×); C: Particle size of saLRIG1-NBs (562.5±23.1nm) D: Scanning electron microscope image of saLRIG1-NB (5000×).

[0025] Figure 2 The effect of PLL and saRNA concentration on the amount of gene carrier. ⑴PLL (1 mg / ml) + saLRIG1 (20 nmol); ⑵PLL (2 mg / ml) + saLRIG1 (20 nmol); ⑶PLL (3 mg / ml) + saLRIG1 (20 nmol); ⑷PLL (4 mg / ml) + saLRIG1 (20 nmol); ⑸PLL (5 mg / ml) + saLRIG1 (20 nmol); ⑹PLL (3 mg / ml) + saLRIG1 (30 nmol); ⑺PLL (3 mg / ml) + saLRIG1 (40 nmol); ⑻PLL (3 mg / ml) + saLRIG1 (50 nmol).

[0026] Figure 3 To detect the gene quantity of saLRIG1-NBs.

[0027] Figure 4 Fluorescence image of HeLa cells uptake of SaLRIG1-NBs (100×); A: Fluorescence image of SaLRIG1-NBs cell uptake (100×); B: LRIG1 protein expression.

[0028] Figure 5 MTT assay was used to detect the inhibition of HeLa cell proliferation by saLRIG1-NBs. *: P<0.05, **: P<0.01, ***: P< 0.001 .

[0029] Figure 6 A is a crystal violet staining observation of saLRIG1-NBs inhibiting the proliferation of HeLa cells (100×), and B is a crystal violet staining statistical analysis chart. *: P<0.05, **: P<0.01, ***: P<0.001 .

[0030] Figure 7 A is a graph showing the inhibition of HeLa cell proliferation by saLRIG1-NBs in clonal formation, and B is a graph showing the statistical analysis of clonal formation. *: P<0.05, **: P<0.01, ***: P<0.001 .

[0031] Figure 8 A is a graph showing the cell scratch wound healing ability observation of saLRIG1-NBs inhibiting the migration of HeLa cells (100×), and B is a statistical analysis graph showing the cell scratch wound healing ability; *: P<0.05, **: P<0.01, ***: P<0.001 .

[0032] Fig. 9 Transwell assay to observe the inhibition of HeLa cell migration by saLRIG1-NBs (100×). DETAILED DESCRIPTION

[0033] Example 1 Preparation of nanobubbles NBs DPPC, PEG2K and DC-Chol were weighed in a ratio of 5:2:0.5 and placed in a spherical glass bottle. An appropriate amount of chloroform was added to completely dissolve the above reagent powders. The mixture was evaporated in a rotary evaporator at 40°C for 1 h. At this time, a uniform film-like substance is formed on the wall of the glass bottle. Add an appropriate amount of PBS glycerol mixture (volume ratio 9:1) to the spherical glass bottle after film formation, place it in a constant temperature water bath at 45°C for 1 hour, and after the lipid film is completely dissolved in the PBS glycerol mixture, use a pipette to dispense it into glass vials, 400μL per bottle. After sealing the vial, use a homemade inflation and ventilation equipment to inflate and ventilate the vial, insert the empty needle connected to the inflation and ventilation equipment into the bottle through the vial cap (note that it cannot be extended into the liquid surface), use a negative pressure vacuum pump to pump for 25 seconds, then inflate with perfluoropropane for 25 seconds. After four cycles, fix the vial filled with perfluoropropane to the arm of the high-speed oscillator with tape, and oscillate for a total of 90 seconds to obtain milky white nano-microbubbles (see Figure 1 The Blank NBs and SaLRIG1-NBs prepared in this experiment were spherical, concentrated, and uniform in size (562.5±23.1nm). The NBs concentration was approximately 1.4 ± 0.26 × 10 9 / mL. Fluorescence microscopy showed that saLRIG1-NBs emitted obvious red fluorescence, indicating that saLRIG1 could be successfully loaded onto microbubbles through the principle of positive and negative charge attraction.

[0034] Example 2 Effects of PLL and saLRIG1 concentrations on gene loading PLL with a concentration of 5 mg / mL was diluted with PBS to 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL at a volume of 1:1, and then placed in a 4-degree refrigerator for incubation for 20 minutes with blank NBs (prepared in Example 1). The surface of the blank NBs was positively charged, and saLRIG1-CY3 with a negative charge concentration of 20 nmol was incubated in a 4-degree refrigerator for 20 minutes. After the mixture was diluted to the same ratio, the number of red fluorescent signals after the blank NBs combined with saLRIG1-CY3 was observed under a fluorescence microscope. The results showed that when the concentration of PLL was 1-5 mg / mL, the number of red fluorescent signals was relatively large, and at 4 mg / mL, the number of red fluorescent signals was the largest. Subsequently, the microbubbles carrying 4 mg / mL PLL were incubated with 30 nmol, 40 nmol, and 50 nmol saLRIG1-CY3, respectively, and it was found that the number of red fluorescent signals at 30-50 nmol was relatively large (see Figure 2 ).

[0035] Example 3 Agarose gel detection of gene quantity in saLRIG1-NBs 0, 5, 10, 15, 20, 25 and 30 μL of blank NBs solution were taken and gently mixed with 3 μL of 30 nmol saLRIG1 and placed at 4°C for 15 minutes. After agarose gel electrophoresis (120 mV for 10 minutes), it was found that with Free saLRIG1 as the control group, the amount of saLRIG1 gradually decreased with the increase of NBs solution volume until there was no obvious band shadow at 20 μL. Therefore, 5-30 μL can bind to 3 μL of 30 nmol saLRIG1 (see Figure 3 ).

[0036] Example 4 Cell culture HeLa cells were subcultured by the Key Laboratory of Tumor Microenvironment and Immunotherapy of China Three Gorges University using DMEM culture medium with 10% serum, 1% penicillin and streptomycin added. They were cultured in a cell culture incubator at 37°C and 5% CO2 saturation, and the cells in the logarithmic growth phase were selected for use.

[0037] Example 5 Observation of the cellular uptake characteristics of SaLRIG1-NBs under fluorescence microscopy Take 20x10 HeLa cells in the logarithmic growth phase per well. 5 The cells were seeded in 6-well plates at a high density and cultured for 12 h. After the culture medium was replaced, SaLRIG1-NBs were added and treated with UTMD (ultrasound conditions were ultrasound frequency of 1 MHz and ultrasound intensity of 1.5 W / cm 2 After irradiation for 2 min, the cells were incubated at 37°C for 6 h, the culture medium was discarded, 4% paraformaldehyde was added for fixation for 15 min, the cell nucleus fluorescent dye Hochest was added for staining for 15 min, and PBS was rinsed 3 times. The cell uptake of saLRIG1 was observed under a fluorescence microscope.

[0038] The results showed that under the action of ultrasound of a certain intensity, microbubbles can carry saLRIG1 into cells, and compared with the saLRIG1 Mock group, the red fluorescence of the saLRIG1-NBs+US group increased significantly. The total protein of cells in each treatment group was extracted, and Western Blot detection showed that the LRIG1 protein content in the saLRIG1-NBs+US group increased significantly. Therefore, the action of ultrasound on microbubbles can not only enable saLRIG1 to enter cells, but also improve the transfection efficiency of saLRIG1 and upregulate the LRIG1 protein content. In addition, it was found that the red fluorescence of saLRIG1 overlapped with the cell nucleus stained with the cell dye Hochest blue. Therefore, it shows that SaLRIG1-NBs are mainly taken up in the cell nucleus, and compared with the saLRIG1 Mock group, the red fluorescence of the NBs-saLRIG1+US group increased significantly (see Figure 4 ).

[0039] Example 6 MTT assay Four replicate wells were set up in each group, with 5,000 cells in each well, and the experiment was repeated three times. After culturing the cells for 72 hours, the old culture medium was aspirated and the MTT solution was diluted with DMEM culture medium to a concentration of 10% MTT working solution. 20 μL of MTT working solution was added to each experimental well. The plate was then placed in a cell culture incubator and cultured for another 4 hours in the dark. After culture, the 96-well plate was removed, the supernatant was discarded (sterile operation is not required), 150 μL of DMSO was added to each well, tin foil was used to protect from light, and the plate was gently shaken on a shaker at room temperature for 15 minutes. The plate was placed in a full-wavelength enzyme-linked immunosorbent assay (ELISA) for detection, and the OD value of each well was measured at a wavelength of 490 nm. The formula for calculating the cell growth inhibition rate is: Cell viability (%) = (1-OD value of the experimental group-OD value of the control group / OD value of the control group) × 100%. Results It was found that the cell viability of the NBs-saLRIG1+US group was significantly reduced (see Figure 5 ).

[0040] Example 7 Crystal violet staining Take 1x10 HeLa cells in logarithmic growth phase per well. 5 The cells were inoculated in a 24-well plate at a high density and cultured for 12 hours. After replacing the culture medium, SaLRIG1-NBs were added. After UTMD treatment, the cells were incubated at 37°C for 24 hours, 48 ​​hours, and 72 hours, respectively, and then washed twice with PBS. They were fixed with 4% paraformaldehyde for 15 minutes, washed once with PBS, and then stained with 0.1% crystal violet solution at room temperature in the dark for 15 minutes. Photographs were taken with an optical microscope, and it was found that the cell proliferation rate of the NBs-saLRIG1+US group was significantly inhibited compared with the control group (see Figure 6 ).

[0041] Example 8 Colony formation assay HeLa cells in the logarithmic growth phase were seeded at a density of 500 cells per well in a 6-well plate and cultured for 12 hours. After replacing the culture medium, SaLRIG1-NBs were added. After UTMD treatment, the cells were incubated at 37°C for 7 days, and then the culture medium was discarded. 4% paraformaldehyde was added for fixation for 15 minutes, and then washed once with PBS and 0.1% crystal violet solution was added for staining at room temperature in the dark for 15 minutes. After taking pictures and counting with imaje J, it was found that the number of cell colonies in the NBs-saLRIG1+US group was significantly reduced (see Figure 7 ).

[0042] Example 9 Wound healing assay Ultra-clean bench was sterilized by ultraviolet for more than 15 min. The 6-well cell culture plate, culture medium, trypsin and other reagents were placed in the ultra-clean bench for disinfection and rewarming. A horizontal line was evenly drawn on the bottom of the 6-well plate with a marker pen in the ultra-clean bench (to facilitate finding the same position when taking pictures). Cells in the logarithmic growth phase were taken, and trypsin was added to digest and collect the cell precipitate. According to the cell growth rate, about 40×10 5 cells, the total volume of culture medium in each well was 2 mL, and after 12 hours (overnight) when the cells adhered to the wall and the cell fusion reached 70-80%, a ruler was used as a reference, and a 200 μL pipette tip was used to gently scratch. The pipette tip should be vertical, and it should be noted that the scratching should be completed at one time. Do not scratch back and forth repeatedly. At least 3 lines should be drawn in each well, and each line should be 0.5 cm apart. Then the old culture medium was discarded, and the cells were gently rinsed 3 times with 1× PBS to wash off the cells that fell off due to the scratches. SaLRIG1-NBs were added and cultured with complete culture medium containing 2% FBS. After UTMD treatment, images were taken at 0 h, 24 h, 48 h, and 72 h, and the scale and the time of taking the images were marked, and the analysis results were recorded. Under an optical microscope, it was observed that the wound healing rate of the NBs-saLRIG1+US group was significantly slower than that of the control group (see Figure 8 ).

[0043] Example 10 Transwell experiment The clean bench was sterilized by ultraviolet and the 24-well cell culture plate was sterilized and ready for use. 500 mL of 12% FBS culture medium was added to the new 24-well plate, and a small chamber was gently placed into each well, with the bottom of the chamber close to the surface of the culture medium. After 48 hours of cell treatment, the cells were digested and collected, and the cell pellet was resuspended in the basal culture medium. The cells were counted routinely to a cell concentration of 5×10 5 / mL. Vertically add 100μL of cell suspension into the prepared chamber, shake gently to mix, and continue to culture for 24h. Use tweezers to remove the chamber (no need for sterility), rinse three times with PBS, avoid facing the bottom of the chamber, gently wipe the cells on the wall of the chamber with a cotton swab, put the chamber into a clean methanol well, and fix it for 30 min. Take out the chamber, rinse three times with PBS, put it into a 0.1% crystal violet solution well for staining for 20 min, wash it with running water, observe the number of cells that pass through the membrane under an optical microscope, record the results and analyze them. The results showed that the ability of cells in the NBs-saLRIG1+US group to penetrate the mold was significantly weakened (see Fig. 9 ).

Claims

1. A method for preparing LRIG1 nanobubbles, characterized in that: The steps include: (1) Preparation of blank nanobubbles: Dipalmitoylphosphatidylcholine DPPC, polyethylene glycol 2000-distearoylphosphatidylethanolamine DSPE-PEG2000, and DC-cholesterol DC-Chol were added with anhydrous ethanol and mixed and dissolved, and then rotary evaporated to form a film. The obtained film was heated and dissolved again, and then filled with perfluoropropane under negative pressure, and then ultrasonically oscillated to obtain blank nanobubbles N-NBs; (2) Preparation of saLRIG1-loaded nanobubbles by the poly-lysine PLL method: N-NBs were added to a poly-lysine solution, and saLRIG1 was added after cold incubation. The saLRIG1-loaded nanobubbles were obtained by cold incubation again.

2. The method for preparing LRIG1 nanobubbles according to claim 1, characterized in that: In step (1), the mass ratio of dipalmitoylphosphatidylcholine DPPC, polyethylene glycol 2000-distearoylphosphatidylethanolamine DSPE-PEG2000, and DC-cholesterol DC-Chol is 3-8:1-2:1-2.

3. The method for preparing LRIG1 nanobubbles according to claim 1, characterized in that: In step (2), the concentration of PLL is 1-5 mg / ml; the concentration of saLRIG1 is 20-50 nmol; and the concentration of N-NBs is 5 μL-30 μL.

4. The method for preparing LRIG1 nanobubbles according to claim 3, characterized in that: In step (2), the concentration of PLL is 3-4 mg / ml; the concentration of saLRIG1 is 30-40 nmol; and the concentration of N-NBs is 20-30 μL.

5. The LRIG1 nanobubbles prepared according to the method of any one of claims 1 to 4, wherein saLRIG1 is loaded on the microbubbles, wherein the particle size of the LRIG1 nanobubbles is 500-600 nm, and the concentration of the blank nanobubbles is (1.0-2.0)×10 9 / mL.

6. Use of the LRIG1 nanobubbles prepared by the method according to any one of claims 1 to 4, or the LRIG1 nanobubbles according to claim 5 in preparing a drug for treating cervical cancer.

7. The use according to claim 6, characterized in that: In the process of realizing the application in preparing the drug for treating cervical cancer, ultrasonic treatment is performed, and the ultrasonic conditions are ultrasonic frequency 1-5 MHz and ultrasonic intensity 1.5-3.0 W / cm 2 , duty cycle 40-50%, irradiation time 1-2min.

8. The use according to claim 7, characterized in that: The LRIG1 nanobubbles are used in the preparation of drugs for treating cervical cancer by targeted activation of cervical cancer HeLa cells.

9. The use according to claim 8, characterized in that: The LRIG1 nanobubbles are used in the preparation of drugs for treating cervical cancer by enabling saLRIG1 to enter cervical cancer HeLa cells and upregulating the LRIG1 protein content.

10. The use according to claim 9, characterized in that: The LRIG1 nanobubbles are used in the preparation of drugs for treating cervical cancer by inhibiting the EGFR / AKT pathway to inhibit the proliferation of HeLa cells, and / or increasing the expression of E-cad protein and reducing the expression of N-cad protein to inhibit the migration of HeLa cells.

Images